Although the non-canonical NF-κB pathway has been clearly linked to a number of physiological responses, in some cases the activating receptor/ligand pair has not been clearly identified. Given the restriction of the expression of BAFF receptors to the lymphoid compartment, it is unsurprising that most identified roles for BAFF are in lymphocytes. Indeed the largest body of evidence for the role of BAFF mediated NF-κB2 signaling is in relation to B cell biology, including peripheral B cell survival and maturation, the generation of antibody response and the maintenance of PCs, all of which are further discussed below (**Figure 2**). Interestingly, NF-κB2 signaling is also essential for the development and organization of secondary lymphoid organs such as spleen, lymph node and Peyer's patches [reviewed in Ref. (63)]. However these functions of NF-κB2 appear to be mediated entirely by LT-βR signaling, and do not involve BAFF or its receptors and thus will not be further discussed here.

### **PERIPHERAL B CELL SURVIVAL AND MATURATION**

B cells, like all cells of the hematopoietic lineage develop from hematopoietic stem cells in the bone marrow. The defining feature of B cell development is the expression of a B cell receptor (BCR), composed of immunoglobulin heavy and light chains derived from V(D)J recombination, which combine to define the unique specificity of each BCR and also of the antibody that the B cell will secrete should it eventually differentiate into a plasma cell. Pre-B cells in the bone marrow express only a recombined immunoglobulin heavy chain along with a pseudo-light chain. Final rearrangement of the light chain immunoglobulin genes and expression of a fully functional BCR is the defining feature of an immature B cell in the bone marrow which is then ready to egress into the periphery. In the periphery immature B cells have been further categorized into transitional subsets T1, T2, and T3. There is much controversy of the number of these subsets (some researchers defining two T2 subsets, a T2 follicular and T2 MZ), their position in the schema of development and even their functionality (it is possible that the T3 population represent not a transitional subset of immature B cells, but rather an anergic population). So while the exact developmental pathways of immature B cells in the periphery are not well defined, their outcomes are. Two subsets of mature B cells can be found in the periphery: follicular B cells,found in the spleen, lymph nodes and circulating in the

**BAFF/BAFFR-mediated NF-**κ**B2 activation**. Activation of non-canonical NF-κB signaling in response to BAFF contributes to key

events throughout the lifespan of a B cell. These include facilitating the survival of immature (Imm) B cells in the periphery and activating transcriptional programs which allow them to mature into follicular

and marginal zone (MZ) B cells; contributing to some T-independent immune response; extending the duration of germinal center (GC) reactions; and maintaining long lived plasma cells (PCs) in the bone marrow. Refer to Section "Tissue Responses and Effector Functions: The Outcomes of NF-κB2 Signaling in Response to BAFF" of text for further details.

blood, andMZ B cells, a sessile populationfound only in the spleen. The various genetically modified mice lines discussed below have demonstrated the importance of BAFF/BAFFR-mediated NF-κB2 signaling to the survival of mature peripheral B cell subsets and their maturation (**Figure 2**). It should be noted that a further population of B cells exists, known as B1 B cells. These arise in the fetal liver and mainly populate the peritoneal cavity, though some are also found in the spleen. Their survival appears to be independent of BAFF and BAFFR signaling and thus they will not be further discussed here.

BAFF-deficient (64, 65) and BAFFR-deficient (66, 67) mice exhibit very similar phenotypes, with normal early B cell development in the bone marrow, but a paucity of peripheral B cell populations after transitional stage 1, suggesting that BAFF/BAFFR signaling is essential for B cell maturation and survival in the periphery. In addition a naturally occurring mutant strain A/WySnJ, which lacks peripheral B cells, was identified to have a mutation in the Tnfrsf13c gene that encodes BAFFR (68, 69) further validating the role of BAFF and BAFFR in supporting peripheral B cell survival. In contrast, BAFF transgenic mice display expanded mature B cells in the periphery, demonstrating that the constitutive production of BAFF is limiting and restricts total B cell numbers in wild-type mice (70, 71). It was of course possible that signaling through the other BAFF receptors may contribute to B cell survival. However, the phenotype of BCMA- and TACIdeficient mice, demonstrate that BAFF/BAFFR signaling is the main contributor to this phenotype. BCMA-deficient mice do not display any B cell specific phenotype (65, 72) and TACI-deficient mice actually exhibit a slight hyperplasia of B cells, suggesting negative regulatory role for this receptor in maintaining B cell populations (73, 74).

The accumulation of B cells in BAFF transgenic mice is associated with autoantibody production and autoimmune manifestations (70, 71) suggesting that induced or increased production of BAFF may rescue some self-reactive B cells from elimination from the repertoire. This was verified to be the case using transgenic mouse models of B cell self-reactivity (75, 76) and may contribute to the onset of disease in a number of human autoimmune diseases associated with increased levels of circulating BAFF (21).

As described above, BAFF/BAFFR signaling efficiently activates the NF-κB2 pathway and genetic evidence also demonstrates that it is this pathway that primarily supports peripheral B cell survival. Thus NF-κB2-deficient mice also display a deficit in peripheral B cells (77, 78) and more importantly the survival *ex vivo* of those that do exist cannot be rescued by addition of BAFF, emphasizing that this pathway is required to facilitate BAFF's survival effect (79). The survival of some periphery B cells to maturity in NF-κB2-deficient mice suggests that other signaling pathways also contribute to B cell survival and maturation. Indeed NFκB1/NF-κB2 double-deficient mice show a more severe loss of peripheral B cells (80) and certainly NF-κB1 signaling activated by the BCR contributes to B cell survival. At least part of the canonical pathway's contribution is indirect, as p100 and RelB are NF-κB1 regulated genes (81–83). Thus canonical activation may be required to furnish the cell with sufficient components of the non-canonical pathway to allow it to promote B cell survival (84). Over activation of the canonical pathway can substitute for loss of BAFFR (85). However, it is likely that under physiological conditions both pathways contribute to B cell survival and that the BCR is the primary activator of canonical NF-κB signaling, whilst BAFFR is primarily responsible for activating NF-κB2 signaling.

NF-κB inducing kinase is the central kinase controlling NF-κB2 activity. Deficiency of NIK (86) or mutation in its kinase domain (alymphoplasia – aly mice) (87) results in severe lymphoid abnormalities including loss of lymph nodes and Peyer's patches as well as B cell lymphopenia and splenic disorganization. As mentioned above control of lymphoid organ formation and organization has been attributed to LT-βR mediated NF-κB2 signaling and indeed these mice phenocopy LT-βR-deficient mice with respect to these phenotypes (88). These abnormalities are also more similar to NF-κB1/NF-κB2 double-deficient mice than either of the single deficient mice alone. Indeed it has been suggested that, in addition to its role in NF-κB2 signaling, NIK may also contribute to NFκB1 signaling. Despite the many additional phenotypes present in the aly mice, they retain the B cell lymphopenia common to mice models with defects in BAFF/BAFFR signaling and NF-κB2 signaling. Thus the phenotype of NIK-deficient mice also reflects the role of BAFFR-mediated NF-κB2 activation in B cell survival and maturation.

In contrast to deletion of other components of the NF-κB2 pathway, deletion of IKKα results in perinatal lethality (89) suggesting that this kinase has additional roles outside NF-κB signaling. Bone marrow chimeras for IKKα-deficient fetal liver cells or mice expressing a kinase-inactive version of IKKα, do display B cell abnormalities similar to other mice models with defective NF-κB2, including a deficit in peripheral B cell numbers and impaired GC formation (90, 91). However, given that non-NF-κB2 related roles for IKKα exist and may confound NF-κB2 related phenotypes, further detailed description of these mice will not be undertaken here.

One outcome of NF-κB2 signaling which is thought to promote B cell survival is the upregulation of anti-apoptotic molecules such as B cell CLL/lymphoma 2 (Bcl-2) (92, 93). Indeed transgenic overexpression of Bcl-2 in mice models lacking functional BAFFR signaling was sufficient to restore mature B cell populations (66,94, 95). However while follicular B cells were observed in these models, MZ B cells were not restored. This demonstrated that in addition to providing survival signals to peripheral B cells, BAFF/BAFFR signaling is also essential for the complete maturation of B cells into MZ phenotype.

In line with this finding, mouse models with hyperactive NFκB2 signaling display an expansion of the MZ B cell population, confirming that NF-κB2 signaling promotes this phenotypic outcome. These models include BAFF transgenic mice (71), as well as mice lacking components of the ubiquitin ligase complex which facilitates NIK degradation, namely TRAF2, TRAF3, or the cIAPs (43, 96–98). TRAF2 and TRAF3 play co-operative, but distinct roles in facilitating both the constitutive suppression of NF-κB2 signaling and the BAFFR-mediated activation of this pathway, the molecular details of which are described above. Mice completely lacking either TRAF are perinatally lethal (99, 100). Thus the use of a B cell specific deletion system to investigate their role in B cells has been important in furthering our understanding of these molecules. Lack of either TRAF2 or TRAF3 specifically in B cells

led to increased NF-κB2 signaling and an enlarged mature B cell population, with the largest expansion being in the MZ compartment of the spleen (43). While loss of TRAF2 or TRAF3 from B cells produced a similar phenotype, the implication from *in vitro* work that the two molecules are not performing the same function in regulating BAFFR signaling is supported by evidence from *in vivo* work. Firstly, the fact that loss of either TRAF resulted in the same phenotype, that is one was not able to completely compensate for the loss of the other. Secondly, mice lacking both TRAF2 and TRAF3 from their B cells did not display a more extreme phenotype in terms of levels of NF-κB2 activation or expansion of mature B cell populations, as would be expected if TRAF2 and TRAF3 were able to partially compensate for each other (43).

In contrast to the TRAF proteins, cIAP1 and cIAP2 are able to compensate for each other in their roles in BAFFR signaling. Thus mice completely deficient in cIAP2 or mice lacking cIAP1 in B cells, both displayed normal NF-κB2 activation in their B cells and consequently normal B cell survival and maturation. However mice lacking both cIAP1 and cIAP2 from their B cells had hyperactive NF-κB2 and expanded mature B cell populations (98). cIAP1/2-deficient B cells also contained high levels of TRAF3 (98). This demonstrated definitively that *in vivo* cIAP1 and cIAP2 are able to compensate for each other in facilitating both the degradation of NIK to suppress NF-κB2 activation and the degradation of TRAF3 in order to activate it. Indeed the ubiquitin ligase activity of cIAPs was shown to be essential *in vivo* to mediate these processes by the development of mice in which cIAP2 contained mutations which inactivated its E3 ligase activity (101). These mice displayed increase peripheral B cells and expanded MZs in the spleen. The authors proposed that mutant cIAP2 also had the affect of inhibiting the association of cIAP1 with TRAF2 and thus with the ubiquitin ligase complex which regulates NIK, thus explaining why in this case cIAP1 was not able to compensate for cIAP2 as it is able to in the complete absence of cIAP2.

The loss of either BAFF (43), or BAFFR (98) can be completely compensated for in terms of B cell survival and maturation by disruption of the TRAF/cIAP ubiquitin ligase complex and thus constitutive hyperactivation of NF-κB2. Whilst other evidence presented above shows that BAFF, BAFFR and NF-κB2 signaling are all able to individually contribute to B cell survival and maturation, it is these experiments which definitively demonstrate that activation of NF-κB2 sufficiently compensates for loss of BAFF or BAFFR. That is the primary, perhaps even the exclusive purpose of BAFF/BAFFR signaling in B cells is the activation of NF-κB2 signaling and it is this pathway which facilitates the transcriptional effects required in order for B cells to survive and mature in the periphery. However, because of the tight link between BAFF signaling and B cell survival, the specific genes that are upregulated by BAFF in B cells and their roles in B cell physiology have been difficult to verify and are not well understood.

### **B CELL ANTIBODY RESPONSES**

The main function of B cells is to protect the body from foreign invasion by the production of antibodies. A B cell encountering a foreign antigen that matches the specificity of its BCR can ultimately differentiate into a plasma cell, a specialized antibodyproducing factory capable of making large amounts of secreted antibodies. Most foreign antigens illicit a T cell dependent antibody response, in which activated B cells form GCs where they are able interact with cognate T follicular helper cells as well as receive survival signals from support cells such as follicular dendritic cells (FDC) in order to select cells with high affinity for the foreign antigen that have been generated by somatic hypermutation (SHM) of the immunoglobulin genes [reviewed in Ref. (102)]. Alternatively some antigens are able to illicit maximal responses from B cells even in the absence of T cells and germinal center formation – that is they illicit T cell independent antibody responses. These antigens tend to be generic antigens,for example the repeating carbohydrate units which make up bacterial cell walls. BAFF/BAFFR signaling has been implicated in both these processes (**Figure 2**).

### **The germinal center reaction**

Despite the severe restriction on the survival of peripheral B cell in mice deficient for either BAFF or BAFFR, those remaining are able to form of relatively normal GCs in which SHM can occur (67, 103, 104). Thus BAFF/BAFFR signaling activating NF-κB2 is not required for these processes. However it appears they do play a role in the maintenance of GCs as GCs in mice lacking BAFF/BAFFR signaling dissipate a few days after they form. Interestingly the reasons for the instability differed between the two models. A lack of BAFF meant the FDC reticulum, important for trapping and presenting immune complexes failed to mature, probably leading to a lack of stimulation being received by the B cells present in the GC and subsequent breakdown of the GC (103, 104). However the FDC reticulum was normal in A/WySnJ (BAFFR mutant) mice, and B cells instead exhibited a proliferation defect after initial germinal center formation (103). The most obvious explanation for this discrepancy is that BAFF acts directly on FDCs to promote their maturity and does so through a receptor other than BAFFR. In addition BAFF acting through the BAFFR on B cells is also required for germinal center maintenance. However this explanation is unlikely as both TACI and BCMA-deficient mice (72, 73) display normal GC formation, and therefore were unlikely to be the receptors responsible for the failure of FDC maturation in BAFF-deficient mice. Whatever the precise roles of BAFF are in supporting GC persistence, the fact that activated FDCs are a rich source of BAFF (105) makes them the most likely source of the BAFF required to support the GC.

A second explanation is that the maturity of the B cells entering the GC may impact on FDC maturation as this process is known to require interaction with GC B cells and those lacking signals from BAFF may be too immature to provide these signals to the FDCs. Indeed some B cells in A/WySnJ are able to mature past the transitional stages in contrast to BAFF- or BAFFR-deficient mice (106), thus the greater maturity of the these cells may explain their ability to support maturation of the FDC reticulum, where the B cells from BAFF-deficient mice cannot. The increased maturity of B cells in A/WySnJ mice may be due to the ability of the mutant BAFFR to retain some signaling, as only the last 8 amino acids of this mutant receptor have been replaced with an unrelated transposon sequence (69). An examination of GC formation in BAFFR-deficient mice would help to answer this question and indeed like BAFF-deficient and A/WySnJ mice GCs in BAFFRdeficient mice form, but fail to be maintained (67), However, whether this is due to lack of FDC maturation or a B cell proliferation defect has unfortunately not been investigated. The authors did suggest that the defect they observed in BAFFR-deficient mice was less severe than that observed in BAFF-deficient mice. If this is the case it does suggest that BCMA or TACI, while not playing any essential role in GC maintenance as indicated by the phenotypes of their respective knockout mice, may be able to compensate for some of the functions of BAFFR in its absence.

It is highly likely given the involvement of BAFFR and its strong activation of the NF-κB2 pathway, that NF-κB2 signaling is involved in GC maintenance. However this has been difficult to confirm as NF-κB2-deficient mice completely fail to form GCs and FDC networks suggesting the NF-κB2 is a key signaling pathway involved in the initiation of GCs as well as their maintenance (77, 78). Neither BAFF nor BAFFR are involved in initial GC formation, suggesting that in this case the NF-κB2 pathway is being activated through a different ligand, possibly lymphotoxin αβ. Of course without formation of GC it is impossible to assess their maintenance. Adoptive transfer experiments in RelB-deficient mice showed that in order to restore GC formation, RelB was required in radioresistant stromal cells rather than hematopoietic cells (107). However, as GCs were only examined on day 10 postimmunization,it is impossible to say whether GC maintenance was also defective when RelB was absence from hematopoietic cells. In alymphoplasia (*aly* – NIK mutant) mice, similar adoptive transfer experiments revealed that functional NIK was indeed required in the stromal cells for FDC formation, but was also required in hematopoietic cells for GC formation (108). These apparently conflicting results as to the requirement for NF-κB2 signaling in hematopoietic cells may be explained by functional redundancy of RelB with another member of the NF-κB family, whereas NIK, the central kinase of the NF-κB2 pathway is absolutely required.While these results attempt to identify the cell types in which NF-κB2 signaling is required, it remains difficult to link its usage with the activating receptor, as indeed it is likely that more than one receptor contributes at various stages in GC formation and maintenance. Thus the questions surrounding GC maintenance remain.

Whilst normal NF-κB2 appears necessary for GC formation, even hyperactive NF-κB2 cannot rescue GC formation in the absence of other signals required, for example T cell help in the form of CD40-CD40L interactions. The adapter molecules TRAF2, TRAF3, and cIAP1/2 are all involved in CD40 signaling in addition to their regulation of BAFFR signaling. While indeed they maintain their role as gatekeepers of NF-κB2 signaling, almost certainly through analogous activation methods as BAFFR, they are also variously involved in the JNK, MAPK, and NF-κB1 pathways. Generally TRAF2 and cIAP1/2 are positive mediators of these pathways, whereas TRAF3 is a negative regulator (40, 98, 109). Thus in mice lacking these molecules in their B cells, TRAF3 deficient B cells are able to form and maintain GCs as they retain competent CD40 signaling, whereas GCs are stunted or almost completely absent in the TRAF2 and cIAP1/cIAP2-deficient mice, due to a lack of CD40 signaling, despite high NF-κB2 signaling (98). Likewise over expression of BAFF and thus NF-κB2 in BAFF transgenic mice is unable to rescue GC formation in CD40 deficient mice (110). These results emphasize the complex nature of the signaling required in order to establish and maintain GC

reactions. BAFF/BAFFR signaling contributes to these processes, but it is not a master regulator of them, as it is with B cell survival and maturation.

### **T-independent antibody responses**

The involvement of both TACI and BAFF in T-independent antibody responses seems to be quite clear [reviewed in Ref. (111)]. It is possible that the multimeric BAFF 60mer is the ligand responsible for these functions (112). However, as TACI is not a strong inducer of NF-κB2 either via its interaction with BAFF or APRIL (84), it is unlikely that NF-κB2 signaling plays a role in these processes. For some T-independent antigens the BAFFR, and thus possibly NFκB2 signaling, do appear to be involved. Antibody titers were lower in BAFFR-deficient mice compared to wild-type mice in response to NP-Ficoll or TNF-Ficoll but similar in response to Pneumovax vaccine (66, 67), suggesting that certain specific responses may require BAFFR signaling. In addition, some human patients with BAFFR deficiency also showed defects in mounting T-independent responses (113). However it remains unclear if BAFF/BAFFR signaling is directly required during a T-independent antibody response. It is possible that the defects observed in the absence of BAFFR, may be ascribed at least in part to a dramatic decrease in MZ B cells as this subset is recognized to be the origin of the early responders to T-independent antigens (114).

### **PLASMA CELL MAINTENANCE**

Plasma cells are highly differentiated B cells capable of secreting large amounts of antibody and, along with the production of memory B cells, are the main B cell outcome of an immune response. Whilst large numbers of antigen specific PCs exist during and immediately following an immune response, over time these numbers greatly reduce. However, it is thought that a small number of the PCs resulting from a particular immune response survive long term and are the source of "basal" immunoglobulin found circulating within the body. The bone marrow has been identified as a survival niche for these long lived PCs and a variety of cells including stromal cells, myeloid cells and granulocytes all contribute to the production of factors which attract and promote the survival of PCs in these sites [reviewed in Ref. (115)]. Among these survival factors are APRIL and BAFF. Neutralization of both ligands is required in order to ablate plasma cell survival in the bone marrow (19, 116), however it is likely that under physiological conditions APRIL plays a greater role than BAFF. This is supported by the findings that APRIL is better than BAFF at supporting plasma cell survival *in vitro* (117) and *in vivo* plasma cell survival in APRIL-deficient mice was greatly diminished, whereas it was normal in BAFF-deficient mice (117, 118). BCMA is the receptor which is most thought to contribute to plasma cell survival, with BCMA-deficient mice failing to sustain long lived PCs in the bone marrow (119). It is possible, however, that TACI and/or BAFFR may contribute to these responses.

Although BCMA is not thought to be a strong activator of NF-κB2 signaling, there is evidence that NF-κB2 signaling can contribute to plasma cell survival, at least in the disease state. Multiple myeloma is late stage B cell malignancy that arises from PCs in the bone marrow.Whilst the primary genetic lesions are immunoglobulin gene translocations and hyperdiploidy, a large number of secondary genetic mutations also characterize the progression of the disease and it is amongst these that the NF-κB pathways have been strongly implicated (120–122). Mutations that activate NFκB2 signaling including inactivating mutation in *TRAF2*, *TRAF3*, *cIAP1*, and *cIAP2*, as well as activating mutations or duplications of *NIK* and *NF-*κ*B2* have been identified in patients with multiple myeloma. These mutations are thought to contribute to the ability of the tumor to become independent in terms of its survival from the bone marrow microenvironment, that is no longer requiring BAFF and APRIL and other survival factors produced in the bone marrow. This in turn implies that NF-κB2 signaling is either normally involved in these processes for non-malignant PCs, or at least it can compensate for the signaling pathways involved under non-cancerous conditions. Thus it seems that BAFF induced NFκB2 signaling plays a role at almost every stage of a B cell's life (**Figure 2**).

## **CONCLUSION**

In addition to multiple myeloma, BAFF signaling as been implicated in a variety of autoimmune disorders, B cell malignancies and immunodeficiency disorders. It is also emerging that BAFF plays a role in regulating immune responses to infections [reviewed in Ref. (123)].While in many of these cases there is quite strong evidence that BAFF contributes to disease, further molecular details, such as whether BAFFR is the responsible receptor and whether activation of NF-κB2 also contributes to disease, require additional investigation. Given the central role of BAFF mediated NF-κB2 activation in the life span of B cells and the importance of B cells in attempting to control (in the case of infection) or potentially contributing to (in the case of autoimmunity) disease, it is likely that this signaling pathway indeed impacts greatly on our state of health and disease.

## **ACKNOWLEDGMENTS**

Sandra Gardam and Robert Brink are supported by personal Research Fellowships from the National Health and Medical Research Council of Australia.

### **REFERENCES**

apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. *J Biol Chem* (1999) **274**:15978–81. doi:10.1074/jbc.274.23.15978

BAFF-receptor signals and promotes B cell proliferation upon activation. *Immunity* (2006) **24**:729–39. doi:10.1016/j.immuni.2006.04.005

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 01 November 2013; paper pending published: 02 December 2013; accepted: 24 December 2013; published online: 06 January 2014.*

*Citation: Gardam S and Brink R (2014) Non-canonical NF-*κ*B signaling initiated by BAFF influences B cell biology at multiple junctures. Front. Immunol. 4:509. doi: 10.3389/fimmu.2013.00509*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Gardam and Brink. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# CD40-mediated activation of the NF-κB2 pathway

## **Bruce S. Hostager 1,2\* and Gail A. Bishop2,3,4,5\***

<sup>1</sup> Department of Pediatrics, The University of Iowa, Iowa City, IA, USA

<sup>4</sup> Department of Internal Medicine, The University of Iowa, Iowa City, IA, USA

5 Iowa City VA Medical Center, Iowa City, IA, USA

### **Edited by:**

Linda C. Burkly, Biogen Idec, Inc., USA

### **Reviewed by:**

Fulvio D'Acquisto, Queen Mary University of London, UK Ellen D. Cahir-McFarland, Biogen Idec, Inc., USA

### **\*Correspondence:**

Bruce S. Hostager, Department of Pediatrics, The University of Iowa, Iowa City, IA 52242, USA e-mail<EMAIL_ADDRESS>Gail A. Bishop, Department of Microbiology, The University of Iowa, Iowa City, IA 52242, USA e-mail<EMAIL_ADDRESS>
CD40 is a critical stimulatory receptor on antigen-presenting cells of the immune system. CD40-mediated activation of B cells is particularly important for normal humoral immune function. Engagement of CD40 by its ligand, CD154, on the surface of activated T cells initiates a variety of signals in B cells including the activation of MAP kinases and NF-κB. The transcriptional regulator NF-κB is in reality a family of factors that can promote B cell activation, differentiation, and proliferation. Complex – and only partially understood – biochemical mechanisms allow CD40 to trigger two distinct NF-κB activation pathways resulting in the activation of canonical (NF-κB1) and non-canonical (NF-κB2) NF-κB. This brief review provides a summary of mechanisms responsible for activation of the latter, which appears to be particularly important for enhancing the viability of B cells at various stages in their life cycle and may also contribute to the development of B cell malignancies. CD40 is also expressed by various cell types in addition to B cells, including T cells, macrophages, dendritic cells, as well as certain non-hematopoietic cells. Here too, while perhaps less extensively studied than in B cells, the CD40-mediated activation of NF-κB2 also appears to have important roles in cellular physiology.

### **Keywords: CD40, NF-**κ**B, signal transduction**

The transcriptional regulator NF-κB participates in many important activation events in B cells, including B cell proliferation and differentiation in response to signaling by tumor necrosis factor receptor (TNFR) family members or toll-like receptor (TLR) proteins (1). NF-κB is not a single transcription factor, but rather a family of factors composed of homo- and hetero-dimers of p50, p52, c-Rel, RelA (p65), and RelB. Activation of these dimer pairs occurs via two general mechanisms, sometimes referred to as the canonical and non-canonical pathways. Canonical (NF-κB1) activation is typically rapid as it does not usually require new protein synthesis. Activation of this pathway is mediated largely by degradation of inhibitors present in the cytoplasm, which allows transit of NF-κB dimers (typically p50/RelA or p50/c-Rel heterodimers) into the nucleus. In contrast, activation of non-canonical NF-κB (NF-κB2) often occurs after activation of the canonical pathway, may require new protein synthesis, and ultimately results in the nuclear localization of predominantly p52/RelB heterodimers, although p52/p65 and p52/c-Rel hetero-dimers have also been described (2). Excellent recent reviews discuss current general models of NF-κB2 activation (2–5). Briefly, the activation of the NF-κB2 pathway is largely regulated by the production and posttranslational processing of its precursor, p100. Its proteolytic processing is regulated by IκB kinase α (IKKα), which is in turn regulated by NF-κB-inducing kinase (NIK).

## **CD40-MEDIATED NF-**κ**B2 ACTIVATION IN B CELLS**

CD40 signaling in B cells can modulate the activation of NF-κB2 at several points. First, CD40 signaling strongly activates NF-κB1 in B cells. This activation leads to the enhanced production of p100 (4). In addition, CD40 can regulate the posttranslational processing of p100 by regulating the activity of NIK (6). In resting cells, NIK activity is inhibited by a protein complex that includes TNFR-associated factor (TRAF)2, TRAF3, and cellular inhibitors of apoptosis (cIAP)1/2 (5). The major function of this complex appears to be in mediating the ubiquitination and degradation of NIK in resting B cells.Within the complex,TRAF3 appears to interact with NIK, while TRAF2 mediates interactions between TRAF3 and the cIAP molecules. Engagement of CD40 by its ligand leads to recruitment of TRAF proteins, including TRAFs 2 and 3, to the cytoplasmic domain of CD40. This event disrupts the NIK regulatory function of the TRAF2/TRAF3/cIAP complex, perhaps by promoting the ubiquitination, and degradation of TRAF3. With disruption of the NIK regulatory complex, NIK begins to accumulate (via new protein synthesis) in the cytoplasm to levels where it promotes the phosphorylation and activation of IKKα. In turn, IKKα activity mediates the phosphorylation of p100, which targets the protein for processing by the proteasome, resulting in p52 production (3–5).

A number of genetically modified mouse strains and cell lines have contributed to our understanding of NF-κB2 regulation by CD40 in B cells (7–11). TRAF2, TRAF3, and TRAF6 all significantly contribute to this CD40 signal transduction. Mouse B cell lines deficient in TRAF2 or TRAF6 exhibit little or no defect in CD40-mediated NF-κB2 activation (10). However, B cell lines doubly deficient in TRAF2 and TRAF6 appear defective in the activation of NF-κB2, suggesting that the two molecules have overlapping functions in the activation of this pathway. Potentially, these results are explained by the ability of CD40, through TRAF2 and TRAF6, to activate the NF-κB1 pathway, which in turn activates p100 transcription. Primary B cells from TRAF2-deficient mice exhibit elevated basal NF-κB2 activation (not observed in the cell line studies), which is only weakly augmented by CD40 signaling (9), consistent with a role for TRAF2 in the NIK regulatory complex. In the cell line studies, TRAF2 deficiency did not appear to augment the basal level of p52 production (10), although this may have been due to limited production of p100 in non-activated cells. B cells from mice conditionally deficient in TRAF3 specifically in B cells also exhibit strong constitutive activation of the NF-κB2 pathway (7, 11), which is again consistent with the model in which TRAF3 is a major component of the NIK regulatory complex. While the phenotypes of TRAF2-, cIAP-, and TRAF3-deficient primary B cells are somewhat similar with respect to the constitutive activation of NF-κB2, there are instructive differences between the strains [reviewed in (5)]. CD40-mediated differentiation of B cells into germinal center B cells is augmented in TRAF3-deficient mice (7, 11), but TRAF2 or cIAP-deficient mice have a significant defect in germinal center B cell development (8). This likely reflects the role of TRAF2/cIAP in activation of the NF-κB1 pathway, which TRAF3 does not share.

Interestingly, in B cell-specific TRAF3-deficient mice, the elevated constitutive activation of NF-κB2 can be enhanced somewhat by CD40 stimulation (11),indicating the existence of TRAF3 independent mechanisms of NIK/NF-κB2 regulation. This possibility is further supported by the observation that CD40 signaling can activate NF-κB2 in TRAF3-deficient B cell lines reconstituted with a mutant TRAF3 molecule that binds NIK robustly, but is not degraded following CD40 stimulation (Lin et al., this issue). These observations demonstrate that the regulation of NF-κB2 by CD40 in B cells is only partially understood. Recently, the protein kinase TANK-binding kinase 1 (TBK1) was shown to negatively regulate NF-κB2 activation and IgA Ig isotype switching in primary B cells, potentially through phosphorylation/degradation of NIK (12). The de-ubiquitinating enzyme OTUD7B was also shown recently to potentially negatively regulate NF-κB2 (and primary B cell activation) by inhibiting activation-induced degradation of TRAF3 (13). Additionally, zinc finger protein 91 has recently been shown to promote NIK stability in a CD40-stimulated epithelial tumor cell line, perhaps through K63-linked polyubiquitination of NIK (14). Whether this mechanism is relevant to the CD40 mediated activation of B cells remains to be demonstrated. It is likely that additional regulatory mechanisms will come to light in the next few years, and will contribute to our understanding of normal B cell biology as well as the physiology of B cell malignancies.

### **CD40-MEDIATED NF-**κ**B2 ACTIVATION IN NON-B CELLS**

Compared to the extensive research performed in B cells, a cell type in which NF-κB activation is a major regulatory pathway, relatively little investigation has been performed on CD40-mediated NF-κB2 signaling in other cell types. Most studies of CD40 functions in non-B cells do not examine NF-kB activation. In those that do, canonical NF-κB1 activation is typically the focus, and/or methods used (reporter gene activation or electrophoretic mobility shift assay, EMSA) do not allow results to distinguish between NF-κB1 and NF-κB2 induction. We summarize below the information that is currently available on CD40-mediated induction of the NF-κB2 pathway in non-B cells.

## **OTHER HEMATOPOIETIC CELLS**

### **T lymphocytes**

The physiologic importance of direct signaling to T cells by CD40 has at times been a controversial topic (15). However, there is no doubt that normal activated CD4<sup>+</sup> and CD8<sup>+</sup> T cells can express CD40, and it appears to play significant biological roles in mouse models of T cell-dependent immune responses (16, 17), including autoimmune responses [reviewed in Ref. (15)]. However, the role of CD40-mediated NF-κB activation, and in particular NF-κB2 activation in these roles is unclear, and may be context-dependent. CD3+CD8+CD40<sup>+</sup> T cells in Balb/c mice exhibit cytotoxic activity toward CD4+CD25<sup>+</sup> T regulatory cells during *Leishmania* infection – this activity is CD40-dependent, but unaffected by inhibitors of NF-κB (18). A CD4<sup>+</sup> mouse T cell line stably expressing transfected CD40 utilizes CD40 as an effective co-stimulatory signal with T cell receptor signals to activate T cell functions. In this model, both NF-κB1 and NF-κB2 pathways in T cells are activated by CD40 (19). The ultimate biological importance of NF-κB2 activation in T cell CD40 signaling remains to be explored.

## **Myeloid cells**

Monocytes and macrophages, like B cells, constitutively express CD40, which activates mitogen-activated protein (MAP) kinases and NF-κB in these cells [reviewed in Ref. (20)]. However, very little is known about the potential involvement of the NF-κB2 pathway in macrophage CD40 signaling. Revy et al. performed a direct comparison between human monocytes and B cells activated through CD40. EMSA demonstrated that nuclear translocation of NF-κB binding activity is induced by CD40 in both cell types, and the p50 subunit involved in NF-κB1 activation is part of these nuclear complexes. Interestingly, the p65 subunit was only present in complexes from B cells (21). However, as the CD40 stimulation was only of short duration in these experiments (30 min); the activation of NF-κB2 was not assessed at later times.

CD40 also delivers potent and important signals to dendritic cells (DC) [reviewed in Ref. (22)]. Following signals from CD40 that stimulate the ability of DC to cross-present antigen to T cells, the nuclear translocation of p52 characteristic of NF-κB2 signaling is seen. Mice bearing a mutant NIK that disrupts NF-κB2 activation by various signals, the NIKaly/aly mouse, show DC functional defects (23). CD40 fails to induce cross presentation of antigens to CD8 T cells in the NIKaly/aly mouse, implicating NF-kB2 in this particular DC function. However, the evidence is indirect, as the NIK mutation is expressed in all cells of the mouse, and will impact multiple pathways of cell development and function. In another experimental model, human monocyte-derived DC treated with siRNA for NIK or IKKα are unable to produce the cytokines IDO and IL-6 following *in vitro* stimulation with the CD40 ligand, CD154. This also renders the treated DC unable to promote the

induction of T regulatory cells (24). Thus, NF-κB2 signaling plays important roles in CD40 functions in DC.

## **NON-HEMATOPOIETIC CELLS**

### **Epithelial cells**

CD40 was first described as an antigen expressed on the surface of bladder carcinoma cells (25), and can be expressed on a variety of types of epithelial cells under particular circumstances [reviewed in (26, Lin et al., this issue)]. Unfortunately, by far the most commonly used cells of epithelial origin used to study CD40 signaling are the transformed cell lines HEK 293 and HeLa. In the majority of studies using these cell lines, CD40 and/or the signaling proteins studied are exogenously expressed at highly non-physiologic levels. Thus, because such models can provide misleading results [recently reviewed in (27)], we will not discuss this body of work in this article. Very little is known about the usage of the NF-κB2 pathway by endogenous CD40 expressed by normal epithelial cells. An interesting report using airway epithelial cells, which produce the cytokine RANTES upon CD40 stimulation, examined NF-κB activation by EMSA. Nuclear binding complexes for probes with sites characteristic of both NF-κB1 and NF-κB2 were identified, with the former peaking at 30 min of stimulation, and the latter at 2 h. The p65 subunit was present in both types of complexes (28). The functional importance of this NF-κB2 activation remains to be defined.

## **Fibroblasts**

Although CD40 can be expressed by fibroblasts, no information is currently available on the extent or importance of NF-κB2 activation by CD40 in this cell type. A recent report made the interesting observation that TWEAK, a molecule implicated in various fibrotic processes [reviewed in (29)] inhibits CD40 signaling in the HeLa cell line (30). However, neither fibroblasts nor NF-κB2 pathways were examined in this study.

### **Vascular endothelium**

It is well-known that CD40 can be expressed on activated endothelium, and its interaction with CD154 has been implicated in the pathogenesis of atherosclerosis [reviewed in Ref. (31)]. A specific role for NF-κB2 activation has yet to be explored in this CD40 function. In human vascular endothelial cells, stimulation of CD40 with soluble CD154 stimulates the phosphorylation and nuclear translocation of the p65 subunit of NF-κB at 6 h (32). However,it is unclear whether this represents activation of the NF-κB2 pathway.

While it is clear that the mechanisms of CD40-mediated NF-κB activation are complicated (and still incompletely characterized), the consequences of NF-κB activation in B cells and other cells are orders of magnitude more complex. Many of the genes regulated by NF-κB have been identified by experiment and by computer analysis, but the work of understanding the subsequent roles of these genes and their interactions in cellular physiology is only in its infancy. Nevertheless, continued characterization of NFκB activation mechanisms and consequences of NF-κB-regulated transcription will lead to a more complete understanding of basic immune system biology as well as the identification of molecular events and interactions that can be exploited in the treatment of immune disorders and cancer.

## **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 24 September 2013; accepted: 31 October 2013; published online: 15 November 2013.*

*Citation: Hostager BS and Bishop GA (2013) CD40-mediated activation of the NF-*κ*B2 pathway. Front. Immunol. 4:376. doi: 10.3389/fimmu.2013.00376*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Hostager and Bishop. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# A complex relationship betweenTRAF3 and non-canonical NF-κB2 activation in B lymphocytes

#### **WaiW. Lin<sup>1</sup> , Joanne M. Hildebrand<sup>2</sup>† and Gail A. Bishop1,2,3,4\***

<sup>1</sup> Graduate Program in Immunology, University of Iowa, Iowa City, IA, USA

<sup>2</sup> Department of Microbiology, University of Iowa, Iowa City, IA, USA

<sup>3</sup> Department of Internal Medicine, University of Iowa, Iowa City, IA, USA

<sup>4</sup> VA Medical Center, Iowa City, IA, USA

### **Edited by:**

John Silke, The Walter and Eliza Hall Institute, Australia

### **Reviewed by:**

Christine Loh, Biogen Idec Inc., USA John Silke, The Walter and Eliza Hall Institute, Australia

### **\*Correspondence:**

Gail A. Bishop, Department of Microbiology, University of Iowa, 375 Newton Road, 2193 MERF, Iowa City, IA 52242, USA

e-mail<EMAIL_ADDRESS>
### **†Present address:**

Joanne M. Hildebrand, The Walter and Eliza Hall Institute for Medical Research, Parkville, Australia

The adaptor protein TRAF3 restrains B cell activating factor receptor (BAFFR) and CD40 mediated activation of the NF-κB2 pathway in B cells. Mice lacking TRAF3 specifically in B cells revealed the critical role of TRAF3 in restraining homeostatic B cell survival. Furthermore, loss-of-function mutations of the traf3 gene have been associated with human B cell malignancies, especially multiple myeloma (MM). It has been proposed that receptorinduced TRAF3 degradation leads to stabilization of the NF-κB inducing kinase (NIK), and subsequent NF-κB2 activation. However, it is unclear how receptor-mediatedTRAF3 degradation or loss-of-function contributes to B cell-specific NF-κB2 activation. In the current study, we employed two complementary models to address this question. One utilized a mutant traf3 gene found in a human MM-derived cell line called LP1.The LP1 mutantTRAF3 protein lacks the TRAF-N and TRAF-C domains. Consistent with the paradigm described, expression of LP1 TRAF3 in B cells promoted higher basal levels of NF-κB2 activation compared to Wt TRAF3. However, LP1 did not associate with TRAF2, CD40, or BAFFR, and no LP1 degradation was observed following receptor engagement. Interestingly, LP1 showed enhanced NIK association. Thus, TRAF3 degradation becomes dispensable to activate NFκB2 when it is unable to associate with TRAF2. In a second model, we examined several mutant forms of BAFFR that are unable to induce NF-κB2 activation in B cells. Signaling to B cells by each of these BAFFR mutants, however, induced levels of TRAF3 degradation similar to those induced by Wt BAFFR. Thus, in B cells, receptor-mediated TRAF3 degradation is not sufficient to promote NF-κB2 activation. We thus conclude that there is not a simple linear relationship in B lymphocytes between relative levels of cellular TRAF3, induced TRAF3 degradation, NIK activation, and NF-κB2 activation.

**Keywords:TRAF3, NF-**κ**B, B cell, CD40, BAFF**

## **INTRODUCTION**

Tumor necrosis factor receptor (TNFR) associated factor 3 (TRAF3) is an important adaptor molecule shown to regulate NFκB2 activation induced by the TNFR superfamily molecules CD40 and B cell activating factor receptor (BAFFR), as well as other immune receptors (1–3). In B lymphocytes, CD40 and BAFFRmediated signaling is important for regulation of maturation, survival, Ig class switch recombination, and antibody production (4, 5). Engagement of BAFFR with BAFF or interaction between CD40 and its ligand CD154 triggers the non-canonical NF-κB or NFκB2 pathway [reviewed in Ref. (6)]. TRAF3 negatively regulates NF-κB2 activation (7, 8), and mouse B cells deficient in TRAF3 have substantially enhanced survival, which correlates with constitutive activation of the NF-κB2 pathway (9, 10). Understanding how TRAF3 regulates both NF-κB2 activation and homeostatic B cell survival is thus of great importance.

The NF-κB2 pathway is tightly regulated at steady state, and dysregulation of the pathway is frequently observed in hematological malignancies (11, 12). Gain or loss-of-function mutations of genes encoding key signaling molecules implicated in the NF-κB2 pathway have been observed in such tumors. In particular, mutations in NF-κB inducing kinase (NIK), TRAF3, TRAF2, and cellular inhibitors of apoptosis 1 and 2 (cIAP1, cIAP2) are highly associated with human multiple myeloma (MM) and MMderived cell lines. Loss-of-function mutations of the *traf3* gene have been identified in 9–17% of MM patient cohorts (13, 14). Such mutations have also been identified in different subtypes of B cell lymphoma and Waldenström's macroglobulinemia (15– 17). Consistent with this, a portion of older mice deficient in TRAF3 specifically in B cells develop B cell lymphomas (18). Taken together, these studies provide strong evidence that loss or reduced expression of TRAF3 contributes to B cell malignancies.

A widely held paradigm suggests that the physical association between TRAF3, TRAF2, and NIK allows the TRAF2-cIAP E3 ubiquitin ligase complex to polyubiquitinate NIK (19, 20). This promotes proteasome-mediated degradation of NIK, and by so doing, restrains NF-κB2 activation (21). Engagement of CD40 or BAFFR on B cells is known to recruit TRAFs 2 and 3 to the plasma membrane lipid-raft compartment (22, 23). This recruitment initiates TRAF2-mediated polyubiquitination of both TRAFs 2 and 3, and their subsequent proteasome-mediated degradation (22, 24, 25). The paradigm mentioned above posits that this receptor-mediated TRAF3 degradation allows NIK stabilization, and thus NF-κB2 activation (26, 27). However, it remains unclear whether TRAF3 degradation is necessary and sufficient for NF-κB2 activation, in response to CD40 and BAFFR signaling, in B lymphocytes. The present study was designed to address this important mechanistic question. Utilizing two complementary approaches, we found that association between TRAFs 2 and 3 is important for CD40 and BAFFR-mediated TRAF3 degradation. However, degradation of endogenous TRAF3 was neither sufficient nor always required to induce CD40 or BAFFR-mediated activation of NF-κB2 in B cells.

**MATERIALS AND METHODS**

### **MICE**

A/J, BAFFR−/−, and Bcl2 transgenic (tg) mice with A/J congenic backgrounds were kindly provided by Dr. Colleen Hayes (University of Wisconsin, Madison, WI, USA). A/WySnJ mice, also on the A/J genetic background, were purchased from Jackson Laboratory (Bar Harbor, ME, USA). A/J, A/WySnJ, and BAFFR−/<sup>−</sup> mice were bred with Bcl2 tg mice to circumvent mature B cell developmental defects in A/WySnJ and BAFFR−/<sup>−</sup> mice (28, 29). All mice had one allele of the *Bcl2* transgene, and were used at 10–12 weeks of age as a source of B cells for experiments. Mice were maintained under pathogen-free conditions at the University of Iowa. Use of mice in this study was according to a protocol approved by The University of Iowa Animal Care and Use Committee.

### **CELL LINES**

The mouse B cell lines M12.4.1 (30), CH12.LX, and its TRAF3 deficient subclones have been previously described (31, 32). B cell lines stably transfected with hybrid human CD40-mouse BAFFR constructs described below were maintained in B cell medium containing RPMI 1640 (Life Technology, Grand Island, NY, USA) with 10µM 2-βmercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), 10% heat-inactivated FCS (Atlanta Biologicals,Atlanta, GA, USA), 2 mM l-Glutamine (Life Technologies), 100 U/ml of Penicillin Streptomycin antibiotics (Life Technologies) (BCM10). Medium additionally contained 400µg/ml of G418 disulfate (Research Products International, Mount Prospect, IL, USA) for subclones expressing transfected hCD40-BAFFR constructs, and both G418 and 200µg/ml of hygromycin (Life Technologies) for subclones expressing FLAG-tagged TRAF3 or the LP1 mutant TRAF3.

### **DNA CONSTRUCTS AND TRANSFECTIONS**

Plasmids encoding a mutant BAFFR from the A/WySnJ mouse and a mouse BAFFR lacking the C-terminal eight amino acids (∆C) were kindly provided by Drs. Colleen Hayes and Christopher Mayne. These plasmids were used as a source of BAFFR to produce cDNA encoding a chimeric molecule consisting of the extracellular portion of human CD40 and the transmembrane and intracellular portions of mouse Wt BAFFR, the BAFFR mutant of the A/WySnJ mouse, or mouse BAFFR lacking the Cterminal 8 amino acids (∆C). These chimeric constructs were subcloned into the mammalian expression vector pRSV5.neo for stable expression in CH12.LX or M12.4.1 cells (33). cDNAs encoding FLAG-tagged human Wt TRAF3, or the truncated TRAF3 mutant identified in the human MM cell line LP1 (14) were subcloned into a variant of the pRSV5.neo plasmid with a sequence containing the binding element of the repressor of the bacterial Lac operon in the promoter, as previously described (33). This allows Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible, stable TRAF3 expression in a CH12.TRAF3−/<sup>−</sup> cell line that stably expresses the Lac repressor (32, 33). There is thus no endogenous TRAF3 in cells expressing Wt or LP1 TRAF3 in these studies.

### **ANTIBODIES AND REAGENTS**

Isopropyl β-d-1-thiogalactopyranoside was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit anti-phosphojun kinase (JNK) Ab, rabbit anti-phospho-p38 Ab, rabbit antipIκBαAb, rabbit anti-total IκBαAb, rabbit anti-NIK Ab, and rabbit anti-p100/p52 Ab were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-TRAF2 Ab was purchased from Medical and Biological Laboratories (Woburn, MA, USA). Anti-FLAG M2 Ab for immunoprecipitation, anti-FLAG M2-HRP Ab for immunoblotting, and mouse anti β-actin Ab were purchased from Sigma. Rabbit anti-JNK Ab, mouse anti-YY1 (H-10) Ab, and rabbit anti-mouse CD40 (M-20) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Production of Hi Five (HiV) strain insect cells infected with Wt baculovirus or baculovirus encoding human CD154 or mouse CD154 is described in (34). Rat anti-mouse CD40 mAbs (1C10 and 4F11) were produced in our laboratory from their respective hybridomas as described (32), kindly provided by Dr. Frances Lund (University of Alabama, Birmingham, AL, USA). The isotype control rat total IgG Ab was purchased from Southern Biotech (Birmingham, AL, USA) and mouse IgG1 control Ab from eBioscience (San Diego, CA, USA). Goat anti-BAFFR Ab for immunoprecipitation was purchased from R&D Systems (Minneapolis, MN, USA), and rabbit anti-BAFFR Ab for immunoblotting was purchased from Abcam (Cambridge, MA, USA). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG secondary Abs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

## **CELL ISOLATION AND ASSAYS TO MEASURE RECEPTOR SIGNALING AND TRAF3 DEGRADATION**

Resting splenic B cells were obtained as previously described (9). Briefly, high density splenic B cells were isolated by centrifugation through a Percoll density gradient (GE Life Sciences, Pittsburg, PA, USA) followed by anti-CD43 Ab-mediated negative selection, using a magnetic bead kit (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer's protocol. 5 × 10<sup>6</sup> splenic B cells or 2 × 10<sup>6</sup> CH12.LX or M12.4.1 cells were stimulated as indicated in the Figure legends. After stimulation, cells were lysed and cytosolic and nuclear extracts were prepared as described in Xie et al. (35) to detect NF-κB2 activation via nuclear translocation of p52. For proximal signaling assays and TRAF3 degradation assays, 1–2 × 10<sup>6</sup> cells were stimulated for indicated times at 37°C. After stimulation, cells were lysed in 100µl 2× SDS-PAGE loading buffer (1% SDS, 2% β-mercaptoethanol, 62.5 mM Tris, pH 6.8). Lysates were sonicated using a Branson Sonifier 250 (VWR International, Radnor, PA, USA) with 10 pulses at 90% duty cycle, output 1.5. Samples were boiled for 10 min at 95°C prior to gel loading.

## **IMMUNOPRECIPITATION**

CH12.TRAF3−/<sup>−</sup> cells expressing LacR and stably transfected with inducible FLAG-tagged Wt or LP1 mutant TRAF3 were cultured with BCM10 containing 100µM IPTG overnight to induce TRAF3 expression. 2 × 10<sup>7</sup> cells were resuspended in 1 ml BCM10 and stimulated with 10µg/ml of hamster anti-mouse CD40 mAb (HM40.3, eBioscience) or 500 ng/ml of recombinant BAFF (Peprotech, Rocky Hill, NJ, USA) for the indicated times. Cells were pelleted and lysed in lysis buffer (0.5% Triton X, 40 mM Tris, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2) containing complete protease inhibitor cocktail (Roche, Germany), 0.05 mg of DNAse I (Roche), and 2 mM Na3VO4. Whole cell lyses were incubated with mouse anti-FLAG Ab (Sigma) or control IgG1 Ab overnight at 4°C with constant agitation. The immune complex was precipitated with Dyna Protein G beads (Life Technologies), washed and resuspended in SDS-PAGE loading buffer, and heated to 95°C for 10 min.

## **IMMUNOPRECIPITATION FROM DETERGENT-INSOLUBLE PLASMA MEMBRANE FRACTIONS**

3 × 10<sup>7</sup> CH12.LX cells were stimulated at 37°C with 500 ng/ml recombinant BAFF (Peprotech) or 3 × 10<sup>6</sup> HiV insect cells infected with Wt baculovirus, or baculovirus expressing mouse CD154 (34). These insect cells, which grow at 25°C, die and serve as a source of CD154-expressing membranes at 37°C. Cells were lysed using buffer containing 1% Brij58 (Thermo Scientific, Rockford, IL, USA), 150 mM NaCl, 20 mM Tris, 50 mM β-glycerophosphate, 1 mM MgCl2, 1 mM CaCl2, protease inhibitor cocktail, and 2 mM Na3VO4. The Brij58-insoluble cell fractions were separated by centrifugation at 14,000 × *g* for 30 min at 4°C and solubilized using 1% Triton X-100 and 0.1% SDS-containing buffer. This solubilized cholesterol-rich membrane fraction (22) was subjected to immunoprecipitation using rat anti-mouse CD40 mAbs (an equal mixture of the clones 1C10 and 4F11) (36), goat polyclonal anti-BAFFR Ab, or appropriate isotype control antibodies. To immunoprecipitate the receptor signaling complex, Protein G Dyna beads, pre-incubated with goat anti-rat Ab (Jackson) for 30 min at room temperature, or uncoupled beads were incubated with solubilized membrane fractions for 2 h at 4°C with constant agitation. After washing, the immunoprecipitate was deglycosylated using PNGaseF (NEB) and boiled in SDS-PAGE loading buffer.

## **IMMUNOPRECIPITATION OF HCD40-BAFFR CHIMERIC MOLECULES**

3 × 10<sup>7</sup> cells CH12.LX cells that stably express hCD40-BAFFR (Wt), hCD40-A/WySnJ (A/WySnJ), hCD40-∆C, or hCD40- BAFFR chimeric molecules with a mutated TRAF3 binding site (AVAAA) were stimulated with Protein G Dyna Beads (Life Technologies) conjugated with agonistic mouse anti-hCD40 mAb or isotype control Ab for the indicated time points at 37°C. Abs were crosslinked to the beads using DSS crosslinker (Thermo Scientific) according to the manufacturer's recommended protocol. Immunoprecipitation of hCD40-BAFFR chimeric molecules was performed as previously described (37).

## **WESTERN BLOTS**

Proteins were resolved on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% milk in TBST (120 mM NaCl, 0.08% Tween 20, and 40 mM Tris) for 1 h at room temperature, and incubated overnight at 4°C in primary Ab. Blots were washed in TBST, incubated with secondary Abs for 2 h at room temperature and developed using Supersignal West Pico (Thermo Scientific). Western blot chemiluminescence was read with an LAS-4000 low-light camera and analyzed with Multi Gage software (Fujifilm Life Science, Edison, NJ, USA).

## **STATISTICAL ANALYSIS**

*p*-Values were generated by Student's *t*-test (unpaired, two-tailed, at 95% confidence interval).

## **RESULTS**

## **RECEPTOR-INDUCED DEGRADATION OF Wt VS. LP1 TRAF3**

We and others previously reported that B cell-specific loss of the *traf3* gene enhances the survival of B cells independent of cell proliferation (9, 10). We also demonstrated that this enhanced B cell-specific survival renders B cells independent of the soluble factor BAFF, and results in constitutive activation of the noncanonical NF-κB2 pathway (9). At the same time, several groups reported that loss-of-function mutations in the *traf3* gene are associated with human MM and enhanced NF-κB2 activity (13, 14). These studies identified various forms of TRAF3 mutations, from point mutations leading to single amino acid substitutions to large truncations. These mutations of *traf3* are highly correlated with frequent chromosomal translocations of chromosome 14, commonly seen in MM (13, 14). Among these is a TRAF3 mutation identified in the human MM LP1 cell line. The LP1 TRAF3 missense mutation results in production of a truncated TRAF3 lacking the TRAF-N and TRAF-C domains (**Figure 1A**), which we predict would fail to associate with TRAF2 (19) and thus not undergo receptor-mediated degradation.

To test our prediction that the LP1 TRAF3 mutant fails to undergo receptor-mediated degradation, we established CH12.TRAF3−/<sup>−</sup> subclones with stable, IPTG-inducible expression of FLAG-tagged Wt or LP1 TRAF3. Cells were stimulated via CD40 or BAFFR, known strong activators of TRAF2-dependent TRAF3 degradation (22, 24, 25). Based on the results shown in **Figures 1B,C**, LP1 was only minimally degraded after stimulation with CD40 or BAFFR, compared to Wt TRAF3.

## **RECEPTOR-INDUCED NF-**κ**B2 AND JNK ACTIVATION IN LP1-EXPRESSING CELLS**

A widely held paradigm is that TRAF3 degradation induced by CD40 or BAFFR ligation allows NIK stabilization, which then activates the NF-κB2 pathway (20, 26, 27). This paradigm predicts that TRAF3−/<sup>−</sup> cells expressing LP1 would fail to undergo receptorinduced NF-κB2 activation, as LP1 is not able to undergo receptorinduced degradation. To investigate whether LP1 is inducing NF-κB2 activation independent of receptor-induced degradation, we provided CD40 or BAFF stimuli to CH12.TRAF3−/<sup>−</sup> B cells induced to express either Wt or LP1 TRAF3. Consistent with TRAF3's proposed role in NF-κB2 activation, induction of Wt TRAF3 expression reduced nuclear p52 from its elevated levels in TRAF3−/<sup>−</sup> B cells. Because the LP1 TRAF3 mutant is a lossof-function mutation, we predicted that LP1 TRAF3-expressing

B cells will have constitutive nuclear p52 and there will be no further activation of the NF-κB2 pathway upon stimulation. Interestingly, however,induction of LP1 expression in TRAF3−/<sup>−</sup> B cells also reduced nuclear p52 levels (**Figures 2A,B**). Induction of LP1 expression reduced basal nuclear p52 levels in TRAF3−/<sup>−</sup> B cells. However, p52 levels remained ~twofold higher in LP1 compared to Wt TRAF3-expressing cells (**Figures 2A,B**), which is consistent with previous observations in myeloma cells and fibroblasts that the presence of LP1 TRAF3 mutant led to higher p52 levels (8, 14). Surprisingly, unlike TRAF3-deficient B cells, which further activate NF-κB2 in response to CD40 but not BAFFR stimulation (9), LP1-expressing B cells responded to both CD40 and BAFF stimulation with further nuclear p52 increases (**Figures 2A,B**). These data suggested that the TRAF3 mutant LP1 partially restrains NF-κB2 activation, and upon stimulation activates the NF-κB2 pathway independent of receptor-mediated LP1 degradation. Collectively, published studies and results presented here lead us to propose that induction of NF-κB2 activation in B cells does not solely depend on the absence of TRAF3, whether TRAF3 reduction is caused by receptor-induced degradation, mutation, or gene-targeted deletion.

LP1 expression also resulted in enhanced c-JNK activation following BAFF stimulation (**Figure 2D**), and sustained and significantly enhanced JNK activation in response to CD40 signals (**Figure 2C**). In contrast, neither phosphorylation of the mitogenactivated kinase (MAPK) p38 nor activation of classical NF-κB1 was impacted by the LP1 mutation compared to Wt TRAF3 (**Figure 3**). These data indicate that the C-terminal domains of TRAF3 are important for restraining activation of the JNK pathway. Consistent with our prior report that complete loss of TRAF3 does not impact CD40-mediated NF-κB1 activation (9), LP1 expression did not result in enhanced NF-κB1 or p38 activation in response to CD40 or BAFFR signals. Canonical NF-κB1 activation was unaffected by LP1 TRAF3 expression, indicating that increased nuclear p52 levels in LP1-expressing cells were not due to NF-κB1-induced enhanced p100 expression.

## **ASSOCIATION OF TRAF3 WITH KEY SIGNALING COMPONENTS IN NF-**κ**B2 ACTIVATION**

TRAF3 is typically recruited to the cytoplasmic domains of CD40 and BAFFR, together with TRAFs, to mediate downstream signaling and biological functions (22, 23, 38). Recruitment to receptor

the NF-κB2 precursor protein p100 and its cleavage product p52, with probes for actin and YY1 serving as loading controls for cytoplasmic and nuclear protein levels respectively. YY1 levels were used to normalize nuclear p52 values shown quantitatively in graphs. Representative blots probed for phosphorylated JNK (pJNK), total JNK (JNK), and actin. Representative blots are presented. The corresponding graphs represent mean values for band intensities of pJNK divided by total JNK ± SEM from two (BAFF) or three (CD40) independent experiments.

cytoplasmic domains and association with TRAF2 and/or cIAP1/2 are thought to be required for K48-mediated ubiquitination, followed by proteasomal degradation of TRAF3 after receptor engagement (24, 25, 39, 40). To investigate the reason for the lack of receptor-mediated LP1 degradation, we assessed whether LP1 can associate with TRAF2. CH12.TRAF3−/<sup>−</sup> B cells induced to express Wt or LP1 TRAF3 were stimulated through CD40, and Wt or LP1 TRAF3 was immunoprecipitated from cell lysates. Consistent

with a previous report (19), the C-terminal TRAF-N and TRAF-C domains of TRAF3 were required for TRAF3 to associate with TRAF2, with or without CD40 stimulation (**Figure 4A**). Similar results were obtained with BAFF stimulation (data not shown).

To assess whether LP1 associated with either CD40 or BAFFR after stimulation, we isolated the lipid-raft enriched detergentinsoluble plasma membrane fractions of cells as described in Section "Materials and Methods," and immunoprecipitated the

receptor signaling complex. **Figures 4B,C** reveal that LP1 was not part of the CD40 or BAFFR signaling complexes, but Wt TRAF3 associated with the receptors upon stimulation. These data suggest that the observed lack of receptor-induced LP1 degradation reflects a requirement for recruitment of TRAF3 to the receptor cytoplasmic domains and association with TRAF2, to initiate the degradation process.

antibodies to total p38 also recognize the phosphorylated form). Representative blots from three independent experiments are shown.

Strikingly, despite lack of receptor-mediated degradation of LP1 TRAF3, LP1-expressing B cells were capable of inducing NF-κB2 activation above the already high basal levels following receptor stimulation. To address this discrepancy with the prevailing paradigm, we investigated whether LP1 associates with NIK, a MAP kinase implicated as key to NF-κB2 activation (21). A previous report showed that TRAF3 physically interacts with NIK when both molecules are overexpressed in transformed epithelial cells, and this interaction requires the C-terminal residues from 424 to 543 of TRAF3 (20). The interaction between TRAF3 and NIK is proposed to allow the TRAF2 E3 ubiquitin ligase activity to constantly ubiquitinate NIK for proteosomal degradation (20, 26, 27). Hence, although LP1 was not degraded after receptor stimulation (**Figure 1**), if NIK fails to associate with LP1, this could stabilize NIK in LP1-expressing cells, ultimately leading to enhanced NF-κB2 activation. However, immunoprecipitated LP1 TRAF3 from unstimulated or BAFF-activated B cells displayed 10–16 fold *increased* association with NIK, compared to Wt TRAF3 (**Figure 5**). Notably, there was no drastic difference in total NIK accumulation in LP1 vs. Wt TRAF3-expressing B cells. It is important to note that the function of TRAF3 is highly context-dependent; TRAF3 plays very different roles with regards to distinct receptors and cell types [reviewed in Ref. (2)]. Thus, the discrepancy between the present findings and previous conclusions based upon experiments performed in epithelial cell lines (20) are likely due to these contextual differences. Collectively, the present results suggest that when tumor-derived mutant TRAF3 becomes resistant to receptor-mediated degradation, stabilization of cellular NIK levels is controlled by TRAF3 degradationindependent mechanisms to regulate NF-κB2 activation in B cells.

## **DEFECTIVE NF-**κ**B2 ACTIVATION VIA THE MUTANT BAFFR OF A/WySnJ B CELLS**

B cell activating factor receptor provides an important B cell survival signal, thought to require activation of the non-canonical NF-κB2 pathway (5). As a complementary experimental model to further examine the importance of receptor-mediated TRAF3 degradation for NF-κB2 activation in B cells, we isolated B cells from the A/WySnJ mouse. This mouse contains a spontaneous mutation in the *Baffr* gene called *Bcmd1* (B cell maturation defect-1) that leads to defects in survival and maturation of peripheral B cells (41). *Bcmd1* encodes a protein that shares ≥95% sequence homology with Wt BAFFR. Notably, the TRAF3 binding motif PVPAT remains intact in this mutant BAFFR (**Figure 6A**) (42). However, the last eight amino acids of Wt BAFFR are replaced by a 22-amino acid peptide sequence resulting from a proviral Intracisternal A-particle (IAP) retro-transposon insertion event (41). Although A/WySnJ mice have considerably reduced peripheral B cell numbers due to reduced B cell survival, the expression of tg Bcl2 restores the peripheral B cell compartment to nearnormal levels (29). We thus utilized A/WySnJ-Bcl2 Tg mice for these experiments (designated A/WySnJ in Figures for simplicity). When we stimulated their splenic B cells with BAFF, the NF-κB2 pathway showed markedly defective activation compared to control mice of the parent strain (A/J) expressing tg Bcl2 (**Figure 6B**). Consistent with the concept that BAFF binding to BAFFR preferentially activates the NF-κB2 pathway (43), BAFFR−/<sup>−</sup> B cells were not able to active NF-κB2 in response to BAFF stimulation (**Figure 6B**). In **Figure 6B**, we also noted that A/WySnJ BAFFR signaling induced TRAF3 degradation to a similar extent as did Wt BAFFR. We also examined the endogenous NIK protein levels in B cells of Wt and BAFFR mutant mice, and found that there were no differences in cellular NIK levels in A/WySnJ B cells as compared to Wt B cells (**Figure 6C**). These data suggest that TRAF3 degradation, leading to NIK stabilization, is not sufficient in itself to activate the non-canonical NF-κB2 pathway.

Because BAFF binds to three receptors expressed on B cells, BAFFR (44), BCMA (B cell maturation antigen) (45), and TACI (transmembrane activator and calcium modulator and cyclophilin ligand) (46), it is important to verify that the defective BAFF response of splenic B cells from the A/WysnJ mouse is due to its mutant BAFFR. We thus generated CH12.LX and M12.4.1 mouse B cell lines stably expressing chimeric receptors, containing the human (h)CD40 extracellular and transmembrane domains fused to the cytoplasmic domain of Wt BAFFR (Wt), the A/WySnJ mutant BAFFR, or BAFFR that lacks the last eight

amino acids of the cytoplasmic domain (∆C) (**Figure 6D**). The chimeric receptors allowed us to be certain that we were examining consequences of BAFFR signaling in these B cells. B cells were stimulated with HiV insect cells expressing hCD154, and NF-κB2 activation was analyzed. Stimulation with anti-mouse (m)CD40 agonistic Ab served as an internal control, showing that there was

prepared and immunoprecipitated with anti-FLAG Ab. Western blots of immunoprecipitates were probed for NIK and FLAG. Both short and long exposures of the blot are shown due to differences in NIK association between Wt and LP1 TRAF3. Data shown are representative of two similar experiments.

no global defect in CD40 signaling of cells expressing various chimeric molecules. Consistent with results from primary splenic B cells from A/WySnJ or control mice, B cells expressing hCD40- A/WySnJ BAFFR displayed defective NF-κB2 activation compared to hCD40-Wt BAFFR (**Figure 6E**). This result indicates that the signaling defect observed in splenic B cells is due to the dysfunctional BAFFR. Impaired activation of NF-κB2 was also observed with hCD40-∆C BAFFR, indicating that the last eight amino acids of BAFFR are necessary for NF-κB2 activation, although they have been shown to be dispensable for B cell survival (29). This reveals the interesting information that pathways alternative to NF-κB2 can mediate BAFFR-induced B cell survival.

### **TRAF3 DEGRADATION MEDIATED BY MUTANT BAFFRS**

To explore whether TRAF3 degradation status correlates with the impaired activation of NF-κB2 in cells expressing mutants of BAFFR, we stimulated B cell transfectants shown in **Figure 6C** through hCD40-BAFFR to induce BAFFR-mediated TRAF3 degradation (**Figure 7A**). TRAF3 was degraded similarly, regardless of which BAFFR cytoplasmic domain was expressed. This result reveals that TRAF3 degradation is not sufficient to induce BAFFR-mediated NF-κB2 activation. Results presented in **Figures 6B** and **7A** led us to predict that TRAF recruitment to the A/WySnJ BAFFR cytoplasmic tail would be comparable to TRAF recruitment to Wt BAFFR. Indeed, cells expressing hCD40-A/WySnJ chimeric molecules not only were able to recruit both TRAF2 and 3 to the BAFFR, this mutant BAFFR showed enhanced TRAF recruitment compared to Wt BAFFR (**Figure 7B**). Consistent with previous studies, when the TRAF3 binding site (PVPAT) on BAFFR was disrupted (AVAAA), there was no recruitment of TRAFs to the receptor's cytoplasmic domain (**Figure 7B**). These results show that the defective BAFF-stimulated NF-κB2 activation in A/WySnJ B cells is not due to a failure to induce TRAF3 recruitment or degradation.

Taken together, results from experiments presented in this study indicate that receptor-mediated TRAF3 degradation is not sufficient to induce NF-κB2 activation in B cells and TRAF3 degradation-independent mechanisms can also regulate the NFκB2 pathway.

## **DISCUSSION**

Functions of TRAF molecules vary both between cell types, and between individual types of receptors even within the same cell. This is particularly true for TRAF3 (2). There are many disadvantages of using the experimental model of transient overexpression in transformed cell lines, usually epithelial or fibroblast, to understand how endogenous signaling pathways work in specific hematopoietic cell types [reviewed in Ref. (47)]. Unfortunately, a great deal of the experimental investigation of TRAF3 functions to date has relied upon overexpression models. Largely based upon such models, it was concluded that receptor-induced TRAF3 degradation leading to NIK stabilization is a crucial requirement for downstream activation of the non-canonical NF-κB2 pathway (20, 26, 27).

We wished to further explore this model in B lymphocytes, as our previous findings, and those of others, indicate that TRAF3 plays uniquely important roles in regulation of this cell type. Our results presented here using complementary experimental systems of primary B cells and B cell lines, demonstrate that TRAF3 degradation is not sufficient for downstream NF-κB2 activation. The results from our study further emphasize the importance of studying TRAF functions in a cell type and receptor-specific manner. In this study, we also demonstrated TRAF-N and TRAF-C domains are required for recruitment of TRAF3 to CD40 and BAFFR, and that association with the TRAF2 complex is necessary for receptor-mediated TRAF3 degradation (**Figures 1** and **4**). However, the lack of degradation of the MM-derived TRAF3 mutant LP1 did not prevent activation of downstream signaling pathways; in fact, LP1-expressing B cells showed similar ability to undergo receptor-induced NF-κB2 activation, and enhanced JNK activation. In contrast to a previous study performed in non-hematopoietic cells (20), we found that TRAF3 association with the NF-κB2-activating kinase NIK in B cells does not require the TRAF-N and TRAF-C domains. Although lacking these domains, the LP1 mutant TRAF3 molecule actually bound substantially more NIK than Wt TRAF3. While the explanation for this enhanced binding is currently unknown, it is possible that one or more additional protein–protein interactions are involved. A slight increase in total NIK protein in LP1-expressing cells also suggested that NIK stabilization can be controlled by additional mechanisms. IKKα phosphorylation of NIK also destabilizes NIK (48). Additionally, TBK1 stabilizes NIK protein levels independent of TRAF3 degradation,while in contrast the protein OTUD7B regulates NIK protein levels through TRAF3 degradation in B cells (49, 50). Unlike TRAF3-deficient B cells, LP1 TRAF3-expressing

experiments.

the fold increase over unstimulated control within each strain of mouse, from two independent experiments. **(C)** Equal numbers of splenic B cells isolated

band intensity values of treated/control samples ± SEM of three independent

B cells responded to both BAFF and CD40 stimulation with further increases in nuclear p52 levels. The current proposed model is that TRAF3 association with the TRAF2-cIAP complex prevents activation of the NF-κB2 pathway. Results presented here indicate that the mechanism by which TRAF3 regulates NF-κB2 activation is more complex than this. The degradation-resistant LP1 mutant TRAF3, without complexing with TRAF2-cIAP, can still regulate NF-κB2 activation. Our results also showed that the amino terminal RING and Zinc finger domains of TRAF3 are also quite important for TRAF3 to restrain NF-κB2 activation,in accordance with a published study (8). Taken together, the published and present data demonstrate that there are multiple mechanisms by which TRAF3 regulates the NF-κB2 pathway in B cells, both dependent upon, and independent of receptor-mediated TRAF3 degradation.

B cell activating factor receptor-mediated activation of signaling pathways is important for B cell homeostasis and survival, as exemplified by the deficiencies in mature B cell numbers and

function observed in BAFF−/<sup>−</sup> and BAFFR−/<sup>−</sup> mice, as well as the spontaneous BAFFR mutant-expressing mouse, A/WySnJ (5). The NF-κB2 pathway is robustly activated by BAFFR, and is thought to play a major role in BAFFR-mediated B cell survival signals (21). Thus, the previously presented paradigm of NF-κB2 activation predicts that loss-of-function mutants of BAFFR would show reduced ability to induce TRAF3 degradation in B cells. Consistent with previous reports (28, 51), we observed impaired BAFFmediated NF-κB2 activation in B cells isolated from A/WySnJ and BAFFR−/<sup>−</sup> mice, as well as with B cell lines expressing mutant hCD40-BAFFR chimeric receptors. We further demonstrated that the last eight amino acids of the BAFFR cytoplasmic domain are required for NF-κB2 activation in B cells. This is particularly interesting, because Mayne and Hayes reported that these residues are NOT required for BAFFR-mediated B cell survival (29). Together with the present results, this indicates that NF-κB2 activation is not the only important pro-survival pathway induced by BAFFR signaling.

Several studies have concluded that receptor-induced TRAF3 degradation is sufficient to activate the NF-κB2 pathway (20, 26, 27). Because the A/WySnJ BAFFR retains the TRAF3 binding motif PVPAT, we compared BAFFR-inducedTRAF3 degradation using B cells expressing different forms of BAFFR. Again in contrast to the current paradigm,wefound that signaling through theA/WySnJ or ∆C BAFFRs induced TRAF3 degradation to levels similar to Wt BAFFR, leading to the conclusion that receptor-induced TRAF3 degradation is not sufficient to drive NF-κB2 activation. Additionally, the A/WySnJ BAFFR recruits more TRAF2 and 3 to its cytoplasmic tail, so it is possible that the mutant BAFFR is recruiting important additional, unidentified signaling molecules that regulate NF-κB2 activation.We also found that the last eight amino acids of the C terminus of BAFFR were not required for either receptor-mediated TRAF3 degradation or TRAF recruitment to the receptor, but the impact of this region upon recruitment of other unidentified factors are not yet known.

In summary, our data demonstrate that the relationship between TRAF3 degradation and activation of NF-κB2 in B cells is neither simple nor linear. These findings support the need for further investigation into both how NF-κB2 activation is regulated in B cells, as well as how TRAF3 may regulate B cell survival via mechanisms additional to NF-κB2 activation. The identification of loss-of-function TRAF3 mutations in various types of human B cell malignancies underlines the importance of reaching a clearer and more detailed understanding of these relationships.

### **AUTHOR CONTRIBUTIONS**

Wai W. Lin, Joanne M. Hildebrand, and Gail A. Bishop designed research; Wai W. Lin and Joanne M. Hildebrand performed experiments. Wai W. Lin, Joanne M. Hildebrand, and Gail A. Bishop prepared the manuscript.

## **ACKNOWLEDGMENTS**

The authors are grateful to Drs. Colleen Hayes and Christopher Mayne for provision of some of the mouse strains used in this study, as well as BAFF receptor constructs, as described in Section "Materials and Methods." We thank Dr. Ahmad Halwani for production of the FLAG-tagged human TRAF3 and LP1 TRAF3 plasmids. We also thank Dr. Bruce Hostager for critical review of the manuscript and helpful comments. This work was supported by NIH grants AI028847 and AI049993 and a Research Career Scientist award from the Department of Veterans Affairs to Gail A. Bishop. This material is based upon work supported in part by facilities and equipment provided by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development. Joanne M. Hildebrand received support from an Overseas Biomedical Postdoctoral Fellowship from the Australian NHMRC (#541951).

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The editor declared a potential conflict of interest to the main Topic Editor of this Research Topic, Dr. Linda Burkly, prior to handling this paper because the author Joanne Hildebrand and the editor John Silke are currently employed by the Walter and Eliza Hall Institute of Medical Research. Dr. Burkly was satisfied that the potential for conflict was minor. The editor, John Silke, now further declares that there has been no conflict of interest during the review and handling of this manuscript.

*Received: 16 September 2013; accepted: 07 December 2013; published online: 20 December 2013.*

*Citation: Lin WW, Hildebrand JM and Bishop GA (2013) A complex relationship between TRAF3 and non-canonical NF-*κ*B2 activation in B lymphocytes. Front. Immunol. 4:477. doi: 10.3389/fimmu.2013.00477*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Lin, Hildebrand and Bishop. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Role of the lymphotoxin/LIGHT system in the development and maintenance of reticular networks and vasculature in lymphoid tissues

## **Theresa T. Lu1,2\* and Jeffrey L. Browning<sup>3</sup>\***

<sup>1</sup> Autoimmunity and Inflammation Program and Pediatric Rheumatology, Hospital for Special Surgery, New York, NY, USA

<sup>2</sup> Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY, USA

<sup>3</sup> Department of Microbiology and Section of Rheumatology, Boston University School of Medicine, Boston, MA, USA

### **Edited by:**

Linda C. Burkly, Biogen Idec, Inc., USA

### **Reviewed by:**

Sanjiv A. Luther, University of Lausanne, Switzerland Jorge Caamano, University of Birmingham, UK

### **\*Correspondence:**

Theresa T. Lu, Autoimmunity and Inflammation Program and Pediatric Rheumatology, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA e-mail<EMAIL_ADDRESS>Jeffrey L. Browning, Department of Microbiology and Section of Rheumatology, Boston University School of Medicine, 72 East Concord Street, E-5, Boston, MA 02118, USA e-mail<EMAIL_ADDRESS>
Lymphoid organs are meeting zones where lymphocytes come together and encounter antigens present in the blood and lymph or as delivered by cells migrating from the draining tissue bed. The exquisite efficiency of this process relies heavily on highly specialized anatomy to direct and position the various players. Gated entry and exit control access to these theaters and reticular networks and associated chemokines guide cells into the proper sections. Lymphoid tissues are remarkably plastic, being able to expand dramatically and then involute upon resolution of the danger. All of the reticular scaffolds and vascular and lymphatic components adapt accordingly. As such, the lymph node (LN) is a wonderful example of a physiologic remodeling process and is potentially a guide to study such elements in pathological settings such as fibrosis, chronic infection, and tumor metastasis.The lymphotoxin/LIGHT axis delivers critical differentiation signals that direct and hone differentiation of both reticular networks and the vasculature. Considerable progress has been made recently in understanding the mesenchymal differentiation pathways leading to these specialized networks and in the remodeling that occurs in reactive LNs. In this article, we will review some new advances in the area in terms of developmental, differentiation, and maintenance events mediated by this axis.

**Keywords: lymphotoxin-beta receptor, reticular stroma, high endothelial venules, reactive lymph node, follicular dendritic cell, fibroblastic reticular cells**

## **INTRODUCTION**

The functional specialization of the reticular scaffolds and the vascular and lymphatic vessels in lymphoid organs is essential for the exquisite sensitivity and efficiency of the immune system. These various stromal elements have been increasingly well studied over the last decade with major advances occurring in the description of their developmental lineages and differentiation as well as their activation and adaptation in reactive lymphoid organs. This field has been extensively reviewed (1–12). In general, the stromal cells in the secondary lymphoid organs (SLO) include reticular cells of which there are at least two different kinds, the follicular dendritic cell (FDC) and the fibroblastic reticular cell (FRC). Additional fibroblastoid cells are present in splenic red pulp and in the medullary sinuses of the lymph nodes (LN) (2). Cells of endothelial origin include blood endothelial cells of which there are both conventional "flat" forms as well as high endothelial venules (HEV) and the lymphatic endothelium that lines the afferent and efferent lymphatics, the subcapsular sinus, and the various lymphatic sinuses within the cortex, paracortex, and medulla (13). These structures are relatively stable in the resting state but undergo dynamic remodeling in the case of reactive LN. Reactive LN normally involute and presumably restore homeostasis once the activating trigger is resolved.

The lymphotoxin (LT) system, a member of the TNF family, is a key regulator of lymphoid architecture. For almost two decades now, the roles played by this pivotal system have been studied in the context of the development and maintenance of lymphoid reticular networks, HEVs, neo-angiogenesis, and lymphangiogenesis as well as their impact on immunological function. This pathway has been reviewed in detail (14–20). With a focus on recent advances, we highlight here the role of the LT system in the control of the reticular and endothelial elements within organized lymphoid structures.

## **THE LYMPHOTOXIN SYSTEM**

The original LT protein, called LTα, is a member of the TNF family of ligands and is secreted as a homotrimeric molecule. Like TNF, LTα binds to the TNF receptors. An additional membrane bound form of LTα is composed of a heterotrimeric complex with a second protein called LTβ in a LTα1/β2 stoichiometry (LTα/β). LTα/β binds uniquely to the lymphotoxin-beta receptor (LTβR) (**Figure 1**). A second ligand, LIGHT (TNFSF14), binds to three different receptors; LTβR (TNFRSF3) with high affinity, another TNF family receptor called HVEM (TNFRSF14) and a soluble receptor named DcR3 (TNFRSF6B). LIGHT is found in both membrane tethered and secreted forms. Dimerization of LTβR by either LIGHT or LTα/β is sufficient to induce signal transduction (21, 22); however, it is likely that higher order oligomerization states of the receptor lead to either more intense or qualitatively different signaling (23). HVEM also interacts with several non-TNF family

members, i.e., BTLA and CD160 and these proteins have contextspecific regulatory effects on T and NK cell function (20, 24, 25). Human but not murine LTα can bind to HVEM and LTα binds to Troy (TNFRSF19) (26). However, these LTα interactions are low affinity and their physiological significance is questionable (27).

The soluble human LTα trimer appears to have specific functions. Most notably, LTα and LTβ knockout animals differ in that LTβ <sup>−</sup>/<sup>−</sup> mice retain the mesenteric lymph node (mLN) while LTα <sup>−</sup>/<sup>−</sup> mice lack all nodes (19). Likewise, the two LT knockout animals differ in their ability to survive cerebral malaria (28). The consequences of ectopic LTα gain-of-function in transgenic mice differ depending on whether or not LTβ is expressed with peripheral lymph node addressin (PNAd) display requiring LTβ (29). More recently, a specific role for LTα in the induction of gut IgA responses was defined (30). Indeed, differences between LTα and LTβ deficient mice have been attributed to additional and unique roles for the LTα homotrimer. Whether such LTα-specific functions are physiological can be questioned as the LTβ <sup>−</sup>/<sup>−</sup> mouse represents potentially an LTα gain-of-function mouse. In a study using transfected cells, LTβ expression dramatically rerouted LTα expression into the membrane form to the detriment of secreted LTα (31). Thus, the loss of LTβ could initiate an LTα-driven inflammatory response especially in mucosal environments when TNF expression is minimal. In general, it is believed that the LTα/β heteromeric ligand represents a direct cell-to-cell communication system, whereas LTα3 is only secreted. In early studies, efficient secretion or shedding of the LTα/β complex was not observed (31– 33); however, recently small amounts of shed LTα/β were detected in the blood and synovial fluid from rheumatoid arthritis patients using a highly sensitive assay (34).

The LTα/β-LTβR system has been implicated in many immunological events including maintenance of FDC networks, HEV, specialized macrophages capable of antigen capture in the splenic marginal zone and subcapsular sinus of the LN as well as dendritic

cell (DC) homeostasis and differentiation (19, 35). This system also plays major roles in mucosal microenvironments with multiple consequences for epithelial cell biology and host defense/repair programs (36). LTβR signaling affects the state of specialized epithelial cells within the medulla of the thymus (37). The role of LT in epithelial cell interactions is beyond the scope of this review. Both LTβR and TNF-receptors activate canonical NFκB signaling, yet LTβR is distinguished by the efficient activation of the alternative NFκB pathway (19). Importantly, receptor internalization is required to shift NFκB activation from the classical to the alternative pathway and the nature of the ligand, e.g., soluble LIGHT vs. membrane bound forms of LIGHT or LTα/β may affect the ability to drive receptor internalization (38). The alternative pathway is typically linked to developmental and cell differentiation programs in contrast to the predominantly inflammatory role of the canonical pathway (39). The centerpiece of the alternate NFκB pathway is NIK kinase activation and NIK is required for many if not all of the developmental and differentiation events associated with LTβR (19).

Lymphotoxin-beta receptor is readily observed on most nonhematopoietic cells and on monocytic and DC lineages. Historically expression of the LT ligand was considered to be unique to hematopoietic lineages, especially B cells, activated T cells including Th1 and Th17 subsets, NK cells and innate lymphoid cells (40). This picture has become fuzzier of late, since LTβ RNA is readily observed in several other cell types such as in DC, hepatocytes, and hepatic oval cells (13, 41, 42). As LTβ RNA appears more abundant, LTα expression has been viewed as the limiting element in controlling membrane LTα/β display. It is less clear how often LTβ RNA reflects actual functional membrane LT, which requires expression of both LT proteins. Bona fide surface LTβ protein was observed on DC by histology and FACS; however, surface heteromeric LT α/β protein capable of binding LTβR actually has not been described outside of the hematopoietic system (43). As non-hematopoietic lineage cells and myeloid lineages usually express LTβR, it is theoretically possible that functional ligand produced within such a cell rapidly complexes with receptor, initiates signal transduction and the complex is processed. In such a case, cell autonomous LTβR signaling could be possible without actual transit to the surface and, moreover, surface ligand detection would be difficult. Furthermore, in cells with a high density of LTβR, there is evidence for autonomous signaling in the absence of any ligand (44).

The LT genes lie within a cluster with TNF in the class III region of the MHC and epigenetic control and chromatin organization are key elements of the regulation of cytokine expression within the cluster (45, 46). In general, understanding of the physiological transcriptional control of the LT and LIGHT genes remains incomplete. The LTβ promoter uses Ets1, Sp1, NFκB, and Egr-1/Sp1 transcription factors to drive RNA expression and expression was induced by TNF in a hepatocyte cell line (47). Furthermore, a regulatory element in exon 4 of the LTβ gene appears to interact with multiple sites within the TNF gene cluster (48). In general, LTα is expressed by activated lymphocytes and NFAT, NFκB, Sp1, and STAT elements are found within the promoter region. LTα expression can be induced by interleukin-12 p40 homodimer in myeloid lineages (49). Non-hematopoietic cells can also make LTα, for example, vascular smooth muscle cells secrete LTα in response

to extracellular nucleotide activation of the P2Y<sup>2</sup> receptor (50). Adding to this complexity, LTα RNA can be found in several variants. Interestingly, an alternate core promoter residing within the intron between exons 1 and 2 was predominantly utilized in T cells stimulated with TGFβ1 or FGF-7 (51). Clearly, a better understanding of the physiological regulation of the LT locus is needed.

## **LYMPHOTOXIN AND THE DIFFERENTIATION STATES OF RETICULAR FIBROBLASTS IN RESTING LYMPHOID TISSUES**

The cells forming the lymphoid reticular scaffolds are of mesenchymal lineage and generally are grouped into two broad categories based on their locations within organized lymphoid organs (**Figure 2**). FDC scaffold the B cell follicles and perform a multitude of functions related to antigen-presentation, cell survival, and handling of apoptotic cells (9–12). They possess a unique recycling mechanism to protect captured antigen from degradation and retain it for long-term presentation (52). FDC express the chemokine CXCL13 required for follicular compartmentalization of B cells (10). The second major category of reticular networks encompasses the T cell region of both LN and the spleen and is formed by FRC (2, 5, 8). FRC have multiple functions including the attraction of CCR7-expressing T cells and DC by expression of CCL19 and CCL21, T cell survival signals, and formation of collagen fibers. As such, they are fundamentally different from the FDC networks. A third category called a marginal reticular cell (MRC) has received recent attention. This cell sits under the subcapsular sinus close to B cell follicles, combines elements of both FDC and FRC and has been proposed to be a progenitor cell to the two major lineages (5, 53). Within the FRCs, there is heterogeneity, for example, VEGF-expressing FRC are enriched in vessel-rich areas (54). Also, using a lineage-tracing system for past expression of CD21, a population of mesenchymal cells in the T zone was identified that are similar to FRCs, yet express higher levels of TGFβ and lower amounts of CCL19 and CCL21 relative to FRCs (55). How these cells are related in lineage to the classical FRCs remains to be determined.

To understand reticular cell differentiation pathways, it is important to appreciate the embryological underpinnings of LN development (16). Early LN anlagen are formed from buds of endothelial and mesenchymal tissue. The mesenchymal cells progress through an ICAM1/VCAM1+ intermediate phenotype and at this stage, upon LTβR activation by hematopoietic lymphoid tissue inducer (LTi) cells, they acquire an ICAM1/VCAM1/MAdCAM1+ stromal organizer phenotype (56). This cell is often referred to as a lymphoid tissue organizer (LTo). The LTo cells now express CCL19, CCL21, CXCL13, IL-7, and RelB, and these markers will typify future specialized stromal elements in the mature organ. The reliance on LTβR signaling at the stromal organizer switching point is believed to account for the impaired development of LN in mice deficient in components of the LT pathway. Importantly, ontogeny has provided an important guide in understanding lymphoid tissue changes as embryological paradigms are largely recapitulated during inflammation (57, 58). Studies on the role of the LT system have always been plagued by the complexity of the direct knockout animals, i.e., the lack of LN and the issues of dissociating embryological

**FIGURE 2 | Examples of FDC and FRC networks in the human tonsil**. Top panel shows the podoplanin positive FRC network and lymphatic vessels (both brown) and the surrounding HEV and non-HEV vessels (CD34, blue). Bottom panel shows the FDC network (CD35, brown) in a secondary follicle with a GC. Note light and dark (CD35 positive) regions are readily discerned along with the residual primary follicle or mantle zone surrounding the GC. L, lymphatic vessel; FRC, fibroblastic reticular cell, HEV, high endothelial venule; M, mantle zone or primary follicle; GC, germinal center or secondary follicle, DZ, dark zone; LZ, light zone.

programs including postnatal events from physiological maintenance/homeostasis. Historically, pharmacological inhibition of LTβR signaling has been the most direct method to achieve dissection, however, cell-specific or inducible genetic manipulations now provide powerful tools.

## **RETICULAR NETWORK PROGENITORS**

The current paradigms for reticular cell specialization in the SLO invoke an early progenitor cell that is somewhat similar to a mesenchymal stem cell (MSC) (**Figure 3**) (6). Signals early in development provided by retinoic acid or RET ligand depending on the context lead to an immature organizer cell (16). In most contexts, LTβR signaling is required for progression to the

mature organizer or LTo that releases chemokines and displays surface adhesion molecules to nucleate the emerging LN anlage. Brendolan and colleagues recently were able to use lineage-tracing methods to map this pathway using the transcription factors Nkx2–5 and Islet1 (59). The Nkx2–5/Islet1 cells differentiated into all three major reticular lineages, MRC, FDC, and FRC as well as into classical mural cells or pericytes, but not the endothelial lineages.

progenitor for FRC and FDC lineages based on phenotype, but this relationship has not been formally proven.

## **FDC NETWORKS**

Lymphotoxin-beta receptor signaling is essential for the development, differentiation, and maintenance of FDC networks in the SLO, in mucosal compartments and in chronically inflamed sites, i.e., so-called tertiary lymphoid tissues/organs (TLTs/TLOs) (9–12). TNF signaling is also critical for the differentiation of the FDC phenotype, but not FDC maintenance at least in a secondary follicle. Surface LT on B cells is crucial for the development and maintenance of the FDC networks in the spleen as shown by the use of classical bone marrow chimeras and more specifically with a conditional B cell selective knockout of LTβ (60–63).

Historically, the term "dendritic cell" in this setting was an unfortunate misnomer and contributed to the confusion surrounding their true origin. Obscuring their origin even further, FDC "precursors" could be transferred from bone marrow; however, this observation preceded the discovery of MSCs in the bone marrow. In a breakthrough, the precursor cells to FDCs were recently identified as perivascular cells resembling pericytes (64). Pericytes are members of a subset of fibroblastoid cell types including classical pericytes also called mural cells, vascular smooth muscle cells, adventitial fibroblastoid cells, and stellate cells (hepatic, pancreatic, pituitary, astrocytes, etc.). MSCs are progenitors of both this immediate "pericyte" family as well as a much wider

group of cell types including adipocytes,chondrocytes,osteoblasts, fibroblasts, myofibroblasts, synoviocytes, and podocytes. The exact differences between MSC and perivascular cells or pericyte progenitors remain murky (65, 66). There is some semantic confusion as a pure classical definition of pericytes would include direct contact with an endothelial cell without interruption by a basement membrane. In practice, lacking unique cellular markers, there appears to be a spectrum of related perivascular cells.

Aguzzi and colleagues used multiple approaches to understand the origin of FDCs. They were able to lineage map PDGFRβ+ precursors into FDC and ablation of these same cells led to the loss of FDC. Additionally, transfer of adipose-derived perivascular cells into the renal capsule resulted in the formation of a local FDC network (64). B cells were the sources of LT and TNF for FDC development, yet in the absence of B cells, LTi cells were implicated as a key source of LT. It should be noted that these approaches were not completely definitive, e.g., the markers PDGFRB and MFGE8 are not specific for pericytes and FDC, respectively. This limitation in the study highlights how the pericyte field suffers from the lack of absolutely unique markers (67). Nonetheless, PDGFRβ positive cells in adipose tissue are generally accepted to be a pericyte/MSC and these cells were able to give rise to FDCs. Therefore, the differentiation of mesenchymal progenitor cells into a reticular cell that is highly honed to retain antigen within the follicular microenvironment, requires either LT or TNF signaling provided by local lymphoid cells. This system provides an excellent example of the ability of the lymphoid cells to locally shape mesenchymal lineages.

Established FDC networks exist in both a primary form, i.e., in a follicle lacking a germinal center (GC) reaction or in GC containing secondary follicles wherein the network is further differentiated (10, 68). Maintenance of established networks absolutely requires LTβR signaling as shown by pharmacological inhibition of the pathway (69–73). Mice genetically deficient in elements of the LT system have been instrumental in defining the roles of splenic FDC. For example, spleens from LT-deficient mice displayed vastly reduced CXCL13 expression and pharmacological inhibition gave roughly similar results (74). Using various LT pathway deficient mice or bone marrow chimeric mice, lack of FDC resulted in profound impairment in both class switch recombination and affinity maturation. These results were probably complicated by additional defects in the splenic marginal zone, trafficking of cells to the correct compartments, and DC biology, etc. (14, 75). For example, the impressive loss of immune complex trapping on splenic FDCs in monkeys following LTβR inhibition was potentially a combination of both degradation of antigen trapping machinery in the primate marginal zone as well as FDC collapse (71).

The impact of FDC disruption has been studied by many approaches, but a selective depletion system described recently by Cyster and colleagues provided a relatively clean analysis (76). Loss of FDC networks collapsed follicular organization, reduced CXCL13 chemokine production and led to abortive GC reactions probably due to lack of long-term antigen retention (76). In monkeys, splenic FDC networks were substantially collapsed after only 1 month of pharmacological inhibition of LTβR signaling (71). Curiously, GC reactions in the mLN may be less dependent on proper FDC networks than in the spleen or peripheral LN (pLN) (76). mLN are clearly different from pLN in terms of their developmental requirements (76–79). In general, the role of LTβR signaling in FDC maintenance during a GC reaction remains poorly described.

### **FRC NETWORKS**

Fibroblastic reticular cell are reticular cells that scaffold the T cell rich regions of organized lymphoid structures (1, 2, 4, 7, 8). The chemokines CCL19 and CCL21 are produced by FRC and orchestrate the movement and attachment of lymphocytes along this backbone with DCs being more tightly attached and T cells loosely marching along the network to spatially facilitate T-DC encounters. Clean experiments to dissect immune function in the presence and absence of FRC are lacking and indeed some have questioned how much the FRC actually facilitates encounters (80). FRC also provide the IL-7 for T cell survival and the control of homeostatic compartment size in a manner reminiscent of BAFF and B cell survival (81). FRC have the capacity to directly present antigen to promote peripheral tolerance and they can respond with nitric oxide production during acute inflammation thereby dampening T cell responses (82–84). The scaffold itself is composed of collagen fibers that are made and wrapped by the FRC, forming a functional unit called a "conduit" (85). These conduits are found in all lymphoid tissues, e.g., spleen, thymus, LN, and TLT and appear intrinsic to an organized microenvironment. The conduits allow for very fast transport of low molecular weight proteins, i.e., up to roughly 70 kDa from the lymph or blood to the LN parenchyma. DC residing on the FRC can sample the conduit contents, raising the potential for direct detection of small antigens. Additionally, it is reasonable to propose that conduits allow the LN to sense chemokine/cytokine signals emanating from an inflamed tissue bed.

Recently, three studies have contributed greatly to understanding FRC differentiation. First, the Turley group in conjunction with the Immunological Genome project purified and analyzed the transcriptomes of FRC, blood endothelial, and lymphatic endothelial cells along with a fourth so-called double-negative cell (DNC) population that was negative for the endothelial marker CD31 and the FRC marker podoplanin (86). The DNC population was pericyte-like and most closely resembled FRC. This work provided a unique molecular fingerprint of these stromal populations and suggested a lineage relationship between pericytes and FRC. Caamano and colleagues showed that embryonic progenitor cells resembling preadipocytes can differentiate into lymphoid stromal cells (87). LTβR signaling, via the alternate NFκB pathway, was required to stop a progenitor cell from embarking on an adipocyte program and to direct a shift toward a lymphoid stromal cell differentiation pathway. This capacity was demonstrated using both embryonic and adult progenitors derived from adipose tissue. Moreover, they hypothesized that adipose tissue is a source of lymphoid stromal cells. Consistent with these observations, LIGHT-LTβR interactions can inhibit the program of adipocyte differentiation (88, 89). Third, Ludewig and coworkers exploited a unique CCL19-Cre mouse with expression limited to the FRC (90). Using this tool, LTβR could be selectively deleted from FRClike cells. Surprisingly, these mice developed LN and FRC/conduits normally; however, the LTβR-deficient FRC displayed reduced CCL19, CCL21 chemokine, and podoplanin expression. It is likely that insufficient CCL19 driven Cre is expressed at the early progenitor phase to remove LTβR prior to LTo generation and hence LN development proceeds normally. The mice bearing LTβR-deficient FRC poorly managed a murine herpes virus infection suggesting defective FRC diminished the immune response. These studies indicate that some aspects of FRC are under LT control, both developmentally and upon ectopic relocation, i.e., a setting similar to the induction of TLT.

Therefore, it appears that, like FDC, *de novo* development of FRC requires LTβR signaling. This result was best illustrated in the earlier observations on the formation of FRC networks in the spleen and TLT (74, 91). Despite these elegant studies, the basic question is less clear as to whether established FRC networks in either resting or reactive LN are under continuous LTβR control as observed with FDC. In prior analyses by the Cyster group, there was a clear developmental dependence on LT positive B cells for the splenic T zone FRC and their expression of CCL21 (74, 92). The development of splenic FRC, like FDC, continues from birth out for several weeks and indeed neonatal inhibition of LTβR signaling reduced CCL21 levels (a surrogate for FRC maturation) (92, 93). However, neither pharmacological inhibition of LTβR in adult mice nor transfer of LT-deficient lymphocytes appreciably reduced CCL21 levels in either spleen or mLN (74, 92). As in the spleen, FRC in LN continue to develop for 2–6 weeks post gestation based on CCL19 expression (90). The loss of LTβR on FRC using the CCL19-Cre system caused major disruptions in the nature of the FRC network (90). Likewise, combined TNF and LTβR signaling was synergistic leading to generation of collagen containing conduit-like fibrils in cultures of LN-derived stromal cells (94). One interpretation of this result is that canonical NFκB signaling by TNF is required to resupply the components to drive long-term alternate NFκB signaling by LTβR. In the light of these various observations, it is perhaps perplexing that pharmacological LTβR inhibition in adult mice did not affect CCL21 levels. However, these observations are consistent with a developmental role for LTβR signaling in FRC development, but not in the maintenance of established networks. Further histological analysis of non-matrix markers such as podoplanin, PDGFRβ, etc., expression in FRC networks following LTβR-Ig treatment need more careful examination. Therefore, in contrast to FRC development and FDC networks, the existing data indicate that the maintenance of established FRC networks is not under LT control.

## **THE ROLE OF THE LYMPHOTOXIN SYSTEM IN REGULATION OF THE RESTING LYMPHOID ENDOTHELIUM HIGH ENDOTHELIAL VENULES**

A prominent feature of LN is the presence of specialized postcapillary venules called HEVs that allow for the transit of lymphocytes from the blood into the LN parenchyma (13). The selective display of various adhesion molecules, coupled with specific chemokine triggers for integrin activation differentially gate access to the unique microenvironments of the skin, mucosa, and various LN. The robust transit of lymphocytes through the endothelium presses the endothelial cells into plump shapes giving the venule its characteristic "high" status (95). Development and maintenance of the HEV specifically requires LTβR signaling via the alternative NFκB pathway with a dialog occurring between LT positive DC and LTβR positive endothelial cells to promote the "HEV program" (96–98). The role of LTβR in HEV maintenance was conclusively demonstrated by pharmacological intervention of LTβR signaling in adult mice (99, 100). Developmentally, the requirement for LTβR signaling was less obvious since LN development itself is arrested in knockout mice, although addressin expression was reduced both in the mLN in LTβ deficient mice and in the occasional LN that develop following gestational inhibition of LTβR signaling (101, 102). Recently, deletion of LTβR expression selectively in endothelial lineages curtailed development of a normal repertoire of LN's, yet HEV development was blocked in remaining rudimentary LN (103). This study proved that the HEV program was directly under LTβR control and not a consequence of more indirect events related to a disrupted lymphoid architecture. Although some HEV are also associated with the pericyte-like DNCs (86), FRC appear to extend directly to the HEV, and wrap the abluminal face (104). It is conceivable that the FRC/HEV acts as a unit comparable to the astrocyte/pericyte/endothelial cell neurovascular unit or the relationship between stellate cells, pericytes. and endothelial cells in the liver sinusoids.

The PNAd molecule on HEV is a complex sulfated glycan assembled on specific scaffold proteins in both N- and O-linked forms. PNAd binds to L-selectin on the lymphocyte triggering the initial rolling. The basic LTβR-controlled "HEV program" encompasses the direct induction of scaffold gene expression in LN such as MAdCAM1, GLyCAM1, and CD34, as well as the biosynthetic machinery needed to assemble on the scaffold proteins the unique sulfated glycans that bind to L-selectin (13). MAdCAM1 is an addressin for the integrin α4β7, as well as itself being a scaffold for PNAd attachment. Vascular MAdCAM1 expression in the mLN is LTβR-dependent, although inflammatory signals such as TNF or secreted LTα can also induce MAdCAM1 expression (13). MAdCAM1 expression on the sinus floor of the splenic marginal zone and on FDCs is also LTβR-dependent as is mostly likely the expression on MRCs and the LN subcapsular sinus (105). Vascular MAdCAM1 is elevated in colitis in an LTβR-dependent manner; however, it is unresolved whether this component is indirect and secondary to effects on the disease processes (14).

HEV can readily accumulate radiolabeled sulfate and contain elevated levels of the machinery involved in sulfate uptake and transport (106, 107). In addition to specific sulfated glycan that comprise PNAd, sulfation is also involved in chemokine retention. Heparin sulfate is crucial for endothelial chemokine binding and lymphocyte trafficking as well as DC recruitment to the lymphatics (108). Whether there is any dependence on LTβR signaling for endothelial sulfate capture or sulfated matrix components has not been explored.

While considerable progress has been made uncovering the role of the LT pathway in lymphoid vascular biology, there remain murky aspects of HEV development and specialization within unique environments, e.g., mucosal, peripheral, TLT, tumor, etc. For example, the homeodomain transcription factor Nkx2.3 controls vascular compartmentalization in the spleen yet its deletion led to the formation of splenic HEV-like vasculature complete with expression of PNAd and CCL21 (109). HEV formation remained LTβR-dependent in line with the appearance of splenic PNAd positive structures in a mouse with a constitutive gain in alternative NFκB signaling (110). Nkx2.3 deficiency also led to altered splenic FRC structures and the presence of LYVE1 positive lymphatic-like structures thus pivoting the spleen toward an "LN architecture" (111).

## **LYMPHATICS**

The afferent lymphatics transport cells and soluble substances from the draining tissue to the subcapsular sinus. From the subcapsular sinus, DC find their way into the T zone parenchyma while T cells either flush through or they can enter the LN parenchyma via peripheral medullary sinuses (112). Recirculating lymphocytes leave the LN parenchyma by entering cortical sinuses that feed into medullary sinuses and efferent lymphatics (113). The lymphatic endothelial cells are an important source of sphingosine-1-phosphate that elicits lymphocytes to leave the parenchyma and enter the sinuses (114). Whether LTβR signaling is crucial for lymphatic function is less clear, although defects in lymphatic function were observed in LT-deficient mice (115).

## **LYMPHOTOXIN PATHWAY AND ADAPTATION IN THE REACTIVE STATE**

During immune responses, the reticular networks and the endothelium undergo growth and remodeling with an overall increase in LN cellularity. Such enlarged LN are termed "reactive" and normally the LN involute and return to the resting state once the triggering stimulus is resolved. Experimentally, reactive LN are induced by immunization with an adjuvant such as alum, complete Freund's adjuvant (CFA) or Montanide, or by infection with a pathogen. Total LN cellularity in both the resting and hypertrophic reactive state is LTβR-dependent (99, 116, 117). Whether the stroma or vasculature bears an imprint of prior reactivity is unknown, although gross lymphangiogenesis following inflammation is certainly reversible (118).

## **RETICULAR NETWORK**

Within the T zone, the reticular network expands with the enlarging T zone and the stromal cells undergoing proliferative expansion (119–121). The concentration of reticular fibrils under the follicles where T cells frequently interact with DC is known as the cortical ridge and this ridge becomes more prominent after immunization with ovalbumin (OVA) in alum (119). Following immunization with OVA in CFA, the stromal cells undergo an initial proliferative burst between days 0 and 2 that is dependent on CD11c+ DC cells and independent of lymphocytes. This burst is followed by continued proliferation and expansion in cell numbers that is detectable by day 5. The expansion was abrogated in the absence of lymphocytes and B cells contributed to the full expansion, while T cells contributed to the continued high proliferation rate (120). Recently, Luther and colleagues carefully analyzed changes in the FRC network following immunization with OVA in the adjuvant Montanide in mice containing transferred OVA specific T cells (121). Similar to changes induced with OVA/CFA, OVA/Montanide-induced a rapid CD11c+ celldependent stromal proliferation, and modest stromal expansion could be detected within 40 h. Stromal cells continued to proliferate and expand, and the expansion by day 5.5–6 was abrogated in RAG2−/<sup>−</sup> mice. Significantly, this study revealed that LTβR-Ig treatment did not affect the modest expansion at day 3, but did reduce FRC expansion at day 5.5–6 suggesting a role for LTα/β expressed by lymphocytes. A number of genes were previously shown to be altered in activated FRC and in this recent study, these cells displayed elevated levels of podoplanin and smooth muscle actin (86, 121). While maintenance of the resting FRC is not obviously under LTβR control, it is clear that events occurring in reactive FRC are LT/LIGHT-dependent and this dependence may underlie partially the profound effects of LTβR-Ig treatment on reactive LN. Again, the reticular changes occurring during inflammation appear to recapitulate programs that are utilized during development.

As hinted above, reticular networks may differ in their response to various immunological challenges including acute vs. chronic settings. For example, in contrast to Alum, CFA as an adjuvant leads to disruption of normal ER-TR7 patterning (ER-TR7 is a marker of collagen fibrils that is usually a surrogate for FRC presence), with loss of distinct B and T zones (119). A more detailed analysis of the time course is needed to determine whether the distinctions between Alum and CFA may in part reflect differences in kinetics. During a viral response, such as with LCMV infection, CCL21 and CXCL13 chemokine expression is downregulated during the first 8 days in the spleen and this response was partly dependent on interferon-γ but was minimally impacted by LTβR-Ig (119, 122, 123). The same process occurs in draining LN (123). Although, the LCMV clone-13 strain can infect the FRCs, this phenomenon of chemokine downregulation also occurred with OVA/LPS suggesting that this is a general phenomenon in inflamed lymphoid tissues (123). The loss of chemokine expression with viral infection may be in part attributable to loss of stromal cells (122). After this initial viral-induced stromal disruption, stromal cells recover and lymphoid organization is gradually restored. It is this process of recovery that is accelerated by LTi cells and is at least partially dependent on LTβR (122).

During LN hypertrophy, some of the most notable changes occur in the medulla (121, 124). This compartment normally appears condensed at homeostasis, but upon immune stimulation, the region swells and fills with lymphocytes. By day 6 after NP-OVA/CFA immunization, the parenchyma is enlarged relative to the area covered by the lymphatic marker LYVE1, and areas of collagen IV reticulum-rich and reticulum-poor areas can be identified. B cells are preferentially localized to the reticulum-poor area and newly generated plasma cells localize to the reticulumrich areas. The significance of this compartmentalization remains to be elucidated. Medullary remodeling was reduced by LTβR-Ig treatment in a manner that appeared independent of its effects on FDC and HEV (124).

## **VASCULATURE**

Following immunization, the vasculature also undergoes proliferative expansion that is accompanied by major phenotypic changes in the HEV. HEV, non-HEV blood endothelial cells, and lymphatic endothelial cells all proliferate and expand coordinately with remodeling of the feeding arteriole to deliver more blood flow (100, 120, 125–128). Similar to stromal cell growth, endothelial cells in LNs stimulated with OVA/CFA undergo initial CD11c+ cell-dependent proliferation followed by lymphocyte-dependent expansion (120, 125). HEVs can be seen to grow in length, width, and branching as imaged using optical projection tomography (116, 129). The HEV expansion triggered by LCMV infection is sensitive to LTβR-Ig and partially dependent upon B cell-derived LT. As B cells are not required for HEV maintenance at homeostasis (99, 100), this role of B cell-derived LTβ appears to be specific to the inflamed LN. With inflammation, there is the phenomenon of venularization whereby adjacent endothelium adopts a postcapillary venule phenotype (130). In inflamed LN, this change seems to occur as HEV (i.e., PNAd+) endothelial cells expand to a greater degree than PNAd-blood endothelial cells (120). One unresolved question is whether the reduction of HEV expansion in the absence of LTβR signaling reflects inhibition of HEV differentiation or inhibition of HEV proliferation? Anderson and colleagues observed that endothelial cell proliferation occurred frequently at transitions between high and flat endothelium, consistent with simultaneous proliferation and differentiation (131). Another area that is still poorly understood is whether the LTβRdependent HEV expansion reflects LTβR function in reticular cells, DC, or the endothelial cells.

In the reactive LN,numbers of both blood vessels and lymphatic sinuses increase and this expansion is accompanied by lymphatic endothelial cell proliferation (100, 120, 126). The increase in the number of lymphatic structures was partly sensitive to LTβR-Ig (100), but it is unknown whether LTβR-Ig blocked proliferation or impacted other facets of lymphatic growth. Somewhat surprisingly, LTβ <sup>−</sup>/<sup>−</sup> mice showed greater lymphangiogenesis and angiogenesis in inflamed lung and skin and hence, the general role of LTβR in LN vascular growth remains ill-defined (115). In contrast, LTα <sup>−</sup>/<sup>−</sup> mice showed reduced lymphatic function and reduced lymphangiogenesis with skin inflammation, and transgenic LTα expression drove lymphangiogenesis, suggesting a pro-lymphangiogenic role for LTα3 as well as a role for LTαβ in "sequestering" LTα subunits. In agreement with a role for LTα3,

TNF inhibition or TNFR1 deficiency were shown to partially reduce lymphangiogenesis in lung (132).

In addition to proliferative expansion, the HEVs also undergo phenotypic alterations. The phenotypic changes seen in reactive HEV are illustrated in **Figure 4**. At day 1 after injection of bone marrow derived DCs or OVA/CFA immunization, VCAM1 is upregulated and there is a greater trafficking of lymphocytes inwards via HEVs (104). This early change corresponds to disruption of the tight sheath of FRCs around vessels and the HEV activation is similar to the early ICAM1 upregulation seen after fever-range thermal stress (104, 133). By day 4 after oxazalone stimulation, HEV endothelial cells show reduced expression of proteins associated with the mature HEV program, i.e., LTβR, the scaffold protein GlyCAM1, and the sulfo-transferase HEC-GlcNAc6ST (100, 134). The downregulation of these HEV program proteins was accompanied by upregulation of MADCAM1 perhaps reflecting an immature HEV, since MADCAM1 is normally only expressed in peripheral nodes during development and in mucosal lymphoid tissues (100, 135). In the same time frame, LTβR expression decreases and PNAd and LYVE1 were colocalized despite LYVE1 being a typical lymphatic marker (100). It was unclear from this study whether this colocalization resulted from lymphatics that expressed PNAd or HEVs that expressed LYVE1. However, vessels with the morphology of HEVs in non-Hodgkins lymphoma patients expressed LYVE1 and blood vessels during development can express LYVE1, suggesting that the colocalization of PNAd with LYVE1 represents HEVs that have upregulated LYVE1 (136, 137). This PNAd/LYVE1 colocalization at day 4 was blocked by LTβR-Ig treatment at day 0 (100). By day 7, the mature HEV markers began to rebound while MADCAM1 and LYVE1 regressed, and this rebound phase was dependent on B cells and also sensitive to LTβR-Ig.