The results described above, most of them obtained in CTL, suggest that PKCθ is not mandatory for granule exocytosis. However, it has not been studied yet in detail its possible involvement in NK cell immune synapse signaling and in NK cell granule polarization and/or secretion.

# **CONCLUSION**

PKCθ plays an important role in tumor immune surveillance *in vivo* (Garaude et al., 2008; Aguiló et al., 2009). In the case of CTL-mediated tumor control, this can be explained by its role as a CTL survival factor (Barouch-Bentov et al., 2005), the impaired cytokine response observed in PKCθ deficient animals (Sun et al., 2000) and its implication in FasL expression (Villalba et al., 1999; Villunger et al., 1999; Pardo et al., 2003).

In the case of NK cell-mediated tumor control, several functional consequences of PKCθ deficiency can also contribute to

**FIGURE 3 | Schematic showing possible mechanisms for the role of PKC**θ **in NK cell-mediated anti-tumor immunity.** The following working hypothesis are formulated (see the text for details): **(A)** cytokines produced by macrophages and/or dendritic cells, by signaling through PKC-θ, increase granzyme B expression. An increase in granzyme B expression is directly associated with the augmentation of the cytolytic potential of NK cells; **(B)** cytokines produced by macrophages and/or dendritic cells, by signaling through PKC-θ, induce or increase the expression of a protein (X) implicated in degranulation of NK cells; **(C)** PKC-θ could be directly implicated in degranulation of NK cells through specific KARs.

the final outcome. As depicted in the schematic **Figure 2**, KARinduced TNF-α and IFN-γ secretion is defective in *PKC*θ −/− mice (Tassi et al., 2008), and this can contribute to a defective recruitment of effector cells to the site of tumor development (Aguiló et al., 2009). Also, PKCθ could be implicated in the induction of FasL expression (Pardo et al., 2003), although this has not been demonstrated in NK cells. In addition, the possibilities shown in **Figure 3** may be considered, although they should be taken as working hypothesis that need to be better characterized:

These three possibilities are not mutually exclusive, and all of them can contribute to the regulation of NK cell anti-tumoral

#### **REFERENCES**

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potential. In addition, different signaling and/or functional responses can be activated by different KARs, and PKC-θ could be relevant for signaling elicited by some KARs but not by others. Undoubtedly, uncovering the precise molecular mechanisms that direct these processes will allow a rational improvement of NK cell-mediated anti-tumor immunotherapy protocols.

#### **ACKNOWLEDGMENTS**

The work reflected in this review was supported by the CliNK project from Sudoe (UE), by SAF2010-15341 grant to Alberto Anel, SAF2008-02139 and SAF2011-2539 grants to Julián Pardo, from Ministerio de Economía y Competitividad (Spain). Elena Catalán was supported by an FPI fellowship from MEC. This work was also supported by the program "Chercheur d'avenir" from the Region Languedoc-Rousillon (Martín Villalba), a scientific program from the "Communauté de Travail des Pyrénées" (CTPP10/09 to Martín Villalba and Alberto Anel), the Association pour la Recherche contre le Cancer (Martín Villalba), the Fondation pour la Recherche Medicale (Martín Villalba), a grant FEDER Objectif competitivite (Martín Villalba), and a fellowship from the ARC and Higher Education Commission, Pakistan (M.G. Rathore).

the transcription factor complex AP-1 in T lymphocytes. *Mol. Cell. Biol.* 16, 1842–1850.

and Jacobs, R. (2010). Cytokineactivated NK cells inhibit PMN apoptosis and preserve their functional capacity. *Blood* 116, 1308–1316.

syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. *Blood* 110, 1906–1915.

(2002). *In vivo* evidence for a dependence on interleukin 15 for survival of natural killer cells. *Blood* 100, 3633–3638.

Rodriguez, F., Hernández, J., Hipskind, R. A., Anel, A., and Villalba, M. (2008). Impaired anti-leukemic immune response in PKCθ-deficient mice. *Mol. Immunol.* 45, 3463–3469.

lytic granule polarization and NK cell cytotoxicity. *Proc. Natl. Acad. Sci. U.S.A.* 105, 2568–2573.

Le, C., Defor, T. E., Burns, L. J., Orchard, P. J., Blazar, B. R., Wagner, J. E., Slungaard, A., Weisdorf, D. J., Okazaki, I. J., and McGlave, P. B. (2005). Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. *Blood* 105, 3051–3057.

Velardi, A. (2007b). Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. *Blood* 110, 433–440.

human natural killer cells. *Nat. Immunol.* 7, 524–532.

Green, D. R., and Baier, G. (1999). Synergistic action of protein kinase C θ and calcineurin is sufficient for Fas ligand expression and induction of a CrmA-sensitive apoptosis pathway in Jurkat T cells. *Eur. J. Immunol.* 29, 3549–3561.

and Acute Leukaemia Working Party of the EBMT. (2009). KIRligand incompatibility in the graftversus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia. *Leukemia* 23, 492–500.

Zeng, J., Cannon, J. L., Abraham, R. T., Way, M., Billadeau, D. D., Bubeck-Wardenberg, J., and Burkhardt, J. K. (2003). SLP-76 coordinates Nckdependent WASP recruitment with Vav-1/Cdc42-dependentWASP activation at the T cell–APC contact site. *J. Immunol.* 171, 1360–1368.

**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: 28 March 2012; accepted: 15 June 2012; published online: 05 July 2012. Citation: Anel A, Aguiló JI, Catalán E, Garaude J, Rathore MG, Pardo J and Villalba M (2012) Protein kinase C-*θ *(PKC*θ*) in natural killer cell function and antitumor immunity. Front. Immun. 3:187. doi: 10.3389/fimmu.2012.00187*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Anel, Aguiló, Catalán, Garaude, Rathore, Pardo and Villalba. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Targeting PKC**θ** in alloreactivity and graft-versushost-disease: unanswered questions and therapeutic potential

# *Crystina C. Bronk1,2, Xue-ZhongYu1,3 and Amer A. Beg1,3\**

1 Department of Immunology, Moffitt Cancer Center, Tampa, FL, USA

2 Cancer Biology PhD Program, University of South Florida, Tampa, FL, USA

3 Department of Oncologic Sciences, University of South Florida, Tampa, FL, USA

#### *Edited by:*

Noah Isakov, Ben-Gurion University of the Negev, Israel

#### *Reviewed by:*

Balbino Alarcon, Consejo Superior de Investigaciones Cientificas, Spain Etta Livneh, Ben-Gurion University of the Negev, Israel

#### *\*Correspondence:*

Amer A. Beg, Department of Immunology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA. e-mail<EMAIL_ADDRESS>
Protein kinase C isoform θ (PKCθ) is a key modulator of TCR signaling and mediates activation of NF-κB, NF-AT, and AP-1 transcription factors. Although in vitro studies of PKCθ−/− T cells have shown impaired activation responses, in vivo studies indicate that PKCθ requirement is not universal. While PKCθ is important in induction of experimentally induced autoimmune diseases in mice and generation of Th2 responses, it is not essential for induction of T cell proliferative and cytotoxic responses against influenza virus, LCMV, and vaccinia virus. The context-specific involvement of PKCθ in T cell responses suggests that inhibition of PKCθ may be beneficial in some but not all situations. In the bone marrow transplantation (BMT) setting, we have shown that graft-versus-host-disease (GVHD) cannot be induced in the absence of PKCθ. However, graft-versus-leukemia effects and T cell ability to clear virus infection remains intact. Therefore, PKCθ is a potential therapeutic target in BMT, inhibition of which may prevent GVHD while retaining anti-tumor and anti-infection responses.

**Keywords: alloreactivity, graft-versus-host-disease, protein kinase C,T cell activation, anti-tumor effect**

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Studies of PKCθ−/− mice have shown normal T cell development but greatly impaired *in vitro* proliferative responses (Sun et al., 2000; Pfeifhofer et al., 2003; Grumont et al., 2004). Several studies have shown that PKCθ exerts functions leading to survival of T cells and activation versus tolerance outcomes *in vivo* (Berg-Brown et al., 2004; Barouch-Bentov et al., 2005). In addition, PKCθ is crucial for induction of experimentally induced autoimmune diseases in mice, including encephalomyelitis, arthritis, and myocarditis (Salek-Ardakani et al., 2005; Anderson et al., 2006; Healy et al., 2006; Marsland et al., 2007). It has also been shown that T cells cannot mount Th2 responses in the absence of PKCθ, while induction of Th1 responses against *Leishmania major* are not substantially impacted (Marsland et al., 2004). PKCθ−/− mice can also induce robust infection clearing CD8 T cell responses against multiple viruses including influenza virus, LCMV, and vaccinia virus (Marsland et al., 2004). Although *in vitro* proliferation of PKCθ−/− T cells is typically impaired, why *in vivo* responses to infectious agents remain intact is not well understood. It is thought, however, that stimulation of innate immunity by these infectious agents bypasses the requirement for PKCθ *in vivo* (Marsland et al., 2005, 2007; Marsland and Kopf, 2008). Precisely how this is accomplished and which molecular pathways are involved in PKCθ-independent T cell activation is not clear. The context-specific requirement of PKCθ in T cell activation suggests that inhibition of PKCθ may be beneficial in some but not all situations. Thus, inhibiting PKCθ may be therapeutically beneficial, but these specific circumstances need to be identified.

#### **ESSENTIAL ROLE OF PKCθ IN ALLOREACTIVITY AND GRAFT-VERSUS-HOST-DISEASE**

Alloreactivity is initiated by T cells that specifically recognize mismatched (non-self) MHC/peptide complexes. Graft-versushost-disease (GVHD) is a potentially lethal complication of allogeneic bone marrow transplantation (BMT) in which alloreactive T cells from the donor are activated by mismatched major and/or minor histocompatibility complex antigens of the recipient. Although side-effects of BMT are severe, for many cancer patients this represents a last line of hope to remove residual tumor cells, as the alloantigen response mediating GVHD can also promote donor T cells to exert graft-versus-leukemia (GVL) effects (Shlomchik, 2007; Welniak et al., 2007). In this therapeutic BMT procedure, GVHD is the major complication as it leads to high morbidity and mortality of patients (Appelbaum, 2001; Shlomchik, 2007). To date, no clinical strategy has been established that can selectively prevent GVHD while preserving the GVL effect.

Given the differential requirement for PKCθ in distinct T cellmediated responses, we investigated a potential role for PKCθ in the alloreactive responses of GVHD and GVL (Valenzuela et al., 2009). To investigate the necessity of PKCθ, we used an acute model of GVHD with the donor and recipient mismatchedfor both major and minor histocompatibility complex antigens (Liang et al., 2007). CD4 and CD8 T cells from wild-type (WT), PKCθ−/− or NF-κB p50−/−cRel−/− mice were transferred together with WT T cell-depleted (TCD) BM cells into lethally irradiated MHC mismatched recipients. p50−/−cRel−/− T cells (Zheng et al., 2003) were used to investigate a potential requirement for NF-κB in this setting. As expected, recipients of WT T cells showed typical signs of GVHD with more than 70% mortality within 60 days after BMT. In a poignant divergence, the mice receiving PKCθ−/− or p50−/−cRel−/− T cells showed little evidence of GVHD and survived through the duration of the experiment. Additionally, histologic analysis of the small intestine (a major site of GVHD-induced tissue destruction) was normal in mice receiving PKCθ−/− T cells, while their WT counterparts showed glandular destruction, lymphocytic infiltrate, and loss of mucosa. Although a clear mechanism remains elusive, additional studies indicated that GVHD does not occur in the absence of PKCθ because these T cells have impaired proliferation and increased apoptosis.

# **PRESERVATION OF ANTI-VIRUS AND GVL RESPONSES BY T CELLS LACKING PKCθ**

Allogeneic BMT results in increased risk of life threatening infections due to conditioning regimens these patients must undergo. Previous studies showed that responses to bacterial and viral agents remain intact in the absence of PKCθ (Marsland et al., 2005; Marsland and Kopf, 2008); yet, such studies have not been performed in a post-BMT setting. Because CMV is one of the most prevalent viruses carried by humans, it represents a major threat to BMT patients (Meyers et al., 1986). To recapitulate this disease, we used an MCMV infection model (Hossain et al., 2007, 2008). Importantly, both anti-MCMV T cell response and virus clearance was comparable between WT and PKCθ−/− T cell transplanted mice (Valenzuela et al., 2009). Therefore, PKCθ is dispensable for a successful anti-MCMV response post-BMT.

The purpose of BMT in the cancer treatment settings is for donor T cells to be able to target malignant cells. Therefore, it is imperative that PKCθ−/− T cells retain this GVL activity. To this end, we performed studies (Valenzuela et al., 2009) using the A20 B cell lymphoma line (Liang et al., 2007). Different numbers of WT and PKCθ−/− T cells were transplanted, and evaluated for their ability to induce GVHD and mediate GVL activity. In the absence of T cells, all recipients died from lymphoma growth. All doses of WT T cells induced moderate to severe GVHD but little or no lymphoma growth. In contrast, all recipients of PKCθ−/− T cells survived through the duration of the experiment with mild body weight loss and low GVHD. Furthermore, all the recipients of high PKCθ−/− T cell numbers and most recipients of low or intermediate T cell numbers remained largely free of tumor. This result led us to the conclusion that PKCθ plays a substantial role in the induction of GVHD, but is not essential for GVL (Valenzuela et al., 2009). Importantly, these results indicate that pharmacological PKCθ targeting may impair GVHD without significantly impacting GVL responses.

### **KEY UNRESOLVED ISSUES IN PKCθ FUNCTION**

Previous studies and our findings indicate relatively normal responsiveness of PKCθ−/− cells to infectious agents as well as high affinity antigenic stimulation (e.g., OVA) *in vivo* (Valenzuela et al., 2009). However, PKCθ−/− T cell alloreactivity and GVHDinducing ability is severely impaired, likely due to reduced proliferation and survival in recipient mice (Valenzuela et al., 2009).

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Thisfundamental difference in the requirement of PKCθ in various settings is a key unanswered question, and is central for understanding how detrimental and beneficial functions of T cells in BMT can be separated. The specific inability of PKCθ−/− T cells to induce GVHD can be due to several mutually non-exclusive reasons. First, the conditioning regimen used for BMT may play an important role. Thus, lethal irradiation prior to BMT severely depletes recipient APC required for donor T cell activation. It is possible that reduction in APC impacts PKCθ−/− T cell responses more severely than WT T cells. Interestingly, allograft survival in heart transplantation models showed a relatively small requirement for PKCθ in transplant rejection (Manicassamy et al., 2008; Gruber et al., 2009), likely due to presence of compensatory functions of PKCα (Gruber et al., 2009). Therefore, it is possible that impaired alloreactivity in the absence of PKCθ is more pronounced in the BMT setting. Second, it is possible that defects in CD4 and CD8 T cell migration (Letschka et al., 2008) contribute to lack of GVHD induction in the absence of PKCθ. Thus, impaired migration of PKCθ−/− T cells to GVHD target organs such as the gut, lungs, and skin may be responsible for reduced GVHD. Third, the function of PKCθ in alloreactivity may not be limited to effector T cell responses. Previous studies investigating a role for PKCθ in Tregs suggest that PKCθ function in Treg may also be important in alloreactivity (Zanin-Zhorov et al., 2010). While PKCθ localizes to the immune synapse (IS) in effector T cell, PKCθ is sequestered in a distal complex away from the IS in Treg (Zanin-Zhorov et al., 2010). As such, PKCθ is responsible for mediating a negative effect on the suppressive function of Treg. Consequently, PKCθ inhibition enhances Treg function leading to protection from inflammatory colitis in mice (Zanin-Zhorov et al., 2010). A caveat worth mentioning here is that while PKCθ inhibition leads to enhanced Treg function, PKCθ absence does not have the same effect (Gupta et al., 2008). The underlying reason for this is not completely clear (Zanin-Zhorov et al., 2011).

The easiest albeit simplistic way to understand why PKCθ absence does not impact anti-infection and anti-tumor responses is to consider a role for functionally redundant pathways. As mentioned above, PKCθ is involved in regulating activation of NFκB, AP-1, and NF-AT. Studies by Marsland and colleagues have shown that microbial stimulation through pattern recognition receptors (PRR) can induce NF-κB activation in PKCθ−/− T cells (Marsland et al., 2005, 2007; Marsland and Kopf, 2008). Thus, PRR may play a key functionally redundant role with PKCθ during infection with microbial agents. In contrast, why anti-tumor responses are only slightly reduced in the absence of PKCθ is more difficult to understand. BMT is primarily used for leukemia treatment. Leukemic cells, in particular B lymphocytes, have naturally high expression of MHC and co-stimulatory molecules, reflecting the natural function of these lineages in antigen presentation. In the above-mentioned study (Valenzuela et al., 2009), A20 B cell lymphoma cells were used as tumor targets. Whether PKCθ−/− T cells are specifically (or only) able to eradicate leukemic tumors can be directly tested by determining PKCθ requirement in eradication of non-leukemic tumors. Mechanistically, one possibility is that functionally redundant pathways are strongly activated in PKCθ−/− T cells by A20 and potentially other leukemic tumors. Furthermore, leukemic tumors may represent better targets for PKCθ−/− T cells than epithelial cells targeted during GVHD. Regardless of precise mechanisms, it is likely that both responses to infectious agents and leukemic tumors are maintained in the absence of PKCθ through functionally redundant pathways. A recent study identified a novel rolefor PKCθ as a transcriptional coactivator capable of interacting with promoters of several immune function genes (Sutcliffe et al., 2011). How this function impacts alloreactivity and other known functions for PKCθ remains to be determined.

#### **INVESTIGATING THE EFFECT OF PKCθ INHIBITION IN GVHD AND GVL**

The above-mentioned studies provide strong rationale for targeting PKCθ in a BMT settings (Valenzuela et al., 2009). The PKC family has been implicated in tumor cell proliferation, survival, invasion, metastasis, and tumor angiogenesis. Therefore, targeting PKC isotypes may present an attractive target for novel anticancer therapies. Consequently, several inhibitors of PKC isoforms have been developed by different pharmaceutical companies. Although some selective PKCθ inhibitors have been reported (Cywin et al., 2007; Cole et al., 2008), their *in vivo* toxicity or efficacy remains to be determined. A major advance was achieved recently with AEB071 (developed by Novartis), a very potent and selective inhibitor of both novel and classical PKCs. Skvara

#### **REFERENCES**

Chaudhary, D. (2008). Identification, characterization and initial hit-tolead optimization of a series of 4 arylamino-3-pyridinecarbonitrile as protein kinase C theta (PKCtheta) inhibitors. *J. Med. Chem.* 51, 5958– 5963.

et al. (2008) reported that treatment with AEB071 significantly improved symptoms of patients with severe psoriasis. Another potentially important PKC inhibitor is enzastaurin (Ly317615, from Eli Lilly and Company; Baier and Wagner, 2009). Enzastaurin was identified as an inhibitor of the PKCβ isoform, but it also impacts the AKT pathway (Graff et al., 2005); importantly, enzastaurin inhibits the PKCθ isoform approximately fivefold more potently than the beta isoform (Graff et al., 2005). *In vivo* studies indicate that enzastaurin is very well tolerated with a favorable safety profile, allowing it to be dosed for extended durations (Herbst et al., 2007). Furthermore, enzastaurin has antiproliferative and pro-apoptotic activity in solid tumors as well as hematological malignancies, including leukemia and lymphoma (Herbst et al., 2007). Thus, while not exclusively specific for PKCθ, enzastaurin has the major advantage of being well tolerated *in vivo* and the added benefit of anti-tumor effects. Hence, PKCθ inhibition by enzastaurin may prevent GVHD while preserving GVL responses, which will act cooperatively with the direct antitumor effects of enzastaurin. We expect future studies to define more potent and specific inhibitors of PKCθ and consequently help move this important therapeutic target to the clinical arena.

#### **ACKNOWLEDGMENT**

This work was supported by 5R01AI082685.

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and Waller, E. K. (2007). Chronic GvHD decreases antiviral immune responses in allogeneic BMT. *Blood* 109, 4548–4556.

J., and Rao, S. (2011). Chromatinassociated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes. *Mol. Cell* 41, 704–719.

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deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for NF-kB in regulating mature T cell survival and in vivo function. *J. Exp. Med.* 197, 861–874.

**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: 13 April 2012; accepted: 01 August 2012; published online: 14 August 2012.*

*Citation: Bronk CC, Yu X-Z and Beg AA (2012) Targeting PKC*θ *in alloreactivity and graft-versus-host-disease: unanswered questions and therapeutic potential. Front. Immun. 3:259. doi: 10.3389/fimmu.2012.00259*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Bronk, Yu and Beg. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

**REVIEW ARTICLE** published: 02 August 2012 doi: 10.3389/fimmu.2012.00225

# Intervention of PKC-**θ** as an immunosuppressive regimen

# *Zuoming Sun\**

Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA

#### *Edited by:*

Noah Isakov, Ben Gurion University of the Negev, Israel

#### *Reviewed by:*

Balbino Alarcon, Consejo Superior de Investigaciones Cientificas, Spain Marina Bouche, Sapienza University of Rome, Italy

#### *\*Correspondence:*

Zuoming Sun, Division of Immunology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA. e-mail<EMAIL_ADDRESS>
PKC-θ is selectively enriched in T cells and specifically translocates to immunological synapse where it mediates critical T cell receptor signals required for T cell activation, differentiation, and survival. T cells deficient in PKC-θ are defective in their ability to differentiate into inflammatory effector cells that mediate actual immune responses whereas, their differentiation into regulatory T cells (Treg) that inhibits the inflammatory T cells is enhanced. Therefore, the manipulation of PKC-θ activity can shift the ratio between inflammatory effector T cells and inhibitory Tregs, to control T cell-mediated immune responses that are responsible for autoimmunity and allograft rejection. Indeed, PKC-θ-deficient mice are resistant to the development of severalTh2 andTh17-dependent autoimmune diseases and are defective in mounting alloimmune responses required for rejection of transplanted allografts and graft-versus-host disease. Selective inhibition of PKC-θ is therefore considered as a potential treatment for prevention of autoimmune diseases and allograft rejection.

**Keywords: PKC-θ,T cell activation,T cell differentiation, autoimmunity, allograft rejection**

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# **INTRODUCTION**

T cells that are newly migrated out of thymus are naïve T cells incapable of mediating immune responses. Engagement of antigens with their T cell receptors (TCR) initiates the activation and differentiation programs that transform naïve T cells into inflammatory Th1, Th2, and Th17 effector cells or regulatory T cells (Treg), both of which participate in actual immune responses. The interference with molecules involved in T cell activation and differentiation is therefore considered an effective strategy to control the overwhelming immune responses that mediate autoimmunity and allograft rejection. However, most molecules involved in the processes of T cell activation and differentiation are not likely to be good targets for interference or inhibition because they also play essential roles in other cell types and signaling pathways. The ideal targets should meet at least the following criteria: (1) they selectively regulate T cell function; (2) small molecule inhibitors of them are easy to isolate or develop; (3) rather than inhibit, they actually promote Treg function. Ideally, inhibition of the target molecule should inhibit inflammatory T cells, and at the same time, enhance the suppressive function of Tregs. In this review, we summarize the evidence that supports PKC-θ as an excellent target for development of immunosuppressive agents.

# **PKC-θ IS SELECTIVELY ENRICHED IN T CELLS**

PKC-θ belongs to a family of serine/threonine kinases that consists of 12 different isoforms, each with distinct roles in the regulation of cellular functions (Newton, 1997). Members of this family can be divided into three subfamilies (Newton, 1997): conventional PKCs, including PKC-α, β, and γ, which are activated by Ca2+ and diacylglycerol (DAG); novel PKCs, including PKC-δ, θ, η, and ε, whose activation is dependent on DAG but is independent of Ca2+; and the atypical PKCs, PKC-ζ and λ, whose activation occurs independently of both Ca2+ and DAG. PKC-θ was first cloned as a novel PKC predominantly expressed in skeleton muscle (Osada et al., 1992; Chang et al., 1993) and found to be significantly enriched in hematopoietic compartments and skeleton muscle (Baier et al., 1994; Altman et al., 2000; Bauer et al., 2000). In hematopoietic compartments, PKC-θ is primarily expressed in T cells, but not B cells, and this has also been confirmed by *in situ* hybridization to mouse whole body sections (Bauer et al., 2000). The selective expression pattern of PKC-θ strongly suggests it plays a unique function in T cell compartments, and therefore may be a good immunosuppressive target for controlling T cellmediated immunity. However, it is important to point out that T cells also express other isoforms of PKC (Bauer et al., 2000). Furthermore, although most studies have so far focused on PKC-θ function in T cells, there is evidence supporting that PKC-θ is also expressed and play a role in other tissues including muscle (Kim et al., 2004; Benoit et al., 2009; Paoletti et al., 2010), platelets (Nagy et al., 2009; Harper and Poole, 2010; Cohen et al., 2011), natural killer (NK) cells (Aguilo et al., 2009), and likely mast cells (Kempuraj et al., 2005). Therefore, inhibition or targeting of PKC-θ for immunotherapeutic treatments may also affect other tissues in addition to T cells.

#### **PKC-θ SELECTIVELY TRANSLOCATES TO IMMUNOLOGICAL SYNAPSE**

PKC-θ attracted significant attention when it was shown among all the isoforms of PKC expressed in T cells, PKC-θ selectively translocates to the immunological synapse (IS), the stable cell– cell junction formed between T cells and antigen-presenting cells (Monks et al., 1997, 1998). The IS is a cluster of specialized membrane microdomains where TCR signaling molecules, including the TCR itself, are assembled (Grakoui et al., 1999). Formation of the IS is an active process that requires Lck-mediated signals to initiate re-organization of cytoskeleton (Morgan et al., 2001). Although there is still some controversy (Lee et al., 2002), it is generally believed that the IS serves as the platform that delivers integrated signals essential for T cell activation (Moran and Miceli, 1998). The IS consists of three major compartments: the central supramolecular activation cluster (cSMAC), the peripheral SMAC (pSMAC), and the distal SMAC (dSMAC; Barouch-Bentov et al., 2005; Dustin, 2009). The cSMAC was initially referred to as a signaling structure (Monks et al., 1998; Freiberg et al., 2002) and is located at the center of the IS. Around this center is the pSMAC, a ring of LFA-1/ICAM-1 co-localized with the cytoskeletal integrin linker talin (Monks et al., 1998). The outermost ring is the dSMAC, a zone enriched in the CD45 tyrosine phosphatase (Freiberg et al., 2002). Recently, it was demonstrated that the TCRs initially microcluster in the dSMAC, and then move through the pSMAC into the cSMAC, and is believed to be critical for the generation of continuous TCR signals that are required for T cell activation (Varma et al., 2006). PKC-θ is recruited to the junction between the cSMAC and pSMAC and co-localizes with TCRs in a CD28 co-stimulatory-dependent manner (Monks et al., 1997, 1998; Somersalo et al.,2004). Microscopic studies of IS have shown that T cells expressing PKC-θ periodically break open the pSMAC to create an asymmetric focal zone accumulation pattern that relocates to nearby areas where the pSMAC reformed (Sims et al., 2007). This periodic breaking of the symmetric pSMAC to form a polarized focal zone allows short bursts of migration, facilitating T cell interaction with multiple antigen presenting cells (Lindquist et al., 2004). This observation is also consistent with the asymmetric cell division theory that suggests that the IS leads to asymmetric cell division, a feature that is important for memory/effector differentiation of lymphocytes (Chang et al.,2007). A recent study has identified a unique region of PKC-θ, called the V3 domain, that is responsible for the selective translocation of PKC-θ to the IS (Kong et al., 2011). V3 was found to interact with the SH3 domain of Lck which is in turn, tethered to the phosphorylated tail of CD28 via its SH2 domain. The PKC-θ–Lck–CD28 interaction explains why PKC-θ recruitment to the IS depends on CD28 co-stimulation. However, in a different study the active kinase domain of PKC-θ was reported to be essential for PKC-θ translocation into the IS (Cartwright et al., 2011) and is not clear why there is a discrepancy. One possibility is that the two studies used different sources of T cells: primary T cells transduced with retrovirus and a D10 cell line. In contrast to conventional T cells, PKC-θ does not translocate to IS of Tregs. In fact it is actually sequestered away from the IS (Zanin-Zhorov et al., 2010), suggesting that the function of PKC-θ in Tregs is likely to be different from its functions in conventional T cells. Altogether, the fact that selective translocation of PKC-θ (but not other isoforms of PKC) to the IS is critical for T cell activation, strongly suggests it has unique functions in mediating TCR signals, and that selective inhibition of PKC-θ could specifically interfere with T cell function.

#### **PKC-θ MEDIATES TCR SIGNALS ESSENTIAL FOR T CELL ACTIVATION**

TCR signals are initiated by activation of the Src family protein tyrosine kinase (PTK) Lck (Weiss and Littman, 1994), which leads first to recruitment of ZAP 70 and then the subsequent recruitment of the adaptor proteins LAT, SLP76, and VAV. LAT then

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recruits PLCγ1 (Clements, 2003; Berg et al., 2005) which catalyzes phosphatidylinositol 4,5-biphosphate into inositol triphosphate (IP3), a Ca2+ mobilizer and DAG, the PKC-θ activator (Weiss and Littman, 1994). In addition to DAG, PKC-θ activation seems to also require phosphorylation of threonine 538 (T538) in its activation loop (Liu et al., 2002; Lee et al., 2005). Previously, PDK1 was believed to phosphorylate T538 (Liu et al., 2002). However, a recent study indicates GLK is the upstream kinase responsible for T538 phosphorylation (Chuang et al., 2011). Upon activation, PKC-θ mediates the activation of NF-κB, AP-1, and nuclear factor of activated T cells (NFAT), critical transcription factors that are required for activation of the IL-2 gene (Sun et al., 2000; Pfeifhofer et al., 2003). Several adaptor proteins play a critical role in mediating PKC-θ-induced NF-κB activation including membrane-associated guanylate kinase (MAGUK), CARMA1, B-cell lymphoma 10 (Bcl 10), and mucosa-associated lymphoid tissue 1 (MALT1). Together with PKC-θ, these adaptors facilitate the activation of IKK, leading to the phosphorylation, ubiquitination, and degradation of IκB. Degradation of IκB releases NF-κB to the nucleus, where it participates in the activation of target genes essential for T cell activation (Weil et al., 2003; Lin and Wang, 2004; Weil and Israel, 2004). Studies using PKC-θ-deficient T cells or cells overexpressing the constitutively active PKC-θ or the catalytically inactive form of PKC-θ have demonstrated that PKC-θ also selectively activates the AP-1 signaling pathway in T cells (Sun et al., 2000). AP-1 is composed of c-Jun and c-Fos, which regulate many cellular events (Baier-Bitterlich et al., 1996). Although the exact mechanism responsible for PKC-θ-mediated activation of AP-1 is still unclear, several studies have provided some insight into this process. Ras and MAP kinases: JNK, ERK, and P38 are all involved in PKC-θ-mediated AP-1 activation (Baier-Bitterlich et al., 1996; Shaulian and Karin, 2002). Li et al. (2004) isolated a PKC-θ-interacting upstream MAP kinase, originally termed Ste20/SPS1-related proline and alanine-rich kinase (SPAK or PASK), and demonstrated that SPAK selectively interacts with PKC-θ and participates in PKC-θ-mediated activation of AP-1, but not NF-κB. Activation of T cells through the TCR also leads to IP3-mediated elevation of cytosolic [Ca2+]i by inducing Ca2+ influx. Elevated intracellular Ca2+ ultimately leads to activation of the calcineurin phosphatase, which dephosphorylates NFAT, resulting in its translocation to the nucleus. Translocated NFAT cooperates with AP-1 to activate IL-2 expression and it has been shown that, in the absence of AP-1, NFAT activation can lead to T cell anergy (Macian et al., 2002). Several studies have shown that PKC-θ enhances the activation of NFAT by stimulating Ca2+ influx; TCR-induced Ca2+ influx, and NFAT activation is defective in T cells from *PKC*θ−/− mice (Pfeifhofer et al., 2003; Manicassamy et al., 2006a). Although it is clear that PKC-θ regulates Ca2+ signals via stimulation of PLCγ1, it is not known how PKC-θ stimulates PLCγ1. The Tek kinase family member Itk may be the missing link. Itk-deficient T cells display defective Ca2+ influx and PLCγ1 activation (Liu et al., 1998), whereas overexpression of Itk leads to stimulation of PLCγ1 activity (Tomlinson et al., 2004). Therefore, it is possible that PKC-θ regulates PLCγ1 activation via Itk. Altogether, PKC-θ-mediated TCR signaling regulates multiple signaling pathways including NF-κB, AP-1, and NFAT that are all critical for T cell activation, which were clearly

summarized in our previously published review articles (Manicassamy et al., 2006b; Kwon et al., 2010). Inhibition of PKC-θ is therefore expected to prevent T cell activation by blocking these pathways.

# **PKC-θ ENHANCES T CELL SURVIVAL**

Productive engagement of the TCR leads to T cell activation, resulting in cell proliferation and production of IL-2. Proliferating T cells especially during S phase of the cell cycle are susceptible to apoptosis (Boehme and Lenardo, 1993; Radvanyi et al., 1996). The TCR delivers signals that are required not only for stimulating proliferation but also for enhancing survival (Weiss and Littman, 1994; Boise et al., 1995). Such survival signals ensure the completion of the T cell activation process that is essential for differentiating naïve T cells into effectors that can mediate actual immune responses (Radvanyi et al., 1996). During T cell activation, survival of the T cells is enhanced by IL-2, which acts as an extrinsic survival factor. In addition, activated T cells substantially up-regulate Bcl-xL that intrinsically increases resistance to apoptosis (Noel et al., 1996; Radvanyi et al., 1996; Van Parijs et al., 1996). We and others have shown that PKC-θ is required for the survival of activated T cells (Barouch-Bentov et al., 2005; Manicassamy et al., 2006a). *PKC-*θ−/− T cells undergo apoptosis in response to TCR stimulation, which correlates with the reduced expression of NF-κB-dependent Bcl-xL. Forced expression of Bcl-xL and Bcl-2 restores the survival of *PKC-*θ−/− T cells and exogenous IL-2 can partially overcome the defective survival and proliferation of *PKC-*θ−/− T cells. Similar to primary T cells, PKC-θ is also required for the survival of T cell lines such as Jurkat. It has been shown that PKC-θ promotes Jurkat survival by phosphorylating Bad and thereby inactivating its function (Villalba et al., 2001). However, primary T cells deficient in PKC-θ showed comparable levels of Bad phosphorylation to that of wild-type (WT) T cells (Barouch-Bentov et al., 2005), suggesting that the observed apoptosis of *PKC-*θ−/− T cells is unlikely to be due to lack of Bad phosphorylation. Other pathways may also be involved in the apoptosis of *PKC-*θ−/− T cells. For example, up-regulation of pro-apoptotic molecule Bim might be partially responsible for the observed apoptosis in *PKC-*θ−/− T cells (Barouch-Bentov et al., 2005). Wan and DeGregori (2003) reported that inhibition of NF-κB leads to an increase in the expression of pro-apoptotic protein p73 . Since *PKC-*θ−/− T cells are defective in NF-κB activation, it is possible that down-regulated p73 may contribute to the apoptosis of *PKC-*θ−/− T cells. Because T cell survival ensures T cell-mediated immune responses, one of the mechanisms for PKC-θ to regulate immune responses is to enhance T cell survival and this is confirmed by our allograft rejection study discussed later in the review.

#### **PKC-θ PROMOTES THE DIFFERENTIATION OF NAÏVE T CELLS TO INFLAMMATORY Th17 CELLS**

Differentiation of naïve T cells into specific T helper lineages is a critical checkpoint for controlling immune responses. Altered regulation of this checkpoint can lead to aggravated autoimmunity by overproduction of T helper cells that cause pathogenic inflammation. Similarly, allograft rejection also depends on the effectors that differentiate from allo-specific naïve T cells. *In vitro*

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stimulation of naïve CD4 T cells in the presence of appropriate cytokines up-regulates master transcription factors that instruct their differentiation into Th1, Th2, or Th17 inflammatory T helper cells (Dong, 2010). When stimulated with IL-12 or interferon-γ (INF-γ), naïve T cells express the T-box transcription factor T-bet as their lineage-specific transcription factor and differentiate into Th1 cells that secrete IFN-γ as their signature cytokine. In the presence of IL-4, naïve T cells express GATA3 as their lineage-specific transcription factor and differentiate into Th2 cells that secrete the signature cytokines IL-4, IL-5, and IL-13 (Murphy and Reiner, 2002). TCR stimulation in the presence of IL-6 and TGF-β leads to up-regulation of RORγt and differentiation of naïve T cells into Th17 cells that produce IL-17, IL-21, IL-22, and GM-CSF. We compared the differentiation of WT and *PKC-*θ−/− T cell under Th1, Th2, and Th17 priming conditions *in vitro* to determine the function of PKC-θ in these processes and found that PKCθ preferentially involved in the regulation of Th17 formation (Kwon et al., 2012). We showed that purified naive *PKC-*θ−/− T cells were defective in Th17 differentiation, whereas Th1 and Th2 differentiation appeared normal. Activation of PKC-θ with PMA promoted Th17 differentiation in WT but not *PKC-*θ−/− T cells. Furthermore, *PKC-*θ−/− T cells had notably lower levels of Stat3, a transcription factor required for Th17 differentiation, and PMA markedly stimulated the expression of Stat3 in WT but not *PKC-*θ−/− T cells. In contrast, activation of Stat4 and Stat6, which are critical for Th1 and Th2 differentiation, was normal in *PKC-*θ−/− T cells. Forced expression of Stat3 significantly increased Th17 differentiation in *PKC-*θ−/− T cells, indicating that reduced Stat3 levels were responsible for impaired Th17 differentiation and that Stat3 lies downstream of PKC-θ. Constitutively active PKC-θ or WT PKC-θ activated by either PMA or TCR crosslinking, stimulated the expression of a luciferase reporter gene driven by the Stat3 promoter. PKC-θ-mediated activation of the Stat3 promoter was inhibited by dominant negative AP-1 and IκB kinase-β, but stimulated by WT AP-1 and IκB kinase-β, suggesting that PKC-θ stimulates Stat3 transcription via the AP-1 and NF-κB pathways. Finally, conditions favoring Th17 differentiation induced the highest activation level of PKC-θ. Altogether, the data indicate that PKC-θ integrates the signals from the TCR activated with Th17 priming cytokines to up-regulate Stat3 via NFκB and AP-1, which stimulate Th17 differentiation. The results are also consistent with the observation that PKC-θ-deficient mice are resistant to the induction of experimental autoimmune encephalomyelitis (EAE; Salek-Ardakani et al., 2005; Tan et al., 2006), which is a Th17-associated autoimmune disease. In contrast, and although *PKC-*θ−/− mice have been reported to be defective in development of Th1 and Th2 immune responses, depending on the mouse model used (Marsland et al., 2004; Salek-Ardakani et al., 2004; Healy et al., 2006), our *in vitro* assays have shown that Th1 and Th2 differentiation is normal in the absence of PKC-θ. In addition to the apparently different priming conditions *in vitro* and *in vivo*, defects in other PKC-θ-regulated functions such as survival are likely to contribute to the overall defective Th1 and Th2 immune responses observed in *PKC-*θ−/− mice *in vivo*. Although it is difficult to define which PKC-θregulated functions are responsible for the defective immune responses*in vivo*, we excluded the possibility that PKC-θ-regulated survival affected our differentiation assays *in vitro* by adding exogenous IL-2 which inhibits *PKC-*θ−/− T cell apoptosis (Manicassamy et al., 2006a). Altogether, both *in vitro* and *in vivo* studies indicate that PKC-θ promotes the differentiation of Th17 cells that are associated with multiple autoimmune disorders (Huang et al., 2007).

#### **PKC-θ INHIBITS THE DIFFERENTIATION AND ENHANCES SUPPRESSIVE FUNCTION OF Tregs**

Naïve CD4+ T cells can differentiate into either inflammatory effector T cells or Tregs (iTregs; Bettelli et al., 2006; Zhou et al., 2008), two distinct subsets of T cell helpers with opposite functions. A fine balance between these two opposing T cell types is required for a functional immune system. The activation of naïve T cells in the presence of TGFβ induces expression of Forkhead Box P3 (Foxp3), a master transcription factor instructing iTreg differentiation, and is also a marker for iTreg (Curotto de Lafaille and Lafaille, 2009). In contrast to iTregs, natural Tregs (nTregs) are not induced but develop in the thymus. The fact that naïve T cells can be differentiated into inhibitory iTregs suggests there is a therapeutic value for such a conversion in the treatment of autoimmunity. Our data has demonstrated that PKC-θ-mediated signals inhibit iTreg differentiation (Ma et al., 2012). We found that TGFβ-induced iTreg differentiation was enhanced in *PKC-*θ−/− T cells or WT cells treated with a specific PKC-θ inhibitor, but was inhibited by the PKC-θ activator PMA or by CD28 crosslinking which enhances PKC-θ activation. Further, we showed that *PKC-*θ−/− T cells had reduced activity of the AKT kinase, and that the expression of a constitutively active form of AKT in *PKC-*θ−/− T cells restored their ability to inhibit iTreg differentiation. In addition, knockdown or over expression of the AKT downstream targets FoxO1 and FoxO3a was found to inhibit or promote iTreg differentiation in *PKC-*θ−/− T cells respectively, indicating that the AKT-FoxO1/3A pathway is responsible for the inhibition of iTreg differentiation downstream of PKC-θ. Considering the positive role played by PKC-θ in the activation and differentiation of naïve T cells into inflammatory T effector cells (Altman et al., 2000; Sun et al., 2000; Pfeifhofer et al., 2003; Marsland et al., 2004), together the data indicate that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of effector T cells and inhibitory Tregs.

In addition to Treg differentiation, a recent study also demonstrated a role for PKC-θ in the regulation of the effector function of Tregs (Zanin-Zhorov et al., 2010). Tregs inhibit inflammatory effector T cell function via cell contact-dependent and independent mechanisms (Sakaguchi et al., 2008). Such inhibitory functions of Tregs are important for establishing tolerance mechanisms required to prevent activation of autoreactive T cells that lead to autoimmunity (Sakaguchi et al., 2006; Shevach, 2006; Shevach et al., 2006; Miyara and Sakaguchi, 2007). The depletion of CD4+CD25+ Tregs from mice results in the development of widespread autoimmune/inflammatory diseases such as autoimmune gastritis, thyroiditis, type 1 diabetes, and inflammatory bowel disease. These autoimmune disorders are prevented by reconstituting the mice with CD4+CD25+ Treg cells (Sakaguchi et al., 1995; Singh et al., 2001), indicating a therapeutic potential for Tregs for the treatment of autoimmunity. To increase the efficacy of Treg-mediated inhibition, it is important to enhance the suppressive function of Treg. Inhibition of PKC-θ either by knockdown or a specific PKC-θ inhibitor has been shown to significantly boost the potential of Tregs to inhibit T cell activation (Zanin-Zhorov et al., 2010). However, in the presence of TGFβ neutralizing antibody, the PKC-θ inhibitor fails to enhance the suppressive function of Tregs, suggesting that inhibition of PKC-θ stimulates Tregs to produce the TGFβ that is responsible for inhibition of T cell activation. Interestingly, the suppressive function of Treg was also enhanced by inhibiting the activation of NF-κB, a critical downstream target of PKC-θ (Sun et al., 2000), indicating the possibility that PKC-θ inhibitor enhances Treg function by blocking activation of the NF-κB pathway. Furthermore, PKC-θ inhibitor-treated Tregs were more potent than untreated Tregs in preventing inflammatory colitis *in vivo* (Zanin-Zhorov et al., 2010), supporting the potential clinical application of PKCθ inhibitors for Treg-mediated treatment of autoimmunity. Taken together, inhibition of PKC-θ can interfere with T cell-mediated immunity by inhibiting inflammatory T cell differentiation, by promoting Treg differentiation and by enhancing the suppressive function of Tregs.

#### **PKC-θ PLAYS A CRITICAL ROLE IN T CELL-DEPENDENT AUTOIMMUNITY**

Due to the unique roles played by PKC-θ in the regulation of T cell activation and differentiation, PKC-θ is believed to be a potential drug target and pharmaceutical companies have developed highly specific PKC-θ inhibitors (Cywin et al., 2007; Mosyak et al., 2007). Mouse models of autoimmune diseases have been used to define PKC-θ function in T cell-dependent autoimmunity (Marsland and Kopf, 2008). Two independent studies have shown that *PKC-*θ−/− mice were resistant to the induction of Th2-dependent lung inflammation in airway hyper responsiveness (AHR; Marsland et al., 2004; Salek-Ardakani et al., 2004), supporting a requirement for PKC-θ in Th2 type autoimmunity. In contrast, PKC-θ played a lesser role in the development of a similar lung inflammatory response mediated by Th1 cells (Salek-Ardakani et al., 2004), suggesting different functions of PKC-θ in Th1 and Th2 responses. However, PKC-θ was found to be essential for both methylated BSA and type II collageninduced arthritis, a Th1-mediated autoimmunity disease (Healy et al., 2006). The results suggest that PKC-θ function is dependent on the model used. *PKC-*θ−/− mice were reported by two different groups to be resistant to the development of Th17-mediated EAE, the mouse model for multiple sclerosis (Salek-Ardakani et al., 2005; Tan et al., 2006), indicating a requirement for PKC-θ in Th17-dependent autoimmunity.

The evaluation of PKC-θ function *in vivo* is complicated by several factors. First, PKC-θ function may be compensated for *in vivo*. Most *in vitro* assays clearly indicated essential role of PKC-θ in T cell functions (Manicassamy et al., 2006b). However, we found that the requirement for PKC-θ may be bypassed if T cells are stimulated by overwhelmingly strong TCR signals such as high concentrations of phorbol ester and ionomycin or anti-CD3/28 antibodies (unpublished data). It is therefore possible that PKC-θ

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function *in vivo* may also depend on the strength of TCR stimulation. In contrast to *in vitro* assays using purified T cells, *in vivo* immune responses involve many different types of cells that can produce factors to compensate for PKC-θ function. For example, one of the major signaling pathways that PKC-θ regulates is NF-κB. NF-κB can also be activated by PKC-θ-independent pathways such as TNFα and IL-1 (Sun et al., 2000). Many *in vivo* inflammation conditions produce TNFα and IL-1 inflammatory cytokines and these cytokines are likely compensate for PKC-θ function at least for the activation of NF-κB in T cells. Toll like receptors (TLR) can also activate the NF-κB pathway and it was indeed found that TLR-mediated signals can overcome the requirement for PKC-θ in T cell activation and the development of autoimmune myocarditis (Marsland et al., 2005). Second, autoimmune diseases usually involve more than one type of T helper cell. For example, both Th1 and Th17 responses are likely contribute to the development of EAE (Salek-Ardakani et al., 2005). In humans, it is even more difficult to specifically define the types of T helper cells involved in autoimmunity. Therefore, inhibition of PKC-θ may inhibit one type of T helper, but not other types of T helpers that can induce autoimmunity. Conversely, PKC-θ-mediated T cell differentiation is not the only PKC-θ function essential for the development of autoimmunity; PKC-θ also regulates other T cell functions including activation and survival. Therefore, the observed defects in the development of autoimmunity in *PKC-*θ−/− mice are likely to be due to the disruption of several PKC-θ-regulated functions including those that have not yet been identified. However, despite the possible complications, the potential of PKC-θ as a drug target has been indicated by a trial for the treatment of psoriasis (Skvara et al., 2008). In this study, the clinical severity of psoriasis was reduced up to 69% after 2 weeks of treatment with a PKC inhibitor, AEB071 which inhibits multiple isoforms of PKC with strong specificity for PKC-θ, PKC-α, and PKC-β and lesser specificity for PKC-δ, PKC-ε, and PKC-η. Other clinical trials are expected because many companies have developed PKC-θ inhibitors. One of potential trials will be systemic lupus erythematosus, because patients with this disease show considerably enhanced activation of both PKC-θ and its potential upstream kinase GLK (Chuang et al., 2011).

#### **PKC-θ IS REQUIRED FOR NKT-MEDIATED AUTOIMMUNE HEPATITIS**

Autoimmune hepatitis (AIH) results from the mistaken attack on healthy liver cells by an individual's own immune system (McFarlane, 1999). In mice, acute hepatitis can be induced by treatment with concanavalin A (ConA), which causes rapid activation of CD1d-positive NK T cells. These activated NKT cells produce large amounts of cytokines that cause strong inflammation responsible for damaging liver tissues. Our research has shown that *PKC-*θ−/− mice were resistant to ConA-induced hepatitis due to an essential requirement for PKC-θ during NKT cell development and activation. A dose of ConA (25 mg/kg) that was lethal to WT mice failed to cause death due to liver injury in *PKC-*θ−/− mice (Fang et al., 2012). Correspondingly, the ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in *PKC-*θ−/− mice. In addition,

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upon stimulation with an NKT cell-specific lipid ligand, peripheral *PKC-*θ−/− NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cells to induce hepatitis (Fang et al., 2012), and thus, is a potential drug target for prevention of autoimmune hepatitis. NKT cells are also thought to be involved in liver injury induced by LPS, α-galactosylceramide (α-GalCer), *Salmonella* infection, chronic hepatitis C infection, and primary biliary cirrhosis (Ishigami et al., 1999; Kawano et al., 1999; Kaneko et al., 2000; Kato et al., 2000; Kim et al., 2002). Inhibition of PKC-θ is also likely to have therapeutic value in the treatment of liver injury in patients with these conditions.

#### **PKC-θ PLAYS A CRITICAL ROLE IN ALLOIMMUNE RESPONSES ESSENTIAL FOR TRANSPLANT REJECTION**

Solid organ transplants that benefit end-stage organ failure patients are severely limited by the occurrence of rejection. Alloreactive T cells are critical targets for tolerance induction since they mediate the immune responses required for rejection. The alloreactive T cell pool is very large (Suchin et al., 2001), which explains why immune responses against allografts are at least two orders of magnitude stronger than immune responses against a specific antigen. Therefore, long-term tolerance to allografts is extremely difficult to establish. The initial evidence for requirement of PKC-θ in alloresponses came from the impaired *in vitro* mixed lymphocyte reaction of *PKC-*θ−/− T cells (Sun et al., 2000). Injection of allogeneic cells into the footpad of PKC-θ-deficient mice provoked a significantly diminished local T cell response compared toWT mice similarly challenged, suggesting an essential role for PKC-θ in the allo-reaction *in vivo* (Anderson et al., 2006). We tested PKC-θ function in transplant rejection using a cardiac allograft model (Manicassamy et al., 2008). *Rag1*−/− mice reconstituted with WT T cells readily rejected fully mismatched cardiac allografts, whereas, *Rag1*−/− mice reconstituted with *PKC-*θ−/− T cells failed to promote rejection, suggesting that PKC-θ is required for T cell-mediated allograft rejection. One of the important mechanisms responsible for establishing tolerance to allografts is to reduce the number of alloreactive T cells by inducing apoptosis (Wells et al., 1999). Since PKC-θ is required for survival of activated T cells (Manicassamy et al., 2006a), we therefore tested the role of PKC-θ-regulated survival in cardiac allograft rejection and demonstrated that the transgenic expression of Bcl-xL in *PKC-*θ−/− T cells was sufficient to restore cardiac allograft rejection (Manicassamy et al., 2008). This result suggests that apoptosis of alloreactive T cells in the absence of PKC-θ is responsible for the observed tolerance to cardiac allografts. Alloreactive T cells can be tolerized through anergy, suppression and deletion. Tolerizing mechanisms through anergy and Treg-mediated suppression are unlikely change the size of alloreactive T cell pool which is ready to destroy allografts. This is the problem for cyclosporin A (CsA), the most successful immunosuppressive drug used clinically so far. CsA prevents apoptosis of alloreactive T cells by inhibition of T cell activation (Li et al., 1999), resulting in accumulation of large amounts of alloreactive T cells that destroy allografts once the immunosuppressive drugs are discontinued (Li et al., 2001). Therefore, prevention of allograft rejection usually requires transplant recipients to take life-long immunosuppressive drugs, which can result in complications including infections and malignancy. Whereas, deletion induces tolerance by decreasing the number of alloreactive T cells via apoptosis, and thus avoids the potential risk of accumulating alloreactive T cells. In addition to an adoptive transfer model, we also tested cardiac rejection using intact *PKC-*θ−/− mice. *PKC-*θ−/− mice displayed delayed, but successful cardiac allograft rejection, suggesting there was some compensation for the missing PKC-θ function. Finally, a subtherapeutic dose of anti-CD154 antibody or CTLA4-Ig, which was not sufficient to prevent cardiac allograft rejection in WT mice, prevented heart rejection in *PKC-*θ−/− mice. *PKC-*θ−/− mice treated with sub-therapeutic doses of Anti-CD154 or CTLA4-Ig also accepted donor-type second cardiac allografts but rejected third-party allografts (Wang et al., 2009). Thus, in combination with other treatments, the inhibition of PKC-θ allows longterm survival of cardiac allografts (Manicassamy et al., 2008; Wang et al., 2009).

In addition to heart rejection, the role of PKC-θ was also examined in a bone marrow transplantation (BMT) model (Valenzuela et al., 2009). BMT is used to replace damaged bone marrow with healthy stem cells or used as therapy for hematopoietic malignancies. In the latter case, allogeneic BMT boosts the patient's immune system to aid in fighting against the cancer, which is called the graft-versus-leukemia (GVL) effect. However, graftversus-host disease (GVHD), a potentially lethal consequence of BMT, limits the clinical application of this very effective treatment. Alloreactive donor T cells recognize the mismatched MHC of the recipient and undergo robust activation, expansion, and differentiation, resulting in GVHD, which causes severe damages to multiple tissues including gut, liver, skin, and kidney (Shlomchik, 2007). Immunosuppressive drugs are therefore needed clinically to prevent GVHD-induced damage. However, commonly used immunosuppressive drugs such as CsA and FK506 also inhibit the immune response against pathogens as well as tumors (GVL), and consequently limit the effects of GVL on the elimination of

#### **REFERENCES**

(1994). Expression and biochemical characterization of human protein kinase C-theta. *Eur. J. Biochem.* 225, 195–203.

residual tumor cells (Reddy et al., 2005). The optimal immunosuppressive regimens are able to prevent GVHD, but also preserve the immune responses against infectious pathogens. Similar to WT mice, *PKC-*θ−/− mice have the ability to respond to infection by the listeria bacteria and MCMV virus (Valenzuela et al., 2009). Moreover, *PKC-*θ−/− mice survived the BMT procedure and did not develop GVHD, whereas majority of WT mice died from GVHD. More importantly, *PKC-*θ−/− mice retained their ability to induce rejection of tumors. This study demonstrated that PKC-θ inhibitor-based immunosuppressive regimens are able to prevent GVHD but also preserve the protective immune response against infections and tumors.

#### **CONCLUSION**

PKC-θ controls fundamental functions of T cells including activation, differentiation, and survival via NF-κB, AP-1, and NFAT pathways. PKC-θ also regulates T cell-mediated immune responses *in vivo* and selective PKC-θ inhibitors are believed to have the potential for clinical application in the treatment of autoimmunity and prevention of allograft rejection. However, PKC-θ function in T cell-mediated immune responses is dependent on the mouse models used. Therefore, the mechanisms involved in each of the diseases should be carefully examined. More questions need to be addressed prior to the clinical application of PKC-θ inhibitors including how the inhibition of PKC-θ affects the function of other tissues *in vivo*. It is encouraging to report that many pharmaceutical companies have developed selective PKC-θ inhibitors, and therefore many PKC-θ-regulated functions can be evaluated using these inhibitors instead of *PKC*θ−/− mice, which have potential developmental caveat. With the availability of PKC-θ inhibitors, it is now possible to test their efficacy in mouse models of human autoimmune diseases including EAE and arthritis, which are likely to lead to clinical trials of PKC-θ-based treatments for human diseases.

#### **ACKNOWLEDGMENT**

We thank Dr. Margaret Morgan for expert editorial assistance.

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L., and Kuchroo, V. K. (2006). Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. *Nature* 441, 235–238.

inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells. *Br. J. Pharmacol.* 145, 934–944.

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(Th)2 cell but not Th1 cell responses. *J. Exp. Med.* 200, 181–189.

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C. C., Wang, J., Celis, E., Engelman, R. W., Blazar, B. R., Bevan, M. J., Waller, E. K., Yu, X. Z., and Beg, A. A. (2009). PKCtheta is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice. *J. Clin. Invest.* 119, 3774–3786.

regulatory T cell function. *Science* 328, 372–376.

Zhou, L., Lopes, J. E., Chong, M. M., Ivanov, I. I., Min, R., Victora, G. D., Shen, Y., Du, J., Rubtsov, Y. P., Rudensky, A. Y., Ziegler, S. F., and Littman, D. R. (2008). TGFbeta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. *Nature* 453, 236–240.

**Conflict of Interest Statement:** The author declares 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: 30March 2012; accepted: 10 July 2012; published online: 02 August 2012. Citation: Sun Z (2012) Intervention of PKC-*θ *as an immunosuppressive regimen. Front. Immun. 3:225. doi: 10.3389/ fimmu.2012.00225*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Sun. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

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REVIEW ARTICLE published: 27 June 2012 doi: 10.3389/fimmu.2012.00177

# The role of protein kinase Cη inT cell biology

# **Guo Fu\* and Nicholas R. J. Gascoigne\***

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA

#### **Edited by:**

Amnon Altman, La Jolla Institute for Allergy and Immunology, USA

#### **Reviewed by:**

Nikolai Petrovsky, Flinders Medical Centre, Australia Jonathan Kaye, Cedars-Sinai Medical Center, USA

#### **\*Correspondence:**

Guo Fu and Nicholas R. J. Gascoigne, Department of Immunology and Microbial Science, IMM1, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Protein kinase Cη (PKCη) is a member of the novel PKC subfamily, which also includes δ, ε, and θ isoforms. Compared to the other novel PKCs, the function of PKCη in the immune system is largely unknown. Several studies have started to reveal the role of PKCη, particularly in T cells. PKCη is highly expressed in T cells, and is upregulated during thymocyte positive selection. Interestingly, like the θ isoform, PKCη is also recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell. However, unlike PKCθ, which becomes concentrated to the central region of the synapse, PKCη remains in a diffuse pattern over the whole area of the synapse, suggesting distinctive roles of these two isoforms in signal transduction. Although PKCη is dispensable for thymocyte development, further analysis of PKCη- or PKCθ-deficient and double-knockout mice revealed the redundancy of these two isoforms in thymocyte development. In contrast, PKCη rather than PKCθ, plays an important role for T cell homeostatic proliferation, which requires recognition of self-antigen. Another piece of evidence demonstrating that PKCη and PKCθ have isoform-specific as well as redundant roles come from the analysis of CD4 to CD8 T cell ratios in the periphery of these knockout mice. Deficiency in PKCη or PKCθ had opposing effects as PKCη knockout mice had a higher ratio of CD4 to CD8 T cells compared to that of wild-type mice, whereas PKCθ-deficient mice had a lower ratio. Biochemical studies showed that calcium flux and NFκB translocation is impaired in PKCηdeficient T cells upon TCR crosslinking stimulation, a character shared with PKCθ-deficient T cells. However, unlike the case with PKCθ, the mechanistic study of PKCη is at early stage and the signaling pathways involving PKCη, at least in T cells, are essentially unknown. In this review, we will cover the topics mentioned above as well as provide some perspectives for further investigations regarding PKCη.

**Keywords: development, homeostatic proliferation, immune synapse, immunological synapse, protein kinase C, signaling,T cell,T cell activation**

### **INTRODUCTION**

Protein kinase C (PKC) is a large family of serine/threonine kinases that can be divided into three subfamilies based on their structural homology and requirement of cofactors for activation (Baier, 2003). The conventional PKC subfamily contains α, βI, βII, and γ, requiring calcium and diacylglycerol (DAG) for activation. The novel PKC subfamily contains δ,ε, θ, and η, and requires DAG but not calcium for activation. In contrast, the atypical PKC subfamily (i.e.,ζ and λ/ι) requires neither DAG nor calcium for their activation (Pfeifhofer et al., 2003). Studies using PKC isoform-specific knockout mice have shown differential roles of each isoform in T cell development and function (Sun et al., 2000; Thuille et al., 2004; Gruber et al., 2005a,b; Pfeifhofer et al., 2006). For example, PKCα-deficient mice have a normal T cell development phenotype. In peripheral T cells, PKCα is dispensable for normal T cell activation and IL2 production, but it is required for proliferation and IFN-γ production (Pfeifhofer et al., 2006). PKCβ is dispensable for normal T cell development and function (Thuille et al., 2004), although it was found to be important for LFA-1-mediated T cell locomotion in a PKCβ-deficient cell line (Volkov et al., 2001). PKCδ is a negative regulator of T cell activation, as PKCδ-deficient T cells are hyperproliferative and produce more IL2 cytokine upon

stimulation (Gruber et al., 2005a). This negative regulatory role is also reflected in PKCδ-deficient B cells (Mecklenbrauker et al., 2002; Miyamoto et al., 2002). In striking contrast, PKCθ-deficient T cells completely lose the ability to proliferate or to produce IL2 after stimulation through the T cell receptor (TCR) in *in vitro* assays (Sun et al., 2000; Pfeifhofer et al., 2003), even though both δ and θ have the closest identity (60%) within the novel PKC subfamily (Kong et al., 2011; Quann et al., 2011). PKCε was dispensable for T cell development and activation (Gruber et al., 2005b). In PKCζ-deficient mice, there is no overt defect in T cell development (Leitges et al., 2001), but these mice showed impaired Th2 cell differentiation (Martin et al., 2005). Interestingly, although discovered more than two decades ago (Osada et al., 1990), and like PKCθ, highly expressed in T cells (Baier, 2003; **Figure 1**: data from www.biogps.org (Su et al., 2004; Wu et al., 2009) the role of PKCη had never been thoroughly examined in T cells until the recent study from our group (Fu et al., 2011). This despite the fact that PKCη-deficient mice have existed for almost 10 years (Chida et al., 2003). Meanwhile, although discovered only a little later than PKCη, PKCθ is considered paramount in T cell function. Our recent work on PKCη has significantly filled this gap by showing both isoform-specific and redundant (with PKCθ) roles of PKCη

in T cell development and function (Fu et al., 2011; Fu and Gascoigne, 2012). In this article,we will first briefly review some earlier studies on PKCη, then mainly focus on four subjects currently under study: (1) the recruitment of PKCη to the immunological synapse; (2) its role in T cell development; (3) its role in T cell function; (4) its role in TCR signaling. Finally, we would like to share some of our thoughts with the readers about future investigations regarding PKCη.

### **COMPARISON OF PKC**η **AND PKC**θ **MOLECULES**

In the novel PKC subfamily, PKCδ and PKCθ are closely related (60% identity), as are PKCε and PKCη (also 60% identity; Baier, 2003; Quann et al., 2011). A cross comparison between PKCη and PKCθ reveals that these two isoforms bear 42% identity (**Figure 2A**). The overall domain structure of PKCη and PKCθ proteins shows a high degree of similarity. This domain architecture is shown in **Figure 2B**. In both isoforms, there is a "C2-like" domain near the amino-terminal of the protein, which cannot bind calcium, unlike the C2 domains in conventional PKC isoforms (Baier, 2003). Following the C2-like domain, there are tandem repeats of two DAG binding C1 domains and the V3 hinge region. This is the most different region between PKCη and PKCθ (**Figure 2C**). In PKCθ, V3 is important in association of the kinase with CD28 and as a result is required to mediate PKCθ's localization in the central synapse (Kong et al., 2011). The motif within PKCθV3 domain that is required for CD28 interaction, including the conserved PXXP sequence (Kong et al., 2011), is missing in PKCη (**Figure 2C**). The C2-like, C1, and V3 domains together form a regulatory region,which likely performs the isoform-specific functions, as the carboxyl-terminal serine/threonine kinase domain is rather conserved across all PKC isoforms. The difference between the V3 domains of PKCθ and PKCη suggests that this may be responsible for their different localization in the immunological synapse.

# **A BRIEF HISTORY OF PKC**η **STUDIES**

PKCη was originally identified from a mouse epidermis cDNA library and found to be highly expressed in mouse tissues such as skin, lung, and heart (Osada et al., 1990). Because of this tissue-specific expression pattern, most studies regarding PKCη were historically focused on keratinocyte proliferation and differentiation (Ohba et al., 1998; Cabodi et al., 2000). However, development of skin was normal in PKCη-deficient mice in steady state (Chida et al.,2003). In contrast, under challenging conditions, these PKCη-deficient mice were susceptible to skin tumor induction and showed impaired wound healing (Chida et al., 2003). In immune cells, PKCη is highly expressed in mouse macrophages and T cells, but not B cells (**Figure 1**). However, interestingly, potential roles of PKCη in B cells were suggested in a number of studies (Morrow et al., 1999; Oda et al., 2008). For example, PKCη was shown to be specifically transcribed in pro-B but not pre-B cells, and a pro-apoptotic role of PKCη in B cells was suggested (Morrow et al., 1999). In another study, PKCη was shown to direct IRF4 expression and Igκ gene rearrangement in pre-BCR signaling (Oda et al., 2008). Surprisingly, nothing was known about the specific role of PKCη in T cells until quite recent work from our group and others (Singleton et al., 2009; Fu et al., 2011; Quann et al., 2011; Sewald et al., 2011), which is the topic we address below.

# **RECRUITMENT OF PKC**η **TO THE IMMUNOLOGICAL SYNAPSE**

The immunological synapse or supramolecular activation cluster (SMAC) forms at the interface between a T cell and an

antigen-presenting cell (APC; or a surrogate), and is the site at which early signaling events occur (Grakoui et al., 1999). The widely accepted importance of PKCθ in T cells is largely due to its identification as the only PKC isoform recruited to the immunological synapse (Monks et al., 1997), and particularly to the central synapse region (cSMAC), along with TCR and other molecules (Monks et al., 1998). Since then, PKCθ has served as a landmark for defining the immunological synapse. However, studies from our group and others challenged the view that only PKCθ is recruited to the synapse (Singleton et al., 2009; Fu et al., 2011; Quann et al., 2011). PKCη is recruited to the immunological synapse upon T cell recognition of its cognate antigenic peptide-MHC (pMHC), but not non-stimulatory pMHC, presented by APCs (**Figure 3**; Fu et al., 2011). More interestingly, PKCη and PKCθ showed different recruitment patterns, as PKCη forms a diffuse pattern at the immunological synapse, whereas PKCθ concentrates into the central region (**Figure 3**; Singleton et al., 2009; Fu et al., 2011), suggesting different functions in time and space of these two PKC isoforms. In addition to PKCη and PKCθ, PKCε is also recruited to the immunological synapse (Quann et al., 2011). In this study, polarization of the T cell microtubule-organizing center (MTOC) is directed by diacylglycerol (DAG) at the immunological synapse via three PKC isoforms, in two sequential steps. Initially, PKCε and PKCη accumulate in a broad region of the interface between T cell and APC, followed by PKCθ concentrating in a smaller, central, zone (Quann et al., 2011). It seems that in different cell types, recruitment of PKC isoforms could also be different. For example, it has been shown that, in contrast to the immunological synapse-localization in effector T cells, PKCθ is sequestered away from the immunological synapse in regulatory T cells (Treg), and thus mediates negative feedback on Treg cell function (Zanin-Zhorov et al., 2010). This intriguing observation may be also worth examination for PKCε and PKCη.

#### **PKC**η **IN T CELL DEVELOPMENT**

Our initial speculation that PKCη may play a role in T cell development was based on the finding that PKCη mRNA expression was upregulated during thymocyte positive selection (Mick et al., 2004; Niederberger et al., 2005). These observations were surprising given the established important role of PKCθ in T cell biology, but intriguing because PKCθ-deficient mice have only a very minor defect in thymocyte development. Initial phenotyping of PKCθ-deficient mice did not identify any defects in thymocyte development (Sun et al., 2000; Pfeifhofer et al., 2003), although later studies did find a mild thymocyte development defect in such mice (Morley et al., 2008; Fu et al., 2011). However, phenotyping of PKCη-deficient mice showed rather normal thymocyte development. This was not completely unexpected given the multiple novel PKC isoforms co-expressed in T cells, and redundancy could play a role to compensate for the absence of any particular isoform. We also noted that induction of PKCη mRNA is much higher and earlier in PKCθ-deficient mice than in wildtype mice (i.e., induction during positive selection in wild-type mice, but induction before positive selection in PKCθ-deficient mice), suggesting a compensatory effect due to redundancy of function between PKCη and PKCθ (Fu et al., 2011). In accord

Blue are EL4 cells used as antigen-presenting-cells. Red are OT-I T hybridoma cells transfected with PKCη- or PKCθ-RFP as indicated. Adapted

from Fu et al. (2011).

with this notion, PKCη is recruited to the immunological synapse in immature CD4+CD8+ (DP) thymocytes in the PKCθ –/– mice, as is PKCθ in the PKCθ-sufficient DP cells. In PKCθ-sufficient cells, PKCη is only recruited to the synapse in mature CD4+ or CD8+ (SP) thymocytes. These results are only suggestive of redundant function, but clear redundancy between PKCη and PKCθ in thymocyte development was confirmed when we phenotyped PKCη –/–θ –/– mice. Positive selection of thymocytes in these double-knockout mice was more severely impaired than either single PKC-knockout mice. However, the blockade of thymocyte development in PKCη –/–θ –/– mice was not complete, as SP cell numbers were only reduced by about 50% (**Figure 4A**; Fu et al., 2011). Therefore, it is possible that other PKC isoforms than PKCη and θ can still compensate for their deficiency, perhaps most likely those members within the same subfamily (e.g., PKCε).

It is natural to speculate that in a multimember protein family, there are some overlapping functions between individual members (i.e., redundancy), as well as isoform-specific functions. In our study, we found that PKCη and PKCθ had opposite effects on the CD4 to CD8 T cell ratios in the secondary lymphoid organs (**Figure 4B**). PKCη-deficient mice had a higher CD4/CD8 ratio than wild-type mice, whereas PKCθ-deficient mice had a lower ratio, indicating an isoform-specific role of these PKCs in balancing CD4 and CD8 T cell homeostasis. Interestingly, these effects are "neutralized" by each other in that PKCη –/–θ –/– mice exhibited normal CD4/CD8 ratios. Multiple factors can affect the CD4/CD8 T cell ratio during thymocyte development (Corbella et al., 1994; Suzuki et al., 1995;Sim et al., 1998a,b). The

SP thymocytes from these knockout mice did not show altered CD4/CD8 ratios, indicating that the effects on the CD4/CD8 ratio occur post-thymically (Fu et al., 2011). Another intriguing observation is that PKCη-deficient mice have an irregular distribution of T cells between spleen and peripheral lymph nodes. The total T cell numbers are increased in the lymph nodes of PKCη-deficient mice, which mirrored the phenomenon that the lymph nodes are much larger in size in PKCη-deficient mice compared to wild-type mice. In contrast, the total T cell numbers are reduced in the spleen in PKCη-deficient mice compared to wild-type mice. Enlarged lymph nodes (i.e., lymphadenopathy) were also observed in PKCδ-deficient mice, which was mainly attributed to the increased B cell numbers (Mecklenbrauker et al., 2002). Currently, it is not clear what causes this biased T cell distribution in PKCη-deficient mice. We speculate that altered lymphocyte homing and/or homeostasis could be one of the reasons.

#### **PKC**η **IN PERIPHERAL T CELL HOMEOSTASIS AND RESPONSE TO ANTIGEN**

For the sake of simplicity,we focused on CD8 T cells for most functional studies on PKCη –/– mice (Fu et al., 2011). PKCη-deficient CD8 T cells showed a mild proliferation defect compared to wildtype T cells upon anti-CD3 antibody stimulation. In contrast, under the same conditions, PKCθ-deficient CD8 T cells were completely non-proliferative, as previously reported (Sun et al., 2000; Pfeifhofer et al., 2003). However, this striking difference between PKCη –/– and PKCθ –/– CD8 T cells was blurred under more physiological conditions. For example, when we used APCs pulsed with antigenic peptide to stimulate these PKC-deficient CD8 T cells, both PKCη –/– and PKCθ –/– CD8 T cells still proliferated less well than wild-type cells, but the relative difference between PKCη –/– and PKCθ –/– CD8 T cells is much more subtle than with anti-CD3 crosslinking (Fu et al., 2011). In general, we observed that antigenspecific proliferation of PKCη-deficient T cells was more severely reduced compared to wild-type cells than was anti-CD3 antibody induced proliferation. It may be that this is because the anti-CD3 stimulation does not involve the formation of the immunological synapse, whereas the synapse is important in the antigen-specific responses. The proliferation defect of PKCη –/– CD8 T cells was also confirmed in *in vivo* experiments, where wild-type and PKCη –/– CD8 T cells were co-transferred into recipient mice and stimulated by antigenic peptide (**Figure 5A**; Fu et al., 2011).

Therefore the proliferation defect of PKCη –/– CD8 T cells is consistent both *in vitro* and *in vivo*. However,in the case of PKCθ –/– CD8 T cells, *in vivo* reductions in responses were much less severe than those observed *in vitro*. For instance, the absence of PKCθ does not impair antigen-specific proliferation (Barouch-Bentov et al., 2005) or antiviral immune responses, in which PKCθ –/– CD8 T cells were found to proliferate normally (Berg-Brown et al., 2004; Marsland et al., 2005). The role of PKCθ in the *Listeria* infection model is controversial, with one group showing PKCθ is not important (Valenzuela et al., 2009) and another group claiming the opposite (Sakowicz-Burkiewicz et al., 2008). These conflicting results may be due to the different bacterial infection doses used between these two groups. One common explanation of PKCθ's dispensable role in these infection models is that *in vivo* innate signals can compensate for the absence of PKCθ (Marsland et al., 2005;Valenzuela et al., 2009), however it is also possible that PKCη functions in place of PKCθ in these cases.

In stark contrast, in an experiment to measure T cell homeostatic proliferation, we found that PKCη, but not PKCθ, is required (**Figure 5B**; Fu et al., 2011). In these experiments, no matter whether we used polyclonal T cells or monoclonal TCR transgenic T cells as donor cells, only PKCη –/– CD8 T cells showed impaired proliferation in lymphopenic animals, whereas PKCθ –/– CD8 T cells showed normal homeostatic proliferation. The nonessential role of PKCθ in T cell homeostatic proliferation was also independently reported by others (Valenzuela et al., 2009). This was indeed an unexpected result: one would have assumed that defective homeostatic proliferation might occur in PKCθ –/– T cells, at least as a reflection of strong deficiency in *in vitro* proliferation. Both TCR mediated signaling and the cytokines IL7 and IL15 are required to support normal homeostatic proliferation (Jameson, 2002; Surh and Sprent, 2005). However, we think

altered responsiveness to these cytokines is unlikely to contribute to the defective homeostatic proliferation in PKCη-deficient T cells, because the amounts of IL7Rα (CD127) and IL15R (CD122) on the PKCη-deficient T cells were the same as those of wild-type T cells (Fu et al., 2011). We were also unable to find any difference in the numbers of apoptotic cells between PKCη-deficient and -sufficient mice, suggesting that the requirement for PKCη for homeostatic proliferation is not due to differential cell survival. PKCθ has been found to be a survival factor for CD8 T cells. In contrast to antigen-specific T cell proliferation, which is the clonal expansion of particular T cells recognizing their cognate antigen, homeostatic proliferation is the response of T cells to self-MHCp complexes for survival. Therefore the strength of TCR signaling is different in these two scenarios. It is possible that PKCη and PKCθ play dominant roles in homeostatic and antigenspecific proliferation respectively. PKCθ may be more important in antigen-specific activation because of its reported role in breaking the "symmetry" of the synapse (Sims et al., 2007). This is required for T cell movement, such as during scanning over the surface of an APC.

#### **PKC**η **IN T CELL RECEPTOR SIGNALING**

Compared to the very well characterized mechanisms regarding PKCθ in the molecular signaling machinery in T cells (Egawa et al., 2003; Wang et al., 2004; Roose et al., 2005; Manicassamy et al., 2006), similar studies of PKCη are at a very early stage. In our study, we showed that Ca2+ flux and NFκB nuclear translocation were impaired in PKCη –/– T cells, but that TCR-proximal signaling pathways were intact. These signaling defects are similar to those defects reported in PKCθ –/– T cells (Sun et al., 2000; Pfeifhofer et al., 2003). Thus two questions remain: First, if the signaling defects are the same in PKCη- and PKCθ-deficient T cells, why are the defects in PKCη-deficient T cells not as strong as PKCθ-deficient T cells, at least *in vitro*? One possibility is that more signaling pathways are interrupted by PKCθ-deficiency compared to PKCη-deficiency, in addition to NFκB (Sun et al., 2000) and NFAT (i.e., Ca2+ signaling-related) defects (Pfeifhofer et al., 2003). For example, it was recently shown that PKCθ can bind to CD28 and thus mediates a co-stimulation-driven signaling pathway from the immunological synapse (Yokosuka

#### **Table 1 | Comparison of PKC**η **and PKC**θ **in T cell biology.**

Morley et al. (2008), Fu et al. (2011), Sun et al. (2000), Pfeifhofer et al. (2003), Berg-Brown et al. (2004), Barouch-Bentov et al. (2005), Valenzuela et al. (2009), Marsland et al. (2005), Marsland et al. (2004), Monks et al. (1997), 11Monks et al. (1998), Singleton et al. (2009), Quann et al. (2011), Kong et al. (2011), Zanin-Zhorov et al. (2010).

et al., 2008; Kong et al., 2011). More importantly, are there non-overlapping or distinct pathways between PKCη and PKCθ? The answer is likely yes. First of all, as shown in our study, PKCη and PKCθ have distinct roles in homeostatic proliferation, with η being required but θ being dispensable (Fu et al., 2011). Second, the different spatio-temporal localization of PKCη and

PKCθ in the immunological synapse, with η showing an earlier and more diffuse pattern and θ showing a later and more concentrated pattern in the central region of the synapse (Singleton et al., 2009; Fu et al., 2011; Quann et al., 2011). Finally, there is a study showing PKCη and PKCθ having differential downstream functions in EL4 thymoma cells (Resnick et al., 1998). Collectively, these results strongly indicate the existence of an at least partially independent signaling pathway involving PKCη.

#### **FUTURE DIRECTIONS**

As mentioned earlier, the study of PKCη in T cell biology and the immune system in general, is far behind the state of knowledge we have on its cousin PKCθ (Fu and Gascoigne, 2012). Several recent studies have finally brought PKCη under the spotlight (Singleton et al., 2009; Suzuki et al., 2009; Fu et al., 2011; Quann et al., 2011; Sewald et al., 2011). In **Table 1**, we summarize the available results regarding PKCη in comparison with PKCθ. However, much more work needs to be done before we have a comprehensive understanding of the role of PKCη. First, what molecular machinery is involved in PKCη signaling? Does PKCη share the same signaling complex with PKCθ, such as the CAMA1/MALT1/Bcl10 complex? Second,what drives PKCη to the immunological synapse and what is the importance of differential localization of PKCη compared to PKCθ in the synapse? A recent study shows that the V3 domain is required for PKCθ recruitment to the immunological synapse (Kong et al.,2011). Is this also truefor PKCη, considering their generally similar structures, or is the diffuse synapse-localization of PKCη due to the lack of the relevant motif in the V3 domain? Since

#### **REFERENCES**

PKCθ interacts with CD28 through a V3 motif, does PKCη also interact with CD28, or if not,is it due to the differentV3 sequences? Does PKCη interact with other co-stimulatory molecules? Third, what roles does PKCη have in other T cell subsets or in other immune cells? In mice, it has been shown that PKCθ-deficiency impairs regulatory T cell development (Schmidt-Supprian et al., 2004), and in humans it has been shown that PKCθ plays a negative feedback role in regulatory T cell function, which is in contrast to its positive feedback role in naïve conventional T cells (Zanin-Zhorov et al., 2010). Thus it may be informative to check the role of PKCη in Treg cell development and function. PKCθ-deficiency has been shown to specifically impair Th2 cell responses but not Th1 responses, and thus has various effects in anti-pathogen immune responses (Berg-Brown et al., 2004;Marsland et al., 2004, 2005; Sakowicz-Burkiewicz et al., 2008). Could PKCη play an opposing role in these cases or a redundant role? What effects may PKCη have on CD4 T-helper cell subset differentiation? The role of PKCη in infection models and autoimmune diseases is another area that clearly needs attention. A simple but very informative study would be to directly compare the immune responses in η-, θ-, or ηθdouble deficient mice to the same viral and bacterial pathogens to get a full picture of the role of these two PKC isoforms in immunity. All these questions deserve more systematic studies in the future.

#### **ACKNOWLEDGMENTS**

This work was supported by NIH grants GM065230 and AI074074 to Nicholas R. J. Gascoigne. This is manuscript 21769 from The Scripps Research Institute.

P. M., and Dustin, M. L. (1999). The immunological synapse: a molecular machine controlling T cell activation. *Science* 285, 221–227.

in the impairment of the NF-κB pathway. *Mol. Cell* 8, 771–780.

lung and skin. *J. Biol. Chem.* 265, 22434–22440.

subsets: class discrimination or co-receptor recognition? *Immunol. Today* 19, 276–282.

and infection in mice. *J. Clin. Invest.* 119, 3774–3786.

**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: 19 April 2012; paper pending published: 08 May 2012; accepted: 11 June 2012; published online: 27 June 2012.*

*Citation: Fu G and Gascoigne NRJ (2012) The role of protein kinase C*η *in T cell biology. Front. Immun. 3:177. doi: 10.3389/fimmu.2012.00177*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Fu and Gascoigne. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*

# Th1/Th2 differentiation and B cell function by the atypical PKCs and their regulators

# **Pilar Martin1\* and Jorge Moscat 2**

1 Department of Vascular Biology and Inflammation, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain 2 Sanford-Burnham Medical Research Institute, La Jolla, CA, USA

#### **Edited by:**

Noah Isakov, Ben Gurion University of the Negev, Israel

#### **Reviewed by:**

Kjetil Taskén, University of Oslo, Norway Catalina Ribas, Universidad Autonoma de Madrid, Spain

#### **\*Correspondence:**

Pilar Martin, Department of Vascular Biology and Inflammation, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain. e-mail<EMAIL_ADDRESS>
The members of the atypical Protein Kinase Cs (aPKC) kinase subfamily, PKCζ and PKCλ/ι, as well as their adapters, p62 and Par-6, form part of the PB1-domain-containing group of signaling regulators. Both adapters serve to locate through heterotypic interactions the aPKCs into the NF-κB and cell polarity pathways, respectively. Both signaling cascades have been critically implicated inT cell function in vitro and in vivo.The analysis of gene-knockout (KO) mice deficient in the different PB1 molecules is providing more definitive information on the actual role that the aPKCs and other PB1-containing molecules play in B cell biology and T cell polarity, survival, and differentiation toward the different effector lineages in vivo and at the cellular ex vivo level. Here we discuss recent data generated from the analysis of KO mice linking the control of cell polarity by PKCλ/ι and PKCζ, their adapter p62, and the Par-4 inhibitor, in the control of B and T cell signaling and differentiation. Altogether, these genetic and biochemical evidences reveal the existence of a PB1-orchestrated signaling network that acts to control Th2 differentiation in vitro and in vivo, and the gene transcriptional programs that are essential during the B cell maturation and function and Th2 differentiation.

**Keywords: atypical PKCs, B cells,T cells,Th1,Th2, adaptive immune responses, asthma**

#### **INTRODUCTION**

It is well known that cells from the adaptive immune system need proper activation of the nuclear factor κB (NF-κB) for their function and development. NF-κB provides necessary mediators for the survival of immature T and B cells in antigen receptormediated negative and positive selection of non-reactive clones in thymus and bone marrow, respectively. NF-κB activation is also necessary for mature lymphocyte differentiation and function (Hayden et al., 2006; Schulze-Luehrmann and Ghosh, 2006). However, uncontrolled activation of NF-κB can induce cancer development, autoimmune, and chronic inflammatory diseases as a result of exacerbated lymphocyte function (Karin and Greten, 2005). The Protein Kinase Cs (PKCs) family is an important piece in the puzzle of NF-κB-mediated cell activation and several studies on recent years have demonstrated that proteins of this family are potent mediators of antigen receptor downstream signaling in B and T cells. Hence, these proteins are key in both, the development and the control of innate and adaptive immune responses. Since there are three different families of PKCs; classical PKCs (PKCα, βI, βII, γ), novel PKCs (PKCδ, ε, η, θ), and atypical PKCs (aPKCs; PKCζ, λ/ι), some of the lymphocyte signaling pathways controlled by these kinases can be redundant although they have distinct *in vivo* functions due to their broad substrate specificity. For example, in addition to NF-κB they are also involved in AP-1 or NFAT transcription factors activation in T cells (Tan and Parker, 2003). In this review we discuss the mechanisms by which aPKCs regulate T and B cell signaling after antigen stimulation and the role of the aPKCs-associated molecules Par-4, a potent inhibitor of

aPKCs, and the scaffolding protein p62 in lymphocyte activation and differentiation.

#### **ATYPICAL PKCs IN B CELL BIOLOGY**

B cells are lymphocytes originated from lymphoid precursors in the bone marrow after rearrangement of the immunoglobulin genes to provide the immune system with the specific repertoire of B cells to protect the body against pathogens (Harwood and Batista, 2010). B cells generation from hematopoietic precursors needs four different steps to take place: early pro-B cells, late pro-B cells, pre-B cells, and immature B cells formation. Before immunoglobulin gene rearrangement starts, the early pro-B cell subset emerges from the precursors giving rise to the following subset of late pro-B cells. In this step begins the rearrangement of D and J gene segments to generate pre-B cells with intact immunoglobulin heavy chains. When the rearrangement of the light-chain genes is completed the immature B cell subset is formed, expressing IgM on cell surface (B220low MHC-IIhigh IgMhigh IgDlow). At this step, B cells undergo a selection process to eliminate self-reactive cells to avoid autoimmunity before going out to periphery. Once positioned in secondary lymphoid organs, immature B cells become transitional B cells that are ready to maturate within the follicles in lymph nodes or in the marginal zone of the spleen (marginal zone B cells). After antigen challenge, mature B cells (B220high MHC-IIhigh IgMlow IgDhigh) become antibody-secreting plasma cells in germinal centers or memory B cells distributed elsewhere (Hardy and Hayakawa, 2001). The role of PKCs in these processes as well as in the activation and

function of mature B cells is from diverse nature. Is important to remark that, unlike what is described for other PKCs, PKCδ is involved in the generation of B cell tolerance and anergy (Mecklenbrauker et al., 2002; Miyamoto et al., 2002) whereas PKCβ and PKCζ have a different role in B cell biology. Mice deficient for PKCβ has defective B cell signaling (Leitges et al., 1996) and PKCζ deficiency is associated to defects in B cell development, proliferation, and survival (Leitges et al., 2001; Martin et al., 2002; Moscat et al., 2003).

#### **Role of PKC**ζ **in secondary lymphoid organ maturation and B cell differentiation**

Although the lack of PKCζ in mice generates no apparent abnormality, the formation, and maturation of secondary lymphoid organs is altered during the first weeks after birth. In this regard, 2– 4 weeks old mice fail to develop an appropriate number of Peyer's patches (PP) with an important reduction in follicles size and total cell number within each PP. Moreover, internal structure of PPs is also impaired as B and T cell zones are disrupted, this is due in part to the almost total lack of follicular dendritic cells (FDCs) that organized follicles structure. B cell zones are reduced and there is an imbalance between B220high MHC-IIhigh IgMlow IgDhigh mature and B220low MHC-IIhigh IgMhigh IgDlow immature B cells that is also observed in adult mice (Leitges et al., 2001). Regarding the spleen of these mice, although grossly normal, they present a defect in the formation of B cell follicles in the white pulp. The deficiency of FDCs in this organ induces disruption of the marginal zone architecture. Accordingly there is a decrease in the percentage of mature BCs in lymph nodes. However most of these defects are overcome in adult animals indicating that PKCζ could have an important role in B cell maturation in young animals that can be compensated by other molecules in adult mice (Leitges et al., 2001). In this regard it is important to note that B cell subpopulations in spleens and lymph nodes are normal and also microarchitecture of these organs has no apparent defects in adult animals.

#### **B cell activation and survival is dependent of PKC**ζ **signaling**

In depth study of the role of PKCζ in B cell signaling indicates that this aPKC is required for an optimal survival rate, cell cycle entry, and proliferation after B cell receptor (BCR) stimulation in adult animals (Martin et al., 2002). As stated before, B cell subpopulation and lymphoid organ architecture of 4–6 weeks old animals is quite normal. In contrast to that observed in embryonic fibroblasts (EFs; Leitges et al., 2001), studies with PKCζ-deficient mice indicate that the defects in B cell activation and proliferation correspond to impaired activation of extracellular signal-regulated kinase (ERK), whereas the activation of other mitogen-activated protein kinases (MAPK) is not altered. Regarding transcription factors activation through PKCζ, a small decrease in activating protein-1 (AP-1) transcription factor activation is observed along with an inhibition of c-Fos induction in PKCζ-deficient mice (Martin et al., 2002; **Figure 1A**).Although PKCζis an important mediator of the NF-κB pathway (Leitges et al., 2001), the activation and nuclear translocation of this transcription factor is not inhibited in BCs from PKCζ-deficient mice. However, activation of I*k*B transcription is severely impaired in PKCζ-deficient B cells after BCR stimulation

and more importantly, transcription of NF-κB-dependent genes such as IL-6 or Bcl-XL (important for B cell survival) is also inhibited (**Figure 1A**). These data demonstrate that although PKCζ is important for the transcription of NF-κB-dependent genes associated with proliferation and survival, and consistent with previous data of EF from PKCζ-deficient mice (Leitges et al., 2001), NF-κB nuclear translocation is not affected in the absence of this molecule in B cells (Martin et al., 2002).

#### **Adaptive immune responses are altered in PKC**ζ**-deficient mice**

PKCζ is an essential kinase for B cell survival and function through the activation of I*k*B and Bcl-XL transcription (Martin et al., 2002). These data agree with the notion that Rel, RelA, and NF-κB1 transcription factors are essential in B cell development, survival, proliferation, and immunoglobulin expression (Grossmann et al., 2000; Gugasyan et al., 2000). The lack of Rel/NF-κB genes or the individual subunits of the IκB kinase, IKKα and IKKβ, alters the development of adaptive immune response (Gerondakis et al., 1999). Accordingly, although PKCζdeficient mice develop immune responses against T-independent and -dependent antigens and are able to induce the formation of germinal centers in the spleen, the T-dependent humoral immune responses are very faint. The secretion of IgG1, IgG2a, and IgG2b immunoglobulins, specific for dinitrophenyl-ovalbumin (DNP-OVA) T-dependent antigen, is almost abrogated in PKCζ-deficient mice, indicating that these mice are not able to mount a proper humoral response against T-dependent antigens (Martin et al., 2002; **Figure 1A**).

Even nowadays there is little information on the role of the other family member of aPKCs, the PKCλ/ι, in secondary lymphoid organ development or signaling downstream BCR in mature B cells. This is in part due to the lack of viable homozygous deficient mice for PKCλ/ι as these mice die before birth (Soloff et al., 2004; Yang et al., 2009), which is incompatible with the study of mature B cells. While more effort needs to be done to clarify this issue, PKCλ/ι seems not to be activated after BCR stimulation or after incubation with anti-CD40 indicating that this aPKC could not be playing any role in B cell biology (Martin et al., 2002). In summary, PKCζ plays a major role in lymphoid organ formation and B cell differentiation and function that could be non-redundant with the other aPKCs, the PKCλ/ι.

#### **Role of PKC**λ **in B cell development**

The role of PKCλ/ι in BC development or function has not been studied in such detail so as PKCζ. However it is known that PKCλ is involved in pre-BCR-induced activation of NF-κB in B cells undergoing maturation process (Saijo et al., 2003). Src-family protein tyrosine kinases (SFKs) have an important role in the first steps during B cell development, controlling the generation, and survival of pre-B cells (Wechsler and Monroe, 1995). These effects are mediated by NF-κB thus in the absence of SFKs, IKK phosphorylation is inhibited and the nuclear translocation of NF-κB is impaired inducing defects in the formation of pre-B cells. It has been proposed that the atypical PKCλ is a mediator of SFK induced activation of NF-κB in immature B cells, since PKCλ activation in pro-B cells deficient for SFK restores IKK activation and NF-κB-mediated signaling (Saijo et al., 2003). The mechanisms of

IKK activation by PKCλ are not addressed, however it could be a direct association by the consensus phosphorylation site Ser177 (Lallena et al., 1999), or by an indirect interaction with Bcl10 and Carma1 (Gaide et al., 2002).

#### **T CELL SIGNALING AND ATYPICAL PKCs**

The nature of effector T lymphocytes and their functional properties into inflammatory tissues has been a matter of study during decades. However, in the last few years the paradigms of effector cell populations at the site of inflammation has changed considerably with the emergence of new players. After the infection of invading pathogens or during inflammation, T cell activation, and subsequent differentiation of T helper lymphocyte subsets are crucial for the development of immune responses. Once the antigen presenting cell presents the antigen to naïve CD4 T cells, these cells experienced differentiation toward Th1, Th2, or Th17 T helper cell subsets in the lymph nodes. Afterward, T helper cells migrate to the inflamed tissue and develop an adaptive immune response. Th1 cells are characterized by the expression of T-bet and the release of the Th1-related cytokine IFN-γ. Th1 cells are involved in the generation of autoimmunity and inflammatory disorders mediated by cellular immune responses such as the clearance of pathogens. The effector subset Th2 is essential for the development of inflammatory conditions mediated by antibodies and in the generation of allergic responses such as asthma. Th2 cells produce mainly IL-4, IL-5, and IL-13 among other cytokines and express the transcription factors GATA-3, Stat6, and c-Maf. And the more recently discovered T helper subset, the Th17 cells are characterized by the synthesis of IL-17A, IL-17F, and IL-22. Th17 cell emerged as an independent differentiation pathway as the express a different panel of transcription factors such as Stat3 and RORγt (Harrington et al., 2005; Bettelli et al., 2007; Nakayamada et al., 2012). The role of classical and novel PKCs in T cell receptor (TCR) signaling and T cell differentiation into helper T cells have been addressed extensively. PKCθ is recruited to the T cell synapse after TCR stimulation, what makes this PKC essential for the downstream signaling through NF-κB (Liu et al., 2000). The function of this PKC in T cell signaling is shared with PKCα, since both PKCs mediate T lymphocyte activation (Gruber et al., 2009). However PKCθ have different roles in Th1, Th2, or Th17 differentiation. PKCθ-deficient mice develop normal Th1 responses toward intracellular pathogens whereas Th2 asthmatic response against that this protein acts at the level of antigen receptor signaling. Interestingly Par-4 deficient T cells secrete high amounts of IL-2 *in vitro* after stimulation. As Par-4 is a pro-apoptotic protein, Par-4 deficient T cells are more resistant to apoptosis induced cell death than their WT T cell control (Lafuente et al., 2003). As it was previously mentioned, aPKCs downstream signaling cascade controls NF-κB activation (Leitges et al., 2001) and, in turn, Par-4 inhibits this pathway and proliferation of T cells through the inhibition of aPKCs phosphorylation. Then, it seems that Par-4-mediated inhibition of T cell proliferation is not controlled through PKCζ since the proliferation of T cells from PKCζ-deficient mice is normal (Martin et al., 2002). Hence Par-4 should exert its effect through the other aPKC, the PKCλ/ι. Moreover, primary EFs from Par-4-deficient mice have decreased JNK activation that is observed also in T cells from these mice (Garcia-Cao et al., 2003; Lafuente et al., 2003). JNK is a potent inhibitor of NFATc1 transcription factor essential for T cell differentiation and function and accordingly Par-4 deficient T cells have an increase in NFATc1 activation, which may explain the high levels of IL-2 secretion by these cells that could directly influence the high rate of proliferation of Par-4-deficient T cells (Chow et al., 2000; Crabtree and Olson, 2002). A well as PKCζ and PKCλ/ι play an important role in T cell differentiation toward helper subsets, Par-4 is important for the control of Th2 responses. Par-4 deficiency leads to aPKC hyperactivation and subsequent inhibition of JNK, signaling pathway required for the control of IL-4 secretion, and Th2 differentiation (Dong et al., 1998; **Figure 1B**). Moreover, *in vivo* experiments in a model of T cell-dependent hepatitis have demonstrated that Par-4 deficiency leads to an increase in IL-4 signaling pathway, increasing the levels of Th2-cytokines, eotaxin, apoptosis, and liver injury after treatment with Con-A. These results are consistent with an enhanced signaling downstream IL-4 in EFs from Par-4 deficient mice, where Jak1 and Stat6 phosphorylation is increased demonstrating a connection with an enhanced signaling through aPKC (Duran et al., 2004a; Martin et al., 2005; Yang et al., 2009).

#### **aPKC scaffold protein p62 controls T helper differentiation**

The adapter protein p62 interacts with aPKCs through their PB1 protein interaction domain and is essential in the maintenance of NF-κB activity during osteoclastogenesis (Duran et al., 2004b). Similarly is a key protein for the long-term NF-κB activation after

#### **REFERENCES**

tumor progression. *EMBO J.* 18, 6362–6369.

TCR stimulation of naïve CD4+ T cells (Martin et al., 2006). Unlike PKCζ and PKCλ/ι do, p62 controls Th2 differentiation and function by an IL-4 independent mechanism. Th2 cells deficient for p62 have impaired activation and nuclear translocation of GATA-3 and RelA, playing an important role in NF-κB activation, whereas Stat6 signaling through IL-4 receptor is not affected (Das et al., 2001; Martin et al., 2006). In this regard, proximal TCR downstream signaling pathway is not altered in the absence of p62 as Zap-70, ERK, or AKT activation and Ca2+ flux are normal after antigen receptor triggering. Degradation of IκBα, an early indicator of NF-κB activation, is also intact in p62-deficient cells while IκBα activation is inhibited several hours after TCR triggering along with NF-κB inhibition. Interestingly, p62 expression is induced at later time points after TCR stimulation and particularly under Th2 differentiation conditions. This is possible because p62 establish a connection with Malt-1 and TRAF6, both proteins involved in IKK ubiquitination (Wooten et al., 2005),favoring NFκB activation after long-term TCR stimulation probably through PKCλ/ι (**Figure 1B**). Accordingly with impaired Th2 differentiation *in vitro*, p62-deficient mice also show a dramatically reduction of lung inflammation toward OVA-induced allergic airway disease demonstrating a key role of p62 in Th2 function *in vivo* (Martin et al., 2006).

#### **CONCLUDING REMARKS**

It is clear that over the recent years the aPKCs have emerged as critical players in the control of T and B cell function, which has important implications as potential therapeutic targets in inflammation and immunity. Importantly due to their role also in cancer, they can also be considered critical players in the ability of the tumor cell to grow and proliferate but also as key players in the tumor microenvironment whereby they can be central in orchestration signals controlling the impact that inflammation has in tumor progression and initiation. Future studies using the animal models described here will be instrumental in addressing these important questions.

#### **ACKNOWLEDGMENTS**

This work was funded by grants SAF2011-27330 and INDISNET 01592006 from the Spanish Ministry of Economy and Competitiveness, and Comunidad de Madrid, respectively, to Pilar Martín, and R01AI072581 from the National Institutes of Health to Jorge Moscat.

induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. *Cell* 86, 777–786.

that this protein acts at the level of antigen receptor signaling. Interestingly Par-4 deficient T cells secrete high amounts of IL-2 *in vitro* after stimulation. As Par-4 is a pro-apoptotic protein, Par-4 deficient T cells are more resistant to apoptosis induced cell death than their WT T cell control (Lafuente et al., 2003). As it was previously mentioned, aPKCs downstream signaling cascade controls NF-κB activation (Leitges et al., 2001) and, in turn, Par-4 inhibits this pathway and proliferation of T cells through the inhibition of aPKCs phosphorylation. Then, it seems that Par-4-mediated inhibition of T cell proliferation is not controlled through PKCζ since the proliferation of T cells from PKCζ-deficient mice is normal (Martin et al., 2002). Hence Par-4 should exert its effect through the other aPKC, the PKCλ/ι. Moreover, primary EFs from Par-4-deficient mice have decreased JNK activation that is observed also in T cells from these mice (Garcia-Cao et al., 2003; Lafuente et al., 2003). JNK is a potent inhibitor of NFATc1 transcription factor essential for T cell differentiation and function and accordingly Par-4 deficient T cells have an increase in NFATc1 activation, which may explain the high levels of IL-2 secretion by these cells that could directly influence the high rate of proliferation of Par-4-deficient T cells (Chow et al., 2000; Crabtree and Olson, 2002). A well as PKCζ and PKCλ/ι play an important role in T cell differentiation toward helper subsets, Par-4 is important for the control of Th2 responses. Par-4 deficiency leads to aPKC hyperactivation and subsequent inhibition of JNK, signaling pathway required for the control of IL-4 secretion, and Th2 differentiation (Dong et al., 1998; **Figure 1B**). Moreover, *in vivo* experiments in a model of T cell-dependent hepatitis have demonstrated that Par-4 deficiency leads to an increase in IL-4 signaling pathway, increasing the levels of Th2-cytokines, eotaxin, apoptosis, and liver injury after treatment with Con-A. These results are consistent with an enhanced signaling downstream IL-4 in EFs from Par-4 deficient mice, where Jak1 and Stat6 phosphorylation is increased demonstrating a connection with an enhanced signaling through aPKC (Duran et al., 2004a; Martin et al., 2005; Yang et al., 2009).

#### **aPKC scaffold protein p62 controls T helper differentiation**

The adapter protein p62 interacts with aPKCs through their PB1 protein interaction domain and is essential in the maintenance of NF-κB activity during osteoclastogenesis (Duran et al., 2004b). Similarly is a key protein for the long-term NF-κB activation after

#### **REFERENCES**

tumor progression. *EMBO J.* 18, 6362–6369.

TCR stimulation of naïve CD4+ T cells (Martin et al., 2006). Unlike PKCζ and PKCλ/ι do, p62 controls Th2 differentiation and function by an IL-4 independent mechanism. Th2 cells deficient for p62 have impaired activation and nuclear translocation of GATA-3 and RelA, playing an important role in NF-κB activation, whereas Stat6 signaling through IL-4 receptor is not affected (Das et al., 2001; Martin et al., 2006). In this regard, proximal TCR downstream signaling pathway is not altered in the absence of p62 as Zap-70, ERK, or AKT activation and Ca2+ flux are normal after antigen receptor triggering. Degradation of IκBα, an early indicator of NF-κB activation, is also intact in p62-deficient cells while IκBα activation is inhibited several hours after TCR triggering along with NF-κB inhibition. Interestingly, p62 expression is induced at later time points after TCR stimulation and particularly under Th2 differentiation conditions. This is possible because p62 establish a connection with Malt-1 and TRAF6, both proteins involved in IKK ubiquitination (Wooten et al., 2005),favoring NFκB activation after long-term TCR stimulation probably through PKCλ/ι (**Figure 1B**). Accordingly with impaired Th2 differentiation *in vitro*, p62-deficient mice also show a dramatically reduction of lung inflammation toward OVA-induced allergic airway disease demonstrating a key role of p62 in Th2 function *in vivo* (Martin et al., 2006).

#### **CONCLUDING REMARKS**

It is clear that over the recent years the aPKCs have emerged as critical players in the control of T and B cell function, which has important implications as potential therapeutic targets in inflammation and immunity. Importantly due to their role also in cancer, they can also be considered critical players in the ability of the tumor cell to grow and proliferate but also as key players in the tumor microenvironment whereby they can be central in orchestration signals controlling the impact that inflammation has in tumor progression and initiation. Future studies using the animal models described here will be instrumental in addressing these important questions.

#### **ACKNOWLEDGMENTS**

This work was funded by grants SAF2011-27330 and INDISNET 01592006 from the Spanish Ministry of Economy and Competitiveness, and Comunidad de Madrid, respectively, to Pilar Martín, and R01AI072581 from the National Institutes of Health to Jorge Moscat.

induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. *Cell* 86, 777–786.

distribution of functions between the two atypical protein kinase C isoforms. *J. Immunol*. 173, 3250–3260.

**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: 09 May 2012; accepted: 17 July 2012; published online: 06 August 2012. Citation: Martin P and Moscat J (2012) Th1/Th2 differentiation and B cell function by the atypical PKCs and their regulators. Front. Immun. 3:241. doi: 10.3389/fimmu.2012.00241*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Martin and Moscat. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*