The discovery of IL-17 producing helper T cells (Th17) in mice (89, 90) and humans (91) and their relative instability (11, 92) has led to a profound re-evaluation of the concept of two terminally differentiated helper T cell subsets. The fact that human CD4+ T cells produce IL-17 was known for a long time (93). However, it took a decade to realize that these cells represented an independent differentiation lineage (89, 90), which have unique differentiation requirements and express the lineage-defining transcription factor ROR-γt in mice and RORC2 in humans (94, 95). Th17 cells are important to fight extracellular bacteria and fungi, since patients that lack Th17 cells have uncontrolled infections with *Candida albicans* (*C. albicans*) and *Staphylococcus aureus* (96). The discovery of Th17 cells has been complicated by the fact that T cell differentiation to Th1 and Th17 cells relies on shared components of cytokines and their receptors. Thus, it was known that IL-12p40 and IL-12Rβ1 hetero-dimerize with respectively IL-12p35 and IL-12Rβ2 to induce Th1 cells, but later it was realized that they can also associate with respectively IL-23p19 and the IL-23R to promote Th17 responses (97). The IL-23/IL-23R pathway is involved in many different autoimmune diseases (98–100) and IL-23-induced Th17 cells are thought play a prominent pathogenic role (101–104). Conversely, the contribution of Th1 cells, which were initially thought to drive autoimmune diseases, is now debated. The requirements for Th17 differentiation are more complex than for Th1 and Th2 cells, because IL-17 production in CD4+ T cells can be induced by different cytokine combinations. Initially, TGF-β plus IL-6 was identified in mice (105), while IL-1β, IL-6, and/or IL-23 were proposed in humans (106, 107). The *de novo* Th17 differentiation is very inefficient in humans, and therefore it was suggested that only a cocktail with all four cytokines induces significant Th17 differentiation (108). Although the role of TGF-β in human Th17 differentiation has been a subject of debate (109), it was shown in mice that TGF-β induces ROR-γt, while pro-inflammatory cytokines are required to inhibit TGF-β-induced Foxp3 expression and thus Treg generation (110). The presence of CD4+ T cells co-expressing Foxp3, RORC2, and/or IL-17 in humans is consistent with a role for TGF-β in human Th17 and Treg development (86, 88). An alternative explanation for the positive role of TGF-β in Th17 differentiation is that TGF-β indirectly favors Th17 cell differentiation by inhibiting Th1 cell development (111). Indeed, in the absence of TGF-β1 (106, 107, 112), or in the presence of TGF-β3 in mice (113), pathogenic Th17 cells that co-produce IL-17 and IFN-γ are generated. These Th1/17 cells co-express RORC2 and T-bet, are enriched in autoimmune patients and are specific for both Th1 and Th17-inducing pathogens (114, 115).

*In vitro* stability experiments and fate reporter mice suggested that Th17 cells are partially unstable and can switch completely from IL-17 producing Th17 to IFN-γ producing Th1 cells in chronic immune responses (92, 116). IL-12 can induce this Th17 to-Th1 switch (117), and CD161 was proposed as a marker that distinguishes these ex-Th17 cells from conventional Th1 cells in humans (118). However, *ex vivo* isolated human Th17 cells exhibited stable epigenetic marks at cytokine and transcription factor loci (119), suggesting that *in vivo* generated human Th17 cells are not necessarily unstable. Finally, also a very rare population of human T cells that co-produces IL-17 and IL-4 was identified (120). These Th2/17 cells were proposed to be highly pro-inflammatory in allergic asthma, but their role in immune responses against pathogens remains to be understood.

Th17 cells are highly heterogeneous and produce several effector cytokines besides IL-17. IL-22, a cytokine that promotes epithelial proliferation and barrier function (121), is produced by some Th17 cells (122, 123), and IL-22 and IL-17 co-operate to control gram-negative bacteria in the lung (124). However, a subset of human skin-homing IL-22 producing cells was identified that were distinct from Th17 cells (125, 126). Indeed, in contrast to IL-17, IL-22 is inhibited by TGF-β (127) and thus how Th17 cells acquire IL-22 producing capacities and if they can even switch from IL-17 to IL-22 production is unclear. Some Th17 and Th22 cells also produce IL-26, a pro-inflammatory cytokine that is not expressed in mice (128) and that also acts selectively on non-hematopoietic cells. A particular relevant cytokine in the pathogenesis of experimental autoimmunity is GM-CSF, which is induced by IL-1β, IL-23, and ROR-γt in mice (102, 129). Conversely, GM-CSF is inhibited by IL-1β and IL-23 in humans, and is produced by both Th1 and Th17 cells (130, 131).

Th17 cells also produce high levels of IL-21. IL-6 induces IL-21 in naive T cells upon priming (132), and IL-21 can induce its own expression (133) and promotes Th17 differentiation in an autocrine manner (131, 134–136). Importantly however, IL-21 inhibits GM-CSF and IFN-γ production and promotes instead IL-10 secretion in developing Th17 cells. Consequently, IL-21 promotes the generation of conventional (137) or regulatory Th17 cells (138), but inhibits the generation of pathogenic Th1/17 cells (131). Finally, a subset of skin-homing T cells produces IL-9 and responds to *C. albicans* (139). Some of these cells co-produce IL-9 and IL-17 (60), while others appear to represent Th9 cells. IL-9 production seems however to be transient, suggesting that these skin-homing Th9 cells are largely unstable (139).

In summary, the current knowledge indicates that human Th17 cells are highly heterogeneous and partially unstable (**Figure 2**), and much remains to be learned on the role of different Th17 subsets in immune-mediated diseases.

chronic inflammation.

**REGULATION OF HUMAN T CELL PLASTICITY IN TISSUES: THE NEW FRONTIER**

addition to IL-17, including the Th1 and Th2 marker cytokines IFN-γ and IL-4.

The complex regulation of T helper subsets by cytokines raises the questions where T cells are re-educated and also why this might be important to successfully resist pathogens, since this was a major evolutionary pressure that shaped the human immune system. It was soon realized that stable T cell differentiation often requires repetitive *in vitro* TCR stimulation in the appropriate cytokine condition, suggesting that immature T cells might be more plastic than more differentiated ones (12, 140). *In vivo* primed T cells that are at an intermediate stage of differentiation are central memory T cells (TCM), which similar to naïve T cells have maintained the capacity to home to lymph nodes, produce only low levels of effector cytokines, but produce high levels of IL-2 and IL-21 (131), and expand rapidly to generate secondary waves of effector cells (8). Conversely, effector memory T cells (TEM) are more differentiated cells since they produce high levels of effector cytokines and home preferentially to inflamed non-lymphoid tissues (8)*.* Consistent with the view that plasticity is progressively reduced upon T cell differentiation, pre-committed Th1CM cells are more plastic than fully differentiated Th1EM cells, since Th1CM cells generate a substantial population of *bona fide* Th2 cells upon re-stimulation with IL-4, while Th1EM cells do not revert to Th2 cells, but some acquire IL-4 in addition to IFN-γ producing capacities (24). This plasticity requires TCR stimulation, since antigen-independent proliferation with homeostatic cytokines resulted exclusively in the generation of Th1 effector cells (24). Based on these findings it can be speculated that pre-committed TCM cells that cross-react with a different pathogen can be still partially re-educated to a different lineage in lymph nodes, while TEM cells do not easily switch cytokine production, but rather become polyfunctional (**Figure 1**). Another example of functional plasticity in lymphoid organs is the generation of follicular Foxp3+BCL-6+ Tregs, which are specialized Tregs that control B cell responses (79, 80). Also Tregs in non-lymphoid tissues acquire tissue-specific properties that are important for their functions (76). TEM helper cells that are activated by antigen in non-lymphoid tissues can up-regulate CCR7 (141) and home to inflamed lymph nodes (142) where they can influence the secondary immune response and are exposed to a different cytokine milieu. Conversely, tissue-resident memory (TRM) cells have lost sphingosine-1 phosphate receptors and thus also the capacity to re-circulate through the blood to secondary lymphoid organs (143). TRM belong predominantly to the CD8 compartment, but influenza virus-specific CD4+ TRM can be identified in the lung of humans and mice (144). If tissueresident CD4+ T cells are terminally differentiated effector cells or still possess the plasticity to acquire additional cytokine producing capacities remains to be established (145).

A central organ for the generation of different subsets of Th17 cells is the intestine (146). Thus, upon self-limiting colitis induced by anti-CD3 injections in mice predominantly IL-10 producing Th17 cells with regulatory functions are induced (138). Conversely, under conditions that induce IL-23 in the intestine pathogenic IFN-γ and GM-CSF producing Th17 cells are generated that induce colitis (147, 148). IFN-γ and IL-17 co-producing Th1/17 cells have also been observed in patients with IBD (92), but very little is known about the regulation of Th17 responses in the human intestine. Th1/17 cells that produce IL-17, IFN-γ, and GM-CSF also drive central nervous system (CNS) inflammation in EAE, a standard mouse model of multiple sclerosis (MS) (149). The CNS is separated from pro-inflammatory T cells by the blood–brain barrier (150), but spontaneous JC Virus

re-activations and progressive multifocal leukoencephalopathy in MS patients treated with anti-VLA-4 antibodies, which block lymphocyte extravasation to the CNS, suggest nevertheless a constant immune surveillance by T cells (151). How the microenvironment of the CNS influences the properties of CD4+ T cells is the focus of intensive research in mice, but is largely unknown in humans given the difficulties to analyze T cells in the human CNS.

Thus, accumulating evidence underlines the role of the tissue microenvironment in T cell plasticity, and the identification of tissue-specific factors that control T cell functions is likely to have a major impact on translational medicine.

#### **CONCLUSION AND PERSPECTIVE**

The original concept of two terminally differentiated subsets of Th1 and Th2 cells has been substituted by the view that many different T cell subsets with specific cytokine profiles are required to protect us from the different pathogenic insults that were are continuously exposed to. These various T cell subsets possess different degrees of plasticity to acquire new characteristics and functions in secondary or chronic immune responses. In particular, while the stability of Tregs is debated, it is widely accepted that Th17 cells are largely unstable, although exceptions might exist. In addition, human Th17 cells are highly heterogeneous, but thefunctions of all these different types of Th17 effector cells in protective immune responses and their roles in autoimmune diseases remain to be understood. Another important but poorly understood aspect of T cell plasticity is how different tissue microenvironments impact on human T cell differentiation and stability. The definition of the relative plasticities or stabilities of human T cell subsets in different tissues is highly relevant for future therapeutic interventions in so different immune-related pathologies as chronic viral infections, cancer, and autoimmune diseases.

#### **ACKNOWLEDGMENTS**

Jens Geginat, Massimiliano Pagani, and Sergio Abrignani are supported by the Cariplo foundation and Sergio Abrignani and Massimiliano Pagani by an ERC grant. The INGM is supported by the Romeo ed Enrica Invernizzi foundation.

#### **REFERENCES**

proinflammatory cytokines in driving and modulating human T(H)-17 responses. *Nat Immunol* (2008) **9**(6):650–7. doi:10.1038/ni.1613

which is essential for the effector phase of autoimmune neuroinflammation. *Nat Immunol* (2011) **12**(6):560–7. doi:10.1038/ni.2027

151. Berger JR, Houff S. Progressive multifocal leukoencephalopathy: lessons from AIDS and natalizumab. *Neurol Res* (2006) **28**(3):299–305. doi:10.1179/ 016164106X98198

**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: 03 October 2014; accepted: 25 November 2014; published online: 16 December 2014.*

*Citation: Geginat J, Paroni M, Maglie S, Alfen JS, Kastirr I, Gruarin P, De Simone M, Pagani M and Abrignani S (2014) Plasticity of human CD4 T cell subsets. Front. Immunol. 5:630. doi: 10.3389/fimmu.2014.00630*

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

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

REVIEW ARTICLE published: 15 April 2015 doi: 10.3389/fimmu.2015.00175

## Long intergenic non-coding RNAs: novel drivers of human lymphocyte differentiation

#### **Ilaria Panzeri 1 , Grazisa Rossetti 1 , Sergio Abrignani 1 and Massimiliano Pagani 1,2\***

1 Integrative Biology Unit, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", IRCCS Ospedale Maggiore Policlinico, Milano, Italy 2 Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milano, Italy

#### **Edited by:**

Dragana Jankovic, National Institutes of Health (NIH), USA

#### **Reviewed by:**

Christopher E. Rudd, University of Cambridge, UK John J. Miles, Queensland Institute of Medical Research, Australia Susan Carpenter, University of California San Francisco, USA

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

Massimiliano Pagani, Integrative Biology Unit, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", IRCCS Ospedale Maggiore Policlinico, via Francesco Sforza 35, Milano 20122, Italy e-mail<EMAIL_ADDRESS>
Upon recognition of a foreign antigen, CD4+ naïve T lymphocytes proliferate and differentiate into subsets with distinct functions. This process is fundamental for the effective immune system function, as CD4+ T cells orchestrate both the innate and adaptive immune response. Traditionally, this differentiation event has been regarded as the acquisition of an irreversible cell fate so that memory and effector CD4+ T subsets were considered terminally differentiated cells or lineages. Consequently, these lineages are conventionally defined thanks to their prototypical set of cytokines and transcription factors. However, recent findings suggest that CD4+ T lymphocytes possess a remarkable phenotypic plasticity, as they can often re-direct their functional program depending on the milieu they encounter. Therefore, new questions are now compelling such as which are the molecular determinants underlying plasticity and stability and how the balance between these two opposite forces drives the cell fate. As already mentioned, in some cases, the mere expression of cytokines and master regulators could not fully explain lymphocytes plasticity. We should consider other layers of regulation, including epigenetic factors such as the modulation of chromatin state or the transcription of non-coding RNAs, whose high cell-specificity give a hint on their involvement in cell fate determination. In this review, we will focus on the recent advances in understanding CD4+ T lymphocytes subsets specification from an epigenetic point of view. In particular, we will emphasize the emerging importance of non-coding RNAs as key players in these differentiation events. We will also present here new data from our laboratory highlighting the contribution of long non-coding RNAs in driving human CD4+ T lymphocytes differentiation.

**Keywords: long non-coding RNAs, epigenetic regulation, lymphocyte differentiation**

#### **THE REVOLUTIONS OF REGULATORY NON-CODING RNAs**

At the beginning of this century, the results of the human genome project highlighted the complexity of our genome. What emerged was that the fraction of the genome that is informative is higher than we expected. Subsequent analysis revealed that the vast majority of informative sequences does not encode for proteins. Indeed against a total of 62.1% of the human genome covered by processed transcript (74.7% by primary transcripts), exons of protein-coding genes cover only the 2.94% of the genome (1). From an evolutionary point of view, the genome size is in close relationship with coding potential in prokaryotes, which have haploid genomes primarily composed by protein-coding sequences (~88%). Conversely, in eukaryotes, a correlation lacks between protein-coding gene number and organismal complexity. These observations are likely explained by the evolution of a more sophisticated architecture to control gene expression that includes the expansion of non-coding regulatory RNAs (ncRNAs) (2). Thus, we should clearly reassess the centrality of protein-coding RNAs in favor of non-coding ones.

Non-coding RNAs with fundamental functions within cells are known since the discovery of the first transfer RNA (tRNA) (3) and comprise also ribosomal RNAs (rRNAs). Nonetheless, the interest toward non-coding RNAs with regulatory functions arose with

the discovery of the first human micro-RNA, let-7 (4). In order to apply a theoretical framework to the transcriptome, regulatory ncRNAs are usually classified based on their dimension: "small" ncRNAs being less than 200 nucleotides in length and "long" or "large" ncRNAs (lncRNAs) ranging from more than 200 to tens of thousands of nucleotides (**Table 1**).

Further complicating the picture, lncRNAs seem to be the preferred substrate for the generation of small RNAs (21). This maze of non-coding transcripts was revealed also in a genome-wide identification of lncRNAs in mouse CD8+ T lymphocytes, where 18 of the identified lncRNAs appeared to overlap with annotated miRNAs and 21 with snoRNAs (37).

Both classes can be further classified according to their position relative to known sequences of the genome, like in the case of promoter-associated RNAs (PASRs) or transcription initiation small RNAs (tiRNAs). In particular, long non-coding RNAs are usually classified relative to neighboring protein-coding genes. They can be defined as "sense" if they are transcribed from the same strand of the protein-coding gene or "antisense" if the opposite is true. They can be "divergent" if their promoter and the one of the coding transcript are in close proximity and located in a head to head fashion. They can be "exonic" or "intronic" if they overlap one or more exons, or an intron of the protein-coding

gene respectively. Instead, they can be "intergenic" (or "intervening"; lincRNAs) if they lie within a sequence between two protein-coding genes (38). In this review, we will focus on this last category, which is probably the most studied given that the location of these lncRNAs avoids complications deriving from the overlap with other genes. The majority of known lncRNAs is generated by the same transcriptional machinery of mRNAs. This means that transcribed lincRNAs genomic sequences are marked by RNA polymerase II occupancy and histone modifications that are shared with active protein-coding genes, such as H3K4me3 at promoters and H3K36me3 within gene bodies (39). They are capped by methylguanosine at their 50 , spliced, and polyadenylated, even if the widespread representation of this last property among known lncRNAs could be partially due to the RNA sequencing strategies used for their identification (15, 40). Indeed, broader analysis identified about 39% of lncRNAs to have at least one of the six most common poly(A) motifs, compared to 51% for coding transcripts (1). These properties imply that there are few distinctive biochemical features that allow the distinction of lncRNAs from protein-coding mRNAs. Among

them, lncRNAs have unusual exon structure, with on average 2–5 exons. Intriguingly, lncRNAs are significantly more likely to overlap repetitive elements and particularly RNA-derived transposable elements (TEs). These last account for about 30% of human lncR-NAs nucleotides, often in proximity of their transcriptional start site (TSS), which could suggest that TEs could be important drivers of lncRNAs evolution (see below). Nonetheless, the main difference between lncRNAs and protein-coding genes relies by definition on their coding potential: lncRNAs does not possess open reading frames (ORFs), as evaluated based on: the conservation of ORFs codons (41), ORFs length, the presence of known protein domains, *in vitro* translation (42, 43), and ribosome footprinting (44) assays. However, these conceptual constraints are terribly artificial: short, non-canonical peptides have been found to arise from small ORFs within ncRNA (45–48); lncRNAs genes can also code for proteins and have a double function (49) and ultimately, the coding potential does not necessarily exclude a function as RNA also for known mRNAs (50). Evolution makes boundaries between coding and non-coding genes fainter as ncRNAs can evolve by pseudogenization. This event can follow disruption of the ancestral ORF, but not of the untranslated regulatory regions (UTRs) in protein-coding genes duplicates (50) or can arise without duplication, but from the co-option of ancestral genes to different, non-coding functions (51). This was the case of the long-known Xist RNA, involved in the silencing of the inactive X chromosomes in eutherians. In particular, two exons of the protein-coding gene Lnx3 are homologous to Xist. This gene retained a protein-coding capacity at least in the common ancestor of marsupials and placentals. Conversely, the Xist A-repeat implicated in X-silencing function is not conserved. This sequence likely arose from the insertion of a TE recruited to form a proto-Xist gene (52, 53). Therefore, the difference between dosage compensation in marsupials, eutherians, and monotremes can be ascribed from the presence of a Xist-independent XCI in mammalian ancestor and the peculiar evolution of the proto-Xist gene by pseudogenization in the eutherian ancestor. Intriguingly, other lncRNAs involved in X-inactivation are similarly examples of pseudogenization (54). The boundary between coding and non-coding is even less defined when ncRNAs arise from joining of coding and non-coding exons through alternative splicing (55, 56), from untranslated regions of mRNAs (57, 58) or from the opposite strand of the overlapping protein-coding gene (59). Strikingly, more than a half of protein-coding genes in mammals have a complementary non-coding transcript (60). These findings further challenge our "linear" model of the genome, prompting a re-evaluation of current dogma and genes definitions. Genomic regions indeed are far more complex than previously thought: genes can be used for different purposes and different functional elements can co-locate intermingling coding and non-coding regions.

The interest toward lncRNAs has been rapidly growing and their expressions have been quantitated in many different tissues and cell types by high-throughput sequencing (RNA-seq). These efforts retrieved catalogs with little overlap, so that the number of known lncRNAs is still growing, in contrast with the number of known protein-coding genes that has been remarkably stable over years. Indeed, lncRNAs are far more cell-specific than mRNAs, generally less but also more dynamically expressed at various differentiation stages. For this reason, immune system is an excellent context in which we can deepen our knowledge on lncRNAs. While many excellent reviews cover the recent advances in understanding the role of these molecules within the innate branch (61, 62), little is still known about their importance for the human adaptive immune system. Effector lymphocytes are highly specialized cells that arise from common progenitors through differentiation processes still not completely understood. Besides, lymphocytes can be purified through cell sorting from blood of healthy donors and the existence of *in vitro* differentiation protocols provide the ideal setting for the identification of lncRNAs expressed in the human immune system and for their functional characterization.

Indeed, the growing interest on lncRNAs and the lack of knowledge on their expression patterns in the human immune system prompted us to perform the RNA-seq analysis on 13 human primary lymphocytes subsets purified by FACS sorting from healthy donors (CD4+ naïve, TH1, TH2, TH17, Treg, TCM, TEM, CD8+ naïve, TCM, TEM, B naïve, B memory, B CD25+) and to develop a bioinformatics pipeline for lincRNAs identification.

Through this analysis, we identified long intergenic ncRNAs genes expressed in these subsets and confirmed that lincRNAs cellspecificity is higher than protein-coding genes even when comparing lincRNAs genes with membrane receptor protein-coding genes, which are generally referred as the most accurate markers for lymphocyte subsets definition. Besides, a major outcome of this analysis is the identification through *de novo* transcriptome reconstruction of 563 novel, previously unannotated long intergenic ncRNAs genes, increasing by ~12% the number of lincRNAs known to be expressed in human lymphocytes (63). Intriguingly, a fraction of lincRNAs specific for B cells and a fraction of "pan-T" lincRNAs also exist (63). It would be extremely interesting to study these lincRNAs during lymphocytes development in order to understand their likely peculiar role in thymic or bone-marrow-derived cells development.

These observations imply that the little overlap between available catalogs is a direct consequence of lncRNAs specificity and that we could overcome this limitation only assessing lncRNAs expression in every different, highly purified cell type at different developmental stages, instead of considering tissues as a whole. Moreover, due to their specificity of expression, human lymphocytes lincRNAs that are not yet annotated in public resources would have not been identified without performing *de novo* transcriptome reconstruction. As mentioned before, such tissuespecificity has been linked to the enrichment of TEs in proximity to lincRNAs TSS (64, 65). Moreover, RNA-seq experiments performed in a human CD4+ naïve T cells *in vitro* differentiation time-course suggest that lincRNA-specific expression in human lymphocyte subsets is acquired during their activation-driven differentiation from naïve to memory cells (63).

These findings hint to the involvement of lymphocyte-specific lincRNAs as fine-tuners in cell fate commitment, differentiation, and maintenance of cell identity, as demonstrated by many examples in other cell types (66–68). Also, lincRNAs are functionally involved in cell growth (69, 70), apoptosis (71–74), development (75–77), imprinting (78–80), and dosage compensation (81) in almost every cellular context (**Table 2**).

Long non-coding regulatory RNAs exert their function in these fundamental processes interacting with chromatin or DNA modifiers and transcription factors (TFs) modulating gene expression (82); competing with micro-RNAs acting as sponges (83); modulating subcellular trafficking (84), translation (85), splicing (86), and likely through many other mechanisms still to be discovered (**Figure 1**).

Long non-coding regulatory RNAs functional flexibility derive from their intrinsic propensity to fold into thermodynamically stable secondary and higher orders structures that function as interaction modules (87). Each module can fold independently from another, forming bonds at the level of Watson–Crick, Hoogstein, and ribose face (88, 89). These RNAs can rapidly shift between diverse stable structural conformation, allowing allosteric transitions that can act as switches in response to environmental stimuli. They are also processed faster than mRNA, given that they must not be translated, allowing a rapid response to signals. LncRNAs can also be regulated via more than a hundred different nucleotide modifications, like in the case of tRNAs, rRNAs, and snoRNAs (90–92) that modulate their function and probably their structure. RNAs can generate multiple modules within their structure, allowing the interaction with multiple players, the reception of multiple stimuli, and the generation of multiple outputs. The required pairing is likely extremely flexible, such as in the case of micro-RNAs, and allows mismatches, bulges, and wobblings (93). Many of these interaction modules derive from repetitive elements, such as transposons that took advantage of the fewer constraints that lncRNAs sequences have compared to protein-coding genes (1, 94). Indeed, lncRNAs rate of sequence evolution is higher relative to protein-coding genes, even if also these transcripts exhibit evolutionary signatures of functionality. They evolve under modest but detectable selective pressure, accumulating fewer substitutions than neutrally evolving sequences (95, 96). Likely, conservation of relatively small units of lncRNAs sequences (estimated to be less than 5%) could be sufficient to preserve their function, considering their already mentioned modular structure (97). This

could be the reason why existing bioinformatic approaches fail to detect low level and scattered selective constraint within these loci (97).

Through such a plastic and versatile structure, lncRNAs can exert their functions binding to proteins, other RNAs (98), and probably also DNA, even if there is still little evidence on the existence of RNA:DNA triplex (99, 100). In particular, lncRNAs can act as scaffolds, bridging together different molecules in a coordinated hub, like in the case of NEAT1: a highly abundant lncRNA that controls sequestration of proteins involved in the formation of paraspeckles, nuclear domains associated with mRNA retention and pathologically enriched in influenza and herpes viruses infections (101, 102). LncRNA can also act as guides, recruiting proteins at specific loci: this has been hypothesized in the case of recombination events that mediate genetic diversity in developing lymphocytes as class switch (CS) and V(D)J recombinations that seem to be mediated by sense and antisense transcripts that dictates the locations of combinatorial events (103–105). Again, lncRNAs can act as control devices or riboswitches in response to extracellular stimuli. For example, they can act as decoys, precluding pre-existing interactions such as GAS5 RNA that detach glucocorticoid receptor (GR) from its responsive elements in conditions of growth arrest (106, 107). Nonetheless, the regulatory potential of lncRNAs has been better characterized in the context of the epigenetic regulation of transcription that ultimately defines the cell transcriptome.

#### **THE ROLE OF LONG NON-CODING RNAs IN EPIGENETICS**

Histones and DNA modifications together with the tridimensional chromosomes conformation within the nucleus define, at least in part, the epigenetic landscape of the cell. This extremely dynamic context modulates gene expression and dictates the final transcriptional output in response to environmental stimuli. By definition, these modifications are then propagated throughout cell divisions. This process is important in every moment of cell life, but particularly during differentiation. Indeed, every cell within our body harbor the same genome, but every cell acquires a particular phenotype according to intrinsic and extrinsic cues that ultimately defines its epigenome and therefore its fate during differentiation. Epigenetics also defines to what extent this fate can be irreversible or plastic (108–110).

As mentioned before, human lymphocytes are an interesting model system for understanding the basis of cell fate specification and plasticity. Indeed, although traditionally the broad range of effector lymphocytes has been referred to as composed by distinct lineages, it has become increasingly clear that these cells also have notable features of plasticity. Differentiation of naïve cells into specific helper subsets requires the integration of extrinsic cues that converge into cell-intrinsic changes in the epigenetic landscape on the genome (111, 112). The interest within the field has been focused on the regulation of prototypical cytokine genes for each subset such as *Ifng* gene for TH1 or *Il-4* for TH2 CD4+ lymphocytes. Much work has been done in both cases to define the complex genetic structure of these loci and the *cis* regulatory elements bound by TFs and chromatin modifiers promoting or repressing their transcription (113–115). The importance of the setting of epigenetic memory at these fundamental loci was underlined also by treatment with DNA methylation inhibitors (116, 117) or histones deacetylases inhibitors (118–120) and by deletion of DNA methyltransferase (121–123), which caused respectively: constitutive production of IFN-γ, enhanced production of both TH1 and TH2 prototypic cytokines, and inability to activate the proper pattern of expressed cytokines. The same is true for deletion of components of trithorax group (TrxG) or polycomb repressive complex (PRC) that dictates active or repressive epigenetic marks at fundamental loci for proper T-helper cell differentiation, such as *Il-4*,*Il-5*,*Il-13*, and *Gata3* (124–129). The pattern of chromatin marks is conventional for signature cytokines: active marks are present at prototypical cytokines whereas repressive marks restrain the expression of antagonistic molecules. However, master regulators and other TFs, usually considered as definers of lineage-specific identity, are characterized by bivalent poised domains, in which both active and repressive chromatin marks are present (130, 131). This histone epigenetic status is peculiar also to promoters in embryonic stem cells, where it poises the expression of key developmental genes thus allowing their timely activation in the presence of differentiation signals and concomitantly precluding expression in their absence (132). Indeed, while the expression of master TFs is quite rapid, cell divisions are required for cytokine loci to become accessible or conversely repressed. Indeed, GATA3 and T-bet/STAT proteins initiate the epigenetic changes at IFN-γ and IL-4 loci that follow the initial activation of naïve T cells and differentiation toward TH1 and TH2 cell fate (133, 134). These observations imply that T-helper cells harbor both clear-cut and plastic epigenetic marks. Nonetheless, we must consider that even cytokines genes that are clearly defined epigenetically, can be expressed or repressed in unexpected context, as reported in TH1 cells converted in IL-4-producing cells during strong TH2-polarizing helminth infections (135) or stable TH1/TH2 hybrid cells derived after parasite infections (136). Therefore, other players must be involved to define the degree of plasticity of lymphocytes in response to these ever-changing environmental conditions during differentiation.

Long non-coding regulatory RNAs have been linked to epigenetic control of gene expression since the first studies regarding the already mentioned Xist transcript, involved in X chromosome inactivation in eutherians. Many other lncRNAs have been associated to chromatin or DNA modifiers and even TFs, thanks to specific mechanistic studies or high-throughput screenings (82, 137–139). This interplay can be observed across a broad range of eukaryotic organisms, suggesting that the epigenetic role of lncR-NAs is conserved, even if their mere sequence conservation is often limited (as described previously). It seems that lncRNAs could act as scaffolds, physically associating with proteins that modify chromatin either activating or repressing gene expression. Thanks to the already discussed structural properties of RNA, lncRNAs could organize multiple players in spatially and temporally concerted actions (138). Not only: thanks to their ability to base pair with other nucleic acids, they could recruit these modifiers at specific loci, therefore conferring them specificity of action (98). This property has been an unsolved issue, given that chromatin modifiers do not possess intrinsic bias toward consensus sequences, at least in mammals, while in *Drosophila* these "docking sites" are well defined (140, 141). Interestingly, while many of these enzymes lack DNA-binding properties, they instead possess RNA-binding motifs (142–144).

The majority of reported lncRNAs are involved in the repression of gene transcription, in particular by interacting with polycomb group (PcG) proteins. The first examples of a direct interaction with PRC2 are the already mentioned Xist (145) and Kcnq1ot1, expressed only in the mammalian paternal chromosome and involved in the silencing of 8–10 protein-coding genes (146). In both these cases, lncRNAs are strictly required for the enrichment of PRC2-associated proteins and for the trimethylation of the lysine 27 of histone H3 at specific loci. Furthermore, other lncRNAs such as NEAT2 and TUG1 promote relocation of growth-control genes at foci of PcG proteins (called PcG bodies), therefore likely facilitating the concerted repression/activation of the transcription units in response to mitogenic signal (147). Many other protein complexes have been found to interact with lncRNAs, the majority targeting histones, either methylases or demethylases, but other involved in DNA methylation (148). Indeed, lncRNAs can bind proteins part of the TrxG (68) that antagonize PcG-mediated silencing (149). Interestingly, an antisense lncRNA has been recently involved in recruiting a regulator of DNA demethylation at a specific promoter (150). This process remains still largely unknown and it has only recently been associated to active enzymatic reactions, via TET family of methylcytosine dioxygenases (151, 152). Even in this case, one of the unsolved questions has been how locus-specificity can be achieved. Particularly, DNA demethylation is often restricted to few dinucleotides at the TSS. The precise mechanism, though, through which lncRNAs could direct DNA or chromatin modification has never been described. Indeed in all reported examples, correlations have been described between lncRNA-modifiers associations and loss of modification after lncRNA gene silencing.

Long non-coding regulatory RNAs are supposed to confer binding specificity to modifiers and recruiting them either *in cis* or *in trans*. In the first case, lncRNAs could act directly on sites where they are synthesized without needing to leave DNA. The current hypothesis suggests that the 50 region of the nascent transcript could bind proteins while the 30 is transcriptionally lagging, being still tethered to chromatin by RNA polymerase (153). This model is particularly intriguing as through this mechanism, lncR-NAs could exert an allele-specific effect, like in the well-studied case of Xist. *In trans* regulation is instead achieved when lncRNAs act modulating genes across great distances or even on different chromosomes (154). Regarding this dichotomy, we must underline once again its artificiality. Indeed, chromosomes fold into complex, three-dimensional territories together with specialized subnuclear bodies. Proteins that are part of the transcriptional or splicing machinery and regulators of these processes group at these foci (155, 156). These structures are not static, but on the contrary, large-scale chromosomal repositioning is observed in response to environmental stimuli or during differentiation (157, 158). Subnuclear movements are of key importance in regulating events like transcription and rearrangement that occur at immunoglobulin loci during B lymphocytes development (159). The dynamic folding of the genome into higher order structure encompasses loci belonging to the same chromosome, even hundreds of kilobases apart, or different ones, bringing together regions that are distant if we consider the genome as linear. Therefore, in this context, it is extremely difficult to discern what regulations are *in*

*cis* or *in trans*, especially when they involve long distance interactions. Intriguingly, lincRNAs have been found to regulate the formation of subnuclear structures, such as NEAT1, required for paraspeckles nucleation (101). LncRNAs can also affect directly the three-dimensional organization of chromosomes enhancing the function of proteins involved in looping formation, like the insulator protein CTCF (160). There are also many examples of lncRNAs involved in three-dimensional local chromatin looping that brings together the ncRNA gene with the region that it regulates within the same chromosome (68, 161). Recently, a lincRNA called Firre has been shown to recruit specific gene loci located on different chromosomes, acting as a docking station for organizing *trans*-chromosomal associations. Consistently, genetic deletion of Firre leads to a loss of proximity of several *trans*-interactions (162). A peculiar type of lncRNA has been described that is transcribed from enhancer regions (eRNAs). Classic enhancer elements therefore likely act through transcription of these lncRNAs that upregulate expression at promoters via the recruitment of Mediator complex (163, 164). Finally, there is increasing evidence that even promoters could be transcribed (165), producing lncR-NAs probably involved in the enhancer–promoter loop that was hypothesized years ago but never fully resolved (166).

#### **LONG NON-CODING RNAs IN THE ADAPTIVE IMMUNE SYSTEM**

The adaptive immune system is an extraordinary context for the study of the role of lincRNAs in differentiation. Indeed, upon antigen stimulation, naïve CD4+ T cells differentiate into distinct T-helper subsets that were traditionally considered lineages and defined by a prototypic set of expressed cytokines and master TFs. Recently, this relative simple scenario, although useful, has been subjected to debate. CD4+ T cells demonstrated to exhibit substantial plasticity and it has become increasingly clear that they can change the pattern of cytokines and TFs according to the milieu they encounter through their life (167, 168). Not only, in some cases, they can concomitantly express other cytokines and TFs together with their prototypical set. Best examples include IL-10, once thought to specifically identify TH2 and now known to be produced also by TH1, Treg, and TH17 cells (169) and IFN-γ, the classic TH1 cytokine, frequently released by TH17 cells simultaneously with IL-17 (170, 171). Regarding master TFs, Tregs can express *Foxp3* (their prototypical TF), but also *ROR*γ*t* (TH17 TF) and *Runx3* (172–174); similarly TFH cells can differentiate from FOXP3 positive cells also expressing *Bcl6* (their specific TF) (175, 176). In this context, lncRNAs have a fundamental role in governing flexibility and plasticity or maintenance of cell identity, together with lineage-specific TFs and other ncRNAs. In particular, what is emerging from the literature is that ncRNAs typically act as fine-tuners of fate choices and this seems to be true not only in the immune system. Nonetheless, in the case of CD4+ T-cell subsets that are specified but not fully determined, subtle changes in extrinsic signals can reverberate through responsive ncRNAs inducing changes that alter cell phenotype (38, 177, 178). Usually, the stability of lineage identity is achieved through the implementation and inheritance of epigenetic modification, but as mentioned before, lncRNAs can act directly on histone and DNA modifiers redefining this context.

Conversely, lncRNAs can also buffer this situation in other conditions, acting as maintainers of cell identity. In the cellular system, lncRNAs can be regarded as minor nodes in a huge interconnected network (179), as they usually interact with few other players. This condition allows them to be more flexible and sensitive to variations without disrupting the whole network integrity (180). This is true both over a very short period, as cells can easily and rapidly adapt to environment, and also over long evolutionary periods, as lncRNAs are among the fastest evolving sequences in the genome (95, 181–183). Conversely, master transcription regulators can be considered highly connected hubs, which confer robustness to the network. Indeed, very few protein-coding genes have been lost from worms to human and mutations are most often pathological (184, 185).

Several single-case or genome-wide studies on lncRNAs in the murine adaptive immune system or cell lines are now available in the literature, whereas only few studies have been conducted until now in the human context. The number of studies that unveiled the function and mechanism of a specific lncRNA is so small that can be counted on one hand (**Table 3**).

The importance of the studies in the human immune system is underlined by the fact that the differences between experimental animal models and human are still subject of debate in terms of immunologic responses (199–201). Moreover, there are increasing evidences that ncRNAs are poorly conserved between animal models and human (202, 203). In particular, lncRNAs are really fastevolving elements as demonstrated by the fact that over 80% of the human lncRNAs that arose in the primate lineage, only 3% are

#### **Table 3 | Studies on lncRNAs in the adaptive immune system**.

CM, B cells

Treg cells; neutrophils, basophils, CD8+

conserved across tetrapods and most mammalian lncRNAs lack known orthologs outside vertebrates (97). In detail, even between mouse and human, lncRNAs are poorly conserved (204–206). Despite their rapid evolution, lncRNAs are selected more than neutral sequences and in particular more than intergenic regions, but significantly less than mRNAs (96, 97, 207). It must be underlined that the conservation rate reported could be overestimated: substitution rates are derived from whole-genome alignment and based on the assumption that even segment of homologies imply that that segment belongs to the same RNA class, but this is not necessarily the case. Indeed, it could be that in another genome context a specific lncRNA gene segment is transcribed and processed as part of a protein-coding RNA (208). A striking example is Hotair that is involved in the regulation of the highly conserved cluster of *Hox* genes (68). The human lincRNA is conserved in the mouse genome (209), nonetheless only the 30 region is effectively part of the murine homolog (183). The importance of studying lincRNAs specifically within the human immune system derives from these considerations, but this field is still poorly investigated. The majority of the studies focused on the innate immune system (210–212) or analyzed pathological situations, such as cancerrelated lncRNAs (192, 213) or responses to specific infections (102, 214–216), mostly in mice. The first functional study that focused on the adaptive immune system, and in particular on TH1 and TH2 lymphocytes, involved a lincRNA, Tmevpg1, that is selectively expressed in TH1 cells via STAT4 and T-bet, both in mouse and human. It participates in the induction of IFN-γ expression strictly in response to TH1 differentiation program and not in other cellular contexts. These results highlight once again the complexity of gene expression regulatory network and the specificity of action of lincRNAs (196). Another paper described a lincRNA, GATA3-AS1, specifically expressed in primary TH2 cells and hypothesized its co-regulation with GATA3 (198). GAS5, expressed in human T lymphocytes, is degraded in optimal growth conditions, but it accumulates contributing to growth arrest in starving conditions (107). In this situation, it competes with GRs DNA-binding sequences, suppressing GR-mediated transcription (106). Broader studies have been performed on the CD8+ T cell transcriptome (37), and recently on CD4+ T lymphocytes (189), but still on mice models. In B cells, chromatin remodeling associated with V(D)J recombination has been potentially linked to a widespread antisense intergenic transcription that occurs in the variable (V) region of the immunoglobulin heavy chain (Igh) locus (104, 105). So far, no studies have been published that performed a deep transcriptomic analysis on human primary lymphocytes from healthy donors, identifying lncRNAs fundamental for differentiation processes. These few examples are just clues of the importance that lincRNA could have for the proper function also of the human immune system and prompt to a deeper analysis of their role in this particularly intriguing context.

#### **LONG NON-CODING RNAs AS EPIGENETIC MODULATORS IN LYMPHOCYTE DIFFERENTIATION**

Traditionally, the secretion of IFN-γ and TNF-α characterizes TH1 lymphocytes, whereas IL-4, IL-5, and IL-13 are considered prototypic cytokines secreted by TH2 cells. According to this classic paradigm, these differences underline the different functions exerted by these lymphocytes: TH1 are considered as important to eliminate intracellular bacteria and viruses, whereas TH2 to resist parasitic infections (217). The advantage of solid *in vitro* differentiation protocols allowed a deep understanding of the genetic mechanisms governing these cells. Since the discovery of this dichotomy, other cell subsets have been identified, but this TH1/TH2 paradigm was undoubtedly useful. Therefore, it is not a case that among the few lncRNAs identified in the immune system, many of those functionally characterized have been described in these two cell subsets. Nevertheless as mentioned before, just one lincRNA, Tmevpg1 (also known as NeST or IFNG-AS1) has been characterized in deep. Tmevpg1 is located proximal to IFNγ gene both in mice and humans, antisense and convergently transcribed respect to the neighboring gene and plays a role in chromatin remodeling. This transcript is a TH1-specific lincRNA: it requires STAT4 and T-bet for being transcribed and is also bound by CTCF and cohesin during lineage-specific induction (196). Therefore, Tmevpg1 is directly dependent on the activation of a TH1-polarizing transcriptional program, in which the presence of IL-12 leads to the activation of the JAK/STAT pathway via STAT4 (and STAT1) that induces the expression of T-bet. Interestingly, *Tmevpg1* gene harbor sequences regulated by histone acetylation and DNase I hypersensitive sites found in TH1 but not TH2 cells (218, 219). Tmevpg1, in its turn, plays a direct part in defining the proper TH1 cytokine expression pattern, influencing *Ifng* transcription in the presence of T-bet (196), via H3K4 trimethylation by WDR5 binding in mice models (197).

Given the increasing number of lncRNAs described in different cellular contexts and the high number of specific lincRNAs expressed in the different lymphocytes subsets identified with the aforementioned RNA-seq analysis, many more lincRNAs will likely be characterized in the future with a relevant function in the human immune system. A major limitation, though, in the studies on lncRNAs is that there is little biological knowledge on the biochemical or molecular function of lncRNA genes. Compared to classical protein-coding gene studies, hints on their functions cannot be gained simply by the analysis of their primary sequence and application of computational methods to infer lncRNA function are also still in their infancy. As lincRNAs have been reported to influence the expression of neighboring genes (25, 26, 28, 39), one possible approach to investigate their putative function is to focus on lymphocyte lincRNAs proximal to protein-coding genes involved in key cell-functions.

Through this approach, we identified a TH1-specific lincRNA that localized ~140 kb upstream to *MAF* that was therefore called linc-MAF-4. MAF is a TF involved in TH2 differentiation and required for the efficient secretion of IL-4 by TH17 and the proper development of TFH cells (220–222). Intriguingly, the expression of *linc-MAF-4* is negatively correlated with respect to the expression of *MAF*: linc-MAF-4 expression is high and specific in TH1 lymphocytes, where *MAF* is lowly expressed whereas in TH2 cells the expression of *linc-MAF-4* is extremely low and *MAF* is highly expressed. Coherently, linc-MAF-4 knock-down in naïve CD4+ T cells increased the expression of *MAF* and interestingly induced a more general skewing of the whole transcriptomic profile of these cells toward a TH2-like fate (63). The regulation exerted by linc-MAF-4 on *MAF* gene was analyzed in more detail and this lincRNA

proved to modulate *MAF* expression *in cis*, as hypothesized by expression analysis. Linc-MAF-4 exerts this regulation by exploiting a chromatin loop that brings its genomic region close to the promoter of *MAF* gene. Indeed, the chromatin organization of this region allows linc-MAF-4 transcript to recruit chromatin remodelers that inhibit *MAF* transcription. In particular, linc-MAF-4 was found to associate with EZH2, key enzymatic subunit of the PRC2 complex, and LSD1. These proteins methylate H3K27 and demethylate H3K4, respectively: two histones modifications that code for transcriptional repression. (63). A similar mechanism was described for other lincRNAs, such as HOTAIR and MEG3 (154, 223) but never before for other lncRNAs expressed in the adaptive immune system (**Figure 2**).

Changes of lincRNAs expression during naïve to memory CD8+ T-cell differentiation (37) and during naïve CD4+ T cells differentiation into distinct helper T-cell lineages (189) have been described in the mouse immune system. linc-MAF-4, though, is, to our knowledge, the first example of a lincRNA playing a role in the proper differentiation of human TH1 cells, suggesting that, besides cytokines and TFs, lncRNAs take part in the TH1 differentiation program as already shown in many other cell types. At this point, an obvious question arises: to what extent are these cells plastic? These findings are evidences that it is possible to redirect the differentiation path of naïve CD4+ T cells acting on their lincRNA content. Nevertheless, could it be possible to modulate already committed cells? We would expect that the mere downregulation of a lincRNA would not be sufficient nor a lincRNA knock-out: as mentioned before, lincRNAs are minor nodes in a huge interconnected network composed by feedback mechanisms and epigenetic marks that act stabilizing a pre-existent differentiation status. However, a modulation in lincRNA content may

be sufficient to make these cells more responsive to environmental cues that could overcome stabilizing forces, inducing a sort of trans-differentiation event. Functional characterization of other lncRNAs is required to address this crucial issue and to assess the extent of their contribution to cell differentiation and to the maintenance of cell identity in human lymphocytes. Based on what we discussed so far on lncRNA functions and cell-specificity, we believe that future studies will show how these molecules could be capitalized as new molecular targets for the development of novel and highly specific therapies for diseases, such as autoimmunity, immunodeficiencies, allergy, and cancer.

#### **ACKNOWLEDGMENTS**

We thank V. Ranzani for support in figures preparation and for discussions and critical revision of the manuscript. Supported by Consiglio Nazionale delle Ricerche – Ministero dell'Istruzione dell'Universita e della Ricerca (EPIGEN), Fondazione Cariplo (2013-0955), AIRC Associazione Italiana per la Ricerca sul Cancro (IG2013-ID14596), the European Research Council (269022 to SA; 617978 to MP), and Fondazione Romeo ed Enrica Invernizzi.

#### **REFERENCES**

the 3D structure of the Igh locus in pro-B cells. *Proc Natl Acad Sci U S A* (2012) **109**(42):17004–9. doi:10.1073/pnas.1208398109

tumor suppressor TCF21 via GADD45A. *Mol Cell* (2014) **55**(4):604–14. doi:10.1016/j.molcel.2014.06.031

**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: 22 January 2015; accepted: 28 March 2015; published online: 15 April 2015. Citation: Panzeri I, Rossetti G, Abrignani S and Pagani M (2015) Long intergenic non-coding RNAs: novel drivers of human lymphocyte differentiation. Front. Immunol. 6:175. doi: 10.3389/fimmu.2015.00175*

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

*Copyright © 2015 Panzeri, Rossetti, Abrignani and Pagani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

REVIEW ARTICLE published: 07 October 2014 doi: 10.3389/fimmu.2014.00488

## Cellular plasticity of CD4 T cells in the intestine +

#### **Verena Brucklacher-Waldert † , Edward J. Carr † , Michelle A. Linterman and Marc Veldhoen\***

Laboratory for Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK

#### **Edited by:**

Dragana Jankovic, National Institutes of Health, USA

#### **Reviewed by:**

John J. Miles, Queensland Institute of Medical Research, Australia Sid P. Kerkar, National Institutes of Health, USA

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

Marc Veldhoen, Laboratory for Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK e-mail: marc.veldhoen@ babraham.ac.uk

†Verena Brucklacher-Waldert and Edward J. Carr have contributed equally to this work.

Barrier sites such as the gastrointestinal tract are in constant contact with the environment, which contains both beneficial and harmful components. The immune system at the epithelia must make the distinction between these components to balance tolerance, protection, and immunopathology. This is achieved via multifaceted immune recognition, highly organized lymphoid structures, and the interaction of many types of immune cells. The adaptive immune response in the gut is orchestrated by CD4+ helperT (Th) cells, which are integral to gut immunity. In recent years, it has become apparent that the functional identity of these Th cells is not as fixed as initially thought. Plasticity in differentiated T cell subsets has now been firmly established, in both health and disease.The gut, in particular, utilizes CD4+ T cell plasticity to mold CD4+ T cell phenotypes to maintain its finely poised balance of tolerance and inflammation and to encourage biodiversity within the enteric microbiome. In this review, we will discuss intestinal helperT cell plasticity and our current understanding of its mechanisms, including our growing knowledge of an evolutionarily ancient symbiosis between microbiota and malleable CD4+ T cell effectors.

**Keywords:T cells, plasticity, intestines,Th1 cells,Th17 cell**

#### **INTRODUCTION**

The adult human gastrointestinal tract is the largest surface area of the body that contacts with the environment, covering 200– 300 m2 (1). This intestinal surface is constantly exposed to a diverse range of foreign antigens originating from microorganisms (both commensals and pathogens) and food antigens from the diet (2). Commensal microorganisms play an essential role in extracting nutrients from food that are otherwise inaccessible to the host [such as the metabolism of vitamin K by *E. coli* (3)], they are required for the development of the host's immune system and for the prevention of colonization of the gastrointestinal tract by pathogens. Mucosal pathogens, including viruses, fungi, parasites, and bacteria, can cause pathology either by local effects after mucosal colonization – such as inducing local inflammation or secreting toxins – or, through systemic infection after breeching mucosa. Microorganism-derived antigens, food-derived antigens, and airborne particles, can be potential immunogens. An inappropriate response to these immunogens at the mucosal surface can be detrimental, leading to local or systemic pathology that result in acute or chronic inflammation. Therefore, it is essential that the myriad of antigens present at the intestinal surface is dealt with appropriately to minimize potential danger and maximize host benefit. This protection is achieved by a flexible, multilayered system of physical, and immunological barriers within the gastrointestinal tract.

A central part of the complex host defense system is gutassociated lymphoid tissue (GALT). GALT is a system of highly organized immune structures strategically placed along the entire gastrointestinal tract, containing specialized micro-environments where gut-derived antigens are presented by professional antigen presenting cells (APCs) to lymphocytes [reviewed recently in Ref. (4)]. The broad antigenic sampling within the GALT facilitates the interaction between rare antigen-specific B and T cells leading to the initiation of an appropriate adaptive immune response (5). CD4+ T cells are critical players in the adaptive immune response within the GALT. Naïve T cells egress from the thymus as immature T cells with a broad range of T cell receptors (TCRs) and can be activated in the periphery following encounter with their specific antigen. T cell activation is initiated by ligation of the TCR by peptide–MHC class II complex in conjunction with co-stimulatory signals. During T cell priming, cytokine receptor ligation can skew activated T cells into a particular effector cell type (6). These cell types are commonly referred to lineages or subsets, with each being identified by selected expression of characteristic transcription factors and effector molecules (7). With the exception of thymically derived regulatory T cells (Tregs) [reviewed in Ref. (8)], thymic emigrants lack any predisposition to make effector molecules associated with a particular CD4+ T cell subset, and require signals in the periphery to skew their differentiation into a particular cell subset. Present understanding of T cell lineage commitment is dominated by single fate model, a process whereby a naive T cell differentiates along a terminal fixed expression program, in response to signals at the time of antigen encounter. However, this view has needed revision in light of findings from many groups, which together demonstrate that CD4+ T cell subset fate is not a permanent attribute, but rather a flexible, plastic, feature that can be modified to suit the requirements of the immune system at a particular point in space and time. Thus, the new paradigm of T cell differentiation encompasses the ability of CD4+ T cells to change between expression programs traditionally thought to be mutually exclusive terminal states of differentiation (9). This feature has been reported *in vivo* in several experimental systems, and intriguingly, is a prominent feature of CD4+ T cell biology within the GALT.

The detailed mechanisms underlying T cell plasticity within the GALT remains to be defined, but several factors that facilitate its occurrence have been proposed and can be divided into extrinsic and intrinsic pathways. In this review, we will summarize the recent literature on CD4+ T cell plasticity in the gut, highlight possible underlying mechanisms and discuss its potential benefits for intestinal homeostasis and health.

#### **CD4**+ **T CELL DIFFERENTIATION IN THE GALT**

The GALT contains one of the largest lymphoid cell population found anywhere in the body. GALT is distributed along the intestinal tract and is separated from the luminal content, containing about 100 trillion microorganisms (10) and many dietary products,by a single epithelial layer covered with an intricate network of glycoproteins; the mucous layers. The GALT provides three functions: provides antigenic samples from throughout the GI tract; optimizes the opportunities for naïve lymphocytes to encounter antigen, and finally supports the activated lymphocyte and its initial differentiation. A network of highly organized lymphoid structures comprise the GALT (**Figure 1**) – including mesenteric lymph nodes (mLNs), Peyer's patches (PPs), isolated lymphoid follicles (ILFs), cryptopatches (CPs), and fat-associated lymphoid tissues – as well as the loose connective tissue of the lamina propria (LP). Despite the numerous types of GALT, the organization of all GALT lymphoid tissues shares a basic cellular architecture that facilitates the interaction of APCs with lymphocytes and their subsequent activation and differentiation. However, the GALT tissues

differ from each other both in function and in the physical distance to the intestinal lumen; CP and ILFs are located directly underneath the epithelial layer, PPs are further from the lumen and most distant are mLNs and fat-associated lymphoid tissues. The specialized functions of each the GALT tissues have been recently reviewed in Ref. (4), and therefore will not be discussed in detail in this review.

Naïve L-selectin-expressing CD4+ T cells migrate from the blood through high endothelial venules (HEVs) to the PPs, CP, ILFs, and mLNs. In these inductive sites of the adaptive immune system, they encounter their cognate antigen presented by APCs on MHC class II. Naïve T cells that did not encounter their cognate antigen leave via efferent lymphatic vessels into the systemic circulation to continue their search for their cognate antigen. In PPs, CP, and ILFs, dendritic cells (DCs) receive antigens transported from the mucosal surface by specialized epithelial cells overlying these lymphoid structures, the microfold (M) cells, and Goblet cells [reviewed in Ref. (4)]. mLNs receive antigens via afferent lymphatics that are connected with the PPs, while fat-associated lymphoid tissues obtain antigen by lymphatic drainage directly from the intestine and from the peritoneal cavity (4).

After activation in the intestine CD4+ T cells proliferate, shed L-selectin from their surface, increase surface expression of gut-specific adhesion molecules and differentiate into an effector T cell, of a particular subset. Antigen-experienced effector T cells usually leave via efferent lymphatic vessels and home to effector tissues such as the intestine after re-circulation in the blood, but

some remain in the lymphoid organs to perform their specialized effector functions (11). Upon arriving in the intestine, effector T cells are likely to re-encounter their specific antigen presented by a diverse range of cells such as macrophages, B cells various types of DCs, and stromal and epithelial cells. This second antigenspecific interaction will trigger the T cells to execute their effector function.

It is the nature of antigen presentation and the surrounding micro-environment that largely seems to determine the outcome of CD4+ T cell differentiation and the subsequent spectrum of T cell identities leaving the lymphoid structures (6). The CD4+ T cell subsets currently recognized are: T helper cells of type-1 (Th1), Th2, Th9, Th17, Th22, Tregs, and follicular helper T cells (Tfh). Each subset is characterized by the potential to produce a defined set of mediators such as cytokines and chemokines, respond to particular stimuli via the expression of selected cytokine and chemokine receptors, largely driven by so called"master regulator" transcription factors (summarized in **Table 1**).

#### **CD4**+ **T CELL PLASTICITY**

Plasticity of effector CD4+ T cells has been increasingly recognized in recent years. Initially, there were single reports of observations of particular subsets behaving "non-typically," by the expression of transcription factors, cytokines, or both, of another subset. Together these various reports support a more nuanced view of Tfh differentiation, in place of the monolithic view of distinct, noninterchangeable lineages (12). T helper cells are able to acquire a mixed phenotype or to switch entirely to the transcription and cytokine profile of another lineage (**Table 2**). These hybrid cells or ex-Th lineage cells may either directly differentiate from naïve CD4+ T cells or arise during secondary immunological challenge. Current data suggest that, within the gut, particular transitions between CD4+ T helper cell subsets occur and are important for maintaining gastrointestinal homeostasis, as outlined below.

**Th1/Th2 CELL PLASTICITY**

bacteria and viruses (31). In contrast, Th2 cells are absent in the intestine of laboratory mice and humans under healthy conditions (31, 32); not surprising in view of their role in coordinating host responses to helminths and the absence of these organisms in a healthy intestinal flora (31). Identification of Th1 and Th2 as distinct cell types was the first, time where CD4+ T cell subsets were divided on the basis of phenotypic differences *in vitro* (33). It is therefore interesting that culture of T cells under mixed Th1 and Th2 conditions results in a continuum of cytokine expression, with some single positive IFNγ + or IL-4+ and double positive cells, correlating with expression levels of Tbet and GATA-3 (34), the respective master transcription factors for the Th1 and Th2 subsets, demonstrating these cell fates are not a distinct as originally thought. Importantly, this finding is consistent with *in vivo* observations; during infections with *Heligmosomoides polygyrus*, a parasite that triggers a strong Th2 response, Th1/Th2 hybrid cells were observed (13). In the intestine, mLNs and spleen, these Th1/Th2 hybrid cells express simultaneously the Tbet and GATA-3 and not transcription factors of other subsets [retinoic acid (RA) receptor-related orphan nuclear receptor (ROR)γt or Bcl-6]. In addition, they express the Th1 cell-associated molecules CXCR3 and IFNγ concordantly with the Th2 cell-associated IL-33R, IL-4, IL-13, and IL-5. Furthermore, Th2 cells *in vivo* can express Tbet and IFNγ upon infection with lymphocytic choriomeningitis virus (LCMV), a potent Th1 skewing pathogen. These Th1/Th2 hybrid cells represent a stable phenotype, where attempts at reprograming by stimulation under Th1 or Th2 polarizing conditions result in quantitative changes of Th1/Th2 cytokine production without fully extinguishing either. Upon transfer into wild-type mice, around 20% of LCMV-specific Th1/Th2 cells maintained their expression of GATA-3, IL-4, and IL-13 alongside Tbet and IFNγ

T helper cells of type-1 cells are an abundant effector population in the gut responsible for protection against invading intracellular

### **Table 1 | Effector CD4**+ **T cells in the gut**.

**CD4**+

expression many months after systemic infection with LCMV. Earlier work from the same group has shown that the development of LCMV-specific Th1/Th2 hybrid cells originating from antigenexperienced Th2 cells required TCR engagement, not just a "Th1 milieu" provided by LCMV challenge and are stable *in vivo* for months (35).

Functionally, the Th1/Th2 hybrid cells have the capacity to elicit Th1 and Th2 cell responses but with a decreased Th-specific potency compared their single identity counterpart. Mice that received *in vitro* derived Th1/Th2 cells before the induction of type-1 inflammation showed signs for delayed-type hypersensitivity but not as strong as after a transfer of pure Th1 cells. Similarly, the transfer of Th1/Th2 cells reduced the signs for induced allergic airway inflammation compared to a Th2 cell transfer (13).

#### **Th17/Th1 CELL PLASTICITY**

In health, Th17 cells preferentially home to small intestinal LP (26) and are important for intestinal homeostasis [reviewed in Ref. (36)]. Th17 cells are characterized by the expression of IL-17A, IL-17F,and IL-22 and the lineage-defining transcription factor RORγt (37–39), which acts in co-operation with other transcription factors, including aryl hydrocarbon receptor (AhR) and RORα (40, 41). Shortly after the first descriptions of Th17 cells, cells making both IL-17 and IFNγ, so called double producers, were noted. In the intestine of patients with Crohn's disease (CD), IL-17A IFNγdouble positive CD4+ T cells co-expressing RORγt and Tbet have been found (14), sometimes with frequencies above those of single positive T cells (15). Cosmi et al. characterized these double producers further and found that virtually all IL-17+IFNγ +CD4+ T cells, as well as Th17 cells expressed the lectin-like surface molecule CD161 (15). This led them to conclude that human Th17 and Th17/Th1 cells exclusively originate from an NKT-like CD161+CD4+ T cell precursor. Furthermore, Kleinschek et al. showed that CD161+CD4+ T cells preferentially home to the gut due to their high expression levels of CCR6 and integrin β7. They confirmed that these cells can be induced to produce IFNγ in addition to IL-17 (16). In mice, colitis studies revealed not only the presence of Th1/Th17 hybrid cells (17–19) but also that Th17 cells, initially unable to produce IFNγ, abolish IL-17 production completely and switch on IFNγ (ex-Th17Th1) (18, 20, 42).

Hirota et al. used an IL-17-fate reporter mouse that permanently marks IL-17-producing cells to show that these ex-Th17Th1 cells undergo a near complete switch from Th17 cell to Th1 cell, except for the maintenance of IL-1R and AhR expression as remnants of their former identity (26). Since Th1 cells are not known for the expression of the IL-1R, ex-Th17Th1 cells are unique within the Th1 lineage in the ability to respond to the acute phase reactant IL-1. AhR expression has been shown to increase Th17 cell activity and particularly drive the expression of IL-22 (40). It is currently not known what the role of AhR is in ex-Th17Th1 cells, however, it is tempting to speculate that there is a role in coordinating IL-22 production at barrier sites such as the intestine, where IL-22R is expressed by epithelial cells (43). IL-22 deficiency worsens the colitis induced by either dextran sodium sulfate or by T cell transfer (44), and results in reduced secretion of antimicrobial agents important for maintaining epithelial health (45, 46). Interestingly, AhR has been shown essential for the long-term maintenance of

lymphoid cells, such as innate lymphoid cells (ILC) type 3, intraepithelial lymphocytes (IELs), and tissue resident cells, at epithelial barrier sites (47–49). This suggests that ex-Th17Th1 cells may be maintained in the intestine providing long-term protection or upholding aberrant immunity, in part through IL-22 signaling, with the help of AhR ligands.

Because the frequency and number of IL-17/IFNγ double producers are increased in the gut of CD patients (14), they have been implicated in disease pathogenesis. However, it is unknown if the presence of Th17/Th1 hybrid cells in CD patients are a cause or a consequence of the disease. These hybrid cells could develop as a bystander product of inflammation, or might drive the initial inflammatory response. In colitis models, the pathogenicity of Th1/Th17 hybrid cells and ex-Th17 cells is linked with Tbet. However, the role for Tbet in the pathogenicity of Th17 cells remains controversial (50–53) and switching to Th17/Th1 or IFNγ +ex-Th17 might involve Runx family members in co-operation with Tbet (54).

#### **Th17/Th2 CELL PLASTICITY**

Naïve T cell activation *in vitro* in the presence of IL-4 inhibits RORγt expression and IL-17 production (55), which makes the existence of T cells with a hybrid phenotype of Th17/Th2 cells seem unlikely. However, *in vivo* studies identified CD4+ T cells simultaneously expressing IL-4 and IL-17 (22). These Th17/Th2 hybrid cells were reported to be present in draining LNs and mLNs of mice with experimental autoimmune encephalomyelitis (EAE) that either had a CD4-specific Bcl11b-deficency or were treated with IL-4. In EAE, Th17 cells are pathogenic when they gain access to the central nervous system (CNS), contributing to CNS inflammation. EAE-induced Bcl11b-deficient mice showed a delayed onset and reduced disease severity, attributed to a redirection of T helper cells away from a draining LN/CNS route to the intestine. Even though an accumulation of Th17 cells in the intestine has been reported to cause colitis these mice were asymptomatic.

#### **Th17-TO-Treg CELL PLASTICITY**

A different approach was chosen to address Th17 cell plasticity by Carbo et al. (23). Computational modeling predicated a role of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in a phenotype switch from Th17-to-Treg cell in the murine gut. *In silico*, PPARγ activation of fully differentiated Th17 cells reduced IL-17 and RORγt, while initiating Foxp3 expression. *In vitro*, analysis confirmed these *in silico* predictions. Polarized Th17 cells treated with pioglitazone, a synthetic PPARγ agonist, induced Foxp3 and inhibited RORγt and IL-17A expression. This effect was not observed in PPARγ-deficient Th17 cells. Mice that received pioglitazone orally to activate PPARγ recovered from transferred colitis, with a phenotypic switch in transferred T cells, from a predominantly Th17 phenotype to an iTreg cell phenotype characterized by decreased IL-17 and RORγt and increased expression of Foxp3.

In humans, IL-17+ FOXP3+ T cells were identified in inflamed intestinal mucosa of patients with CD, but not in patients with ulcerative colitis or healthy controls (24). These Treg/Th17 cells shared characteristics of Th17 and Treg cells, expressing *RORC* (encoding RORγt) and CD161 and showing Treg-typical suppressor activity *in vitro*. In addition, the majority these FOXP3+IL-17+ T cells expressed CCR6, a receptor that mediates homing to skin and mucosal tissues (56) and high levels of integrin α4β7 and CD103, markers for gut-homing potential. Analysis of the TCR repertoire suggested that FOXP3+IL-17+ cells develop from FOXP3+ Tregs when exposed to inflammatory signals in the gut (24) and *in vitro* stimulation of FOXP3+ circulating cells can result in IL-17 expression (57). In human tonsils obtained after routine tonsillectomy, up to 25% of the FOXP3+CD4+ T cell population can produce IL-17 upon activation (25). These FOXP3+IL-17 producers are CCR6+, co-express *RORC* and *FOXP3* and display cell-contact dependent suppressive properties (25). Whilst FOXP3+IL-17+ cells in the LP appear unique to areas of intestinal inflammation in CD (24), in tonsil they are much more readily identifiable (25). Together these observations suggest that heterogeneity in the GALT micro-environment can influence Th17– Treg plasticity. Intriguingly, the FOXP3+IL-17+ T cells from CD patients also express IFNγ, IL-22, and IL-21 (24), suggesting plasticity not just between Th17 and Treg, but with other CD4+ effector states.

#### **Th17/Tfh CELL PLASTICITY**

In addition to a Th17-to-Th1 conversion, Th17 cells can acquire a Tfh cell phenotype (26), including expression of Bcl-6, the transcription factor that is both necessary and sufficient for the formation of Tfh (58–60). Tfh provide antigen-specific help to germinal center (GC) B cells via CD40L, IL-4, and IL-21 (61). The only GALT to contain GCs are the PP and mLNs, and within these structures Tfh act to select high-affinity GC B cells to exit the PP GC as long-lived IgA secreting plasma cells and memory B cells (62). An IL-17-fate reporter mouse was used to show that transfer of Th17 cells into a T cell deficient recipient could support high-affinity IgA production in the GCs of PPs by converting into Tfh cells (26). Interestingly, this conversion resulted in the loss of IL-17 expression and other Th17 associated features to enable these cells to effectively switch into Tfh cells in the PP. The physiological relevance of these ex-Th17Tfh cells in host defense or in microbial homeostasis remains to be elucidated. But, the essential role for Th17 cells in the production of high-affinity IgA (26) suggests that this Th17 to Tfh cell switch is critical for IgA-dependent immune responses.

IgA production in the gut can occur outside the GC but the IgA species are low-affinity, with little somatic hypermutation (63). The production of GC-derived IgA is essential for the maintenance of bacterial communities in the gut. Mice that express a mutated form of activation-induced cytidine deaminase (AIDG23S) that allows class switch to IgA but not somatic hypermutation have alterations of the gut microbiota, suggesting that IgA derived from the PP GC response is essential for maintaining homeostasis of the gut microbiota (64). This demonstrates that high-affinity IgA promotes healthy microbiota, rather than target it for elimination like IgG derived from the LN and spleen (62). The production of immunoglobulin that nourishes commensal microbiota is one of the features that discriminates the function of GALT GCs from peripheral GCs, and may be one of the reasons that GALT GC T cells exhibit plasticity that is not observed in the GCs of lymph nodes and spleen. Selective IgA deficiency (OMIM 137100) is a common incidental finding amongst blood donors (with numerous reports since the 1970s), without any particular symptomatology originally appreciated [reviewed in Ref. (65)]. However, it has recently been reported that IgA-deficient persons have increased frequency of gastrointestinal and respiratory infections, and of allergy and autoimmunity (66).

#### **Treg-TO-Tfh CELL PLASTICITY**

In mice, Th17 cells are not unique in their ability to switch to a Tfh cell phenotype, also Foxp3+ cells have been reported to be able to do this in the PP GCs (27, 28). The first study used adoptive transfer of CD4+EGFP+ cells from spleen or LNs of Foxp3–EGFP reporter mice into T cell deficient mice (27). Whilst 80% of Foxp3+ T cells switched off Foxp3 expression in the PPs, about half of the transferred cells kept their Foxp3+ profile in the LP of the small intestine as well as in the spleen. The ex-TregTfh cells that form in the PP expressed Bcl-6 and also IL-21, CXCR5, CD40L, ICOS, PD-1, and CD28, all associated with Tfh cell function (61). Functionally,it has been shown that these ex-TregTfh cells induce GC formation in PPs and facilitate IgA-producing cells in the gut (27). These findings appear in contrast with the work of Hirota et al. that identified ex-Th17Tfh cells (26). In the study by Hirota et al., mucosal Foxp3+ T cells transferred into TCRα −/− hosts did not differentiate into Tfh cells, induce GC B cells or IgA production. It remains unclear whether differences in the models (Tsuji: CD3ε −/− recipients; Hirota: TCRα −/− recipients) or source of cells transferred (Tsuji: spleen and LN, Hirota: mucosal origin) or intestinal environment of the transfer recipients explain the discrepancies. In a separate study, Takahashi et al. transferred iTreg cells into TCRα −/− mice and showed that these cells can lose Foxp3 expression and become Tfh in the PP (67). Intriguingly,transferred iTreg cells were more likely to lose Foxp3 expression in the PP than in the spleen, suggesting that the micro-environment of the gut affects the plasticity of Tregs. The conversion of Treg-to-Tfh in the gut is controlled, in part, by expression of microRNA-10a that suppresses Bcl-6 and Ncor2, thereby repressing of Treg-to-Tfh conversion in the gut (67).

Not all Tregs within the PP convert into Tfh, however, there is a regulatory population of CD4+ T cells in GCs; T follicular regulatory cells (Tfr) (68–70). Tfr are the progeny of Foxp3+CD4+ cells that share phenotypic characteristics of Tfh cells, including CXCR5, PD-1, and Bcl-6 expression. In peripheral secondary lymphoid organs, Tfr control the size and output of the GC response (68–70). In PP GCs, Tfr control the quality of IgA produced, which in turn promotes diversity within the microbial community of the gut (28). Taken together, conversion of Treg-to-Tfh in the gut is finely balanced to ensure the ratio of Tfh to Tfr within the PP supports high-affinity IgA production and homeostasis of commensal bacteria.

#### **Th2-TO-Tfh CELL PLASTICITY**

The gastrointestinal helminth parasite *H. polygyrus* has been shown to give raise to Th1/Th2 hybrid cells but also been suggested give rise to Th2/Tfh hybrid cells (71). These hybrids were described as IL-4 producing CD4+ cells expressing CXCR5, ICOS and PD-1, Bcl-6 and IL-21 and that localized in B cell follicles of mLNs Brucklacher-Waldert et al. Intestinal helper T cell plasticity

after *H. polygyrus* infection. Zaretsky et al. found similar IL-4 +CXCR5+PD-1+ Th cells after immunization with schistosomiasis soluble egg antigen (72). A later study using dual reporters for IL-4 and IL-13 and *N. brasiliensis* infection demonstrated that Tfh phenotype cells express IL-4 but not IL-13, whereas Th2 cells express both prototypic Th2 cytokines. Importantly, IL-4+ Tfh cells are Bcl-6+ and do not express high levels of GATA-3, suggesting that these cells are Tfh cells that produce IL-4 rather than *bona fide* Th2/Tfh hybrids (73).

#### **CD4**+ **T CELL INTO CYTOTOXIC CD4**+ **T CELL CONVERSION**

Among the CD4+ IELs a population of cells has been described that lost its CD4-lineage transcription factor ThPOK and acquired the CD8-lineage transcription factor Runx3 (29, 30). The induction of Runx3 expression by CD4+ T cells was associated with expression of CD8αα, the natural killer cell – and CTL-related molecule 2B4 (CD244) and Tbet (29). In addition, these cells expressed granzyme and the activation-induced degranulation marker CD107a (LAMP-1) (30). As well as closely resembling mature CD8+ CTLs, these cells showed high cytolytic activity. At the same time, these cells lose their ability to produce IL-17 and to express RORγt (29, 30). Fate-mapping and adoptive transfer experiments by Mucida et al. showed that CD4+ CTLs originate from ThPOK+ naive CD4+ T cells (30).

The loss of ThPOK together with the inability to produce IL-17 in the intestine had physiological implications on the development of colitis. Conditional deletion of ThPOK in CD4+ T cells prior to their transfer into lymphopenic Rag−/− deficient mice enhanced the differentiation of CD4+CD8α + CTLs in the gut of the recipients, which, as a consequence, saw a reduction in intestinal inflammation compared with controls (29). By contrast, the transfer of Runx3-deficient CD4+ T cells resulted in an exacerbated intestinal inflammation, but provided recipient mice with increased protection against infection with the enteropathogenic bacterium *Citrobacter rodentium* (29). A role of CD4+ CTL cells in celiac disease is suggested by their restriction to MHC class II, their induction by dietary antigen, responsiveness to IL-15, and cytotoxic potential (30).

#### **MECHANISMS OF CD4**+ **T CELL PLASTICITY**

Very little is known about the underlying mechanisms determining CD4+ T cell plasticity. It is likely that the same factors that are involved in T cell differentiation are implicated in a phenotype switch, but this remains largely untested.

The development of some CD4+ T hybrid cells or ex-Th lineage cells seem to predominantly take place in the intestine, indicating that the micro-environment has a major influence on CD4+ T cell plasticity and identity. For example, the micro-environmental cues contributing to a phenotype switch between Th17 and Tfh seem to be unique to the PP, as the plasticity of Th17 cells toward a Tfh cell fate was restricted to the micro-environment of PPs and did not occur in peripheral LNs (26). Likewise, transferred Foxp3+ T cells lost Foxp3 expression and acquired Tfh features preferentially in the PPs (27, 65). Further evidence of the unique nature of the PP comes from *in vitro* stimulation assays in which APCs from PP and spleen generate different CD4+ effectors (74, 75).

Factors enabling CD4+ T cell plasticity could, in part, originate from the microbiota or dietary products that effect CD4+ T cells either directly or indirectly via innate immune cells. These extrinsic cues can alter transcription factor and microRNA expression and epigenetic markers such as histone modifications (**Figure 2**). For example, microRNA-10a dampens the conversion of Treg cells to both Th17 and Tfh cell fates (67). In addition, the cellular composition of the GALT, enriched in non-conventional lymphocytes such as ILCs, IEL populations of TCRγδ+, and CD8αα+TCRαβ+ T cells and mucosal associated innate NKT cells, is substantially different compared with other secondary lymphoid organs and will contribute to a different environment in which T cells encounter antigen and are maintained.

#### **MICROBIOTA**

The nature of antigen or metabolic products provided by the gut microbiota plays a role in T cell plasticity. There are a number of examples where CD4+ T cell subsets are expanded in response to particular microbial species and their products. Using germ-free mice, a mixture of 17 *Clostridia* species were selected from human gut flora for their ability to induce mucosal Tregs (76). Introduction of these 17 strains mitigated colitis and allergic diarrhea models (76). This protective effect is mediated by short chain fatty acids, products of these 17 bacteria (and of *Bacteroides fragilis*, also shown to induce Tregs) (77). The microbiome is shaped, in part by induced Tregs, and mice lacking induced Tregs are more susceptible to colitis and asthma (78). Thus the gut microbiome is implicated in the protection of autoimmunity and autoinflammation both in the gut and at distant sites. Recent work provides evidence for a symbiosis between CD4+ T cell subsets and the microbiome (28). Lack of Tfr resulted in poor quality IgA production and a limited biodiversity of the microbiome. The presence of Tfr permitted a more diverse microbiome including a larger representation of nonpathogenic *Clostridia* (28), which establishes a positive feedback loop between CD4+ cells and the microbiome mediated through SCFA, Foxp3 expression, and IgA production. This narrowing of the microbiome and subsequently SCFA deplete environment might explain the association between IgA deficiency and allergy and autoimmunity (66). Taken together, these studies provide evidence that the gut microbiota and CD4+ T cell fate within the gut are inter-dependent, with each affecting the composition of the other.

CD4+CD8α + CTLs are absent from the intestines of germ-free mice and of mice mono-colonized with segmented filamentous bacteria (SFB), but appear in the intestines following reconstitution with specific non-pathogenic microorganisms (30). Little is known about the mechanisms how antigens originating from microbiota induce a phenotype switch. However, data on Th17 cell differentiation indicate that SFB colonization promotes Th17 cell commitment by the expression of inflammation-associated genes, such as the gene for serum amyloid A (SAA) (79). Some Th17 cells from murine gut have TCR specific for SFB-derived antigen (80). In addition, it has been shown that ATP derived from commensal bacteria activates CD70high CD11clow cells in the LP, leading to enhanced differentiation of Th17 cells (81). Therefore, changes in the concentration of SAA or ATP could either induce a switch toward Th17 phenotype or destabilize Th17 cell commitment toward another phenotype.

#### **DIETARY PRODUCTS**

Dietary products, mainly after being processed by microorganisms, can induce phenotype switch. PPARγ has been identified to induce a Th17-to-Treg cell switch (23). This nuclear receptor regulates fatty acid storage and glucose metabolism. Therefore, consumption of food containing certain fatty acids may promote a phenotype switch from Th17-to-Treg cells. Several dietary products have been reported to promote or inhibit lineage commitment [reviewed in Ref. (2)]. It is likely that these factors are also candidates to induce a phenotype switch of CD4+ T cells. In addition, dietary products could influence a phenotype switch by affecting metabolic and signaling pathways and epigenetic status.

#### **INNATE IMMUNE CELLS: PROVIDERS OF COSTIMULATION AND CYTOKINES**

Innate immune cells are abundant in the GALT. They have the potential to influence CD4+ T cell plasticity via their determination of the micro-environment via the secretion of soluble mediators, expression of co-stimulatory molecules, and via their potential to act as APCs. Mucida et al. demonstrated that continuous activation of CD4+ T cells by oral administration of an antigen is necessary for a phenotype switch to CD4+ CTLs (30). It has been reported previously that antigen dose and peptide/TCR affinity can influence Th commitment (82).

Co-stimulatory factors have been shown to play a role in Tregto-Tfh plasticity. Blocking the interaction of CD40, expressed on APCs such as B cells and DCs, with CD40L, found on T cells, is able to prevent a phenotype switch from Treg cell to Tfh cell (27).

The role of cytokines in Th17 cell plasticity has been studied by several investigators but has not yet been fully elucidated. However, it is clear that IL-23-dependent pathway does play an important role. In *Helicobacter hepaticus*-induced typhlocolitis, mRNA levels for IL-23p19 were elevated after bacterial inoculation and *ex vivo* IL-17A+ cells isolated from the colitic intestine expressed both subunits of the IL-23R, indicating that IL-23 acts on Th17 cells to induce a program resulting in IFNγ production (18). T cell transfer studies also showed that IL-23 is required for the appearance of IL-17A+IFNγ + double-producing T cells in the intestine. Transferred naive CD4+ T cells developed to Th1/Th17 hybrid cells in Rag−/− mice, but not in p19−/− RAG−/− mice [lacking IL-23; (21)]. These studies were extended by Ahern et al. demonstrating that in the intestine the emergence of IL-17A+IFNγ + double-producing Th cells, but not IL-17A single producing Th cells, requires T cell-intrinsic IL-23 signaling by transferring IL-23R−/−CD45RBhigh CD4+ T cells into full hosts (17). In humans, CD161+CD4+ T cells from CD patients readily produce IL-17 and IFNγ upon stimulation with IL-23, whereas, in healthy subjects, IL-1β was required alongside IL-23 (16).

The role of IL-12, the primary cytokine inducing Th1 cell differentiation, in mediating Th17-to-Th1 cell conversion remains controversial.*In vitro*, obtained results differ from those data being observed in *in vivo* models. Upon transfer of naive CD4+ T cells, Th17/Th1 cells were detected in the mLNs of IL12p35-Rag-double deficient mice (lacking IL-12), demonstrating that IL-12 is negligible in Th1/Th17 induction in the intestine (21). In contrast, Lee et al. showed *in vitro* that in both IL-23 and IL-12 are able to switch off IL-17 and enhance IFNγ production in a STAT4- and Tbet-dependent manner. It has also been shown that *ex vivo* Th17 cells can be converted into a Th1-like phenotype following IFNγinduced expression of Tbet and acquisition of IL-12Rβ2 surface expression (83, 84). Lee et al. investigated the effect of additional cytokines on Th17 cell plasticity and found that Th17 cells require TGFβ for sustained expression of IL-17F and IL-17A (20).

Although the precise role of IL-23 in the Th17-to-Th1 cell conversion requires further investigation, it does not seem to play an essential role in a phenotype switch from Th17 to Tfh cell. Th17 cells can give rise to Tfh cells in IL-23-competent and -deficient mice (26). For a phenotype switch from Tregs to Tfh cells it has been shown that the cytokine IL-6 can to down-regulate Foxp3, and this may be relevant to Treg plasticity in the IL-6 rich PPs (85).

#### **TRANSCRIPTION FACTORS**

Transcription factors maintain and instruct lineage programs by complex mechanisms, often involving several cooperating transcription factors, and their binding and access to specific DNA sequences. The expression of lineage determining transcription factors is induced by multiple factors in the micro-environment, including cytokines. Their regulation falls outside the scope of this review, but their interactions are of importance in maintaining Th

identity and are likely to play a prominent role in ultimately generating hybrid Th cells and Th identity conversions. Co-expression of IFNγ and Th2-associated cytokines in the same T cell seems to be enabled by signals that keep both Tbet and GATA-3 expression in balance, which is in contrast to studies examining chromatin modification at *Tbx21* and *Gata3* (86, 87).

The loss of a single transcription factor can tip the balance in favor of an alternative lineage. In Bcl11b-deficient mice, GATA-3 expression in Th17 cells is unrestrained, resulting in GATA-3 mediated IL-4 production (22). This change in cell phenotype feeds back to the micro-environment, and has further implications for T cell biology. For example, the cytokine mix produced by Th2/Th17 hybrid cells triggers gut-imprinting properties in DCs. IL-4 together with GM-CSF enhance the expression of the enzyme RALDH2 in DCs (88, 89), leading to elevated levels of RA (90) that imprint gut-homing properties on T cells, such as α4β7 and CCR9 (91). Similarly, the termination of ThPOK in mature CD4+ T cells enables the de-repression of the CTL program (30), resulting in MHC class II restricted CD4+ T cells with a cytotoxic effector function. This process can be further promoted by Runx3 (29).

The factors responsible for carefully balancing two transcriptional programs within a single T cell may be present together from the moment of T cell differentiation or may be induced later. *In vitro*, data have shown that expression of one differentiation program is often found to be mutually exclusive with factors from another (55). Yet, differentiation of some programs are characterized by co-expression of two pathways from the start, such as seen with iTreg differentiation in which T cells co-express Foxp3 and RORγt from the start (25). These *in vitro* observations are often not recapitulated *in vivo* and vice versa. This highlights missing links in our understanding of which factors are required to determine full Th effector cell differentiation, which factors enable T cells to postpone the identity decision, as well as those that can still interfere at later stages.

#### **EPIGENETICS**

The observed stability or plasticity of Th subsets is governed by the epigenetic regulation of the key transcription factors and cytokines and their access to downstream genes (92). Whether a particular gene is poised for expression is controlled by the chromatin structure and histone or DNA modification state. Wei et al. evaluated the histone H3 methylation status over the entire genome in a variety of defined Th subsets (87). Promoter regions for *Tbx21* (encoding Tbet) in Th1 cells and *Rorc* (encoding RORγt) in Th17 cells displayed a permissive methylation state (H3K4me3) associated with full expression of these master regulators in each lineage. However, the promoter region for *Tbx21* in Th17 cells had a bivalent status characterized by H3K4me3/H3K27me3, reflecting the relative instability of these cells and their potential to acquire another Th cell phenotype. In addition, it has been shown that treatment of naïve T cells under Treg polarizing conditions with the fatty acid, butyrate, enhances permissive acetylation in the promoter region of the *Foxp3* locus. Butyrate, being a large bowel fermentation product, provides a link between microbial products and Treg cells by epigenetic mechanisms. Beyond histone modifications, miRNAs offer a further tuning of gene expression and CD4+ phenotype, as illustrated earlier.

#### **FUNCTION**

CD4+ T cell plasticity is important in the resolution of infections but can contribute to immunopathogenesis. The ability to switch or combine effector phenotypes can generally be seen as a very useful feature in the arsenal of the adaptive immune system. It is an additional tool providing flexibility to adequately control rapidly changing microorganisms. It allows the retention of TCRs with a useful track record while displaying a high degree of flexibility and adaptation depending on local environmental cues. Therefore, a phenotype switch may facilitate the ability to respond more quickly to changing immunological challenges and to successfully terminate immune responses after pathogen clearance. This flexibility may be especially important and useful at epithelial interfaces, where luminal content is constantly changing and the largest population of microorganism is encountered.

The ability to switch from one phenotype to another or obtain features from two effector programs increases the efficiency of a response. Combining effector mechanisms would enable the control of complex organisms that have evolved ways to deal with one predominant effector subset. This implies that effector programs can co-exists within one T cell, while the co-existences of different effector subset within the same micro-environment may be harder to achieve and maintain. It is worth noting that reports on Th cells displaying a hybrid phenotype highlight that such cells are less effective in dealing with situations where one part of their identity is required compared with the single identity T cells. This indicates that each identity is weakened by the presence of the transcriptional program of the other, a feature that could be abused by microorganisms. Conversely, producing these "weakened" T cells may be method of peripheral tolerance – in an inflamed milieu, T cells are stimulated under polarizing conditions and develop a single identity, whereas non-inflamed milieu offers conditions less favorable to any single identity and the "weakened" T cells are tolerized.

An additional physiological role for CD4+ T cell plasticity is the gaining of specific functional features that require aspects of the previous full identity combined with a very specific trade. This is the case for Tfh cells that can develop from committed Th17 or Treg cells. In case of ex-Th17Tfh cells, they represent a subset of Tfh cells that are specialized to promote high-affinity IgA production in the GCs of PPs. A similar specialized role in GC B cell support is likely the case for ex-TregTfh. These Tfh adopters have only been found in the PP and only support IgA production, demonstrating that this plasticity is unique to the gut environment.

Lastly, the transcriptional programs of each T cell effector subset may not be compatible with long-term survival. Effector cells that switch to a Th1 cell phenotype, may be maintained better as memory cells due to the activity mediated by Tbet. Recent reports describe a role of Tbet expression gradient as a regulator of CD4+ T cell memory formation, with highly Th1-polarized Tbet+ cells displaying end-effector features and a short lifespan, whereas Th cells with a lower expression of Tbet are able to enter the long-lived memory T cell pool (93, 94). This is to some degree mimicked by the finding that long-lived ex-Th17 cells acquired Tbet expression, albeit at levels lower than their Th1 counterparts.

From a therapeutic viewpoint, contemplating a deliberately induced phenotype switch could be used to up- or down-regulate Brucklacher-Waldert et al. Intestinal helper T cell plasticity

the different effector arms of the adaptive immune response. Dampening CD4+ T cell function would be useful for autoimmune disorders including CD and multiple sclerosis. In a colitis model, oral pioglitazone induces a switch from Th17 to an iTreg phenotype, improving disease and in EAE (a model of MS) pathogenic Th17 cells can be redirected to gut, again improving disease. Enhanced CD4+ T cell responses could improve cancer surveillance and clearance. Whilst phenotypic switching appears to occur in very specific circumstances, some of these conditions are encountered spontaneously in healthy animals. Thus strategies to induce phenotype switching, with minimal side effects or co-morbidities, might be identifiable.

#### **CONCLUSION**

The nuanced view of Th differentiation suggested that within any CD4+ effector population there are varying ratios of lineage determining transcription factors in each cell, with correspondingly graded cellular phenotypes. In GALT these ratios integrate a number of different signals – microbial products, dietary influences, costimulation, cytokines, and epigenetic modifications. Variation in these signals and their interpretation is the substrate for heterogeneity of CD4+ T cells. As these microbial and dietary cues are unique to GALT, perhaps even unique to the most luminal GALT, such as PP, they could together provide a micro-environment permissive for plasticity, in contrast to other secondary lymphoid organs, where plasticity is less frequently reported and where much of the rigid *in vivo* definitions of the CD4+ subsets were derived. Whether the phenotypic switches described herein are unique to an *in entero* environment is unknown. It is possible that similar blurring of CD4+ subsets occurs at other mucosal sites, balancing tolerance, protection, and immunopathology with the need to maintain a diverse microbiome. To understand the precise factors underlying plasticity is relevant for our understanding of host (and microbiomial) health, host defense, pathogenesis of various gastrointestinal diseases and also for development and optimization of vaccines, where an enforced Tfh phenotype is desired.

#### **ACKNOWLEDGMENTS**

Verena Brucklacher-Waldert is supported by the Deutsche Forschungsgemeinschaft (DFG BR 4253/1-1 Forschungsstipendium). Edward J. Carr is an academic clinical trainee, funded by the NIHR. Michelle A. Linterman is supported by the BBSRC. Marc Veldhoen is supported by a BBSRC ISPG grant and the ERC (No. 280307 – Epithelial\_Immunol).

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

*Received: 24 July 2014; paper pending published: 08 September 2014; accepted: 22 September 2014; published online: 07 October 2014.*

*Citation: Brucklacher-Waldert V, Carr EJ, Linterman MA and Veldhoen M (2014) Cellular plasticity of CD4*+ *T cells in the intestine. Front. Immunol. 5:488. doi: 10.3389/fimmu.2014.00488*

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

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

## MemoryT follicular helper CD4 T cells

### **J. Scott Hale\* and Rafi Ahmed**

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA

#### **Edited by:**

Dragana Jankovic, National Institutes of Health, USA

#### **Reviewed by:**

António Gil Castro, University of Minho, Portugal Remy Bosselut, National Cancer Institute, USA

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

J. Scott Hale, Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Road, Atlanta, GA 30322, USA e-mail<EMAIL_ADDRESS>
T follicular helper (Tfh) cells are the subset of CD4T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity.This specializedT helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whetherTfh cells differentiated into memory cells and whether they maintain Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall theirTfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to otherT helper lineages (Th1,Th2,Th17, etc.) will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.

**Keywords:T follicular helper cells, memory T cells, Bcl6, CXCR5, helper T cells**

#### **INTRODUCTION**

Effective B cell responses to infectious diseases or immunization require the assistance of CD4+ helper T cells. A specialized subset of CD4 T cells named T follicular helper (Tfh) cells are required for providing this help to antigen-specific B cells. Without cognate Tfh help, activated B cells are unable to generate and maintain the germinal center response that is required for efficient somatic hypermutation of immunoglobulin genes and the selective processes that facilitate affinity maturation of antibodies (1, 2). Furthermore, the germinal center reaction is the origin of longlived memory B cells and long-lived plasma cells that populate the periphery and bone marrow (respectively), and provide long-term antibody-mediated protection against (re)exposure to pathogens (3). Thus, Tfh cells play a critical role in the generation of effective and long-lived humoral immune responses to antigens (1).

T follicular helper cells were first identified as a subset of CD4 T cells isolated from human tonsils (4, 5). These cells expressed the B cell follicle homing chemokine receptor CXCR5 and the inducible costimulator (ICOS), and localized within the germinal center (4, 5). Furthermore, these human tonsil CXCR5+ cells (compared to CXCR5−CD45RO+ T cells) efficiently promoted production of class switched immunoglobulin (Ig)G and IgA in T cell:B cell co-culture assays (4–6). Interestingly, CXCR5+ CD4 T cells from human blood, which were presumed to be the memory counterparts of CXCR5+ cells in tonsils and lymph nodes, did not efficiently produce the Th1 signature cytokine IFNγ or the Th2 cytokines IL-4, IL-5, and IL-13 (5). Together, these studies suggested that the CXCR5+ Tfh cells represented a novel subset of helper T cells with the specific functions of providing help for B cell responses and that are distinct from Th1 and Th2 cells. Since these initial seminal reports describing Tfh cells, extensive studies have demonstrated that while Tfh cells share certain similarities with Th1, Th2, and Th17 cells (depending upon context of infection or vaccination), these cells have unique developmental requirements and distinct phenotypic, homing, and functional qualities compared to other T helper cell lineages (Th1, Th2, Th17, Treg) (1).

Upon activation with cognate antigen by dendritic cells, antigen-specific CD4 T cells can differentiate to become various types of effector CD4 T cells with specific roles in promoting anti-pathogen immune responses (**Figure 1**). Early differentiation toward the Tfh lineage requires ICOS expression and signaling to induce expression of the transcriptional repressor Bcl-6 (7). Bcl6 is required for Tfh cell generation, maintenance, and function, establishing Bcl6 as a central regulator in Tfh cell lineage development (**Figure 1**) (8–10). Bcl6 expression promotes Tfh differentiation, at least in part by suppressing transcription of the transcriptional regulators Tbet, RORγt, GATA3, and Blimp-1 (8–10), and through other potential mechanisms, such as the repression of microRNAs (10). In addition, cytokines such as IL-6 and IL-21 (11), and other molecules such as SAP are important for Tfh differentiation and function (12,13). Importantly,interactions with cognate B cells were required for amplifying the expression of Bcl6 for the maintenance of the Tfh phenotype during the immune response (7). During Tfh differentiation, Bcl6 plays an important role in suppressing Blimp1 (8), which is a regulator of Th1, Th17, and Th2 lineage cells (1). Thus, the promotion of Bcl6 coupled to

**FIGURE 1 |T follicular helper cell differentiation and context-dependent Tfh cell heterogeneity**. Following activation of naïve CD4 T cells, cells proliferate and undergo fate decisions in response to cytokines and other differentiating factors. Tfh cell differentiation is influenced by IL-6 and IL-21, and dependent upon ICOS signaling for expression of the transcription factor Bcl6. Cytokines including IL-12, IL-4, IL-1β, and many others, direct **(A)** Th1, **(B)** Th2, and **(C)** Th17 cell differentiation, respectively. The context-dependent cytokine milieu also influences Tfh cell differentiation; thus, Tfh effector cells can express some low/intermediate levels of the transcription factors Tbet, Gata3, and RORγt, which are associated with the **(A)** Th1, **(B)** Th2, and **(C)** Th17 cell lineages, respectively.

the repression of Blimp1 plays a role in the differentiation, function, and possibly the stability of Tfh cells in relation to other T helper cell subsets. Additional transcription factors such as Maf (14, 15), Ascl2 (16), and others play important roles in Tfh cell differentiation and/or function (1).

T follicular helper cells have developmental requirements that differ from those that promote Th1, Th2, and Th17 effector cell development (1). However, depending upon the type of infection (viral, helminth, fungal, etc.) or immunization and the inflammatory environment that is generated, Tfh cells can express low to intermediate levels of Tbet, Gata3, or RORγt (**Figure 1**) (17–20). This intriguing context-dependent transcription factor expression in Tfh cells results in a variety of Tfh cell subsets that can express low levels of specific cytokines that can influence antibody class switching (17, 21, 22). Thus, studying the complex relationship between Tfh cells and their non-Tfh cell counterparts, and whether they remain phenotypically and functionally distinct throughout the immune response and beyond is critical to understanding Tfh cell commitment and flexibility and to determining their biologically important roles during specific types of immune responses.

#### **LINEAGE-COMMITTED MEMORY T HELPER CELLS**

The adaptive immune system responds to infectious challenge with two major goals. The first goal is to generate sufficient numbers of antigen-specific effector cells to limit and clear the pathogen. The second priority is to provide long-lasting immunity that will defend the host from subsequent exposure to the pathogen (23). The activation-driven proliferation and lineage differentiation of CD4 T cells *in vivo* is accompanied by the progression of memory differentiation. Following clearance of antigen, the majority (approximately 90–95%) of antigen-specific effector T cells undergo apoptosis, leaving behind a population of memory cells. In some experimental models, antigen-specific CD4 memory T cells gradually decline over long periods of time (24, 25). For example, *Listeria monocytogenes* infection-induced memory CD4 T cells are present at relatively high frequencies 90 days postinfection; however, by approximately 250 days post-infection, the population has largely disappeared from the spleen and lymph nodes (25). In contrast, human studies reveal that long-lived vaccinia-specific memory CD4 T cells are relatively stable for at least several decades after smallpox vaccination (26, 27).

Memory T cells possess many important features compared to their naïve CD4 T cell precursors. First, antigen-specific memory cells are found in increased numbers relative to their naïve antigen-specific precursors, providing better coverage and a more rapid cellular response upon pathogen rechallenge. Second, memory cells are not restricted to blood circulation and secondary lymphoid organs, but instead may also traffic to and reside in non-lymphoid tissues, where they may rapidly exert effector functions if their specified pathogen gains entry to that particular anatomical site. Third, memory T cells have undergone changes in cell-intrinsic programing, allowing them to rapidly recall their effector functions, such as prompt expression of specific effector cytokines, chemokines, and cytotoxic molecules. Finally, memory cells are long-lived, and a central feature of their longevity is dependent on their ability to undergo homeostatic proliferation in the absence of antigen (23, 28).

Combining the study of T helper lineage differentiation and T cell memory differentiation *in vivo* following vaccination or infection is incredibly complex. However, it provides the opportunity to gain vital understanding into the heterogeneity and lineage commitment and flexibility of the resulting antigen-specific memory CD4 T cells that will be informative for ongoing and future vaccine discovery/development efforts. It has become clear that among the vast heterogeneity of memory CD4 T cells, many memory cells demonstrate commitment to a previously defined T helper lineage. The existence of Th1-commited long-lived memory CD4 T cells was demonstrated in BAC transgenic mice that used a reporter to indicate transcription of the *Ifng* gene. In this study, Harrington et al. demonstrated that these memory cells were derived from the effector Th1 cells, and rapidly recalled IFNγ expression at the effector phase (29). Several other studies similarly found that subsets of LCMV-specific and *Listeria*-specific memory CD4 T cells with distinct phenotypes were committed Th1 memory cells that recalled IFNγ expression and expression of other Th1 effector molecules (20, 30, 31). One study utilized an IL4 IRES EGFP reporter mouse to demonstrate that Th2 effector cells (EGFP+) generated from *N. brasiliensis* infection could provide anti-parasite protective immunity after adoptive transfer into immunocompromised recipient mice and 30 days resting before parasite challenge (32). Similarly, *Trichuris*-specific Th2 memory cells recall their Th2 effector functions and mediate anti-parasite immunity (33). While Th17 CD4 T cells generated by intranasal *Listeria* infection (a Th1 pathogen) do not form memory cells (25),*Candida* and other fungal vaccines, as well as other conditions have been shown to induce Th17 memory cells *in vivo* (34–36). Together, these studies demonstrate the characteristics and programs of polarized effector Th1, Th2, and Th17 cells that are generated early during effector differentiation are preserved in resting memory cells. Importantly, these effector programs are recalled after reactivation *in vivo* to infectious challenge in an antigen-specific manner, and with the appropriate T helper effector response to effectively eliminate the pathogen.

#### **T FOLLICULAR HELPER MEMORY CELLS**

The establishment of Tfh cells as an independent effector T helper subset, and the factors that drive Tfh differentiation being defined, provides a strong rationale for exploring whether Tfh cells that progress to become memory cells maintain their Tfh attributes following resolution to the immune response. However, given the potential flexibility/plasticity of Tfh cells toward repolarization (37), one might predict that Tfh cells generate non-committed memory CD4 T cells. Several fundamental questions exist regarding the relationship of Tfh cells and memory cells. First, do Tfh cells survive to become memory cells? Second, do memory cells derived from Tfh cells maintain their commitment/programing to recall Tfh effector cells, or, instead possess pluripotency/plasticity to become cells of other T helper lineages? The answer to these questions provides profound insight into the importance of how Tfh differentiation during primary immune responses to natural infection and vaccination have the potential to influence secondary antibody responses. Several recent studies have provided insight into the existence and characteristics of memory Tfh cells and their capacity to recall Tfh-specific effector functions following reactivation with antigen.

One early study investigating memory Tfh cells in mice described a population of CXCR5+ICOS+ cells in the draining lymph nodes 30–56 days following immunization with pigeon cytochrome C in adjuvant. Compared to day 7 Tfh effector cells, day 30 PCC-specific cells had decreased ICOS and OX40 expression. The authors reported that these were a subset of memory Tfh cells with enhanced recall capacity upon immunization 6–8 weeks after priming (38). However, this study further reported the persistence of antigen for more than 75 days after immunization and the maintenance of CD69 on these PCC-specific CD4 Tfh cells indicate that this experimental system promotes long-lived antigen depots. Thus, the CXCR5+ICOSlo cells identified in this study cannot be clearly distinguished as true memory cells that survive in the absence of antigen (38).

A study by MacLeod and Marrack provided a stronger basis for the existence of memory CD4 T cells with accelerated Tfh function during recall responses (39). Their study demonstrated that on a per cell basis, antigen-specific memory CD4 T cells (compared to Ag-specific naïve cells) provided accelerated B cell responses and antibody class switching. Interestingly,this accelerated B cell helper capacity was contained within the CXCR5+ subset of memory CD4 T cells, resulting in higher OVA-specific IgG1 titers following adoptive cell transfer and followed by immunization. The authors suggested that CXCR5 chemokine receptor expression promotes their more rapid migration of reactivated memory CD4 T cells to B cell follicles, allowing them to provide accelerated help to the B cell response (39).

It was unknown whether Tfh cells were able to survive and become memory cells. To address this question, Weber et al. used protein immunization in CFA to induce TCR transgenic CD4 effector T cells, sorted them into CXCR5− and CXCR5+ subsets, and then adoptively transferred these into naïve recipient mice (40). Fourteen days later, adoptively transferred CXCR5+ effector cells had become CXCR5 low/negative. After resting these cells in naïve recipient mice for several weeks, a large proportion of transferred Tfh cells quickly recalled a Tfh phenotype within 2.5 days following re-immunization, expressing higher levels of PD-1, Bcl6, CXCR5, and IL-21 compared to non-Tfh cells and primary effector cells (40). While the time periods that these cells were rested in the absence of antigen were relatively brief, this study was highly suggestive that effector Tfh cells could be maintained in the absence of antigen and preferentially recall a Tfh phenotype. A study by Choi and colleagues addressing the fate commitment of Tfh cells demonstrated that day 3 LCMV-specific Tfh effector cells, following adoptive transfer into day 3 LCMV-infected (infectionmatched) recipient mice, were maintained up to 45 days later and retained CXCR5 surface expression (41). An additional study demonstrated that CXCR5+RFP+ (from Bcl6 RFP reporter mice) OTII cells induced by Ova/CFA immunization can persist following adoptive transfer for 20 days, and upon OVA+CFA immunization preferentially recall Tfh cells in the draining lymph node (42). Together, these studies clearly demonstrate that not all Tfh effector cells are terminally differentiated and fated to die. Instead, some Tfh effector cells progress to become memory cells that have some similar features compared to their Tfh effector predecessors.