Relationship Between CD4 Regulatory T Cells and Anergy in Vivo Lokesh A

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Relationship between CD4 Regulatory T Cells and Anergy In Vivo Lokesh A. Kalekar and Daniel L. Mueller This information is current as J Immunol 2017; 198:2527-2533; ; of October 1, 2021. doi: 10.4049/jimmunol.1602031 http://www.jimmunol.org/content/198/7/2527

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Relationship between CD4 Regulatory T Cells and Anergy In Vivo Lokesh A. Kalekar1 and Daniel L. Mueller + Selective suppression of effector CD4 T cell functions Treg cells control tolerance to some tissue-restricted self-Ags is necessary to prevent immune cell–mediated damage Two recent studies took advantage of experimental Ags to healthy tissues. This appears especially true during (peptides derived from either the P1 bacteriophage Cre pregnancy or in individuals predisposed to autoimmu- recombinase [Cre] or the Aequorea victoria enhanced GFP + nity. Foxp3 regulatory T (Treg) cells and induction of [eGFP]) whose expression in mice was directed by transgenic anergy, an acquired state of T cell functional unrespon- 2 tissue-speciﬁc promoters, and additionally made use of pep- siveness in Foxp3 cells, have both been implicated as tide MHC class II (pMHCII) tetramers to detect polyclonal +

mechanisms to suppress dangerous immune responses CD4 T cells that respond to these model self-Ags (4, 5). Downloaded from to tissue-restricted self-Ags. Anergic CD4+ T cells and Both experiments demonstrated that ubiquitous (both thy- Treg cells share a number of phenotypic and mechanis- mic and peripheral) expression of self-Ag leads to deletion of tic traits—including the expression of CD73 and folate 60–95% of the highest affinity self-specific CD4+ T cells, receptor 4, and the epigenetic modification of Treg cell with the remaining low-affinity cells left functionally unre- signature genes—and an interesting relationship be- sponsive to Ag (Fig. 1). In contrast, restriction of self-Ag

tween these two subsets has recently emerged. In this expression exclusively to nonthymic tissues leads to a toler- http://www.jimmunol.org/ review, we will compare and contrast these two subsets, ance that appears to rely solely on ignorance by naive auto- as well as explore the role of anergy in the generation of reactive T cells. The strongest evidence for an active peripheral self-tolerance mechanism in these studies came in peripheral Treg cells. The Journal of Immunology, 2017, 198: 2527–2533. the form of self-Ags that were only weakly expressed in the thymus and, in particular, were otherwise restricted to mucosal tissues (e.g., Cre protein expression in the intestinal he self-tolerance mechanisms of T cells can be broadly brush border driven by a Vil1-Cre transgene, or expression in characterized as central or peripheral (Fig. 1). Cen- airway Clara cells driven by a Scgb1a1-Cre transgene). These tral tolerance mechanisms destroy high-affinity self- Ags induce little central deletion, and instead self-tolerance by guest on October 1, 2021 T + reactive T cells during thymic development, or else induce relies on the generation of self pMHCII-binding Foxp3 Treg their differentiation into a regulatory T (Treg)celllineage(1). cells (5). Importantly, these Treg cells appear to suppress the Nonetheless, central tolerance appears to be insufficient to conventional CD4+ T cell responses to self-Ag, because sys- + clear all self-reactive T cells, and other peripheral tolerance temic ablation of Foxp3 Treg cells at the time of a self-Ag mechanisms are necessary (2, 3). Peripheral tolerance may immunization led to a restoration of the conventional T cell rely on: 1) ignorance, wherein autoreactive T cells never clonal expansion and cytokine production response to near- encounter their cognate Ag; 2) deletion, whereby self-specific control levels (5). peripheral T cells are destroyed after TCR engagement; 3) In addition to Treg cells, several other suppressor cells have anergy, which is a state of functional unresponsiveness in- been described, such as the Tr1 cells, myeloid-derived sup- + duced upon self-Ag recognition; and/or 4) Foxp3 Treg cell– pressor cells, as well as the regulatory B cells. However, it is + mediated suppression of dangerous T cell responses against well established that Foxp3 Treg cells maintain immune ho- self-Ag. Each of these potential tolerance mechanisms has meostasis and prevent adverse immune responses throughout been clearly defined in numerous in vivo experimental sys- the life span of an individual. Foxp3 is an essential lineage- tems using TCR-transgenic responder T cells, typically at defining transcription factor of Treg cells (6, 7), because abnormally high cell frequencies. However, much less is mutations or deletion of the Foxp3 gene lead to impaired + known about self-tolerance in the natural polyclonal CD4 generation of Treg cells and cause severe autoimmunity in T cell repertoire. humans and mice (6, 8). Furthermore, instability of Foxp3

Department of Medicine, University of Minnesota Medical School, Minneapolis, MN Address correspondence and reprint requests to Dr. Daniel L. Mueller, University of 55455; and Center for Immunology, University of Minnesota Medical School, Minne- Minnesota Medical School, 2101 6th Street SE, 3-186 WMBB, Campus Delivery Code: apolis, MN 55455 2641, Minneapolis, MN 55455. E-mail address: [email protected] 1Current address: Department of Dermatology, University of California, San Francisco, Abbreviations used in this article: CNS, conserved noncoding DNA sequence; Cre, Cre San Francisco, CA. recombinase; DC, dendritic cell; eGFP, enhanced GFP; FR4, folate receptor 4; InsB, insulin B chain; Nrp1, neuropilin 1; pMHCII, peptide MHC class II; pT , peripheral Received for publication December 2, 2016. Accepted for publication January 9, 2017. reg Treg;Treg, regulatory T; Treg-me, Treg cell methylome; tTreg, thymic Treg. This work was supported by a Within Our Reach: Finding a Cure for Rheumatoid Arthritis campaign grant from the Rheumatology Research Foundation and by National Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Institutes of Health Grant P01 AI35296.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602031 2528 BRIEF REVIEWS: ANERGIC T CELLS AS Treg PROGENITORS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. The role of anergy in pTreg cell generation: a model. CD4 single-positive thymocytes speciﬁc for tissue-restricted self-Ags can experience one of several fates. Abundant thymic self pMHCII presentation leads to central deletion. If self pMHCII abundance is very low, thymocytes escape to the periphery and often ignore the Ag. For the case of intermediate self pMHCII complex abundance, some thymocytes will die, some will differentiate to a Treg cell fate, and some will escape the thymus. Self-Ag–speciﬁc CD4+ T cells that escape into the periphery can recognize peripheral self pMHCII, and through the suppressive actions of Treg cells become anergic cells that express CD73 and FR4. Persistent Ag encounter induces Nrp1 expression and partial demethylation of the Treg-me in some of + the anergic T cells. Nrp1 anergic T cells then become precursors for Treg cell differentiation. Upon conversion to a stable pTreg cell lineage, anergy-derived Treg cells join the tTreg cell pool to suppress immunopathology and reinforce anergy induction.

+ expression allows for the transdifferentiation of Treg cells to Most Foxp3 Treg cells undergo their terminal differentia- T effector cell lineages capable of causing autoimmunity (9, tion in the thymus and are referred to as thymic Treg (tTreg) 10). Although Foxp3 is considered to be the master regulator cells, whereas others originate in the periphery (particularly at of Treg suppressive function, the expression of Foxp3 is not mucosal barrier surfaces exposed to food Ags and commensal 2 + sufficient to maintain a stable Treg cell lineage (11, 12). organisms) from conventional Foxp3 CD4 T cells and are Numerous Treg cell–specific genes are in fact expressed in- consequently called peripheral Treg (pTreg) cells (15, 16). It is dependently of Foxp3 protein, including Il2ra (the gene for believed that the self pMHCII specificity and suppressive CD25), Ctla4, Ikzf4 (Eos), and neuropilin 1 (Nrp1) (11, 13, functions of these two Treg subsets complement one another 14). These data suggest that additional lineage-defining in preventing immunopathology (17). tTreg cells are primarily factors act with Foxp3 to ensure the generation of a stable, responsible for maintaining general T cell immune homeo- functional Treg cell compartment. stasis, whereas pTreg cells control immunopathology that is The Journal of Immunology 2529 directed against tissue-restricted Ags in mucosal tissues such as plays some role in the establishment of self-tolerance in concert + the lung and gut (17). with Ag-specific Foxp3 Treg cells? Natural Treg (nTreg) cells are defined as all Treg cells gen- Historically, anergy has been defined as a state of functional + erated in vivo, and thus include tTreg cells and some pTreg inactivation wherein CD4 T cells lose the capacity to pro- cells (16). For the case of most natural Treg cells, epigenetic duce growth factors and proliferate in response to pMHCII changes associated with the development of a unique Treg cell recognition (21–23). But it has been unclear how long this methylome (Treg-me) are thought to be necessary for stable state lasts and/or whether it is an intermediate state in vivo. + Foxp3 expression. The Treg-me consists of demethylated Anergy can be induced in CD4 T cells when TCR signaling DNA CpG motifs at four Treg cell–related gene loci, in- is unaccompanied by strong CD28 costimulatory receptor cluding Tnfrsf18 (the gene for GITR), Ctla4, Ikzf4, and the ligation (24–26). The binding of coinhibitory receptors such Foxp3 conserved noncoding DNA sequence (CNS) 2 (18). as CTLA4 and PD-1 reinforces the induction and/or mainte- Therefore, the expression of Foxp3 and the development of nance of anergy (27, 28). In vivo, anergy is observed in CD4+ the Treg-me are independent and complementary events. TCR T cells after systemic, repeated exposure to a soluble Ag or signaling is required for Treg-me induction, which in turn superantigen in the absence of infection or adjuvant (29). + helps maintain the function and stability of Treg cells (12, 18). Likewise, the adoptive transfer of naive self-Ag–specific CD4 It has been proposed that within the thymus, the strength of T cells to normal mice bearing relevant self pMHCII com- TCR signaling controls Foxp3 expression, whereas the dura- plexes leads to the development of functional unresponsiveness tion of signaling establishes the demethylations that are as- (30, 31). This is in contrast with the adoptive transfer of self- + Downloaded from sociated with the Treg-me (12, 18). In contrast, quantitative reactive CD4 T cells into lymphopenic hosts lacking a + demethylation of the Treg-me loci (including Foxp3 CNS2) population of Foxp3 Treg cells, whereby a failure of anergy appears unnecessary for some pTreg cell differentiation. Rather, induction typically leads to dangerous T effector cell clonal Foxp3 transcription in mucosal tissue Treg cells can be stabilized expansion and differentiation, with consequent severe immu- by Tgfb1-andSmad3-dependent activation of the CNS1 en- nopathology (31, 32). It is important to note that there are hancer element (19, 20). many independent paths to anergy, which result in different http://www.jimmunol.org/ Even though pTreg cells have been found to play an essential types of functional unresponsiveness. The different ways that role in the suppression of tissue-specific immunopathology in assays are performed lead to varied readouts because the mea- adoptive transfer experiments (17), the experimental data of surement of anergy has not been standardized. We define Malhotra et al. (4) and Legoux et al. (5) have seemed to point anergy in this review as defective proliferation in response to toward a thymic origin for the increased self-Ag–specific Treg pMHCII recognition by CD4 T cells. cells observed when tissue-restricted Ag expression in the Multiple biochemical signaling defects have been ascribed to thymus is only modest. Vil1 promoter–driven Cre-specific the CD4+ T cell anergic state, including blocked signaling to Treg cells express high levels of Helios and Nrp1, both Ras and MAPKs 1 and 8, and impaired expression of functional markers compatible with a tTreg cell origin (5). In addition, AP-1 transcription factor complexes (21, 33, 34). Upregulation by guest on October 1, 2021 genetic disruption of the CNS1 sequence from the Foxp3 of counterregulatory gene products such as Cblb, Dgka, Rap1, + locus fails to inhibit the generation of Cre-specific Foxp3 Rnf128, Itch, Dtx1,andNdrg1 (which in most cases lie Treg cells or restore the functional responsiveness of the downstream of NFAT- and Egr2/Egr3-dependent trans- + conventional Cre-reactive CD4 T cell population in Vil1- activation) are understood to underlie this development of + Cre transgenic mice (5). Interestingly, Foxp3 Treg cells spe- functional unresponsiveness (35–39). Despite the presence of cific for eGFP are more frequently detected in the thymus of multiple counterregulatory molecules and signal transduction Ins2 promoter–driven eGFP transgenic mice (4), whereas the defects, anergic CD4+ T cells retain their capacity to recognize number of Cre-specific Treg cells in Vil1-Cre and Scgb1a1-Cre pMHCII and undergo anergy reversal in its absence (29). transgenic mice is similar to the wild type (5). Taken together, Anergy is also reversed in the setting of T cell lymphopenia, the experiments suggest that weak expression of self-Ag in regardless of the continued presence of Ag (40). This may the thymus (presumably as a consequence of Aire-mediated relate to Treg cell deficiency, as well as to increased availability transcription in medullary thymic epithelial cells) is insuffi- of homeostatic cytokines in lymphopenic hosts. Consistent cient to elicit central deletion of all high-affinity autoreactive with this, anergy induction and maintenance are strongly + CD4 T cells, yet facilitates the maintenance of self-tolerance antagonized by IL-2R signaling that leads to downstream by inducing the differentiation of highly functional self PI3K, Akt, and mTORC1 activation, and that promotes a pMHCII-specific Treg cells that suppress peripheral responses. shift in metabolism away from oxidative phosphorylation and In contrast, CNS1-dependent pTreg cells may be more im- toward aerobic glycolysis (41, 42). The activation, clonal ex- portant to suppress mucosal T cell reactivity to food and pansion, and differentiation of aggressive CD4+ T effector commensal Ags introduced only after thymic development cells is associated with unique metabolic demands that require has completed. mTORC1 activity to integrate environmental, nutrient, and intracellular signaling cues (43). If activated CD4+ T cells fail Anergy induction versus pTreg cell generation: differences and to increase their glycolytic metabolism because of inefficient similarities mTORC1 activation, they can become anergic (44). Notably, neither of these elegant studies suggested a role It is worth noting that inhibition of mTORC1 signaling not for anergy in the development of tolerance to peripherally only induces anergy, but also promotes the differentiation of + expressed self-Ags. Should one conclude, therefore, that the Foxp3 Treg cells from conventional precursors and stabilizes the induction and maintenance of anergy are unimportant to expression of Foxp3 (45–47). Not surprisingly, Treg cells them- natural peripheral self-tolerance? Or is it possible that anergy selves appear to be anergic, because they do not produce IL-2 or 2530 BRIEF REVIEWS: ANERGIC T CELLS AS Treg PROGENITORS proliferate when stimulated unless exogenesis IL-2 is provided. conventional polyclonal CD4+ Tcellswerefoundtobenaive Nonetheless, mTORC1 activity is also necessary for proliferation in phenotype and generally ignorant of self-Ag (49, 50), con- by Treg cells. In fact, it has been suggested that alternate acti- sistent with the results of Legoux et al. (5) and Malhotra et al. vation and subsequent inhibition of mTORC1 facilitate optimal (4). In contrast, conventional InsB/I-Ag7 tetramer-binding Treg cell function and lineage stabilization (48). polyclonal T cells isolated from the pancreas-draining lymph Our own investigations have led to the discovery of two node of nondiabetic NOD mice demonstrated: 1) evidence of additional anergy factors: ecto 59-nucleotidase (Nt5e; hereafter previous Ag recognition (increased CD44 expression); 2) referred to as CD73) and folate receptor 4 (FR4; Izumo1r). upregulation of FR4 and CD73; and 3) defective IFN-g Both molecules are found expressed at moderate levels on production, all consistent with the induction of anergy (49, + g7 + Foxp3 Treg cells (as well as at lower levels on conventional 50). Notably, InsB/I-A –specific Foxp3 Treg cells were also + CD4 naive T cells and T follicular helper cells), but CD73 identified and found to be similar in number in both synge- and FR4 are most highly expressed on conventional Ag- neic strains. These data, therefore, generally supported the hi + experienced CD44 CD4 T cells after the induction of model that normal naive CD4+ T cells often ignore tissue- anergy (31, 49, 50). Little is understood about the function of restricted self-Ags in the presence of a stable self-Ag–specific FR4 on T cells; however, the gene encoding FR4 (Izumo1r) Treg cell compartment. Nonetheless, NOD mice predisposed was recently shown to encode Juno, the receptor for Izumo1 to autoimmune disease development apparently reveal InsB/ (51). In reproductive biology, Izumo1 expression on sperm I-Ag7 complexestothenaiveperipheralTcellrepertoire; and Juno on eggs guide normal sperm–egg fusion during then the anergy mechanism becomes available to maintain Downloaded from fertilization (51). It remains unclear how the expression of self-tolerance. FR4 relates to anergy. Of course, these observations in disease-prone NOD mice CD73 is an ectoenzyme that normally acts in tandem with begged the question whether anergy develops only when other CD39 to convert extracellular ATP to adenosine (52). Ex- immune tolerance mechanisms fail. This point was addressed tracellular ATP is secreted by activated T cells and also ac- in a series of experiments that examined fetal tolerance in cumulates at sites of tissue ischemia and necrosis (53–56). healthy B6 pregnant mice, after mating to syngeneic B6 males http://www.jimmunol.org/ Although the exact mechanisms remain unclear, it is thought made transgenic for a ubiquitously expressed 2W self-Ag (62, that the CD39/CD73-mediated depletion of extracellular + 63). Clonal expansion of 2W pMHCII-specific Foxp3 Treg ATP limits the triggering of purinogenic receptors such as cells is known to be necessary for fetal success in this system P2X7. Activation of P2X7 in some systems stabilizes the ex- (64), with Foxp3 expression apparently stabilized by the a pression of the glycolytic gene positive regulator Hif1 (57). CNS1 enhancer element (65). Given that the niche for Treg CD73-dependent production of extracellular adenosine may cells having any particular Ag specificity appears limited (66, also serve to resist glycolytic reprogramming through the 67) and the observations of similar metabolic programming suppressive effects of the adenosine A2a receptor and its in- for both the anergic and Treg fates (48), we hypothesized that

+ by guest on October 1, 2021 tracellular second messenger cAMP on mTORC1 activity during pregnancy both anergic T cells and Foxp3 Treg cells (54). Note that CD39 is not expressed on anergic conven- will be present among the 2W-specific T cells. 2W/I-Ab + tional CD4 T cells, and the role that CD73 plays in the tetramer-binding CD4+ T cells were observed to undergo a induction or maintenance of anergy remains uncertain. 5-fold clonal expansion during pregnancy that resulted in Nevertheless, these findings may indicate one mechanism by approximately equal numbers of unresponsive CD44hi FR4hi + hi + which CD39 Treg cells facilitate the induction of anergy CD73 anergic T cells and Foxp3 Treg cells (49). Interest- through their conversion of extracellular ATP to the more ingly, most of the anergic compartment disappeared during tolerogenic nucleotide adenosine (58, 59). the postpartum period, perhaps reflecting a requirement for b + continuous TCR recognition of fetus-derived 2W/I-A com- An anergic polyclonal CD4 T cell compartment in healthy mice plexes to maintain anergy or cell survival. Therefore, normal Tissue-restricted expression of Aire-regulated transgenic model pregnancy is associated with CD4+ T cell anergy to fetal Ags. + hi hi Ags has clearly defined self pMHCII-specific Foxp3 Treg cells The discovery of functionally unresponsive CD44 FR4 as an important barrier to peripheral self-reactivity by CD4+ CD73hi CD4+ T cells specific for self InsB/I-Ag7 or fetal 2W/ T cells, whereas no evidence for peripheral anergy induction I-Ab complexes lends support to the notion that peripheral was obtained. Nonetheless, extrathymic Aire-regulated ex- self-tolerance can rely on CD4+ T cell anergy. To investigate pression of self-Ags within secondary lymphoid organs has the generality of these observations, we characterized the also been shown to functionally inactivate naive conventional natural repertoire of polyclonal conventional CD4+ T cells autoreactive CD4+ TCR-transgenic T cells, as well as promote that express these anergy markers in combination. A sub- hi hi hi 2 the accumulation of self-Ag–specific Treg cells (60). We have population of CD44 FR4 CD73 Foxp3 cells was found questioned whether the detection of naturally occurring to make up 2–5% of the polyclonal CD4+ T cell repertoire in anergic CD4+ T cells has been hindered by the lack of sen- the secondary lymphoid organs (but not thymus) of multiple sitive and specific markers for detection of anergic T cells in normal mouse strains, and this subpopulation increased with the polyclonal repertoire. age (49). Furthermore, expression of these markers was found Our previous discovery of CD73 and FR4 as reliable surface to strongly correlate with proliferative arrest and defective markers of anergic conventional T cells, together with the cytokine production, the two hallmarks of anergy. Finally, development of Ag tetramer technologies by our collaborators, loss of Aire-dependent gene expression and central deletion in 2 2 offered us the opportunity to thoroughly investigate tolerant the thymus of mutant Aire / mice led to an increase in the polyclonal CD4+ T cells (31, 49, 50, 61). In healthy B6.g7 proportion of peripheral CD4+ T cells that have this anergic mice, insulin B chain (InsB) pMHCII tetramer-binding phenotype (49). The Journal of Immunology 2531

Evidence of anergy in this polyclonal CD44hi FR4hi CD73hi that are specific for peripheral self pMHCII complexes, but CD4+ T cell compartment cannot be taken as proof of self-Ag these TCRs also make them ideal progenitor cells for the + reactivity. However, loss of the anergic phenotype in fetal Ag- peripheral differentiation of Foxp3 Treg cells. Furthermore, specific T cells postpartum after the expulsion of fetal tissues these experiments indicate that anergy-derived Treg cells cannot did suggest a requirement for continuous TCR engagement to be readily distinguished from tTreg cells based on phenotype or + maintain the anergic state (49). Consistent with this, steady- Treg-me. Therefore, it is conceivable that some of the Nrp1 + state polyclonal anergic T cells were shown to express high Foxp3 Treg cells that preferentially expanded during the course levels of PD-1, CTLA4, CD69, and Nrp1, all molecules of self-Ag immunization by Malhotra et al. (4) and Legoux whose expression can be induced and/or maintained by per- et al. (5) were originally conventional CD4+ T cells that had sistent TCR engagement. Uniformly increased levels of CD5 undergone anergy induction after peripheral recognition of and a Nur77 reporter gene in anergic T cells also suggested the same tissue-restricted self-Ag. that these cells have high-affinity TCRs specific for available self pMHCII complexes, similar to bona fide self-Ag–specific Role of Nrp1 in the generation of anergy-derived Treg cell progenitors Treg cells (68–70). In addition, the frequency and number of Nrp1 is a transmembrane glycoprotein on the surface of many anergic cells was not different between germ-free mice and cell types, including Treg cells, dendritic cells (DCs), NKT cells, specific pathogen-free mice from our colony, suggesting that neurons, and endothelial cells (71, 72, 75, 76), and its function the functional inactivation observed here was not solely in is important for axonal guidance in the developing nervous response to commensal Ags (L.A. Kalekar, unpublished ob- system (77). Nrp1 is a coreceptor for the soluble class 3 sem- Downloaded from servations). Therefore, we now hypothesize that many or all aphorins (78), but can also promote angiogenesis by binding to + anergic phenotype CD4 T cells in secondary lymphoid or- vascular endothelial growth factor (76). More recently, Nrp1+ gans have recently recognized self pMHCII. Treg cells have been shown to bind to semaphorin-4a on plasmacytoid DCs, with subsequent recruitment of PTEN Anergy reversal can result in immunopathology or alternatively lead to and inhibition of downstream Akt and mTORC1 signaling protective Treg cell differentiation pathways (79, 80). The stability of the T cell lineage is reg http://www.jimmunol.org/ Additional experiments were designed to formally test the self- maintained by a Nrp1:semaphorin-4a axis, and Nrp1 can + reactivity of anergic phenotype CD4 T cells by transferring induce the expression of Treg cell lineage-related genes in- 2 2 them into autoimmune disease–prone Tcra / mice and dependently of Foxp3 (79, 81). observing for the development of immunopathology. Initial As described earlier, a majority of polyclonal anergic CD4+ experiments failed to demonstrate reliable autoimmune dis- T cells express high levels of Nrp1 and demonstrate a unique ease development, but instead led to the discovery that pattern of partial Treg-me DNA demethylation. Consistent polyclonal anergic T cells can transdifferentiate into Foxp3+ with this coordinate expression of Nrp1 and demethylation of + + + Treg cells (49). After the adoptive transfer of highly purified the Treg-me, FR4 CD73 anergic CD4 T cells sorted for

2 2 2 by guest on October 1, 2021 Foxp3 CD44hi FR4hi CD73hi CD4+ T cells into lympho- high Nrp1 expression and transferred to lymphopenic Tcra / 2 2 penic Tcra / mice, as many as 25% of the resulting pe- hosts were found to be most efficient for the generation of + + 2 ripheral CD4 T cells expressed Foxp3. Analogous to tTreg Foxp3 Treg cells (49). Conversely, the Nrp1 fraction of cells that experience persistent high-affinity TCR engage- anergic T cells was found to be a poor source of Treg cell ments during their differentiation in the thymus, most of progenitors, and preferentially differentiated toward a Th17 these anergy-derived Treg cells also expressed Nrp1 and dem- lineage that caused wasting disease after adoptive transfer to 2/2 onstrated a fully demethylated Treg-me (including the Foxp3 Tcra mice (49) (L.A. Kalekar, unpublished observations). CNS2). A previous study similarly showed that autoreactive CD4+ Nrp1 was originally thought to distinguish tTreg cells from T cells lacking Nrp1 would induce a more severe form of 2 peripherally differentiated pTreg cells (71, 72). However, this experimental autoimmune encephalitis, with their Nrp1 + notion has since been challenged because activated Treg cells CD4 T cells also appearing biased toward the Th17 lineage (which include both tTreg cells and pTreg cells), as well as (80). Loss of Nrp1 expression on Treg cells limits the nuclear + inducible Treg cells generated in vitro from naive CD4 localization of Foxo1/3a, leads to a failure of Foxp3 expres- T cells, can express Nrp1 (73, 74). Similar to natural poly- sion, and allows for the upregulation of transcription factors clonal Treg cells, anergy-derived polyclonal Treg cells demon- such as RORgt that facilitate Th17 differentiation (79). Thus, 2 2 2 strated an ability to protect lymphopenic Tcra / mice from the skewing of Nrp1 polyclonal anergic CD4+ T cells toward inflammatory bowel disease (49). Anergy-derived Treg cells a Th17 fate suggests that a similar Nrp1-directed genetic also suppressed the development of autoimmune arthritis and program may govern lineage selection in both anergic T cells demonstrated a capacity to induce anergy in other self-Ag– and Treg cells. specific CD4+ T cells (49). It remains unclear whether Nrp1 expression is essential for 2 2 Interestingly, the treatment of Tcra / recipients of the induction and/or the maintenance of the anergic pheno- Foxp3DTR anergic T cells with diphtheria toxin to destroy any type. Nrp1 was previously implicated in immunological developing Foxp3-expressing cells not only prevented the ac- synapse formation, and on Treg cells Nrp1 enhances the du- cumulation of anergy-derived Treg cells, but also led to the ration of the Treg interactions, with DCs resulting in higher development of severe wasting disease and the generation of sensitivity to small amounts of Ag (82). How the TCR rep- tissue-specific autoantibodies, further demonstrating the self- ertoire and self pMHCII specificity of anergic T cells relates to reactivity of naturally anergic polyclonal CD4+ T cells (49). this expression of Nrp1 remains uncertain. Nrp1 has also been Taken together, the data suggest that anergic phenotype shown to directly induce the expression of CD73 in Treg cells polyclonal CD4+ T cells have potentially dangerous TCRs (79). Nevertheless, our analysis of fetal Ag-specific anergic 2532 BRIEF REVIEWS: ANERGIC T CELLS AS Treg PROGENITORS

T cells suggested that Nrp1 is expressed only after the anergic hi hi Disclosures phenotype (CD73 FR4 ) is established. Only half of fetal The authors have no financial conflicts of interest. Ag-specific FR4hi CD73hi CD4+ T cells expressed Nrp1 at day 10 of gestation, whereas Nrp1 expression on anergic References phenotype T cells increased to 80–90% by day 18 (49) (L.A. 1. Klein, L., B. Kyewski, P. M. Allen, and K. A. Hogquist. 2014. Positive and negative Kalekar, unpublished observations). Thus, the level of Nrp1 selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol. 14: 377–391. on anergic T cells may simply indicate the degree of unre- 2. Mueller, D. L. 2010. Mechanisms maintaining peripheral tolerance. Nat. Immunol. sponsiveness. Alternatively, Nrp1 expression on anergic T cells 11: 21–27. may reinforce longer interactions with self pMHCII on DCs to 3. Davis, M. M. 2015. Not-so-negative selection. Immunity 43: 833–835. 4. Malhotra, D., J. L. 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