Ana Agua-Doce

Regulation of the Germinal Center Reaction by Foxp3+ Follicular Regulatory T Cells

Follicular helper T (TFH) cells participate in humoral responses providing selection signals to germinal center B cells. Recently, expression of CXCR5, PD-1, and the transcription factor Bcl-6 has allowed the identification of TFH cells. We found that a proportion of follicular T cells, with phenotypic characteristics of TFH cells and expressing Foxp3, are recruited during the course of a germinal center (GC) reaction. These Foxp3+ cells derive from natural regulatory T cells. To establish the in vivo physiologic importance of Foxp3+ follicular T cells, we used CXCR5-deficient Foxp3+ cells, which do not have access to the follicular region. Adoptive cell transfers of CXCR5-deficient Foxp3+ cells have shown that Foxp3+ follicular T cells are important regulators of the GC reaction following immunization with a thymus-dependent Ag. Our in vivo data show that Foxp3+ follicular T cells can limit the magnitude of the GC reaction and also the amount of secreted Ag-specific IgM, IgG1, IgG2b, and IgA. Therefore, Foxp3+ follicular regulatory T cells appear to combine characteristics of TFH and regulatory T cells for the control of humoral immune responses.

Identification of Regulatory Foxp3+ Invariant NKT Cells Induced by TGF-β

Invariant NKT (iNKT) cells were shown to prevent the onset of experimental autoimmune encephalomyelitis in mice following administration of their specific TCR agonist α-galactosylceramide. We found that this protection was associated with the emergence of a Foxp3+ iNKT cell population in cervical lymph nodes. We demonstrate that the differentiation of these cells is critically dependent on TGF-β in both mice and humans. Moreover, in vivo generation of Foxp3+ iNKT cells was observed in the TGF-β–rich environment of the murine gut. Foxp3+ iNKT cells displayed a phenotype similar to that of Foxp3+ regulatory T cells, and they suppress through a contact-dependent, glucocorticoid-induced TNFR-mediated mechanism. Nevertheless, Foxp3+ iNKT cells retain distinctive NKT cell characteristics, such as promyelocytic leukemia zinc finger protein expression and preferential homing to the liver following adoptive transfer, where they stably maintained Foxp3 expression. Our data thus unveil an unexpected capacity of iNKT cells to acquire regulatory functions that may contribute to the establishment of immunological tolerance.

Induced IL-17–Producing Invariant NKT Cells Require Activation in Presence of TGF-β and IL-1β

IL-17 production by innate-like lymphocytes, including γδ and invariant NKT (iNKT) cells, have been ascribed to specific lineages that are endowed with this functional specialization during thymic differentiation. IL-17–producing iNKT cells have been described as a CD4−NK1.1− lineage in mice and CD161+ in humans. We found that, in mice, noncommitted iNKT cells can be induced to produce IL-17 when activated in presence of TGF-β and IL-1β. This peripheral induction of IL-17 expression could be observed in any subset irrespectively of CD4 and NK1.1 expression, the process leading to loss of NK1.1 expression and partial CD4 downmodulation. Furthermore, induced IL-17–producing iNKT cells were sufficient to drive neutrophilic airways inflammation upon intratracheal adoptive cell transfer into congenic mice. Taken together, our data show that similarly to regulatory T cells, which have a natural and peripherally induced subset, IL-17 production by iNKT cells can also be imprinted in natural iNKT17 cells or peripherally induced.

Modulation of IL-17 and Foxp3 Expression in the Prevention of Autoimmune Arthritis in Mice

Background Rheumatoid Arthritis (RA) is a chronic immune mediated disease associated with deregulation of many cell types. It has been reported that different T cell subsets have opposite effects in disease pathogenesis, in particular Th17 and Treg cells. Methodology and Findings We investigated whether non-depleting anti-CD4 monoclonal antibodies, which have been reported as pro-tolerogenic, can lead to protection from chronic autoimmune arthritis in SKG mice – a recently described animal model of RA – by influencing the Th17/Treg balance. We found that non-depleting anti-CD4 prevented the onset of chronic autoimmune arthritis in SKG mice. Moreover, treated mice were protected from the induction of arthritis up to 60 days following anti-CD4 treatment, while remaining able to mount CD4-dependent immune responses to unrelated antigens. The antibody treatment also prevented disease progression in arthritic mice, although without leading to remission. Protection from arthritis was associated with an increased ratio of Foxp3, and decreased IL-17 producing T cells in the synovia. In vitro assays under Th17-polarizing conditions showed CD4-blockade prevents Th17 polarization, while favoring Foxp3 induction. Conclusions Non-depleting anti-CD4 can therefore induce long-term protection from chronic autoimmune arthritis in SKG mice through reciprocal changes in the frequency of Treg and Th17 cells in peripheral tissues, thus shifting the balance towards immune tolerance.

Monoclonal Anti-CD8 Therapy Induces Disease Amelioration in the K/BxN Mouse Model of Spontaneous Chronic Polyarthritis

OBJECTIVE CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. METHODS CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. RESULTS CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 mAb-treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb-treated mice. In anti-CD8 mAb-treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti-glucose-6-phosphate isomerase antibodies did not change. CONCLUSION These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function

Circulating human T follicular regulatory (Tfr) cells are distinct from tissue Tfr cells. Suppressing Sjögren syndrome T follicular regulatory (Tfr) cells regulate antibody production in the germinal center, yet individuals with the autoimmune disease Sjögren syndrome have increased numbers of circulating Tfr cells compared with healthy individuals. Fonseca et al. compared blood Tfr cells with tissue Tfr cells and found that blood Tfr cells were phenotypically distinct from their tissue counterparts. Moreover, blood Tfr cells did not preferentially suppress humoral responses and had a naïve-like phenotype. These cells were not thymically derived but were generated during germinal center responses, exiting the tissue to enter the blood. These data explain why increased number of blood Tfr cells does not correlate with increased suppression potential and suggest that, instead, increased numbers of blood Tfr cells indicate ongoing humoral activity. Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high-affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cells defined as CXCR5+Foxp3+ T cells remains elusive. We found that blood Tfr cells are increased in Sjögren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titers, as well as in healthy individuals upon influenza vaccination. Although blood Tfr cells correlated with humoral responses, they lack full B cell–suppressive capacity, despite being able to suppress T cell proliferation. Blood Tfr cells have a naïve-like phenotype, although they are absent from human thymus or cord blood. We found that these cells were generated in peripheral lymphoid tissues before T-B interaction, as they are maintained in B cell–deficient patients. Therefore, blood CXCR5+Foxp3+ T cells in human pathology indicate ongoing humoral activity but are not fully competent circulating Tfr cells.

Adjuvant facilitates tolerance induction to factor VIII in hemophilic mice through a Foxp3-independent mechanism that relies on IL-10

Current treatment of hemophilia consists of the administration of recombinant clotting factors, such as factor VIII (FVIII). However, patients with severe hemophilia can mount immune responses targeting therapeutically administered FVIII through inhibitory immunoglobulins that limit treatment efficacy. Induction of immune tolerance to FVIII in hemophilia has been extensively studied but remains an unmet need. We found that nondepleting anti-CD4 monoclonal antibodies (mAbs) are effective in inducing long-term tolerance to FVIII in different strains of hemophilic mice. Tolerance induction was facilitated when anti-CD4 mAbs were administered together with FVIII adsorbed in an adjuvant (alum). The observed state of tolerance was antigen specific, with mice remaining immune competent to respond to different antigens. Importantly, we found that following immunization with FVIII, the primed cells remained susceptible to tolerance induction. Studies with Foxp3-deficient and interleukin 10 (IL-10)-deficient mice demonstrated that the underlying tolerance mechanism is Foxp3 independent but requires IL-10. Our data show that an adjuvant, when administered together with a tolerizing agent such as nondepleting anti-CD4, can facilitate the induction of long-term tolerance to recombinant proteins, possibly not only in hemophilia but also in other diseases that are treated with potentially immunogenic therapeutics.

T Follicular Regulatory Cells in Mice and Men

It has long been known that CD4 T cells are necessary to provide help to B cells, triggering a germinal centre (GC) reaction where affinity maturation and isotype switching occur. However, the nature of the dedicated CD4 helper T cells, known as T follicular helper (Tfh), was only recently described. Here, we review the biology and function of the recently described T follicular regulatory (Tfr) cells, another CD4 T‐cell population also found within GCs but with regulatory function and characteristics. Tfr cells have been identified in mice and humans as simultaneously presenting characteristics of T follicular cells (namely CXCR5 expression) and regulatory T cells (including Foxp3 expression). These Tfr cells have been implicated in the regulation of the magnitude of the GC reaction, as well as in protection from immune‐mediated pathology.

IL-9 Expression by Invariant NKT Cells Is Not Imprinted during Thymic Development

Invariant NKT (iNKT) cell thymic development can lead to distinct committed effector lineages, namely NKT1, NKT2, and NKT17. However, following identification of IL-9–producing iNKT cells involved in mucosal inflammation, their development remains unaddressed. In this study, we report that although thymic iNKT cells from naive mice do not express IL-9, iNKT cell activation in the presence of TGF-β and IL-4 induces IL-9 secretion in murine and human iNKT cells. Acquisition of IL-9 production was observed in different iNKT subsets defined by CD4, NK1.1, and neuropilin-1, indicating that distinct functional subpopulations are receptive to IL-9 polarization. Transcription factor expression kinetics suggest that regulatory mechanisms of IL-9 expression are shared by iNKT and CD4 T cells, with Irf4 and Batf deficiency deeply affecting IL-9 production. Importantly, adoptive transfer of an enriched IL-9+ iNKT cell population leads to exacerbated allergic inflammation in the airways upon intranasal immunization with house dust mite, confirming the ability of IL-9–producing iNKT cells to mediate proinflammatory effects in vivo, as previously reported. Taken together, our data show that peripheral iNKT cells retain the capacity of shaping their function in response to environmental cues, namely TGF-β and IL-4, adopting an IL-9–producing NKT cell phenotype able to mediate proinflammatory effects in vivo, namely granulocyte and mast cell recruitment to the lungs.

Prevention of house dust mite induced allergic airways disease in mice through immune tolerance.

Allergic airways disease is a consequence of a Th2 response to an allergen leading to a series of manifestations such as production of allergen-specific IgE, inflammatory infiltrates in the airways, and airway hyper-reactivity (AHR). Several strategies have been reported for tolerance induction to allergens leading to protection from allergic airways disease. We now show that CD4 blockade at the time of house dust mite sensitization induces antigen-specific tolerance in mice. Tolerance induction is robust enough to be effective in pre-sensitized animals, even in those where AHR was pre-established. Tolerant mice are protected from airways eosinophilia, Th2 lung infiltration, and AHR. Furthermore, anti-CD4 treated mice remain immune competent to mount immune responses, including Th2, to unrelated antigens. Our findings, therefore, describe a strategy for tolerance induction potentially applicable to other immunogenic proteins besides allergens.