Human pregnancy levels of estrogen and progesterone contribute to humoral immunity by activating TFH/B cell axis

Circulating T_FH (cT_FH) cells express CXCR5, PD-1, and, when activated, ICOS, and release IL-21. According to the production of IFN-γ, IL-4, and IL-17 and expression of FoxP3, these cells are also classified as cT_FH1, cT_FH2, cT_FH17, and cT_FR cells, respectively. This CD4⁺T-cell subset is pivotal to efficient humoral immunity, and pregnancy appears to favor IgG production. Here, not only pregnancy amplified the in vivo production of anti-HBsAg IgG in HBV immunized women, but the frequency of cT_FH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-β-estradiol (E2) directly increased the percentage of different cT_FH subsets. While E2 and progesterone (P4) increased the proportion of differentiated T_FH cells derived from naïve CD4⁺T-cells, only E2 amplified the release of IL-21 in those cell cultures. In addition, E2 and P4 increased the proportion of memory B cells and plasma cells, respectively. In SEB-activated B/T_FH cell co-cultures, E2, in the presence of P4, increased the production of total IgG. Finally, among the hormones, P4 was stronger in upregulating the percentage of IL-10⁺T_FR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cT_FH and B cell subsets.     

Langerhans cells promote early germinal center formation in response to Leishmania‐derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen‐primed CD4⁺ follicular helper T cells (T_FH). A tight and stable formation of T_FH/B cell conjugates is required for cytokine‐driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of T_FH/B cell conjugates is crucial for B‐cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for T_FH‐cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin⁺ DC subsets in vivo, we show that the functionality of T_FH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC‐depleted mice show a reduction in somatic hypermutation in B cells isolated from T_FH/B cell conjugates and markedly reduced GC reactions within skin‐draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B‐cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.     

Phenotypic analysis of T follicular helper and T follicular regulatory cells in primary selective IgM deficiency

Selective IgM deficiency (SIgMD) is a rare immunodeficiency characterized by serum IgM below two standard of mean, and normal IgG and IgA levels. Both in human and mice with selective IgM deficiency, germinal centers cells are decreased. The development of germinal center and humoral immunity are regulated in part by follicular helper T (T_FH) and follicular regulatory T (T_FR) cells. However, the analysis of circulating T_FH (cT_FH) and T_FR (cT_FR) cells in the pathogenesis of SIgMD has not been explored. We observed lower percentage of cT_FR cells in SIgMD patients than in control group. However, we did not observe any significant difference in the percentage of cT_FH cells and their subsets between both experimental groups. When data were analyzed according to specific antibody response to pneumococcal polysaccharide, we observed a higher percentage of cT_FH cells in SIgMD patients with specific antibody deficiency than in SIgMD patients with normal specific antibody response. Our results suggest that cT_FH cells and their subsets are preserved in SIgMD patients. However, the role of lower percentage of cT_FR cells in the pathogenesis of this immunodeficiency is not clear.     

Roles of follicular helper and regulatory T cells in allergic diseases and allergen immunotherapy

Allergic diseases are characterized by overactive type 2 immune responses to allergens and immunoglobulin E (IgE)-mediated hypersensitivity. Emerging evidence suggests that follicular helper T (T_FH) cells, rather than type 2 T-helper (T_H2) cells, play a crucial role in controlling IgE production. However, follicular regulatory T (T_FR) cells, a specialized subset of regulatory T (T_REG) cells resident in B-cell follicles, restricts T_FH cell-mediated help in extrafollicular antibody production, germinal center (GC) formation, immunoglobulin affinity maturation, and long-lived, high-affinity plasma and memory B-cell differentiation. In mouse models of allergic asthma and food allergy, CXCR5⁺ T_FH cells, not CXCR5⁻ conventional T_H2 cells, are needed to support IgE production, otherwise exacerbated by CXCR5⁺ T_FR cell deletion. Upregulation of T_FH cell activities, including a skewing toward type 2 T_FH (T_FH2) and IL-13 producing T_FH (T_FH13) phenotypes, and defects in T_FR cells have been identified in patients with allergic diseases. Allergen immunotherapy (AIT) reinstates the balance between T_FH and T_FR cells in patients with allergic diseases, resulting in clinical benefits. Collectively, further understanding of T_FH and T_FR cells and their role in the immunopathogenesis of allergic diseases creates opportunities to develop novel therapeutic approaches.     

Flow cytometric analysis of TCR Vβ repertoire in patients with 22q11.2 deletion syndrome

In 22q11.2 deletion patients, the normal decrease in T lymphocyte counts after 1–2 years is blunted such that relatively T lymphocyte numbers increase over early childhood, probably via post‐thymic expansion of peripheral lymphocytes. This may leave less T lymphocyte receptor (TCR) diversity than when derived from naive thymic emigrants. We analysed TCR Vβ repertoire on 27 22q11.2 chromosome deletion patients. No patient had infection at sampling. CD3⁺CD4⁺ recent thymic emigrants (RTEs) were identified by CD45RA and CD31 expression. TCR Vβ repertoire was determined using four‐colour flow cytometry. Patients and controls showed significant TCR Vβ family usage differences between CD3⁺CD4⁺ and CD3⁺CD4^? T lymphocyte subpopulations. Vβ family abnormalities (±3 SD of controls) were identified in 18/27 (67%) patients and 12/47 (25%) controls. In patients, the magnitude of expansions was increased, with some Vβ families representing 37% of the cells present in the subpopulations. There was a significant increase in frequency of abnormalities in CD3⁺CD4⁺ (P < 0.001) and CD3⁺CD4^? T lymphocytes (P < 0.05) in patients. A total of 11/16 patients had an abnormal CD4⁺CD25^Bright TCR Vβ repertoire. There was no difference in expansions/contractions between CD4⁺CD25^Bright and CD4⁺ T lymphocyte repertoires (P = 0.575) for individual patients but significant differences in expansions/contractions between CD4⁺CD25^Bright and CD8⁺ T lymphocytes repertoires (P = 0.011). There was bias in Vβ usage between CD3⁺CD4⁺ and CD3⁺CD4^? T lymphocyte subsets. A total of 67% patients had TCR Vβ repertoire abnormalities, with a trend towards increased repertoire abnormalities with fewer RTEs, suggesting thymic output plays an important role in TCR repertoire diversity. There was no correlation between skewed repertoire and symptoms of infection or autoimmunity.     

Expansion of follicular helper T cells in the absence of Treg cells: Implications for loss of B‐cell anergy

The maintenance of B‐cell anergy is essential to prevent the production of autoantibodies and autoimmunity. However, B‐cell extrinsic mechanisms that regulate B‐cell anergy remain poorly understood. We previously demonstrated that regulatory T (Treg) cells are necessary for the maintenance of B‐cell anergy. We now show that in Treg‐cell‐deficient mice, helper T cells are necessary and sufficient for loss of B‐cell tolerance/anergy. In addition, we show that the absence of Treg cells is associated with an increase in the proportion of CD4⁺ cells that express GL7 and correlated with an increase in germinal center follicular helper T (GC‐T_FH) cells. These GC‐T_FH cells, but not those from Treg‐cell‐sufficient hosts, were sufficient to drive antibody production by anergic B cells. We propose that a function of Treg cells is to prevent the expansion of T_FH cells, especially GC‐T_FH cells, which support autoantibody production.     

The hippo kinase MST1 negatively regulates the differentiation of follicular helper T cells

Follicular helper T (T_FH) cells are essential for inducing germinal centre (GC) reactions to mediate humoral adaptive immunity and antiviral effects, but the mechanisms of T_FH cell differentiation remain unclear. Here, we found that the hippo kinase MST1 is critical for T_FH cell differentiation, GC formation, and antibody production under steady-state conditions and viral infection. MST1 deficiency intrinsically enhanced T_FH cell differentiation and GC reactions in vivo and in vitro. Mechanistically, mTOR and HIF1α signalling is involved in glucose metabolism and increased glycolysis and decreased OXPHOS, which are critically required for MST1 deficiency-directed T_FH cell differentiation. Moreover, upregulated Foxo3 expression is critically responsible for T_FH cell differentiation induced by Mst1^−/−. Thus, our findings identify a previously unrecognized relationship between hippo kinase MST1 signalling and mTOR-HIF1α-metabolic reprogramming coupled with Foxo3 signalling in reprogramming T_FH cell differentiation.     

Identification of novel markers for mouse CD4+ T follicular helper cells

CD4⁺ T follicular helper (T_FH) cells are central for generation of long‐term B‐cell immunity. A defining phenotypic attribute of T_FH cells is the expression of the chemokine R CXCR5, and T_FH cells are typically identified by co‐expression of CXCR5 together with other markers such as PD‐1, ICOS, and Bcl‐6. Herein, we report high‐level expression of the nutrient transporter folate R 4 (FR4) on T_FH cells in acute viral infection. Distinct from the expression profile of conventional T_FH markers, FR4 was highly expressed by naive CD4⁺ T cells, was downregulated after activation and subsequently re‐expressed on T_FH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory T_FH cells. Comparative gene expression profiling of FR4^hi versus FR4^lo Ag‐specific CD4⁺ effector T cells revealed a molecular signature consistent with T_FH and T_H1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto‐enzyme CD73, were enriched in T_FH cells compared with T_H1 cells, and phenotypic analysis confirmed expression of CD73 on T_FH cells. As there is now considerable interest in developing vaccines that would induce optimal T_FH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of T_FH cells following vaccination and infection.     

Evidence for the persistence of monoclonal expansions of CD8+ T cells following primary simian immunodeficiency virus infection

A longitudinal study of the CD8⁺ TCR variable (Vβ) chain repertoire was performed in rhesus macaques experimentally infected with simian immunodeficiency virus (SIV) using both TCR Vβ chain-specific monoclonal antibodies and TCR β chain CDR3 length analysis. Expansions of subpopulations of CD8⁺ T cells were detected during the acute phase of SIV infection. In all monkeys studied, monoclonal expansions persisted for at least 18 months and increasingly dominated the repertoire of CD8⁺ T cells expressing the relevant Vβ chain. This study shows that persistent CD8⁺ T cell expansions develop in response to a virus infection. This is important not only for our understanding of the T cell response to viruses but also for understanding the factors that determine the normal CD8⁺ TCR repertoire.     

High-throughput sequencing reveals restricted TCR Vβ usage and public TCRβ clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes

Islet-reactive memory CD4⁺ T cells are an essential feature of type 1 diabetes (T1D) as they are involved in both spontaneous disease and in its recurrence after islet transplantation. Expansion and enrichment of memory T cells have also been shown in the peripheral blood of diabetic patients. Here, using high-throughput sequencing, we investigated the clonal diversity of the TCRβ repertoire of memory CD4⁺ T cells in the pancreatic lymph nodes (PaLN) of non-obese diabetic (NOD) mice and examined their clonal overlap with islet-infiltrating memory CD4 T cells. Both prediabetic and diabetic NOD mice exhibited a restricted TCRβ repertoire dominated by clones expressing TRBV13-2, TRBV13-1 or TRBV5 gene segments. There is a limited degree of TCRβ overlap between the memory CD4 repertoire of PaLN and pancreas as well as between the prediabetic and diabetic group. However, public TCRβ clonotypes were identified across several individual animals, some of them with sequences similar to the TCRs from the islet-reactive T cells suggesting their antigen-driven expansion. Moreover, the majority of the public clonotypes expressed TRBV13-2 (Vβ8.2) gene segment. Nasal vaccination with an immunodominat peptide derived from the TCR Vβ8.2 chain led to protection from diabetes, suggesting a critical role for Vβ8.2⁺ CD4⁺ memory T cells in T1D. These results suggest that memory CD4⁺ T cells bearing limited dominant TRBV genes contribute to the autoimmune diabetes and can be potentially targeted for intervention in diabetes. Furthermore, our results have important implications for the identification of public T cell clonotypes as potential novel targets for immune manipulation in human T1D.     

T cell development and repertoire of mice expressing a single T cell receptor α chain

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) α chain derived from the H-2D^b -lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these α P14 mice, mice transgenic for the P14 TCR α chain were backcrossed to TCR α-deficient mice. Thymi from α P14 mice exhibited a marked decrease of mature CD4⁺8^? and CD8⁺4^? single-positive thymocytes comparable to thymi from TCR α-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from α P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with Vβ-specific antibodies showed a diverse endogenous TCR Vβ repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR α chain.     

Analysis of T cell receptor Vβ diversity in peripheral CD4+ and CD8+ T lymphocytes in patients with autoimmune thyroid diseases

Autoimmune thyroid diseases are characterized by intrathyroidal infiltration of CD4⁺ and CD8⁺ T lymphocytes reactive to self‐thyroid antigens. Early studies analysing T cell receptor (TCR) Vα gene usage have shown oligoclonal expansion of intrathyroidal T lymphocytes but not peripheral blood T cells. However, TCR Vβ diversity of the isolated CD4⁺ and CD8⁺ T cell compartments in the peripheral blood has not been characterized fully in these patients. We performed complementarity‐determining region 3 (CDR3) spectratyping as well as flow cytometric analysis for the TCR Vβ repertoire in peripheral CD4⁺ and CD8⁺ T cells from 13 patients with Graves' disease and 17 patients with Hashimoto's thyroiditis. Polyclonal TCR Vβ repertoire was demonstrated by flow cytometry in both diseases. In contrast, CDR3 spectratyping showed significantly higher skewing of TCR Vβ in peripheral CD8⁺ T cells but not CD4⁺ T cells among patients with Hashimoto's thyroiditis compared with healthy adults. We found trends towards a more skewed CDR3 size distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves' disease exhibited no skewing both in CD4⁺ and CD8⁺ T cells. These findings indicate that clonal expansion of CD8⁺ T cells in Hashimoto's thyroiditis can be detected in peripheral blood and may support the role of CD8⁺ T cells in cell‐mediated autoimmune attacks on the thyroid gland in Hashimoto's thyroiditis.     

The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)⁺ T regulatory (T_reg) cells and T helper type 1 (Th1) cells. Murine CD4⁺ T cells isolated from Ptpn22 knock‐out (Ptpn22^KO) mice cultured in T_reg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild‐type (WT) cells, and subsequently reduced FoxP3 expression at optimal‐to‐high levels of activation. However, at lower levels of TCR activation, Ptpn22^KO CD4⁺ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock‐down by specific oligonucleotides compromises the differentiation of naive CD4⁺ T cells into T_reg cells. Notably, in vivo T_reg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of T_reg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22^KO mice, thus suggesting PTPN22 as a FoxP3‐specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3⁺ T_reg cell differentiation.     

Oligoclonal T cell expansions in patients with Behçet's disease

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vβ gene products in CD4⁺ and CD8⁺ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4⁺ T cell compartment, oligoclonal TCR Vβ expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8⁺ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vβ5.1 subset was observed in five BD patients among CD8⁺ T cells. Other elevations of TCR Vβ subsets were heterogeneously distributed with one to three different Vβ subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vβ group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments.     

Preselection TCR repertoire predicts CD4+ and CD8+ T-cell differentiation state

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4⁺ naive, CD4⁺ memory, CD8⁺ naive and CD8⁺ memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4⁺ and CD8⁺ T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation.     

TFH cells in bystander and cognate interactions with B cells

Follicular T‐helper (T_FH) cells play a crucial role in three aspects of the germinal center (GC) response. They promote GC formation, arbitrate competition among GC B cells to determine the outcome of affinity maturation, and regulate GC output of memory and plasma cells to shape the long‐lived humoral immune memory. Of fundamental importance are dynamic physical interactions between T_FH and B cells, which are the main platform for T_FH cells to deliver “help” factors to B cells and also for reciprocal signaling from B cells to maintain the helper state of T_FH cells. Recent work has significantly expanded our understanding of how T‐B interactions are spatiotemporally regulated and molecularly orchestrated to fulfill those T_FH functions. In this review, we elaborate two modes of T‐B interactions, the antigen‐specific or cognate mode in which T_FH cells engage individual antigen‐presenting B cells and the antigen nonspecific bystander mode in which T_FH cells are engaged with the ensemble of follicular B cells. We discuss findings that indicate how short‐lived cognate T‐B contacts coupled with an intercellular positive feedback drive affinity‐based selection and how bystander interactions between T and B cells regulate follicular T‐cell recruitment and maintenance of an appropriate helper state. We argue that this combination of bystander and cognate interactions with B cells constantly shapes the internal state of T_FH cells and provides the platform to execute their helper functions.     

Central tolerance spares the private high‐avidity CD4+ T‐cell repertoire specific for an islet antigen in NOD mice

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4⁺ T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes‐resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β_1‐15 epitope, as compared to wild‐type NOD mice, due to intrathymic negative selection. In both TSSP‐deficient and wild‐type NOD mice, the TCRαβ repertoire of S100β‐specific CD4⁺ T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate‐ to low‐avidity. In contrast, high‐avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild‐type NOD mice. Hence, in NOD mice, the autoreactive CD4⁺ T‐cell compartment has two major components, a dominant and public low‐avidity TCRα repertoire and a private high‐avidity CD4⁺ T‐cell repertoire; the latter is deleted by re‐enforced negative selection.     

Diversity and clonotypic composition of influenza‐specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.