Tfh Effector Research Articles

Modeling uncertainty: the impact of noise in T cell differentiation

BackgroundThe regulatory mechanisms guiding CD4 T cell differentiation are complex and are further influenced by intrinsic cell variability along with that of microenvironmental cues, such as cytokine and nutrient availability.ObjectiveThis study aims to expand our understanding of CD4 T cell differentiation by examining the influence of intrinsic noise on cell fate.MethodologyA model based on a complex regulatory network of early signaling events involved in CD4 T cell activation and differentiation was described in terms of a set of stochastic differential equation to assess the effect of noise intensity on differentiation efficiency to the Th1, Th2, Th17, Treg, and TFH effector phenotypes under defined cytokine and nutrient conditions.ResultsThe increase of noise intensity decreases differentiation efficiencies. In a microenvironment of Th1-inducing cytokines and optimal nutrient conditions, noise levels of 3%, 5% and 10% render Th1 differentiation efficiencies of 0.87, 0.76 and 0.62, respectively, underscoring the sensitivity of the network to random variations. Further increments of noise reveal that the network is relatively stable until noise levels of 20%, where the resulting cell phenotypes becomes heterogeneous. Notably, Treg differentiation showed the highest robustness to noise perturbations. A combined Th1-Th2 cytokine environment with optimal nutrient levels induces a dominant Th1 phenotype; however, removal of glutamine shifts the balance towards the Th2 phenotype at all noise levels, with an efficiency similar to that obtained under Th2-only cytokine conditions. Similarly, combinations of Th1/Treg and Treg/Th17-inducing cytokines along with the removal of either tryptophan or oxygen shift the dominant Th1 and Treg phenotypes towards Treg and Th17 respectively. Model results are consistent with differentiation efficiency patterns obtained under well-controlled experimental settings reported in the literature.ConclusionThe stochastic CD4 T cell mathematical model presented here demonstrates a noise-dependent modulation of T cell differentiation induced by cytokines and nutrient availability. Modeling results can be explained by the network topology, which assures that the system will arrive at stable states of cell functionality despite variable levels of biological intrinsic noise. Moreover, the model provides insights into the robustness of the T cell differentiation process.

Effector-Phase IL-2 Signals Drive Th1 Effector and Memory Responses Dependently and Independently of TCF-1.

Following viral infection, CD4+ T cell differentiation is tightly regulated by cytokines and TCR signals. Although most activated CD4+ T cells express IL-2Rα after lymphocytic choriomeningtis virus infection, by day 3 postinfection, only half of activated T cells maintain expression. IL-2Rα at this time point distinguishes precursors for terminally differentiated Th1 cells (IL-2Rαhi) from precursors for Tfh cells and memory T cells (IL-2Rαlo) and is linked to strong TCR signals. In this study, we test whether TCR-dependent IL-2 links the TCR to CD4+ T cell differentiation. We employ a mixture of anti-IL-2 Abs to neutralize IL-2 throughout the primary CD4+ T cell response to lymphocytic choriomeningitis virus infection in mice or only after the establishment of lineage-committed effector cells (day 3 postinfection). We report that IL-2 signals drive the formation of Th1 precursor cells in the early stages of the immune response and sustain Th1 responses during its later stages (after day 3). Effector-stage IL-2 also shapes the composition and function of resulting CD4+ memory T cells. Although IL-2 has been shown previously to drive Th1 differentiation by reducing the activity of the transcriptional repressor TCF-1, we found that sustained IL-2 signals were still required to drive optimal Th1 differentiation even in the absence of TCF-1. Therefore, we concluded that IL-2 plays a central role throughout the effector phase in regulating the balance between Th1 and Tfh effector and memory cells via mechanisms that are both dependent and independent of its role in modulating TCF-1 activity.

A novel memory-like Tfh cell subset is precursor to effector Tfh cells in recall immune responses.

T follicular helper (Tfh) cells, essential for germinal center reactions, are not identical, with different phenotypes reported. Whether, when, and how they generate memory cells is still poorly understood. Here, through single-cell RNA-sequencing analysis of CXCR5+Bcl6+ Tfh cells generated under different conditions, we discovered, in addition to PD-1hi effector Tfh cells, a CD62L+PD1low subpopulation. CD62L-expressing Tfh cells developed independently from PD-1+ cells and not in direct contact with B cells. More importantly, CD62L+ Tfh cells expressed memory- and stemness-associated genes, and with better superior long-term survival, they readily generated PD-1hi cells in the recall response. Finally, KLF2 and IL7R, also highly expressed by CD62L+ Tfh cells, were required to regulate their development. Our work thus demonstrates a novel Tfh memory-like cell subpopulation, which may benefit our understanding of immune responses and diseases.

Distinct Minor Neoplastic Follicular T-Cell Subsets Dictate Lymphoma Fates through Functional Specialization

Background: The presence and role of follicular T-cell populations other than T follicular helper (Tfh) cells, such as T follicular regulatory (Tfr) and cytotoxic (Tfc) cells, are gaining increasing attention in certain pathological states. However, the ecosystem of follicular T cells in the tumor microenvironment (TME) has not been fully elucidated. In particular, the significance of minor follicular T-cell subsets in the neoplastic follicular environment remains elusive. Here, we aimed to reveal the landscape of follicular T-cell alterations in various cancers, with a particular emphasis on the follicular lymphoma (FL) TME. Methods: We analyzed single-cell RNA/TCR sequencing data of >500,000 human T cells from FL (obtained from four cohorts) and 25 other cancer types, as well as homeostatic and reactive lymph nodes (LNs), to construct a comprehensive single-T-cell atlas. We investigated differentially expressed genes, RNA velocity, and TCR clonality using this atlas. To determine the functions of neoplastic follicular regulatory (Tnfr) and cytotoxic (Tnfc) T cells, we performed in vitro cytokineproduction and co-culture assays, in combination with cell activation/suppression, cell division, and apoptosis assays, using human FL samples. With the PhenoCycler-Fusion system, we conducted multiplex digital spatial profiling (DSP) of 169 FL samples from two independent cohorts (now being extended to 242 FL samples from three cohorts) for >25 antibodies. We also performed single-cell spatial and protein expression profiling and prognostic analysis. Results: In FL, distinct minor neoplastic follicular T-cell subsets-Tnfr and CD4 (Tnfc4) and CD8 (Tnfc8) Tnfc cells-increased relative to homeostatic LNs. The TCR repertoire analysis revealed that Tnfr cells shared clonotypes with conventional effector regulatory T (Trg) and Tfh cells, whereas Tnfc4 and Tnfc8 cells shared clonotypes with Tfh cells and effector and exhausted (Tcex) cytotoxic CD8 T cells, respectively. In line with these findings, the RNA velocity survey suggested that Tnfr, Tnfc4, and Tnfc8 cells originated from Trg, Tfh, and naïve-like CD8 T cells, respectively. Tnfr and Tnfc cells expressed higher levels of effector genes, including those involved in cytokine release, chemokine response, migration, and PD-1 signaling, than their reactive LN counterparts. The pan-cancer survey revealed that Tfr and CD4 Tfc cells were exclusive to FL, whereas the prevalence and gene expression profiles of CD8 Tfc cells varied across cancers. Tnfr cells were marked by abundant expression of IL10 and IL21, whereas Tnfc cells displayed a unique phenotype, as they concomitantly expressed markers of effector Tfh (e.g., CXCL13, CXCR5, and PDCD1), naïve/stem (e.g., CCR7 and TCF7), central memory (e.g., CD27, CD28, and SELL), and tissue-resident memory (e.g., ITGAE) cells. Hierarchical clustering demonstrated that Tnfc8 cells had transcriptional profiles similar to those of melanoma TCF1 +PD-1 +CD8 + stem-like T cells. DSP of FL detected Tnfr and Tnfc cells frequently localized within and around neoplastic follicles, forming a cellular neighborhood that allowed them to interact closely. Tnfr cells were distributed predominantly near Tfh cells. The functional co-culture assays demonstrated that Tnfr cells suppressed Tfh-cell activation and division, thereby inhibiting Tfh-mediated malignant B-cell activation and survival. Tnfc8 cells showed a higher cell division capability than that of Tcex cells, suggesting that Tnfc8 cells function as a pool of CD8 T cells in neoplastic follicles. The prognostic analysis revealed that Tnfr and Tnfc cell proportions correlated with early disease relapse (i.e., POD24) and predicted a significantly longer time-to-relapse ( P <0.05 for Tnfr and <0.001 for Tnfc cells) in FL. In the multivariate analysis, the prognostic impact of these two cell subsets was independent of the FLIPI. The prognostic analysis findings were confirmed using a validation cohort. Conclusions: Our multi-omics approach identified the expansion of minor neoplastic follicular T-cell subsets that carry unique transcriptional and functional profiles and robust prognostic impacts. These findings deepen our understanding of the biological and immunological roles of non-Tfh follicular T cells in the lymphoma TME and highlights their clinical potential for patient risk stratification and future therapeutic interventions.

IL-21-producing effector Tfh cells promote B cell alloimmunity in lymph nodes and kidney allografts.

Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21-producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21-producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21-producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21-producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.

Dysregulation of circulating T follicular helper cell subsets and their potential role in the pathogenesis of syphilis.

The role of the host immune response could be critical in the development of Treponema pallidum (Tp) infection in individuals with latent syphilis. This study aims to investigate the alterations in T follicular helper T (Tfh) cell balance among patients with secondary syphilis and latent syphilis. 30 healthy controls (HCs), 24 secondary syphilis patients and 41 latent syphilis patients were enrolled. The percentages of total Tfh, ICOS+ Tfh, PD-1+ Tfh, resting Tfh, effector Tfh, naïve Tfh, effector memory Tfh, central memory Tfh,Tfh1, Tfh2, and Tfh17 cells in the peripheral blood were all determined by flow cytometry. The percentage of total Tfh cells was significantly higher in secondary syphilis patients compared to HCs across various subsets, including ICOS+ Tfh, PD-1+ Tfh, resting Tfh, effector Tfh, naïve Tfh, effector memory Tfh, central memory Tfh, Tfh1, Tfh2, and Tfh17 cells. However, only the percentages of ICOS+ Tfh and effector memory Tfh cells showed significant increases in secondary syphilis patients and decreases in latent syphilis patients. Furthermore, the PD-1+ Tfh cells, central memory Tfh cells, and Tfh2 cells showed significant increases in latent syphilis patients, whereas naïve Tfh cells and Tfh1 cells exhibited significant decreases in secondary syphilis patients when compared to the HCs. However, no significant change was found in resting Tfh and effector Tfh in HCs and secondary syphilis patients or latent syphilis patients. Dysregulated ICOS+ Tfh or effector memory Tfh cells may play an important role in immune evasion in latent syphilis patients.

The Differentiation and Maintenance of SARS-CoV-2-Specific Follicular Helper T Cells.

Upon acute viral infection, virus-specific CD4+ T cells differentiate into either TH1 cells or follicular helper T (TFH) cells. The molecular pathways governing such bimodal cell fate commitment remain elusive. Additionally, effector virus-specific TFH cells further differentiate into corresponding memory population, which confer long-term protection against re-infection of same viruses by providing immediate help to virus-specific memory B cells. Currently, the molecular mechanisms underlying the long-term maintenance of memory TFH cells are largely unknown. In this review, we discuss current understanding of early differentiation of virus-specific effector TFH cells and long-term maintenance of virus-specific memory TFH cells in mouse models of viral infection and patients of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

BCR Affinity Influences T-B Interactions and B Cell Development in Secondary Lymphoid Organs.

B cells produce high-affinity immunoglobulins (Igs), or antibodies, to eliminate foreign pathogens. Mature, naïve B cells expressing an antigen-specific cell surface Ig, or B cell receptor (BCR), are directed toward either an extrafollicular (EF) or germinal center (GC) response upon antigen binding. B cell interactions with CD4+ pre-T follicular helper (pre-Tfh) cells at the T-B border and effector Tfh cells in the B cell follicle and GC control B cell development in response to antigen. Here, we review recent studies demonstrating the role of B cell receptor (BCR) affinity in modulating T-B interactions and the subsequent differentiation of B cells in the EF and GC response. Overall, these studies demonstrate that B cells expressing high affinity BCRs preferentially differentiate into antibody secreting cells (ASCs) while those expressing low affinity BCRs undergo further affinity maturation or differentiate into memory B cells (MBCs).

T Follicular Regulatory (TFR) Cell Deficiency during Chronic Graft-Versus-Host Disease Is Improved By Low-Dose IL-2 Therapy

Background: Chronic Graft versus Host Disease (cGVHD) reflects the inability to achieve immune tolerance after allogeneic stem cell transplantation (allo-SCT) resulting in the persistence of allo and auto-reactive T cells and B cells. We previously showed that regulatory T cells (Treg) are decreased in patients with cGVHD and may be rescued by low dose IL-2 therapy resulting in improved Treg number and survival and clinical improvement of cGVHD symptoms. More recently, we showed that circulating T follicular helper cells (cTFH) have increased function during cGVHD, thus stimulating B cells to produce auto and allo-antibodies involved in cGVHD. T follicular regulatory cells (TFR) regulate the germinal center (GC) reaction and suppress B cell differentiation and immunoglobulin secretion. TFR reside primarily in lymphoid tissues but also circulate in small numbers. In the present study we characterized the numbers and function of blood CD3+CD4+CD45RA-CD25hiFoxP3+CXCR5+ TFR in healthy donors (HD; N=18) and allo-SCT patients (N=64).Results: Patients undergoing allo-SCT showed a significant reduction of circulating TFR (cTFR) compared to HD (median: 0.08% vs 0.34% of CD4+T respectively, p<10-4). Moreover, patients with active cGVHD (N=37) had significantly lower cTFR frequency compared to patients with no cGVHD (N=15) (median: 0.034 vs 0.18% of CD4+, p=0.0012) and resolved cGVHD (N=12) (median: 0.034 vs 0.14, p=0.02). Interestingly, cTFR values tended to be negatively associated with cGVHD grade (median cTFR %: 0.11 vs 0.029, p=0.004 for mild vs moderate; 0.11 vs 0.012, p=0.005 for mild vs severe; 0.029 vs 0.012, p=0.2 for moderate vs severe). The numeric reduction of cTFR in cGVHD patients appears to be a consequence of decreased survival as attested by lower BCL-2 expression (median: 1831 vs 1939, p=0.037 in active cGVHD and without cGVHD, respectively). This favors the survival of TFH effector cells (CD4+CD45RA-CXCR5+ excluding CD25hiFoxP3+) as measured by the ratio of expression of BCL-2 in effectors/regulators in active compared to no cGVHD (BCL-2 E/R ratio: 1.26 vs 1.14, p=0.0013 respectively). Similar to tonsil TFR, blood TFR exhibit suppressive functions in in vitro assays. Purified TFR were co-cultured with TFH and memory B cells at different ratios. The addition of TFR was associated with a significant reduction of plasmablast (PB) generation (CD27hiCD38hiCD19+ cells) (Figure 1A, t-test: p=0.0087) and IgG production (Figure 1B, t-test: p=0.0087). Human cTFR in vivo display similar suppressive function toward B cells, as analysis of active cGVHD patients revealed an inverse correlation between TFR and PB frequency in the peripheral blood (Spearman test: r = -0.49, p=0.003) (Figure 1C). As IL-2 has been shown to enhance regulatory T cells, we assessed its impact on the GC reaction in vitro . Purified TFH and memory B cells from HD were co-cultured at different doses of IL-2 from 0 to 100 IU/mL, simulating a germinal center reaction in vitro . Low dose IL-2 starting at 10 IU/mL was associated with a suppressive effect on PB generation and IgG production that peaked at 50 IU/mL reflecting the high expression level of IL-2 high affinity receptor (CD25). We also evaluated the capacity of IL-2 to restore in vivo cTFR:cTFH ratio using mass cytometry (CyTOF) in a cohort of patients receiving IL-2 (1x106 IU/M2/day) for 12 weeks as previously described (Koreth et al, Blood 2016). We observed an improvement of blood cTFR:cTFH ratio over the course of IL-2 therapy, with a maximum ratio observed at 2 weeks, and subsequently decreasing to reach baseline values after stopping IL-2 (Figure 2).Conclusion: Our results confirm that blood TFR exert a suppressive function as evidence by an inverse correlation with PB and in vitro suppression of memory B cell Ig production. This suppressive subset is deficient in patients with cGVHD due to altered TFR survival, thus favoring imbalance toward TFH effectors which may promote a GC reaction in vivo resulting in PB and memory B cell pathogenic Ig production. By enhancing TFR, low-dose IL-2 therapy promotes effective control of the GC reaction. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Millennium Pharmaceuticals: Research Funding; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Roche: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sigma Tau: Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Tensha: Research Funding; Genmab: Consultancy. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Helios expression in CD4+Foxp3− T cells

Abstract The transcription factor Helios is expressed in the majority of Foxp3+ Treg cells, where it is necessary for the maintenance of self-tolerance. Mice with a Foxp3-specific deficiency in Helios develop systemic immune activation with increased T follicular helper (Tfh) and T helper 1 (Th1) effector responses. Notably, mice with a CD4-specific deletion of Helios do not exhibit autoimmunity, pointing to an additional role for Helios in CD4+ T cells. In fact, Helios is expressed in a minor population of conventional CD4+ T cells and its expression is upregulated upon activation of CD4+ T cells, yet the role of Helios in these cells remains unknown. We generated TCR Tg mice with a CD4-specific Helios deletion and have shown that Helios deficient naive CD4+ T cells are fully able to differentiate into Th1, Th2 and Tfh effector cells in vivo. Helios deficient CD4+T cells show no defect in expansion or the production of cytokines. However, Helios deficiency increased the propensity of naive T cells to differentiate into antigen-specific pTregs under inflammatory as well as tolerizing conditions and attenuated the induction of a memory response. To further characterize CD4+Foxp3−Helios+ cells, Next-generation sequencing (NGS) analysis was performed. This analysis has yielded evidence for a heterogenous population that is comprised of Tfh, Tmem, iNKT and a population of cells that have Treg-like features. In vitro activation assays and qPCR have validated differentially expressed cytokines between Helios+ and Helios− CD4+ T cell populations, in agreement with the NGS data. Together, these findings suggest that Helios may play a role in stabilizing effector function.

Plasma IL-5 but Not CXCL13 Correlates With Neutralization Breadth in HIV-Infected Children.

Children may be the optimal target for HIV vaccine development as they generate substantially more frequent and more potent broadly HIV neutralizing antibodies (bnAbs) than adults. Development of a biomarker that correlates with neutralization breadth in this group could function as a powerful tool to facilitate the development of an HIV vaccine. Previously, we observed that this preferential ability in HIV-infected children over adults to generate bnAbs is associated with an enrichment of circulating follicular helper T-cells (TFH) with an effector phenotype, and the presence of IL-21 secreting HIV-specific TFH within lymphoid tissue germinal centers (GC). In adults, bnAbs development has been linked with high plasma levels of CXCL13, a chemoattractant for CXCR5-expressing TFH cells to the lymph node GC. We sought to test this relationship in HIV-infected children, but found no association between neutralization breadth and plasma levels of CXCL13, or with the Th2 cytokines IL-4 and IL-13, or the TFH associated factor Activin A. However, we did find an unexpected association between plasma IL-5 levels and bnAb development in these children. Importantly, although CXCL13 correlated with total circulating TFH cells, it was not associated with effector TFH. Additionally, raised CXCL13 expression was associated with a lower CD4 percentage, higher viral load and a loss of immune function, implying it is associated with progressive disease rather than HIV-specific GC activity in these subjects. Taken together, our data suggests that IL-5 should be evaluated further as a candidate plasma biomarker for HIV neutralization breadth and for monitoring vaccine responses in the pediatric age group.

Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses.

CD4+ T follicular helper (Tfh) cells are essential for inducing efficient humoral responses. T helper polarization is classically orientated by dendritic cells (DCs), which are composed of several subpopulations with distinct functions. Whether human DC subsets display functional specialization for Tfh polarization remains unclear. Here we find that tonsil cDC2 and CD14+ macrophages are the best inducers of Tfh polarization. This ability is intrinsic to the cDC2 lineage but tissue dependent for macrophages. We further show that human Tfh cells comprise two effector states producing either IL-21 or CXCL13. Distinct mechanisms drive the production of Tfh effector molecules, involving IL-12p70 for IL-21 and activin A and TGFβ for CXCL13. Finally, using imaging mass cytometry, we find that tonsil CD14+ macrophages localize in situ in the B cell follicles, where they can interact with Tfh cells. Our results indicate that human lymphoid organ cDC2 and macrophages play complementary roles in the induction of Tfh responses.

Using single-cell RNA-seq to lineage trace effector to memory transitions in CD4+ T cells during malaria

Abstract Hallmark features of adaptive CD4+ T-cells include differentiation into multiple effector T helper (Th) fates and subsequent emergence of memory states. Due to the heterogeneous nature of CD4+ T-cells, determining possible developmental relationships between effector and memory states in vivo has remained a challenge. Previously we employed single-cell RNA-sequencing (scRNA-seq) to delineate the molecular trajectories of Plasmodium-specific TCR transgenic CD4+ T cells during the first week of experimental malaria. We observed a fate bifurcation as cells progressed from an intermediate, activated state to either effector Th1 or T follicular helper (Tfh) cells. My current project tests the hypothesis that CD4+ Th cells directly convert into memory CD4+ T cells during malaria, and aims to define the underlying transcriptomic processes. Tracking endogenous TCR sequences via TraCeR analysis firstly confirmed that sibling clones populated both Th1 and Tfh fates. Next, by comparing transcriptomes, and performing computational lineage tracing of the scRNA-seq data, we observed a direct transition from Th1 and Tfh effector states to memory states. This was characterised by gradual transcriptomic quiescence during the first month of infection, as well as transcriptomic coalescence of Th1 and Tfh fates, but with retention of Th1/Tfh bifurcated structure in memory cells. Finally, premature cure of infection with anti-malarial drugs altered transcriptomic pathways taken by cells towards those with better Th1 recall and germinal centre Tfh phenotypes, respectively. Thus, by examining transcriptional dynamics using scRNA-seq, I am tracing the molecular pathways taken by effector CD4+ T cells towards functional memory states.

IL-10-producing regulatory B cells restrain the T follicular helper cell response in primary Sjögren's syndrome.

Increased numbers of T follicular helper (Tfh) cells have been implicated in the development of autoimmune diseases including primary Sjögren's syndrome (pSS), but how the Tfh cell response is regulated during autoimmune pathogenesis remains largely unclear. Here, we first found negative correlations between IL-10+ regulatory B (Breg) cell numbers and Tfh cell responses and disease activity in patients with pSS and mice with experimental Sjögren's syndrome (ESS). Moreover, we detected high expression of IL-10 receptor on Tfh cells and their precursors in both humans and mice. In culture, IL-10 suppressed human and murine Tfh cell differentiation by promoting STAT5 phosphorylation. By using an adoptive transfer approach and two-photon live imaging, we found significantly increased numbers of Tfh cells with enhanced T cell homing into B cell follicles in the draining cervical lymph nodes of RAG-2-/- mice transferred with IL-10-deficient B cells during ESS development compared with those of RAG-2-/- mice transferred with wild-type B cells. In ESS mice, CD19+CD1dhiCD5+ Breg cells with decreased IL-10 production exhibited severely impaired suppressive effects on T cell proliferation. Consistently, CD19+CD24+CD38hi Breg cells from pSS patients showed significantly reduced IL-10 production with defective inhibitory function in the suppression of autologous Tfh cell expansion. Furthermore, the adoptive transfer of IL-10-producing Breg cells markedly suppressed the Tfh cell response and ameliorated ESS progression in ESS mice. Together, these findings demonstrate a critical role for IL-10-producing Breg cells in restraining the effector Tfh cell response during pSS development.

Increased CCR7loPD-1hiCXCR5+CD4+ T Cells in Peripheral Blood Mononuclear Cells Are Correlated with Immune Activation in Patients with Chronic HBV Infection.

T follicular helper cells (Tfh cells) affect essential immune pathogenesis in chronic hepatitis B virus (HBV) infection. The CCR7loPD-1hi Tfh subset has a partial Tfh effector phenotype and is associated with active Tfh differentiation, whereas the CCR7hiPD-1lo Tfh subset is a resting phenotype. We recruited 20 healthy volunteers and 77 patients with chronic HBV infection, including those in the immune tolerant (IT) phase (n=19), immune clearance (IC) phase (n=20), low replicative (LR) phase (n=18), and reactivation (RA) phase (n=20). The expression of CD4, CXCR5, PD-1, and CCR7 was detected in T cells from peripheral blood by flow cytometry. The frequency of the CCR7loPD-1hi T subset was significantly higher in the patients than in the healthy controls (14.92±4.87% vs 12.23±2.95%, p=0.018). The frequency of this Tfh subset in the IC group (18.42%±3.08) was increased compared with the IT group (11.94±2.87%, p=0.001) and LR group (13.65±4.93%, p=0.031) and was higher in the RA group than in the IT group (16.03±5.37% vs 11.94±2.87%, p=0.030). We observed a weak positive correlation between the CCR7loPD-1hi Tfh subset population and the alanine transaminase (ALT) level (r=0.370, p=0.001). The CCR7loPD-1h Tfh subset in the chronic HBV-infected patients was elevated to various degrees among the different immune phases. CCR7loPD-1hiCXCR5+CD4+ T cells are correlated with the immune status of chronic HBV infection patients and may be developed as a potential indicator for antiviral treatment.

Increased circulating Tfh17 and PD-1+Tfh cells are associated with autoantibodies in Hashimoto’s thyroiditis

Objective: Hashimoto’s thyroiditis (HT) is characterized by autoantibodies targeting the thyroid. Abnormal CD4+CXCR5+T cell levels were previously shown to be associated with HT. However, Tfh cells consist of heterogeneous subpopulations, and which T follicular helper (Tfh) cell subpopulation participates in the pathogenesis of HT remains poorly understood.Methods: Thirty healthy controls (HCs) and 52 HT patients were enrolled in the study. The percentages of Tfh, ICOS+Tfh, PD1+Tfh, Tfh1, Tfh2, Tfh17, effector Tfh, resting Tfh, effector memory Tfh, central memory Tfh, and naïve Tfh cells in the peripheral blood were all determined via flow cytometry, and the associations between the percentages of these cells and thyroid function indices were also investigated.Results: The percentage of Tfh cells was significantly higher in HT patients than in HCs. Examination of the Tfh cell subsets revealed that the percentages of Tfh1, Tfh2, and resting Tfh cells were significantly decreased, while those of the ICOS+Tfh, PD1+Tfh, Tfh17, and effector Tfh cells were significantly increased in HT patients. No significant differences in effector memory, central memory or naïve Tfh cell percentages were noted between the HC and HT groups. Furthermore, the percentage of PD1+Tfh cells was positively correlated with anti-thyroglobulin antibody levels. Most importantly, only Tfh17 cell percentages were positively correlated with anti-thyroglobulin and anti-thyroid peroxidase antibody levels and were negatively correlated serum free T3 and free T4 levels in HT patients.Conclusions: Increased circulating Tfh17 cell and PD1+Tfh percentages are associated with higher autoantibody levels in HT patients, which imply that Tfh17 or PD1+Tfh cells may play a pathogenic role in the development of HT.

CD8 Follicular T Cells Promote B Cell Antibody Class Switch in Autoimmune Disease.

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.

The impact of age and gut microbiota on Th17 and Tfh cells in K/BxN autoimmune arthritis

BackgroundAge is an important risk factor for rheumatoid arthritis (RA), which often develops in middle age. However, how age-associated changes in immunity impact RA is poorly understood. Gut microbiota are known to be involved in the pathogenesis of RA, but the effects of microbiota in older subjects remain mostly unknown.MethodsWe used segmented filamentous bacteria (SFB), a gut commensal species with immunomodulatory effects, and K/BxN mice, a T cell receptor (TCR) transgenic model, to study the effect of age and microbiota on autoimmune arthritis. Comparing young and middle-aged K/BxN T cells of the same TCR specificity allows us to study T cells with an age focus eliminating a key variable: TCR repertoire alteration with age. In addition to joints, we also studied pathological changes in the lung, an important extra-articular RA manifestation. We used flow cytometry to evaluate T follicular helper (Tfh) and T helper 17 (Th17) cells, as they both contribute to autoantibody production, a key disease index in both RA and K/BxN arthritis.ResultsMiddle-aged K/BxN mice had aggravated arthritis and pathological changes in the lung compared to young mice. Middle-aged mice displayed a strong accumulation of Tfh but not Th17 cells, and had defective Th17 differentiation and low expression of interleukin-23, a critical cytokine for Th17 maintenance. Although a soaring Tfh cell population accompanied by robust germinal center B cell responses were found in middle-aged mice, there was decreased cycling of Tfh cells, and SFB only induced the non-Tfh cells to upregulate Bcl-6, the Tfh master transcription factor, in the young but not the middle-aged group. Finally, the accumulated Tfh cells in middle-aged mice had an effector phenotype (CD62LloCD44hi).ConclusionAge-dependent Tfh cell accumulation may play a crucial role in the increased autoimmune disease phenotype in middle-age. SFB, a potent stimulus for inducing Tfh differentiation, fails to promote Tfh differentiation in middle-aged K/BxN mice, suggesting that most of the middle-aged Tfh cells with an effector phenotype are Tfh effector memory cells induced at an earlier age. Our results also indicate that exposure to immunomodulatory commensals may allow the young host to develop an overactive immune system reminiscent of that found in the middle-aged host.

Targeting Antigen to Clec9A Primes Follicular Th Cell Memory Responses Capable of Robust Recall

Targeting Ags to dendritic cell (DC) surface receptors can induce a variety of responses depending on the DC type targeted, the receptor targeted, and the adjuvant used. Clec9A (DNGR-1), which is expressed by CD8(+) DCs, has been shown to bind F-actin exposed on damaged cells. Targeting Ag to this receptor in mice and nonhuman primates induces strong humoral immunity even in the absence of adjuvant, a process seen for a few select DC receptors. In contrast with other receptors, however, targeting Clec9A induces long-lived, affinity-matured Ab responses that are associated with efficient CD4(+) T cell responses shown to possess properties of follicular Th cells (TFH). In this article, we provide definitive evidence that Clec9A targeting promotes the development of TFH by showing that responding CD4 T cells express CXCR5, PD1, the TFH transcription factor Bcl6, and the cytokine IL-21, and that these cells localize to germinal centers. Furthermore, we extend studies from the model Ag OVA to the viral Ag glycoprotein D of HSV-1 and examine the capacity of primed TFH to form functional memory. We show that targeting glycoprotein D to Clec9A even in the absence of adjuvant induced long-lived memory CXCR5(+) PD1(hi) CD4(+) T cells that proliferated extensively upon secondary challenge and rapidly developed into effector TFH. This was associated with enhanced germinal center B cell responses and accelerated Ab production. Our study indicates that targeting Ags to Clec9A in the absence of adjuvant routinely generates TFH responses that form long-lived memory capable of robust secondary TFH responses.

T cell expression of the histone demethylase UTX promotes clearance of a persistent virus infection by modulating Tfh cell differentiation (VIR1P.1155)

Abstract T cell differentiation and memory formation are impacted by epigenetic changes that modulate nucleosome structure to regulate transcription factor accessibility. The enzymes and specific targets that mediate these processes within T cells are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, promotes the formation of the CD4+ T follicular helper cell (Tfh) phenotype during a chronic virus infection. Mice with T cell specific UTX deletion had defects in Tfh cell generation and function that resulted in reduced germinal center responses and virus-specific immunoglobulin production. Furthermore, clearance of the chronic virus infection was specifically dependent on T cell expression of UTX. H3K27me3-ChIP-sequencing and RNA expression analysis revealed a subset of genes that were specifically regulated by UTX via H3K27 demethylation. This group included IL-6Ra, a known regulator of Tfh responses, as well as other genes, which are potentially novel Tfh effector genes. Thus, we identify a critical link between UTX-dependent H3K27 demethylase activity in T cells and immunity to infection.