Tfh-like Cells Research Articles

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells

SummaryMurine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

An Enhanced Expression Level of CXCR3 on Tfh-like Cells from Lupus Skin Lesions Rather Than Lupus Peripheral Blood

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, and the etiopathogenesis is unclear. Follicular helper T (Tfh) cells have been reported as an important pathogenic cell type in SLE. CXCR3 was reported to be decreased on lupus peripheral CD4+T cells. However, the expression level of CCR4, CCR6 and CXCR3 on Tfh-like cells in SLE peripheral blood and skin lesions is unknown. In this study, we detected CCR4, CCR6 and CXCR3 expression level on Tfh-like cells in the peripheral blood and skin lesions from SLE patients and normal controls (NCs). A decreased expression level of CXCR3 on Tfh-like cells was found in lupus peripheral blood. However, an increased CXCR3 expression was observed on total CD4+T and Tfh-like cells from lupus skin lesions. Moreover, we observed a higher expression level of CXCR3 in Tfh cells from human tonsils. These findings indicate that CXCR3 might help Tfh-like cells to migrate into the inflammatory sites.

Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3

BackgroundAutoantibody production against endogenous cellular components is pathogenic feature of systemic lupus erythematosus (SLE). Follicular helper T (TFH) cells aid in B cell differentiation into autoantibody-producing plasma cells (PCs). The IL-6 and IL-21 cytokine-mediated STAT3 signaling are crucial for the differentiation to TFH cells. Niclosamide is an anti-helminthic drug used to treat parasitic infections but also exhibits a therapeutic effect on autoimmune diseases due to its potential immune regulatory effects. In this study, we examined whether niclosamide treatment could relieve lupus-like autoimmunity by modulating the differentiation of TFH cells in two murine models of lupus.Methods10-week-old MRL/lpr mice were orally administered with 100 mg/kg of niclosamide or with 0.5% methylcellulose (MC, vehicle) daily for 7 weeks. TLR7 agonist, resiquimod was topically applied to an ear of 8-week-old C57BL/6 mice 3 times a week for 5 weeks. And they were orally administered with 100 mg/kg of niclosamide or with 0.5% MC daily for 5 weeks. Every mouse was analyzed for lupus nephritis, proteinuria, autoantibodies, immune complex, immune cell subsets at the time of the euthanization.ResultsNiclosamide treatment greatly improved proteinuria, anti-dsDNA antibody levels, immunoglobulin subclass titers, histology of lupus nephritis, and C3 deposition in MRL/lpr and R848-induced mice. In addition, niclosamide inhibited the proportion of TFH cells and PCs in the spleens of these animals, and effectively suppressed differentiation of TFH-like cells and expression of associated genes in vitro.ConclusionsNiclosamide exerted therapeutic effects on murine lupus models by suppressing TFH cells and plasma cells through STAT3 inhibition.

Bronchoalveolar Lavage Fluid Reflects a TH1-CD21low B-Cell Interaction in CVID-Related Interstitial Lung Disease.

BackgroundAbout 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool.ObjectiveTo gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD).MethodsSixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology.ResultsBoth sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (TFH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This TFH-like cell subset is clearly skewed toward TH1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17.ConclusionUnlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of TFH- and TPH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described TH1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

Identification of Follicular T-Cell Subsets in Murine and Human Tissues.

Follicular helper T (Tfh) cells play a key role in B cell activation and differentiation. Within recent years, distinct subsets of follicular T cells, including regulatory and cytotoxic T cells, have been identified. Apart from classical Tfh cells in secondary lymphoid organs, Tfh-like cells are found in chronically inflamed nonlymphoid tissues. Here, we provide protocols to identify different follicular T cell subsets in murine and human tissues by flow cytometry. This chapter also contains an immunization protocol for the induction of large numbers of Tfh cells in mice.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren’s syndrome with ectopic germinal centres and MALT lymphoma

ObjectivesTo explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren’s syndrome (SS)...

Dynamics of NK, CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID-19.

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8+ T, CD4+ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8+ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3+ Tfh‐like cell and CD226+ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS+ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8+ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4+ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.

Abstract LB-352: CD4+ follicular helper-like T cells are key players in anti-tumor immunity

Abstract CD4+ T helper cells (TH) are central orchestrators of an efficient immune response. However, within the tumor microenvironment (TME), it is unknown what type of CD4+ TH cells or CD4 helper-derived signals are essential to generate robust CD8+ CTL and TRM anti-tumor immune responses, which are positively associated with patient prognosis. To determine the nature of CD4+ T cells that provide ‘help' for generating robust anti-tumor CD8+ CTL responses, we profiled the transcriptomes of patient-matched CD4+ and CD8+ T cells present in the TME of treatment-naïve patients with lung cancer and analyzed them jointly using a novel approach, integrated weighted gene correlation network analysis. We found that the follicular helper T cell (TFH) program in CD4+ T cells was strongly associated with proliferation and tissue-residency in CD8+ CTLs. Single-cell transcriptomic analysis demonstrated the presence of tumor-infiltrating TFH-like cells, a subset of which lacked CXCR5 expression but displayed superior functional properties such as provision of CD8+ ‘help', cytotoxicity and proliferation. Because these anti-tumor functions were not previously ascribed to TFH cells which are known to support B cell responses, we validated our findings using independent in vitro and in vivo approaches. Multi-parametric immunohistochemistry showed co-localization of TFH cells with CD8+ TRM cells within tumors, the density of which was positively correlated with that of TFH cells. Adoptive transfer or induction of TFH cells in mouse tumor models resulted in augmented CD8+ CTL responses and impairment of tumor growth, indicating an important role of TFH-like CD4+ T cells in anti-tumor immunity. Furthermore, tumor-infiltrating TFH-like cells expressed PD-1 and were enriched in tumors following checkpoint blockade, suggesting that they may respond to anti-PD-1 therapy in addition to CD8+ CTLs. Our findings provide insights into the molecular identity and functional status of CD4+ T cells within tumors that mount robust CTL responses and uncover potential targets for tumor immunotherapy. Citation Format: Anusha Preethi Ganesan. CD4+ follicular helper-like T cells are key players in anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-352.

Thyroid cancer metastasis is associated with an overabundance of defective follicular helper T cells.

Metastatic thyroid cancers are more difficult to treat and have a significantly worse prognosis than localized thyroid cancers. Previous studies have shown that follicular helperT cells (Tfh) may participate in antitumor immune responses. Here, we investigated the characteristics of Tfh cells in patients with differentiated thyroid cancer (DTC) at various severities, including patients with localized disease, cervical metastasis, and distant metastasis. In circulating CD4 T cells, the proportion of CD4+ CXCR5+ Tfh-like cells was significantly higher in patients with distant metastasis than in healthy controls, patients with local disease, and patients with cervical metastasis. Also, the expression of Tfh cell-associated surface molecules, such as PD-1, ICOS, and BTLA, tended to be higher in patients with cervical and distant metastasis than in healthy controls. However, the expression of secreted molecules, such as IL-10, IL-21, and CXCL13, was significantly lower in patients with distant metastasis than in healthy controls and patients with local disease. Additionally, circulating Tfh-like cells from patients with distant metastasis were less capable of supporting B-cell growth and IgM secretion. We also examined the CD4+ CXCR5+ Tfh-like cells in tumor samples. Tumor-infiltrating Tfh-like cells were highly enriched in the pulmonary metastasis compared to the local tumor and the cervical metastasis. However, tumor-infiltrating Tfh-like cells from pulmonary metastasis displayed higher PD-1, TIM-3, and lower IL-21 expression than those from the local tumor. Together, this study identified that the metastasis of DTC patients was associated with an overabundance of defective Tfh cells.

NonO Is a Novel Co-factor of PRDM1 and Regulates Inflammatory Response in Monocyte Derived-Dendritic Cells.

Proper expression of the transcription factor, Positive regulatory domain 1 (PRDM1), is required for maintaining homeostasis of human monocyte derived-dendritic cells (MO-DCs). The molecular mechanisms and gene targets of PRDM1 in B and T lymphocytes have been identified. However, the function of PRDM1 in dendritic cells (DCs) remains unclear. We investigate co-regulators of PRDM1 in MO-DCs identified by mass spectrometry (MS) and co-immunoprecipitation (Co-IP). Notably, non-POU domain-containing octamer-binding protein (NonO) was found to be a PRDM1 binding protein in the nucleus of MO-DCs. NonO is recruited to the PRDM1 binding site in the promoter region of IL-6. Knockdown of NonO expression by siRNA lessened suppression of IL-6 promoter activity by PRMD1 following LPS stimulation. While NonO binding to PRDM1 was observed in human myeloma cell lines, an effect of NonO on IL-6 expression was not observed. Thus, loss of NonO interrupted the inhibitory effect of PRDM1 on IL-6 expression in MO-DCs, but not plasma cells. Moreover, MO-DCs with low expression of PRDM1 or NonO induce an increased number of IL-21-producing TFH-like cells in vitro. These data suggest that low level of PRDM1 and NonO lead to enhanced activation of MO-DCs and the regulation of MO-DC function by PRDM1 is mediated through cell lineage-specific mechanisms.

Characterization of circulating and cultured Tfh-like cells in sickle cell disease in relation to red blood cell alloimmunization status

BackgroundPeople living with sickle cell disease (SCD) are prone to red blood cell (RBC) alloimmunization. We hypothesized that subjects with alloantibodies (responders) would have differences in circulating T-follicular helper (Tfh)-like cells compared to subjects without alloantibodies (non-responders). Materials and methodsPeripheral blood mononuclear cells were collected from 28 subjects, including those with SCD and controls. Circulating CD4 T-cell subsets were first evaluated at baseline. CD4 T-cell subsets were also evaluated after naïve CD4 T-cells were differentiated into Tfh-like cells following in vitro culture with CD3/CD28 beads, IL-7, IL-12, and Activin A. Transfusion and alloantibody histories were extracted from the electronic medical record. ResultsNon-responders had a lower percentage of CD45RA negative Tmemory cells than responders or controls (p<0.05). Notably, there were no differences in circulating Tfh-like cells between any group. However, naïve CD4 T-cells from subjects with SCD were more likely to express CXCR5 after in vitro culture than cells from controls. After culture, CXCR5 expressing cells from responders were more likely to express PD1 and ICOS (16.43 %, sd. 20.23) compared to non-responders (3.69 %, s.d. 3.09) or controls (2.78 %, s.d. 2.04). DiscussionThe tendency for naïve CD4 T-cells from responders to differentiate into Tfh-like cells after in vitro culture may suggest these cells are prepared to assist B-cells with antibody production regardless of antigen specificity. Further studies are needed, but it is possible that these results may explain why some responders form RBC alloantibodies with multiple specificities, in addition to RBC autoantibodies and HLA alloantibodies.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

Biological characteristics and prognostic value of Tfh-like cells in the tumor tissue of breast cancer.

5 Background: T follicular helper (Tfh) cells formed in germinal center are CXCR5+ICOS+PD-1hiBcl-6+CXCL13+IL-21+CD4+ T cells. They have been identified in different types of tumors. However, it still remains unclear in breast cancer, with one indicating the positive prognostic value of 8-gene Tfh signature. Methods: Freshly resected invasive breast cancer tissues from Fudan University Shanghai Cancer Center of all 386 patients were collected during 03/2015 to 05/2017. Data was analyzed based on the result from Multiparameter Flow Cytometry and IHC. The correlation between Tfh-like cells and clinicopathological characteristics in breast cancer was examined with t test or one way ANOVA with Tukey’s multiple comparisons test. Results: A subpopulation of PD1hiCD4+ T cells in the tumor tissues of breast cancer was identified as Tfh-like cells, with the specific expression of Bcl-6 and CXCL13. Tfh-like cells were of a high level of ICOS but negative for CXCR5. They had high levels of activated molecules such as CD38, CD71, CD95 and HLADR, high levels of suppressive markers as Tim3, TIGIT, and LAG3 and high level of proliferation marker Ki-67, specifically secreting cytokine IL-21 with stimulation. Data showed that high grade (N = 166; mean±SEM, 17.62±0.87) compared with low grade (N = 220; mean±SEM, 12.41±0.46) or high Ki-67 level (N = 300; mean±SEM, 15.38±0.58) with low Ki-67 level (N = 86; mean±SEM, 12.10±0.67), and negative ER expression (N = 115; mean±SEM, 17.47±1.05) compared with positive ER expression (N = 271; mean±SEM, 13.46±0.50) or negative PR expression (N = 177; mean±SEM, 16.87±0.83) compared with positive PR expression (N = 209; mean±SEM, 12.77±0.50) was associated with higher frequency of Tfh-like cells (P &lt; 0.01 to all). Patients with triple negative had higher frequency of Tfh-like cells than those with Luminal A (difference, 6.83; P = 0.0006) and Luminal B (HER2-) (difference, 4.61; P = 0.0124). These results indicate that higher frequency of Tfh-like cells could have negative prognostic influence. Conclusions: Our study defined a PD1hiBcl6+CXCL13+CD4+IL-21+ T cell subpopulation. Higher frequency of Tfh-like cells is more likely to be seen in patients with poor prognosis.

Immunological Fingerprints of Controllers Developing Neutralizing HIV-1 Antibodies

SummaryThe induction of broadly neutralizing antibodies (bnAbs) is highly desired for an effective vaccine against HIV-1. Typically, bnAbs develop in patients with high viremia, but they can also evolve in some untreated HIV-1 controllers with low viral loads. Here, we identify a subgroup of neutralizer-controllers characterized by myeloid DCs (mDCs) with a distinct inflammatory signature and a superior ability to prime T follicular helper (Tfh)-like cells in an STAT4-dependent fashion. This distinct immune profile is associated with a higher frequency of Tfh-like cells in peripheral blood (pTfh) and an enrichment for Tfh-defining genes in circulating CD4+ T cells. Correspondingly, monocytes from this neutralizer controller subgroup upregulate genes encoding for chemotaxis and inflammation, and they secrete high levels of IL-12 in response to TLR stimulation. Our results suggest the existence of multi-compartment immune networks between mDCs, Tfh, and monocytes that may facilitate the development of bnAbs in a subgroup of HIV-1 controllers.

Inhibition of T Helper Cell Differentiation by Tacrolimus or Sirolimus Results in Reduced B-Cell Activation: Effects on T Follicular Helper Cells

The effect of immunosuppressive drugs on the generation of T follicular helper (Tfh) cells, specialized in supporting B-cell differentiation, is largely unknown. We examined whether the calcineurin inhibitor tacrolimus (TAC) and the mammalian target of rapamycin (mtor) inhibitor sirolimus (SRL) inhibit Tfh cell differentiation, and affect subsequent B-cell functions. Isolated naive T cells were polarized into Tfh-like cells in the presence of TAC or SRL. To demonstrate their functionality, we co-cultured these cells with isolated B cells in the presence of alloantigen and studied the activation and differentiation of these B cells. Tfh-like cells were defined as CD4+CXCR5+ T cells, expressing immunoinhibitory programmed death protein 1 (pd1) and inducible T-cell costimulator (icos). We found that TAC and SRL significantly inhibited Tfh-like cell differentiation. Therapeutic concentrations of TAC and SRL reduced the percentage of pd1+ and icos+ Tfh cells compared to controls. In addition, T cells grown in the presence of TAC or SRL expressed less IL-21 and provided less B-cell help. TAC and SRL both inhibited Tfh-dependent alloantigen-activated B-cell proliferation and differentiation into plasma cells and transitional B cells.In conclusion, TAC and SRL inhibited the differentiation of naive T cells into functional Tfh-like cells, a finding that can be extrapolated to immunosuppressive regimens in transplant patients.

Fli-1 Regulates Multiple T-Cell Subsets during Inflammatory Responses and Experimental Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a procedure undertaken to cure hematological malignancies, especially leukemia. Graft-Versus-Host Disease (GVHD) is a serious condition that often appears following allo-HCT. Friend Leukemia Virus Integration 1 (Fli-1) is a transcription factor highly expressed in cancers including erythroleukemia and acute myeloid leukemia while also implicated in pathogenesis of systemic lupus. We have interrogated the role of Fli-1 in T-cell responses by generating a novel T-cell specific conditional disruption of fli-1 where essential exons 3 and 4 of the gene are floxed and excised in the presence of CD4-Cre recombinase. Models of acute GVHD (aGVHD) and chronic GVHD (cGVHD) were tested utilizing hematopoietic cells from mice with a heterozygous (fli-1-/+ or Het) or homozygous (fli-1-/-or Null) disruption of the fli-1 gene, or from wild-type (fli-1+/+ or WT) littermate controls. At baseline, T cells among each of these three mouse strains showed no significant difference in CD44/CD62L expression or CD4+FoxP3+ (nTreg) frequencies. In the cGVHD model, BALB/c (H2Kd) recipients were infused with allogeneic B6 (H2Kb) genotype-respective bone marrow and splenocytes in order to induce cGVHD. Recipients that received fli-1-/+CD4Cre+ marrow and splenocytes demonstrated improved survival and mild cGVHD, whereas those that received fli-1-/-CD4Cre+ or WT donor cells developed serve cGVHD (Fig. 1 a-f). Cellular studies from lymphoid organs of cGVHD allo-HCT recipients revealed that disruption of fli-1 was associated with decreased frequencies of donor CD4+ cells expressing IL-17A, IFN-γ, T follicular-like (TFH-like) cell markers, and CD8+ cells expressing PD-1. In aGVHD settings, donor fli-1-/+CD4Cre+ T cells had a decreased ability to induce aGVHD compared to WT donor T cells and fli-1-/- donor T cells (Fig. 2 a-b). When investigating cellular mechanisms in aGVHD settings, we found that fli-1-/+CD4Cre+ T cells produced significantly lower IFN-γ early after T-cell activation in vivo compared to WT and fli-1-/-CD4Cre+ T cells (Fig. 2 c). To then investigate the role of Fli-1 in T cells beyond GVHD, we utilized a colitis model by transferring naïve CD4+ T cells (CD44-CD25-) into Rag2-/- syngeneic recipients. While fli-1+/+ T cells induced severe colitis as expected, fli-1-/- and fli--/+ T cells caused moderate and mild colitis, respectively. These results were consistent with those observed in GVHD models. To elucidate underlying mechanisms, we tested the effects of Fli-1 on antigen-specific T-cells using a MHC-II restricted TCR transgenic (Tg) mouse strain specific for HY-peptide. CD4+CD25- T cells from fli-1+/+, fli-1-/+, or fli-1-/- CD4Cre+ TCR-Tg mice were polarized in vitro with endogenous antigen presenting cells from spleen in presence of HY-peptide under iTreg- or Th17-polarizing conditions. Both fli-1-/+ and fli-1-/- TCR-tg T cells exhibited significant increases in iTreg (CD4+FoxP3+) frequencies and surface expression of iTreg functional markers (CD25, CD39, CD73, NRP-1), while also having significantly decreased frequencies of IL-17A producing T cells compared to WT-TCR-tg T cells. To further explore the molecular mechanisms, we retrieved fli-1+/+, fli-1-/+, or fli-1-/- donor T cells from recipient spleens after allo-HCT and did RNAseq analysis on these cells. RNAseq data reveals significant differences in mRNA expression within acute inflammatory response and positive regulation of the immune response enrichment pathways between fli-1-/+ T cells and littermate fli-1+/+ T cells, and to a lesser extent in fli-1-/- T cells (Fig. 2 d-f). Thus, reducing Fli-1 transcriptional activity represents a potential therapeutic concept toward ameliorating GVHD after allo-HCT, while simultaneously targeting cancers such as leukemia which typically overexpress Fli-1. Disclosures No relevant conflicts of interest to declare.

IL-12 signaling drives the differentiation and function of a TH1-derived TFH1-like cell population

CD4+ T follicular helper (TFH) cells provide help to B cells and promote antibody-mediated immune responses. Increasing evidence supports the existence of TFH populations that secrete cytokines typically associated with the effector functions of other CD4+ T cell subsets. These include T helper 1 (TH1)-biased TFH (TFH1) cells that have recognized roles in both immune responses to pathogens and also the pathogenesis of autoimmune disease. Given their apparent importance to human health, there is interest in understanding the mechanisms that regulate TFH1 cell formation and function. However, their origin and the molecular requirements for their differentiation are unclear. Here, we describe a population of murine TH1-derived, TFH1-like cells that express the chemokine receptor Cxcr3 and produce both the TH1 cytokine interferon-γ and the TFH-associated cytokine interleukin-21 (IL-21). Furthermore, these TFH1-like cells promote B cell activation and antibody production at levels indistinguishable from conventional IL-6-derived TFH-like cells. Regarding their regulatory requirements, we find that IL-12 signaling is necessary for the differentiation and function of this TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, promotes increased expression of IL-21 and the TFH lineage-defining transcription factor Bcl-6 in TFH1-like cells. Taken together, these findings provide insight into the potential origin and differentiation requirements of TFH1 cells.

IL-21 and IL-21-producing T cells are involved in multiple sclerosis severity and progression

Multiple sclerosis is a common neuroinflammatory disease of the central nervous system causing nervous system defects and severe physical disability. IL-21 is a proinflammatory cytokine produced mainly by Th-17 and Tfh cells which its exact role in MS was not yet clearly understood. In the present study we aimed to investigate the possible correlation of IL-21 gene expression, methylation, and its serum levels with MS severity and progression. The results showed that IL-21 mRNA level and serum level were significantly increased in patient group compared with control group (p = 0.02 and p < 0.0001 respectively). Moreover, we found a strong positive correlation between IL-21 mRNA levels and EDSS scores (r = 0.637, P < 0.0001), IL-21 mRNA levels and Progression Index (r = 0.540, P < 0.0001), IL-21 serum levels and EDSS scores (r = 0.617, P < 0.0001), and IL-21 serum levels and Progression Index (r = 0.527, P < 0.0001) in MS patients. Additionally, we found that the methylation level of IL-21 promoter region was decreased in patient group compared with the control group (p < 0.0001). We also found that methylation level of IL-21 gene promoter is negatively correlated with the IL-21 mRNA level (r = −0.263, p = 0.02), serum level (r = −0.249, p = 0.03), EDSS scores (r = −0.276, p = 0.01) and Progression Index (r = −0.430, p = 0.0001). Data showed that the increased percentages of IL-21-producing Tfh-like, Th-17 and Th1 cells in patients are positively correlated with MS severity and progression. The results of our study suggest a pro-inflammatory and booster role for IL-21 in the MS pathogenesis and progression.

Circulating T follicular helper cells are a biomarker of humoral alloreactivity and predict donor-specific antibody formation after transplantation.

Donor-specific antibodies (DSAs) contribute to renal allograft loss. However, biomarkers to guide clinical management of DSA posttransplant or detect humoral alloimmune responses before alloantibodies develop are not available. Circulating T follicular helper (cTfh) cells are CD4+ CXCR5+ Tfh-like cells in the blood that have been associated with alloantibodies in transplant recipients, but whether they precede antibody formation for their evaluation as a predictive biomarker in transplant is unknown. To evaluate the ability of cTfh cells to predict DSA, we used murine transplant models to determine the temporal relationship between cTfh cells, germinal center formation, and DSA development. We observed that donor-reactive CD4+ CXCR5+ cTfh cells expand after allotransplant. These cTfh cells were equivalent to graft-draining lymph node-derived Tfh cells in their ability to provide B cell help for antibody production. cTfh cell expansion and differentiation into ICOS+ PD-1+ cells temporally correlated with germinal center alloreactivity and preceded the generation of DSAs in instances of modified and unmodified alloantibody formation. Importantly, delayed costimulation blockade initiated after the detection of ICOS+ PD-1+ cTfh cells prevented DSAs. These findings suggest that cTfh cells could serve as a biomarker for humoral alloreactivity before the detection of alloantibodies and inform therapeutic approaches to prevent DSAs.

TLR-2 and TLR-4 agonists favor expansion of CD4+ T cell subsets implicated in the severity of neuromyelitis optica spectrum disorders

High frequency of circulating Th17 cell subsets expressing TLR2, TLR4 and TLR9 was observed in Neuromyelitis optica spectrum disorder (NMOSD) patients, a severe humoral autoimmune disease of the central nervous system. Our objective was to evaluate the direct effects of different TLR ligands on CD4+ T-cells form those patients. CD4+ T-cell cultures from NMOSD and healthy individuals were stimulated with different TLR ligands and the cell proliferation and cytokine profile was analyzed by [3H] TdR up take and ELISA/ cytometry, respectively. The plasma levels of CD14 were determined by ELISA. Here, Pam3C (TLR2) and LPS (TLR4) induced significant cell proliferation and IL-6, IL-17 and IL-21 production by CD4+ T-cells from NMOSD. Additionally, while both TLR ligands were more potent in favoring the expansion of TFH-like cells, Pam3C reduced the frequency of IL-10-secreting FoxP3+and FoxP3- CD4+ T-cells. With regard to disease severity, the levels of IL-6, IL-17 and IL-21 produced by CD4+ T-cells, as well as the frequency of TFH-like cells, in response to TLR2 and TLR4 agonists were positively correlated with neurological disabilities and the occurrence of new acute relapses during follow up. Finally, circulating levels of CD14, an indirect marker of microbial translocation, were positively correlated with IL-6, IL-17 and IL-21 release by Pam3C- and LPS-activated CD4+ T-cells. In summary, our data suggest that microbial antigens may affect NMOSD outcomes by favoring an imbalance between Th17 and TFH-like cells and regulatory T cell subsets.