Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Toll-like receptor 7 agonist, GS-986, is an immune-stimulant inducing follicular helper T cells and expanding HBs antigen-specific B cells in vitro.

BACKGROUNDS AND AIMS: Toll-like receptor (TLR) agonists have been developed as adjuvants to efficiently induce antiviral immune responses. Specificity and potency of these compounds are essential requirements for clinical trial applications. In patients with hepatitis B virus (HBV) infections, sustained loss of hepatitis B surface antigen (HBsAg) is a therapeutic goal, which may be achievable by the sequential activation of follicular helper T cells (Tfh) and antibody-secreting B cells. We aimed to elucidate whether novel TLR7 agonist, GS-986, could activate immune responses involved in HBV elimination. METHODS: To clarify the impact of GS-986 on pDCs, we quantified the expression levels of surface markers and evaluated for Tfh induction in a culture model consisting of human pDCs with allogeneic naïve CD4+ T cells. In addition, we examined whether GS-986 could enhance HBs antibody production capacity using PBMC from CHB patients. RESULTS: pDCs from CHB patients had lower OX40L expression and as well as impaired capacity for Tfh induction compared with those from healthy donors. However, GS-986-stimulated pDCs from CHB patients expressed OX40L and produced IL-6 and IL-12, resulting in the induction of IL-21-producing Tfh cells (CXCR5+ PD-1+ CD4+ ) from naïve CD4+ T cells. The Tfh-inducing capacity of GS-986 was reduced in the presence of an anti-OX40L blocking antibody. Furthermore, GS-986 promoted HBsAg-specific antibody production in PBMCs from CHB patients. CONCLUSIONS: GS-986 is an adjuvant that stimulates pDCs to induce Tfh differentiation and antigen-specific B-cell production. This immune profile may be beneficial for therapeutic application as an immune modulator in CHB patients.

Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction.

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4+ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4+ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.

T cell depletion increases humoral response by favoring T follicular helper cells expansion.

Antibody-mediated rejection is a major cause of long-term graft loss in kidney transplant patients. T follicular helper (Tfh) cells are crucial for assisting B cell differentiation and are required for an efficient antibody response. Anti-thymocyte globulin (ATG) is a widely used lymphocyte-depleting induction therapy. However, less is known about how ATG affects Tfh cell development and donor-specific antibody (DSA) formation. We observed an increase in circulating Tfh cells at 6 months after kidney transplant in patients who received ATG. Using an NP-OVA immunization model, we found that ATG-treated mice had a higher percentage of Tfh cells, germinal center B cells, and higher titers of antigen-specific antibodies compared to controls. ATG-treated animals had lower levels of IL-2, a known Bcl-6 repressor, but higher levels of IL-21, pSTAT3 and Bcl-6, favoring Tfh differentiation. In a mouse kidney transplant model, ATG-treated recipients showed an increase in Tfh cells, DSA and C4d staining in the allograft. Although ATG was effective in depleting T cells, it favored the expansion of Tfh cells following depletion. Concomitant use of IL-2, tacrolimus, or rapamycin with ATG was essential to control Tfh cell expansion. In summary, ATG depletion favors Tfh expansion, enhancing antibody-mediated response.

A review of signaling and transcriptional control in T follicular helper cell differentiation.

T follicular helper (Tfh) cells are a critical component of adaptive immunity and assist in optimal Ab-mediated defense. Multiple effector functions of Tfh support germinal center B cell survival, Ab class switching, and plasma cell maturation. In the past 2 decades, the phenotype and functional characteristics of GC Tfh have been clarified allowing for robust studies of the Th subset including activation signals and environmental cues controlling Tfh differentiation and migration during an immune response. A unique, 2-step differentiation process of Tfh has been proposed but the mechanisms underlying transition between unstable Tfh precursors and functional mature Tfh remain elusive. Likewise, newly identified transcriptional regulators of Tfh development have not yet been incorporated into our understanding of how these cells might function in disease. Here, we review the signals and downstream transcription factors that shape Tfh differentiation including what is known about the epigenetic processes that maintain Tfh identity. It is proposed that further evaluation of the stepwise differentiation pattern of Tfh will yield greater insights into how these cells become dysregulated in autoimmunity.

Follicular Helper T Cells in the Immunopathogenesis of SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a serious infectious disease that has led to a global pandemic with high morbidity and mortality. High-affinity neutralizing antibody is important for controlling infection, which is closely regulated by follicular helper T (Tfh) cells. Tfh cells play a central role in promoting germinal center reactions and driving cognate B cell differentiation for antibody secretion. Available studies indicate a close relationship between virus-specific Tfh cell-mediated immunity and SARS-CoV-2 infection progression. Although several lines of evidence have suggested that Tfh cells contribute to the control of SARS-CoV-2 infection by eliciting neutralizing antibody productions, further studies are needed to elucidate Tfh-mediated effector mechanisms in anti-SARS-CoV-2 immunity. Here, we summarize the functional features and roles of virus-specific Tfh cells in the immunopathogenesis of SARS-CoV-2 infection and in COVID-19 vaccines, and highlight the potential of targeting Tfh cells as therapeutic strategy against SARS-CoV-2 infection.

Critical regulation of follicular helper T cell differentiation and function by Gα13 signaling.

GPCR-Gα protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how Gα proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that Gα13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific Gα13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, Gα13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, Gα13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific Gα13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of Gα13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Dexamethasone reduces autoantibody levels in MRL/lpr mice by inhibiting Tfh cell responses.

Previous studies have shown that dexamethasone (Dex) reduces the levels of anti-nuclear (ANA) and anti-dsDNA antibodies in MRL/lpr mice (a mouse model of SLE). However, the effect of Dex on T follicular helper (Tfh) cells is less documented. Here, using the MRL/lpr mouse model, we investigated the influence of Dex on Tfh cells and potential underlying mechanisms. The data showed that the proportion of Tfh cells, identified as CD4+ CXCR5+ ICOS+ , CD4+ CXCR5+ PD-1+ or CD4+ BCL-6+ cells, markedly decreased after treatment with the Dex, in both Balb/c mice and MRL/lpr mice. Dex significantly inhibited IL-21 expression at both the mRNA and the protein levels. Dex also significantly reduced the proportion of germinal centre B cells and decreased serum IgG, IgG2a/b and IgA levels. Moreover, a positive correlation between the proportion of Tfh cells (CD4+ CXCR5+ ICOS+ , CD4+ CXCR5+ PD-1+ or CD4+ BCL-6+ ) and autoantibodies was observed. Dex significantly increased the Prdm1 and Stat5b mRNA expression and decreased the Bcl-6 and c-Maf mRNA expression of CD4+ T cells. In brief, Dex inhibited the Tfh development, which relies on many other transcription factors in addition to Bcl-6. Our data indicate that Dex can be used as a Tfh cell inhibitor in SLE.

Spontaneous Differentiation of T Follicular Helper Cells in LATY136F Mutant Mice.

Mice with a mutation at the LAT-PLCγ1 binding site (Y136) have a defect in thymocyte development due to dampened TCR signaling. CD4+ T cells that do reach the periphery are hyper-activated and skewed to Th2. Over time, these mice develop an autoimmune-like syndrome, characterize by overproduction of Th2 cytokines, T cell infiltration into various organs, and B cell activation, isotype switching, and autoantibody production. In this study, we examined IL4 production by CD4+ T cells in the LATY136F mice using the KN2 reporter mice, in which human CD2 expression marks T cells that are actively producing IL4 protein. We showed that these mice had spontaneous Tfh differentiation. Despite the fact that the majority of CD4+ T cells were skewed to Th2 and were GATA3+, only a small subset of them were actively secreting IL4. These T cells were Tfh cells that expressed BCL6 and were localized to B cell-rich germinal centers within the spleen. Interestingly, these Tfh cells expressed high levels of both BCL6 and GATA3. By using LAT conditional knockout mice that inducibly express only the LATY136F allele, we further showed that Tfh cell differentiation was likely the result of defective LAT-PLCγ1 signaling in the periphery. In addition, B cells were required for spontaneous development of Tfh cells and uncontrolled T cell expansion in these mice. Together, these results indicated a novel role for tonic LAT-PLCγ1 signaling in modulating Tfh cell differentiation during development of autoimmune syndrome.

Protein/AS01B vaccination elicits stronger, more Th2-skewed antigen-specific human T follicular helper cell responses than heterologous viral vectors.

Interactions between B cells and CD4+ T follicular helper (Tfh) cells are key determinants of humoral responses. Using samples from clinical trials performed with the malaria vaccine candidate antigen Plasmodium falciparum merozoite protein (PfRH5), we compare the frequency, phenotype, and gene expression profiles of PfRH5-specific circulating Tfh (cTfh) cells elicited by two leading human vaccine delivery platforms: heterologous viral vector prime boost and protein with AS01B adjuvant. We demonstrate that the protein/AS01B platform induces a higher-magnitude antigen-specific cTfh cell response and that this correlates with peak anti-PfRH5 IgG concentrations, frequency of PfRH5-specific memory B cells, and antibody functionality. Furthermore, our data indicate a greater Th2/Tfh2 skew within the polyfunctional response elicited following vaccination with protein/AS01B as compared to a Th1/Tfh1 skew with viral vectors. These data highlight the impact of vaccine platform on the cTfh cell response driving humoral immunity, associating a high-magnitude, Th2-biased cTfh response with potent antibody production.

Transcriptome and chromatin landscape of iNKT cells are shaped by subset differentiation and antigen exposure.

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.

Dynamic regulation of TFH selection during the germinal centre reaction.

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.

Role of CD4+ T Cells in the Control of Viral Infections: Recent Advances and Open Questions.

CD4+ T cells orchestrate adaptive immune responses through their capacity to recruit and provide help to multiple immune effectors, in addition to exerting direct effector functions. CD4+ T cells are increasingly recognized as playing an essential role in the control of chronic viral infections. In this review, we present recent advances in understanding the nature of CD4+ T cell help provided to antiviral effectors. Drawing from our studies of natural human immunodeficiency virus (HIV) control, we then focus on the role of high-affinity T cell receptor (TCR) clonotypes in mediating antiviral CD4+ T cell responses. Last, we discuss the role of TCR affinity in determining CD4+ T cell differentiation, reviewing the at times divergent studies associating TCR signal strength to the choice of a T helper 1 (Th1) or a T follicular helper (Tfh) cell fate.

Immune cell infiltration features and related marker genes in lung cancer based on single-cell RNA-seq.

PURPOSE: Immune cells in the immune microenvironment of lung cancer have a great impact on the development of lung cancer. Our purpose was to analyze the immune cell infiltration features and related marker genes for lung cancer. METHODS: Single cell RNA sequencing data of 11,485 lung cancer cells were retrieved from the Gene Expression Omnibus. After quality control and data normalization, cell clustering was performed using the Seurat package. Based on the marker genes of each cell type from the CellMarker database, each cell was divided into G1, G2M, and S phases. Then, differential expression and functional enrichment analyses were performed. CIBERSORT was used to reconstruct immune cell types. RESULTS: Following cell filtering, highly variable genes were identified for all cells. 14 cell types were clustered. Among them, CD4 + T cell, B cell, plasma cell, natural killer cell and cancer stem cell were the top five cell types. Up-regulated genes were mainly enriched in immune-related biological processes and pathways. Using CIBERSORT, we identified the significantly higher fractions of naïve B cell, memory CD4 + T cell, T follicular helper cell, T regulatory helper cell and M1 macrophage in lung cancer tissues compared to normal tissues. Furthermore, the fractions of resting NK cell, monocyte, M0 macrophage, resting mast cell, eosinophil and neutrophil were significantly lower in tumor tissues than normal tissues. CONCLUSION: Our findings dissected the immune cell infiltration features and related marker genes for lung cancer, which might provide novel insights for the immunotherapy of lung cancer.

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

Circulating TFH (cTFH ) 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 cTFH 1, cTFH 2, cTFH 17, and cTFR 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 cTFH cells was directly correlated with estradiol levels. In vitro, pregnancy-related dose of 17-ß-estradiol (E2) directly increased the percentage of different cTFH subsets. While E2 and progesterone (P4) increased the proportion of differentiated TFH 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/TFH 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+ TFR cells. Collectively, our findings suggested that E2 and P4 cooperate in the humoral immune response by favoring the expansion of different cTFH and B cell subsets.

Functional subsets of circulating follicular helper T cells in patients with atherosclerosis.

Frequencies of circulating T follicular helper (cTfh) functional subsets vary in autoimmune diseases. We evaluated the frequencies and clinical relevance of functional subsets of cTfhs in patients with different degrees of stenosis. Blood samples were collected from high (≥50%) (n = 12) and low (<50%) stenosis (n = 12) groups and healthy controls (n = 6). Three subsets of cTfh cells including cTfh1 (CXCR3+ CCR6- ), cTfh2 (CXCR3- CCX6- ), and cTfh17 (CXCR3- CCR6+ ) were detected by flow cytometry. The frequency of cTfh1 cells was higher in control (p = .0006) and low-stenosis groups (p = .005) compared to high-stenosis group. The percentages of cTfh2 and cTfh17 cells were increased in high-stenosis compared to low-stenosis (p = .002 and p = .007) and control groups (p = .0004 and p = .0005), respectively. The frequency of cTfh1 cells negatively correlated with cholesterol (p = .040; r = -.44), C-reactive protein (CRP) (p = .015; r = -.68), erythrocyte sedimentation rate (ESR) (p = .002; r = -.79), neutrophil/lymphocyte ratio (NLR) (p = .028; r = -.67), and cTfh17 (p = .017; r = -.7244) in the high-stenosis group. The percentages of cTfh2 and cTfh17 cells positively correlated with cholesterol (p = .025; r = .77 and p = .033; r = .71), CRP (p = .030; r = .61 and p = .020; r = .73), ESR (p = .027; r = .69 and p = .029; r = .70), NLR (p = .004; r = .76 and p = .005; r = .74), and with each other (p = .022; r = .7382), respectively, in the high-stenosis group. The increased frequencies of cTfh2 and cTfh17 subsets and their correlation with laboratory parameters in patients with atherosclerosis may suggest their role in promoting the inflammatory response and atherosclerosis progression.

Foxo3 Promotes the Differentiation and Function of Follicular Helper T Cells.

Follicular helper T cells (Tfhs) are essential for germinal center (GC) B cell maturation and antibody development. However, the intrinsic mechanisms that regulate Tfh differentiation are largely unknown. Here, we demonstrate that the frequencies of Tfhs and GC B cells, as well as interleukin-21 (IL-21) and anti-ovalbumin (OVA) antibodies, are markedly decreased in forkhead box O3 (Foxo3) knockout mice immunized with OVA. Using mixed bone marrow chimeras and lymphocyte-repopulated Rag1-/- mice proves that wild-type (WT), but not Foxo3-deficient T cells provoke GC B cell maturation and antibody production. Deficiency of Foxo3 inhibits inducible T cell co-stimulator (ICOS)-induced Tfh differentiation. Chromatin immunoprecipitation assay results suggest that Foxo3 is able to bind to the IL-21 promoter and regulate IL-21 secretion. In conclusion, our study unveils a critical role of Foxo3 in the regulation of Tfh differentiation and IL-21 production. Modulating Foxo3 activity may be beneficial for enhancing or preventing antibody-mediated immune responses.

Bcl-6-directed follicular helper T cells promote vascular inflammatory injury in diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-threatening complication of diabetes mellitus characterized by chronic retinal microvascular inflammation. The involvement of CD4+ T cells in retinal vascular inflammation has been considered, but the specific subset and mechanism of T cell-mediated response during the process remains unclear. Here, we aim to investigate the potential role of follicular helper T (Tfh) cells, a newly identified subset of CD4+ T cells in retinal vascular inflammation in DR. Methods: Patients with DR were enrolled and the PD-1+CXCR5+CD4+ Tfh cells were detected in the peripheral blood by flow cytometry. The streptozotocin (STZ)-induced DR model and oxygen-induced retinopathy (OIR) model were established, and 79-6, an inhibitor of Bcl-6, was injected intraperitoneally to suppress Tfh cells. The Tfh cells-related genes were investigated in the spleen, lymph nodes, and retina of mice by flow cytometry, immunofluorescence, and qPCR. Results: The Tfh cells expanded in the circulation of patients with DR and also increased in circulation, lymph nodes and retinal tissues from the STZ-induced DR mice and OIR mice. Notably, inhibition of Bcl-6, a critical transcription factor for Tfh cells development, prevented upregulation of Tfh cells and its typical IL-21 cytokine, and ameliorated vascular leakage in DR mice or retinal angiogenesis in OIR mice, indicating that Bcl-6-directed Tfh cells could promote vascular inflammation and angiogenesis. Conclusions: Our results suggested that excessive Bcl-6-directed Tfh cells represent an unrecognized feature of DR and be responsible for the retinal vascular inflammation and angiogenesis, providing opportunities for new therapeutic approaches to DR.