ATP-P2X7R pathway activation limits the Tfh cell compartment during pediatric RSV infection.

Background: Follicular helper T cells (Tfh) are pivotal in B cell responses. Activation of the purinergic receptor P2X7 on Tfh cells regulates their activity. We investigated the ATP-P2X7R axis in circulating Tfh (cTfh) cells during Respiratory Syncytial Virus (RSV) infection. Methods: We analyzed two cohorts: children with RSV infection (moderate, n=30; severe, n=21) and healthy children (n=23). We utilized ELISA to quantify the levels of PreF RSV protein-specific IgG antibodies, IL-21 cytokine, and soluble P2X7R (sP2X7R) in both plasma and nasopharyngeal aspirates (NPA). Additionally, luminometry was employed to determine ATP levels in plasma, NPA and supernatant culture. The frequency of cTfh cells, P2X7R expression, and plasmablasts were assessed by flow cytometry. To evaluate apoptosis, proliferation, and IL-21 production by cTfh cells, we cultured PBMCs in the presence of Bz-ATP and/or P2X7R antagonist (KN-62) and a flow cytometry analysis was performed. Results: In children with severe RSV disease, we observed diminished titers of neutralizing anti-PreF IgG antibodies. Additionally, severe infections, compared to moderate cases, were associated with fewer cTfh cells and reduced plasma levels of IL-21. Our investigation revealed dysregulation in the ATP-P2X7R pathway during RSV infection. This was characterized by elevated ATP levels in both plasma and NPA samples, increased expression of P2X7R on cTfh cells, lower levels of sP2X7R, and heightened ATP release from PBMCs upon stimulation, particularly evident in severe cases. Importantly, ATP exposure decreased cTfh proliferative response and IL-21 production, while promoting their apoptosis. The P2X7R antagonist KN-62 mitigated these effects. Furthermore, disease severity positively correlated with ATP levels in plasma and NPA samples and inversely correlated with cTfh frequency. Conclusion: Our findings indicate that activation of the ATP-P2X7R pathway during RSV infection may contribute to limiting the cTfh cell compartment by promoting cell death and dysfunction, ultimately leading to increased disease severity.

Dynamics of circulating follicular helper T cell subsets and follicular regulatory T cells in rheumatoid arthritis patients according to HLA-DRB1 locus.

B cells, follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are part of a circuit that may play a role in the development or progression of rheumatoid arthritis (RA). With the aim of providing further insight into this topic, here we evaluated the frequency of different subsets of Tfh and Tfr in untreated and long-term treated RA patients from a cohort of Argentina, and their potential association with particular human leukocyte antigen (HLA) class-II variants and disease activity. We observed that the frequency of total Tfh cells as well as of particular Tfh subsets and Tfr cells were increased in seropositive untreated RA patients. Interestingly, when analyzing paired samples, the frequency of Tfh cells was reduced in synovial fluid compared to peripheral blood, while Tfr cells levels were similar in both biological fluids. After treatment, a decrease in the CCR7loPD1hi Tfh subset and an increase in the frequency of Tfr cells was observed in blood. In comparison to healthy donors, seropositive patients with moderate and high disease activity exhibited higher frequency of Tfh cells while seropositive patients with low disease activity presented higher Tfr cell frequency. Finally, we observed that HLA-DRB1*09 presence correlated with higher frequency of Tfh and Tfr cells, while HLA-DRB1*04 was associated with increased Tfr cell frequency. Together, our results increase our knowledge about the dynamics of Tfh and Tfr cell subsets in RA, showing that this is altered after treatment.

Effects of Calcium Hydroxide Intracanal Medications on T Helper (Th1, Th2, Th9, Th17, and Tfh) and Regulatory T (Treg) Cell Cytokines in Apical Periodontitis: A CONSORT RCT.

INTRODUCTION: This Consolidated Standards of Reporting Trials randomized clinical trial investigated T helper (Th1, Th2, Th9, Th17, and Tfh) and regulatory T (Treg) cell-type cytokines and their networks in apical periodontitis (AP). We also assessed the effects of calcium hydroxide [Ca(OH)2] intracanal medications (ICMs) on helper T and Treg cell-type cytokines. METHODS: Twenty teeth with primary endodontic infection and apical periodontitis were randomly divided into two groups: Ca(OH)2 + saline solution (n = 10) and Ca (OH)2 + 2% chlorhexidine-gel (n = 10). Samples were collected from the periradicular tissue fluid (PTF) before (PTFs1) and after 14 days of ICMs (PTFs2). The Human High Sensitivity T Cell Panel was used to quantify target T-helper (Th)1: interelukin (IL)-2, IL-12, and interferon-gamma (IFN-γ); Th2: IL-4, IL-5, and IL-13; Th9: IL-9; Th17: IL-17; T follicular helper cells (Tfh): IL-21; and Treg-cell-type cytokine: IL-10. RESULTS: Th1-type cytokines were higher than Th2-type ones, at PTFs1. Positive (+) associations were found among all Th1-type cytokines and all Th2-type cytokines. There were negative (-) correlations between all Th1- and Th2-type cytokines. Size of radiolucent lesions and symptoms (tenderness to percussion and/or pain on palpation) were positively correlated with Th1-type cytokines, IL-17, and IL-21 but negatively correlated with Th2-type cytokines and IL-10 (all, P < .001). Both ICMs increased Th2-type cytokines and IL-10 (P < .05) and decreased Th1-type cytokines, IL-17, and IL-21 (P < .05), with no differences among them (P > .05). CONCLUSIONS: Complex T-cell cytokine networks are involved in AP. Both Ca(OH)2 ICMs effectively increased IL-4, IL-5, IL-10, and IL-13 and lowered IL-2, IL-12, IL-17, IL-21, and IFN-γ.

CD8 Treg-Mediated Suppression of Naive CD4+ T Cell Differentiation into Follicular Helper T Cells.

BACKGROUND: The regulatory CD8+ T (CD8+ Treg) cells play an important role in immune tolerance and have been implicated in several human autoimmune diseases. In this context, follicular helper T (TFH) cells contribute by controlling the antibody production. In mice, CD8+ Treg cells control the number and function of TFH cells however the role of human CD8+ Treg cells on the differentiation of naive CD4+ T cells into TFH cells has not been studied. OBJECTIVES: Here, we evaluated the ability of human CD183+ CD8+ Treg cells to suppress TFH cell differentiation in vitro. METHODS: Activated CD183+CCR7+CD45RA-CD8+ Treg and CD183+CD25highICOS+CD8+ Treg cells were sorted and cocultured with naïve CD4+ T cells under TFH differentiation condition. The differentiation of TFH cells was evaluated by flow cytometry. RESULTS: Our results showed that activated CD183+CD8+ Treg cells upregulated the expression of Forkhead box P3 transcription factor, inducible T-cell co-stimulator (ICOS), and CD25 compared to CD183-CD8+ T cells. The CD183+CD25highICOS+CD8+ Treg cells suppressed TFH cell differentiation and CD4+ T cell proliferation in vitro which was not observed when CD183+CCR7+CD45RA-CD8+ Treg were cocultured with naïve CD4+ T cells under TFH cell differentiation condition. CONCLUSION: These results suggest that CD25highICOS+CD183+CD8+ Treg cells may regulate autoimmune and inflammatory responses mediated by TFH cells.

T follicular helper cells: Their development and importance in the context of helminthiasis.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

Cellular and molecular regulation of the programmed death-1/programmed death ligand system and its role in multiple sclerosis and other autoimmune diseases.

Programmed Cell Death 1 (PD-1) receptor and its ligands (PD-Ls) are essential to maintain peripheral immune tolerance and to avoid tissue damage. Consequently, altered gene or protein expression of this system of co-inhibitory molecules has been involved in the development of cancer and autoimmunity. Substantial progress has been achieved in the study of the PD-1/PD-Ls system in terms of regulatory mechanisms and therapy. However, the role of the PD-1/PD-Ls pathway in neuroinflammation has been less explored despite being a potential target of treatment for neurodegenerative diseases. Multiple Sclerosis (MS) is the most prevalent, chronic, inflammatory, and autoimmune disease of the central nervous system that leads to demyelination and axonal damage in young adults. Recent studies have highlighted the key role of the PD-1/PD-Ls pathway in inducing a neuroprotective response and restraining T cell activation and neurodegeneration in MS. In this review, we outline the molecular and cellular mechanisms regulating gene expression, protein synthesis and traffic of PD-1/PD-Ls as well as relevant processes that control PD-1/PD-Ls engagement in the immunological synapse between antigen-presenting cells and T cells. Also, we highlight the most recent findings regarding the role of the PD-1/PD-Ls pathway in MS and its murine model, experimental autoimmune encephalomyelitis (EAE), including the contribution of PD-1 expressing follicular helper T (TFH) cells in the pathogenesis of these diseases. In addition, we compare and contrast results found in MS and EAE with evidence reported in other autoimmune diseases and their experimental models, and review PD-1/PD-Ls-targeting therapeutic approaches.

Impact of HIV-1 Infection and Antigen Class on T Follicular Helper Cell Responses to Pneumococcal Polysaccharide-Protein Conjugate Vaccine-13.

Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of Ab responses to T cell-independent type 2 pneumococcal capsular polysaccharides (PPS). The pneumococcal polysaccharide-protein conjugate vaccine-13 (PCV-13) contains PPS conjugated to the T cell-dependent protein (diphtheria toxoid [DT] [CRM197]). We investigated the differential response to PPS and DT by human Ab-secreting B cells (ASC) after immunization with PCV-13 in newly diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls. However, DT-specific IgG ASC increased in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (Tfh) cells, the predominant T cell subset providing B cell help. Expression of inducible T cell costimulator (ICOS), which sustains Tfh function and phenotype, increased significantly among controls, when compared with the HIV+ group. Increases in ICOS+ Tfh correlated with changes in T-dependent, DT-specific IgG ASC in controls but not in HIV+ In contrast, ICOS expression did not correlate with T cell-independent type 2 PPS-specific ASC in either group. Of note, upon optimized ex vivo stimulation, CD4 T cells from HIV+ subjects differentiated into Tfh cells and formed synapses with Raji B cells at frequencies similar to that of controls. In summary, PCV-13-induced increase in ICOS expression on Tfh was associated with responses to DT, which was compromised in recently diagnosed healthy HIV+ adults and can be restored ex vivo by providing effective Tfh-differentiating signals.

Targeting T Follicular Helper Cells to Control Humoral Allogeneic Immunity.

Humoral allogeneic immunity driven by anti-HLA donor-specific antibodies and antibody-mediated rejection (AMR) significantly impede prolonged survival of organ allografts after transplantation. Although the importance of T follicular helper (TFH) cells in controlling antibody responses has been long established, their role in directing donor-specific antibody generation leading to AMR was only recently appreciated in the clinical setting of organ transplantation. In this review, we provide a comprehensive summary of the current knowledge on the biology of human TFH cells as well as their circulating counterparts and describe their pivotal role in driving humoral alloimmunity. In addition, we discuss the intrinsic effects of current induction therapies and maintenance immunosuppressive drugs as well as of biotherapies on TFH cells and provide future directions and novel opportunities of biotherapeutic targeting of TFH cells that have the potential of bringing the prophylactic and curative treatments of AMR toward personalized and precision medicine.

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.

IL-21 treatment recovers follicular helper T cells and neutralizing antibody production in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children under 1 year. RSV vaccines are currently unavailable, and children suffering from multiple reinfections by the same viral strain fail to develop protective responses. Although RSV-specific antibodies can be detected upon infection, these have limited neutralizing capacity. Follicular helper T (Tfh) cells are specialized in providing signals to B cells and help the production and affinity maturation of antibodies, mainly via interleukin (IL) 21 secretion. In this study, we evaluated whether RSV could inhibit Tfh responses. We observed that Tfh cells fail to upregulate IL-21 production upon RSV infection. In the lungs, RSV infection downregulated the expression of IL-21/interleukin-21 receptor (IL-21R) in Tfh cells and upregulated programmed death-ligand 1 (PD-L1) expression in dendritic cells (DCs) and B cells. PD-L1 blockade during infection recovered IL-21R expression in Tfh cells and increased the secretion of IL-21 in a DC-dependent manner. IL-21 treatment decreased RSV viral load and lung inflammation, inducing the formation of tertiary lymphoid organs in the lung. It also decreased regulatory follicular T cells, and increased Tfh cells, B cells, antibody avidity and neutralization capacity, leading to an overall improved anti-RSV humoral response in infected mice. Passive immunization with purified immunoglobulin G from IL-21-treated RSV-infected mice protected against RSV infection. Our results unveil a pathway by which RSV affects Tfh cells by increasing PD-L1 expression on antigen-presenting cells, highlighting the importance of an IL-21-PD-L1 axis for the generation of protective responses to RSV infection.

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.

Conventional type 1 dendritic cells induce TH 1, TH 1-like follicular helper T cells and regulatory T cells after antigen boost via DEC205 receptor.

Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory TH 1 cell response. Antigen targeting to cDC2s induced TFH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the TH 1 cell response and induced TH 1-like TFH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs.

Thymic B Cells Promote Germinal Center-Like Structures and the Expansion of Follicular Helper T Cells in Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE.

Circulating T Follicular Helper Cell Abnormalities Associated to Different Clinical Forms of Chronic Chagas Disease.

Multiple perturbations of the immune response affecting a range of cells have been reported in Trypanosoma cruzi-infected individuals and associated to clinical manifestations of chronic Chagas disease. There is a paucity of knowledge about the role of T follicular helper (Tfh) cells in this infection. Here, we sought to characterize circulating Tfh (cTfh) cells in chronic Chagas disease patients and to identify potential associations with disease severity in humans. cTfh cells were characterized by flow cytometry in freshly isolated PBMCs from 7 T. cruzi-infected asymptomatic patients (ASYMP), 5 patients with chronic chagasic dilated cardiomyopathy (CCC) and 8 healthy controls, using antibodies against chemokine receptors CXCR5, CXCR3, CCR6, and CCR7. Our results showed significant expansion of CD4+CD45RO+CXCR5+CCR6+ cells in ASYMP and CCC patients, along with a contraction of CD4+CD45RO+CXCR5+CXCR3-CCR6- (cTfh2) cells. ASYMP patients further exhibited decreased CD4+CD45RO+CXCR5+CXCR3+CCR6- (cTfh1) cells and expanded CD4+CD45RO+CXCR5+CXCR3-CCR6+ (cTfh17) cells while CCC patients exhibited significantly increased frequencies of CD4+CD45RO+CXCR5+CCR7+ cells. Linear regression analysis revealed a positive trend of CD4+CD45RO+CXCR5+CXCR3+CCR6+ (cTfh1/17) cells and negative trends of cTfh1 and cTfh2 cells as disease was more severe. There was no correlation between the frequencies of cTfh cells and circulating CD19+IgD-IgG+ cells or serum levels of T. cruzi-specific IgG. These results demonstrate that the cTfh compartment of humans chronically infected with T. cruzi comprises expanded CCR6-expressing cells and reduced cTfh2 cells. The association of discrete phenotypic changes in cTfh subsets with different clinical forms suggests the potential contribution of T follicular helper cells to Chagas heart disease progression.

Pregnancy favors circulating IL-21-secreting TFH -like cell recovery in ARV-treated HIV-1-infected women.

PROBLEM: Pregnancy appears to favor maternal antibody production. In contrast, by damaging follicular helper T cells (TFH ), HIV-1 infection compromises protective humoural immune response. Therefore, we aimed to investigate the frequency of different TFH -like cells in HIV-infected pregnant women (PW) before and after antiretroviral (ARV) therapy. METHOD OF STUDY: Peripheral blood mononuclear cells, CD4+ T and B cells, were obtained from asymptomatic HIV-1-infected non-PW and PW just before and after ARV therapy. In some experiments, healthy HIV-1-negative PW were also tested. The frequency of different TFH -like cell subsets was determined by flow cytometry. The plasma titers of IgG anti-tetanus toxoid (TT), anti-HBsAg, and anti-gp41 were determined by ELISA. The in vitro production of total IgG, IL-21, and hormones (estrogen and progesterone) was quantified also by ELISA. RESULTS: Our results demonstrate that antiretroviral (ARV) therapy was more efficient in elevating the percentage of circulating IL-21-secreting TFH cells in HIV-1-infected pregnant women (PW) than in non-pregnant patients (nPW). Moreover, in co-culture systems, CD4+ T cells from ART-treated PW were more efficient in assisting B cells to produce IgG production. The in vivo anti-HBsAg IgG titers after ARV therapy were also significantly higher in PW, and their levels were directly associated with both IL-21+ TFH frequency and plasma concentration of estrogen. CONCLUSION: In summary, our results suggest that pregnancy favors the recovery of TFH -like cells after ARV therapy in HIV-1-infected women, which could help these mothers to protect their newborns from infectious diseases by transferring IgG across the placenta.