cTfh Cells Research Articles

Young but not older adults exhibit an expansion of CD45RA+CCR7+CD95+ T follicular helper cells in response to tetanus vaccine.

A subset of CD4+ T cells, known as T follicular helper (Tfh), provides co-stimulating signals required to establish long-term humoral immunity. Recent studies have shown a reduced frequency and functionality of this population in older adults in comparison to young adults, in response to vaccination. To evaluate whether memory generation of circulating Tfh (cTfh) cells contributes to this phenomenon, the memory subpopulations of cTfh, and their activation degree, were evaluated both ex-vivo and in-vitro, in response to the model antigen tetanus toxoid (TT) after the first dose of tetanus vaccine. Here, we report a lower frequency of cTfh after vaccination in older adults compared to young adults. Moreover, whereas cTfh from older adults preferably expanded with an effector memory phenotype, young adults experienced a temporal increase of CCR7+CD45RA+ cTfh cells, which also displayed higher levels of CD95, CD40L, CXCR3, and Bcl-6 upon antigen re-encounter. This phenotype was confirmed using automatized algorithm. In conclusion, our results suggest that an age-related loss of heterogeneity and an expansion of more differentiated memory cells within the cTfh compartment could affect the responsiveness of older individuals to vaccines, making this phenotype a characteristic feature of immunosenescence.

Rapid Emergence of T Follicular Helper and Germinal Center B Cells Following Antiretroviral Therapy in Advanced HIV Disease.

Low nadir CD4 T-cell counts in HIV+ patients are associated with high morbidity and mortality and lasting immune dysfunction, even after antiretroviral therapy (ART). The early events of immune recovery of T cells and B cells in severely lymphopenic HIV+ patients have not been fully characterized. In a cohort of lymphopenic (CD4 T-cell count < 100/µL) HIV+ patients, we studied mononuclear cells isolated from peripheral blood (PB) and lymph nodes (LN) pre-ART (n = 40) and 6-8 weeks post-ART (n = 30) with evaluation of cellular immunophenotypes; histology on LN sections; functionality of circulating T follicular helper (cTfh) cells; transcriptional and B-cell receptor profile on unfractionated LN and PB samples; and plasma biomarker measurements. A group of 19 healthy controls (HC, n = 19) was used as a comparator. T-cell and B-cell lymphopenia was present in PB pre-ART in HIV+ patients. CD4:CD8 and CD4 T- and B-cell PB subsets partly normalized compared to HC post-ART as viral load decreased. Strikingly in LN, ART led to a rapid decrease in interferon signaling pathways and an increase in Tfh, germinal center and IgD-CD27- B cells, consistent with histological findings of post-ART follicular hyperplasia. However, there was evidence of cTfh cells with decreased helper capacity and of limited B-cell receptor diversification post-ART. In conclusion, we found early signs of immune reconstitution, evidenced by a surge in LN germinal center cells, albeit limited in functionality, in HIV+ patients who initiate ART late in disease.

Circulating T Follicular Helper Subsets in Human Blood.

CXCR5+ memory CD4+ T cells in human blood represent a circulating compartment of T follicular helper (Tfh) cells (termed circulating Tfh: cTfh cells). cTfh cells are composed of subsets that differ in phenotype and functions, and include resting and activated cell populations. Analysis of cTfh cell subsets have proven to be valuable to assess the ongoing global Tfh cell responses in vivo in distinct medical conditions including autoimmune diseases, infectious diseases, and after vaccination. Herein we will describe a detailed protocol to analyze cTfh subsets in human blood samples and to assess their helper capacity in culture with B cells.

Assessment of the TCR Repertoire of Human Circulating T Follicular Helper Cells.

Every T cell clone has its unique T cell receptor that results from somatic recombination of V(D)J genes in developing T cells. This process leads to a highly diverse TCR repertoire of naïve T cells, which is selected, upon antigenic recognition,to form the repertoires of effector and memory T cells. The advent of next-generation sequencing (NGS) technology allows for the high-throughput analysis of the TCR repertoires in the different T cell populations. TFH cells, since their initial discovery in human tonsils and in mouse lymphoid organs,have become the subject of intense investigations due to their essential role in regulating B cell responses and the process of antibody affinity maturation.Circulating follicular helper T cells (cTFH) are considered a helper T cell linage in the blood that to some extent relates with bona fide TFH cells in the germinal centers of secondary lymphoid organs. Due to the limited access to the secondary lymphoid organs, cTFHhave become a more accessible immunological readout.The assessment of the TCR repertoires of TFH and ofcTFH cells is of both fundamental and clinical importance being instrumental to define the linage relationship of cTFH with other T cell subsets and to monitor response to infections or vaccination or disease states. In this chapter, we will provide detailed methods for isolation of antigen-specific cTFH cells in vitro and subsequent protocols for the high-throughput TCR sequencing, followed by repertoire data analysis.

Evaluation of Human Circulating T Follicular Helper Cells in Influenza- and SARS-CoV-2-Specific B Cell Immunity.

Generation of effective immune protection against viral infection and vaccination depends greatly on a successful engagement and stimulation of adaptive immune B cells and a specialized CD4+ T cell subset called T follicular helper cells (TFH cells). Since TFH cells primarily reside in lymphoid tissues, they can be challenging to study in human settings. However, a counterpart of these cells, circulating TFH (cTFH) cells, can be detected in peripheral blood. Assessment of cTFH cells serves as an informative marker of humoral responses following viral infection and vaccination and can be predictive of antibody titers. Here, we describe a comprehensive flow cytometry detection method for dissecting cTFH subsets and activation, together with the assessment of antibody-secreting cells (ASCs), from a small volume of human whole blood. This approach allows the investigation of cellular events that underpin successful immune responses following influenza and SARS-CoV-2 infection/vaccination in humans and is applicable to other viral disease settings.

Multicolor Flow Cytometry and High-Dimensional Data Analysis to Probe Complex Questions in Vaccinology.

Vaccines induce a highly complex immune reaction in secondary lymphoid organs to generate immunological memory against an antigen or antigens of interest. Measurement of postimmunization immune responses generated by specialized lymphocyte subsets requires time-dependent sampling, usually of the blood. Several T and B cell subsets are involved in the reaction, including CD4 and CD8 T cells, T follicular helper cells (Tfh), and germinal center B cells alongside their circulating (c) counterparts; cTfh and antibody secreting cells. Multicolor flow cytometry of peripheral blood mononuclear cells (PBMC) coupled with high-dimensional analysis offers an opportunity to study these cells in detail. Here we demonstrate a method by which such data can be generated and analysed using software that renders multidimensional data on a two dimensionalmap to identify rare vaccine-induced T and B cell subsets.

T follicular helper cells in the humoral immune response to SARS-CoV-2 infection and vaccination.

Vaccination remains the most effective mechanism to reduce the impact of COVID‐19. Induction of neutralizing antibodies is a strong correlate of protection from infection and severe disease. An understanding of the cellular events that underpin the generation of effective neutralizing antibodies is therefore key to the development of efficacious vaccines that target emerging variants of concern. Analysis of the immune response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) infection and vaccination has identified circulating T follicular helper cells (cTFH) as a robust correlate of the neutralizing antibody response. Here, we discuss the analysis of cTFH cells and their lymphoid counterparts in human humoral immune responses during COVID‐19, and in response to vaccination with SARS‐CoV‐2 spike. We discuss the phenotypic heterogeneity of cTFH cells and the utility of cTFH subsets as informative biomarkers for development of humoral immunity. We posit that the analysis of the most effective cTFH will be critical to inducing durable immunity to new variants of SARS‐CoV‐2.

Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans.

Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4+ T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people.

Robust and prototypical immune responses toward influenza vaccines in the high-risk group of Indigenous Australians

SignificanceIndigenous populations worldwide are highly susceptible to influenza virus infections. Vaccination with inactivated virus is highly recommended to protect Indigenous populations, including Indigenous Australians. There is no study to date that assessed immune responses induced by the inactivated seasonal influenza vaccine in the Indigenous population. Vaccine recommendations are thus based on data generated for non-Indigenous populations and might not be representative for Indigenous people. We found robust antibody responses to influenza vaccination induced in Indigenous Australians, with activation profiles of cTFH1 cells at the acute response strongly correlating with total change of antibody vaccine titers induced by vaccination. Our work strongly supports the recommendation of influenza vaccination to protect Indigenous populations from severe seasonal influenza virus infections and subsequent complications.

Th1-Biased Hepatitis C Virus-Specific Follicular T Helper-Like Cells Effectively Support B Cells After Antiviral Therapy.

Circulating Th1-biased follicular T helper (cTfh1) cells have been associated with antibody responses to viral infection and after vaccination but their B cell helper functionality is less understood. After viral elimination, Tfh1 cells are the dominant subset within circulating Hepatitis C Virus (HCV)-specific CD4 T cells, but their functional capacity is currently unknown. To address this important point, we established a clone-based system to evaluate CD4 T cell functionality in vitro to overcome experimental limitations associated with their low frequencies. Specifically, we analyzed the transcription factor expression, cytokine secretion and B cell help in co-culture assays of HCV- (n = 18) and influenza-specific CD4 T cell clones (n = 5) in comparison to Tfh (n = 26) and Th1 clones (n = 15) with unknown antigen-specificity derived from healthy donors (n = 4) or direct-acting antiviral (DAA)-treated patients (n = 5). The transcription factor expression and cytokine secretion patterns of HCV-specific CD4 T cell clones indicated a Tfh1 phenotype, with expression of T-bet and Bcl6 and production of IFN-γ and IL-21. Their B helper capacity was superior compared to influenza-specific or Tfh and Th1 clones. Moreover, since Tfh cells are enriched in the IFN-rich milieu of the HCV-infected liver, we investigated the impact of IFN exposure on Tfh phenotype and function. Type I IFN exposure was able to introduce similar phenotypic and functional characteristics in the Tfh cell population within PBMCs or Tfh clones in vitro in line with our finding that Tfh cells are elevated in HCV-infected patients shortly after initiation of IFN-α therapy. Collectively, we were able to functionally characterize HCV-specific CD4 T cells in vitro and not only confirmed a Tfh1 phenotype but observed superior Tfh functionality despite their Th1 bias. Furthermore, our results suggest that chronic type I IFN exposure supports the enrichment of highly functional HCV-specific Tfh-like cells during HCV infection. Thus, HCV-specific Tfh-like cells after DAA therapy may be a promising target for future vaccination design aiming to introduce a neutralizing antibody response.

T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome

AbstractPediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor β chain (TCR-β) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

Evolution and long-term outcomes of combined immunodeficiency due to CARMIL2 deficiency.

Biallelic loss-of-function mutations in CARMIL2 cause combined immunodeficiency associated with dermatitis, inflammatory bowel disease (IBD), and EBV-related smooth muscle tumors. Clinical and immunological characterizations of the disease with long-term follow-up and treatment options have not been previously reported in large cohorts. We sought to determine the clinical and immunological features of CARMIL2 deficiency and long-term efficacy of treatment in controlling different disease manifestations. The presenting phenotypes, long-term outcomes, and treatment responses were evaluated prospectively in 15 CARMIL2-deficient patients, including 13 novel cases. Lymphocyte subpopulations, protein expression, regulatory T (Treg), and circulating T follicular helper (cTFH ) cells were analyzed. Three-dimensional (3D) migration assay was performed to determine T-cell shape. Mean age at disease onset was 38±23months. Main clinical features were skin manifestations (n=14, 93%), failure to thrive (n=10, 67%), recurrent infections (n=10, 67%), allergic symptoms (n=8, 53%), chronic diarrhea (n=4, 27%), and EBV-related leiomyoma (n=2, 13%). Skin manifestations ranged from atopic and seborrheic dermatitis to psoriasiform rash. Patients had reduced proportions of memory CD4+ T cells, Treg, and cTFH cells. Memory B and NK cells were also decreased. CARMIL2-deficient T cells exhibited reduced T-cell proliferation and cytokine production following CD28 co-stimulation and normal morphology when migrating in a high-density 3D collagen gel matrix. IBD was the most severe clinical manifestation, leading to growth retardation, requiring multiple interventional treatments. All patients were alive with a median follow-up of 10.8years (range: 3-17years). This cohort provides clinical and immunological features and long-term follow-up of different manifestations of CARMIL2 deficiency.

Regulatory B cell imbalance correlates with Tfh expansion in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease with fibrosis, microangiopathy and immune dysfunction. B cell abnormalities characterised by autoantibody production and polyclonal B cell activation play an important role in the pathogenesis of SSc. We previously identified an expansion of functional and activated circulating T follicular helper (cTfh) cells in SSc patients. The aim of this study was to analyse the frequency of regulatory B (Breg) cell subsets and the correlation with Tfh in SSc patients. Circulating Breg cells CD24hiCD38hi and CD27+CD24hi levels and cTfh cells CD4+CXCR5+PD1+ were determined by cytometry in 50 SSc patients and 32 healthy subjects. The frequency of Breg cells CD24hiCD38hi and CD24hiCD27+ was significantly reduced in patients with SSc as compared to controls (p=0.02 and p<0.001, respectively). In contrast, when examining the CD21low B cell subset, the frequency was significantly increased in SSc patients compared to healthy controls, (p<0.001). There was no difference in Breg cell levels in patients with diffuse SSc and limited SSc. However, CD24hiCD27+ Breg cell frequency was significantly decreased in SSc patients with pulmonary arterial hypertension (p=0.014), but not in patients with interstitial lung disease (p=0.058). Furthermore, we observed a negative correlation between cTfh and CD24hiCD27+ Breg cell levels in SSc patients but not in healthy controls (p=0.02). These results suggest that Breg cell subsets may participate in the regulation of cTfh and disease severity. Decreased CD24hiCD27+ Breg cell frequency may contribute to the development of SSc.

Reduced Energy Metabolism Impairs T Cell-Dependent B Cell Responses in Patients With Advanced HBV-Related Cirrhosis.

Background and AimsPatients with decompensated HBV-related liver cirrhosis (HBV D-LC) showed compromised immune responses, which manifested as a proneness to develop infections and hyporesponsiveness to vaccines, resulting in accelerated disease progression. The alterations in T cell-dependent B cell responses in this pathophysiological process were not well understood. This study aimed to investigate T cell-dependent B cell responses in this process and discuss the mechanism from the perspective of metabolism.MethodsChanges in phenotypes and subsets of peripheral B cells between HBV D-LC patients and healthy controls (HCs) were compared by flow cytometry. Isolated B cells were activated by coculture with circulating T follicular (cTfh) cells. After coculture, the frequencies of plasmablasts and plasma cells and immunoglobin levels were analyzed. Oxidative phosphorylation (OXPHOS) and glycolysis were analyzed by a Seahorse analyzer. Mitochondrial function and the AKT/mTOR pathway were analyzed by flow cytometry.ResultsThe proliferation and differentiation capacities of B cells after T cell stimulation were impaired in D-LC. Furthermore, we found that B cells from D-LC patients showed reductions in OXPHOS and glycolysis after activation, which may result from reduced glucose uptake, mitochondrial dysfunction and attenuated activation of the AKT/mTOR pathway.ConclusionsB cells from HBV D-LC patients showed dysfunctional energy metabolism after T cell-dependent activation. Understanding the regulations of B cell metabolic pathway and their changes may provide a new direction to rescue B cell hyporesponsiveness in patients with HBV D-LC, preventing these patients be infected and improving sensitivity to vaccines.

Characterization of hepatitis C virus-specific Th1 and Th1-biased circulating T follicular helper CD4+ T cells that arise following pregnancy

Abstract Chronic hepatitis C virus (HCV) infection is marked by a loss of HCV-specific CD4+ T cells and CD8+ T cell exhaustion. However, recovery of antiviral CD4+ T cells and decreased viremia occurs in some women after pregnancy. We hypothesize that recovery of HCV-specific Th1 and Tfh biased CD4+ T cell populations contributes to viral control postpartum. Here we undertook a detailed phenotypic analysis of lineage-defining chemokine receptor expression on recovering HCV-specific CD4+ T cells using tetramer staining and flow cytometry of PBMCs from 14 women with chronic HCV infection. Seven women had &amp;gt;10-fold decreases in viremia by 3 months postpartum (3PP) (controllers) and the remaining half did not (non-controllers). At 3PP, tetramer+CD4+T cells were predominantly CXCR5+/CXCR3+/CCR6− (cTfh1) with most of the remaining HCV-specific CD4+ T cells being CXCR5−/CXCR3+/CCR6−(Th1). The proportions of cTfh1 cells within tetramer+CD4+ T cell populations were higher in controllers compared to non-controllers (median 74.3% vs. 52.8%, p=0.0262, Mann-Whitney), while the proportions of Th1 cells were higher in non-controllers (p=0.007, Mann-Whitney). Higher cTfh1 frequencies correlated with viral control (p=0.0072, Spearman). In all mothers, over 85% of HCV-specific cTfh1 cells were PD-1+. ICOS expression correlated with reduced viral control. Together, these data suggest that a shift of HCV-specific CD4+ T cell responses towards a cTfh1 phenotype contributes to the unique viral control that occurs in some women after pregnancy.

Normalization of B Cell Subsets but Not T Follicular Helper Phenotypes in Infants With Very Early Antiretroviral Treatment.

Introduction: Infant HIV-1-infection is associated with high morbidity and mortality if antiretroviral treatment (ART) is not initiated promptly. We characterized development of circulating T follicular helper cells (cTfh) and their relationship to naïve/memory B cell subsets in a cohort of neonates initiating ART within the first week of life.Methods: Infants were diagnosed within 48 hours of birth and started ART as soon as possible. The frequency and phenotype of cTfh and B cells were analyzed at enrollment (birth −19 days) and at 4, 12, and 72 weeks of age in blood of 27 HIV-1-intrauterine-infected and 25 HIV-1 exposed uninfected (HEU) infants as part of a study in Johannesburg, South Africa. cTfh cells were divided into Tfh1, Tfh2, and Tfh17 subsets. B cell phenotypes were defined as naïve, resting memory, activated memory and tissue-like memory cells.Results: HIV-1-infected infants had higher frequencies of cTfh cells than HEU infants up to 12 weeks of age and these cTfh cells were polarized toward the Tfh1 subset. Higher frequencies of Tfh1 and lower frequencies of Tfh2 and Tfh17 correlated with lower CD4+ T cell percentages. Lower frequencies of resting memory, with corresponding higher frequencies of activated memory B cells, were observed with HIV-1 infection. Importantly, dysregulations in B cell, but not cTfh cell, subsets were normalized by 72 weeks.Conclusion: Very early ART initiation in HIV-1-infected infants normalizes B cell subsets but does not fully normalize perturbations in cTfh cell subsets which remain Tfh1 polarized at 72 weeks. It remains to be determined if very early ART improves vaccine antibody responses despite the cTfh and B cell perturbations observed over the time course of this study.

Altered distributions in circulating follicular helper and follicular regulatory T cells accountable for imbalanced cytokine production in multiple sclerosis.

Follicular T helper (Tfh) and regulatory (Tfr) cells are distinct subsets of CD4+ T lymphocytes, regulating humoral immune responses in the germinal center. It is widely accepted that dysregulated Tfh and Tfr cells are associated with autoimmunity. In this study, we evaluated the frequencies of circulating chemokine receptor (CXCR)5+ programmed cell death 1 (PD-1+ ) Tfh (cTfh) and CXCR5+ PD-1+ forkhead box protein 3 (FoxP3+ ) CD25+ Tfr (cTfr) cells, and their corresponding cytokines from the peripheral blood mononuclear cells of 28patients with relapsing-remitting multiple sclerosis (MS) and 16 age- and sex-matched healthy controls (HC). Subsets of cTfh cells by Th1- and Th17-related surface markers (CXCR3 and CCR6) were also evaluated. We found that the frequency of cTfh cells was significantly higher in MS patients compared to that of HC. Conversely, the frequency of cTfr cells was lower in MS patients than that of HC. Interleukin (IL)-21-producing cTfh cells were significantly increased in MS patients, while IL-10-secreting cTfr cells were lower in MS patients compared to levels in HC. Among cTfh cells, cTfh17.1 cells were the major subtypes that were significantly increased in MS patients compared to HC, with the frequency of IL-21-secreting cells being the highest. These results suggest that an imbalanced distribution of cTfh and cTfr exist in MS patients, which contributes to the reciprocally altered IL-21 and IL-10 production.

Mechanistic understanding of the combined immunodeficiency in complete human CARD11 deficiency

Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.

Circulating Tfh cell and subsets distribution are associated with low\u2010responsiveness to hepatitis B vaccination

BackgroundAn estimated 5–10 % of healthy vaccinees lack adequate antibody response following receipt of a standard three-dose hepatitis B vaccination regimen. The cellular mechanisms responsible for poor immunological responses to hepatitis B vaccine have not been fully elucidated to date.MethodsThere were 61 low responders and 56 hyper responders involved in our study. Peripheral blood samples were mainly collected at D7, D14 and D28 after revaccinated with a further dose of 20 µg of recombinant hepatitis B vaccine.ResultsWe found low responders to the hepatitis B vaccine presented lower frequencies of circulating follicular helper T (cTfh) cells, plasmablasts and a profound skewing away from cTfh2 and cTfh17 cells both toward cTfh1 cells. Importantly, the skewing of Tfh cell subsets correlated with IL-21 and protective antibody titers. Based on the key role of microRNAs involved in Tfh cell differentiation, we revealed miR-19b-1 and miR-92a-1 correlated with the cTfh cell subsets distribution and antibody production.ConclusionsOur findings highlighted a decrease in cTfh cells and specific subset skewing contribute to reduced antibody responses in low responders.

The Imbalance of Circulating Follicular T Helper Cell Subsets in Primary Sjögren's Syndrome Associates With Serological Alterations and Abnormal B-Cell Distribution.

Since B-cell hyperactivity and pathologic antibody response are key features in the immunopathogenesis of primary Sjögren’s syndrome (pSS), the role of follicular T helper (TFH) cells as efficient helpers in the survival and differentiation of B cells has emerged. Our aim was to investigate whether a change in the balance of circulating (c)TFH subsets and follicular regulatory T (TFR) cells could affect the distribution of B cells in pSS. Peripheral blood of 38 pSS patients and 27 healthy controls was assessed for the frequencies of cTFH cell subsets, TFR cells, and certain B cell subpopulations by multicolor flow cytometry. Serological parameters, including anti-SSA, anti-SSB autoantibodies, immunoglobulin, and immune complex titers were determined as part of the routine diagnostic evaluation. Patients with pSS showed a significant increase in activated cTFH cell proportions, which was associated with serological results. Frequencies of cTFH subsets were unchanged in pSS patients compared to healthy controls. The percentages and number of cTFR cells exhibited a significant increase in autoantibody positive patients compared to patients with seronegative pSS. The proportions of transitional and naïve B cells were significantly increased, whereas subsets of memory B cells were significantly decreased and correlated with autoantibody production. Functional analysis revealed that the simultaneous blockade of cTFH and B cell interaction with anti-IL-21 and anti-CD40 antibodies decreased the production of IgM and IgG. Imbalance in TFH subsets and TFR cells indicates an ongoing over-activated humoral immune response, which contributes to the characteristic serological manifestations and the pathogenesis of pSS.