EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.

NSG-Mice Reveal the Importance of a Functional Innate and Adaptive Immune Response to Overcome RVFV Infection.

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNARtmAgt, C.129S7(B6)-Ifngtm1Ts/J, B6-TLR3tm1Flv, B6-TLR7tm1Aki, NOD/ShiLtJ), helper T-cell- (CD4tm1Mak), cytotoxic T-cell- (CD8atm1Mak), B-cell- (Igh-Jtm1DhuN?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-PrkdcscidIl2rgtm1WjI/SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNARtmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses.

Equine herpesvirus-1 is the cause of respiratory disease, abortion, and equine herpesvirus myeloencephalopathy (EHM) in horses worldwide. EHM affects as many as 14% of infected horses and a cell-associated viremia is thought to be central for EHM pathogenesis. While EHM is infrequent in younger horses, up to 70% of aged horses develop EHM. The aging immune system likely contributes to EHM pathogenesis; however, little is known about the host factors associated with clinical EHM. Here, we used the "old mare model" to induce EHM following EHV-1 infection. Peripheral blood mononuclear cells (PBMCs) of horses prior to infection and during viremia were collected and RNA sequencing with differential gene expression was used to compare the transcriptome of horses that did (EHM group) and did not (non-EHM group) develop clinical EHM. Interestingly, horses exhibiting EHM did not show respiratory disease, while non-EHM horses showed significant respiratory disease starting on day 2 post infection. Multiple immune pathways differed in EHM horses in response to EHV-1. These included an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1 and responses skewed towards a T-helper 2 phenotype. Further, a dysregulation of coagulation and an upregulation of elements in the progesterone response were observed in EHM horses.

Hallmarks of the aging T-cell system.

The adaptive immune system has the enormous challenge to protect the host through the generation and differentiation of pathogen-specific short-lived effector T cells while in parallel developing long-lived memory cells to control future encounters with the same pathogen. A complex regulatory network is needed to preserve a population of naïve cells over lifetime that exhibit sufficient diversity of antigen receptors to respond to new antigens, while also sustaining immune memory. In parallel, cells need to maintain their proliferative potential and the plasticity to differentiate into different functional lineages. Initial signs of waning immune competence emerge after 50 years of age, with increasing clinical relevance in the 7th-10th decade of life. Morbidity and mortality from infections increase, as drastically exemplified by the current COVID-19 pandemic. Many vaccines, such as for the influenza virus, are poorly effective to generate protective immunity in older individuals. Age-associated changes occur at the level of the T-cell population as well as the functionality of its cellular constituents. The system highly relies on the self-renewal of naïve and memory T cells, which is robust but eventually fails. Genetic and epigenetic modifications contribute to functional differences in responsiveness and differentiation potential. To some extent, these changes arise from defective maintenance; to some, they represent successful, but not universally beneficial adaptations to the aging host. Interventions that can compensate for the age-related defects and improve immune responses in older adults are increasingly within reach.

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice.

T Follicular Helper (Tfh) cells are an independent CD4+ T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

3CL hydrolase-based multiepitope peptide vaccine against SARS-CoV-2 using immunoinformatics.

The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.

Follicular Helper T Cells Are Major Human Immunodeficiency Virus-2 Reservoirs and Support Productive Infection.

Follicular helper T cells (Tfh), CD4 lymphocytes critical for efficient antibody responses, have been shown to be key human immunodeficiency virus (HIV)-1 reservoirs. Human immunodeficiency virus-2 infection represents a unique naturally occurring model for investigating Tfh role in HIV/acquired immune deficiency syndrome, given its slow rate of CD4 decline, low to undetectable viremia, and high neutralizing antibody titers throughout the disease course. In this study, we investigated, for the first time, Tfh susceptibility to HIV-2 infection by combining in vitro infection of tonsillar Tfh with the ex vivo study of circulating Tfh from HIV-2-infected patients. We reveal that Tfh support productive HIV-2 infection and are preferential viral targets in HIV-2-infected individuals.

X4-Tropic Latent HIV-1 Is Enriched in Peripheral Follicular Helper T Cells and Is Correlated with Disease Progression.

Follicular helper T (TFH) cells have been shown to support productive human immunodeficiency virus type 1 (HIV-1) replication and to serve as a key component of the latent viral reservoir. However, the viral characteristics of this latent reservoir and the clinical relevance of this reservoir remain unclear. In this study, we assessed the tropic composition of latent viruses from peripheral TFH (pTFH), non-TFH memory, and naive CD4+ T cells from individuals with HIV-1 infections on suppressive combined antiretroviral therapy (cART). X4-tropic latent HIV-1 was preferentially enriched in pTFH cells compared to levels in the other two subsets. Interestingly, the ratio of X4-tropic latent HIV-1 in pTFH cells not only was robustly and inversely correlated with blood CD4+ T cell counts across patients but also was prognostic of CD4+ T cell recovery in individuals on long-term cART. Moreover, patients with higher X4-tropic latent HIV-1 ratios in pTFH cells showed greater risks of opportunistic coinfections. These findings reveal the characteristics of latent HIV-1 in TFH cells and suggest that the ratio of X4-tropic latent HIV-1 in pTFH cells is a valuable indicator for disease progression and cART efficacy.IMPORTANCE TFH cells have been shown to harbor a significant amount of latent HIV-1; however, the viral characteristics of this reservoir and its clinical relevance remain largely unknown. In this study, we demonstrate that X4-tropic latent HIV-1 is preferentially enriched in pTFH cells, which also accurately reflects the viral tropism shift. The ratio of X4-tropic proviruses in pTFH cells but not in other memory CD4+ T cell subsets is inversely and closely correlated with blood CD4+ T cell counts and CD4+ T cell recovery rates with cART. Our data suggest that the ratio of X4-tropic provirus in peripheral TFH cells can be easily measured and reflects disease progression and treatment outcomes during cART.

CD4+CD19+ conjugates favor HIV-1 infection and latency during chronic HIV-1 infection.

OBJECTIVE: CD4CD19 conjugates play an important role in regulating antibody responses and follicular helper T cells development in animal models. However, little is known regarding the characteristic of CD4CD19 conjugates in humans with chronic HIV-1 infection. METHODS: The numbers of CD4CD19 conjugates were counted in 86 HIV-1-infected patients, including 66 typical progressors and 20 complete responders. CD4CD19 conjugates were sorted by flow cytometry and dissociated into CD4 T singlets and CD19 B singlets. The phenotypes of these cells were analyzed in both typical progressors and complete responders, and the levels of HIV-1 DNA in CD4CD19 conjugates were measured in 10 complete responders. RESULTS: We identified CD4CD19 cells as one type of T-B conjugate in peripheral blood, and the numbers and percentages of CD4CD19 conjugates decreased with HIV-1 disease progression. Phenotypic analysis showed CD4CD19 conjugates expressed higher levels of surface CD32. mRNA analysis found that the mRNA levels for CD32b were significantly higher compared with CD32a in CD4CD19 conjugates. Further analysis found that CD4CD19 conjugates expressed higher levels of CCR7 and CXCR5 than CD4 T and CD19 B singlets. A virus infectivity assay showed that CD4CD19 conjugates expressed higher levels of HIV-1-p24 than CD4CD19 cells. CD4CD19 conjugates in lymph node from typical progressors expressed higher levels of HIV-1-p24 than CD4CD19 conjugates in respective peripheral blood. Importantly, CD4CD19 conjugates from complete responders contained higher levels of HIV-1 DNA than total CD4 T cells. CONCLUSION: Our study indicates that CD4CD19 conjugates actively participate in HIV-1 infection and latency, and may serve as a new cellular target to eliminate latency.

High numbers of activated helper T cells are associated with better clinical outcome in early stage vulvar cancer, irrespective of HPV or p53 status.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. METHODS: Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. RESULTS: Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8-Foxp3-), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161-CD38+HLA-DR+ and CD8+CD103+CD161-NKG2A+/-PD1++CD38++HLA-DR+) effector memory T cells. CONCLUSION: This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated.

T Cell-Intrinsic IL-6R Signaling Is Required for Optimal ICOS Expression and Viral Control during Chronic Infection.

The pleiotropic cytokine IL-6 plays an integral role not only in innate inflammatory responses but also in the activation and differentiation of lymphocyte subsets. In this study, by using a conditional knockout (cKO) model with selective IL-6 receptor deletion in T cells (IL-6R-cKO), we demonstrated that T cell-specific IL-6R signaling is essential for viral control during persistent lymphocytic choriomeningitis virus clone 13 infection. Strikingly, we observed that in contrast to previous studies with ubiquitous IL-6 deletion or blockade, specific IL-6R deletion in T cells did not affect T follicular helper (Tfh) cell accumulation unless IL-6R-deficient T cells were competing with wild-type cells in mixed bone marrow chimeras. In contrast, Tfh cells from IL-6R-cKO-infected mice exhibited reduced ICOS expression in both chimeric and nonchimeric settings, and this sole identifiable Tfh defect was associated with reduced germinal centers, compromised Ig switch and low avidity of lymphocytic choriomeningitis virus-specific Abs despite intact IL-6R expression in B cells. We posit that IL-6R cis-signaling is absolutely required for appropriate ICOS expression in Tfh cells and provides a competitive advantage for Tfh accumulation, enabling generation of optimal B cell and Ab responses, and ultimately viral control during in vivo chronic infection.

Context-Dependent Role for T-bet in T Follicular Helper Differentiation and Germinal Center Function following Viral Infection.

Following infection, inflammatory cues upregulate core transcriptional programs to establish pathogen-specific protection. In viral infections, T follicular helper (TFH) cells express the prototypical T helper 1 transcription factor T-bet. Several studies have demonstrated essential but conflicting roles for T-bet in TFH biology. Understanding the basis of this controversy is crucial, as modulation of T-bet expression instructs TFH differentiation and ultimately protective antibody responses. Comparing influenza and LCMV viral infections, we demonstrate that the role of T-bet is contingent on the environmental setting of TFH differentiation, IL-2 signaling, and T cell competition. Furthermore, we demonstrate that T-bet expression by either TFH or GC B cells independently drives antibody isotype class switching. Specifically, T cell-specific loss of T-bet promotes IgG1, whereas B cell-specific loss of T-bet inhibits IgG2a/c switching. Combined, this work highlights that the context-dependent induction of T-bet instructs the development of protective, neutralizing antibodies following viral infection or vaccination.

Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers.

CD32 expressing doublets in HIV-infected gut-associated lymphoid tissue are associated with a T follicular helper cell phenotype.

Gut-associated lymphoid tissue (GALT) is a key location for the HIV reservoir. The observation that B-cell-T-cell doublets are enriched for CD32a (a low-affinity IgG receptor) in peripheral blood raises interesting questions, especially as these cells have been associated with HIV DNA in some studies. We sought to determine if similar doublets were present in GALT, the significance of these doublets, and their implications for the HIV reservoir. Given the importance of GALT as a reservoir for HIV, we looked for expression of CD32 on gut CD4 T cells and for evidence of doublets, and any relationship with HIV DNA in HIV + individuals initiated on antiretroviral therapy (ART) during primary HIV infection (PHI). Tonsil tissue was also available for one individual. As previously shown for blood, CD32high CD4 cells were mainly doublets of CD4 T cells and B cells, with T-cell expression of ICOS in tonsil and gut tissue. CD4 T cells associated with CD32 (compared with 'CD32-' CD4 cells) had higher expression of follicular markers CXCR5, PD-1, ICOS, and Bcl-6 consistent with a T follicular helper (TFH) phenotype. There was a significant correlation between rectal HIV DNA levels and CD32 expression on TFH cells. Together, these data suggest that CD32high doublets are primarily composed of TFH cells, a subset known to be preferentially infected by HIV.

Cutaneous lesions of angioimmunoblastic T-cell lymphoma: Clinical, pathological, and immunophenotypic features.

BACKGROUND: Angioimmunoblastic T-cell lymphoma (AITL) is a systemic peripheral T-cell lymphoma with a follicular helper T-cell (TFH ) immunophenotype that frequently involves the skin. However, the histopathology of cutaneous involvement by AITL has not been fully established. METHODS: We reviewed the clinicopathological features of 19 patients seen at our institution with AITL involving the skin. Pan-T-cell and TFH marker expression was evaluated by immunohistochemistry. Epstein-Barr virus (EBV) was detected using in situ hybridization (ISH) for Epstein-Barr virus-encoded small RNA (EBER). T-cell receptor (TCR) gene rearrangement was evaluated by PCR. RESULTS: AITL affected both trunk and extremities in 15/19 cases (79%). Perivascular infiltration by small and/or medium-sized lymphocytes was seen in 18/19 (95%). Granulomatous inflammation was identified in 4/19 (21%). Aberrant loss of CD2, CD5, or CD7 was identified in 1/18 (6%), 2/18 (11%), or 7/19 (37%) cases, respectively. Seventeen of eighteen evaluable cases (95%) expressed 2 to 3 TFH markers: PD-1 in 19/19 (100%), BCL6 in 94% (17/18), and CD10 in 37% (7/19). EBV-positive cells were detected in 3/18 (17%) with varying density. Clonal TCR gene rearrangement was identified in 9/11 (82%). CONCLUSIONS: Cutaneous involvement by AITL shows relatively non-specific histopathological features. However, an immunohistochemical panel including TFH markers and EBER ISH is useful in differential diagnosis.

Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice.

Seasonally recurrent influenza virus infections are a significant cause of global morbidity and mortality. In murine models, primary influenza infection in the respiratory tract elicits potent humoral responses concentrated in the draining mediastinal lymph node and the spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary infection is also associated with formation of ectopic inducible bronchus-associated tissues (iBALT) in the lung. These structures display a lymphoid organization, but their function and protective benefits remain unclear. Here we examined the phenotype, transcriptional profile and antigen specificity of B cell populations forming iBALT in influenza infected mice. We show that the cellular composition of iBALT was comparable to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh) cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization. Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of anatomical localization. The architecture of iBALT was pleiomorphic and less structurally defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC reactions that contribute to the diversity of the humoral response targeting influenza.

Simian Immunodeficiency Virus Infects Functionally Polarized Memory CD4 T Cells Equivalently In Vivo.

Among the numerous immunological abnormalities observed in chronically human immunodeficiency virus (HIV)-infected individuals, perturbations in memory CD4 T cells are thought to contribute specifically to disease pathogenesis. Among these, functional imbalances in the frequencies of T regulatory cells (Tregs) and interleukin 17 (IL-17)/IL-22-producing Th cells (Th17/Th22) from mucosal sites and T follicular helper (Tfh) cells in lymph nodes are thought to facilitate specific aspects of disease pathogenesis. However, while preferential infection of Tfh cells is widely thought to create an important viral reservoir in an immunologically privileged site in vivo, whether immunological perturbations among memory CD4 T cell populations are attributable to their relative infectivity by the virus in vivo is unclear. Here we studied peripheral blood and lymphoid tissues from antiretroviral (ARV)-treated and ARV-naive Asian macaques and isolated functionally defined populations of memory CD4 T cells. We then assessed the degree to which these populations were infected by simian immunodeficiency virus (SIV) in vivo, to determine whether particular functionally identified populations of memory CD4 T cells were preferentially infected by the virus. We found that SIV did not preferentially infect Th17 cells, compared to Th1 cells, Th2 cells, or Tregs. Moreover, Th17 cells contributed proportionately to the total pool of infected cells. Taken together, our data suggest that, although Tfh cells are more prone to harbor viral DNA, other functionally polarized cells are equally infected by the virus in vivo and Th17 cells are not preferentially infected.IMPORTANCE Functional perturbations of memory CD4 T cells have been suggested to underlie important aspects of HIV disease progression. However, the mechanisms underlying these perturbations remain unclear. Using a nonhuman primate model of HIV, we show that SIV infects functionally defined populations of memory CD4 T cells equally in different anatomic sites. Thus, preferential infection by the virus is unlikely to cause functional perturbations.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV.

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.