Chronic HIV-1 Infection Impairs Superantigen-Induced Activation of Peripheral CD4+CXCR5+PD-1+ Cells, With Relative Preservation of Recall Antigen-Specific Responses.
ABSTRACT: Peripheral CD4+CXCR5+PD-1+ T cells are a putative circulating counterpart to germinal center T follicular helper (TFH) cells. They show both phenotypic and functional similarities to TFH cells, which provide necessary help for the differentiation of B cells to antibody-secreting plasmablasts. In this study, we evaluated the frequency, phenotypes, and responses of peripheral TFH-like (pTFH) cells to superantigen and recall antigen stimulation in 10 healthy and 34 chronically infected treatment-naive HIV-1+ individuals. There was no difference in the frequency of pTFH cells between HIV+ and HIV- individuals. Surface expression of ICOS, but not CD40L, was higher on pTFH cells at baseline in HIV+ individuals. Compared with HIV- individuals, pTFH cells from HIV+ individuals had decreased maximal expression of ICOS and CD40L in response to in vitro superantigen stimulation. This decreased response did not correlate with viral control, CD4 T-cell count, duration of infection, or the degree of neutralizing antibody breadth. Despite a decreased maximal response, pTFH responses to HIV Gag and tetanus toxoid recall antigens were preserved.
Project description:Antigen-primed cluster of differentiation (CD) 4+ T follicular helper (Tfh) cells interact with B cells in the germinal centers (GCs) of lymph nodes to generate vaccine-induced antibody (Ab) responses. In the circulation, peripheral Tfh (pTfh) cells, a subset of memory CD4 T cells, serve as surrogates for GC Tfh because of several functional and phenotypic similarities between them. We investigated features of H1N1 influenza antigen-specific pTfh (Ag.pTfh) in virologically controlled HIV+ volunteers on antiretroviral therapy (ART) and healthy control (HC) participants selected from a seasonal influenza vaccine responsiveness study. Selection of the participants was made based on age, defined as young (18-40 y) and old (>60 y) and on their classification as a vaccine responder (VR) or vaccine nonresponder (VNR). VRs demonstrated expansion of CD40L+ and CD69+ Ag.pTfh, with induction of intracellular interleukin 21 (IL-21) and inducible costimulator (ICOS) post vaccination; these responses were strongest in young HC VRs and were less prominent in HIV+ individuals of all ages. Ag.pTfh in VNRs exhibited dramatically different characteristics from VRs, displaying an altered phenotype and a cytokine profile dominated by cytokines IL-2, tumor necrosis factor alpha (TNF-?), or IL-17 but lacking in IL-21. In coculture experiments, sorted pTfh did not support the B cell IgG production in VNRs and were predominantly an inflammatory T helper 1 (Th1)/T helper 17 (Th17) phenotype with lower ICOS and higher programmed cell death protein 1 (PD1) expression. Induction of IL-21 and ICOS on Ag.pTfh cells are negatively affected by both aging and HIV infection. Our findings demonstrate that dysfunctional Ag.pTfh cells with an altered IL-21/IL-2 axis contribute to inadequate vaccine responses. Approaches for targeting inflammation or expanding functional Tfh may improve vaccine responses in healthy aging and those aging with HIV infection.
Project description:Background:T follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation. Methods:In a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART-) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV-), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA. Results:ART- children had reduced levels of pTfh cells compared with HIV- children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios. Conclusion:Low memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies.
Project description:In this study, we examined the peripheral blood (PB) central memory (TCM) CD4(+) T cell subsets designated peripheral T follicular helper cells (pTfh cells) and non-pTfh cells to assess HIV permissiveness and persistence. Purified pTfh and non-pTfh cells from healthy HIV-negative donors were tested for HIV permissiveness using green fluorescent protein (GFP)-expressing HIV-1NL4-3/Ba-L, followed by viral reactivation using beads coated with anti-CD3/anti-CD28 monoclonal antibodies. The role of pTfh cells in HIV persistence was analyzed in 12 chronically HIV-1 infected patients before and 48 weeks after initiation of raltegravir-containing combination antiretroviral therapy (cART). Total cellular HIV-1 DNA and episomes containing two copies of the viral long terminal repeat (2LTR circles) were analyzed in using droplet digital PCR in the purified pTfh and non-pTfh cells. Activation-inducible HIV p24 expression was determined by flow cytometry. Results indicate that pTfh cells, in particular PD1(+) pTfh cells, showed greater permissiveness for HIV infection than non-pTfh cells. At week 48 on cART, HIV DNA levels were unchanged from pre-cART levels, although a significant decrease in 2LTR circles was observed in both cell subsets. Inducible HIV p24 expression was higher in pTfh cells than in non-pTfh cells, with the highest frequencies in the PD1(+) CXCR3(-) pTfh cell subset. Frequencies of HLADR(+) CD38(+) activated CD4 T cells correlated with 2LTR circles in pTfh and non-pTfh cells at both time points and with p24(+) cells at entry. In conclusion, among CD4 TCM cells in PB of aviremic patients on cART, pTfh cells, in particular the PD1(+) CXCR3(-) subset, constitute a major HIV reservoir that is sustained by ongoing residual immune activation. The inducible HIV p24 assay is useful for monitoring HIV reservoirs in defined CD4 T cell subsets.Identification of the type and nature of the cellular compartments of circulating HIV reservoirs is important for targeting of HIV cure strategies. In lymph nodes (LN), a subset of CD4 T cells called T follicular helper (Tfh) cells are preferentially infected by HIV. Central memory (TCM) CD4 T cells are the major cellular reservoir for HIV in peripheral blood and contain a subset of CD4 TCM cells expressing chemokine receptor CXCR5 similar in function to LN Tfh cells termed peripheral Tfh (pTfh) cells. We found that the circulating pTfh cells are highly susceptible to HIV infection and that in HIV-infected patients, HIV persists in these cells following plasma virus suppression with potent cART. These pTfh cells, which constitute a subset of TCM CD4 T cells, can be readily monitored in peripheral blood to assess HIV persistence.
Project description:The generation of Ab-secreting plasma cells depends critically on CD4 T-follicular helper (TFH) cells during the germinal center reaction. Germinal center TFH cells share functional properties with circulating CXCR5(+) CD4 T cells, referred to herein as peripheral TFH (pTFH) cells. Because deficient Ab production and CD4 T-cell loss are recognized features of HIV infection, in the present study, we investigated pTFH cells in 25 HIV-infected patients on antiretroviral therapy. pTFH frequency was equivalent in patients and healthy controls (HCs), and these cells displayed a central memory phenotype. Sixteen patients and 8 HCs in this group were given a single dose of H1N1/09 influenza vaccine during the 2009 H1N1 influenza outbreak. In the vaccine responders (n = 8) and HCs, pTFH cells underwent expansion with increased IL-21 and CXCL13 secretion in H1N1-stimulated PBMC culture supernatants at week 4 (T2). These changes were not seen in vaccine nonresponders (n = 8). In coculture experiments, sorted pTFH cells supported HIN1-stimulated IgG production by autologous B cells only in vaccine responders. At T2, frequencies of pTFH were correlated with memory B cells, serum H1N1 Ab titers, and Ag-induced IL-21 secretion. Characterization of pTFH cells may provide additional insight into cellular determinants of vaccine-induced Ab response, which may have relevance for vaccine design.
Project description:Follicular helper CD4 T (Tfh) cells provide B cells with signals that are important for the generation of high-affinity Abs and immunological memory and, therefore, are critical for the protective immunity elicited by most human vaccines. Transcriptional regulators of human Tfh cell differentiation are poorly understood. In this article, we demonstrate that Bcl6 controls specific gene modules for human Tfh cell differentiation. The introduction of Bcl6 expression in primary human CD4 T cells resulted in the regulation of a core set of migration genes that enable trafficking to germinal centers: CXCR4, CXCR5, CCR7, and EBI2. Bcl6 expression also induced a module of protein expression critical for T-B interactions, including SAP, CD40L, PD-1, ICOS, and CXCL13. This constitutes direct evidence for Bcl6 control of most of these functions and includes three genes known to be loci of severe human genetic immunodeficiencies (CD40L, SH2D1A, and ICOS). Introduction of Bcl6 did not alter the expression of IL-21 or IL-4, the primary cytokines of human Tfh cells. We show in this article that introduction of Maf (c-Maf) does induce the capacity to express IL-21. Surprisingly, Maf also induced CXCR5 expression. Coexpression of Bcl6 and Maf revealed that Bcl6 and Maf cooperate in the induction of CXCR4, PD-1, and ICOS. Altogether, these findings reveal that Bcl6 and Maf collaborate to orchestrate a suite of genes that define core characteristics of human Tfh cell biology.
Project description:Dysfunction of T follicular-helper (TFH) cells is a possible cause of impaired germinal centre (GC) and IgG antibody responses in individuals with human immunodeficiency virus-1 (HIV-1) infection and might contribute to decreased magnitude and isotype diversification of IgG antibodies to pneumococcal polysaccharides (PcPs). We examined the production of IgG1 and IgG2 antibodies to PcPs 4, 6B, 9V and 14 by enumerating antibody secreting cells (ASCs) at day (D) 7 and determining fold-increase in serum antibody levels at D28 after vaccination with unconjugated PcPs in HIV seronegative subjects (n = 20) and in HIV patients who were receiving antiretroviral therapy (ART) (n = 28) or who were ART-naive (n = 11) and determined their association with ICOS+ and ICOS- circulating memory TFH (cmTFH) cells (CD4+CD45RA-CD27+CXCR5+PD-1+) and short lived plasmablasts (SPBs) at D7, and with PcP-specific and total IgM+ and IgG+ memory B cells at D0. In HIV seronegative subjects, production of IgG1+ and IgG2+ ASCs was consistently associated with the frequency of ICOS+ cmTFH cells but not ICOS- cmTFH cells or memory B cells. In contrast, post-vaccination ASCs in HIV patients, regardless of ART status, were lower than in HIV seronegative subjects and not associated with ICOS+ cmTFH cells, the expansion of which was absent (ART-naive patients) or much lower than in HIV seronegative subjects (ART-treated patients). Production of SPBs was also lower in ART-naive patients. Fold-increase in IgG2 antibodies at D28 also correlated with ICOS+ cmTFH cells at D7 in HIV seronegative subjects but not in HIV patients. These novel findings provide evidence that ICOS+ cmTFH cells contribute to the regulation of PcP-specific IgG antibody responses, including isotype diversification, and that TFH cell dysfunction may be a cause of impaired PcP-specific IgG antibody responses and increased susceptibility to pneumococcal disease in HIV patients.
Project description:Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4(+) cells did not change, and CCR6(+) cells decreased. CXCR3(+), but not CXCR3(-), GC Tfh cells produced IFN-? (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4(+)IFN-?(+) T cells within the hyperplastic follicles during chronic SIV infection. CXCR3(+) GC Tfh cells also expressed higher levels of ICOS, CCR5, and ?4?7 and contained more copies of SIV DNA compared with CXCR3(-) GC Tfh cells. However, CXCR3(+) and CXCR3(-) GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs.
Project description:Background:Follicular helper T cells (TFH) are specialized CD4 T cells required for B-cell help and antibody production. Methods:Given the postulated role of immune activation in dengue disease, we measured the expansion and activation of TFH in the circulation (peripheral TFH [pTFH]) collected from Thai children with laboratory-confirmed acute dengue virus (DENV) infection. Results:We found significant expansion and activation of pTFH subsets during acute infection with the highest frequencies of activated pTFH (PD1hi pTFH and PD1+CD38+ pTFH) detected during the critical phase of illness. Numbers of activated pTFH were higher in patients with secondary compared with primary infections and in patients with more severe disease. We also found a positive correlation between the frequencies of activated pTFH and the frequencies of plasmablasts. Conclusions:To our knowledge, this is the first ex vivo analysis of pTFH activation during acute DENV infection. Overall, our study supports the model that pTFH contribute to disease evolution during the critical stage of illness.
Project description:The majority of HIV-infected individuals fail to produce protective antibodies and have diminished responses to new immunizations. We report here that even though there is an expansion of follicular helper T (TFH) cells in HIV-infected individuals, the cells are unable to provide adequate B cell help. We found a higher frequency of programmed cell death ligand 1 (PD-L1)(+) germinal center B cells from lymph nodes of HIV-infected individuals suggesting a potential role for PD-1-PD-L1 interaction in regulating TFH cell function. In fact, we show that engagement of PD-1 on TFH cells leads to a reduction in cell proliferation, activation, inducible T-cell co-stimulator (ICOS) expression and interleukin-21 (IL-21) cytokine secretion. Blocking PD-1 signaling enhances HIV-specific immunoglobulin production in vitro. We further show that at least part of this defect involves IL-21, as addition of this cytokine rescues antibody responses and plasma cell generation in vitro. Our results suggest that deregulation of TFH cell-mediated B cell help diminishes B cell responses during HIV infection and may be related to PD-1 triggering on TFH cells. These results demonstrate a role for TFH cell impairment in HIV pathogenesis and suggest that enhancing their function could have a major impact on the outcome and control of HIV infection, preventing future infections and improving immune responses to vaccinations.
Project description:Aging and HIV infection are independently associated with excessive immune activation and impaired immune responses to vaccines, but their relationships have not been examined.For selecting an aging population we enrolled 28 post-menopausal women including 12 healthy volunteers and 16 HIV-infected women on antiretroviral treatment with <100 HIV RNA copies/ml. Antibody titers to trivalent influenza vaccination given during the 2011-2012 season were determined before and 4 weeks after vaccination.Seroprotective influenza antibody titers (? 1:40) were observed in 31% HIV(+) and 58% HIV-uninfected women pre-vaccination. Following vaccination, magnitude of antibody responses and frequency of seroprotection were lower in HIV(+) (75%) than in HIV(-) (91%) women. Plasma IL-21, the signature cytokine of T follicular helper cells (Tfh), and CD4 T cell IL-21R were upregulated with seroconversion (? 4 fold increase in antibody titer). Post-vaccine antibody responses were inversely correlated with pre-vaccination plasma TNF? levels and with activated CD4 T cells, including activated peripheral (p)Tfh. Plasma TNF? levels were correlated with activated pTfh cells (r=0.48, p=0.02), and inversely with the post-vaccination levels of plasma IL-21 (r=-0.53, p=0.02). In vitro TNF? blockade improved the ability of CD4 T cells to produce IL-21 and of B cells to secrete immunoglobulins, and addition of exogenous IL-21 to cell cultures enhanced B cell function. Higher frequencies of activated and exhausted CD8 T and B cells were noted in HIV(+) women, but these markers did not show a correlation with antibody responses.In aging HIV-infected and uninfected women, activated CD4 and pTfh cells may compromise influenza vaccine-induced antibody response, for which a mechanism of TNF?-mediated impairment of pTfh-induced IL-21 secretion is postulated. Interventions aimed at reducing chronic inflammation and immune activation in aging, HIV-infected patients may improve their response to vaccines.