Dysfunctional phenotype of T cells and their contribution to impaired B cell function during HIV-1 infection

Microbial translocation and increased immune activation have been involved in functional T cell impairments and disease progression during HIV-1 infection. The impact of microbial translocation on the phenotype of memory B cells in HIV-1 infected patients was studied in paper I. The expression of activation marker IL-21R was higher in HIV-1 infected patients compared with controls. An inverse correlation was observed between IL-21R expression and frequency of resting memory (RM) B cells in blood; IL-21R+ RM B cells were more sensitive to apoptosis and their frequency correlated with sCD14, a marker of microbial translocation. Furthermore, TLR triggering by microbial products resulted in IL- 21R expression on memory B cells in vitro. These results suggest a direct link between microbial translocation and an impaired B cell phenotype. In paper II we showed that IL-7 induced upregulation of CD70 expression on T cells. Increased CD70 expression, by triggering the CD27 receptor on B cells, can lead to alteration of the B cell phenotype and IgG production. In addition, IL-7 led to an increased production of BAFF by T cells, which enhanced B cell survival in vitro. In the context of HIV-1 infection, the mechanisms mediated by increased CD70 expression on T cells might be implicated in establishment of B cell activation, a characteristic of immune pathology in infected patients. The role of CD70 in B cell dysfunction during HIV-1 infection was further studied in paper III. We found an increased expression of CD70 on CD4+ T cells which correlated with CD4+ T cell depletion and viremia in HIV-1 infected patients. CD4+ CD70+ T cells expressed pro-inflammatory cytokines and, based on their chemokine profile, it was predicted that they can migrate to sites of inflammation. A potential role for CD4+ CD70+ T cells in B cell activation in HIV-1 infected individuals was suggested by the association with CD38 and CD95 expression in memory B cells, with increased B cell proliferation and plasma IgG levels. The mechanism leading to CD70 up- regulation on T cells during HIV-1 infection remains elusive. Although treatment with ART can lead to a nearly complete suppression of HIV-1 replication, ART does not fully target the increased immune activation found in HIV-1 infected patients. We showed in paper IV that ART initiation during primary HIV-1 infection (PHI) (early ART=EA) did not prevent the establishment of phenotypical changes of T cells, previously reported in HIV-1 infected patients starting treatment during the chronic phase of infection (late ART=LA). The phenotypical changes of T cells, comparable in the EA and LA groups, consisted in increased expression of immune activation markers HLA-DR and CD38 and reduced expression of CD127, which characterizes differentiated CD8+ T cells. It is worrisome that ART initiation during PHI does not correct for abnormal immune activation. It is however interesting that the number of HIV-1 DNA copies in blood of EA patients was significantly lower compared to LA patients; the correlation between T cell phenotype and size of the HIV-1 reservoir should be studied further. The frequency of B cell sub-populations in blood of EA and LA patients did not differ (preliminary results) and was not significantly altered compared to non-infected controls

Impaired B cells survival upon production of inflammatory cytokines by HIV-1 exposed follicular dendritic cells

Additional file 3. Activation of B and T cells upon different stimuli. The frequency of activated CD69 + cells among PBMCs (A), B cells (B) and T cells (C) are shown when PBMCs were exposed to different activation stimuli

IL-7 Promotes CD95-Induced Apoptosis in B Cells via the IFN-γ/STAT1 Pathway

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals

IL-7 and CD4 T Follicular Helper Cells in HIV-1 Infection

IL-7 was previously shown to upregulate the expression of molecules important for interaction of CD4+ T cells with B cells. It is poorly studied whether IL-7 has a role in the biology of T follicular helper (Tfh) cells and whether IL-7 dysregulates the expression of B-cell costimulatory molecules on Tfh cells. We review the literature and provide arguments in favor of IL-7 being involved in the biology of human Tfh cells. The CD127 IL-7 receptor is expressed on circulating Tfh and non-Tfh cells, and we show that IL-7, but not IL-6 or IL-21, upregulates the expression of CD70 and PD-1 on these cells. We conclude that IL-7, a cytokine whose level is elevated during HIV-1 infection, may have a role in increased expression of B cell costimulatory molecules on Tfh cells and lead to abnormal B cell differentiation

Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+) T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+) T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+) T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF) and was more prominent on gut-homing compared to lymph node-homing CD4(+) T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+) T cells to HIV infection and target cell availability in the gut in some infected individuals

Dysfunctional phenotypes of CD4+ and CD8+ T cells are comparable in patients initiating ART during early or chronic HIV-1 infection

11sinoneEarly initiation of antiretroviral therapy (ART) is becoming a common clinical practice according to current guidelines recommending treatment to all HIV-1-infected patients. However, it is not known whether ART initiated during the early phase of infection prevents the establishment of abnormal phenotypic features previously reported in CD4+ and CD8+T cells during chronic HIV-1 infection. In this cross-sectional study, blood specimens were obtained from 17 HIV-1-infected patients who began ART treatment shortly after infection (early ART [EA]), 17 age-matched HIV-1-infected patients who started ART during chronic phase of infection (late ART [LA]), and 25 age-matched non-HIV-1-infected controls. At collection of specimens, patients in EA and LA groups had received ART for comparable periods of time. Total HIV-1 DNA was measured in white blood cells by quantitative PCR. The concentration of 9 inflammatory parameters and 1 marker of fibrosis, including sCD14 and b-2 microglobulin, was measured in plasma. Furthermore, expression of markers of abnormal immune activation (human leukocyte antigen-antigen D related [HLA-DR] and CD38), exhaustion (programmed death 1, CD28, CD57) and terminal differentiation (CD127) was measured on CD4+ and CD8+T cells. T-cell proliferation was measured through Ki67 expression. The copies of total HIV-1 DNA in blood were significantly lower (P=0.009) in EA compared with that in LA group. Only the expression of HLA-DR on naïve CD4+ T cells distinguished EA from LA, whereas expression of 3 surface markers distinguished T-cell populations of HIV-1-infected patients from controls. These included HLA-DR distinguishing CD4+ T cells from EA compared with controls, and also CD38 and CD127 on CD4+ and CD8+ T cells, respectively, distinguishing both groups of patients from controls. The sCD14 levels were significantly higher in EA patients, and b-2 microglobulin levels were higher in LA group compared with that in controls. Our results demonstrate an equivalent abnormal expression of activation (HLA-DR and CD38 on CD4+ T cells) and terminal differentiation (CD127 on CD8+ T cells) markers in T cells from both EA and LA patients. The size of total HIV-1 DNA copies in blood of EA was lower compared with LA patients. These findings suggest that some abnormalities taking place in the T-cell compartment during primary HIV-1 infection may not be corrected by early ART.openAmu, Sylvie; Graham, Rebecka Lantto; Bekele, Yonas; Nasi, Aikaterini; Bengtsson, Carina; Rethi, Bence; Sorial, Sam; Meini, Genny; Zazzi, Maurizio; Hejdeman, Bo; Chiodi, FrancescaAmu, Sylvie; Graham, Rebecka Lantto; Bekele, Yonas; Nasi, Aikaterini; Bengtsson, Carina; Rethi, Bence; Sorial, Sam; Meini, Genny; Zazzi, Maurizio; Hejdeman, Bo; Chiodi, Francesc

IL-7 induces CD95 upregulation on B cells via IFN-γ released from T cells.

<p><b>A.</b> Purified T cells were cultured in the presence or absence of 25 ng/ml IL-7 and IFN-γ concentrations were measured at the indicated time points using ELISA. Mean values and standard deviations are calculated from the results of 7 independent experiments. <b>B.</b> IFN-γ expression was analyzed by flow cytometry in naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), effector memory (CD45RA-CCR7−) and TEMRA (CD45RA+CCR7−) T cell subsets cultured in the presence (black line) or absence (grey line) of 25 ng/ml IL-7 for five days. Dashed lines represent isotype control staining. Data are representative of 3 independent experiments. <b>C.</b> IFN-γ mRNA levels were measured in IL-7 treated or untreated T cells by real time PCR. <b>D.</b> IFN-γ concentrations measured in the IL-7 treated or non-treated T cell supernatants are correlated with the ability of the same supernatants to induce CD95 expression on B cells (using the samples described in panel A). Black dots represent IL-7 treated, white dots represent non treated T cell supernatants collected at day 2, 5, 8 or 11. <b>E.</b> CD95 expression is shown on freshly isolated B cells (dashed line) or following a 12 h (grey lines) or 48 h (black lines) treatment with IL-7 treated T cell supernatants or 20 ng/ml recombinant human IFN-γ. <b>F.</b> STAT-1 phosphorylation is measured using flow cytometry in sorted B cells treated with supernatants of IL-7 treated T cells in combination with 10 mg/ml neutralizing anti-IFN-γ or isotype control antibodies. Data are representative of 3 experiments. <b>G.</b> B lymphocytes were cultured for five days with IL-7 treated or non treated T cell supernatants or were left untreated in the presence of 10 mg/ml neutralizing anti-IFN-γ or isotype control Abs. Thereafter the expression of CD95 was analyzed by flow cytometry. Data are representative of 3 different experiments.</p

GPR15 is strongly up-regulated on gut homing CD4⁺Tcells and is highly expressed on colon CD4⁺Tcells.

<p>TLR3 stimulation up-regulates GPR15 on gut homing (α4β7-integrin⁺) (A, C) and on CD4⁺ T cells homing to lymph nodes (CD62L⁺) (B, D). The different symbols in the Figures C and D specify different donors. Before TLR3 stimulation both subsets express GPR15 to a similar level (E). The different symbols describe individual donors (E). PBMCs were isolated from whole blood by Lymphoprep gradient centrifugation and co-stained for CD4, GPR15 and CD62L or β7-integrin. The cells were gated on lymphocytes, CD4⁺ as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1 A</a>. The graphs show GPR15 expression as a percent of CD62L⁺ CD4⁺ T cells or β7⁺ CD4⁺ T cells expressing the co-receptor. The experiments were done at least two times including two donors each time. Statistical analysis was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g003" target="_blank">Figure 3</a> using paired t-test. Human colon intraepithelial mononuclear cells (IEMC) and lamina propria mononuclear cells (LPMC) express GPR15 on high level. IEMC and LPMC were isolated following the described protocol and co-stained for CD45, CD3, CD4 and GPR15. Cells were gated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1A</a> with the accretion that CD45 were gated out to exclude epithelial cell contamination (F). Colon biopsies of HIV-1 infected and uninfected individuals were immunofluorescently stained for GPR15, CD4 and cell nuclei using DAPI. Slides were analysed by confocal microscopy. Three biopsies per patient and 15–20 images per biopsy were acquired at 63×. Cells were enumerated using ImageJ cell counting software for % of CD4⁺ cell expressing GPR15 (G).</p

Enhanced CD95 mediated apoptosis of T and B cells upon IL-7 treatment.

<p>Kinetics of CD95 expression (left panels) and CD95 mediated apoptosis sensitivity (right panels) of CD3+ T cells (<b>A</b>) and CD19+ B cells (<b>B</b>) are shown, measured in PBMC cultures treated with 25 ng/ml IL-7, with supernatants of IL-7 treated or non treated T cell cultures or left untreated. Apoptosis was triggered using recombinant CD95L added for 24 hours to cultures. Data are representative of 3 independent experiments.</p