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CD32 Expression Is Associated to T Cell Activation and Is Upregulated By CROI2016 Boston CD32 expression is associated to T cell activation and is upregulated by HIV #385 E-mail: [email protected] Roger Badia, Edurne García-Vidal, Maria Pujantell, Bonaventura Clotet, Miguel A. Martínez, Ester Ballana, Eva Riveira-Muñoz, José A. Esté Phone: 34 934656374 IrsiCaixa AIDS Research Institute and Health Research Institute Germans Trias i Pujol (IGTP), Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona (Badalona, Catalonia) Fax: 34 934653968 ABSTRACT CD32 IS A MARKER OF T CELL ACTIVATION HIV-1 INFECTION INDUCES UPREGULATION OF CD32 EXPRESSION CD32 EXPRESSION IS ASSOCIATED TO HLA-DR ACTIVATION MARKER IN HIV+ INDIVIDUALS BACKGROUND: HIV infection establishes a subset of latently infected CD4+ T cells thought not to a) c) d) a) Fig 4. CD32 is upregulated after produce viral proteins and remain indistinguishable from uninfected cells. However, the productive HIV-1 infection. (a) a) overexpression of the gene encoding for the transmembrane protein FCGR2A (CD32a+) has been CD32 expression in PHA/IL-2 proposed as a potential marker of HIV+ latently infected cells. The study of molecular signatures stimulated CD4+ T cells infected that allow the identification of resting, latently infected cells would facilitate the development of with HIV-1 (a representative new therapeutic approaches donor out of 5 is shown). The left METHODOLOGY: Cell surface markers, including CD32 and activation markers HLA-DR and CD69, panel indicates the ratio of were measured in PBMC and CD4+ T lymphocytes from healthy donors and HIV+ individuals by HIV+(GFP+) to CD32- or to CD32+ flow cytometry and mRNA by qPCR. Effect of HIV-1 infection on CD32 expression was determined cells (N:5) (b) Percentage of in FACS-sorted CD4+ T lymphocytes stimulated with or without IL-2 (16 U/ml) and PHA (4 µg/ml) b) CD32 cell surface expression and/or infected with an HIV-1 NL4-3-GFP virus modified to express Vpx (NL4-3*GFP-Vpx). measured by flow cytometry and SSC Contribution of CD32+ cells to the viral reservoir was determined in sorted CD4+ T cells from infected with different CD32 healthy donors infected in vitro or from HIV+ patients by qPCR of integrated HIV-1 DNA. multiplicities of infection of HIV- b) c) d) RESULTS: Stimulation of CD4+ T cells with IL-2/PHA, αCD3/αCD28 or IL-7 induced the expression b) e) 1 NL4-3 or uninfected (UN). (c) of CD32 concomitant to the activation markers HLA-DR and/or CD69 respectively. Infection with Upregulation of CD32 expression (a) HIV-1 NL4-3*GFP-Vpx of non-stimulated or activated CD4 T cells increased CD32 expression that after HIV-1 infection (INF) is was strongly associated to HLA-DR or CD69 expression. Addition of the NNRTI efavirenz inhibited NL4-3*GFP (Vpx) reduced concomitant to CD32 expression, indicating a virus replication induced effect. CD32 expression in CD4+ T cells c) d) blockade of HIV-1 infection with from HIV+ individuals (n: 19) under antiretroviral treatment indicated that a mean of 85% (70-94) efavirenz (INF+EFV). (d) of cells were CD32+/HLA-DR+. We found higher proviral DNA copies/cell in resting CD4+/CD32- T Percentage of HIV-1+ (GFP+) cells cells (n:5) infected in vitro with HIV-1 NL4-3*GFP-Vpx, except in one donor with significantly in the CD32+ compartment. Data higher basal CD4 T cell activation (HLA-DR+/CD69+ cells). There were no statistically significant represent mean ± SD of 5 (p:0.76, n:10) differences in the mean viral DNA copies/cell in CD32+ or CD32- CD4+ T cells from different donors. HIV+ individuals under therapy but 2 of 10 patients showed higher DNA copies/cell in CD32+ cells. Fig 6. CD32 expression in HIV-1+ individuals is associated to HLA-DR activation marker in CD4+ T cells. (a) Representative dot- However, total DNA copies were higher in the CD32- compartment in all patients (mean 18-fold). plots of CD32 staining in HIV-1 infected individuals (N:19). Individual isotype control labeling was set to a stringent criteria (less or equal to 0.1% positive cells).(b) Expression of cell surface CD32 measured by flow cytometry in CD4+ T cells from healthy CONCLUSIONS: CD32 expression is a marker of CD4+ T cell activation in healthy donors and HIV+ CD32+ CELLS ARE NOT ENRICHED WITH PROVIRAL HIV FOLLOWING donors (HIV-1Neg) and HIV-1+ individuals (HIV-1Pos) (c) Percentage of CD32 positive cells in HIV-1 infected patients. CD32 cell patients. The viral reservoir lay outside the CD32+ component as the majority of HIV DNA copies INFECTION IN CELL CULTURE surface expression was measured in 19 HIV-1+ individuals by flow cytometry in combination with the HLA-DR (left panel) or are harbored in CD32- cells. HIV-1 latency may not be preferentially associated to CD32+cells. CD69 (right panel) activation markers. (d) Contribution of the CD32+ cells to the HLA-DR and CD69 compartments in CD4+ T a) cells from HIV-1+ individuals. Data represents mean ± SD from healthy donors (N=10) and HIV-1+ individuals (N=19). ** p<0.01 Fig 2. Evaluation of CD32 and T cell activation markers. (a) CD32 expression CD4+ T cells stimulated with IL-2, PHA/IL-2 or α- *** p<0.001. CD3/α-CD28 from 5-4 different donors. Cells were cultured in the presence of different stimuli for 72h and protein levels of the GATING STRATEGY AND DETERMINATION OF CD32 EXPRESSION cell surface marker CD32 were evaluated by flow cytometry. (b) Dot plots for representative donor. (c) CD32 gene expression a) Lymphocytes Isotype levels after T cell activation with PHA/IL-2 as measured as mRNA levels of CD32a expression in CD4+ T cells activated with PHA/IL2 CONTRIBUTION OF HIV-1 PROVIRAL TO CD32 COMPARTMENT IN HIV+ INDIVIDUALS or with IL2. Relative mRNA expression of CD32a was measured by quantitative PCR and normalized to GAPDH expression. Data Monocytes represents 1/ΔCt*100 in 5 distinct donors. (d) Upregulation of the CD32 correlates with the expression of activation marker HLA- DR and CD69 after stimulation with PHA/IL2 and α-CD3/α-CD28 (representative donor is shown). (e) CD32+ cells are located Fig 7. Determination on the HIV-1 within the HLA-DR+ compartment after CD4+ T cell activation through PHA/IL2 or α-CD3/α-CD28 (left panel) and within the CD69 proviral DNA in the CD32+ A A - - 3 compartment of CD4+ T cells SSC CD compartment for PHA/lL2 stimulation (right panel). Data represents mean ± SD of 4-5 different donors. * p<0.05, ** p<0.01. SSC FSC from HIV patients under ART. (a) FSC-H FSC-A CD14 CD32 b) c) Quantification of proviral HIV-1 b) Isotype DNA copies per cell in CD32- INTERLEUKIN-7 ACTIVATION IS ASSOCIATED TO CD32 EXPRESSION (black bars) and CD32+ (white Limf. T CD3+CD4+ bars) CD4 T cells from 10 ART Limf. a) b) B Fig 3. Co-expression of CD32 and treated individuals. (b) Individual CD3+CD8+ activation markers HLA-DR and HIV-1 DNA copies per cell data in CD69 in CD4+ T cells from CD32- and CD32+ CD4 T cells. DR 3 3 - healthy donors induced by c) Median values are presented. NS, CD CD d) CD8 CD19 HLA CD32 interleukin-7. (a) Percentage of d) e) not significant (p = 0.3). (c) Total CD32 expression Ki67+ cells in IL-2, α-CD3/α-CD28 number of HIV pDNA copies in Isotype Isotype Isotype or IL-7 –activated CD4+ T the CD32- (black bars) and CD32+ Activation markers lymphocytes. (b) CD32 (white bars) CD4 T cell c) expression is CD4+ T cells compartment from 10 ART stimulated with IL-2 or IL-7 in 3 treated individuals. (d) Mean different donors. Dot plots from contribution of proviral HIV-1 one representative donor are DNA in the CD32- and CD32+ CD4 shown on the right. Upregulation Fig 5. Contribution of HIV-1 proviral DNA in the CD32+ compartment of CD4+ T cells from in vitro T cell compartment. CD69 of CD32 expression in CD4+ T HLA-DR infections. (a) Infection of CD4+ T cells stimulated with IL-2 infected with NL4-3GFP, NL4-3*GFP and NL4- cells correlates with the 3*GFP carrying Vpx. Percentage of infection was evaluated by flow cytometry. Representative dot plots DR 69 - expression of activation markers are shown on the left. (b) Percentage of CD32 cell surface expression measured by flow cytometry and SSC CD HLA HLA-DR (c) and CD69 (d) after CD32 CD32 CD32 d) infected with different multiplicities of infection of HIV-1 NL4-3 carrying HIV-2 Vpx or uninfected (UN). stimulation with IL-7. Dot plots (c) Upregulation of CD32 expression after infection (INF) is reduced concomitant to blockade of HIV-1 CONCLUSIONS Fig 1. Gating strategy in the IL-‐2, PHA/IL-‐2 and α-‐CD3/α-‐CD28 treated PBMCs or CD4+T cells from one representative donor infection with efavirenz (INF+EFV). (d) Integrated HIV-1 DNA copy number in sorted CD32+ and CD32- In summary, our data suggests that CD32 expression represents a marker of activation of a subset of CD4+ T cells, are shown on the right. Data from donors. (a) Cell doublets were removed from the analysis (FSC-‐A versus FSC-‐H) and cells of five different donors infected with NL4-3*(Vpx). Measurement of integrated proviral DNA was rather than a marker of the latent HIV reservoir. However, taking into account the observed association between represents mean ± SD of 3-5 lymphocytes were gated by using the forward and side scatter areas (FSC and SSC). Monocytes performed by pre-amplifying an LTR DNA fragment with equal amount of genomic DNA input (100 ng) immune activation and viral persistence that suggests that these two phenomena may be reciprocally connected, (upper/right) and B lymphocytes (b) were excluded by labelling CD14 and CD19 Cell surface different donors.
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