Semen CD4+ T cells and macrophages are productively infected at all stages of SIV infection in macaques.

International audienceThe mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4(+) T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4(+) T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure

Viral shedding in blood and semen in macaques.

<p>(A–B) Longitudinal follow-up of RNA viral loads in blood (A) and seminal plasma (B) in 8 macaques infected intravenously with high doses of SIVmac251 (5,000 AID₅₀); each animal is represented by different dot. The solid black line indicates the mean PVL. The dotted horizontal line represents the limit of quantification (111 and 37 copies/ml in blood and semen plasma respectively).</p

Semen leukocytes in macaques.

<p>(A) Seminal concentrations of six pro-inflammatory molecules in normal and leukocytospermic animals. (B) Proportion of each studied leukocyte subset among total CD45⁺ cells. (C) Semen vRNA load in SIV⁺ macaques with normal (<i>n</i> = 11, blue circle) or leukocytospermic (<i>n</i> = 8, half plein black circles) semen. (D) Number of CD45⁺ events acquired by flow cytometry per sample since the total collected semen cells were analyzed, for control uninfected (<i>n</i> = 20), and SIV-infected macaques, at 10 and 14 dpi (<i>n</i> = 7) and during chronic infection. The dotted line represents the leukocytospermia threshold (10,000 CD45⁺ events acquired) (<i>n</i> = 13). (E) Proportion of each subset among total CD45⁺ events in SIV⁻ macaques (<i>n</i> = 12, blue circle) and SIV⁺ macaques during primary infection (<i>n</i> = 7, red triangle) and chronic infection (<i>n</i> = 9, red diamond). (F) Seminal concentrations of IP-10, RANTES and IL-8 in SIV⁻ macaques (<i>n</i> = 8 for IP-10 and <i>n</i> = 12 for RANTES and IL-8, blue circle) and SIV⁺ macaques during primary infection (<i>n</i> = 6 for IP-10 and <i>n</i> = 10 for RANTES and IL-8, red triangle) and chronic infection (<i>n</i> = 8 for IP-10 and <i>n</i> = 16 for RANTES and IL-8, red diamond).</p

Semen CD4⁺ T-cell phenotype.

<p>(A) Gating of central memory CD4⁺ T cells (CD95⁺CD28⁺⁾ (B) Comparison of CD4⁺ T-cell differentiation markers between blood (half plain dot) and semen (plain dot) (<i>n</i> = 13 uninfected macaques). (C) Gating strategy for CD69 and HLA-DR expression. (D) CD69 and HLA-DR expression in CD4⁺ T cells in blood (half plain dots) and semen (plain dots) from SIV⁻ macaques (<i>n</i> = 13). (E) CD69 and HLA-DR expression in semen CD4⁺ T cells from SIV⁻ (<i>n</i> = 6, empty dots) and SIV⁺ macaques, at different stages of infection (<i>n</i> = 10, plain dots). (F) Longitudinal follow-up of CD69 (solid line, plain dot) and HLA-DR (solid line, plain box) expression in semen CD4⁺ T cells during infection (<i>n</i> = 6). Dotted lines represent PVL (plain grey triangle base down) and SVL (plain grey triangle base up) (G). CCR5 and CXCR4 expression in CD4⁺ T cells from a representative uninfected macaque. From left to right: contour plots representing outliers, overlay of CCR5 and CXCR4 staining (black line) and isotype control (solid gray curve). (H) Comparison of CCR5⁺, CXCR4⁺ and CCR5⁺ CXCR4⁺ CD4⁺ T cells between SIV⁻ (<i>n</i> = 6, open dot) and chronically SIV-infected macaques (<i>n</i> = 6, plain dot). (I) Gating strategy for LFA-1 integrin expression on CD4⁺ T cells. From left to right: contour plots representing the outliers, overlay of CD11a and CD18 staining (black line) and isotype control (solid gray curve) (J) Comparison of CD11a⁺ CD18⁺ (LFA-1) CD4⁺ T cells between SIV⁻ (<i>n</i> = 6, open dot) and chronically SIV-infected macaques (<i>n</i> = 7, plain dot). Mean and SEM are represented.</p

SIV antigens in semen CD4⁺ T cells and macrophages.

<p>(A–D) Immunocytofluorescence staining targeting CD3 (red), CD163 (green) and SIV Nef (pink) on cytospun CD45⁺-enriched semen cells from SIV⁺ (B,D) and uninfected macaques (A,C). Nuclei are stained using DAPI (blue), visible on merged figures (B) SIV⁺ macaque at 28 dpi. (D) SIV⁺ macaque at 65 dpi.</p