Characterizing the effects of seminal plasma on anti-HIV immune responses
AuthorJohn Selva, Kevin
Document TypePhD thesis
Access StatusOpen Access
© 2019 Dr. Kevin John Selva
Introduction: Semen — an overlooked aspect in models of HIV-1 infection — not only serves as the major vehicle for HIV-1 transmission, but also potently modulates immune responses at the mucosa. Here, we have explored the ability of seminal plasma (SP) to modulate general as well as anti-HIV-1-specific responses by natural killer (NK) cells, cytotoxic T-lymphocytes (CTL), monocytes and neutrophils. These effector cells are potentially important for establishing immunity to HIV-1. Method: The effects of SP on “missing-self” and antibody-dependent responses by NK cells were explored using healthy PBMCs and uncoated 721.221 cells or rituximab-coated 721.221 cells, respectively. In-vitro functional assays were performed in the presence or absence of HIV-1-uninfected and HIV-1-infected SP. Anti-HIV-1-specific NK cell activation and cytolysis were measured by co-incubating healthy PBMCs with gp120-coated CEM.NKr-CCR5 cells in the presence of anti-HIV-1 immune globulin (HIVIg). HIV-1-infected whole blood was stimulated with superantigen Staphylococcus enterotoxin B (SEB) or HIV-1 15-mer Gag peptides to study CTL activation. Redirected cytolysis through CD3 was assessed using healthy PBMCs and P815 target. Activation and cytolysis was assessed utilizing intracellular cytokine staining (ICS) and lactate dehydrogenase (LDH) release assays, respectively. The phagocytic potential and oxidative burst responses of monocytes and granulocytes were measured in healthy whole blood using the PhagoTest and PhagoBurst kits, respectively. The RFADCC assay was used to quantify anti-HIV-1 antibody-mediated responses by monocytes and purified neutrophils through co-incubation with gp120-coated CEM.NKr-CCR5 cells in the presence of HIVIg. The impact of active HIV-1 or bacterial infections on the immunosuppressive capacity of SP was studied using NK cell activation assays. Paired SP samples were collected from HIV-1-infected individuals pre-ART initiation or during-ART, and during the presence or absence of chlamydia and/or gonorrhea infection(s). Finally, through a series of biochemical fractionation and mass spectrometry analyses, we narrowed down the suppressive factor(s) involved in SP-mediated immune inhibition. Results: SP (1:100 dilution) potently suppressed “missing-self” and antibody-mediated activation of NK cells by uncoated 721.221 cells and rituximab-coated 721.221 cells, respectively. Likewise, SP (1:1000 dilution) significantly impaired NK cell-mediated cytolysis against either uncoated 721.221 cells or rituximab-coated 721.221 cells. This inhibition of NK cell activity was observed with both HIV-1-uninfected and HIV-1-infected SP in a dose-dependent manner. Anti-HIV-1 antibody-dependent NK cell activation and cytolysis was potently inhibited by SP (1:100 and 1:1000 dilutions respectively). SP (1:100 dilution) also suppressed CTL activation towards both SEB and HIV-1 15-mer Gag peptide. T-cell mediated cytolysis was also impaired by the presence of SP (1:1000 dilution). In contrast, a higher SP concentration (1:10 dilution) was needed to suppress the phagocytic and oxidative burst responses mediated by granulocytes. This was also true for phagocytic (SP 1:1) and oxidative burst responses (SP 1:10) by monocytes. RFADCC responses by granulocytes and monocytes were also only inhibited by SP 1:1. Active HIV-1 or bacterial infection did not impact the suppression of NK cells by SP. Lastly, we narrowed down SP factor(s) involved in the suppression of NK cells to 15 putative metabolites derived largely from four classes – glycerophosphocholines (GPC), polar metabolites of retinol, polar steroids and polar eicosanoids such as prostaglandins. Conclusion: The presented findings highlight that SP may attenuate vaccine-induced effector responses upon HIV-1 mucosal exposure. This possibility should be considered for HIV-1 vaccine development. Further identification of the factor(s) in SP associated with this potent immunosuppression may help with both understanding susceptibility to HIV-1 infection and identifying novel immunomodulatory agents.
KeywordsHIV; semen; seminal plasma; inhibitory factor; inhibition; NK cells; phagocytes
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