Human seminal plasma fosters CD4(+) regulatory T-cell phenotype and transforming growth factor-β1 expression

Abstract

Problem: Semen mediates expansion of CD4(+) regulatory T cells (Treg) in the murine female reproductive tract. The impact of semen on Treg in humans, however, remains unclear.

Method of study: Using seminal plasma (SP) from 20 healthy donors, we investigated the impact of human semen on CD4(+) T-cell expression of CD127 and CD49d as well as CD4(+) CD127(low) CD49d(low) Treg proliferation, apoptosis, and intracellular expression of FoxP3, TGF-β1, and IL-10.

Results: SP reduced CD4(+) T-cell expression of CD127 and CD49d and increased the proportion of CD127(low) CD49d(low) Treg. This increase was non-proliferative, mediated in part via the conversion of CD4(+) helper T cells into FoxP3(-) but not FoxP3(+) Treg. Additionally, SP induced an increase in intracellular expression of the immunosuppressive cytokine TGF-β1 by the FoxP3(-) but not FoxP3(+) Treg. SP had no impact on Treg intracellular expression of IL-10.

Conclusion: Human seminal plasma fosters the non-proliferative increase in the proportion and immunoregulatory activity of FoxP3(-) Treg.

Figure 1. Gating strategy for Th and Treg

We initially identified lymphocytes within the peripheral blood mononuclear cells (PBMC), followed by the identification of CD4+ T cells as lymphocytes that were positive for CD3 and CD4 (A). Subsequently, we identified CD127+CD49d+ Th cells as CD4+ T cells that were positive for the alpha chains of IL-7 receptor (CD127) and integrin α4β1 (CD49d), and CD127-CD49d-Treg as CD4+ T cells that were negative for both, CD127 and CD49d (B). The identity of Treg and Th was further confirmed via presence or absence of intracellular FoxP3 expression, respectively (C and D, n=20).

Figure 2. SP reduces Th expression of CD127 and CD49d

We incubated PBMC with or without 0.5% SP followed by assessment of Th surface expression of CD127 and CD49d. Incubation with SP for 24 hours triggered a decrease in Th surface expression of both CD127 (A, n=20) and CD49d (B, n=20). *** P < 0.001

Figure 3. SP increases the proportion of FoxP3− Treg and enhances FoxP3+ Treg expression of FoxP3

72 hours incubation with SP significantly increased the percentage of Treg (A, n=20). However, SP reduced the percentage of Treg expressing FoxP3 (B, n=20). Within the FoxP3+ Treg, SP induced a small and transient increase in FoxP3 expression (C, n=20). Upon separate analysis of the FoxP3+ compared to FoxP3− Treg, we observed that the SP-mediated increase in the percentage of Treg was primarily confined to the FoxP3− but not FoxP3+ Treg (D, n=10). Similarly, SP did not impact the proportion of CD4+CD25+ T cell (E, n=20) and induced a small and transient increase in CD4+CD25+ T cell expression of FoxP3 (F, n=20). NS = Not significant, * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 4. SP mediates a non-proliferative increase in the proportion of FoxP3− Treg

We assessed whether the observed SP mediated increase in the percentage of Treg was mediated via increased proliferation or ongoing conversion of Th into Treg by comparing the intracellular expression of Ki67 and activated caspase-3 between Treg and Th, also between Treg subsets after 72 hours of incubation with 0.5% SP. No difference was observed in the intracellular expression of Ki67 (A and B, n=6) or activated caspase-3 (C and D, n=10) between Treg and Th, or between FoxP3− and FoxP3+ Treg, suggesting an increase in Treg via ongoing conversion of the pre-existing Th into Treg. NS = Not significant.

Figure 5. SP induces Treg secretion of TGF-β1

We examined the impact of SP on the suppressive function of the Treg via assessment of Treg expression of the immunosuppressive cytokines TGF-β1 via intracellular cytokine staining (ICS), 72 hours after incubation with or without 0.5% SP. SP induced a profound and significant induction of Treg but not Th intracellular expression of TGF-β1 (A, n=5). Importantly, this increase was mostly observed within the FoxP3− Treg compared to FoxP3+ Treg (B, n=5). * P < 0.05, ** P < 0.01

Figure 6. SP induces a time-dependent increase in FoxP3− Treg secretion of TGF-β1

We investigated the time dynamics of SP mediated induction of FoxP3− Treg intracellular expression of TGF-β1. SP induced a time-dependent increase in TGF-β1 expression by the FoxP3− Treg, peaking at 72 hours (n=5). Error bars represent standard error of mean, NS = Not significant, ** P < 0.01