This could mean that the absence of gD-HVEM interaction in HVEM?/? animals further augments the scenario of diminished FoxP3 expression on Tregs already existing due to lack of HVEM. PLN are shown in WT and HVEM?/? animals at day 5.5 post HSV kos infection. (H) Viral titers in the foot pad of WT and HVEM deficient animals is shown following HSV- kos infection NIHMS607526-supplement-02.ppt (457K) GUID:?600F6A01-B436-4C6C-A737-3CCB64406DC5 Abstract In many infections, especially those that are chronic such as Herpes Simplex Virus-1 (HSV-1), the outcome may be influenced by the activity of one or more types of regulatory T cells (Tregs). Some infections can cause Treg expansion, but how viruses might promote preferential Treg expansion is has been unclear. In this report, we demonstrate a possible mechanism by which HSV (Herpes Simplex virus-1) infection could act to signal and expands the Treg population. We show that CD4+ FoxP3+ Tregs up- regulate HVEM (herpes virus entry mediator), which is a binding site for major viral glycoprotein HSVgD, following HSV infection, which is Fst a binding site for major viral glycoprotein HSVgD. Recombinant HSVgD enhanced the proliferation of CD4+ FoxP3+ Tregs cells in vitro. Furthermore, compared to wild type (WT), HVEM deficient mice (HVEM?/?) generated a weaker Treg responses represented by significantly diminished ratios of CD4+FoxP3+/CD4+FoxP3? cells along with diminished proportions of FoxP3+ Tregs cells co-expressing Treg activation markers and a reduced MFI of FoxP3 expression on CD4+ T cells. Consistent with defective Treg responses, HVEM?/? animals were more susceptible to HSV-1 induced ocular immunopathology, with more severe lesions in HVEM?/? animals. Our results indicate that HVEM regulates Treg responses, and its modulation could represent a useful approach to control HSV induced corneal immunopathology with either UV inactivated HSV-1 or anti-CD3/anti-CD28 for 72 hours. HVEM expression was analyzed on CD4+ WS 12 FoxP3+ and CD4+ FoxP3? WS 12 cells by flow cytometry. Our results demonstrated that HVEM expression was further up-regulated on FoxP3+ CD4+ T cells (Fig. WS 12 3A upper panel) upon stimulation with UV inactivated HSV-1, but not on CD4+ FoxP3? cells (Fig. 3A lower panel). The highest MFI of HVEM expression after UV-inactivated HSV stimulation was observed when the cells were obtained after day 6 pi (Fig. 3A). Stimulation with anti-CD3/ anti-CD28 did not result in altered HVEM expression levels on either the FoxP3+ or FoxP3? CD4+ T cells (Fig. 3B). Open in a separate window Figure 3 Further up regulation of HVEM on FoxP3+ Tregs upon in-vitro re-stimulation of primed cells with UV inactivated HSV kosFoxP3-GFP knock in mice were infected with 2105 PFU of HSV kos in a 30 ul drop in the footpad. Draining PLN cells were isolated at indicated time points p.i and stimulated with either UV inactivated HSV kos or anti-CD3 (1 mg/ml) anti-CD28 (0.5 mg/ml) for 72 hours. (A) The cells were then analyzed flow cytometrically for HVEM expression on CD4+FoxP3+ (upper panel) and CD4+FoxP3? (lower panel) cells. Histograms depicting HVEM expression by un-stimulated (shaded area) and UV inactivated HSV kos stimulated (black line). (B) FACS analysis for HVEM expression on CD4+FoxP3+ (upper panel) and CD4+FoxP3? (lower panel) cells. Histograms depicting HVEM expression by un-stimulated (shaded area) and anti-CD3 anti-CD28 stimulated (black line). (C) HVEM expression on CD4+FoxP3+ cells following foot pad immunization of FoxP3GFP knock in animals with UV WS 12 inactivated HSV kos is shown. The expression of HVEM on Tregs in draining PLN populations after foot pad infection WS 12 with UV inactivated HSV was also measured. As shown in Fig. 3C, around 50C58% FoxP3+ cells.