FIG 5.

The HCMV-specific CD4⁺ T cell response to HCMV-infected cells is polyfunctional. (A) moDCs from each donor were prepared and then mock or lytically infected with HCMV strain TB40/E-UL32-GFP at an MOI of 0.1 for 7 days. Autologous CD4⁺ T cells were incubated overnight with either uninfected, HCMV-infected, or UV-irradiated HCMV-infected moDCs in the presence of an anti-CD107a antibody, monensin, and brefeldin A. CD4⁺ T cells were then stained with a polyfunctional flow cytometry antibody panel, acquired, and analyzed. Virus-specific CD4⁺ T cells were identified by the upregulation of CD40L and 4-1BB above the background response. The total specific response to CMV and the proportion of the specific response, composed of MIP-1β, granzyme A and B (GrzAB), CD107a, and IFN-γ production or the production of no functional marker, in 12 donors are shown. (B) The mean proportion of virus-specific CD4⁺ T cells from all donors generating polyfunctional responses is summarized as a pie chart indicating the proportion of HCMV-specific CD4⁺ T cells producing 4, 3, 2, 1, or no functions. (C) The composition of the HCMV-specific CD4⁺ T cell response as a proportion of the antigen-specific population for all donors is illustrated. (D) The proportion of virus-specific cells expressing different memory cell phenotype markers (CD27, CD45RA, CD28, and CD57) is shown. (E) A direct comparison of the size of the specific T cell response to live virus (CMV) versus UV-inactivated virus (UV Virus) in 3 donors is shown. (F) A representative example of UL32 (late gene)-tagged GFP expression in moDCs infected with live virus versus UV-inactivated virus indicating GFP expression in live virus-infected moDCs only.