CMV antigen-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation responses in healthy CMV-seropositive individuals

Abstract

CMV-specific CD4+ and CD8+ T cell IFNgamma expression and proliferation were measured in healthy volunteers by flow cytometry after CMV lysate or CMV pp65 or IE peptide pool stimulation. Cutoff values were set to maximize specificity (i.e., no false positive CMV-seronegatives). Sensitivity (defined as a positive response in CMV-seropositives to at least one of the 3 antigen preparations used) was 100% for CMV-specific CD4+ and CD8+ T cell IFN expression and CD4+ T cell proliferation and 95.4% for CMV-specific CD8+ T cell proliferation. All 22 CMV-seropositive individuals had positive responses by at least three of these four measurements. These findings support the concept that a multiplicity of antigen-specific functional immune responses and persistence of robust virus-specific CD4+T cells are important components of protective immunity in this chronic viral infection.

Figures

FIG. 1

Flow cytometric measurement of antigen-specific proliferation. In the gating strategy to quantify the percentage of proliferating T cells (A), monocytes are excluded (upper left) before a lymphocyte/blast gate is drawn (upper right), and then CD3+CD4+ and CD3+CD4− gates are drawn (lower left). Proliferating cells are CFSElo with high forward scatter (lower right). (B) Typical antigen-specific proliferation patterns in a CMV-seropositive donor are shown. CD4+ T cell responses are shown on the right and CD8 (CD3+ CD4−) are shown on the right in response to stimulation with pp65 peptide pool (i), IE peptide pool (ii), CMV lysate (iii), and unstimulated (iv).

FIG. 2

CD4+ and CD8+ T cell responses of CMV-seropositive and CMV-seronegative donors to stimulation with CMV lysate (i), pp65 peptide pool (ii), and IE peptide pool (iii). CD4+ and CD8+ T cell proliferation (A) was significantly higher in the CMV-seropositive donors, compared with the CMV-seronegatives for all three antigens (p < 0.0001 for all comparisons except CD8+ IE response, p = 0.0005). Likewise CD4+ and CD8+ T cell IFNγ expression, measured in the CFC assay (B), was significantly higher in the CMV-seropositives (p < 0.0001). IFNγ expression and proliferation were quantified as a percentage of the total CD4+ (gated on CD3+CD4+) or CD8+ (gated on CD3+CD4−) T cell population.