Sustained high frequencies of specific CD4 T cells restricted to a single persistent virus

Abstract

Replication of cytomegalovirus (CMV) is largely controlled by the cellular arm of the immune response. In this study the CMV-specific CD4 T-cell response was characterized in a cohort of apparently healthy individuals. In 11% of all individuals, extremely high frequencies, between 10 and 40%, were found. High-level frequencies of CMV-specific CD4 T cells persisted over several months and were not the result of an acute infection. Specific T cells were oligoclonal and were phenotypically and functionally characterized as mature effector cells, with both cytokine-secreting and proliferative potential. These high-level frequencies do not seem to compromise the immune response towards heterologous infections, and no signs of immunopathology were observed. Whereas a large temporary expansion of virus-specific T cells is well known to occur during acute infection, we now show that extremely high frequencies of virus-specific T cells may continuously exist in chronic CMV infection without overtly compromising the remaining protective immunity.

FIG. 1.

Heterogeneous frequencies of CMV-specific CD4 T cells in healthy seropositive individuals (n = 50) (black circles) and long-term renal transplant recipients (n = 111) (open circles). Frequencies of CMV-specific CD4 T cells were analyzed using flow cytometry and may range from 0.1 to up to 43.8% (median, 1.87%). Neither median T-cell frequencies nor the frequency distribution differed between control individuals (median, 1.57%) and long-term transplant patients (median, 1.90%; P = 0.11). Renal transplant recipients tested negative for CMV DNA. No CMV-specific T cells were detectable in CMV-seronegative individuals (n = 77).

FIG. 2.

Detection of CMV-specific CD4 T cells. (A) Whole blood of a CMV-positive individual (#0320) was stimulated with control antigen, CMV antigen, or SEB, and specifically induced cytokine induction in CD4 and CD8 T cells was analyzed using flow cytometry. Numbers indicate percentages of specifically stimulated CD4 T cells. (B) Determination of CMV-specific T cells using the ELISPOT assay. IFN-γ secretion from 2 × 10⁵ PBMC/well stimulated with control antigen, CMV antigen, or SEB was analyzed. The lower panel shows IFN-γ secretion from CD8-depleted PBMC. The mean numbers of spot-forming units per well after stimulation with control antigen, CMV antigen, and SEB were 23, ≫500, and >>500 (upper panel) and 19, ≫500, and 404 (lower panel), respectively. Given the percentage of 52% CD4 T cells among CD8-depleted PBMC, 404 spots correspond to an SEB-reactive CD4 T-cell frequency of 0.4%. Thus, the ELISPOT assay leads to an approximately sevenfold underestimation of frequencies compared to the respective frequencies obtained by flow cytometry (A) 2.85%). (C) PBMC were stimulated with control antigen (o), CMV antigen (•), and SEB (▪), and proliferative responses were assayed after 1, 2, 3, 4, or 5 days. The left diagram corresponds to the individual analyzed in panels A and B, and the middle and right panels represent proliferative responses in individuals with CMV-specific CD4 T-cell frequencies of 2.9 and 0%, respectively.

FIG. 3.

Lack of antigen-nonspecific CD4 T-cell activation. PBMC from pairs of three CMV-positive and three CMV-negative blood donors in all combinations were mixed and stimulated for 6 h using CMV antigen and control antigen. To distinguish cells of the respective donor within the mixture, PBMC of one donor were prestained using FITC-conjugated anti-CD45 antibody (prestained cells are denoted with an asterisk). After stimulation, IFN-γ production was flow-cytometrically analyzed in T cells from each donor in the mixture. (A) PBMC from a CMV-negative (n) and a CMV positive (p) donor were mixed (*p/p, *n/n, *p/n; 10⁶ PBMC from each donor) and stimulated. (B) PBMC from three CMV-positive (prestained) and three CMV-negative donors were mixed in all possible combinations (n = 9; *CMV+/CMV−). Six different combinations of PBMC from CMV-negative (*CMV−/CMV−) and CMV positive donors (*CMV+/CMV+) were stimulated as controls.

FIG. 4.

Extremely high frequencies of CMV-reactive T cells are stable over time. Respective T cells in eight individuals were followed during at least 1.2 to up to 2.3 years using flow cytometry. CMV-DNA was not detectable in any of the patients (hybrid capture assay; data not shown).

FIG. 5.

CMV-specific CD4 T cells are oligoclonal. CMV-specific T cells were found among T cells expressing different T-cell receptors. (A) Shown are the respective specific T cells among cells expressing TCR-Vβ5.2, -Vβ8.0, -Vβ13.1, -Vβ16, -Vβ20, and -Vβ21.3 in a representative example (individual #0320). A particularly high percentage of CMV-specific T cells express TCR-Vβ13.1 or -Vβ16, whereas no specific T cell expresses TCR-Vβ8.0. The two numbers in each diagram indicate the percentages of TCR-Vβ-positive cells among all CD4 T cells and among CMV-specific CD4 T cells, respectively. (B) TCR-Vβ distribution among CMV-specific CD4 T cells in different individuals. + and / indicate the presence or absence, respectively, of IFN-γ-positive CD4 T cells within the respective T-cell population bearing a particular TCR-Vβ subunit.

FIG. 6.

Characterization of CMV-specific CD4 T cells. CMV-specific CD4 T cells are mainly positive for CD45RO and are largely negative for CD27, CD62L, and CCR7. In contrast, a considerable percentage of SEB-responsive T cells are CD27 positive (16.8%). Numbers indicate percentages of T cells positive (CD45R) or negative (CD27, CD62L, and CCR7) for the respective cell surface molecule among IFN-γ-positive CD4 T cells. The percentage of Th1 cells was determined after polyclonal stimulation with PMA-ionomycin (lower right diagram).