Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation

Abstract

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.

FIG. 1.

CMV-specific CD8 T cells are more activated than the overall CD8 T-cell population. (A) Expression of CD45RA and CD45R0 by tetramer⁺ and tetramer⁻ CD8 T cells, showing the line of best fit and the cutoff used to define the CD45RA^bright CD45R0⁻ population. (B) Mean percentage of CD45RA^bright CD45R0⁻ CD8 T cells in tetramer⁺ and total populations for each sampling time. (C) Mean CD45 ratios of tetramer⁺ and total CD8 T cells at each sample time. (D) Expression of CD45R0 and HLA-DR on tetramer⁺ and tetramer⁻ CD8 T cells, showing the cutoffs used to define the CD45R0⁺ and HLA-DR⁺ populations. (E) Mean percentages of HLA-D⁺ and (F) CD45R0⁺ HLA-DR⁺ CD8 T cells among tetramer⁺ and total populations at each sample time. The percentages in the scatter plots refer to cells within that quadrant. The boxes indicate medians and interquartile ranges.

FIG. 2.

CMV-specific CD8 T cells divide more than the overall CD8 T-cell population during the early stages of infection. (A) Expression of Ki-67 by tetramer⁺ and tetramer⁻ CD8 T cells in a representative sample collected immediately postdiagnosis. (B) The expression of Ki-67 for all subjects where tetramer staining was carried out, with differences in expression of Ki-67 between tetramer⁺ cells indicated. There were no differences between the overall populations. The boxes indicate medians and interquartile ranges.

FIG. 3.

CMV-specific CD8 T cells have a more apoptosis-prone phenotype than the overall CD8 T-cell population. (A) Expression of CD62L and CD95 on tetramer⁺ and tetramer⁻ CD8 T cells, showing the definition of the CD62L⁺ and CD95⁺ CD62L⁺ populations. (B) Mean percentages of CD62L⁺ CD8 T cells among tetramer⁺ and tetramer⁻ populations at each sample time. (C) Expression of Bcl-2 on tetramer⁺ and tetramer⁻ CD8 T cells, showing the definition of the Bcl-2^high and Bcl-2^low populations. The percentages refer to proportions of Bcl-2^low cells. (D) Mean percentages of Bcl-2^low CD8 T cells among tetramer⁺ and tetramer⁻ populations at each sample time. Differences between phenotypes of the total CD8 T-cell population at different sampling times are indicated. The boxes indicate medians and interquartile ranges. The percentages in the scatter plots refer to adjacent regions.

FIG. 4.

CMV-specific CD8 T cells are more differentiated and show higher cytotoxic potential than the remainder of the CD8 T-cell population. (A) Scatter plots of representative samples of tetramer⁺ and tetramer⁻ CD8 T cells stained for CD27 and CD28 with the indicated populations and the percentage of cells within each population, box plots of all samples indicating medians and interquartile ranges, and differences between the distributions of all populations between sample times indicated. (B) The same data for cells stained for CD27 and perforin. (C) The same data for cells stained for CD27 and granzyme A. The populations are identified, and the percentages indicate the proportion of cells in each of them.

FIG. 5.

The magnitude of IFN-γ responses was greater in older infants. There was no correlation between age at the time of diagnosis and the magnitude of IFN-γ response in those cases where a peptide induced a peptide-specific response (A) immediately after diagnosis or (B) 4 months later, but (C) there was a positive correlation by 12 months following diagnosis. (D) Samples collected from subjects between 40 and 60 weeks of age who had been infected 4 months previously had an IFN-γ response similar to that of infants who had been infected 12 months previously. The bars indicate means. (E) Twelve-month samples were collected from infants diagnosed with CMV at 4 to 10 weeks when they reached 40 to 60 weeks of age, and 4-month samples were collected from infants diagnosed at 17 to 39 weeks when they reached the same age. When the 4- and 12-month samples from subjects who fell into either of these two categories were compared, a greater IFN-γ response was elicited from the 12-month sample in nearly all cases. The absence of a bar indicates that the sample was collected but no response was detected.

FIG. 6.

The CD8 T-cell population of CMV-infected infants is more activated and differentiated than that of uninfected infants. (A) Expression of CD27 and CD28 in representative samples and all infants sampled, drawn from the groups of CMV-infected and non-CMV-infected infants at 12 months of age. (B) Expression of CD27 and perforin in representative samples and all infants sampled, drawn from the groups of CMV-infected and non-CMV-infected infants at 12 months of age. (C) Expression of CD27 and granzyme A in representative samples and all infants sampled, drawn from the groups of CMV-infected and non-CMV-infected infants at 12 months of age. (D) Expression of CD62L and CD95 in representative samples and all infants sampled, drawn from the groups of CMV-infected and non-CMV-infected infants at 12 months of age. The percentages in the scatter plots refer to cells within the adjacent region. The boxes indicate medians and interquartile ranges.