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. 2012 Jan;86(2):1001-9.
doi: 10.1128/JVI.00873-11. Epub 2011 Nov 9.

Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses

Raskit Lachmann  1 , Martha Bajwa, Serena Vita, Helen Smith, Elizabeth Cheek, Arne Akbar, Florian Kern
Affiliations

Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses

Raskit Lachmann et al. J Virol. 2012 Jan.

Abstract

Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (20 to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ(+) IL-2(+/-) TNF-α(+)) and CD4 (CD40L(+/-) IFN-γ(+) IL-2(+) TNF-α(+)) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people.

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Figures

Fig 1
Fig 1
Frequencies and absolute numbers of pp65-specific T cells are not closely correlated with age. Responding T cells were defined as those displaying at least one of the activation markers CD40L, IL-2, IFN-γ, and TNF-α. Frequencies (left) and absolute numbers (right) of responding CD8/pp65 (top) and CD4/pp65 (bottom) T cells are displayed. Absolute and relative values followed a lognormal distribution and were log transformed to better visualize changes with age. There was no detectable correlation of T-cell responsiveness to pp65 with age.
Fig 2
Fig 2
Large pp65-specific T-cell responses show a more advanced memory T-cell subset composition. (A and B) The absolute counts of CMV pp65-specific CD8 and CD4 T cells do not correlate significantly with the absolute numbers of CD8 and CD4 T cells in peripheral blood. (C and D) There is an overproportioned (nonlinear) relative increase in pp65-specific TEM cells and decrease in TNAIVE cells within these responses. (E and F) The proportions of the entire TNAIVE and TEM CD8 and CD4 T-cell compartments (C and D) are changed in the presence of large pp65-specific responses. ns, not significant.
Fig 3
Fig 3
Large CMV-specific CD8 T-cell responses have large polyfunctional components. Data are presented as dots correlating the absolute numbers of CD8/pp65 T cells displaying the indicated combinations of activation markers to the absolute number of all responding CD8/pp65 T cells (displaying at least one of the activation markers) (n = 39). The Spearman rank correlation coefficient for nonparametric data (RS) is indicated for significant correlations (RS of >0.50). Certain donors did not express certain functional subsets, resulting in what appears to be two groups with respect to each subset, but the nonresponding donors varied. However, the IFN-γ+ IL-2+ TNF-α+ subset was expressed by most donors. Cell counts were log transformed, with zeroes assigned the value 0.01 prior to transformation. The size of responding subsets is given as log(cells/ml of blood).
Fig 4
Fig 4
Large CMV-specific CD4 T-cell responses have large polyfunctional components. Data are presented as dots correlating the absolute numbers of CD4/pp65 T cells displaying the indicated combinations of activation markers to the absolute number of all responding CD4/pp65 T cells (displaying at least one of the activation markers) (n = 38). The Spearman rank correlation coefficient for nonparametric data (RS) is indicated for significant correlations (RS of >0.50). Certain donors did not express certain functional subsets, resulting in what appears to be two groups with respect to each subset, but the nonresponding donors varied. However, the CD40L+ IFN-γ+ IL-2+ TNF-α+ subset was expressed by most donors. Cell counts were log transformed, with zeroes assigned the value 0.01 prior to transformation. The size of responding subsets is given as log(cells/ml of blood).
Fig 5
Fig 5
Example of original data for an older individual. Representative dot plots show different views of the same data. CD4 and CD8 T cells were gated from single lymphocytes (FCS-H/FSC-A). Activated cells were gated individually for each T-cell subset and activation marker, and then Boolean gating combinations were computed. For the purpose of illustration only, all cytokines were plotted against CD40L. Plots show log fluorescence intensities for all markers. Side scatter (SSC) and forward scatter (FSC) are shown on a linear scale. More than 400,000 lymphocytes were gated in the example.
Fig 6
Fig 6
Downregulation of CD3, CD8, and CD4 on T cells after pp65-specific stimulation. Line curves (medians ± 95% confidence intervals [95% CI]) represent CD3 and CD8 expression on CD8/pp65 T cells (top panels), as well as CD3 and CD4 expression on CD4/pp65 T cells (bottom panels), as a function of the number of activation markers displayed simultaneously following overnight stimulation with a pp65 peptide pool. Lineage marker expression is represented by the standardized MFIs of CD3, CD8, and CD4 within the respective populations. There was a nearly linear correlation between TCR downregulation and the number of displayed activation markers (RS is indicated; n = 20 for all diagrams).
Fig 7
Fig 7
Polyfunctional T cells display larger quantities of each activation marker except for IL-2. Line curves (medians ± 95% CI) show standardized MFIs of IFN-γ, IL-2, TNF-α, and CD40L. MFIs for T cells expressing one, two, three, or four activation markers are shown (n = 20). Note that although IL-2 was displayed by the major polyfunctional CD8/pp65 and CD4/pp65 subsets, its levels in responses including more than 2 markers were lower, which probably reflects its role in early antigen-dependent T-cell differentiation/proliferation.
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