Abstract
Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot® assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-γ and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of TH1, TH2, and TH17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-γ and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the TH1, TH2, and TH17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.
Highlights
Asymptomatic infections by human cytomegalovirus (HCMV), a β-herpesvirus, are common with a prevalence between 60% and 100% in industrial and developing countries, respectively [1]
T cells maintain their antigen receptor specificity over time and, the frequency of T cells with a given antigen specificity is a result of the expansion of reactive clones and can be measured precisely. This rule applies to HCMV-specific TH 1, TH 2, and TH 17 cells. When such frequencies are established by a functional assay – in this case by measuring the numbers of antigen-specific T cells that secrete the respective signature cytokines for the TH 1, TH 2, and TH 17 lineages – the assay conditions need to be optimized so that all antigen-specific T cells become activated, and that the analyte secreted is measured at single cell resolution
In preparation for establishing the frequency of HCMV specific TH 1, TH 2, and TH 17 cell in our donor library, we needed to determine (a) the time point of maximal secretion for each cytokine, (b) the distributional properties of the respective cytokine spots for accurate counting, (c) the linearity of measurements, (d) the optimal antigen dose, and (e) that the antigen-triggered cytokine is secreted by antigen-specific CD4 cells as opposed to resulting from bystander reactions
Summary
Asymptomatic infections by human cytomegalovirus (HCMV), a β-herpesvirus, are common with a prevalence between 60% and 100% in industrial and developing countries, respectively [1]. Various cell types including neuronal-, endothelial-, epithelial-, smooth muscle cells, and leucocytes can be infected during an acute phase of HCMV infection [2]. The virus is able to persist during the entire lifetime of the infected host, mostly in a dormant state. Activation and expansion of HCMV-specific lymphocytes will force the virus back into its dormant state [3]. While cycles of dormancy and reactivation are characteristic for HCMV infections, little is known about the frequency of these cycles and the timing of reactivations since they mostly occur subclinically and go unobserved
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