Postnatal acquisition of primary rhesus cytomegalovirus infection is associated with prolonged virus shedding and impaired CD4+ T lymphocyte function

Abstract

Background: Although virus-specific CD4(+) T lymphocytes emerge rapidly during primary cytomegalovirus (CMV) infection in humans, they exhibit a state of prolonged functional exhaustion of unknown etiology. To investigate the suitability of rhesus macaques as a model of primary human CMV infection, we examined the virologic and immunologic features of naturally acquired primary CMV infection in rhesus macaques.

Methods: CMV-specific CD4(+) T lymphocytes and CMV load in blood, saliva, and urine were evaluated in a cohort of simian immunodeficiency virus (SIV)-negative rhesus macaques stratified by age into infant, juvenile, and adult groups.

Results: CMV infection was detected in juvenile and adult monkeys but not in infant monkeys. CMV loads and shedding frequency in urine and saliva were significantly higher in the 2-3-year old juvenile monkeys, compared with the adult monkeys. The increased CMV load in juvenile monkeys was associated with lower polyfunctionality, impaired proliferation, and increased expression of the inhibitory receptor PD-1 in CMV-specific CD4(+) T lymphocytes. The proliferative defect was partially reversible by exogenous PD-1 blockade or addition of interleukin 2.

Conclusions: Postnatal acquisition of primary CMV infection in rhesus macaques results in prolonged virus excretion and impaired CMV-specific CD4(+) T-lymphocyte function, findings that recapitulate key features of primary CMV infection in humans.

Keywords: CD4+ T lymphocyte; IFN-γ; IL-2; PD-1; TNF-α; cytomegalovirus; functional exhaustion; primary cytomegalovirus infection; rhesus macaque; viral excretion.

Figure 1.

A, Viral loads in bodily fluids and plasma according to age. Cytomegalovirus (CMV) DNA was detected in urine, saliva, and plasma specimens from 1-year-old, 2-year-old, 3-year-old, and adult group-housed rhesus macaques. Individual data are represented. Two-year-old, 3-year-old, and adult shedders were compared using the Kruskal–Wallis test (P < .001 for excretion in urine and saliva specimens). The Dunn posttest was used to compare adult excretors to 2-year-old and 3-year-old monkeys, by pairs (P < .01, for all combinations). B, Viral excretion in urine and saliva in 2-year-old, 3-year-old, and adult rhesus macaques. Abbreviation: ND, not done.

Figure 2.

Frequency of cytokine-producing CD4⁺ T lymphocytes. Cytokine-producing cells among memory CD4⁺ T lymphocytes were measured by intracellular cytokine staining following stimulation with lysate of rhesus cytomegalovirus (CMV) whole-virus antigen (A) and rhesus CMV peptide pools (B). Individual data and median values are shown. Juvenile and adult rhesus macaques were compared using the Mann–Whitney test (P > .05 for pp65 [total] and IE2 [total]; P < .05 for CMV [total], CMV [interferon γ {IFN-γ}], CMV [tumor necrosis factor α {TNF-α}], and IE1 [total]; P < .01 for CMV [interleukin 2 {IL-2}]).

Figure 3.

A, Distribution of CMV-specific CD4⁺ T cells among functional subsets. CD4⁺ T lymphocytes responding to the whole cytomegalovirus (CMV) lysate are distributed in functional subsets according to the production of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and interleukin 2 (IL-2). Individual data after subtraction of background and median values are shown. The Mann–Whitney test was used for group comparison (P < .05 for IFN-γ⁺ IL-2⁻ TNF-α⁺ CD4⁺ T cells, P < .001 for triple-positive CD4⁺ T cells). B, Polyfunctionality of CMV-specific CD4⁺ T cells. The pie charts in the left panel illustrate the proportion of cells producing 1 (pale grey), 2 (medium grey), and 3 (dark grey) cytokines among total responding cells in juvenile and adult rhesus macaques. Colored arcs show individual cytokines. In the right panel, the polyfunctionality index was calculated for juvenile and adult macaques in response to the whole CMV lysate, and values were compared using the Mann–Whitney test (individual data and medians are shown; P < .01). C, Relationship between urinary CMV excretion and CD4⁺ T-cell polyfunctionality. Animals have been stratified according to the polyfunctionality index of CMV-specific CD4⁺ T cells (low: index below median; high: index above median). Viral excretion in urine specimens was compared between these subgroups, using the Mann–Whitney test. Individual data and median values are shown (P < .01 for juveniles, P > .05 for adults).

Figure 4.

A, Cytomegalovirus (CMV)–specific CD4⁺ T-cell proliferative responses. Proliferating CD4⁺ T cells in response to whole CMV lysate were detected using the BrdU incorporation assay. Frequencies of Ki67⁺BrdU⁺ CD4⁺ T cells are corrected for the proportion of memory cells among the total CD4⁺ T lymphocyte population. Individual data and median values are shown. The Mann–Whitney test was used for group comparison (P < .0001). B, Relationship between cytokine-producing CD4⁺ T cells and proliferative response to whole CMV lysate. Data on individual monkeys are shown. C, Relationship between urinary CMV excretion and CMV-specific CD4⁺ T-cell proliferative responses. Animals have been stratified according to the CD4⁺ T-cell proliferative response (low: proportion below median; high: proportion above median). Viral excretion in urine specimens was compared between these subgroups, using the Mann–Whitney test. Individual data and median values are shown (P < .05 for juveniles, P < .07 for adults).

Figure 5.

A, Effect of exogenous interleukin 2 (IL-2) on cytomegalovirus (CMV)–specific CD4⁺ T-cell proliferative responses. CD4⁺ T-lymphocyte proliferative responses to whole CMV lysate were measured in the absence or presence of exogenous IL-2. Median and interquartile ranges are shown. Values for juveniles and adults are compared at each concentration, using the Mann–Whitney test (P < .05 in the absence of exogenous IL-2). NS, not significant. B, PD-1 and Tim-3 expression on CMV-specific CD4⁺ T cells. Representative overlay histograms showing PD-1 (left) and Tim-3 (right) staining on interferon γ (IFN-γ)–producing CD4⁺ T cells stimulated by CMV lysate. Data are for 1 representative adult and 2 juvenile monkeys. Fluorescence minus one (FMO) staining–negative controls were used to establish background signal. C, Assessment of PD-1 impact on CMV-specific CD4⁺ T-cell function. PD-1 expression was evaluated by median fluorescence intensity (MFI) measurement on CMV-specific IFN-γ–producing CD4⁺ T cells (left; P < .05, by the Mann–Whitney test). The effect of PD-1 blockade on CD4⁺ T-cell proliferative responses to CMV lysate was assessed (right; P < .01 for juveniles and P > .05 for adults, by the Wilcoxon test). D, Assessment of Tim-3 impact on CMV-specific CD4⁺ T-cell function. Tim-3 expression was evaluated by MFI measurement on CMV-specific IFN-γ–producing CD4⁺ T cells (left panel, P > .05 using the Mann–Whitney test). The effect of Tim-3 blockade on CD4⁺ T-cell proliferative responses to CMV lysate was assessed (right; P > .05 for juveniles and adults, using the Wilcoxon test).