Expression of CD8alpha identifies a distinct subset of effector memory CD4+ T lymphocytes

Abstract

Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8alphaalpha homodimer, while the minor population was CD4lo CD8hi and expressed the CD8alphabeta heterodimer. The majority of CD4hi CD8alphalo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7- CD28- HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8alphalo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8alphalo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8alphalo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8alpha represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection.

Figure 1

Differential expression of CD8β on different subsets of CD4⁺ CD8⁺ T cells. Peripheral whole blood from an uninfected rhesus macaque was analysed by four-colour flow cytometry for expression of CD8β on the indicated populations of lymphocytes. (a) Expression of CD4 and CD8α on CD3⁺ T lymphocytes. (b) Expression of CD8β on different subsets of T lymphocytes. The percentage of CD8β⁺ lymphocytes is indicated for each histogram. Dotted lines represent the isotype control. Results are representative of a total of 36 separate experiments.

Figure 2

Phenotypic analysis of CD4^hi CD8α^lo T cells. CD4 SP, CD4^hi CD8α^lo and CD8 SP lymphocytes were analysed for the coexpression of (a) CD28 and CD95; (b) CD45RA and CD62L; (c) CD28 and β7. Gates used for the identification of central/memory, effector/memory and naive lymphocytes are shown, along with the percentage of cells in each gated population. Data are representative of 12 separate animals.

Figure 3

Expression of CD7, Ki67 and CD28 on CD4 SP, CD4^hi CD8α^lo and CD8 SP lymphocytes. (a) Analysis of CD7 and CD28 expression. The percentages of cells corresponding to each quadrant are indicated on the top right side of the plot. Data are representative of five independent experiments from different animals. (b) Analysis of Ki67 and CD28 expression. The indicated gates for expression of Ki67 were determined based on the analysis of lymphocytes stained with an isotype control anti-mouse-FITC after fixation and permeabilization of surface-stained cells. Plots are representative of eight separate animals.

Figure 4

Expression of CCR5 on CD4^hi CD8α^lo lymphocytes. (a) Expression of CCR5 on CD4 SP, CD4^hi CD8α^lo and CD8 SP lymphocytes. The dotted line indicated staining obtained with an isotype control. The percentage of cells expressing CCR5 is shown, along with the geometric mean fluorescent intensity of the total population of CD4 SP, CD4^hi CD8α^lo and CD8 SP lymphocytes in parentheses. Results are representative of nine animals.

Figure 5

Detection of CMV-specific responses by CD4^hi CD8α^lo lymphocytes using intracellular cytokine staining. PBMC from an SIVΔnef-vaccinated, CMV-seropositive monkey were stimulated with control antigen from uninfected (UI) cells, whole virus-rhCMV antigen, or superantigen (SEA/SEB). The percentages of cells expressing CD69 and TNF-α are indicated in the right upper quadrant of each plot. Results are representative of nine animals.

Figure 6

Analysis of TRECs in CD4^hi CD8α^lo T cells. Purified populations of CD4^hi CD8α^lo, CD4 SP and CD8 SP T cells were obtained by cell sorting from five rhesus macaques. CD4 SP T cells were further sorted into memory and naive subsets based on their CD28 and CD95 expression. TREC analysis was performed by real-time PCR on 200 ng of DNA extracted from the indicated subpopulations of T cells. The difference in the level of TRECs between CD4^hi CD8α^lo and CD4 SP naive cells is significant (P = 0·001, Fisher's PLSD). Sj: signal joint.

Figure 7

Effect of SIVmac239 infection on the frequency of CD4 SP and CD4^hi CD8α^lo lymphocytes. (a) Cross-sectional analysis of the frequency of CD4^hi CD8α^lo and CD4 SP T cells in uninfected and wild-type SIV-infected macaques. P-values calculated by the Mann–Whitney U-test are shown. (b) Longitudinal analysis of the frequency of CD4⁺ and CD4^hi CD8α^lo lymphocytes at the indicated times after infection in rhesus macaques inoculated with pathogenic SIVmac239.