CD4 downregulation by memory CD4+ T cells in vivo renders African green monkeys resistant to progressive SIVagm infection

Abstract

African green monkeys (genus Chlorocebus) can be infected with species-specific simian immunodeficiency virus (SIVagm) but do not develop AIDS. These natural hosts of SIV, like sooty mangabeys, maintain high levels of SIV replication but have evolved to avoid immunodeficiency. Elucidating the mechanisms that allow natural hosts to coexist with SIV without overt disease may provide crucial information for understanding AIDS pathogenesis. Here we show that many CD4(+) T cells from African green monkeys downregulate CD4 in vivo as they enter the memory pool; that downregulation of CD4 by memory T cells is independent of SIV infection; that the CD4(-) memory T cells maintain functions that are normally attributed to CD4(+) T cells, including production of interleukin-2 (IL-2), production of IL-17, expression of forkhead box P3 and expression of CD40 ligand; that loss of CD4 expression protects these T cells from infection by SIVagm in vivo; and that these CD4(-) T cells can maintain major histocompatibility complex class II restriction. These data show that the absence of SIV-induced disease progression in natural host species may be partially explained by preservation of a subset of T cells that maintain CD4(+) T cell function while being resistant to SIV infection in vivo.

Figure 1. Phenotypic analysis of T-cell populations in vervet African green monkeys

(a) Phenotype of T-cells in adult AGM. (b) Phenotype of individual subsets of T-cells in adult AGM. (c) Comparison of percent of CD3⁺ T-cells that express CD4 in peripheral blood in SIV⁺ and SIV⁻ adult AGM, adult SIV⁺ and SIV⁻ SM, adult SIV⁻ RM, and HIV⁻ adult humans. (d) Characterization of CD4⁻CD8α^dim T-cells as CD8αβ⁻ by analysis of CD8β expression. Cells were gated on live CD3⁺ small lymphocytes and analyzed for both CD4 and CD8β expression. (e) Ki67 expression by different subsets of memory T-cells. (f) Negative correlation between the frequencies of CD4⁺ T-cell and CD4⁻CD8α^dim T-cells in adult AGM. (g) Correlation of the frequency of CD4⁺ T-cells and the frequency of CD4⁻CD8α^bright T-cells in adult AGM. White circles represent SIV-infected AGM and black circles represent uninfected AGM. White diamonds represent SIV-infected SM and black diamonds prepresent uninfected SM. Black triangles represent SIV-uninfected RM and black squares represent HIV-uninfected humans. A Mann-Whitney U test was performed for c. A Spearman rank correlation was calculated for f and g.

Figure 2. CD8α and CD4 expression by naive and memory CD4⁺ T-cells

(a) Representative staining of CD4⁺ memory and naive T-cells for CD4. (b) Representative staining of CD4⁺ memory and naive T-cells for CD8. (c) Fluorescence minus one control lacking antibodies against CD8α. (d) Median fluorescence intensity for CD4 expression in naive and memory CD4⁺ T-cells. (e) Median fluorescence intensity for CD8 expression in naive and memory CD4⁺ T-cells. A Wilcoxon matched pairs test was performed for 2d–e.

Figure 3. CD4⁻CD8α^dim T-cells can develop from memory CD4⁺ T-cells

(a) Down-regulation of CD4 by stimulated in vitro PBMC from AGM after 5 days of stimulation with SEB. (b) Maintenance of CD4 expression by stimulated PBMC from pigtail macaques in vitro after 5 days of stimulation with SEB. (c) CD4 mRNA expression in CD14⁺, CD4⁻CD8α^dim, CD4⁻CD8α^bright, or CD4⁺ lymphocyte subsets of an adult AGM. (d)Negative correlation between the frequency of memory CD4⁺ T-cells and the frequency of the total CD4⁺ T-cells in adult AGM. (e) Comparison of percent CD4⁺ T-cells in peripheral blood in SIV⁺ and SIV⁻ adult and SIV⁻ juvenile AGM. White circles represent SIV-infected AGM and black represent uninfected AGM. A Spearman rank correlation was calculated for 3c. A Mann Whitney U test was performed for 3e.

Figure 4. CD4⁻CD8α^dim T-cells can preserve CD4⁺ T-cell function

(a) Comparison of the frequency of memory CD4⁺, CD4⁻CD8α^dim, and CD4⁻CD8α^bright T-cells performing various functions: IL-2 and IL-17 production, and FoxP3 and CD40L expression. (b) Comparison of the relative numbers of memory CD4⁺, CD4⁻CD8α^dim, and CD4⁻CD8α^bright T-cells from different memory T-cell subsets performing various functions: IL-2 and IL-17 production, and FoxP3 and CD40L expression (c) Responses of CD4⁻CD8α^dim T-cells to CMV whole antigen in the presence and absence blocking antibodies to MHC-II or MHC-I. White circles represent SIV-infected AGM and black represent uninfected AGM. A Wilcoxon matched pairs test was performed for a–b.

Figure 5. Infection frequency of lymphocyte subsets

Infection frequency of sorted lymphocyte subsets from SIVagm-infected adult AGM as determined by PCR. White circles represen T-cells with detectable viral DNA and black circles represent an undetectable infection frequency reported as one half the lower limit of detection based on the number of cells within each PCR reaction. A Wilcoxon matched pairs test was performed for this analysis.