HIV immune activation drives increased Eomes expression in memory CD8 T cells in association with transcriptional downregulation of CD127

Abstract

Background: During HIV infection distinct mechanisms drive immune activation of the CD4 and CD8 T cells leading to CD4 T-cell depletion and expansion of the CD8 T-cell pool. This immune activation is polyclonal and extends beyond HIV-specific T cells. One consequence of this immune activation is a profound decrease in IL-7Rα (CD127) expression on memory CD8 T cells. The mechanisms leading to this are unknown and because of the potential impact of reduced IL-7 signaling in memory T cells specific to HIV and other pathogens, in the present study we examined the molecular mechanisms implicated in this downregulation of CD127.

Methods: Membrane bound (mIL7RA) and soluble (sIL7RA) mRNA expression was determined by qRT-PCR. CD127, Eomesodermin (Eomes) and T-bet expression in healthy controls and HIV-infected patients were studied by flow cytometry.

Results: CD127 downregulation occurs at the transcriptional level for both mIL7RA and sIL7RA alternative spliced forms in the CD127 memory CD8 T cells. CD127 memory CD8 T cells exhibited increased Eomes expression and an 'effector-like' gene profile. These changes were associated with higher HIV-RNA levels. Following combination antiretroviral therapy (cART), there was an increase in CD127 expression over an extended period of time (>5 months) which was associated with decreased Eomes expression.

Conclusion: CD127 is downregulated at a transcriptional level in memory CD8 T cells in association with upregulation of Eomes expression.

Fig. 1.. Expression of IL7RA membrane and soluble transcripts in sorted CD8 T cells.

(a) Cell sorting of CD8 T-cell subsets from healthy controls (n = 6, gray symbol) and HIV-infected patients with viral load more than 50 copies/ml (n = 4, red symbol) and viral load less than 50 copies/ml (n = 4, blue symbol) was performed based on surface expression of CD45RA and CD27. The naive CD8 T-cell subset was CD45RA⁺CD27⁺CD127^highHLADR⁻; the memory CD8 T-cell subset was divided by expression of CD127 into memory CD127^high (CD45RA⁻CD27⁺CD127^highHLADR⁻), and memory CD127^low (CD45RA⁻CD27⁺CD127^lowHLADR⁺). The terminal effector memory (TEMRA) subset was defined as CD45RA⁺CD27⁻CD127^lowHLADR⁺. The figure shows a representative example of the sorted cohorts pregated on live CD3⁺CD8⁺ cells. (b) Relative fold increase of membrane bound (mIL7RA) and soluble (sIL7RA) forms of the IL7RA as determined by qRT-PCR. The results were normalized to RPL13 expression. No significant differences were noted between the groups at the P ≤ 0.01 level.

Fig. 2.. Expression of Eomes and CD127 in CD8 T-cell subsets from healthy controls and HIV-infected patients.

(a) The analysis of mRNA expression of the transcription factor EOMES and the effector molecules granzyme B (GZMB) and perforin (PRF1) were measured as described in Fig. 1. (b) The protein expression of Eomes and CD127 in CD8 T-cell subsets were analyzed by flow cytometry in PBMCs from healthy controls (n = 13), HIV-infected patients with viral load less than 50 copies/ml (n = 13) and HIV-infected patients with viral load more than 50 copies/ml (n = 14). Median fluorescence intensities for CD127 and Eomes were calculated using FlowJo. A representative example from each of the three cohorts is displayed (gating strategy is shown in Figure S2). (c) Comparisons of Eomes expression in CD8 T-cell subsets between HIV-infected groups and healthy controls. (d) Correlations between Eomes expression and HIV-RNA levels and (e) Correlations between CD127 and Eomes expression.

Fig. 3.. Protein expression of T-bet and CD127 in CD8 T-cell subsets from healthy controls and HIV-infected patients.

(a) Representative dot plots from the three cohorts studied (Fig. 2 for T-bet and CD127 expression). (b) T-bet median fluorescence intensity in CD8 T-cell subsets (as above). (c) Correlations between T-bet expression and HIV-RNA levels; and (d) Correlations between CD127 and T-bet expression. (e) Correlations between Eomes and T-bet expression in CD8 T-cell subsets.

Fig. 4.. Effect of cART on T-box transcription factors T-bet and Eomes and CD127 expression in CD8 T-cell subsets.

(a) Representative contour plots for CD127, Eomes and T-bet expression over three time points: more than 50 copies/ml (T0), first month less than 50 copies/ml (T1), and 5–8 months later (T2). (b) Changes over time of the MFI of Eomes, T-bet and CD127 in CD8 T-cell subsets. Median change denoted by red line.