The CD8+ HLA-DR+ T cells expanded in HIV-1 infection are qualitatively identical to those from healthy controls - PubMed (original) (raw)

. 2012 Oct;42(10):2608-20.

doi: 10.1002/eji.201142046. Epub 2012 Aug 6.

Richard A Lempicki, Joseph W Adelsberger, Rebecca B Hasley, Alice Rosenberg, Gregg Roby, Catherine A Rehm, Amy Nelson, Sonya Krishnan, Mark Pavlick, Christian J Woods, Michael W Baseler, H Clifford Lane

Affiliations

The CD8+ HLA-DR+ T cells expanded in HIV-1 infection are qualitatively identical to those from healthy controls

Hiromi Imamichi et al. Eur J Immunol. 2012 Oct.

Abstract

HIV-induced immune activation leads to expansion of a subset of human CD8(+) T cells expressing HLA-DR antigens. Expansion of CD8(+) HLA-DR(+) T cells can be also observed in non-HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent "immune exhaustion" and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8(+) HLA-DR(-) counterpart, the CD8(+) HLA-DR(+) T-cell pool contained an increased fraction of cells in S-phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8(+) pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8(+) HLA-DR(+) cells from HIV(+) and HIV(-) donors, indicating that the generation of CD8(+) HLA-DR(+) T cells is a part of normal immune regulation that is exaggerated in the setting of HIV-1 infection.

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1

Figure 1. Analysis of activation markers on CD8+ T cells from HIV-infected patients reveals no evidence of recent T cell receptor engagement

PBMCs isolated from control (n=14), HIV+ <50 copies/ml (n=17) and HIV+ ≥50 copies/ml (n=9) individuals were analyzed by 4-color flow cytometry for cell surface expression of HLA-DR, CD25, CD38 and CD69 activation markers. Percentages of HLA-DR expressing cells were calculated on CD3+CD8+ T cells in the lymphocyte gate. Percentages of CD25, CD38 and CD69 expression were calculated separately for CD8+HLA-DR− and CD8+HLA-DR+ T cell subsets. HLADR depleted CD8+ T cells from healthy control donors that were stimulated with anti-CD3+CD28 for 24 hours in vitro (n=3) were used as a model for antigen-activated CD8+ T cells. The proportions of cells expressing HLA-DR, CD25, CD38 or CD69 are indicated by the dark gray regions of the pie charts. Mean + SEM values are shown. A nonparametric Mann-Whitney test was performed for comparison of the frequencies of cells expressing the activation markers between different groups. Representative plots of cell surface expression of CD25, CD38 and CD69 on CD8+HLA-DR− and CD8+HLA-DR+ T cells are shown in Supporting Information Fig.7. Data are representative of control (n=14), HIV+ <50 copies/ml (n=17) and HIV+ ≥50 copies/ml (n=9) individuals.

Figure 2

Figure 2. Wright-Giemsa staining of CD8+HLA-DR− and CD8+HLA-DR+ T cells

Purified CD8+HLA-DR− and CD8+HLA-DR+ T cells were freshly isolated from PBMCs. Wright-Giemsa staining pictures of CD8+HLA-DR− T cells cultured in vitro in medium alone or with anti-CD3+CD28 for 2 days are also shown. Bar = 10 μm. Data are representative of 3 individuals for each group.

Figure 3

Figure 3. The CD8+HLA-DR+ pool of cells incorporates higher levels of BrdU (or EdU) than CD8+HLA-DR− cells

Ex vivo BrdU (or EdU) labeling of PBMCs was performed. The percentages of the CD8+HLA-DR− and CD8+HLA-DR+ T cells that were positive for BrdU (or EdU) were determined. Bars indicate median and inter-quartile range (IQR). Statistical significance was determined by the Mann-Whitey test. The P values represent differences in the fraction of cells in the S-phase of the cell cycle between CD8+HLA-DR− and CD8+HLA-DR+ subsets. The results were obtained from control (n=4), HIV+ <50 (n=14) and HIV+ ≥50 (n=9) individuals.

Figure 4

Figure 4. TCR repertoire and TREC content of CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells

(A) The TCR repertoire patterns (Vβ11) are shown. Due to insufficient RNA from each individual sample, the TCR repertoire analysis was performed only for CD8+HLA-DR− and CD8+HLA-DR+ cells. The spectratype profiles shown in (A) are representative sets from control (n=6) and HIV-infected (n=10) individuals. (B) The number of sjTREC/1×106 cells was determined for FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ T cell subsets. Solid bars indicate median values. The level of statistical significance was determined by the Mann-Whitey test. DR− and DR+ stand for HLA-DR negative and HLA-DR positive cells respectively. Na and Me stand for naive and memory cells respectively. The results were obtained from control (n=9), HIV+ <50 (n=9) and HIV+ ≥50 (n=7) individuals.

Figure 5

Figure 5. The peripheral pool of CD8+HLA-DR+ cells contains more cycling cells than that of CD8+HLA-DR− naive and memory cells

Cell cycle analysis was performed by flow cytometry using simultaneous staining with 7-AAD (DNA) and pyronin Y (PY, RNA). Definitions of G0, early G1, late G1, and S+G2/M phases of the cell cycle are described in the Materials and methods and the diagram on the right helps identify positions of G0, early G1, late G1 and S+G2/M of the cell cycle. The values in the plots indicate the percentages of cells in the regions corresponding to G0, early G1, late G1 and S+G2/M. Representative patterns from 2 different donors are shown for control (n=5), HIV+ <50 (n=6), and HIV+ ≥ 50 (n=8) groups.

Figure 6

Figure 6. The proliferative capacity and death rates of CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells

DNA and RNA content were determined for cells freshly isolated by microbeads (Day 0) and for cells harvested following 1-3 days of culture with medium alone or in the presence of anti-CD3+CD28. The regions corresponding to G0, early G1, late G1 and S+G2/M are the same as those in Figure 5. The percentages of dead cells were determined by flow cytometry with Live/Dead cell staining dye. Plots shown are from an HIV-infected individual with HIV-RNA <50 copies/ml representative of 3 such individuals. Gating strategy used to analyze the cell cycle status is shown in Supporting Information Fig.8.

Figure 7

Figure 7. Expression of genes associated with cell survival in CD8+HLA-DR-naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells

(A) Expression levels of Bcl-2 and Survivin in FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR-memory and CD8+HLA-DR+ cells. Mean + SEM values were calculated using cells from control (n=11), HIV+ <50 (n=9), and HIV+ ≥50 (n=7) individuals. The level of statistical significance was determined by the Mann-Whitey test. *P<0.001. (B) Levels of Bcl-2 and Survivin proteins as determined by western blot. Actin was used as a loading control. Results are representative of 3 independent experiments.

Figure 8

Figure 8. The CD8+HLA-DR+ pool of cells is enriched for cells actively involved in cell cycle progression compared with CD8+HLA-DR− naive and memory cells

(A) Expression levels of 11 genes involved in cell cycle control were examined in FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells. The mRNA expression levels were normalized to the value of beta-2 microglobullin (B2M) expression. Mean + SEM values were calculated using cells from control (n=9), HIV+ <50 (n=10), and HIV+ ≥50 (n=9) individuals. Statistical significance was determined by the Mann-Whitey test. *P<0.001. Genes that were found to be upregulated in CD8+HLA-DR+ cells compared with CD8+HLA-DR− naive and CD8+HLA-DR− memory cells are shown in red and those found to be downregulated are in blue. (B) Protein levels of the genes depicted in (A) as determined by western blot. Cell lysates from FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells were used. In order to achieve adequate levels of proteins, cell lysates cells from 3-4 individuals were pooled (approximately 1 million cells from each individual). Representative patterns from 3 separate experiments are shown. Actin and TFII-I were used as loading controls for whole cell lysate and nuclear fraction, respectively. Images presented are from a 1-minute film exposure.

Figure 8

Figure 8. The CD8+HLA-DR+ pool of cells is enriched for cells actively involved in cell cycle progression compared with CD8+HLA-DR− naive and memory cells

(A) Expression levels of 11 genes involved in cell cycle control were examined in FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells. The mRNA expression levels were normalized to the value of beta-2 microglobullin (B2M) expression. Mean + SEM values were calculated using cells from control (n=9), HIV+ <50 (n=10), and HIV+ ≥50 (n=9) individuals. Statistical significance was determined by the Mann-Whitey test. *P<0.001. Genes that were found to be upregulated in CD8+HLA-DR+ cells compared with CD8+HLA-DR− naive and CD8+HLA-DR− memory cells are shown in red and those found to be downregulated are in blue. (B) Protein levels of the genes depicted in (A) as determined by western blot. Cell lysates from FACS-sorted CD8+HLA-DR− naive, CD8+HLA-DR− memory and CD8+HLA-DR+ cells were used. In order to achieve adequate levels of proteins, cell lysates cells from 3-4 individuals were pooled (approximately 1 million cells from each individual). Representative patterns from 3 separate experiments are shown. Actin and TFII-I were used as loading controls for whole cell lysate and nuclear fraction, respectively. Images presented are from a 1-minute film exposure.

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