HLA-DR⁺ CD38⁺ CD4⁺ T Lymphocytes Have Elevated CCR5 Expression and Produce the Majority of R5-Tropic HIV-1 RNA In Vivo

Percentages of DR/38 lymphocyte subsets in non-virus-producing (GFP⁻) and virus-producing (GFP⁺) PHA-stimulated PBMC inoculated with R5-tropic (A) and X4-tropic (B) HIV-1 GFP reporter viruses. Mean fluorescence intensity (MFI) of GFP within virus-producing DR/38 subsets of cells inoculated with R5-tropic (C) and X4-tropic (D) HIV-1 reporter viruses. Percentages of GFP⁺ cells within DR/38 subsets inoculated with R5-tropic (E) and X4-tropic (F) HIV-1 reporter viruses. PBMC from 10 individuals at low risk for HIV-1 infection were depleted of CD8⁺ cells, cultured for 2 days with PHA and IL-2, inoculated with HIV-1 GFP reporter viruses, and evaluated 48 h later by flow cytometry. For panels A through D, the percentages of DR/38 subsets of cells were determined after first gating on cells in the lymphocyte gate and then on GFP⁺ and GFP⁻ cells. For panels E and F, the percentages of GFP⁺ cells were determined after first gating on cells in the lymphocyte gate and then on each DR/38 subset. Lines link the values from an individual subject.

Percentages of CCR5⁺ (A) and CXCR4⁺ (C) and cell surface concentrations (B and D) of these receptors, respectively, on DR/38 CD4⁺ T-cell subsets in whole-blood (WB) samples, PBMC isolated by density centrifugation, and CD8-depleted PHA-stimulated PBMC cultured for 2 days. Peripheral blood samples were obtained from 6 individuals at low risk for HIV-1 infection and stained with antibodies to cell surface markers at each time point, and results were determined by flow cytometry. Chemokine receptor expression on DR/38 subsets was determined after first gating on cells in the lymphocyte gate, then on CD3⁺ CD4⁺ cells, and finally on each DR/38 subset. Asterisks indicate the values for PHA-stimulated PBMC that were different from the values for whole blood (P = 0.03 in all instances).

Percentages of DR/38 CD4⁺ T-cell subsets in PBMC after 2 and 4 days of culture with PHA. PBMC from 6 individuals at low risk for HIV-1 infection were depleted of CD8⁺ cells and cultured with PHA. Using flow cytometry, the percentages of DR/38 subsets of cells were determined by first gating on cells in the lymphocyte gate and then on CD3⁺ CD4⁺ cells. Lines indicate paired values from one subject. Differences between day 2 and day 4 were significant within all subsets (P = 0.03).

Representative flow cytometry plots of lymph node cells. The cells were acquired from the lymphocyte gate of the forward- versus side-scatter profile. A dot plot was used to define CD3⁺ CD4⁺ cells (A) and gates for CD38 (B) and HLA-DR (C) set with fluorescence minus one (FMO) controls. CD4⁺ CD3⁺ lymphocytes were evaluated in a CD38 versus HLA-DR plot (D), and CCR5 expression was evaluated in four DR 38 subsets using histograms (E). Data were analyzed using FlowJo software (Tree Star).

Percentages of CCR5⁺ (A) and CXCR4⁺ (B) and concentrations of these HIV-1 coreceptors (C and D), respectively, in DR/38 CD3⁺ CD4⁺ subsets of lymph node cells from HIV-1-seropositive individuals (n = 18) and HIV-1-seronegative individuals (n = 6). Each symbol represents the value for an individual. Short horizontal lines indicate median values for groups of individuals. P values indicate comparisons between DR⁺ 38⁺ cells and other subsets in seropositive subjects. Asterisks denote P values that were no longer statistically significant after Bonferroni's correction for multiple comparisons (cutoff, P = 0.017). Differences between DR⁺ 38⁺ cells and other subsets in seronegative subjects and differences between seropositive and seronegative subjects were not statistically significant after adjusting for multiple comparisons.

HIV-1 RNA measurements within subsets of CD4/CD8 CD3⁺ lymph node cells (n = 6). (A) Numbers of HIV-1 RNA copies/10⁵ cells; (B) percentages of CD4/CD8 subsets within CD8⁻ CD3⁺ lymph node cells; (C) percentages of total RNA copies contributed by each CD4/CD8 subset after adjusting for percentages of subsets shown in panel B. For each box-and-whisker plot, the line indicates the median and the whiskers (error bars) indicate the range. Lymph node cells from untreated HIV-1-seropositive subjects were sorted into subsets on a cell sorter, and aliquots of 10⁵ cells were frozen as pellets. HIV-1 RNA was then extracted and measured by PCR.

HIV-1 RNA and DNA measurements within subsets of DR/38 CD8⁻ CD3⁺ lymph node cells. (A) Numbers of HIV-1 RNA copies/10⁵ cells; (B) percentages of DR/38 subsets within CD8⁻ CD3⁺ lymph node cells; (C) percentages of total RNA copies contributed by each DR/38 subset after adjusting for percentages of subsets shown in panel B; (D) numbers of HIV-1 DNA copies/10⁵ cells; (E) percentages of total DNA copies contributed by each DR/38 subset after adjusting for percentages of subsets shown in panel B. Lymph node cells from untreated HIV-1-seropositive subjects were sorted into subsets on a cell sorter, and aliquots of 10⁵ cells were frozen as pellets. HIV-1 RNA and DNA were then extracted and measured by PCR.