Figure 1.
Distribution of Human CD4+ T Cell Subsets in HIV-Infected and HIV-Uninfected Individuals
(A) PBMCs from HIV-uninfected and HIV-infected individuals were stained with purified CCR7 antibody, followed by allophycocyanin-conjugated anti-mouse IgG. After washing, cells were co-stained with CD4-FITC, CD3-Percp.Cy5.5, and CD45RO-PE. Expression of CCR7 and CD45RO were analyzed after gating on CD4+CD3+ T cells. Representative flow cytometry data from over 60 individuals are shown. The abbreviated definition of T cell subsets based on this staining profile is shown above the flow cytometry plots.
(B) CD4+ T cell subsets were analyzed in HIV-uninfected and HIV-infected individuals. Between 300,000 and 500,000 events were collected for each sample, and electronic gates were set on CD4+CD3+ T cells. Distribution and median percentages of TN (CD45RO−CCR7+), TCM (CD45RO+CCR7+), TEMRO (CD45RO+CCR7−), and TEMRA (CD45RO−CCR7−) cells in 33 HIV-infected and 30 HIV-uninfected individuals are shown.
Figure 2.
Phenotype of Distinct CD4+ Human T Cell Subsets
Purified CD4+ T cells were stained with CD45RO and CCR7 in conjunction with the antibodies shown. Electronic gates were set on TN, TCM, TEMRO, and TEMRA cells as described in Figure 1A, and expression of various T cell surface markers was analyzed. A representative profile from an HIV-infected individual is shown. Similar phenotype was observed in all four subsets of T cells from six HIV-infected and HIV-uninfected individuals.
Figure 3.
Proliferation, Apoptosis, and Cytokine Profile of CD4+ T Cell Subsets
(A) Sorted CD4+ T cell subsets from an HIV-uninfected individual were activated using DCs pulsed with SEB (10 ng/ml) and expanded in IL-2–containing medium for 12 d. T cell subsets were then counted to determine fold expansion.
(B) Viability of purified CD4+ T cell subsets was determined using Annexin V staining after 18 h post-activation. Samples were analyzed by flow cytometry. Statistical significance was determined using the Student's two-tailed t test. TN: p = 0.4, TCM: p = 0.002, TEMRO: p = 0.15, and TEMRA: p = 0.3.
(C) Sorted CD4+ T cell subsets were activated using plate-bound anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies. Supernatants were collected 18–24 h post-activation and analyzed for cytokine production using the cytometric bead assay. The results show the mean of four different experiments from four different individuals.
Figure 4.
Expression of HIV-1 Coreceptors on CD4+ T Cell Subsets and Their Susceptibility to HIV-1 Infection
(A) CD4+ T cells from an HIV-uninfected individual were stained with CD45RO-PE and CCR7-FITC in conjunction with CCR5 and CXCR4 antibodies. CD4+ T cell subsets were gated as described in Figure 1, and expression of CCR5 and CXCR4 was analyzed.
(B) Purified CD4+ T cell subsets from an HIV-uninfected individual were activated using plate-bound anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies and concurrently infected with VSV-G.HIV, R5.HIV, or X4.HIV. Percent infected cells was determined by GFP expression of T cells on day 5 and 12 post-infection (p.i.). The results are representative of one of five independent experiments using T cell subsets isolated from five different individuals. The median infection values at day 5 were: R5-HIV: TN = 1.1, TCM = 9.5, TEMRO = 5.4, and TEMRA = 0.85; X4-HIV: TN = 17, TCM = 36, TEMRO = 43, and TEMRA = 33; and VSV-G.HIV: TN = 44, TCM = 48, TEMRO = 19, and TEMRA = 6.5.
Figure 5.
Replication of HIV-1 Strains in CD4+ T Cell Subsets
Purified CD4+ T cell subsets were activated through TCR as in Figure 4 and concurrently infected with replication-competent HIV-1.
(A) R5.HIV and X4.HIV infection time course of CD4+ T cell subsets from HIV-uninfected and HIV-infected individuals.
(B) Supernatants from purified CD4+ T cell subsets infected with R5.HIV and X4.HIV cultures were collected at different time points and HIV p24 levels were measured by ELISA. The percentage of infected cells was determined by GFP expression at different time points post-infection by flow cytometry. The results are representative of one of five independent experiments from different individuals.
(C) Infection of T cell subsets with HIV-1 expressing different primary isolate envelopes. Percent infected T cells was determined by intracellular p24 staining of T cells on day 4, 6, and 8 post-infection. The T cell subsets in this experiment were also infected with VSG-V.HIV, where the percent infected cells was determined by GFP expression at 4 day post-infection. The VSV-G-HIV infection was 33% for TN, 33% for TCM, and 9% for TEMRA cells.
Figure 6.
Replication of HIV-1 strains in CD57+ CD4+ T cell subsets
Effector memory T cell subsets were subdivided into CD57+ and CD57− portions by sorting, then activated and concurrently infected with R5.HIV or X4.HIV. The percentage of infected cells was determined by GFP expression at different time points post-infection by flow cytometry. The results are representative of one of five independent experiments from different individuals.
Figure 7.
Identifying the Block in R5-Tropic and VSV-G Pseudotyped Infection of TEMRA Cells
Purified CD4+ T cell subsets were activated using DCs pulsed with SEB (10 ng/ml) and expanded in IL-2–containing medium for 10 d.
(A) Day 12 expanded subsets were stained for CCR5 expression. Cells were then reactivated with SEB-pulsed DCs and subsequently infected with R5.HIV at an MOI of 5. At day 6 post-infection, cells were then stained for CCR5, and infection was analyzed as determined by GFP expression by flow cytometry.
(B) R5.HIV, X4.HIV, and VSV-G.HIV carrying a β-lactamase reporter protein were incubated with expanded CD4+ T cell subsets, TN cells, and Jurkat T cell line at 37 °C for 2 h to allow virus–cell fusion, CCF2/AM (20 μM) was added, and fluorescence was measured as described in Materials and Methods. Fluorescence ratios were calculated after subtraction of the average background fluorescence.
(C) Expanded subsets were reactivated with SEB-pulsed DCs and subsequently infected with R5.HIV, X4.HIV, or VSV-G.HIV at an MOI of 5 for 12 h. Cells were then lysed and processed for real-time PCR analysis using late HIV transcript primers.
(D) Reactivated expanded T cell subsets were lysed and processed for real-time PCR analysis using early HIV transcript primers. All infections were performed at an MOI of 5. The results are representative of one of five independent experiments using T cell subsets isolated from five different individuals.
Figure 8.
Association between TEM Subsets and CD4 Numbers in HIV-Infected Individuals
HIV-infected individuals were separated into three groups based on percent of their CD4+ TEM cells. The medium TEM population was subdivided into TEMROhighTEMRAlow and TEMROlowTEMRAhigh, and the number of total CD4 cells and TN cells in each group was calculated. We set the cut-off percentage for defining TEMRO high and low proportions as 28% of CD4+ T cells and for TEMRA cells, 8% of CD4+ T cells. Statistical analysis was performed with only the medium group since the bottom group did not contain any TEMRAhigh cells (range: 1.6%–5.5%) and within the top group all individuals contained high TEMRO cells (range: 31%–59%). Statistical significance between groups was determined by Wilcoxon rank sum test.