Identification of a CCR5-expressing T cell subset that is resistant to R5-tropic HIV infection

Abstract

Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naïve T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(-)CD45RO(-)CD45RA(+) (referred to as TEMRA cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, TEMRA cells were strikingly resistant to infection with CCR5 (R5)-tropic HIV-1, but remained highly susceptible to CXCR4 (X4)-tropic HIV-1. The resistance of TEMRA cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of TEMRA cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1-resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within TEMRA cells that restrict R5-tropic viruses and endow HIV-1-specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection.

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 T_N (CD45RO⁻CCR7⁺), T_CM (CD45RO⁺CCR7⁺), T_EMRO (CD45RO⁺CCR7⁻), and T_EMRA (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 T_N, T_CM, T_EMRO, and T_EMRA 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. T_N: p = 0.4, T_CM: p = 0.002, T_EMRO: p = 0.15, and T_EMRA: 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: T_N = 1.1, T_CM = 9.5, T_EMRO = 5.4, and T_EMRA = 0.85; X4-HIV: T_N = 17, T_CM = 36, T_EMRO = 43, and T_EMRA = 33; and VSV-G.HIV: T_N = 44, T_CM = 48, T_EMRO = 19, and T_EMRA = 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 T_N, 33% for T_CM, and 9% for T_EMRA 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 T_EMRA 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, T_N 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 T_EM Subsets and CD4 Numbers in HIV-Infected Individuals

HIV-infected individuals were separated into three groups based on percent of their CD4⁺ T_EM cells. The medium T_EM population was subdivided into T_EMRO ^highT_EMRA ^low and T_EMRO ^lowT_EMRA ^high, and the number of total CD4 cells and T_N cells in each group was calculated. We set the cut-off percentage for defining T_EMRO high and low proportions as 28% of CD4⁺ T cells and for T_EMRA cells, 8% of CD4⁺ T cells. Statistical analysis was performed with only the medium group since the bottom group did not contain any T_EMRA ^high cells (range: 1.6%–5.5%) and within the top group all individuals contained high T_EMRO cells (range: 31%–59%). Statistical significance between groups was determined by Wilcoxon rank sum test.