Volume 47, Issue 4, Pages 766-775.e3 (October 2017) Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection  Liang Shan, Kai Deng, Hongbo Gao, Sifei Xing, Adam A. Capoferri, Christine M. Durand, S. Alireza Rabi, Gregory M. Laird, Michelle Kim, Nina N. Hosmane, Hung-Chih Yang, Hao Zhang, Joseph B. Margolick, Linghua Li, Weiping Cai, Ruian Ke, Richard A. Flavell, Janet D. Siliciano, Robert F. Siliciano  Immunity  Volume 47, Issue 4, Pages 766-775.e3 (October 2017) DOI: 10.1016/j.immuni.2017.09.014 Copyright © 2017 Terms and Conditions

Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 1 Latent Infection by CCR5-tropic HIV-1 Is Not Efficient in Naive, Resting Memory, or Activated CD4+ T Cells (A and B) Replication competent HIV-1 (A) was isolated from resting CD4+ T cells of eight patients using a limiting dilution virus outgrowth assay. The patient from whom only CXCR4-tropic virus was recovered was highlighted in blue. Genomic DNA (B) was isolated from resting CD4+ T cells of 11 patients. Viral env sequences were analyzed by the PSSM system. (C and D) Bcl-2-transduced resting or activated CD4+ T cells were infected using a pseudotyped HIV-1 (NL4-3-Δenv-drEGFP) with a R5 (Yu-2) or X4 (NL4-3) envelope. HIV-1 gene expression was assessed by flow cytometry. Productive infection (C) was measured 3 days after infection. To generate latent infection, infected cells were cultured in basal medium without supplement of antibodies or cytokine for another 25 days before removal of GFP-positive cells. Latent infection (D) of Bcl-2-transduced CD4+ T cells was measured 2 days after reactivation of latent virus by stimulation with anti-CD3 and anti-CD28 antibodies. The limit of detection of latent HIV-1 infection by flow cytometry is 0.01%. Blood samples from 12 healthy donors were used for the analysis. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 2 Latent Infection of CCR5-tropic HIV-1 Is Not Efficient in CCR5+ or CCR5− Resting Memory CD4+ T Cells (A) CD4+ T cells isolated from a healthy donor were stimulated with anti-CD3 and anti-CD28 antibodies or left untreated for 3 days. Activated and resting (CD25−, CD69−, and HLA-DR−) CD4+ T cells were analyzed for CD45RO, CCR5, or CCR7 expression. (B) Resting memory CCR5+ or CCR5− CD4+ T cells were purified by sorting from freshly isolated healthy donor CD4+ T cells and cultured for 24 hr. Culture medium was then collected for the measurement of cytokine production. Activated CD4+ T cells were used as control. Error bars represent SEM, n = 3. (C) Productive and latent infection of resting memory CCR5+ CD4+ T cells. Three populations of Bcl-2-transduced CD4+ T cells were purified by FACS for infection experiments: (1) CCR5− activated cells, (2) CCR5+ memory cells, and (3) CCR5− memory cells. Purified cells were infected with NL4-3-Δenv-drEGFP pseudotyped with HIV-1-X4 or -R5 envelope. Error bars represent SEM, n = 3. Flow data were presented in standard, 4 log biexponential plots. In (B) and (C), data were pooled from three healthy donors. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 3 EMT CD4+ T Cells Are More Susceptible to CCR5-tropic HIV-1 Infection due to Upregulation of CCR5 Expression (A) HIV-1 co-receptor expression and markers of T cell activation and proliferation were measured and compared in naive (CD25−, CD69−, HLA-DR−, and CD45RA+), fully activated, EMT, and resting memory (CD25−, CD69−, HLA-DR−, and CD45RO+) CD4+ T cells generated from freshly isolated healthy donor CD4+ T cells. (B and C) CCR5 gene transcription and surface expression in EMT CD4+ T cells. In (B), blood samples from ten healthy donors were used for CCR5 staining. In (C), data were pooled from two donors. (D and E) R5-tropic HIV-1 enters EMT CD4+ T cells expressing high level of CCR5. Data were pooled from two donors. (D) Total CD4+ T cells; (E) CCR5+ CD4+ T cells. Error bars represent SEM, n ≥ 3. t test values: ∗∗∗p < 0.001; ∗∗p < 0.01. See also Figures S1–S4. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 4 CCR5-tropic HIV-1 Preferentially Infects EMT CD4+ T Cells CD4+ T cells isolated from healthy donor were used to generate resting, activated, and EMT CD4+ T cells. (A) 24 hr or 72 hr after infection of different CD4+ T cell populations with X4-tropic virus, late reverse transcription products were measured by qPCR and normalized to levels in activated cells. (B and C) Productive expression by R5-tropic HIV-1 was assessed by GFP expression 72 hr after infection. (D) CD4+ T cells at different activation states were co-transfected by electroporation (Lonza) with a proviral construct (pNL4-3-Δenv-drEGFP) with eGFP in the env ORF, and a control expression vector in which BFP expression was driven by SV40 promoter. GFP and BFP expression was measured 24 hr after transfection. Error bars represent SEM, n = 3. t test values: ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05. Data were pooled from three independent infections or transfections. See also Figure S5. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 5 Establishment of HIV-1 Latent Infection Occurs Preferentially in EMT CD4+ T Cells Freshly isolated CD4+ T cells from healthy donors were used in (A)–(C). Bcl2-transduced CD4+ T cells from healthy donors were used in (D) and (E). (A) Microarray analysis of gene transcription profile was performed in CD4+ T cells at different activation states. The top 100 most upregulated (comparing to resting cells) and top 50 most upregulated NF-κB responsive genes in activated CD4+ T cells were represented. Three healthy donor CD4+ T cells were used for microarray analysis. (B) Cytokine production was measured from CD4+ T cells at different activation states. Data were pooled from three independent experiments. (C) CD4+ T cells at different activation states were infected with NL4-3-Δnef-EGFP pseudotyped with X4 envelope. Individual GFP-positive cells were purified by single cell sorting 24 hr after infection for mRNA isolation and quantification. Quantitative measurement of pol2a and HIV-1 gag transcripts was performed in individual HIV-1-infected cells. Data were pooled from two healthy donor CD4+ T cells. (D and E) NL4-3-Δenv-drEGFP pseudotyped with HIV-1-X4 or -R5 envelope was used to infect CD4+ T cells at different activation states. Productive (D) and latent (E) infection was measured as describe in Figure 1. Data were pooled from six healthy donor CD4+ T cells. Error bars represent SEM, n ≥ 3. t test values: ∗∗p < 0.01; ∗p < 0.05. See also Figures S6 and S7. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 6 Latent HIV-1 Is Enriched in CCR5+ Resting Memory CD4+ T Cells from HIV-1 Patients (A) Frequency of HIV-1 proviral DNA in naive, CCR5−CD45RO+, and CCR5+CD45RO+ resting CD4+ T cells was measured by qPCR. t test values: ∗∗∗p < 0.001; ∗p < 0.05. (B) Frequency of latent HIV-1 in CCR5− and CCR5+ resting memory CD4+ T cells was measured by limiting dilution virus outgrowth assay. Ratio-paired t test was performed for statistical analysis. (C) Relative contribution of CCR5+ and CCR5− subsets to the reservoir for latent HIV-1 in memory CD4+ T cells in each patient was calculated according to the frequency of latently infected cells in each subset and the frequency of each subset in the whole memory CD4+ T cell population. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions

Figure 7 HIV-1-Specific CTLs Inhibits Latent HIV-1 Infection in EMT CD4+ T Cells (A) Patient-derived Bcl-2-transduced EMT CD4+ T cells were infected with NL4-3-Δenv-drEGFP pseudotyped with HIV-1 R5 envelope. Autologous CD8+ T cells pre-stimulated with Gag peptides mixture were added to the culture 24 hr after infection at the ratio of 1:1. Productive infection with (dotted line) or without (solid line) the presence of autologous CD8+ T cells was monitored over time by FACS. (B) GFP-positive cells as well as CD8+ T cells described in (A) were removed from the co-culture 15 days after infection. The remaining cells were stimulated with CD3&CD28 antibodies for the measurement of latent infection. Data were pooled from three HIV-1 patient samples. Error bars represent SEM, n = 3. t test values: ∗p < 0.05. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014) Copyright © 2017 Terms and Conditions