1. 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 e3 (October 2017)
DOI: /j.immuni
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2. Immunity 2017 47, 766-775.e3DOI: (10.1016/j.immuni.2017.09.014)
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3. 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.
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4. 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.
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5. 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 < See also Figures S1–S4.
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6. 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.
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7. 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 < See also Figures S6 and S7.
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8. 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.
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9. 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  , e3DOI: ( /j.immuni )
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