Fig 2: Incoming Vpr is sufficient to drive TRM induction in resting memory CD4+ T cells(A) CD69 (left) and CD69/CXCR6 (right) co-expression in response to IL-7 in the presence of integrase inhibitor raltegravir (n = 6).(B) Quantification of integrated provirus and 2LTR circles in FACS-sorted target CD4+ memory T cells after 72 h of cell-to-cell spread in the presence or absence of raltegravir.(C) Schematic depicting the viruses and VLPs used in (D–H).(D) Western blot showing Vpr packaging into virions of HIV-1 WTPM, VprPM, WT, ?Vpr, and ?Vpr complemented with FLAG-tagged Vpr in trans (?Vpr+Vprtrans).(E) CD69 (left) and CD69/CXCR6 (right) upregulation in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h infected with the indicated HIV-1 viruses (n = 8).(F) Expression of CD69 (left) and CD69/CXCR6 (right) in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h post spinoculation of HIV-1 WT, Vpr, and ?Vpr+Vprtrans (n = 10).(G) Western blot showing packaging of FLAG-tagged Vpr into Env-VLPs or full-length HIV-1 WT or ?Vpr.(H) Expression of CD69 (left) and CD69/CXCR6 (right) in response to IL-7 on Gag+ resting memory CD4+ T cells at 72 h post spinoculation of Env-VLPs with or without Vpr (n = 5). All measurements were made after 72 h or at the indicated time post co-culture or spinoculation. Data are the mean ± SEM. Paired two-tailed t test or one-way ANOVA with Bonferroni or Dunnett’s post test was used. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. EV, empty vector.

Fig 3: Vpr drives HIV-1-induced TRM induction in resting memory CD4+ T cellsResting memory CD4+ T cells were co-cultured with primary CD4+ T cells infected with HIV-1 wild-type (WT) or mutant viruses or with uninfected donor cells (mock).(A) CD69 upregulation in response to IL-7 compared with mock (n = 9).(B) CD69 expression on HIV-1-infected Gag+ resting memory CD4+ T cells compared with uninfected Gag- bystander cells (n = 9).(C) Quantification of cell-to-cell spread of HIV-1 WT and ?Vpr to resting naive and memory CD4+ T cells (n = 9).(D) CD69/CXCR6/CD49a co-expression on resting memory CD4+ T cells infected with HIV-1 WT or ?Vpr (n = 9).(E) CD69, CXCR6, and PRDM1 (Blimp1) mRNA levels from FACS-sorted infected resting memory CD4+ T cells. Fold change relative to uninfected (mock) is shown (n = 5).(F) IFN-? expression by HIV-1-infected resting memory CD4+ T cells at 72 h in response to IL-7 (n = 3).(G) Total lymphocytes from cellularized tonsils were co-cultured with HIV-1 WT- or ?Vpr-infected Jurkat T cells in the presence of IL-7. Expression of CD69 (left) or CD69/CXCR6 (right) was measured in Gag+ infected and Gag- uninfected bystander cells (n = 4).(H) Western blot showing Vpr packaging into HIV-1 WT and Vpr-mutant virions. Values indicate Vpr levels normalized to p24, relative to WT.(I) CD69 upregulation in response to IL-7 on resting memory CD4+ T cells infected with HIV-1 WT, ?Vpr, or Vpr mutants (n = 9).(J) Co-expression of CD69 and CXCR6 from (I) (n = 9). All measurements were made after 72 h or at the indicated time post co-culture. Data are the mean ± SEM. Paired two-tailed t test or one-way ANOVA with Bonferroni or Dunnett’s post test was used. 2LTR circles (I) were compared by unpaired one-tailed t test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

Fig 4: Vpr enhances STAT5 activation to drive a TRM-like resting memory CD4+ T cell phenotype(A) GSEA enrichment plot of the hallmark IL-2 STAT5 signaling pathway for HIV-1 WT-infected resting memory T cells versus mock.(B) CD69 expression on infected resting memory CD4+ T cells ± ruxolitinib at 72 h (n = 4).(C) CD127 MFI on infected resting memory CD4+ T cells ± IL-7 (n = 7) at 72 h.(D) Representative histogram of intracellular STAT5 phosphorylation in resting memory T cells infected by cell-to-cell spread at 72 h.(E) Quantification of (D) shown as P-STAT5 MFI (n = 10).(F) P-STAT5 MFI (left) and %P-STAT5+ (right) in Gag+ resting memory T cells 24 h post spinoculation with the indicated viruses (n = 12).(G) Representative western blot analysis of P-STAT5 and total STAT5 levels in resting T cells at 0 and 24 h post spinoculation with HIV-1 WT and ?Vpr virus (n = 2). Values indicate P-STAT5 or total STAT5 levels normalized to ß-actin and mock at 0 h.(H) Quantification of P-STAT5/STAT5 levels normalized to ß-actin from western blots of total CD4+ T cells at 24 h post spinoculation with HIV-1 WT and ?Vpr virus (n = 4).(I and J) Quantification of (I) P-STAT5 and (J) total STAT5 levels by single-cell immunofluorescence analysis of Gag+ resting T cells 24 h post spinoculation with HIV-1 WT and ?Vpr virus ± IL-7 (P-STAT5 n = 1,829–2,000 cells/condition; STAT5 n = 223–900 cells/condition). Normalized mean intensities (quantifications) and representative images of P-STAT5 in HIV-1 WT- and ?Vpr-infected cells without IL-7 (I, right) are shown. P-STAT5, green; Gag, red; Hoechst 33342, blue. Scale bars, 10 µm.(K) P-STAT5 MFI and %P-STAT5+ (right) in Gag+ resting memory T cells 24 h post spinoculation with VLPs with or without Vpr (n = 7).(L) CD69 (left) and CD69/CXCR6 (right) expression on infected resting memory CD4+ T cells in the presence of IL-7 ± STAT5-inhibitor AC-4-130 at 72 h (n = 6). All measurements were made after 72 h or at the indicated time post co-culture or spinoculation. Data are the mean ± SEM. Paired two-tailed t test or one-way ANOVA with Bonferroni or Dunnett’s post test was used. For (I) and (J), median is indicated and groups were compared using Kruskal-Wallis test with Dunn’s post test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. MFI, median fluorescence intensity.

Fig 5: HIV-1 infection induces a TRM-like phenotype in resting memory CD4+ T cells(A) CD69 expression on resting memory CD4+ target T cells following co-culture with HIV-1-infected primary donor T cells or uninfected donor T cells (mock) (n = 17).(B) Representative flow cytometry plots from (A).(C) CD69 expression on infected resting memory CD4+ T cells ± IL-7 and T20 (n = 7).(D) CD69 expression on infected resting memory CD4+ T cells ± IL-7 and ruxolitinib (n = 8).(E) CD69 expression on infected resting memory CD4+ T cells in response to IL-7 and IL-15 (n = 11).(F) CD69 expression on infected Gag+ resting memory CD4+ T cells and uninfected Gag- bystander cells in response to IL-7 and IL-15 (n = 11).(G) CXCR6 surface expression from (F) (n = 11).(H) Representative flow cytometry plots of CD69 and CXCR6 co-expression in the presence of IL-7.(I) Co-expression of CD69 with CXCR6, CD49A, or PD-1 on infected resting memory CD4+ T cells (n = 5–7).(J) As for (I) in the presence of IL-7 (n = 4–7).(K) As for (I) comparing infected Gag+ memory CD4+ T cells and uninfected Gag- bystander cells.(L and M) Blimp-1 expression in CD69+ HIV-infected resting memory CD4+ T cells and infected CD69- cells in the presence of IL-7 (n = 8).(N) Total lymphocytes from cellularized tonsils co-cultured with HIV-1-infected Jurkat T cells. Infection of resting CD4+ T cells shown as CD45RO versus Gag.(O) Representative flow cytometry plots of CD69 and CXCR6 co-expression on infected Gag+ and uninfected Gag- tonsil resting memory CD4+ T cells ± IL-7.(P) Recall cytokine response by HIV-1-infected Gag+ resting memory T cells. At 72 h of co-culture, expression of IFN-?, IL-2, or TNF was measured after stimulation with PMA/ionomycin and brefeldin A for the indicated duration. Gag+ cells were categorized by CD69 expression (n = 6).(Q) Mean proportion of Gag+ resting memory T cells expressing one, two, or three of the cytokines IFN-?, IL-2, or TNF after 6 h of PMA/ionomycin stimulation in the presence of brefeldin A, categorized by CD69 expression (n = 8). All measurements were made after 72 h or at the indicated time post co-culture. Data are the mean ± SEM. Paired two-tailed t test or one-way ANOVA with Bonferroni or Dunnett’s post test were used. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. MFI, median fluorescence intensity.