Fig 2: 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 3: 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.

Fig 4: Human CD69+CD16- lung NK cell subsets have unique characteristics. a Representative overlays and b summary of data of expression of NKG2A and CD57 on CD16- human lung CD69-CD49a-CD103- (n = 20 for both), CD69sp (n = 20 for both), CD69+CD49a+CD103- (n = 14 and n = 13, respectively), and CD69+CD49a+CD103+ (n = 17 and n = 15, respectively) NK cells. CD56dimCD16+ NK cells are shown for comparison (n = 20 and n = 19, respectively). Numbers in a indicate %NKG2A+ and %CD57+ NK cells, respectively. c Representative overlays and d summary of mean fluorescence intensity (MFI) of CD56 (n = 18 for CD16-CD69-CD49a-CD103-NKG2A+CD57-, n = 21 for CD69+CD49a-CD103-CD16-, n = 16 for CD69+CD49a+CD103-CD16-, n = 19 for CD69+CD49a+CD103+CD16-, and n = 20 for CD56dimCD16+), CXCR3 (n = 3) and CXCR6 (n = 7) on human lung CD69-CD49a-CD103-NKG2A+CD57-, CD69sp, CD69+CD49a+CD103-, and CD69+CD49a+CD103+ NK cells within the CD16- subset. e Representative overlay and f summary of MFI of perforin expression in CD16-CD69-CD49a-CD103-NKG2A+CD57-, CD69spCD16-, CD69+CD49a+CD103-CD16-, CD69+CD49a+CD103+ CD16-, and CD69-CD49a-CD103-CD16+ NK cells in human lung (n = 5). CD127+CD161+ cells from lung tissue are shown as a comparison (n = 5). g Representative overlays and h summary of MFI of Eomes (left panel) (CD16-CD69-CD49a-CD103-NKG2A+CD57- (n = 5, other subsets n = 7) and T-bet (right panel) (CD16-CD69-CD49a-CD103-NKG2A+CD57- (n = 4, other subsets n = 6) expression in CD16- human lung CD69-CD49a-CD103-NKG2A+CD57-, CD69sp, CD69+CD49a+CD103-, and CD69+CD49a+CD103+ NK cells. Expression in CD16+CD69-CD49a-CD103- NK cells and non-NK cell ILCs (CD56-CD16-NKG2A-KIR-CD161+) are shown as a reference for Eomes (n = 4), and T-bet (n = 6), respectively. i Principal component analysis based on 1,000 most variable genes across subsets. j Euler diagram based on the 230 differentially expressed genes (DEGs, p < 0.05, log2 fold change > 1) between CD69+CD49a+CD103+NKG2A+CD16-, CD69+CD49a+CD103-NKG2A+CD16-, CD69spNKG2A+CD16- and CD69-NKG2A+CD16- NK cells. DEGs among CD69+CD49a+CD103+NKG2A+CD16- and CD69+CD49a+CD103-NKG2A+CD16- were pooled in the analysis. k Heatmap of 142 DEGs (padj < 0.01, log2 fold change > 1) with genes ordered by hierarchical clustering. Genes involved in regulation of tissue-residency, homing, and/or NK cell function are highlighted. Two replicates per donor and subsets are shown. Significant DEGs identified using deSeq2. Colour scale depicts z-score. b, d, f, h Kruskal-Wallis test, Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Means ± SD are shown. Source data are provided as a Source Data file

Fig 5: Human lung trNK cells are functional upon stimulation. a Counts per million (CPM) reads for selected genes in human lung CD69-NKG2A+CD16-(dark grey), CD69spNKG2A+CD16- (orange), CD69+CD49a+CD103-NKG2A+CD16-(light blue), and CD69+CD49a+CD103+NKG2A+CD16-(dark blue) NK cells. CD56dimCD16+CD57-NKG2A+ NK cells (light grey) are shown for comparison. Two biological replicates from two donors are shown. Mean ± SEM is shown. b Representative histograms and c summary of data for degranulation and cytokine production of PMA/ionomycin-stimulated lung NK cells. CD49a- and CD49a+ NK cells were gated, and background responses in unstimulated controls were subtracted (CD107a, n = 4; GM-CSF, n = 6; IFN-?, n = 5; MIP-1ß, n = 6; TNF, n = 5). Box and Whiskers, min to max with central line indicating the median and individual data points shown, the mean is indicated as ‘ + ’. Wilcoxon matched-pairs signed rank test, *p < 0.05. Numbers in b indicate percentage of marker-positive NK cells. d Degranulation and cytokine production by sorted CD16- and CD16+ human lung NK cells after PMA/ionomycin stimulation. CD49a and CD103 were used to identify distinct NK cell subsets (n = 3) after stimulation. e Representative overlays and f summary of data for expression of Ki67, perforin, granzyme B, and CCL5 in sorted CD16+, CD16-CD49a-, and CD16-CD49a+ lung NK cells, respectively, following stimulation with IL-15 for 3 days. (n = 4). Mean ± SD is shown. n.a. = not analyzed. The majority of the sorted CD16-CD49a+ NK cells also co-expressed CD103. Numbers in e indicate percentage of marker-positive NK cells. g SPICE analysis for proliferation and effector molecule expression in sorted CD16+, CD16-CD49a-, and CD16-CD49a+ lung NK cell subsets following stimulation with IL-15 for 3 days (n = 3). Floating bars, min-max. Individual data points are shown. Source data are provided as a Source Data file