Fig 2: Functional investigation of differentially expressed genes in ?d[2] versus ?d[T2] cells(A) IL-9 was measured in supernatants harvested on day 15 from ?d[2] and ?d[T2] cells (values for individual donors are linked by a line; unpaired 2-tailed Student’s t test).(B) IL-9 receptor expression was quantified on ?d[2] and ?d[T2] cells on day 15 using flow cytometry (unpaired 2-tailed Student’s t test).(C) PTGER2 protein expression in ?d[2] and ?d[T2] cells (n = 3 independent donors) was determined by western blotting under reducing conditions. Quantification of band pixel density was performed using ImageJ. Data were normalized for each ?d[T2] cell sample with respect to ?d[2] cells expanded from the same donor.(D) CD300a was quantified on day 15 from ?d[2] and ?d[T2] cells using flow cytometry (unpaired Student’s t test).(E) The indicated ffLuc-expressing tumor cell lines were co-cultivated with ?d[2] and ?d[2-ITGAE] cells for 24 h at 1:1 E:T ratio. Tumor cell viability is shown in comparison with tumor cells that had been cultured alone (means ± SDs; 1-way ANOVA).(F) Supernatants were collected from co-cultures described in (E) after 72 h and analyzed for IFN-? (means ± SDs; unpaired Student’s t test).(G) Effect of exogenous PGE2 on cytotoxicity (means ± SEMs, n = 10 from 5 independent donors) by ?d[2] and ?d[T2] cells when co-cultivated with U937 and KG-1 cell lines for 72 h at 1:1 E:T ratio. Data were normalized with respect to cytotoxicity by ?d[2] and ?d[T2] cells in the absence of added PGE2 (2-way ANOVA). NS, not significant.(H) Effect of exogenous PGE2 on IFN-? production (means ± SEMs, n = 8 from 4 independent donors) by co-cultures described in (G). Data were normalized with respect to IFN-? production by ?d[2] and ?d[T2] cells in the absence of added PGE2 (2-way ANOVA).“All” in (E) and (F) refers to pooled data from solid tumor and leukemic cell lines, respectively.

Fig 3: M1hot tumor-associatedmacrophages (TAMs) support tissue-resident memory T cells (TRM) cell maintenance by allowing uptake of fatty acids. (A) Frequency of CD103high, CD103intermediate and CD103low tumors among those categorized by chemokine (C-X-C motif) ligand 9 (CXCL9) expression in TAMs (M1hot, M1intermediate, M1cold; numbers of patients above each bar). (B) Heatmap showing expression of genes related to Tissue Resident Memory features (TRM phenotype) in CD8+ T cells isolated from the same tumors from patients with non-small cell lungcancer (NSCLC) were M1hot TAMs and M1cold TAMs were isolated and studied. (C) Gene set enrichment analysis (GSEA) of 246 gene sets in the transcriptome of CD8+ T cells from patients with NSCLC with M1hot TAMs versus those with M1cold TAMs, presented as the running enrichment score. Statistical significance by Kolmogorov-Smirnov test. (D) Expression of FABP3, FABP4 and FABP5 in non-tumor-associatedmacrophages (NTAMs), M1hot TAMs and M1cold TAMs (log2 normalized counts). Statistical significance by ordinary one-way analysis of variance. (E) Macrophages derived from blood (BDM) treated with interfern (INF)-?/lipopolysaccharides (LPS) (M1hot-like BDM, red) or interleukin (IL)-4 (M1cold-like BDM, blue) cultured with bodipy lipid probe for 30 min (n=7). Lipid uptake measured by flow cytometry and expressed as mean fluorescence intensity. Wilcoxon test, two-tailed. *P=0.05 (F) M1hot-like BDM (red) or M1cold-like BDM (blue) were co-cultured with CD3+ cells and bodipy lipid probe for 30 min (n=6). Uptake of lipid represented as Mean fluorescence intensities (MFI) in TRM (CD8+CD103+), CD8+ (CD8+CD103-) and CD3+ (CD8-CD103-) cells was determined by flow cytometry. Wilcoxon test, two-tailed. *P=0.05. ns, non-significant for p value >0.05, *p=0.05, **p=0.01 and ****p=0.00001. Related to online supplementary figure S6 and tables S5, S6.

Fig 4: Expansion and characterization of ?d[T2] cells(A) Healthy donor PBMCs were activated with zoledronic acid (ZOL, n = 27) or immobilized anti-?d TCR antibody (n = 15) and then cultured for 14–17 days in SFM containing IL-2 alone (?d[2] cells) or IL-2 + TGF-ß (?d[T2] cells). The percentage of ?d T cells was measured in PBMCs (day 1) and after expansion in the indicated cytokines.(B) Absolute number of ?d T cells on day 1 and post-expansion. Median fold expansion is indicated for ?d[2] and ?d[T2] cells.(C–I) Post-?d T cell expansion, the following markers were assessed in ?d[2] and ?d[T2] cells by flow cytometry: (C) % CLA; (D) % CXCR4; (E) binding to E-selectin-Fc fusion protein; (F) state of differentiation: naive (CD45RA+ CD27+), central memory (CM; CD45RA- CD27+), effector memory (CD45RA- CD27-), and terminally differentiated with CD45RA re-expression (TEMRA; CD45RA+ CD27-; (G) viability, apoptosis, and necrosis; (H) % CD103; and (I) % KLRG1.(J) ?d[T2] T cells and ?d T cells expanded from the same donor in human serum + IL-2 (?d[2S]) were engineered to co-express firefly luciferase (ffLuc) and red fluorescent protein (RFP) and then analyzed by flow cytometry. GFP-expressing Jurkat cells were injected i.v. in NSG mice. After 4 days, 6 mice each were treated with 10 million RFP/ffLuc-expressing ?d[2S] or ?d[T2] T cells. Untrans., untransduced.(K) Mice were analyzed by BLI after 24 and 48 h to determine persistence of ?d T cells in the whole body and in a region of interest drawn around the femora (means ± SDs; n = 6 at 24 h; n = 3 at 48 h; 2-way ANOVA).(L) After each imaging session, 3 mice per group were culled. The % RFP+ (?d) T cells present in spleen and bone marrow were determined (means ± SDs).(M) The % GFP+ (leukemic) cells present in bone marrow was also determined (means ± SDs). NS, not significant.(C)–(E) and (G)–(I) show means ± SDs and (A), (B), and (F) show medians ± interquartile ranges, in which data were or were not normally distributed, respectively. Accordingly, statistical analysis was performed using a Student’s t test or Wilcoxon signed-rank test, respectively.

Fig 5: CAF-induced CD39 expression can be inhibited by neutralizing IFN-? and TNF-a or by inhibition of TGF-ß signaling a) Expression of CD39, LAG-3, PD-1 and Tim3 on CD8 + T cells activated either alone (PBMC-only) or in co-culture with CAF (+CAF) in the presence of neutralizing antibody together IL-6 or IL-27. b) Expression of CD39, CD49b, Tim3, PD-1 LAG3 and CD103 on activated CD8+ T cells either cultured alone (media) or in co-culture with CAF (+CAF) and neutralizing antibodies to IFN-? and TNF-a, anti-PD-L1, anti-PD-1, SB431542, SB431542+ anti-PD-1 or isotype control antibody as indicated. Error bars show standard deviation of triplicate wells. Significance shown for each parameter when compared to +CAF. Results for SB431542 are representative of those from three repeat experiments using different PBMCs. For PD-1, NA where PD-1 blockage used with PD-1 surface levels. One way ANOVA was used for all statistical analysis with Tukey’s multiple comparisons posttest (ns = not significant,* P = <0.05, ** P = <0.01, *** P = <0.001, ****P = <0.0001)