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: 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 4: Induction of airway tissue TRM following aerosol or i.m. vaccination.(A) Representative dot plots of airway antigen–specific CD4+ and CD8+ TRM at wk2 in LD aerosol participants. (B) Frequencies of airway antigen–specific IFN-?+CD4+ TRM coexpressing CD69 and CD103 surface markers at various time points in LD aerosol, HD aerosol, and i.m. vaccine cohorts. (C) Frequencies of airway antigen–specific IFN-?+CD8+ TRM coexpressing CD69 and CD103 at various time points in LD aerosol, HD aerosol, and i.m. vaccine cohorts. (D) Comparison of frequencies of airway antigen–specific CD4+ and CD8+ TRM coexpressing CD69 and CD103 at 8 weeks after LD and HD aerosol vaccination. (E) Representative dot plots of peripheral blood antigen–specific IFN-?+CD4+ T cells expressing CD49d at wk2 from LD aerosol participants, and frequencies of circulating antigen-specific CD4+ T cells expressing CD49d at various time points in LD aerosol, HD aerosol, and i.m. vaccine cohorts. (F) Representative dot plots of airway antigen–specific IFN-?+CD4+ T cells expressing CD49d at wk2 from LD aerosol participants, and frequencies of airway antigen–specific CD4+ T cells expressing CD49d at various time points in LD aerosol, HD aerosol, and i.m. vaccine cohorts. (G) Comparison of frequencies of airway antigen–specific IFN-?+CD4+ T cells coexpressing CD49d at the peak time point in LD aerosol, HD aerosol, and i.m. vaccine cohorts. Data in dot plots are expressed as the mean value (horizontal line) with 95% CI. Wilcoxon matched pairs signed-rank test (B, C, E, and F) was used to compare various time points with baseline values within the same vaccination group. Mann-Whitney U test (D and G) was used when comparing between vaccination groups.

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)