Fig 2: CD103+ CD8+ intestinal T cells demonstrate greater capacity for cytokine production(A–D) Cytokine production by small intestinal CD8+ T cells. Representative histograms of expression, and group summaries of proportion of CD8+ T cells expressing TNF-a (A), IFN-? (B), CCL4 (C), and IL-2 (D) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10).(E) Mean proportion of CD8+ T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-a, IFN-?, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10).(F) Mean percentage (± SEM) of CD8+ T cells co-expressing TNF-a, IFN-?, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression.Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 3: A CD39neg and GZMKhigh population characterizes CD8+ T cells infiltrating CRC.A Representative contour plot and quantification of CD39neg and CD39pos frequency within CD8+ TEM (n = 34, p < 0.0001). B Representative contour plot and quantification of CD39neg and CD39pos frequency within GZMKhigh CD8+ TEM. (p < 0.0001). C Expression level of indicated markers within GZMKhigh and GZMKlow CD8+ TEM (p = 0.0032 for CD25, p < 0.0001 for CD39, CD69, CD103, CD161, CX3CR1, PD-1, CD28, p = 0.3752 for CD127, p = 0.0012 for GZMß, p = 0.1035 for HLA-DR, p = 0.4775 for TIGIT, p = 0.0362 for CD27. Bars represent mean ± SEM or box and whisker plots indicate Min to Max value, two-tailed paired Wilcoxon test (A, B), two-tailed unpaired multiple t test (C). Source data are provided as a Source Data file.

Fig 4: Treg cell mRNAs are translated in a DAP5/eIF3d cap-dependent manner.a Human naive CD4+ T cells from four different donors were differentiated from PBMCs into Treg cells with TGF-beta and RAD001, then isolated as described in Fig. 2a legend. Equal protein amounts of cell lysates from each donor Treg cell population were pooled and subjected to immunoblot analysis as shown, comparing IL-2 activated but otherwise untreated CD4+ T cells prior to differentiation to their post-differentiated Treg cells. Quantification of individual bands by digital pixel intensity is shown below each blot, normalized to untreated CD4+ T cells. Results shown are representative of two independent studies from different donors. b HEK 293 cells were transfected with non-silencing (Nsi) siRNAs or siRNAs to eIF4E or eIF3d for 72 h, cytoplasmic protein extracts prepared and equal amounts subjected to m7G-Sepharose cap-chromatography. Input, cap-bound, and unretained flow-through proteins were identified by immunoblot analysis. Representative immunoblots are shown of two independent studies from two donors each. c In vitro translation extracts were prepared from 293 T cells22 (see Methods). Renilla luciferase mRNAs containing the 5'-UTR of ITGß1, CD101 or CD103 mRNAs were generated by in vitro transcription with T7 RNA polymerase containing an m7GTP cap structure or non-functional cap analog (ApppG) and polyadenylated. Equal amounts of mRNAs were used to program translation extracts, and relative luciferase activity was determined in four independent experiments. d Translation extracts prepared as above from Nsi or DAP5 silenced 293 T cells and programmed with equal amounts of m7GTP-capped Renilla luciferase mRNAs containing the 5'-UTR of ITGß1, CD101, or CD103 mRNAs. Luciferase activity was determined from three independent experiments, repeated twice showing mean values. Immunoblot of Nsi and DAP5 silenced 293 T cell extracts, representative of two experiments. e 293 T cells were engineered to stably express doxycycline (Dox) inducible non-silencing (Nsi) or eIF3d silencing shRNAs, Dox-induced for 72 h, then cells transfected with expression vectors for HA-tagged WT eIF3d (HA), or cap-binding deficient mutants a5 or a11. Equal protein amounts of cell extracts were subjected to immunoblot. Representative immunoblots shown from two independent studies. f 293 cell translation extracts were prepared and programmed as above with m7GTP-capped Renilla luciferase reporter mRNAs containing the 5'-UTR of ITGß1, CD101 or CD103. Luciferase activity determined in triplicate from two independent studies. IRES-driven Renilla luciferase was used for normalization. g 293 cells were transfected with Nsi, DAP5 or eIF4E siRNAs for 72 h. Equal protein amounts of cell extracts were subjected to immunoblot. Representative immunoblots shown from two independent studies. h 293 cells silenced as above were transfected with plasmids expressing Renilla luciferase reporter mRNAs containing the 5'-UTR of ITGß1, CD101, CD103, FOXP3, or PRICKLE mRNAs. Light intensity values shown for Renilla luciferase activity normalized to Renilla mRNA determined by qRT-PCR. Mean values of triplicate assays of six independent experiments shown with SEM. (c, d, h) P values determined by statistical analysis using the one-way ANOVA test with Tukey’s multiple comparison test determination. Source data are provided as a Source Data file.

Fig 5: CD103+ and CD103- CD4+ T cells display analogous phenotypic and functional differences to their CD8+ counterparts(A) MFI (CD161, CD7, CD127, ß2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4+ T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69-CD103- cells; red, CD69+CD103- cells; black, CD69+CD103+ cells.(B) MFI of ß2-integrin on recipient-derived CD4+ T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars.(C–H) Cytokine production by small intestinal CD4+ T cells. Representative histograms of expression, and group summaries of proportion of CD4+ T cells expressing IL-17A (C), TNF-a (D), IFN-? (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10).(I) Mean proportion of CD4+ T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-a, IFN-?, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10).Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. *p = 0.05, **p = 0.01, ***p = 0.001.