Fig 2: TFH–B–TRM lymphocyte cooperation and their co-localization in lung tumors.a The dot plot summarizing the cell–cell interactions among CD8-C2 (TRM-like), B, and CD4-C9 (TFH-like) cells by CellPhoneDB analysis. Only significant ligand–receptor interactions are presented here. The size of the circle indicates the statistical significance, and the color gradient indicates the mean expression value of gene pairs. b Representative multiplexed immunofluorescence (IF) of TLS-like lesion in EGFR-WT (n = 14) (upper) (scale bar 100 μm: left, scale bars 50 μm: right magnified) and EGFR-MT (n = 14) (lower) (scale bar 100 μm: left, scale bars 50 μm: right magnified) tumors. Summarized graph for the area of TLS-like lesions in EGFR-WT (n = 5) and EGFR-MT (n = 4) tumors for single-cell analysis (**p ≤ 0.005, two-sided test) (upper panel) and in EGFR-WT (n = 9) and EGFR-MT (n = 10) tumors from independent patients for validation (*p ≤ 0.05, two-sided test) (lower panel). c Representative multiplexed IF of CD4, CXCL13, CD103, CD8, CXCR5, and CD20 in tumor specimens from EGFR-WT (n = 14) (left) (scale bar 100 μm) and EGFR-MT (n = 14) (right) (scale bar 100 μm) patients. d Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT (n = 5) and EGFR-MT (n = 4) patients. The number of CD4+, CXCL13+CD4+ T cells, CD8+ T cells, CD103+ CD8+ T cells are significantly higher in EGFR-WT than EGFR-MT (*p ≤ 0.05, **p ≤ 0.005, two-sided test). e Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT (n = 9) and EGFR-MT (n = 10) tumors from independent patients for validation. Consistently, the number of CD4+, CXCL13+CD4+ T cells, CD8+ T cells, CD103+ CD8+ T cells were significantly higher in EGFR-WT than EGFR-MT (**p ≤ 0.005, ***p ≤ 0.0005, two-sided test). f An illustrative summary of the proposed model for TFH–B–TRM lymphocyte cooperation in TLS for antitumor immunity in lung cancer. Data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig 3: DC3s Prime Naive T Cells and Drive Acquisition of the CD103+ TRM Phenotype(A and B) Representative flow cytometry plots and quantification of CD4+ and CD8+ naive T cells cultured for 5 days with flow cytometry-sorted blood cDC2s, DC3s, or CD14+ monocytes after overnight activation with TLR agonists (25 µg/mL poly(I:C), 1 µg/mL R848, and 10 ng/mL LPS) in the presence of a synthetic superantigen (Cytostim). Absolute numbers and frequencies of cytokine-producing and other activated T cells (A) and CD103+ T cells (B) are shown (n = 5 healthy donors in 5 independent experiments; a line represents the median; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test).(C) Representative flow cytometry plots and quantification showing CD103 expression on CD8+ naive T cells cocultured with blood DC3s sorted by fluorescence-activated cell sorting (FACS) in the presence of 10 µg/mL of neutralizing antibodies against TNF-a or TGF-ß or an isotype CTRL (n = 4 healthy donors in 3 independent experiments; a line represents the median; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way paired ANOVA test).(D and E) Bulk RNA-seq analysis of CD8+CD103- T cells (n = 3) and CD8+CD103+ T cells (n = 3) sorted by flow cytometry after 5 days in vitro coculture of naive blood CD8+ T cells with blood DC3s activated overnight by TLR agonists.(D) GSEA of pairwise comparisons of CD8+CD103+ T cells with CD8+CD103- T cells. Gene signatures (gene set) defining genes upregulated in breast or lung CD103+CD69+CD8+ TRM cells were used (Hombrink et al., 2016; Kumar et al., 2017; Savas et al., 2018).(E) Heatmap displaying 56 representative genes significantly upregulated in CD8+CD103+ cells compared with CD8+CD103- induced by blood DC3s in vitro (of 205 DEGs). Selected genes are shared with at least one of the previously reported gene signatures defining human TRM cells (Hombrink et al., 2016; Kumar et al., 2017; Savas et al., 2018).(F–H) Correlative analysis of TRM cell content in luminal breast cancer primary tumors.(F) Representative flow cytometry plots showing the gating strategy for CD103+CD69+CD8+ T cells in 21 human luminal breast cancer primary tumors.(G) Quantification of CD103+CD69+CD8+ T cells in different stages of human breast tumors (stage I, n = 3; stage II, n = 13; stage III, n = 5).(H) Pearson correlation of the frequencies of the macrophages and cDC1, CD1c+ CD14-, and CD1c+ CD14- cells and the frequencies of CD103+CD69+CD8+ T cells in human breast cancer primary tumors (red, significantly correlated p < 0.05; black, not correlated).See also Figure S7 and Tables S1 and S2.

Fig 4: Induction of NKG2A by TCM is mediated by a small and heat-stable factor and is pH-independent.(A and B) Percentage of (A) NKG2A+ and (B) CD103+ of CD8+ T cells derived from PBMCs and stimulated with CD3/CD28 cultured for 5 d in cRPMI, TCM, or boiled TCM from Detroit 562 or Cal27 cells. (C and D) Percentage of (C) NKG2A+ and (D) CD103+ of CD8+ T cells derived from PBMCs and stimulated with CD3/CD28 cultured for 5 d in cRPMI, TCM, or filtered TCM from Detroit 562 or Cal27 cells. (E and F) Percentage of (E) NKG2A+ and (F) CD103+ of CD8+ T cells derived from PBMCs and stimulated with CD3/CD28 cultured for 5 d in cRPMI, TCM, or pH-neutralized TCM (TCM + KOH) from Detroit 562 or Cal27 cells. Data were quantified by FACS. Results are shown as mean ± SD. In (A)–(F), data were analyzed using one-way ANOVA with a Tukey multiple comparison test. In (G) and (H), the ratio is shown of the relative mRNA expression of (G) KLRC1 (gene for NKG2A) and (H) ITGAE (gene for CD103) normalized to T cells cultured in cRPMI. Data were quantified by quantitative PCR. Results are shown as mean ± SD. Statistical analyses were performed on technical replicates for each independent experiment, and each experiment was performed three independent times. *p < 0.05, **p < 0.01, ****p < 0.001, ****p < 0.0001.

Fig 5: Cancer cell secretome induces expression of NKG2A, exhaustion markers, and tissue-resident memory markers on CD8+ T cells.(A and B) Frequency of NKG2A+ and the NKG2A expression level. (C–H) Frequencies of exhaustion markers (C and D) PD-1+ and (E and F) CD39+, and the frequency of tissue-resident memory marker (G and H) CD103+ of CD3/CD28-stimulated CD8+ T cells derived from PBMCs and cultured for 5 d at different ratios of cRPMI to TCM. Data were quantified by FACS. Results are shown as mean ± SD. Data were analyzed using one-way ANOVA with a Dunnett multiple comparison test. Statistical analyses were performed on technical replicates for each independent experiment, and each experiment was performed three independent times. **p < 0.01, ****p < 0.001, ****p < 0.0001.