Functional Heterogeneity of CD4+ Tumor-Infiltrating Lymphocytes With a Resident Memory Phenotype in NSCLC

Abstract

Resident memory T cells (T_RM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103⁺ T_RM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103⁺CD8⁺ tumor infiltrating lymphocytes (TILs), with T_RM properties, are a positive prognostic marker. To better understand the role of T_RM in tumors, we performed a detailed characterization of CD8⁺ and CD4⁺ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8⁺ and CD4⁺ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in T_RM ratios compared to surrounding lung tissue. The majority of both CD4⁺ and CD8⁺ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of T_RM. While CD103⁺CD8⁺ T cells were enriched in tumors, CD103⁺CD4⁺ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103⁺CD4⁺ and CD103⁺CD8⁺ TILs showed multiple characteristics of T_RM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8⁺ and CD4⁺ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103⁺ TILs. Strikingly, CD103⁺CD4⁺ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103⁺CD4⁺PD-1^low TILs produced the most effector cytokines, CD103⁺CD4⁺PD-1⁺⁺ and CD69⁺CD4⁺PD-1⁺⁺ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung T_RM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103⁺CD4⁺ and CD69⁺CD8⁺ TILs. Our findings thus provide a rationale to target CD103⁺CD4⁺ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

Keywords: NSCLC; TILs; TRM; co-stimulation; cytokines; differentiation; exhaustion.

Figure 1

Distribution of CD103 and CD69 expression on CD4⁺ and CD8⁺ T cells of paired blood, lung, and tumor samples. (A) Frequencies of CD4⁺ (black circles) and CD8⁺ (gray circles) cells of total CD3⁺ T cells of paired blood, lung, and tumor tissue was analyzed by flow cytometry. (B–D) The expression of CD69 and CD103 was analyzed on paired blood, lung and, tumor CD4⁺ and CD8⁺ T cells. (B) Contour plots show representative examples of CD69 and CD103 expression on blood (left panels), lung (middle panels), and tumor (right panels) CD4⁺ (top panels) and CD8⁺ (bottom panels) T cells. (C,D) Frequencies of CD103⁺CD69⁺ (black circles), CD103⁻CD69⁺ (dark gray circles), and CD103⁻CD69⁻(light gray circles) cells of total blood, lung, and tumor CD4⁺ (C) and CD8⁺ (D) T cells were quantified. (E,F) Correlation between CD103⁺CD8⁺ and CD103⁺CD4⁺ lung (E) and tumor (F) T cells was determined. (A–F) n = 33. Open circles, solid circles, solid square indicate adeno-, squamous, and large cell carcinoma, respectively. (A,C,D) Quantifications are shown as dot plots with the horizontal line indicating the mean and each point represents a unique sample. (E,F) Correlation shown as X-Y graph where each point represents a unique sample. (C,D) ^***p < 0.001, ^****p < 0.0001; 2-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. (E,F) r, Pearson's rank coefficient; p < 0.05.

Figure 2

Differentiation status of lung T_RM and TILs. (A–D) The expression of CD45RA, CD27, and CD28 on CD4⁺ and CD8⁺ lung T_RM and TILs was determined. (A,B) The expression of CD27, CD45RA, and CD28 tumor CD103⁺ (top panels), CD69⁺ (middle panels), and CD69⁻ (bottom panels) CD4⁺ (A) and CD8⁺ (B) T cells shown by representative contour plots (CD45RA on y-axis, CD27 on x-axis) and histograms overlays (maximum set to 100%) show the expression of CD28 on the different subsets (black CD27⁻CD45RA⁻, gray CD27⁺CD45RA⁻, blue CD27⁻CD45RA⁺, purple CD27⁺CD45RA⁺). (C,D) The frequencies of CD27⁺CD45RA⁺CD28⁺ (light purple), CD27⁺CD45RA⁺CD28⁻ (medium purple), CD27⁺CD45RA⁻CD28⁺ (dark purple), CD27⁺CD45RA⁻CD28⁻ (light gray), CD27⁻CD45RA⁻CD28⁺ (medium gray), CD27⁻CD45RA⁻CD28⁻ (black), CD27⁻CD45RA⁺CD28⁺ (light blue), CD27⁻CD45RA⁺CD28⁻ (dark blue) of CD103⁺, CD69⁺, and CD69⁻ lung CD4⁺ (C; left bar graph), tumor CD4⁺ (C; right bar graph), lung CD8⁺ (D; left bar graph), and tumor CD8⁺ (D; right bar graph). (C,D) The quantifications are shown as bar graphs with the mean. n = 15.

Figure 3

Expression of T_RM homing molecules by TILs. (A-D) The expression of chemokine receptor CXCR6 and integrin CD49a were analyzed on CD4⁺ and CD8⁺ T_RM and TILs. The expression of CXCR6 (top panels) and CD49a (bottom panels) on lung (left panels) and tumor (right panels) CD4⁺ (A) and CD8⁺ (B) T cell subsets is shown by representative histogram overlays (maximum set to 100%) (CD103⁺ black, CD69⁺ dark gray, CD69⁻ solid light gray). The frequencies of CXCR6⁺ (C) and CD49a⁺ (D) CD103⁺ (black circles), CD69⁺ (dark gray circles), and CD69⁻ (light gray circles) cells of lung and tumor CD4⁺ T cells (left graphs) and CD8⁺ T cells (right graphs). (C,D) The quantifications are shown as dot plots with the horizontal line indicating the mean and each point represents a unique sample. n = 15–17. Open circles and solid circles indicate adeno- and squamous carcinoma, respectively. ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001; 2-way ANOVA with Tukey's multiple comparisons test.

Figure 4

Expression of T-bet, Eomes and GZMB on T_RM and TILs. (A–D) The expression of T-bet, Eomes, and GZMB was analyzed on CD4⁺ and CD8⁺ T_RM and TILs. The expression of T-bet (top panels), Eomes (middle panels), and GZMB (bottom panels) on lung (left panels) and tumor (right panels) CD4⁺ (A) and CD8⁺ (B) T cells is shown by representative histogram overlays (maximum set to 100%) (CD103⁺ black, CD69⁺ dark gray, CD69⁻ solid light gray). The expression of T-bet (geometric mean fluorescence intensity; GeoMFI) (C) and frequencies of Eomes⁺ (D) and GZMB⁺ (E) CD103⁺ (black circles), CD69⁺ (dark gray circles), and CD69⁻ (light gray circles) cells of lung and tumor CD4⁺ (left graphs) and CD8⁺ (right graphs) T cells. (C–E) The quantifications are shown as dot plots with the horizontal line indicating the mean and each point represents a unique sample. n = 17. Open circles and solid circles indicate adeno- and squamous carcinoma, respectively. ^**p < 0.01, ^****p < 0.0001; 2-way ANOVA with Tukey's multiple comparisons test.

Figure 5

CD103⁺ TILs express the highest levels of inhibitory receptors. (A–I) The expression of inhibitory receptors PD-1, CTLA-4, and 2B4 was analyzed on CD4⁺ and CD8⁺ T_RM and TILs. The expression of PD-1 (A), CTLA-4 (D), and 2B4 (G) on lung (left panel) and tumor (right panel) on CD4⁺ (top panel) and CD8⁺ (bottom panel) T cells is shown by representative histogram overlays (maximum set to 100%) (CD103⁺ black, CD69⁺ dark gray, CD69⁻ solid light gray). The frequencies and geoMFI of PD-1⁺ (B,C), CTLA-4⁺ (E,F) and 2B4⁺ (H,I) were quantified for CD103⁺ (black circles), CD69⁺ (dark gray circles), and CD69⁻ (light gray circles) cells of lung and tumor CD4⁺ (left graphs) and CD8⁺ (right graphs) T cells. (B,C,E,F,H,I) The quantifications are shown as dot plots with the horizontal line indicating the mean and each point represents a unique sample. n = 17. Open circles and solid circles indicate adeno- and squamous carcinoma, respectively. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; 2-way ANOVA with Tukey's multiple comparisons test.

Figure 6

Cytokine and chemokine production of T_RM and TILs. (A–C) Cytokine production by lung T_RM and TILs was determined after overnight αCD3/αCD28 stimulation. (A) The production of TNF-α and IFN-γ by CD103⁺ (top panels), CD69⁺ (middle panels), and CD69⁻ (bottom panels) tumor CD4⁺ (left panels) and CD8⁺ (right panels) TILs shown by representative contour plots (TNF-α on y-axis, IFN-γ on x-axis). (B) TNF-α⁺IFN-γ⁺ CD103⁺, CD69⁺, and CD69⁻ cells of lung and tumor CD4⁺ (top graph) and CD8⁺ (bottom graph) T cells. (C) PD-1 expression (geometric mean fluorescence intensity; GeoMFI) was quantified on cytokine⁺ (TNF-α⁺ and/or IFN-γ⁺) (black circles) and cytokine⁻ (TNF-α⁻IFN-γ⁻) (gray circles) CD103⁺, CD69⁺, and CD69⁻ lung and tumor CD4⁺ (top graph) and CD8⁺ (bottom graph) T cells. (D) The expression of CXCL13 was determined by flow cytometry in CD103⁺ (top panels), CD69⁺ (middle panels), and CD69⁻ (bottom panels) tumor CD4⁺ (left panels) and CD8⁺ (right panels) TILs and is shown by representative contour plots (PD-1 on y-axis, CXCL13 on x-axis). (E) CXCL13⁺ of CD103⁺, CD69⁺, and CD69⁻ cells was quantified in lung and tumor CD4⁺ (top graph) and CD8⁺ (bottom graph) T cells. (F) PD-1 expression (GeoMFI) was quantified on the CXCL13⁺ (black circles) and CXCL13⁻ (gray circles) CD103⁺, CD69⁺, and CD69⁻ CD4⁺ TILs. n = 4–6 paired lung-tumor samples; all adenocarcinoma. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001; 2-way ANOVA with Tukey's multiple comparisons test.

Figure 7

Co-stimulation enhances cytokine production of TILs. (A–F) The effect of co-stimulation on cytokine production of TILs was assessed with overnight stimulation. (A–C) The frequencies of IFN-γ⁺ (A), TNFα⁺ (B), or TNF-α⁺IFN-γ⁺ (C) CD103⁺, CD69⁺, and CD69⁻ CD4⁺ TILs stimulated with αCD3 with control cell line (CL) (black circles), αCD3 and αCD28 with control CL (black squares), or αCD3 and αCD28 with CD70-expressing CL (black triangles) was quantified. (D–F) The frequencies of IFN-γ⁺ (D), TNFα⁺ (E), or TNF-α⁺IFN-γ⁺ (F) CD103⁺, CD69⁺, and CD69⁻ CD8⁺ TILs stimulated with αCD3 with control CL (black circles), αCD3 and αCD28 with control CL (black squares), or αCD3 and αCD28 with CD70-expressing CL (black triangles) was quantified. n = 5. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001; 2-way ANOVA with Tukey's multiple comparisons test.