Intratumoral immune triads are required for immunotherapy-mediated elimination of solid tumors

Abstract

Tumor-specific CD8⁺ T cells are frequently dysfunctional and unable to halt tumor growth. We investigated whether tumor-specific CD4⁺ T cells can be enlisted to overcome CD8⁺ T cell dysfunction within tumors. We find that the spatial positioning and interactions of CD8⁺ and CD4⁺ T cells, but not their numbers, dictate anti-tumor responses in the context of adoptive T cell therapy as well as immune checkpoint blockade (ICB): CD4⁺ T cells must engage with CD8⁺ T cells on the same dendritic cell during the effector phase, forming a three-cell-type cluster (triad) to license CD8⁺ T cell cytotoxicity and cancer cell elimination. When intratumoral triad formation is disrupted, tumors progress despite equal numbers of tumor-specific CD8⁺ and CD4⁺ T cells. In patients with pleural mesothelioma treated with ICB, triads are associated with clinical responses. Thus, CD4⁺ T cells and triads are required for CD8⁺ T cell cytotoxicity during the effector phase and tumor elimination.

Keywords: CD4 T cell; CD8 T cell; Cancer; adoptive T cell transfer; cancer immunotherapy; dendritic cell; immune checkpoint blockade; triad; tumor; tumor antigen.

Figure 1.. Tumor-specific CD4⁺ T cells prevent and reverse CD8⁺ T cell dysfunction within solid tumors and mediate tumor elimination

(A) Scheme: tumor models, adoptively transferred effector T cells, and experimental schemes. (B) B16 OVA-GP_61–80 (B16-OG) tumor growth (right) and Kaplan-Meier survival curve (left) of tumor-bearing B6 WT mice (CD45.2; Thy1.2) receiving effector TCR_OTI CD8⁺ T cells alone (CD45.1) (black; TCR_OT1) or together with TCR_SMARTA CD4⁺ T cells (Thy1.1) (red; TCR_OT1^(+CD4)) (ACT = adoptive T cell transfer). Data are representative of 5 independent experiments (n = 5 mice/group). Data are represented as mean ± SEM. Significance is calculated by two-way ANOVA with Bonferroni correction (**p ≤ 0.0001). Kaplan-Meier curve; **p = 0.00021; Mantel-Cox test. (C) MCA205 OVA-GP_61–80 (MCA-OG) tumor outgrowth (significance is calculated by two-way ANOVA with Bonferroni correction (**p ≤ 0.0001), and survival in B6 mice treated as described in (B); **p = 0.0003; Mantel-Cox test. Data are representative of 2 independent experiments (n = 5–6 mice/group). (D) TCR_OT1 (% of total of CD8⁺ T cells) within progressing B16-OG tumors 8–9 days post transfer +/− TCR_SMARTA CD4⁺ T cells. Data pooled from 2 independent experiments (n = 8 mice/group). Each symbol represents an individual mouse. (E) IFNγ and TNFα production of TCR_OT1 isolated from B16-OG tumors 8–9 days post transfer +/− TCR_SMARTA CD4⁺ T cells. Cytokine production was assessed after 4-h peptide stimulation ex vivo. Gates are set based on negative controls (no stimulation or non-antigen-specific T cell population controls). Data show 2 pooled independent experiments (n = 5–7); representative flow plots are shown. (F and G) Inhibitory receptor expression, and (G) TOX expression of B16-OG tumor-infiltrating TCR_OT1 isolated 8–9 days post transfer +/− TCR_SMARTA. Graphs depict relative MFI normalized to naive TCR_OT1 (N); two pooled independent experiments (n = 5–7 mice/group). (D–G) Values are mean ± SD. ***p < 0.0001; **p < 0.001; *p < 0.05. (H) Mice with B16-OG tumors received effector TCR_OTI CD8⁺ T cells 14 days post tumor transplantation; 8 days later, TCR_SMARTA CD4⁺ T cells were adoptively transferred (red); B16-OG tumor growth in mice receiving only TCR_OT1 are shown in black. Data are representative of 2 independent experiments (n = 8 mice/group). Values are mean ± SEM. Significance is calculated by two-way ANOVA with Bonferroni correction (**p ≤ 0.0001). (I) IFNγ and TNFα production of TCR_OT1 isolated from B16-OG tumors which did receive TCR_SMARTA CD4⁺ T cells 6 days later (red); TCR_OT1 which did not receive TCR_SMARTA CD4⁺ T cells are shown in black. Data are representative of 2 experiments. Values are mean ± SEM; *p = 0.04. See also Figure S1.

Figure 2.. Tumor-specific CD4⁺ T cells transcriptionally and epigenetically reprogram tumor-specific CD8⁺ T cells and prevent terminal differentiation

(A) MA plot (scatterplot of log2 fold changes (M, on the y axis) versus the average expression signal (A, on the x axis)) of RNA-seq data showing the relationship between average expression and expression changes of TCR_OT1 and TCR_OT1^(+CD4) TIL. Statistically significantly DEG (false discovery rate (FDR) < 0.05) is shown in red and blue, with select genes highlighted for reference. (B) Heatmap of RNA-seq expression (normalized counts after variance stabilizing transformation, centered and scaled by row for DEG) (FDR <0.05) in TCR_OT1 and TCR_OT1^(+CD4) TIL. (C) Selected GO terms enriched for genes upregulated in TCR_OT1 (blue) and TCR_OT1^(+CD4) (red) TIL. (D) Chromatin accessibility (ATAC-seq); (left) heatmap of log2-transformed normalized read counts transformed with variance stabilization per for regions with differential chromatin accessibility; (right) each row represents one peak (differentially accessible between TCR_OT1 and TCR_OT1^(+CD4) TIL; FDR <0.05) displayed over a 2-kb window centered on the peak summit; regions were clustered with k-means clustering. Genes associated with the two major clusters are highlighted. (E) ATAC-seq signal profiles across the Tox, Pdcd1, Lag3, Tcf7, and Lef1 loci. Peaks significantly lost or gained are highlighted in red or blue, respectively. (F) Top 10 most-significantly enriched transcription factor motifs in peaks with increased accessibility in TCR_OT1^(+CD4) TIL (red) or TCR_OT1 TIL (blue). (G) Enrichment of gene sets in TCR_OT1^(+CD4), described for human tumor infiltrating CD8⁺ TIL; (CD69⁻ CD39⁻) stem-like TIL (responders) or (CD69⁺ CD39⁺) terminally differentiated TIL (non-responders) from metastatic melanoma patients receiving ex vivo expanded TIL for ACT. NES, normalized enrichment score. See also Figure S2.

Figure 3.. Tumor elimination requires unique spatial orientation of tumor-specific CD8⁺ T cells, tumor-specific CD4⁺ T cells, and CD11c⁺ dendritic cells (DC) within tumors

(A) B16-OG tumor outgrowth in CD11c-DTR/GFP bone marrow (BM) chimeras (scheme, top; DTR→WT or WT→WT) treated with diphtheria toxin (DT). In vitro activated TCR_OTI and TCR_SMARTA were adoptively transferred into lymphodepleted tumor-bearing BM chimeras. 5 days post ACT, mice were treated with DT. Representative of 2 independent experiments (n = 3 mice/group). Values are mean ± SEM. Significance is calculated by two-way ANOVA with Bonferroni correction (p ≤ 0.0001). (B) (Top) Experimental scheme of tumor models A and B: 2.5 × 10⁶ B16-OG cancer cells (B16-OG; model A) or 1.25 × 10⁶ B16-OVA (B16-O) mixed with 1.25 × 10⁶ B16-GP_61–80 cancer cells (B16 O+G; model B) were transplanted into B6 WT mice. (Bottom), Tumor outgrowth of B16-OG or B16 O+G tumors after TCR_OTI and TCR_SMARTA ACT. Kaplan-Meier survival curve. Representative of 2 independent experiments (n = 7 mice/cohort). Data are shown as mean ± SEM. Significance is calculated by two-way ANOVA with Bonferroni correction (p ≤ 0.0001). (Right) Kaplan-Meier curve; **p = 0.0002; Mantel-Cox test. (C) Percentage of TCR_OT1^(+CD4) (out of total CD8⁺ TIL) 9 days post ACT. (D) Percentage of TCR_SMARTA (out of total CD4⁺ TIL) 9 days post ACT. Data represent 2 pooled, independent experiments (n = 8 mice/tumor model). Each symbol represents an individual mouse. (E and F) IFNγ, TNFα, CD107, granzyme B production of TCR_OT1^(+CD4) isolated from B16-OG or B16 O+G tumors, or (F) isolated from tumor-draining lymph nodes of B16-OG or B16 O+G tumor-bearing hosts. Cytotoxic molecules and cytokine production assessed after 4-h peptide stimulation ex vivo. Representative of 2 independent experiments (n = 3 mice/tumor). Data are shown as mean ± SEM. ***p < 0.0005, **p < 0.005, *p < 0.05, unpaired two-tailed Student’s t test. ns, not significant. See also Figures S3 and S4.

Figure 4.. Intratumoral immune triads (three-cell-type clusters; CD4⁺ T cell::CD8⁺ T cell::DC) are required for CD8⁺ T cell reprogramming, cytotoxicity, and tumor elimination

(A) Proposed model: Triad formation (three-cell-type clusters; CD4⁺ T cells::CD8⁺ T cells::DC) form in B16-OG tumors (model A) where CD8 and CD4 tumor antigens/epitopes are linked and co-presented on the same APC within tumors; tumor-specific CD8⁺ and CD4⁺ T cells engage on same DC/APC; CD4⁺ T cells reprogram CD8⁺ T cells. Model B: B16 O+G; triads cannot form because CD8 and CD4 tumor antigens are presented on distinct APC. (B) Color-coded mouse models to determine intratumoral immune triad formation (models A and B). B16-OG (model A) or B16 O+G (model B) tumors were established in CD11c-YFP mice (yellow); effector TCR_OT1-RFP (red) and TCR_SMARTA-EGFP T cells (green) were adoptively transferred into tumor-bearing hosts. Confocal microscopy analysis of frozen tumor tissue sections (tumors were analyzed 8–9 days post T cell transfer). Arrows indicate cell-cell interactions within B16-OG tumors. Scale bars indicate 50 μm (top); 10 μm (bottom). (C and D) Numbers of triads per field of view (FOV), and (D) (left) Fold increase of triads normalized to total numbers of CD11c-YFP⁺ cells/FOV (right). (E) Quantification of fold increase of numbers of CD4⁺ T cell-DC dyads normalized to total number of infiltrating CD11c-YFP⁺ cells/FOV. Each symbol represents an individual frozen tumor section (n = 3 mice/group/model). Data are shown as mean ± SEM. ***p < 0.0001, **p < 0.001, unpaired two-tailed Student’s t test. (F) Growth of B16-OG tumors (solid lines; right) and contralateral B16-O tumors (dashed lines; left) in B6 WT mice (n = 6) receiving adoptively transferred in vitro activated TCR_OT1 and TCR_SMARTA T cells 14 days post tumor transplantation. Data are representative of 3 independent experiments. (G) Outgrowth of B16-OG (green) and B16 O+G (red) tumors in B6 WT mice receiving anti-PD-1 and anti-PD-L1 blocking monoclonal antibodies (mAb) at indicated time points (days 12, 14, 16, 18, 20, and 22 post tumor transplantation; black arrows). Data are representative of 2 independent experiments. (Right) Tumor volume at day 22 post tumor implantation following ICB. Data are shown as mean ± SEM. *p = 0.02, using unpaired two-tailed Student’s t test. See also Figure S4.

Figure 5.. Intratumoral triads are associated with pathologic responses to immune checkpoint blockade in patients with malignant pleural mesothelioma (MPM)

(A) Treatment regimen and methodology used to determine triads (CD4⁺ T cell::CD8⁺ T cell::APC) and dyads (CD4⁺::APC). Pipeline of co-localization detection by multiplexed imaging mass cytometry (IMC; see STAR Methods for more details). Briefly, FFPE tumor tissues were stained with 35 target-specific antibodies. Automated cluster detection estimated cluster boundaries by expanding the perimeter of nuclei, identified by Cell ID Intercalator-iridium (191Ir). IMC images were quantified through FIJI, and protein expression data extracted through mean intensity multiparametric measurements performed on individual clusters. Acquired cluster data were normalized with CytoNorm tools, and normalized cytometric data transferred into additional Spanning-tree Progression Analysis of Density-normalized Events (SPADE) to generate automated clustering algorithm and applied cytometric analysis in FlowJo. (B) Representative multiplexed mass cytometry images of triads and dyads. Scale bars indicate 50 μm. (C) Fold change of triads and dyads of pre- and post-immune checkpoint therapy density (numbers/mm²) in pathologic responders (R) and non-responders (NR). In box and whisker plots, the box spans from the lower quartile to the upper quartile, encompassing the interquartile range; a horizontal line within the box marks the median value. The whiskers extend from the box to the minimum and maximum values, showing the overall range. *p = 0.02; p = 0.34 (ns, not significant). (D) Phenotypic analysis of intratumoral CD8⁺ T Cells using IMC. Expression of CD45RO, PD-1, IFNγ, and granzyme B (GZMB) on single CD8⁺ T cells within tumors (orange) or CD8⁺ T cells within triads (red). Data are shown as mean ± SEM; ****p < 0.0001, using paired two-tailed Student’s t test. (E) Analysis of intratumoral CD4⁺ T Cells. FOXP3 expression in single CD4⁺ T cells (green), dyads (blue), and triads (red) compositions using IMC. CD4⁺ T cells in triads exhibit lower expression of FOXP3. Data are shown as mean ± SEM. Two-way ANOVA, p = 0.0141, with Tukey’s multiple comparisons tests as post hoc testing (single vs. dyad, ns (p = 0.777); single vs. triad, **p = 0.0013; dyad vs. triad ****p < 0.0001). (F) Proposed model of TRIAD-associated cancer elimination. See also Figure S5.