NK Receptor Signaling Lowers TCR Activation Threshold, Enhancing Selective Recognition of Cancer Cells by TAA-Specific CTLs

Abstract

Cytotoxic CD8+ T lymphocyte (CTL) recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells and also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes and long-term survival of patients with melanoma. Using MART1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior co-stimulatory effects of DNAM1 over CD28 involved enhanced TCR signaling, CTL killer function, and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in antigen specificity and selectivity of killer function. In addition, the elevation of NKR ligand expression on cancer cells due to chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAAs. Our data help explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T-cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.

Figure 1.. CTL-associated DNAM-1 and NKG2D are associated with the effector status of tumor-infiltrating CD8⁺ T cells and predict clinical outcomes of melanoma patients.

(A) The correlation between DNAM-1/NKG2D gene expression and CTL/NK cell lineage markers. Size and color of the squares represent correlation coefficient and p values, respectively. (B) The 10-year overall survival probability of metastatic melanoma (SKCM) patients with different expression of CD8A, DNAM-1, and NKG2D. (C) Hazard ratios of CD8A, DNAM-1, NKG2D and their combination to estimate overall survival (means and upper/lower limits). (D) Expression of DNAM-1 and NKG2D on effector and exhausted CD8⁺ TILs from melanoma patients. Left: A representative dot plot showing effector (Teff) and exhausted (Texh) identified by TIM-3 and PD-1 expression. Right: Expression of DNAM-1 and NKG2D on Teff vs. Texh CD8⁺ TILs from a representative donor are shown in histograms and their mean florescent index (MFI) are compared (n=6 donors, each pair of dots represents TILs from an individual donor). (E) Expression of DNAM-1 and NKG2D on effector vs. exhausted MART-1–specific melanoma TILs. (F) Expression of DNAM-1 and NKG2D on different CD8⁺ T-cell populations in the PBMCs of healthy donors. Populations of naive (Tn), central memory (Tcm), effector memory (Tem), and terminally differentiated effector memory (Temra), were gated based on their expression of CD62L and CD45RA. Expression of DNAM-1 (left) and NKG2D (right) on CD8⁺ cells from a representative donor are shown in histograms and their MFI are shown in bar graphs (n=11 donors, mean ± SEM). Data in (A) were modeled by simple linear regression and analyzed by Spearman correlation. Kaplan-Meier survival curves in (B) were analyzed by log-rank test. Data in (D and F) were analyzed by two-tailed ratio paired t test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, not significant (ns): P > 0.05.

Figure 2.. DNAM-1 and NKG2D set the threshold for TCR-dependent recognition and killing of cancer cells presenting low levels of MHC I/TAA peptide complexes.

(A) Different levels of endogenous MART-1/Melan A in melanoma cells are associated with their different levels of (re)activation of DC-primed MART-1-specific CTLs. Left: Expression of MART-1/Melan A in cancer cells are shown in histograms with MFI. Right: IFN-γ ELISPOT (n=4 donors, mean ± SEM). (B) IFN-γ secretion by DC-primed MART-1–specific CTLs against melanoma cells expressing different levels of MART-1/Melan A in the absence or presence of NKG2D/DNAM-1 blockade (n=4 donors, each pair of dots represents means of paired triplicate cultures from each individual donor). (C) Correlation between the inhibitory effect of NKG2D/DNAM-1 blockade and the strength of effector response in the recognition of melanoma cells by DC-primed MART-1-specific CTLs. (D and E) IFN-γ secretion by (D) DC-primed MART-1–specific CTLs and (E) a patient-derived MART-1–specific clone in response to cancer cells (SW620) loaded with high- or low-dose MART-1 peptides (1 or 0.01 μg/ml), in the absence or presence of NKR blockers. (D) Representative images of ELISPOT (triplicate wells per condition) and quantification of spots (n=5 donors, mean ± SEM). (E) Data with MART-1-specific clone from a representative experiment performed in triplicate cultures per condition. (F) IFN-γ secretion by NY-ESO-1–specific TCR-transduced CD8⁺ T cells (19305DP and CD8SP) against cancer cells loaded with decreasing doses of NY-ESO-1 peptides, with or without NKR blockade (triplicate cultures per condition, mean ± SD). (G) Representative brightfield pictures of CTL–cancer cell conjugates, and fluorescent pictures showing CFSE labelled cancer cells, CD3, LFA-1, NKG2D or DNAM-1, F-actin and their colocalization. (H) Degranulation of CTLs under indicated conditions was monitored by surface expression of CD107a on CTLs and presented as histograms with mean fluorescent index (MFI) (left) or the ratios of CD107a MFI in each of the indicated conditions to the MFI of unloaded control (right, n=3 donors). (I) Killing of target cells by DC-primed MART-1–specific CTLs under the indicated conditions was analyzed by LDH cytotoxicity assay (n=3 donors). Each pair of dots represents means of paired triplicate cultures from each individual donor. Data were analyzed by two-tailed ratio paired t test (A, B, D, H, and I), or two-tailed unpaired t test (E and F). Data in (C) were modeled by simple linear regression and analyzed by Pearson correlation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, not significant (ns): P > 0.05. ND: not detected.

Figure 3.. DNAM-1, and to a lesser extend NKG2D, enhance CTL polyfunctionality.

Dynabead-induced CTLs were activated by immobilized antibodies. (A) Intracellular IFN-γ levels in CTLs activated by increasing concentrations of OKT3 in the absence or presence of NKR costimulation. Left: Representative flow cytometry histograms showing intracellular IFN-γ levels of differentially stimulated CTLs. Right: Percentages of IFN-γ–producing CTLs activated by low-dose OKT3 in the absence or presence of NKR costimulation (n=5 donors; mean ± SEM). (B to E) Single-cell secretome of CTLs activated under the indicated conditions were analyzed using Adaptive Immune ISOCODE chips and isoLight system (IsoPlexis), showing summary results of 2 separate experiments using different donors. (B) 3D-UMAP projection showing clusters of CTLs activated by OKT3 alone and CTLs activated by OKT3 plus NKR-costimulation. (C) Expression of effector cytokines was overlayed on t-SNE projections, showing intensity of cytokines in CTLs activated by low-dose OKT3 either alone or in combination with the indicated NKR-costimulation. (D) Polyfunctionality was calculated as the percentages of activated CTLs secreting ≥ 2 types of proteins. (E) Polyfunctional Strength Index (PSI) was computed as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Proteins were grouped and color-coded based on their functions: Effector: Granzyme B, IFN-γ, Perforin, TNFα; Stimulatory: GM-CSF, IL-2, IL-12, IL-15; Chemo-attractive: CCL4, CCL5; Modulatory: IL4, IL10, sCD40L. Data in (A) were analyzed by two-tailed ratio paired t test. ∗∗P < 0.01.

Figure 4.. DNAM-1 provides superior costimulation of effector CTLs, lowering the TCR activation threshold, enhancing metabolic reprograming, and all major TCR signaling pathways.

(A-E) RNA sequencing: Gene expression profiles of CTLs from 3 donors stimulated by OKT3 either alone (control) or in combination with DNAM-1, NKG2D, or CD28 costimulation. (A) Principal component analysis (PC3 and PC4) comparing patterns of gene expression in CTLs activated under the indicated conditions. (B) Volcano plot showing the differentially expressed genes (DEGs) in CTLs activated with the indicated costimulation versus control CTLs. DEGs with log2 fold change > 1 and adjusted P value< 0.05 were defined as significant. (C) Heatmap showing DNAM-1-unique DEGs which are associated with CTL effector and regulatory functions. (D) Top enriched pathways in DNAM-1-costimulated CTLs compared to control CTLs from Gene Set Enrichment Analysis (GSEA). (E) Enrichment of mTORC1 signaling and glycolysis in DNAM-1-costimulated CTLs compared to control CTLs. (F and G) CTLs were stimulated by streptavidin crosslinking of biotinylated αCD3 (OKT3) with or without biotinylated αDNAM-1. (F) Induction of calcium flux by OKT3 with or without DNAM-1 costimulation. Left: Intracellular calcium levels over time in CTLs stimulated with OKT3 and different concentrations of αDNAM-1 (the arrow indicated the addition of streptavidin). Right: Results from 3 independent experiments were normalized by Fluo-4 Peak MFI Ratio calculated as Fluo-4 Peak MFI divided by baseline MFI (n=3 donors; mean ± SEM). (G) Western blot of phosphorylated AKT and ERK in CTLs stimulated with or without DNAM-1 costimulation at different timepoints. Data in (F) were analyzed by multiple paired t test. ∗P < 0.05, not significant (ns): P > 0.05.

Figure 5.. Effector versus naïve CD8⁺ T cells rely on DNAM-1 versus CD28 as the dominant costimulatory pathways.

(A) Intracellular calcium levels over time in CTLs stimulated with OKT3, OKT3 and αDNAM-1, or OKT3 and αCD28. The arrow indicated the addition of streptavidin for crosslinking of biotinylated antibodies. (B) Effects of DNAM-1 versus CD28 costimulation in activation of naïve CD8⁺ T cells. Left: CFSE dilution of naïve CD8⁺ T cells activated by immobilized OKT3 with different costimulatory signals in a representative donor. Right: Percentages of CFSE^low activated cells (n=3 donors; mean ± SEM). (C) IFN-γ secretion by naïve CD8⁺ T cells (left, n=3 donors) or Dynabead-induced CTLs (right, n=4 donors) activated by immobilized OKT3 with the indicated costimulatory signals (mean ± SEM). (D) Expression of DNAM-1 ligands (PVR and Nectin2) and CD28 ligands (B7.1 and B7.2) on DCs. (E and F) DCs were pulsed with Staphylococcus Enterotoxin (SEB) to induce polyclonal T cell activation. %inhibition = [%Activated T cells (control) - %Activated T cells (blockade)] / [%Activated T cells (control) - %Activated T cells (no SEB)] × 100. (E) Activation of naïve CD8⁺ T cells by SEB-pulsed DCs in the presence of blocking antibodies against DNAM-1 or CD28. Left: Representative flow cytometry histograms of T cell CFSE dilution. Right: Summary data of triplicate cultures from 3 donors showing inhibitory effect of each blocker on naïve cell activation. (F) Re-activation of Dynabead-induced CTLs by SEB-pulsed DCs in the presence of blocking antibodies against DNAM-1 or CD28. Left: Representative contour plots of side scatter and IFN-γ expression. Right: Summary data of triplicate cultures from 3 donors showing inhibitory effect of each blocker on CTL re-activation. (G) A model of NKR-mediated “alternative signal 2” supporting the specificity of CTL anti-cancer function. Data in (B and C) were analyzed by two-tailed ratio paired t test. ∗P < 0.05, ∗∗P < 0.01. ND: not detected.

Figure 6.. Enhancing NKR-mediated recognition improves CTL functional avidity and cancer cell elimination.

(A) Overexpression of NKG2D and DNAM-1 on TCR transduced CD8⁺ T cells. Upper: Design of the NKG2D/DNAM-1 co-expressing vector (IC: intracellular domain; TM: transmembrane domain; EC: extracellular domain; SS: spacer sequence). Lower: Contour plots comparing the expression of NKG2D and DNAM-1 on blood-isolated CD8⁺ T cells transduced with the TCR construct (single-transduced) or with the TCR construct and the additional NKG2D/DNAM-1 co-expressing vector (double-transduced). (B) Survival of SW620 cell loaded with increasing concentrations of NY-ESO-1 (0, 0.01, and 0.1 μg/ml) after 24-hour co-culture with single-transduced T cells or double-transduced T cells. Data show representative contour plots of AnnexinV and DAPI (left) and summary results of independent experiments quantifying the percentage of AnnexinV^–DAPI^– surviving cancer cells (right, n=3 donors; mean ± SEM). (C) Comparison of expression of NKG2D and DNAM-1 ligands on untreated or oxaliplatin-treated (100 μM; 72 hours) SW620 cells. (D) IFN-γ secretion by DC-primed MART-1–specific CTLs against untreated or oxaliplatin-treated SW620 cells loaded with MART-1 peptide (low-dose: 0.01 μg/ml, high-dose: 1 μg/ml). Data show a representative image of ELISPOT performed in triplicate wells per condition and quantification of spot number from the same experiment (mean ± SD). (E) IFN-γ secretion by DC-primed MART-1–specific CTLs against SW620 untreated or pre-treated with oxaliplatin for 72 hours and loaded with 0.001 μg/ml MART-1 peptide. Data show summary result of 5 independent experiments performed in triplicate wells per condition (means and individual data points). (F) Schematic depiction of sensitizing chemo-resistant cancer cells to immune recognition through upregulation of NKR ligands. Data in were analyzed by multiple paired t test (B), or two-tailed unpaired t test (D). ∗P < 0.05, ∗∗P < 0.01, not significant (ns): P > 0.05.