Differential impact of genetic deletion of TIGIT or PD-1 on melanoma-specific T-lymphocytes

Abstract

Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1^KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGIT^KO T-cells. Functional analyses showed that PD-1^KO and TIGIT^KO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGIT^KO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1^KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.

Keywords: Adoptive cell transfer; CD8+ T cell clones; PD-1; TIGIT; gene editing; immunotherapy; melanoma.

Figure 1.

Phenotypic characterization of WT and gene-edited T-cell clones. (a) PD-1 and TIGIT expression was measured by flow cytometry after CD3 activation. Colored groups (purple, blue and yellow) identify T-cell clones sharing the same TCR. Grey T-cell clones express unique TCR. (b) Comparison of the % of PD-1 expression (left) and median fluorescence intensity (right) between WT and TIGIT^KO T-cell clones (Unpaired T-test, ***untailed p-value <0.001, * <0.05). (c) Western-blot analysis of TIGIT and PD-1 expression on CD3-activated WT and TIGIT^KO T-cell clones. Actin was used as a loading control.

Figure 2.

PD-1^KO T-cells exhibit a lower proliferation ability than WT and TIGIT^KO T-cells. (a) Number of divisions of WT4, PD-1^KOP6, TIGIT^KOT1 T-cell clones, after 14 days of amplification on feeder cells (n = 2). Data are represented as mean ± SD. Number of divisions was calculated according to this formula: n = log₂(N/N₀), where n= number of divisions, N = final number of T-cells and N₀=initial number of seeded T-cells (*p < 0.05, One-way ANOVA and Tukey’s multiple comparison test). (b) WT4, PD-1^KOP6, TIGIT^KOT1 T-cell clones were labeled with CFSE (85 nM) and cultivated with a range of anti-CD3 mAb OKT3 (0-800ng/µL). Proliferation was analyzed after 5 days by flow cytometry. (c) Percentage of proliferating T-cells (n = 2), following CD3 activation, represented as mean ± SEM (****p < 0.0001, Two-way ANOVA and Tukey’s multiple comparison test).

Figure 3.

PDCD1 knock out has a detrimental impact on gene expression profile of activated T-cells in contrast to TIGIT knock-out. (a) Heatmap reporting scale expression of up- and down-regulated genes between WT and TIGIT^KO (upper panel) and WT and PD-1^KO T-cell clones (lower panel). RNAseq was performed on CD3-activated T-cell clones sharing the same TCR in biological triplicates (Maximum FDR p-value = 0.001 and minimum absolute fold-change = 2). (b) Expression of cell cycle regulation genes, and (c) expression of genes involved in PDCD1 modulation, measured by RT-qPCR in WT (black, n = 4), PD-1^KO (red, n = 6) and TIGIT^KO (blue, n = 5) activated T-cell clones. qPCR on each gene was performed on all CD3-activated T-cell clones, whatever TCR expressed, in biological triplicates (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, One-way ANOVA and Tukey’s multiple comparison test).

Figure 4.

Functional properties of PD-1^KO and TIGIT^KO T-cells are enhanced in response to ligand-expressing target cells.(a) Dot plot and histograms illustrating the expression of PD-L1 and CD155 on WT (dark brown), and CD155^pos (light brown), PD-L1^pos (orange) and PD-L1^pos/CD155^pos (yellow) T2 cells. Table illustrates the % of PD-L1 and CD155 expression on each type of T2 cells. (b) Functional avidities of WT4, PD-1^KOP6 and TIGIT^KOT1 T-cell clones were evaluated by measuring CD107a degranulation in response to T2 cells loaded with a range of Melan-A peptide. Table illustrates the EC50 (M) of peptide concentration for each tested T-cell clone (n = 2, data are presented as mean ± SD). (c) WT and PD-L1 and/or CD155 expressing T2 cells were loaded with 100 nM of Melan-A peptide. After 24 h of co-culture with T-cell clones, IFN-γ production was measured by ELISA. % of inhibition was determined according to IFN-γ production in response to WT T2 cells (Data are represented as mean ± SD, *p < 0.05, Two-way ANOVA and Tukey’s multiple comparison test).

Figure 5.

TIGIT^KO T-cells significantly delayed the growth of melanoma tumors and persist longer within tumors, compared to WT and PD-1^KO T-cells. (a) Melanoma tumor (CD155⁺/PD-L1⁺) growth curves in NSG mice receiving 3 i.V. injections (Day 7, 14 and 21) of either DPBS (black line), 5 × 10⁶ WT4 (gray line), PD-1^KOP6 (red line) or TIGIT^KOT1 (blue line) melanoma specific T-cell clones (n = 9/group). Tumor volumes were measured from D0 to Day 36. Arrows represent T-cell injections and data are represented as mean ± SEM. (*p < 0.05 ****p < 0.0001, Two-way ANOVA and Tukey’s multiple comparison test). (b) Example of Ki67 staining of tumor cells from each treated group. Counterstaining was done with hematoxylin. (c) Percentages of Ki67⁺ tumor cells in each treated group (data are represented as mean ± SD; n = 4/group; *p = 0.055; One-way ANOVA and Kruskal-Wallis’ multiple comparison test). (d) T-cell infiltrate revealed by CD3 staining in tumors harvested at Day 22 (30 h after the third injection of T-cells) and (e) at day 36 (2 weeks after the third injection). (f) Percentage of CD3⁺ T-cells in tumors from each group, 30 hrs (D22, n = 1) and 2 weeks after the third injection of T-cells (D36, n = 3). Immunofluorescence staining was performed on 5 levels of sections/tumor, **p < 0.01; One-way ANOVA and Kruskal-Wallis’ multiple comparison test).