TIGIT expression on CD8⁺T cells increases during MM development and is associated with decreased effector cell functions. (A) TIGIT expression on BM CD8⁺ T cells was analyzed by flow cytometry in naïve C57BL/6 WT mice (N) and 1, 2, 3, and 4 weeks after IV challenge with 2 × 10⁶ Vk12653 MM cells. Data are presented as mean ± standard error of the mean (SEM) of 2 pooled experiments with n = 6 to 14 mice per group. (B) Percentages of BM TIGIT⁺CD8⁺ T cells from Vk12653-MM–challenged mice shown in panel A were plotted against percentages of BM MM cells in these mice. (C) Graphs showing the mean ± standard deviation percentage of TIGIT⁺ CD8⁺ T cells in BM CD8⁺ T cells from healthy donors (HDs) (n = 20) and MM patients at diagnosis (n = 86) or after relapse (n = 32). (D) The expression of immune checkpoints was analyzed on MM patient BM CD8⁺ T cells by flow cytometry. Graphs show mean ± SEM from n = 16 to 26 patients with MM. (E) Representative histogram showing the expression of TIGIT on MM patient BM CD8⁺ T-cell subsets T_N (CD62L⁺CD45RA⁺), T_CM (CD62L⁺CD45RA⁻), T_EM (CD62L⁻CD45RA⁻), and T_EMRA (CD62L⁻CD45RA⁺) and graph recapitulating the percentage of TIGIT⁺ cells shown as mean ± SEM from n = 59 MM patients. (F) MM patient CD138⁻ BM cells were stimulated with anti-CD3/CD28/CD2 microbeads for 6 hours. Activation (CD69), intracellular TNF-α, and IFN-γ content and degranulation (CD107a) of TIGIT⁻ and TIGIT⁺ CD8⁺ T cells was determined by flow cytometry. Representative dot plots are shown as well as graphs displaying mean ± SEM from n = 31 patients with MM. (G) MM patient CD138⁻ BM cells were stained with CTV and stimulated with anti-CD3/CD28/CD2 microbeads for 5 days. Representative histogram showing the proliferation of TIGIT⁻ (dark red) and TIGIT⁺ (light red) CD8⁺ T cells and graph recapitulating the percentage of divided TIGIT⁻ and TIGIT⁺ CD8⁺ T cells from the same culture (n = 20 MM patients). (H) TIGIT⁻ and TIGIT⁺ CD8⁺ T cells were sorted by flow cytometry and stimulated with anti-CD3/CD28/CD2 microbeads for 6 hours. TNF-α and IFN-γ production as well as degranulation were determined by flow cytometry. Data shown are from n = 8 to 12 MM patients. (I) Representative dot plot of CD8⁺ BM T cells from HLA-A*02⁺ patients with MM stained with HLA-A*02-NYESO1-PE multimers (left) and histogram showing the TIGIT expression in NY-ESO1–specific CD8⁺ T cells (right). (J) MM patient CD138⁻ BM cells were stimulated with HLA-A*02-NY-ESO-1–specific peptide (1 μg/mL) for 6 hours. Representative dot plots as well as pooled data from n = 6 HLA-A2⁺ MM patients with positive NY-ESO response are shown. Statistical differences between multiple groups were determined by (A,D-E) 1-way analysis of variance (ANOVA) with Tukey posttest analysis, (B) correlations were assessed using a Pearson rank correlation test, and (C) analyses between 2 groups were performed using a Mann-Whitney U test or (F-H,J) Wilcoxon matched-pairs signed rank test. *P < .05, **P < .01, ***P < .001, ****P < .0001. T_CM, central memory; T_EM, effector memory; T_EMRA, terminal effector memory; T_N, naïve.