CD69⁺ Bone Marrow-Resident CD8⁺ T Cells Represent a Functionally Impaired Tumor-Reactive Population and Can be Restored By Blocking PD-1 and Tigit in Multiple Myeloma

Recently, novel anti-myeloma therapies utilizing T cell-mediated immune responses, including chimeric antigen receptor (CAR) T cells or bispecific antibodies engaging T cells to target tumor cells, have been actively studied in clinical trials and shown significant clinical efficacy. To enhance the clinical activity of T cell-exploiting immunotherapies in MM further, a better understanding of immune evasion mechanisms by characterizing myeloma-specific T cells and the strategic reinvigoration of the impaired T cell functions are essential prerequisites. In the present study, we demonstrated that bone marrow (BM)-resident CD69-expressing CD8⁺ T cells showed more prominent pathologic features including tumor-antigen associated exhaustion and impaired function in MM patients. In addition, using direct ex vivo experimental techniques, we examined the strategy how to restore anti-myeloma responses of the tumor-specific T cells, based on the characteristics of BM-resident CD69⁺ CD8⁺ T cell population.

We first tried to evaluate various immunophenotypes of CD8⁺ T cells in BM and peripheral blood (PB) compartments of MM patients and found that the frequency of CD69⁺ cells among CD8⁺ T cells was significantly higher in BM than in peripheral circulation. These CD69⁺CD8⁺ T cells in BM had an effector memory phenotype negative for CCR7. Also, CD8⁺ T cells obtained from BM of normal controls contained similar frequency of CD69⁺ cells to the CD8⁺ T cells from BM of MM patients, suggesting that the CD69⁺ population could represent a BM-resident memory population even in a physiologic condition. To this end, we isolated CD69⁺ and CD69^- memory CD8⁺ T cells from BM of MM patients and analyzed their transcriptomic difference using RNA-sequencing analysis. CD69, EPAS1, EOMES, PDCD1, TIGIT, and TOX were significantly upregulated in CD69⁺ cells when compared to CD69^- cells among memory CD8⁺ T cells. On the contrary, S1PR1, KLF2, KLF4, SELL, GZMB, and GNLY were downregulated in CD69⁺ cells when compared to CD69^- cells. These results indicate that transcriptomic signatures of CD69⁺ cells were similar to previously known features of tissue-resident memory T cells and functionally exhausted CD8⁺ tumor-infiltrating lymphocytes.

We then compared the surface expression level of immune checkpoint receptors between CD69⁺ and CD69^- cells among BM CD8⁺ T cells. CD69⁺ cells showed significantly higher positive frequencies of PD-1, Lag-3 and TIGIT than CD69^- cells, but expression of Tim-3 did not differ between two populations. Notably, TOX, the key transcription factor regulating T cell exhaustion, was higher in CD69⁺ cells than in CD69^- cells. When we examined the expression of other transcription factors as indicators of T cell exhaustion, the Eomes^hiT-bet^lo cells were significantly enriched in CD69⁺ population, indicating that CD69⁺ cells have features of terminally exhausted CD8⁺ T cells. More importantly, the myeloma antigen (NY-ESO-1_157-165 and HM1.24_22-30)-specific CD8⁺ T cells in BM of MM patients, identified with the MHC-multimer technique, also enriched in CD69⁺ population. When we compared the expression of immune checkpoint receptors among myeloma antigen-specific CD8⁺ T cells, the frequencies of PD-1⁺, TIGIT⁺ and PD-1⁺TIGIT⁺ cells were higher in CD69⁺ cells than CD69^- cells. In fact, CD69⁺CD8⁺ T cells from BM of MM patients showed reduced production of effector cytokines in response to direct ex vivo TCR stimulation, compared to CD69^- counterpart.

Next, we investigated whether blocking PD-1 and/or TIGIT could restore the function of BM-resident CD8⁺ T cells of MM patients. Although a single blockade using either anti-PD-1 or anti-TIGIT antibody was not effective, their combination resulted in significant increase of IFN-γ⁺ cells in total CD8⁺ T cells and CD69⁺CD8⁺ T cells but not in CD69^-CD8⁺ T cells

Taken together, we demonstrated that BM-resident CD69⁺CD8⁺ T cells represent a population of exhausted T cells which are functionally impaired but potentially restorable. Indeed, we provide an ex vivo evidence of dual blockade of immune checkpoint receptors to enhance anti-tumor T cell responses in MM. Detailed understanding of BM-resident CD8⁺ T cells could provide more fundamental information for improving the current T cell-based immunotherapeutic approaches against MM.

No relevant conflicts of interest to declare.