Targeting of the CD161 Inhibitory Receptor Enhances Bone Marrow-Resident Memory CD8+ T Cell-Mediated Immunity Against Multiple Myeloma

Multiple myeloma (MM), an incurable hematologic malignancy, presents significant challenges in managing its recurrence. The bone marrow tumor microenvironment (BM-TME) is critical to MM progression. However, classical immune checkpoint blockade (ICB) therapies, like anti-PD1, have shown limited efficacy in MM. Therefore, elucidating the compositional features and regulatory mechanisms of the TIME in MM is crucial for developing novel immunotherapeutic targets. Tissue-resident memory CD8⁺ T cells (CD8⁺ TRMs) reside indefinitely at multiple anatomical locations like the skin, intestine, and bone marrow, functioning as frontline responders against reencountered pathogens and tumors. Whether CD8⁺ TRMs within BM-TME are involved in the MM cell immune escape and pathogenesis has not been systematically elucidated.

Firstly, we leverage single-cell RNA sequencing (scRNA-seq) to dissect the BM CD8⁺ T cells across patients at different stages of MM, and identify that CD69⁺CD8⁺ T subsets were BM residents, and the proportion of BM CD8⁺ TRMs between health donors and MM patients was comparable. Moreover, our findings indicated that BM CD8⁺ TRMs exhibit exhausted features and enrichment of NK genes with MM progresses. Importantly, we observed a considerable elevation of the NK gene KLRB1 and its encoded protein CD161 in BM CD8⁺ TRMs in MM patients. Immunofluorescence labeling of BM biopsy samples from MM patients confirmed the infiltration of the BM-TME with CD3⁺CD8⁺CD69⁺CD161⁺ cells.

Then, we found that KLRB1 expression in BM CD8⁺ TRMs was higher than that of other classical inhibitory receptor genes (e.g., PDCD1, CTLA4, LAG3), and CLEC2D (encodes the ligand of CD161) was also markedly expressed in patients' MM cells, exhibiting considerably higher than classical immunosuppressive receptor ligand genes (e.g., PDL1, CD80, CD86). Moreover, patients with higher CLEC2D expression had unfavorable overall survival.

We further found that the CD161 expression was higher in CD69⁺CD8⁺ TRMs compared to circulating CD69^-CD8⁺ T cells, and the percentage of BM CD161⁺CD8⁺ TRMs in MM patients was significantly higher than in healthy donors, while there was no difference for PD-1, LAG3, and TIM3 expression. Notably, only a small fraction of BM CD8⁺ TRMs have mucosal-associated invariant T cell phenotypes (identified by CD161⁺TCR-Vα7.2⁺). Additionally, the CLEC2D expression of BM cells was analyzed, and the highest level of CLEC2D expression was observed in B cells from MM patients, followed by plasma cells. Importantly, CLEC2D expression in MM patients was significantly higher than in plasma cells from healthy donors. Next, to investigate whether the blockade of CD161 could restore the function of CD8⁺ TRMs, the primary BM cells from MM patients were isolated and stimulated ex vivo. Compared to the anti-PD-1 and isotype group, CD161 blocking presents enhanced CD8⁺ TRMs proliferation and cytokine secretion levels (TNF-α, IFN-γ, IL2), increasing MM cells apoptosis.

Chimeric antigen receptor T (CAR-T) cell therapy has achieved impressive success in treating relapsed/refractory MM. How to prevent CAR-T cell exhaustion and enhance function remains a significant challenge. Therefore, we next set out to investigate the impact of CD161 on CAR-T cells. Our results demonstrated that CD161 was highly expressed in early peripheral blood (PB) CAR-T cells after infusion in MM patients but decreased with time. Moreover, the CD161 expression of CAR-T cells was considerably higher than endogenous T cells. Notably, BM-derived CAR-T cells have higher CD161 expression than PB counterparts for the same patient, suggesting CD161 may have BM-specific expression enrichment. After co-culturing MM and CAR-T cells in vitro for 24 hours, we found that blocking CD161 led to an augmented proportion of CAR-T cells, increased IFN-γ secretion, and a notable elevation in tumor cell apoptosis compared to the anti-PD-1 and isotype groups.

Taken together, we demonstrated that CD8⁺ TRMs constitute a vital anti-tumor cell subset in the MM BM-TME, which exhibits a high expression of the inhibitory CD161 receptor, binding to the CLEC2D expressed by MM cells, thereby mediating the dysfunction of CD8⁺ TRMs. Blocking the CD161-CLEC2D pathway is expected to become a novel target for MM immunotherapy. The combination of CD161 inhibitors and CAR-T cell therapy can potentially enhance the anti-myeloma function of CAR-T cells.