CD4 Cytotoxic T Cells Promote Antitumor Immunity in Multiple Myeloma

Background:

Recent advancements in single-cell RNA sequencing have enhanced our understanding of the immunological dysfunctions during multiple myeloma (MM) progression from monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Immunotherapies employing T cells have demonstrated promise in treating MM. However, a better understanding of effector T cells is necessary due to their limited response durability. While most forms of cancer immunotherapy have primarily focused on CD8⁺ cytotoxic T lymphocytes (CTLs), CD4⁺ T cells are emerging as important contributors to antitumor responses beyond their traditional roles as helpers or regulators. However, the specific subset of CD4⁺ T cells contributing to beneficial outcomes in MM remains unclear, and it is unknown whether CD4⁺ T cells mediate direct antitumor effector functions against myeloma cells.

Aims:

This study aims to characterize CD4⁺ CTLs in MM by investigating their phenotypic and functional properties, assessing their impact on patient outcomes, and identifying their mechanisms and therapeutic potential.

Methods:

We performed scRNA-seq and TCR-seq on CD4⁺ T cells sorted from bone marrow (BM) aspirates of 29 individuals across different stages of myeloma, including healthy donors (n=5) and patients with MGUS (n=4), SMM (n=4), and MM (n=16). We validated the identified subsets and further characterize their immunophenotype by multicolor flow cytometry, while cytotoxicity assays and blocking experiments evaluated their cytotoxic capabilities.

Results:

Droplet-based scRNA-seq and paired scTCR-seq were performed to infer the functional states of BM CD4⁺ T cells, and a total of 105,398 CD4⁺ T cells were obtained from 29 individuals using the 10X Genomic platform. By unsupervised clustering, we identified 14 distinct BM CD4⁺ T cell subsets: naive (CCR7⁺TCF7⁺FAS^-HNRNPLL^-), early activated (early.act, CCR7⁺TCF7⁺FAS^-NR4A1⁺), central memory (CM, CCR7⁺FAS⁺), follicular helper T (Tfh, CXCR5⁺BCL6⁺), Th2 (GATA3⁺CCR4⁺), Th17 (RORC⁺CCR6⁺), Th1 (TBX21⁺CXCR3⁺IFNG⁺), two subsets of regulatory T cells (Treg, FOXP3⁺CTLA4⁺), resting Tregs (rTreg, CCR7⁺) and activated Treg (aTreg, HLA-DRA⁺TNFRSF9⁺), IFN-stimulated genes-enriched (ISGs^hi, OAS3⁺IRF7⁺), and activated and proliferating (act.proli, MKI67⁺CD38⁺). Of note, we identified two distinct states of BM CD4⁺ T subsets that highly express cytolytic effector molecules: cells expressing GZMB and cells expressing GZMK. The GZMB-expressing subset exhibited high levels of a core set of cytotoxic molecules (GZMA⁺NKG7⁺GNLY⁺PRF1⁺) and markers associated with terminal differentiation (B3GAT1⁺KLRG1⁺CD27^-), which was designated as terminal effector CTL (TE.CTL). In contrast, the GZMK-expressing subset had lower levels of cytolytic molecules and instead demonstrated higher expression of Th1 or Th17 signature genes, and we clustered these subsets as Th1.CTL and Th17.CTL, respectively. Importantly, TE.CTL cluster was significantly increased and clonally expanded in MM patients. When we combined CTL clusters and their TCR clonotype-sharing clusters, we identified a major fraction of these cells as CX3CR1^hi cells, which exhibited higher expression of cytotoxic markers such as CX3CR1, KLRK1 (NKG2D), SLAMF7 (CD319), ITGB1 (CD29), ADGRG1 (GPR56). Flow cytometry revealed the effector memory phenotype of CD4+ CTLs and their robust production of perforin and degranulation upon stimulation. Functional assays demonstrated that CD4⁺ CTLs could directly kill myeloma cells ex vivo, with this cytotoxicity significantly reduced by NKG2D blockade, indicating an NKG2D-dependent mechanism. Patients with a higher frequency of NKG2D⁺CD4⁺ CTLs in their BM showed improved progression-free survival, underscoring their clinical relevance.

Conclusions:

CD4⁺ CTLs are important players in the antitumor immunity of MM. They are increased in frequency, clonally expanded, and possess robust cytotoxic capabilities and the abundance of NKG2D⁺CD4⁺ CTLs correlate with improved clinical outcomes. The NKG2D dependency of their cytotoxic function presents a potential target for therapeutic intervention. Harnessing CD4⁺ CTLs offers a promising strategy for enhancing immunotherapy outcomes in MM, paving the way for the development of novel CD4+ CTL-based therapies and better prediction of treatment responses by monitoring NKG2D⁺CD4⁺ CTLs.