P-065: CyTOF and single cell RNA sequencing reveal altered T cell phenotypes in Multiple Myeloma patients: implications for immunotherapy

Abstract

<h3>Background</h3> Immune therapies have had a major impact in multiple myeloma (MM). Responses induced in relapsed and/or refractory MM (RRMM) patients (pts) are unprecedented; however the key challenge is their durability. Most immunotherapeutic approaches rely on the fitness of pts' T cells but exposure to multiple therapies may alter immune cell numbers and function. Here we analyzed and compared the transcriptome and phenotype of T cells from healthy donors (HD), newly diagnosed MM (NDMM) and RRMM pts. <h3>Methods</h3> Peripheral Blood Mononuclear Cells (PBMCs) from age-matched HD, NDMM and RRMM pts were collected before the start of a new line of treatment. Single cell RNA sequencing (scRNAseq) was performed and proportion of cell types were inferred using a reference PBMC dataset with Seurat. Cell type proportions were also estimated with CyTOF and conventional flow cytometry <h3>Results</h3> PBMCs from 10 HDs (median age 69, range 53-79), 20 NDMM pts (median age 71, range 54-88), 20 RRMM pts (median age 71, range 54-80) were analyzed. RRMM pts were exposed to 1 (n=8), 2 (n=6) or 3 (n=6) lines of treatment. All RRMM pts were exposed to a proteasome inhibitor, 75% were exposed to an immunomodulatory drug, and 65% received autologous stem cell transplantation. 48 out of 50 samples were processed by scRNAseq, 45 by CyTOF and 33 by flow cytometry. NDMM pts demonstrated a trend towards lower frequency of CD4 T cells compared to HD. This decreased number was accompanied by a decrease in the expression of genes involved in mitochondrial oxidative phosphorylation and an increase in expression of immediate early response genes (e.g. FOS, JUN, JUNB). In the CD8 compartment, CD8 naive cells were lower in NDMM compared to HD. No differences were found on CD8 central memory, effector memory and/or terminal effector populations In RRMM, the proportion of naive CD4 cells was significantly lower compared to NDMM (p < 0.05 by scRNAseq and CyTOF). This was confirmed by conventional flow cytometry (p < 0.05). No differences in CD4 central memory, effector memory and/or terminal effector proportions were observed. Decreases in the proportion of naive CD4 cells were observed as early as after 1 line of treatment and did not correlate with age, absolute lymphocyte count, previous exposure to lenalidomide or cyclophosphamide cumulative dose. Exposure to higher melphalan doses corelated with lower proportion of CD4 naive T cells. Compared with T cells from NDMM, T cells harvested from RRMM pts expressed higher levels of activation/exhaustion markers such as PD1 (p < 0.05) and HLA-DR (p < 0.05). <h3>Conclusion</h3> T cell phenotypes in MM pts are altered, especially after treatment. These findings may provide the rationale for the investigation of immunotherapy in NDMM and less heavily pretreated MM. Ongoing functional studies will demonstrate whether these phenotypic and transcriptomic changes lead to less fit T cells with consequent impact on effectiveness of T cell directed therapies.