Immunome Single Cell Profiling Reveals T Cell Exhaustion with Upregulation of Checkpoint Inhibitors LAG3 and Tigit on Marrow Infiltrating T Lymphocytes in Daratumumab and IMiDs Resistant Patients

Background: The addition of immunomodulatory (IMiDs) drugs to monoclonal antibodies targeting the transmembrane glycoprotein CD38 has demonstrated very encouraging and durable responses in myeloma patients. It is believed that the synergistic effects observed with anti-CD38 antibodies and IMiDs are derived from their co-modulation of the host adaptive and innate immunity and therefore it is plausible to speculate that acquired resistance to daratumumab and IMiDs may be largely immune-mediated. The aim of the present study was to 1) interrogate at the single cell level the bone marrow immune repertoire of daratumumab sensitive and resistant patients, 2) identify cellular mediators of resistance to anti-CD38 antibodies and 3) define potential means to reinstate sensitivity to daratumumab and IMiDs.

Methods and Results: Serial BM aspirates (n=44) were collected from patients treated with single agent daratumumab (MMY3012 trial) or daratumumab + pomalidomide (MM014 trial) prior to initiation of therapy, C3D1 and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD138^pos and CD138^neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Paired-end sequencing was performed on Illumina NEXTseq and NOVAseq platforms. Cell Ranger Single and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting and data analysis. Sequencing data were analyzed by principal component analysis (PCA), clustering with multi-sample batch correction and then visualized by t-distributed stochastic neighbor embedding (t-SNE) projection. Comparison of the single cell transcriptomes of CD138^neg cells from responding patients pre- and post- treatment revealed that Daratumumab and Pomalidomide dramatically modify the immune cells composition (immunome) of the bone marrow niches leading to: 1) expansion of effector T cells (KLRG1^high, GZMA^high, CCL5^high), 2) significant depletion of CD38^high / FCGR3A^high NK cells with retained population of cytotoxic NK cells (CD27^high, KLRB1^high, NCR3^high, GZMA^pos, PRF1^pos), 3) depletion of FCGR3A^high / CD14^low monocytes, 4) expansion of M1 inflammatory macrophages and depletion of plasmacytoid dendritic cells. Similar changes were seen in patients treated with single agent daratumumab (without IMiDs) however with a lesser expansion of effector T cells and in particular reduced marrow infiltrating inflammatory macrophages. In contrast, the immunome of daratumumab and pomalidomide resistant patients was characterized by a reduced central memory T cells (T_CM), and a largely exhausted effector T cells populations that are CD28^neg and expressing checkpoint inhibitors (LAG3and TIGIT significantly more than PDCD1) as well as high expression of TIM3 (HAVCR2) on marrow macrophages. Upregulation of LAG3 and TIGIT expression on T cells was also confirmed at the antigenic level by flow cytometry. Consistent with the non-bystander and suppressive effect of LAG3 and TIGIT on the function of effector T cells, activation (CD107a expression) and proliferation of LAG^pos and/or TIGIT^pos sorted bone marrow T cells from resistant patients were significantly reduced in response to autologous myeloma cells stimulation or CD3/CD28 crosslinking. Lastly, a higher proportion and number of clonal T cell (through single cell TCR sequencing) was also observed in responding (≥ PR) vs non-responding (< PR) patients. Interrogation of the myeloma cells transcriptome, showed little to no loss of CD38 transcript at the time of acquired resistance with rather upregulation of complement inhibitory molecule CD59 and NFκB signature genes.

Conclusion: A systematic unsupervised interrogation of the bone marrow immunome of daratumumab and IMiDs treated MM patients demonstrated a significant activation of adaptive and innate immunity in responding patients and revealed an expansion of exhausted T cells with upregulation of the checkpoint inhibitors LAG3 and TIGIT in resistant patients. Our findings warrant the exploration of LAG3- and/or TIGIT-blocking strategies as potential means to reinstate sensitivity to daratumumab and IMiDs in myeloma patients.