Immune restoration in long-term myeloma survival revealed by single-cell RNA-sequencing

Abstract Background: Multiple myeloma (MM) remains incurable despite advances in modern therapies. A small subset of patients achieves long-term survival (LTS; ≥10 years) post diagnosis, but mechanisms of durable disease control remain unclear. While clinical factors and minimal residual disease status partially explain outcome, growing evidence highlights the immune bone marrow microenvironment (IBME) as a critical mediator. To identify LTS mediators, we employed single-cell RNA sequencing (scRNA-seq) to test two hypotheses: (i) malignant plasma cells (mPCs) from LTS patients adopt an indolent, MGUS/SMM-like profile; and (ii) immune cells in LTS patients retain surveillance programs observed in precursor stages. Methods: Bone marrow (BM) aspirates from patients with smoldering multiple myeloma (SMM, n = 3), newly diagnosed multiple myeloma (NDMM, n = 3), early relapsed multiple myeloma (RMM, n = 3), and long-term survival multiple myeloma (LTS MM, n = 10) were obtained from the MCWFH Cancer Center with IRB approval. Bone marrow mononuclear cells (BMMCs) were isolated, and single-cell R NA-seq was performed using the 10x Genomics Chromium 3' v3.1 platform. To enhance our analysis, we incorporated published scRNA-seq datasets from various disease stages (MGUS, SMM, NDMM), including myeloma cells (CD138⁺) and the IBME (CD138⁻), as well as data from healthy donors. Quality control and clustering were performed using Seurat; cell annotation utilized SingleR and the Human Primary Cell Atlas. Pseudobulk differential expression was conducted with DESeq2; Plasma B-Cell Malignancy (PBM) scores were calculated using ssGSEA; copy number variations (CNV) inference was performed with inferCNV. Immune pathway enrichment was assessed using fgsea (MSigDB C7), and leading-edge gene analysis identified key drivers. Cell-cell communication was analyzed with CellChat. Results: Malignant plasma cells (mPCs) from LTS patients did not form a distinct cluster but overlapped with those from MGUS, SMM, NDMM, and RMM. LTS mPCs retained high plasma cell burden/malignancy PBM scores and widespread CNVs) similar to NDMM, indicating persistent malignant features. However, limited similarity to MGUS suggests that these cells may retain or re-acquire transcriptional programs typically seen in precursor states, reflecting a hybrid or restrained malignant state. These findings suggest mPCs persist in LTS patients with NDMM-like profiles despite long-term disease control, raising the possibility that immune-mediated mechanisms such as a functionally restored IBME, may contribute to maintaining an MRD-like state. In contrast, the LTS IBME exhibited transcriptional and functional resemblance to precursor stages. LTS patients showed expansion of CD8⁺ effector memory RA (EMRA), CD4⁺ central memory, CD4⁺ effector memory, and IL-2–responsive NK cells, with significant transcriptional enrichment for effector activation, memory differentiation, and interferon response pathways (adjusted p 0.05). Leading-edge gene analysis confirmed coordinated restoration of immune programs. IL-2–responsive NK cells in LTS resembled those in MGUS/SMM, enriched for oxidative stress and innate immune pathways absent in NDMM. Monocytes in LTS also resembled precursor-stage profiles, suggesting restoration of innate immune competence. We next investigated cell-cell communication using CellChat to assess whether enhanced immune interactions contribute to disease control in the LTS. This analysis revealed a marked increase in both the number and strength of immune cell-cell interactions in LTS compared to all other stages, including MGUS, SMM, and NDMM. Notably, CD8⁺ EMRA in LTS exhibited 20 outgoing interactions (vs. 10 in NDMM) and autocrine signaling strength of 2.0 (vs. 0.9), while IL-2–responsive NK cells had 20 interactions and a strength of 1.1 (vs. 8 and 0.3, respectively). CD8⁺ EMRA cells in LTS showed expression of signaling molecules such as MHC-I, CD99, LCK, and CD160 that were significantly increased and reduced in NDMM, indicating reactivation of antigen presentation, TCR signaling, adhesion, and cytotoxic effector programs. Conclusion: Despite retaining transcriptionally and genomically malignant plasma cells, LTS MM patients exhibit a functionally re-engaged, precursor-like immune bone marrow microenvironment. This immune restoration likely contributes to long-term disease control and provides a basis for identifying biomarkers and immunotherapeutic targets in MM.