By integrating single-cell RNA sequencing with clonal tracking technology, this study systematically characterizes the transcriptomic landscape of plasma cells and the bone marrow microenvironment in patients with AL amyloidosis. It reveals novel mechanisms such as activation of inflammatory signaling pathways, expansion of non-malignant plasma cell subsets, and upregulation of genes related to proteostasis, providing critical insights into disease pathogenesis and potential therapeutic targets.
Literature Overview
This article, 'Single cell and clonal analysis of AL amyloidosis plasma cells and their bone marrow microenvironment,' published in the journal Blood, reviews and summarizes a 5’ single-cell RNA sequencing study conducted on bone marrow samples from treatment-naïve patients with AL amyloidosis and age- and sex-matched healthy controls. The study systematically analyzed the transcriptomic profiles of plasma cells and their bone marrow microenvironment, revealing widespread suppression of hematopoiesis in AL patients, alongside aberrant expansion of monocytes and specific CD4+ T-cell subsets. The research identifies a unique transcriptional state in AL-associated plasma cells, marked by enhanced TNF-α and interferon responses, and discovers a distinct non-malignant plasma cell subset expressing CRIP1 that is significantly expanded in patients. Furthermore, clonal tracking analysis demonstrates significant upregulation of proteostasis-related genes in AL plasma cells, with inter-patient heterogeneity. This study provides a high-resolution atlas for understanding disease mechanisms, microenvironmental remodeling, and potential therapeutic targets in AL amyloidosis.Background Knowledge
AL amyloidosis is a systemic disorder caused by organ dysfunction due to deposition of misfolded immunoglobulin light chains (LC) produced by monoclonal plasma cells. It presents with diverse clinical manifestations, frequently affecting vital organs such as the heart and kidneys. Although related to multiple myeloma (MM), the pathophysiology of AL amyloidosis is more focused on the direct toxicity of light chains rather than the proliferation of plasma cells themselves. Current understanding of the transcriptomic features of these plasma cells and their impact on the bone marrow microenvironment remains limited. Single-cell RNA sequencing (scRNA-seq) has recently revolutionized the study of hematologic diseases, enabling high-resolution dissection of cellular heterogeneity, clonal evolution, and microenvironmental interactions. However, previous studies on AL amyloidosis have been constrained by 3’ sequencing technologies, which are inadequate for precise tracking of B-cell receptor rearrangements, thus limiting accurate identification of malignant clones. This study overcomes this technical limitation by employing 5’ scRNA-seq combined with VDJ sequencing, enabling precise annotation of clonal status in plasma cells. This allows for clear distinction between malignant and non-malignant plasma cells and systematic comparison of their transcriptional programs. This methodological advance enables deep insights into AL-specific microenvironmental alterations and intrinsic plasma cell features, filling a critical knowledge gap and providing a theoretical foundation for future targeted therapies—such as targeting proteostasis, the immune microenvironment, or specific surface molecules like B7-H3.
Research Methods and Experiments
The study enrolled treatment-naïve patients with AL amyloidosis and healthy controls matched for age and sex. Bone marrow samples were collected, and mononuclear cells were isolated. Plasma cells were enriched via CD138-positive selection, followed by 5’ single-cell RNA sequencing and VDJ sequencing on both CD138⁻ and CD138⁺ cell populations. The study integrated cohort data from multiple centers, including the Dana Farber/Brigham and Women’s Hospital (DFCI/BWH) cohort and the Tufts cohort, to enhance statistical power. SNP-based genotype deconvolution was used to assign mixed samples to individuals, and tools such as Symphony were employed for cell type annotation of CD138⁻ cells. TCR/BCR clonal tracking was performed to assess T- and B-cell receptor diversity. Differential gene expression analysis was conducted using PyDESeq2, and gene set enrichment analysis (GSEA) was based on Hallmark pathways. Key findings were validated at both protein and transcript levels using multiplex immunofluorescence staining, Western blot, and qRT-PCR. Additionally, TMT-based mass spectrometry proteomics was performed on bone marrow plasma samples to evaluate changes in soluble microenvironmental factors.Key Conclusions and Perspectives
Research Significance and Prospects
This study presents the first comprehensive single-cell resolution map of the bone marrow microenvironment and plasma cell transcriptome in AL amyloidosis, uncovering disease-specific immune remodeling and intrinsic molecular features of plasma cells. The findings of activated inflammatory signaling, elevated sB7-H3, and expanded CRIP1⁺ plasma cells provide new perspectives on disease progression and highlight several potential therapeutic intervention points. For example, antibody therapies targeting B7-H3 could modulate the immune microenvironment, while enhancing or inhibiting proteostasis pathways may affect plasma cell survival.
The study underscores the importance of clonal information in plasma cell disorder research, avoiding the misclassification of transcriptionally similar but non-malignant plasma cells as malignant clones—a limitation of previous studies. Future work should explore the antigen specificity of CRIP1⁺ plasma cells and their role in disease evolution, as well as the clinical utility of sB7-H3 in disease staging and prognosis. Moreover, the analytical framework established here can be applied to pre- and post-treatment samples to dynamically monitor microenvironmental and clonal evolution, offering tools for developing early diagnostic markers and assessing therapeutic responses.
Conclusion
This study integrates single-cell transcriptomics with clonal reconstruction to systematically dissect the molecular features of the bone marrow microenvironment and plasma cells in AL amyloidosis. It reveals a complex microenvironment characterized by suppressed hematopoiesis alongside selective expansion of specific immune cell subsets, widespread activation of inflammatory signaling, and elevated soluble factors such as sB7-H3. The study identifies for the first time a CRIP1⁺ non-malignant plasma cell subset expanded in AL patients, suggesting a possible non-specific B-cell response to amyloid-related antigens. More importantly, precise analysis of clonal plasma cells shows universal upregulation of proteostasis-related genes and a significantly increased IgL transcriptional burden, supporting a central role of proteotoxic stress in AL pathogenesis. This work not only deepens our understanding of the disease mechanism but also provides a solid foundation for developing novel biomarkers and targeted therapeutic strategies, with broad translational potential in immune microenvironment modulation and proteostasis intervention.
