Single cell and clonal analysis of AL amyloidosis plasma cells and their bone marrow microenvironment

AL amyloidosis is a disorder characterized by expansion of clonal plasma cells in the bone marrow and distant end organ damage mediated by misfolded immunoglobulin free light chains. There are currently limited data regarding the functional characteristics of AL amyloidosis plasma cells and their surrounding bone marrow microenvironment. We performed 5’ single cell RNA sequencing on 9 newly diagnosed, treatment naive AL amyloidosis patients and 8 healthy subjects. We identified generalized suppression of normal bone marrow hematopoiesis with distinct expansion of CD16 monocytes and subsets of CD4+ T cells in AL amyloidosis patients. We detected significant transcriptional changes broadly occurring among immune cells with increased interferon α and γ response and decreased TNF-α signaling. T and B cell receptor profiling revealed no overt clonal expansion of B or T cells in AL amyloidosis patients. However, we noted a disproportionate expansion of a distinct population of non-malignant plasma cells in AL amyloidosis patients. Finally, clonal AL amyloidosis plasma cells were identified based on their unique VDJ rearrangement and they showed increased expression of genes involved in proteostasis and antigen processing when compared to autologous, polyclonal plasma cells. Inter-patient transcriptional heterogeneity was evident, with transcriptional states reflective of common genomic translocations easily identifiable. Overall, this study defines the transcriptional characteristics of AL amyloidosis plasma cells and their surrounding bone marrow microenvironment, identifying transcriptional signatures that serve as candidates in early diagnosis in larger studies, and potential molecular targets for therapy. Bone marrow aspirates were obtained from patients with newly diagnosed AL amyloidosis seen at our center between 2019 and 2023. Healthy samples were obtained from discarded surgical material of patients undergoing primary, elective total hip arthroplasty. On the day of analysis, samples were rapidly thawed in a 37 degree C water bath, transferred to a 15 ml conical tube and washed in RPMI with 50% fetal bovine serum medium (Gibco). Cells were spinned for 5 minutes at 300 rcf and resuspended in PBS without calcium or magnesium (Gibco) with 0.4% BSA (Miltenyi). Cells were counted and viable cells enriched via Dead Cell Removal MicroBeads (Miltenyi) according to the manufacturer's protocol. Cells were resuspended in PBS with 0.4% BSA prior to viability check and encapsulation. Samples with viability less than 70% were not included in downstream processing. 10X Chromium Next GEM Single Cell 5’ reagent kits and BCR/TCR amplification kits were used for joint transcriptional and immune profiling of bone marrow samples. For the CD138+ samples, a combined profiling of BCR and 5’RNA was performed. For CD138- samples, a combined profiling of BCR, TCR, and 5’RNA was performed. The samples were aggregated into a fewer number of fraction-specific pools (details in associated publication) and loaded at a concentration of 20,000 cells per lane. The resulting libraries were sequenced on Illumina NovaSeq S1. *************************************************************** Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access. ***************************************************************