Bridging the identity and function of human plasma cells 3270

Plasma cells are unique immune effectors, capable of producing large amounts of high-affinity antibodies that protect against pathogenic infections. Although most plasma cells have short lifespans, certain conditions or vaccinations can give rise to long-lived plasma cells (LLPCs) that provide individuals with lifelong protection against pathogen exposure. The nature of these LLPCs is poorly understood; however, recent studies have shed new light on the ontogeny, diversity, maturation and survival of these unique cells. Whereas LLPCs had been thought to arise preferentially from germinal centres, novel genetic tools have revealed that they can originate from various stages throughout the humoral response. Furthermore, new single-cell analyses have shown that mouse and human plasma cells are heterogeneous and may undergo further maturation in situ in the bone marrow niche. Finally, plasma cells were previously considered to be sessile cells maintained in fixed survival niches, but new data show that plasma cell subsets can differentially migrate and organize into clusters that may be associated with survival niches. These descriptive findings provide new insights into how cell-intrinsic programmes and extrinsic factors may regulate the longevity of plasma cells in various contexts, which suggest new research avenues for their functional validation.

In Vitro Generation of “Long-Lived” Human Plasma Cells

Purpose:Although the association between cytotoxic T lymphocytes and favorable prognosis in colorectal cancer is well established, the prognostic significance of B lymphocytes remains more ambiguous. This study aimed to assess the characteristics and significance of various B-cell and plasma cell subsets in colorectal tumors.Experimental Design:We designed a seven-plex IHC assay, combined with machine learning–based image analysis, to identify various B-cell and plasma cell populations and applied it to study a cohort of 912 colorectal tumors. We assessed the prognostic significance of B-cell and plasma cell densities using Kaplan–Meier estimators and Cox regression models. Additionally, we designed a more clinically applicable three-plex assay, which we used to study B-cell and plasma cell densities in a separate validation cohort of 737 patients.Results:High plasma cell density in the center of the tumor was associated with longer cancer-specific survival independent of disease stage, mismatch repair status, T-cell densities, and other covariates. In the study cohort, the multivariable HR for high (vs. low) plasma cell density was 0.48 (95% confidence interval, 0.32–0.72; Ptrend = 0.0005), whereas the corresponding HR in the validation cohort was 0.37 (95% confidence interval, 0.21–0.65; Ptrend = 0.0003). Of the specific subsets, IgG1–IgG2– plasma cells showed the strongest association with improved survival. High B-cell densities were not independently associated with a better prognosis.Conclusions:Plasma cell densities in the center of the tumor represent a relevant tumor-immune biomarker in colorectal cancer, complementing T-cell density measurements.

Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function

Description High expression of the IRF4 transcription factor (TF) is required for upregulation of the Blimp-1 TF to commit an activated B cell toward the plasma cell (PC) fate. Our lab has shown in B cell-specific IRF4 haploinsufficient mice, not only is PC quantity expectedly diminished, but surprisingly, PC quality was impaired as well. Although these cells display the hallmarks of a functional PC, they fail to develop into complete PCs, suggesting there exists unknown genetic barriers to full PC development. We propose to use the IRF4 haploinsufficient model as a novel tool to investigate unknown genetic PC checkpoints. We hypothesize that dynamic accumulation of IRF4 fine-tunes the expression of agonistic and antagonistic PC regulators to dictate full PC differentiation and activity. We will collect genome-wide measurements of gene expression pattern, chromatin accessibilities, and IRF4 binding of wildtype and IRF4+/- PCs using RNA-, ATAC-, and ChIP-sequencing technologies. We will then integrate these datasets to establish a whole genome landscape of IRF4 activity and investigate differential patterns between wildtype and IRF4+/- PCs. Through differential analyses, we have identified several putative regulators including, Junb and miR-155, and continued investigation will enable us to determine direct or indirect IRF4 regulation. The goal of this study is to establish a more extensive gene regulatory network of PC differentiation and find yet-unknown barriers to PC differentiation. Funding Sources Supported by the Lodric Madox Fellowship, Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Contrary to short-lived plasma cells, which survive only 3-5 days, long-lived plasma cells (LLPCs) contribute to the humoral memory of the body and thus also to many antibody-related diseases. The ability of plasma cells to persist over months, years, and even a lifetime has been demonstrated in vivo. Yet, the in vitro culture of human primary bone marrow-derived plasma cells has been limited to a few days. Here, we establish culture conditions for human primary bone marrow-derived plasma cells for 21 days. Plasma cells and stromal cells are isolated from human bone marrow and cultured in 2D or a 3D ceramic scaffold. The plasma cells' survival and antibody secretion depend on direct contact with stromal cells. The culture promotes CD19-negative PCs. Inhibition of the PI3K or NF-kappaB pathways using chemical inhibitors reduced the survival of the plasma cells. These results underline the supportive role of the stromal cells for the survival of the LLPC and confirm mechanisms that were identified in mouse LLPCs also for human LLPCs. The culture described here will promote further studies to deepen our understanding of the human LLPC.

B cell development and longevity of IgE plasma cells.

Waldenström macroglobulinemia (WM) is characterized by monoclonal gammopathy, usually IgM, in association with lymphoplasmacytic lymphoma (LPL). Little is known of the expression of nuclear proteins involved in B-cell development in LPL/WM. In this study, the expression patterns of PAX5/BSAP, MUM1/IRF4, and PRDM1/BLIMP1 were analyzed in plasma cells and lymphocytes in 29 cases of newly diagnosed LPL/WM by double immunohistochemical staining with CD138 and CD22. These patterns were compared with the expression profiles seen in normal bone marrow samples, reactive tonsils, and cases of plasma cell myeloma and marginal zone lymphoma. The median percentage of plasma cells coexpressing CD138 and PAX5 was significantly higher in LPL/WM compared with benign tissues (P = .001), marginal zone lymphoma (P = .002), and plasma cell myeloma (P < .0001), whereas the median percentage of plasma cells coexpressing CD138 and MUM1 was lower in LPL/WM than plasma cells in benign tissues (P = .02), marginal zone lymphoma (P = .001), and plasma cell myeloma (P = .0002). These findings show that a subset of plasma cells in LPL/WM demonstrates a nuclear protein expression pattern characteristic of the B-cell developmental program. Thus, the results better define the immunophenotypic profile of the neoplastic cells in LPL/WM.

Very late antigen (VLA) expression by normal and neoplastic human plasma cells; including an assessment of antibodies submitted to the Vth International Workshop on Leucocyte Differentiation Antigens using human myeloma cell lines

Multiple myeloma (MM) is characterized by the malignant proliferation of plasma cells within the bone marrow. The transcriptional mechanisms governing plasma cell differentiation and MM pathogenesis are regulated by an intricate network of transcription factors, the role of RUNX1 in this process remains poorly defined. This study aimed to characterize plasma cell subsets in MM and evaluate the expression and functional role of RUNX1 during B cell differentiation. Bone marrow and peripheral blood samples were collected from 61 MM patients and 18 healthy donors. Flow cytometry was used to identify B cell and plasma cell subsets and measure RUNX1 expression across B cell maturation stages. Functional validation was conducted via siRNA-mediated RUNX1 knockdown in CD19+ B cells followed by in vitro plasma cell differentiation assays. Our data showed that MM patients exhibited significantly increased proportions of plasma cells in bone marrow compared to healthy controls. Intriguingly, RUNX1 expression was low in naive B cell subsets but increased progressively through plasmablast, pre-plasma, and plasma stages. Although RUNX1 expression did not significantly differ across MM stages or between newly diagnosed and relapsed/refractory cases, plasmablasts from MM patients showed higher RUNX1 levels than those from controls. Knockdown of RUNX1 in vitro using siRNA delayed B cell differentiation transiently. In summary, RUNX1 expression is dynamically upregulated during terminal B cell differentiation and is elevated in MM-derived plasmablasts. These findings provide new insights into a potential role for RUNX1 in the B cell differentiation axis and MM disease progression.

P-064: Heterogeneity of bone marrow biopsy and bone marrow aspirate (BMA) in patients with heavily pretreated relapsed/refractory Multiple Myeloma (RRMM)

THERE is considerable evidence that lymphocytes bearing immunoglobulins on their surfaces are present in the peripheral blood of various animal species as well as man1–3. Takahashi et al.4 indicated that neoplastic, as well as normal mouse plasma cells, contained specific surface alloantigens. These plasma cells do not, however, contain surface immunoglobulin determinants5. Harris et al.6,7 demonstrated that neoplastic plasma cells of the mouse could be used to prepare specific antisera against normal plasma cells. These anti-plasma cell sera (APS) were found to suppress specifically primary and secondary humoral immunity without apparently affecting cellular immunity. Recently, we established a long term culture of human plasma cells8 from a localised plasmacytoma tumour of a patient with multiple myeloma. The cultured cells morphologically resembled plasma cells, and they secreted an IgG immunoglobulin antigenically identical to the patient's myeloma protein. Using cells from this source, an anti-human plasma cell serum (HuAPS) has been prepared which recognises a surface antigen on human plasma cells.

Therapeutic B cell depletion with monoclonal antibodies targeting CD20 forced a rethink about the pathogenic role of B cells and plasma cells in autoimmune diseases; however, it was tempered by frequent clinical relapses or nonresponse to CD20-directed therapy. Here, we re-evaluate B cell depletion strategies in autoimmunity prompted by 4 recent advances. The first is analysis of clonal accumulations of CD20- CD19+ plasma cells making autoantibodies in patients with anti-CD20 refractory autoimmune disease. The second is the remarkable clinical remissions induced by anti-CD19 chimeric antigen receptor T cells in cases of anti-CD20 refractory autoimmunity. The third is evidence that CD19+ plasma cells comprise the majority of plasma cells in humans, are not terminally differentiated, are long-lived, and if self-reactive have potent capacity to capture autoantigens via their surface immunoglobulin and present major histocompatibility complex class II-bound peptides. The fourth is the role of autoantigen-binding B cells and CD19+ plasma cells as key antigen-presenting cells in "T cell-mediated" autoimmune disorders, type 1 diabetes and celiac disease. Viewing human memory B cells and plasma cells from this alternative perspective offers an explanation for why deep CD19 compartmental depletion may be effective at achieving complete and durable remissions in the autoantibody-positive autoimmune diseases as a group, irrespective of whether the autoantibody is pathogenic.

Healthy Donor Whole Bone Marrow Cells Preconditioned With Myeloma Patient Serum Support Long-Term Survival Of Primary Myeloma and Reveal Altered Microenvironmental Pathways

IgA-Producing Plasma Cells Originate From Germinal Centers That Are Induced by B-Cell Receptor Engagement in Humans