Mature Plasma Cells Research Articles

Flower-Shaped Plasma Cells in Multiple Myeloma with Morphological Heterogeneity.

Flower-shaped nuclei in plasma cells are rare in multiple myeloma. We report on an 88-year-old male who presented with a mass lesion in the clavicular region. A biopsy of the mass revealed an increase in mature plasma cells with round nuclei. In contrast, a bone marrow examination showed increased plasma cells with flower-shaped nuclei. The patient tested negative for human T-lymphotropic virus type-1 and was diagnosed with multiple myeloma. While multiple myeloma is known for intra-tumor heterogeneity, reports of morphological heterogeneity based on the site of tumor sampling are limited. In this case, the presence of plasma cells with flower-shaped nuclei enabled the identification of site-dependent morphological tumor heterogeneity.

Increased autoreactivity and maturity of EBI2+ antibody-secreting cells from nasal polyps.

Elevated numbers of antibody-secreting cells (ASCs) and anti-double-stranded DNA (anti-dsDNA) antibodies are found in nasal polyp (NP) tissue. The presence of anti-dsDNA IgG in tissue prospectively predicts recurrent NP but the characteristics of the source ASCs are unknown. Here, we investigated whether NP B cells expressing the extrafollicular marker EBI2 have increased propensity for autoantibody production and evaluated the molecular characteristics of NP ASCs. NPs showed increased frequencies of anti-dsDNA IgG and total IgG ASCs compared with tonsils, with more pronounced differences among EBI2+ cells. In NPs, EBI2+ cells were frequently double negative (IgD-CD27-) and ASCs. Single-cell RNA-Seq analysis of tonsils and NPs revealed substantial differences in B lineage composition, including differences in percentages of ASCs, germinal centers, proliferative cells, and non-ASCs. NPs exhibited higher expression of specific isotypes (IGHE, IGHA1, IGHA2, and IGHG4) and mature plasma genes, including SDC1 and XBP1, than tonsils. Gene Ontology biological processes indicated upregulated NF-κB and downregulated apoptosis pathways in NP ASCs. Together, these data indicate that NP EBI2+ ASCs secret increased total and anti-dsDNA IgG compared with those from tonsils and had molecular features of mature plasma cell differentiation.

Correlation between Morphological Typing and Monoclonality of Bone Marrow Plasma Cells and Its Diagnostic Value for High-Risk Smoldering Multiple Myeloma

To investigate the correlation between morphological typing and monoclonality of bone marrow plasma cells, and explore the diagnostic value of plasma cell morphological typing for high-risk smoldering multiple myeloma(HR-SMM). The correlation between the morphological characteristics and the monoclonality of bone marrow plasma cells was analyzed in 84 patients with HR-SMM who treated in our hospital. The consistency of morphologically abnormal bone marrow plasma cells with serum free light chain (sFLC) ratio, next-generation sequencing (NGS) detection results, and its correlation with monoclonal plasma cells detected by flow cytometry (FCM) were further verified. The immunoglobulin types and levels of non-involved immunoglobulins in serum of the patients were detected, and the distribution of plasma cell clusters in patients with different disease was observed. The mean percentage of mature plasma cells were decreased successively in the order of reactive plasmacytosis (RP) group, monoclonal gammopathy of undetermined significance (MGUS) group, smoldering multiple myeloma (SMM) group, HR-SMM group and multiple myeloma (MM) group; while the mean percentage of immature, primitive, reticular and flaming plasma cells were increased successively in the order of RP group, MGUS group, SMM group, and HR-SMM group, and the difference between any two groups was statistically significant (P < 0.05).The average proportion of abnormal plasma cells in the bone marrow of HR-SMM patients was 96.2% of the total plasma cells. The proportion of abnormal plasma cells were in good agreement with the sFLC ratio and the results of NGS detection in HR-SMM patients (kappa=0.879 and kappa=0.891, both >0.75)，and showed good correlation with the monoclonal plasma cells with immunophenotype of CD45-/CD38+/CD138+/CD56+/CD19-( γ=0.825). The levels of non-involved immunoglobulin in IgG, IgA and IgM type HR-SMM patients were all decreased by more than 25% compared with the normal reference range, and the differences were statistically significant (P < 0.05). There was no significant difference in the distribution ratio of plasma cell clusters among different disease groups (P >0.05). In HR-SMM patients, the immature, primitive, reticular and flaming plasma cells in bone marrow are considered as abnormal plasma cells, and they are correlated with monoclonal plasma cells. The proportion of abnormal plasma cells in total plasma cells of bone marrow and the reduction extent of non-involved immunoglobulin level in patients have certain reference value for the diagnosis of HR-SMM.

Cutaneous Plasmacytosis Treated with Tocilizumab.

Cutaneous plasmacytosis is a rare disorder of unknown etiology characterized by the proliferation of mature plasma cells in the skin and polyclonal hypergammaglobulinemia. Cutaneous plasmacytosis is associated with an elevated Interleukin-6 level. Anti-IL-6 therapy has not been reported as a therapy for idiopathic cutaneous plasmacytosis. This article presents a case of a Caucasian woman with idiopathic cutaneous plasmacytosis who was successfully treated with tocilizumab, a monoclonal anti-IL-6 receptor antibody. The patient experienced improvement in both her cutaneous lesions as well as subjective symptoms after 2 months on tocilizumab.

CCR6+ T helper cells and regulatory T cells in the blood and gastric mucosa during Helicobacter pylori infection.

Helicobacter pylori (H. pylori) can evade the host's immune response and persist for a long time on the gastric mucosa. T helper (Th) cells appear to be involved in the control of H. pylori bacteria but promote mucosal inflammation. In contrast, regulatory T cells (Tregs) may reduce inflammation but promote H. pylori persistence. CC motif chemokine receptor 6 (CCR6) is involved in the migration of various cells into inflamed gastric mucosa. In this study, we examined CCR6+ Th cells and CCR6+ Tregs during H. pylori infection in humans. Isolation of cells from blood and mucosal biopsies, magnetic separation of В cells, CD4+ and CD4+CCR6+CD45RO+ T cells, antigen-specific activation, B cell response in vitro, flow cytometry, determination of CD4+CD25hiFoxP3+ Tregs and various groups of Th cells. CD4+CCR6+ blood lymphocytes from healthy donors included Th cells and Tregs. These CCR6+ Th cells produced proinflammatory cytokines and also stimulated plasma cell maturation and antibody production in vitro. H. pylori gastritis and peptic ulcer disease were associated with an increase in the number of circulate CD4+CCR6+CD45RO+ cells and the percentage of Th1, Th17 and Th1/17 cells in this lymphocyte subgroup. In H. pylori-positive patients, circulating CD4+CCR6+ cells contained a higher proportion of H. pylori-specific cells compared with their CD4+CCR6- counterparts. H. pylori infection strongly increased the content of CD4+ lymphocytes in the inflamed gastric mucosa, with the majority of these CD4+ lymphocytes expressing CCR6. CD4+CCR6+ lymphocytes from H. pylori-infected stomach included Tregs and in vivo activated T cells, some of which produced interferon-γ without ex vivo stimulation. H. pylori infection causes an increase in the number of mature CD4+CCR6+ lymphocytes in the blood, with a pro-inflammatory shift in their composition and enrichment of the gastric mucosa with CD4+CCR6+ lymphocytes, including CCR6+ Th1 cells and Tregs.

Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation

AbstractPlasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high throughput sequencing (scATAC-seq) to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to activating protein 1 (AP-1) transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription–mediated reprogramming events that sustain PC differentiation.

New insights into the ontogeny, diversity, maturation and survival of long-lived plasma cells.

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.

Unveiling the impact of tertiary lymphoid structures on immunotherapeutic responses of clear cell renal cell carcinoma.

Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that form under pathological conditions. However, the predictive value of TLS in clear cell renal cell carcinoma (ccRCC) for immunotherapies remains unclear. We comprehensively assessed the implications for prognosis and immunological responses of the TLS spatial and maturation heterogeneity in 655 ccRCC patients. A higher proportion of early-TLS was found in peritumoral TLS, while intratumoral TLS mainly comprised secondary follicle-like TLS (SFL-TLS), indicating markedly better survival. Notably, presence of TLS, especially intratumoral TLS and SFL-TLS, significantly correlated with better survival and objective reflection rate for ccRCC patients receiving anti-Programmed Cell Death Protein-1 (PD-1)/Programmed Cell Death-Ligand-1 (PD-L1) immunotherapies. In peritumoral TLS cluster, primary follicle-like TLS, the proportion of tumor-associated macrophages, and Treg infiltration in the peritumoral regions increased prominently, suggesting an immunosuppressive tumor microenvironment. Interestingly, spatial transcriptome annotation and multispectral fluorescence showed that an abundance of mature plasma cells within mature TLS has the capacity to produce IgA and IgG, which demonstrate significantly higher objective response rates and a superior prognosis for ccRCC patients subjected to immunotherapy. In conclusion, this study revealed the implications of TLS spatial and maturation heterogeneity on the immunological status and clinical responses, allowing the improvement of precise immunotherapies of ccRCC.

Atypical Morphological Presentation of Neoplastic Plasma Cells: A Series of Five Cases

The diagnosis of Multiple Myeloma (MM) is made by demonstration clonal plasma cells in Bone Marrow (BM) aspiration/biopsy, in addition to assessing serum biochemical parameters, conducting radiological examinations, and considering the clinical presentation. In most cases, predominantly mature plasma cells are observed, along with scattered immature forms in the BM. Several morphological variants of plasma cells have been reported, including Auer rod-like inclusions, small lymphocyte-like cells, hairy cell-like cells, anaplastic variants, promonocyte-like cells, crystal-storing histiocytes, Burkitt-like cells, and blastoid cells. In this series of five cases, most showed a typical clinical presentation and laboratory findings suggestive of plasma cell dyscrasia. However, the morphology of each case exhibited unusual morphological variants, posing diagnostic challenges. These variants included Auer rod-like inclusions, small lymphocytes/lymph-plasmacytoid cells, hairy-like cells, multilobulated nuclei, and anaplastic variants mimicking dysplastic megakaryocytes, leading to various differential diagnoses. The age range of these cases was 57-76 years. Most of the cases presented with generalised dull aching body pain. Imaging studies revealed lytic lesions involving various parts of the bone, including the skull, ribs, vertebrae, and femur. Biochemical assays suggested the possibility of plasma cell dyscrasia. Two of the cases had primary Plasma Cell Leukaemia (PCL), which is a rare and highly aggressive plasma cell neoplasm. The anaplastic variant is associated with a poor prognosis, aiding in predicting treatment responses. However, due to the unusual morphological presentation, the diagnosis of MM or PCL was made after conducting ancillary studies such as Serum Protein Electrophoresis (SPEP), serum free light chain assay, Immunofixation Electrophoresis (IFE), and immunophenotyping through Immunohistochemistry (IHC) or flow cytometry

Chemical chaperone TUDCA selectively inhibits production of allergen-specific IgE in a low-dose model of allergy.

The cellular response to endoplasmic reticulum (ER) stress accompanies plasma cell maturation and is one of triggers and cofactors of the local inflammatory response. Chemical chaperones, low-molecular substances that eliminate pathological ER stress, are proposed as means of treating pathologies associated with ER stress. The aim of this study was to evaluate the effect and mechanisms of influence of chemical chaperones on the humoral response in a low-dose model of allergy. The allergic immune response was induced in BALB/c mice by repeated administration of ovalbumin at a dose of 100 ng for 6 weeks. Some animals were injected with both the antigen and the chemical chaperones, TUDCA (tauroursodeoxycholic acid) or 4-PBA (4-phenylbutyrate). Administration of TUDCA, but not 4-PBA, suppressed production of allergen-specific IgE (a 2.5-fold decrease in titer). None of the chemical chaperones affected the production of specific IgG1. The effect of TUDCA was associated with suppression of the switch to IgE synthesis in regional lymph nodes. This phenomenon was associated with suppressed expression of genes encoding cytokines involved in type 2 immune response, especially Il4 and Il9, which in turn could be caused by suppression of IL-33 release. In addition, TUDCA significantly suppressed expression of the cytokine APRIL, and to a lesser extent, BAFF. Thus, TUDCA inhibition of the allergy-specific IgE production is due to suppression of the release of IL-33 and a decrease in the production of type 2 immune response cytokines, as well as suppression of the expression of the cytokines APRIL and BAFF.

MORPHOFUNCTIONAL STATE AND MICROSTRUCTURE OF REGIONAL LYMPH NODES IN EXPERIMENTAL HYPOTHYROIDISM AND UNDER CORRECTION

Lymph nodes play an important role in the life of humans and animals. In the modern concept, the lymph nodes are the main homeostatic organs of the internal environment of the body, the lymph nodes are part of the lymphatic bed, on the other hand, they are the organs of the lymphoid (immune) system. Purpose: The work presents data on the morphofunctional state and microstructure of regional lymph nodes in experimental hypothyroidism and under correction. Materials and Methods: Experimental hypothyroidism in animals was induced by per os administration of mercazolil at a dose of 20 mg per 100 g of body weight with drinking water for 21 days. To correct thyroid disorders, starting from the 22nd day of the experiment, the animals received for 30 days an iodine-containing dietary supplement "Renaissance Plus" balm in powder at the rate of 2 μg/100 g of body weight and a decoction of the roots of the white cinquefoil (Potentilla alba L.) 50 ml per 100 g per day with drinking water. Results: The results of the study showed that in conditions of hypothyroidism, the lymph node becomes compacted with a decrease in the main structural and functional zones against the background of reduced lymphopoietic function. In the lymphoid nodule, there is a decrease of 1.43 times in the number of lymphoblasts, 1.56 times in the number of medium lymphocytes, 1.50 times in the number of small lymphocytes due to an increase of 1.29 times in the number of macrophages per unit area of the lymphoid nodule in the lymph node. There was a static decrease in the number of small lymphocytes in all structural and functional areas; the number of blasts decreases in the lymphoid nodule and increases in the paracortex and Billroth's strand; the number of macrophages increases in the lymphoid nodule, cerebral sinus, the number of mature plasma cells decreases in the paracortex and Billroth's strand, and there is also inhibition of cellular reactions in the lymph node, which reflects a decrease in the lymphopoietic function of immune activity and as a result of the immunosuppressive action of mercazolil. Conclusions: After an administration of corrigents, the area of the medulla and paracortex was restored to the control level and the ratio of cortical and medulla in the overall structure of the lymph node was leveled.

Novel SLC5A6 mutations lead to B lymphocyte maturation defects with metabolic abnormality rescuable by biotin replenishment

We characterized a family diagnosed with immunodeficiency disease presenting with low immunoglobulin levels and skin dyskeratosis. Exome sequencing revealed compound heterozygous missense variants in SLC5A6, the gene encoding a cellular sodium-dependent multivitamin transporter (SMVT) responsible for transporting vitamins, including biotin (vitamin B7). We showed that the biotin deficiency was caused by the SLC5A6 variants resulting in defective B cell differentiation and antibody deficiency. Altered cellular metabolic profiles, including aberrant mitochondrial respiration and reliance on glycolysis, may underlie the failure in plasma cell maturation. Replenishment of biotin improved plasma cell maturation and recovered the antibody producing activity in the patient and in a CRISPR-Cas9 gene-edited mouse model bearing a patient-specific SLC5A6 variant. Our results demonstrate the critical role of metabolic reprogramming in the maturation of plasma cells and nominate SLC5A6 as a causative gene for immunodeficiency that may be treated by biotin replenishment.

Assessing a New Paradigm in Defining High Risk Precursor Disease State for Patients with Plasma Cell Dyscrasia: Fluctuating Monoclonal Protein

Multiple Myeloma (MM) is a malignancy originating from mature plasma cells in the bone marrow, typically preceded by clinically asymptomatic precursor conditions. Early identification of the risk of progression from these precursor states, like Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Myeloma, to full-blown MM is critical for improving patient outcomes. The current risk models for MM rely on limited clinical and laboratory parameters, unable to fully encompass the complex nature of the disease continuum. To overcome these challenges, developing and validating a more comprehensive risk model is imperative, incorporating a wider array of clinical, genetic, and immune-related factors. This approach will enhance predictive accuracy and clinical applicability significantly. Notably, changes in serum monoclonal protein spike (M-spike) levels can reflect dynamic fluctuations in the net clonal mass, representing the ongoing battle between the plasma cell clone and the anti-myeloma immune response within patients. However, the impact of this highly dynamic state on the risk of progression to MM remains largely unexplored. Our study aims to bridge this knowledge gap by investigating the association between the dynamic M-spike pattern and the risk of progression from a precursor clone to MM, offering valuable insights into a potential novel prognostic marker for disease advancement. Methods: This study aimed to redefine high-risk precursor states in patients with plasma cell dyscrasias, focusing on positive serum electrophoresis (SPEP) results recorded between January 2011 and January 2021. We categorized precursor states as patients with positive SPEP, excluding those who had received active multiple myeloma (MM) treatment within 90 days of their first positive SPEP, showed M-spike values exceeding 3g/l, or displayed evident signs of disease progression. To assess M-spike patterns, we classified them as stable or fluctuating, using a 20% difference criterion to differentiate between the two (i.e., fluctuations less than 20% from baseline were labeled as stable). The time-to-treatment (TTT) was calculated from the first positive SPEP to the initiation of treatment, with death considered a competing risk. Results: The study included 565 patients, among whom 372 showed a stable M-spike pattern, while 193 exhibited a fluctuating pattern. The racial composition of the study population was predominantly white (75.58%), followed by black (21.77%) and other races (2.65%). The distribution of races was virtually identical in both the fluctuation and stable groups (p=0.977). The fluctuation group had a slightly lower proportion of females (49.7%) than the stable group (53.2%), but this difference was not statistically significant (p=0.432). Univariate analysis revealed that patients in the fluctuating group had a significantly higher odds ratio (OR) of requiring treatment (OR: 4.5, 95% CI: 1.37 - 14.18, p = 0.013) than those in the stable group. Interestingly, the prevalence of autoimmune conditions differed significantly between the groups. While 11.86% of the whole group had an autoimmune disorder, the proportion was lower in the fluctuation group (9.8%) than in the stable group (16.7%) (p=0.028).Confirming these findings, the multivariate time-to-event analysis further demonstrated that patients with fluctuating M-spike patterns had a greater likelihood of needing therapy (Hazard Ratio: 3.73, 95% CI: 1.19 - 11.66, p = 0.024) compared to those with stable patterns. Conclusion: Our study introduces a novel approach to defining high-risk precursor states in patients with plasma cell dyscrasias, emphasizing the significance of undulating monoclonal protein patterns. The dynamic nature of these fluctuations, representing the delicate interplay between pro-growth myeloma clones and anti-tumor immunity, emerges as a strong predictor of progression to MM. Further investigation and validation of these findings in more extensive studies hold promise for advancing personalized risk assessment and the development of targeted therapeutic strategies to prevent or delay the onset of multiple myeloma.

The Therapeutic Efficacy of CD19 Chimeric Antigen Receptor T Cells in Immune Thrombocytopenia Model

Abstract Introduction: ITP is a hemorrhagic autoimmune disease characterized by excessive platelet destruction and impaired platelet production,resulting in a high risk of bleeding. The pathogenesis of ITP involves immune intolerance towards platelet antigen, with platelet autoantibodies produced by plasma cells playing a significant role. Current treatments targeting this pathogenesis mainly rely on CD20 monoclonal antibodies, such as rituximab. However, as CD20 expression is present at the pre-B cell and mature B cell stages but absent on the surface of mature plasma cells. This can explain why a subset of autoantibody-positive patients do not respond to B-cell depletion therapy. CAR-T cell therapy, a form of adoptive cellular immunotherapy, has shown great potential in hematological malignancies and solid tumors. Recent studies have demonstrated its potential effectiveness of CAR-T cell therapy in various autoimmune diseases. CD19, expressed at all stages of B cell differentiation, presents an opportunity for CAR-T cell therapy in ITP. This study aims to evaluate the efficacy of CD19 CAR-T cell immunotherapy in an active murine ITP model. Methods: CD42-knockout mice were immunized with wild type (WT) C57BL/6 mice and splenocytes from these immunized mice were transplanted into irradiated WT mice to construct a murine model of active ITP. The ITP mice were then randomized into two groups, with the experimental group receiving an intraperitoneal injection with 1×10 6 CD19 CAR-T cells. Platelet counts were monitored weekly, and after 3 weeks, the ITP mice were euthanized and sacrificed. Single-cell suspensions were prepared from harvested spleens for analysis. Anti-murine antibodies were analyzed using flow cytometry (Beckmann Coulter, Brea, CA, USA). All data were analyzed by Statistical Package for the Social Sciences (SPSS) software, version 25.0 (IBM Corp., Armonk, NY, USA). P &amp;lt; 0.05 was considered statistically significance. Results: Our results demonstrated that the platelet counts of ITP mice reached their lowest level on day 7 after spleen cell transplantation, gradually recovering within a week. Platelet counts were significantly higher in the CD19 CAR-T cell therapy group compared to the control group on day 7 and persisted until day 21. There were no significant differences in weight between two groups, indicating no severe complication or CRS were identified. In the third week CD19 CAR-T cell therapy, the proportion of plasma cells in the spleen was determined, showing a marked decrease in the percentage of CD138+ plasma cells in the CAR-T group compared to the control group. Additionally, at day 14, the titers of anti-platelet CD42-specific antibodies were significantly lower in the CAR-T cell treated group compared to the control group. Furthermore, the mean fluorescence intensity (MFI) of CD19+ B cells in the spleen cells of the CD19 CAR-T cell immunotherapy group was significantly decreased compared to the control group. Conclusion: This study demonstrates the effectiveness of CD19 CAR-T cell therapy in treating thrombocytopenia by suppressing plasma and B cells in a mouse model of active ITP. These findings provide valuable insights into the potential therapeutic application of CD19 CAR-T cell immunotherapy for immune thrombocytopenia. Disclosure: No conflict of interest.

Optimizing Ex-Vivo Expanded NK Cell- Mediated Cellular Cytotoxicity By Obinutuzumab Combined with NKTR-255 in Burkitt Lymphoma (BL)

Background: We have previously reported the success of treating children with Burkitt lymphoma (BL) resulting in a ≥90% long-term EFS utilizing short intensive rituximab containing chemoimmunotherapy (Cairo et al, Blood, 2007; Goldman /Cairo et al, Leukemia, 2012, Goldman /Cairo et al, Br J Haematol., 2014; Goldman /Cairo et al, Leukemia, 2021). However, the prognosis is dismal in children who are refractory or progress after chemoimmunotherapy (Cairo et al Br J Haematol. 2018). The mechanisms associated with this poor prognosis are mainly due to chemoradioimmunotherapy resistance and an immunosuppressive BL microenvironment (TME) (Cairo et al Br J Haematol. 2016; Cairo et al Br J Haematol. 2019) and represent an urgent unmet need. The CD20 molecule is universally expressed by normal B cells in all stages of development, from the pre-B cell up to the mature plasma cell as well as by most B cell malignancies including CLL, FL and BL (Chu/Cairo, BJH, 2016). Obinutuzumab is a humanized, type II anti-CD20 monoclonal antibody glycoengineered to enhance Fc receptor affinity. It has lower complement-dependent cytotoxicity than rituximab but greater ADCC, phagocytosis and direct B-cell killing effects (Chu/Cairo, BJH, 2018). Obinutuzumab has been successfully utilized in front-line therapy in FLL (Marcus, et al, NEJM, 2017) and CLL (Goede, et al, NEJM, 2014; Moreno, et al, Lancet, 2019). Our group has successfully ex-vivo expanded functional and active peripheral blood NK cells (PBNK) with irradiated feeder cells APC (K562-IL21-41BBL) (Chu/Cairo, et al, JITC 2020). We previously demonstrated that obinutuzumab has significantly enhanced ex-vivo expanded PBNK mediated cytotoxicity against BL and pre-B-ALL cell lines compared to rituximab (Tiwari/Cairo et al, BJH, 2015). NKTR-255 is an IL-15 receptor agonist designed to activate the IL-15 pathway and NK cells and promote the survival and expansion of memory CD8+ T cells without inducing suppressive regulatory T cells (Kuo/Zalevsky, Cancer Res. 2017). NKTR-255 stimulates proliferation and survival of NK, CD8+ T cells, which may lead to sustained anti-tumor immune response. Objective: To investigate the anti-BL effects of NKTR-255 on the ADCC of ex-vivo expanded NK cells with obinutuzumab against rituximab-resistant BL in vitro and in vivo using human BL xenografted NSG mice. Methods: NK cells were expanded with lethally irradiated K562-mbIL21-41BBL cells as previously described (Denman/Lee, et al, PLoS One, 2012). Expanded PBNK cells were isolated using Miltenyi NK cell isolation kit. NKTR-255 was generously provided by Nektar Therapeutics. In vitro cytotoxicity was examined using luminescence reporter-based assays. IFN-g, granzyme B and perforin levels were examined by standard enzyme-linked immunosorbent assays as we previously described (Chu/Cairo, Front. Immunol, 2022). Rituximab-resistant BL cells Raji-2R and Raji-4RH were used as target cells. Luciferase expression Raji-4RH cells were xenografted to NSG mice as we previously described (Chu/Cairo, et al, JITC 2021). Results: NKTR-255 significantly enhanced the proliferation of ex-vivo expanded NK cells (p&amp;lt;0.0001) at day 7. NKTR-255 significantly enhanced the in vitro cytoxicity of expanded NK cells when combined with obinutuzumab against rituximab-resistant BL cells like Raji-2R (E:T=3:1, p &amp;lt;0.01), and Raji-4RH (E:T=3:1, p&amp;lt;0.01) as compared to the control groups. NKTR-255 also significantly enhanced IFN-g, granzyme and perforin release from expanded NK cells when combined with obinutuzumab against Raji-2R (E:T=3:1, IFN-g: p&amp;lt;0.001, granzyme: p&amp;lt;0.001 and perforin: p&amp;lt;0.001) and Raji-4RH (E:T=3:1, IFN- g: p&amp;lt;0.001, granzyme: p&amp;lt;0.01 and perforin: p&amp;lt;0.01) as compared to controls. Our in vivo study demonstrated that the combination of NKTR-255, Obinutuzumab and expanded NK cells significantly improved the survival of mice xenografted with Raji-4RH compared to controls (Fig.1). Conclusion: We found that NKTR-255 significantly enhanced the ADCC of expanded NK cells with Obinutuzumab against rituximab-resistant BL cells in vitro with enhanced IFN- g, granzyme B and perforin release. The in vivo effects of NKTR-255 with expanded NK cells and Obinutuzumab against rituximab-resistant BL cells using humanized NSG models are very promising. Mechanisms studies of BL relapsed from the combination therapy are under investigation. (Supported by HHOW and St Baldrick grants).

The Potent Dihydroorotate Dehydrogenase Inhibitor, Hosu-53, Exhibits Compelling Monotherapy Efficacy in Multiple Myeloma and Augments CD47 Targeted Therapy

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Alleviates Chronic Graft- Versus-Host-Disease Via Modulating the CXCR5/CXCL13 Axis

Introduction: Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic hematopoietic cell transplant (allo-HCT) often leading to transplant-mediated mortality. Studies have shown that T follicular helper cells (Tfh) upregulate CXCR5 and migrate into the lymphoid organ follicles in response to CXCL13 promoting germinal center (GC) B cell proliferation and differentiation of plasma cells resulting in pathogenic antibody production and profibrotic pathways associated with cGVHD. In contrast, T follicular regulatory cells (Tfr) modulate the magnitude of the GC response; thus, the balance of Tfr/Tfh could be crucial to mitigating cGVHD.Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers influencing transcription of a multitude of inflammatory genes and our lab has previously reported pharmacological inhibition of BET, through OPN-51107, improves survivability and limits disease progression in murine models of acute GVHD that led to opening the first-in-human clinical trial of OPN-51107 in steroid refractory acute GVHD. We demonstrated BET proteins to be important in modulating acute GVHD pathogenesis, however, mechanisms of BET regulation within cGVHD are largely unknown. Here, using OPN-51107 we aim to further investigate mechanisms of cGVHD pathogenesis and identify successful BET inhibition-based therapeutic approaches. Methods: We used a Bronchiolitis Obliterans model (B6-&amp;gt;B10.br) of cGVHD. Mice were administered 120mg/kg cyclophosphamide (D-3 and D-2) and 8Gy irradiation (D-1) as a conditioning regimen followed by B6 T-cell depleted bone marrow cells with and without CD45.1 B6 splenocytes. Mice that did not receive allogeneic splenocytes served as controls and did not develop GVHD. Recipients of allogeneic splenocytes were treated with a BET inhibitor - OPN-51107 at 10mg/kg 3x/weekly, or vehicle, from D28-54(+ 4) via oral gavage. Resting lung elastance and resistance was measured using the Flexivent system. At the time of lung function analysis, lung and liver tissue was harvested and stained via Hesson's Trichrome to identify and quantify collagen deposition. We performed immunofluorescence to quantify alveolar alloantibody deposition. Spleen and lung cells were stimulated with PMA/I and analyzed via spectral flow cytometry. An inflammatory murine cytokine array (#ARY006, R&amp;D Systems) was used to analyze circulating plasma cytokine levels. Results: BET inhibition via OPN-51107 treatment decreased lung resistance (vehicle vs OPN-51107 =1.16Rrs vs 0.83Rrs p&amp;lt;0.05) and elastance (vehicle vs. OPN-51107=62Ers vs. 45Ers p&amp;lt;0.05). OPN-51107 treatment also significantly reduced alveolar antibody (Ig) deposition per area compared to vehicle (vehicle vs. OPN-51107= 4.91% vs. 3.3% p=0.01) and collagen deposition (vehicle vs. OPN-51107=19.0% vs. 12.9% p&amp;lt;0.05) and liver (vehicle vs. OPN-51107=19.3% vs. 2.35% p&amp;lt;0.0001). Congruently, BET inhibition decreased B220-CD19- CD138+mature plasma cells (vehicle vs. OPN-51107=15% vs 9.1% p&amp;lt;0.05) in the lung. Serum cytokine array analysis revealed a significant decrease in circulating CXCL13, relevant to Tfh migration as well as a decrease in other proinflammatory cytokines - CXCL9, MCP-1, IL3, RANTES and G-CSF important for macrophage activation and pathogenicity in OPN-51107 compared to vehicle treated mice. BET inhibition reduced splenic Tfh while increasing Tfr, thereby increasing Tfr:Tfh ratio (vehicle vs. OPN-51107= 5% vs. 9% p&amp;lt;0.05). Furthermore, total splenic CD19+ B-cells (vehicle vs. OPN51107= 70% vs. 33% p&amp;lt;0.001) as well as CXCR5 expression on B-cells were decreased in OPN-51107 treated mice (MFI vehicle vs. OPN-51107= 4744 vs. 571.5 p&amp;lt;0.0001). Conclusion: OPN-51107 improved lung function which correlated with significantly decreased lung fibrosis and antibody deposition in a BO model of cGVHD. BET inhibition reduces cGVHD immune pathology through decreased inflammatory cytokine production, promotion of GC Tfr cell population and modulates B-cell proliferation and/or trafficking. Significance: Using OPN-51107 in a BO model of cGVHD we demonstrate that BET inhibition is a feasible therapeutic strategy to reduce disease severity and improve lung function.

Multiple Myeloma B Cells and Pre-Plasma Cells Are Important Reservoirs for Myeloma Relapse Following Plasma Cell-Directed Therapy and Prevent Cure with Standard Therapies

Multiple Myeloma (MM) is a B cell neoplasm characterized by the accumulation of mature plasma cells (PCs) within the bone marrow (BM). Despite treatment advances, MM remains incurable in the vast majority of patients. Disease relapse typically occurs regardless of treatment with proteasome inhibitors (such as bortezomib and carfilzomib), immunomodulatory drugs (such as lenalidomide and pomalidomide), anti-CD38 monoclonal antibodies (such as daratumumab and isatuximab) or myeloablative melphalan and autologous stem cell transplantation, and also occurs following treatment with immunotherapeutics such as bispecific antibodies and/or CAR-T cells (targeting BCMA, FcRH5 or GPRC5D). This failure to achieve reliable cure in MM, despite the routine attainment of deep treatment responses against PCs, indicates the existence of important intra-tumor heterogeneity and the presence of rare drug-resistant MM cells with full tumorigenic capacity. To identify intra-clonal MM cell subpopulations, we examined BM samples from MM patients using a combination of FACS, single cell resolved immunoflourescence-FISH (IF-FISH), whole exome sequencing (WES), custom-capture targeted deep sequencing (CC-Seq) and single cell RNA sequencing (scRNAseq). We sought to characterize the genomic landscape and to track the dynamic interconnectedness of these intra-clonal subpopulations in patients over time. Using sequential FACS-IF-FISH studies of MM BM samples (n=140) we identified MM clone cellular subpopulations within the BM of MM patients that recapitulate the normal maturation stages between non-malignant post germinal centre B cells and mature PCs. These subpopulations include rare MM cells that resemble CD20 +CD38 -CD138 -Bcma -Irf4 - Xbp1s - B cells, CD20 -CD38 -CD138 - Bcma -Irf4 -Xbp1s - pre-plasmablasts and CD38 +CD138 -Bcma -/lowIrf4 -/+Xbp1s + pre-plasma cells, as well as predominating tumor-bulk CD38 +CD138 +Irf4 +Bcma +Xbp1s + plasma cells. Importantly, MM progenitor cells were typically found to possess all of the chromosomal translocations and copy number variations (CNV) present in MM PCs including whole chromosome ploidy changes, translocations such as t(11;14), t(4;14) and t(14;16), and secondary aberrations such as gain(1q), del(1p) and del(17p). To examine if MM progenitor cells also possess the single nucleotide variations (SNV) present within MM PCs, and are thus fully malignant, and to track MM progenitor subpopulations and their inter-relatedness within patients over time, we performed WES (n=60) plus targeted deep CC-Seq (n=210) on purified cell subpopulations from 17 patients, including 5 patients whom we sampled serially over &amp;gt;3 years. A median of 5 cellular subpopulations were isolated from each BM sample, enriched by factors of up to 1000-fold or more, and each subpopulation was then sequenced to a depth of 4,000-20,000x. Significantly, SNVs that were present in tumor-bulk MM PCs were also regularly detected in MM progenitor cells, although the low frequency of the clonal progenitor cells often prevented capture of complete SNV profiles. At the same time, analyses of serial BM samples from repeat donor patients taken pre- and post-treatment demonstrated that relapsing MM PCs typically did not appear to derive linearly from pre-treatment PCs, as the relapsing PCs commonly lacked a number of SNVs that had been present in pre-treatment PCs. Importantly, we instead observed that relapsed MM plasma cells appeared to consistently derive from MM progenitor cell populations, frequently B cells, as emergent relapse-specific SNVs that were exclusively detected in relapsing post-treatment PCs (but in not pre-treatment PCs) could often also be detected within pre-treatment CD138- CD38- progenitor cells isolated from BM samples that had been collected up to 3 years prior to relapse. We conclude that MM B cells and pre-plasma cell progenitors possess all of the clonal genomic aberrations required for full malignant potential. Tracking of these cells in vivo in patients over time by their SNV profile implicates them as the cellular origin of PC relapse and recurrent MM disease. We propose that cure of MM requires eradication of MM progenitor cells alongside plasma cells. We are currently conducting scRNA-seq and CITE-seq studies of primary MM samples to further characterize MM progenitor cells for gene and protein expression in order to define their optimal therapeutic targets.

THU369 A Rare Case Of IgG4-related Autoimmune Pancreatitis And Diabetes Mellitus

Abstract Disclosure: M. Zahid: None. J. Shakil: None. Introduction: IgG4 related disease (IgG4-RD) is a rare multiorgan inflammatory condition characterized by dense lymphoplasmacytic infiltrates, presence of IgG4+ plasma cells and fibrosis. Here we present a case of young male with painless jaundice and hyperglycemia diagnosed with IgG4 related autoimmune pancreatitis and DM. Clinical Case: 46-year-old male with no significant history presented to outpatient clinic for pruritis and dark-colored urine of 2-week onset. He reported 17-pounds weight loss in the preceding month. While undergoing outpatient workup, patient developed epigastric pain prompting visit to the hospital. On examination, patient was afebrile, tachycardiac, icteric and had epigastric tenderness. Labs revealed normal electrolytes and renal function. LFTs were significant for total protein 7.6 g/dl (n=6.3-8.3 g/dl), AST 77 U/L (n = 10-50 U/L), ALT 127 U/L (n = 5-50 U/L), alkaline phosphatase 458 U/L (n = 40-129 U/L) and direct bilirubin &amp;gt;10.0 (n = 0-0.3 mg/dl). Lipase was 99 U/L (n - 13-60 U/L), blood glucose was 292 mg/dl and A1C was 6.5% (n = 4.0-5.6%). US abdomen revealed diffuse gallbladder wall thickening with moderate sludge and intrahepatic and extrahepatic distal biliary obstruction and dilatation. MRCP showed common bile duct and intrahepatic biliary ductal dilation. Pancreas was diffusely irregular with areas of hemorrhagic pancreatitis. ERCP showed dilatation of upper and middle third of main bile duct, left and right hepatic ducts and all intrahepatic branches with a stricture in distal bile duct. Biliary sphincterotomy and placement of temporary plastic biliary stent was performed and ampullary biopsies were taken. IgG4 level was checked due to suspicion for IgG4-RD and was 293 mg/dl (n: 1-123 mg/dl). Pathology revealed mucosa with epithelial reactive changes. CD138 stain showed several mature plasma cells within the inflammatory infiltrate. In addition, immunostain for IgG4 shows several IgG4 positive plasma cells forming small clusters confirming diagnosis of IgG4-RD. Post procedure, his abdominal pain and LFTs started improving. Patient was started on prednisone for IgG4-RD and insulin for persistent hyperglycemia. His prednisone dose was later tapered after starting methotrexate. Patient’s most recent LFTs have normalized except mildly elevated ALT and A1C improved to 6.2%. Conclusion: Autoimmune pancreatitis and cholangiopathy are the pancreatobiliary manifestation of IgG4-RD and most often present as painless jaundice mimicking pancreatic cancer. Endocrine insufficiency usually manifests as diabetes mellitus/pre-diabetes and may also lead to autoimmune hypophysitis and Reidel’s fibrosis. Other organ systems that may also get involved include lacrimal and salivary glands, lungs, kidneys, nervous system, and lymphatic/vascular system. Early recognition and ongoing surveillance are key to appropriate management of this condition. Presentation: Thursday, June 15, 2023