B-cell Lines Research Articles

Mosaic chromosomal alterations (mCAs) in individuals with monoclonal B-cell lymphocytosis (MBL).

MBL is a precursor condition to chronic lymphocytic leukemia (CLL), characterized by monoclonal B-cells in blood. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis that include gains, losses, and copy-neutral loss-of-heterozygosity of large DNA segments. Both MBL and mCAs have been found to increase the risk of CLL and lymphoid malignancies, and the aim of our study was to investigate how mCAs relate to MBL, which is currently unknown. We analyzed genetic, flow cytometric, and hematologic data from 4632 individuals from the Mayo Clinic Biobank and CLL Database. MBL was detected using flow cytometry and classified as high-count (HC) or low-count (LC) MBL based on clone size. mCAs were detected primarily from whole blood DNA using sensitive SNP-array-based analyses. mCAs commonly altered in CLL (deletion of 6q, 11q, 13q, 17p, and trisomy 12) were specific (>99%) to individuals with MBL and CLL. HC-MBL and LC-MBL individuals were 881-fold and 8-fold, respectively, more likely to harbor CLL-associated mCAs than those without MBL. The cell fraction bearing these mCAs typically exceeded the B-cell fraction, suggesting their origin prior to the B-cell lineage. Integrating genetic and blood count data enabled detecting HC-MBL with high specificity in a biobank sample. These results quantify the contribution of mCAs to MBL and could enable large studies of HC-MBL without the need for flow cytometric screening.

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the DNA of the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of IGV gene subgroup-specific primers recognizing nearly all IGV genes together with primers for the J genes. By sequence analysis of IGV region genes from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D and J gene usage. Moreover, the presence and pattern of somatic IGV gene mutations give valuable insight into the differentiation stage of the B cells.

An unusual case of a de novo high-grade B cell neoplasm with MYC and BCL2 rearrangements, strong TdT expression, and BRAF V600E mutation

Abstract Introduction/Objective Aggressive de novo B cell neoplasms can rarely exhibit overlapping features of B- lymphoblastic leukemia/lymphoma (B-ALL) and high-grade B cell lymphoma (HGBL) making their definitive classification challenging. The distinction between mature and immature B cell neoplasms is critical as treatment approaches differ. We report a unique case of an aggressive B cell neoplasm that not only posed diagnostic difficulties but was also found to harbor a BRAF V600E mutation, an unusual finding in such cases. Methods/Case Report A 33-year-old man without any significant past medical history presented for evaluation of a large tonsil mass and diffuse lymphadenopathy. The tonsil was excised, and histologic examination showed a diffuse atypical infiltrate composed of medium to large monomorphic lymphoid cells with high-grade cytology. By immunohistochemistry, the atypical cells were positive for CD19, CD79a, CD22, PAX5, CD10 (strong), CD45 (strong), BCL2, MYC, and TdT (strong). Ki67 showed a high proliferation index (80%). The atypical cells were negative for CD20, CD34, CD1a, CD30, Cyclin D1, T cell markers, myeloid markers, HHV8, and EBER. Flow cytometry detected a CD10 positive B cell population that was negative for CD20, CD34, and surface light chain expression. Fluorescence in-situ hybridization (FISH) detected BCL2 and MYC rearrangements. Staging marrow showed diffuse involvement by a neoplastic infiltrate with similar features to those observed in the tonsil. Chromosome analysis showed a complex karyotype. The patient was treated with two cycles of da-EPOCH-R. A neck lymph node biopsy two months later showed persistent disease. Next-generation sequencing (NGS) on this sample demonstrated a BRAF V600E mutation at 31% allele frequency. A mutation-specific BRAF-VE1 immunostain was diffusely positive. Results (if a Case Study enter NA) NA Conclusion This was a diagnostically challenging case of a de novo B-cell lineage neoplasm with high-grade features, MYC and BCL2 rearrangements, and strong diffuse TdT expression. There is limited literature on mutational profiles and prognostic data for such cases, and whether they are best classified (and treated) as HGBL or B-ALL remains challenging and controversial. In our case, we also discovered an unexpected finding of a BRAF V600E mutation, which has not been reported in similar cases in the existing literature and could serve as a potential therapeutic target. Additional reporting and genetic profiling of such cases may help further elucidate their biology and guide treatment protocols.

YY1 knockout in pro-B cells impairs lineage commitment, enabling unusual hematopoietic lineage plasticity.

During B-cell development, cells progress through multiple developmental stages, with the pro-B-cell stage defining commitment to the B-cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We found here that knockout of YY1 at the pro-B-cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9-DL4 feeder system and in vivo after injection into sublethally irradiated Rag1-/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T-cell lineage profile. Single-cell RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells in vitro, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages, indicating unusual lineage plasticity. In addition, YY1 KO pro-B cells in vivo can give rise to other hematopoietic lineages in vivo. Evaluation of RNA-seq, scRNA-seq, ChIP-seq, and scATAC-seq data indicates that YY1 controls numerous chromatin-modifying proteins leading to increased accessibility of alternative lineage genes in YY1 knockout pro-B cells. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 may regulate commitment in multiple cell lineages.

Microfluidic Affinity Selection of B-Lineage Cells from Peripheral Blood for Minimal Residual Disease Monitoring in Pediatric B-Type Acute Lymphoblastic Leukemia Patients.

Assessment of minimal residual disease (MRD) is the most powerful predictor of outcome in B-type acute lymphoblastic leukemia (B-ALL). MRD, defined as the presence of leukemic cells in the blood or bone marrow, is used for the evaluation of therapy efficacy. We report on a microfluidic-based MRD (MF-MRD) assay that allows for frequent evaluation of blood for the presence of circulating leukemia cells (CLCs). The microfluidic chip affinity selects B-lineage cells, including CLCs using anti-CD19 antibodies poised on the wall of the microfluidic chip. Affinity-selected cells are released from the capture surface and can be subjected to immunophenotyping to enumerate the CLCs, perform fluorescence in situ hybridization (FISH), and/or molecular analysis of the CLCs' mRNA/gDNA. During longitudinal testing of 20 patients throughout induction and consolidation therapy, the MF-MRD performed 116 tests, while only 41 were completed with multiparameter flow cytometry (MFC-MRD) using a bone marrow aspirate, as standard-of-care. Overall, 57% MF-MRD tests were MRD(+) as defined by CLC numbers exceeding a threshold of 5 × 10-4%, which was determined to be the limit of quantitation. Above a threshold of 0.01%, MFC-MRD was positive in 34% of patients. The MF offered the advantage of the opportunity for efficiently processing small volumes of blood (2 mL), which is important in the care of pediatric patients, especially infants. The minimally invasive means of blood collection are of high value when treating patients whose MRD is typically tested using an invasive bone marrow biopsy. MF-MRD detection can be useful for stratification of patients into risk groups and monitoring of patient well-being after completion of treatment for early recognition of potential impending disease recurrence.

Epigenomic, transcriptomic and proteomic characterizations of reference samples.

A variety of newly developed next-generation sequencing technologies are making their way rapidly into the research and clinical applications, for which accuracy and cross-lab reproducibility are critical, and reference standards are much needed. Our previous multicenter studies under the SEQC-2 umbrella using a breast cancer cell line with paired B-cell line have produced a large amount of different genomic data including whole genome sequencing (Illumina, PacBio, Nanopore), HiC, and scRNA-seq with detailed analyses on somatic mutations, single-nucleotide variations (SNVs), and structural variations (SVs). However, there is still a lack of well-characterized reference materials which include epigenomic and proteomic data. Here we further performed ATAC-seq, Methyl-seq, RNA-seq, and proteomic analyses and provided a comprehensive catalog of the epigenomic landscape, which overlapped with the transcriptomes and proteomes for the two cell lines. We identified >7,700 peptide isoforms, where the majority (95%) of the genes had a single peptide isoform. Protein expression of the transcripts overlapping CGIs were much higher than the protein expression of the non-CGI transcripts in both cell lines. We further demonstrated the evidence that certain SNVs were incorporated into mutated peptides. We observed that open chromatin regions had low methylation which were largely regulated by CG density, where CG-rich regions had more accessible chromatin, low methylation, and higher gene and protein expression. The CG-poor regions had higher repressive epigenetic regulations (higher DNA methylation) and less open chromatin, resulting in a cell line specific methylation and gene expression patterns. Our studies provide well-defined reference materials consisting of two cell lines with genomic, epigenomic, transcriptomic, scRNA-seq and proteomic characterizations which can serve as standards for validating and benchmarking not only on various omics assays, but also on bioinformatics methods. It will be a valuable resource for both research and clinical communities.

Prevalence, clinical features, and survival outcome trends of 627 patients with primary cutaneous lymphoma over 29 years: a retrospective review from single tertiary center in Korea

The relative frequency of primary cutaneous lymphoma (PCL) subtypes shows wide variation across different geographical regions. This retrospective study was conducted in a tertiary referral center located in Korea to describe the relative frequency, demographics, survival outcomes, and temporal trend in PCL. A total of 627 PCL cases diagnosed between January 1994 and December 2022 were included. The majority of PCL cases (87.2%) were of T-/NK-cell lineage (CTCL), while the remaining cases (12.8%) were B-cell lineage lymphomas (CBCL). The prevalence of mycosis fungoides (MF) in CTCL increased significantly over time, while other CTCL subtypes, including primary cutaneous extranodal NK/T-cell lymphoma and subcutaneous panniculitis-like T-cell lymphoma (SPTCL), decreased in frequency. Notably, the prevalence of CD4-positive small/medium T-cell lymphoproliferative disorder showed a substantial increase over time. Primary cutaneous marginal zone lymphoma was consistently the commonest CBCL subtype. Survival analysis demonstrated that CTCL had a more favorable 5-year overall survival (OS) than CBCL. OS rate of MF, SPTCL, and primary cutaneous peripheral T-cell lymphoma, NOS improved significantly over time. This study provides comprehensive insights into the dynamic change in the relative frequency and overall survival of PCL subtypes over time.

Nonequilibrium Antigen Recognition during Infections and Vaccinations

The initial immune response to an acute primary infection is a coupled process of antigen proliferation, molecular recognition by naive B cells, and their subsequent clonal expansion. This process contains a fundamental problem: the recognition of an exponentially time-dependent antigen signal. Here, we show that an efficient immune response must be stringently constrained to B-cell lineages with high antigen binding affinity. We propose a tuned proofreading mechanism for primary recognition of new antigens, where the molecular recognition machinery is adapted to the complexity of the immune repertoire. We show that this process produces potent, specific, and fast recognition of antigens, maintaining a spectrum of genetically distinct B-cell lineages as input for affinity maturation. Our analysis maps the proliferation-recognition dynamics of a primary infection to a generalized Luria-Delbrück process, akin to the dynamics of the classic fluctuation experiment. This map establishes a link between signal recognition dynamics and evolution. We derive the resulting statistics of the activated immune repertoire: Antigen binding affinity, expected size, and frequency of active B-cell clones are related by power laws, which define the class of generalized Luria-Delbrück processes. Their exponents depend on the antigen and B-cell proliferation rate, the number of proofreading steps, and the lineage density of the naive repertoire. We extend the model to include spatiotemporal processes, including the diffusion-recognition dynamics of a vaccination. Empirical data of activated mouse immune repertoires are found to be consistent with activation involving about three proofreading steps. The model predicts key clinical characteristics of acute infections and vaccinations, including the emergence of elite neutralizers and the effects of immune aging. More broadly, our results establish infections and vaccinations as a new probe into the global architecture and functional principles of immune repertoires. Published by the American Physical Society 2024

Characterizing the splice map of Turkey Hemorrhagic Enteritis Virus

BackgroundHemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures.MethodsAfter infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions.ResultsResearchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3’ rapid amplification of cDNA ends (3’ RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated.ConclusionsSimilar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs.

Overcoming the age-dependent SARS-CoV-2 vaccine response through hybrid immunity: analysis of humoral and cellular immunity with mass cytometry profiling

BackgroundAge-dependent immune responses to coronavirus disease 2019 (COVID-19) vaccinations and breakthrough infections (BIs) in young and middle-aged individuals are unclear.MethodsThis nationwide multicenter prospective cohort study analyzed immune responses in participants of the ChAdOx1 (ChAd)-ChAd-mRNA vaccine group using cytometry by time-of-flight, anti-spike protein antibody (Sab) and anti-nucleocapsid antibody (Nab) titers, plaque reduction neutralization tests (PRNTs), and interferon-gamma (IFN-γ) release assays at various time points.ResultsWe evaluated 347 participants with an average age of 38.9 ± 9.4 years (range: 21–63). There was a significant inverse correlation between age and Sab levels after the second dose (slope − 14.96, P = 0.032), and this was more pronounced after the third dose (slope − 208.9, P < 0.001). After BIs, older participants showed significantly higher Sab titers (slope 398.8, P = 0.001), reversing the age-related decline observed post-vaccination. This reversal was also observed in PRNTs against wild-type SARS-CoV-2 and the BA.1 and BA.5 variants. IFN-γ responses increased markedly after the third dose and Bis, but showed a weak positive correlation with age, without statistical significance. Immune cell profiling revealed an age-dependent decrease in the proportions of B-cell lineage cells. The proportions of naive CD4+ and CD8+ T cells were inversely correlated with age, whereas the proportions of mature T cell subsets with memory function, including memory CD4+ T, CD8+ TEM, CD8+ TEMRA, and TFH cells, increased with age.ConclusionsAge-dependent waning of the serologic response to COVID-19 vaccines occurred even in middle-aged individuals, but was reversed after BIs. IFN-γ responses were preserved, compensating for the decrease in naive T cell populations, with an increase in memory T cell populations.

RAG1 and RAG2 non-core regions are implicated in leukemogenesis and off-target V(D)J recombination in BCR-ABL1-driven B-cell lineage lymphoblastic leukemia.

The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2' C-terminus absence in Tp53-/- mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1c/c and Rag2c/c in BCR-ABL1+ B-lymphoblastic leukemia (BCR-ABL1+ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1+ B-ALL in mice expressing full-length RAG (Ragf/f), core RAG1 (Rag1c/c), and core RAG2 (Rag2c/c). The Ragc/c (Rag1c/c and Rag2c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Ragf/f cells. Additionally, Ragc/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Ragf/f. We also revealed decreased RAG cleavage accuracy in Ragc/c cells and a smaller recombinant size in Rag1c/c cells, which could potentially exacerbate off-target V(D)J recombination in Ragc/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1+ B-ALL.

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

A pre-B acute lymphoblastic leukemia cell line model reveals the mechanism of thalidomide therapy-related B-cell leukemogenesis

Lenalidomide, a thalidomide derivative, is prescribed as maintenance therapy in multiple myeloma (MM). MM patients receiving lenalidomide were found to develop a distinct therapy-related B cell acute lymphoblastic leukemia (B-ALL). However, the molecular mechanism by which lenalidomide drives B-ALL is unknown. We show that thalidomide treatment of B cell lines increased CD34 expression and fibronectin adhesion. This resembled the effects of Ikzf1 loss of function mutations in B-ALL (1). IKZF1 is a transcription factor that can act as both transcriptional activator as well as a repressor depending upon the target loci. In our experiments, thalidomide-induced degradation of IKZF1 increased the expression of its transcriptional repression targets Itga5 and CD34 explaining the increased adhesion and stemness. Strikingly, withdrawal of thalidomide leads to the mis-localization of IKZF1 to the cytoplasm. Moreover, chromatin immunoprecipitation data showed a long-term effect of thalidomide treatment on IKZF1 target loci. This included decreased chromatin occupancy at early B cell factor 1 (EBF1) and Spi1 (PU.1). Consequently, B-cell lineage specifying transcription factors including Pax5, Spi1 and EBF1 were downregulated even after 7 days of thalidomide withdrawal. Our study thus provides a molecular mechanism of thalidomide-induced B-ALL whereby thalidomide alters the chromatin occupancy of IKZF1 at key B-cell lineage transcription factors leading to a persistent block in B-cell differentiation.

Release of Exosomal PD-L1 in Bone and Soft Tissue Sarcomas and Its Relationship to Radiotherapy.

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT.

Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating advanced B-cell malignancies by targeting CD19, but antigen-negative relapses and immune responses triggered by murine-derived antibodies remain significant challenges, necessitating the development of novel humanized multitarget CAR-T therapies. Here, we engineered a second-generation 4-1BB-CD3ζ-based CAR construct incorporating humanized CD19 single-chain variable fragments (scFvs) and BAFFR single-variable domains on heavy chains (VHHs), also known as nanobodies. The resultant CAR-T cells, with different constructs, were functionally compared both invitro and invivo. We found that the optimal tandem and bicistronic (BI) structures retained respective antigen-binding abilities, and both demonstrated specific activation when stimulated with target cells. At the same time, BI CAR-T cells (BI CARs) exhibited stronger tumour-killing ability and better secretion of interleukin-2 and tumour necrosis factor-alpha than single-target CAR-T cells. Additionally, BI CARs showed less exhaustion phenotype upon repeated antigen stimulation and demonstrated more potent and persistent antitumor effects in mouse xenograft models. Overall, we developed a novel humanized CD19/BAFFR bicistronic CAR (BI CAR) based on a combination of scFv and VHH, which showed potent and sustained antitumor ability both invitro and invivo, including against tumours with CD19 or BAFFR deficiencies.

Therapeutic potential and recent progression of BTK inhibitors against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex chronic inflammatory illness that affects the entire physiology of human body. It has become one of the top causes of disability worldwide. The development and progression of RA involves a complex interplay between an individual's genetic background and various environmental factors. In order to effectively manage RA, a multidisciplinary approach is required, as this disease is complicated and its pathophysiological mechanism is not fully understood yet. In majority of arthritis patients, the presence of abnormal B cells and autoantibodies, primarily anti-citrullinated peptide antibodies and rheumatoid factor affects the progression of RA. Therefore, drugs targeting B cells have now become a hot topic in the treatment of RA which is quite evident from the recent trends seen in the discovery of various B cell receptors (BCRs) targeting agents. Bruton's tyrosine kinase (BTK) is one of these recent targets which play a role in the upstream phase of BCR signalling. BTK is an important enzyme that regulates the survival, proliferation, activation and differentiation of B-lineage cells by preventing BCR activation, FC-receptor signalling and osteoclast development. Several BTK inhibitors have been found to be effective against RA during the invitro and invivo studies conducted using diverse animal models. This review focuses on BTK inhibition mechanism and its possible impact on immune-mediated disease, along with the types of RA currently being investigated, preclinical and clinical studies and future prospective.

Using adjusted local assortativity with Molecular Pixelation unveils colocalization of membrane proteins with immunological significance.

Advances in spatial proteomics and protein colocalization are a driving force in the understanding of cellular mechanisms and their influence on biological processes. New methods in the field of spatial proteomics call for the development of algorithms and open up new avenues of research. The newly introduced Molecular Pixelation (MPX) provides spatial information on surface proteins and their relationship with each other in single cells. This allows for in silico representation of neighborhoods of membrane proteins as graphs. In order to analyze this new data modality, we adapted local assortativity in networks of MPX single-cell graphs and created a method that is able to capture detailed information on the spatial relationships of proteins. The introduced method can evaluate the pairwise colocalization of proteins and access higher-order similarity to investigate the colocalization of multiple proteins at the same time. We evaluated the method using publicly available MPX datasets where T cells were treated with a chemokine to study uropod formation. We demonstrate that adjusted local assortativity detects the effects of the stimuli at both single- and multiple-marker levels, which enhances our understanding of the uropod formation. We also applied our method to treating cancerous B-cell lines using a therapeutic antibody. With the adjusted local assortativity, we recapitulated the effect of rituximab on the polarity of CD20. Our computational method together with MPX improves our understanding of not only the formation of cell polarity and protein colocalization under stimuli but also advancing the overall insight into immune reaction and reorganization of cell surface proteins, which in turn allows the design of novel therapies. We foresee its applicability to other types of biological spatial data when represented as undirected graphs.

Abstract PO-006: ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies

Abstract Glucocorticoids such as prednisone (P) and dexamethasone (D) are key components in standard-of-care immuno-chemotherapy regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for the treatment of B-cell malignancy. However, prolonged systemic glucocorticoid treatment is associated with several adverse events that often result in dose reduction and reduced therapeutic efficacy. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) with a glucocorticoid receptor modulator (GRM) payload engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) CD19 receptor-mediated delivery of potent glucocorticoid receptor modulator (GRM) payload to induce apoptosis, (2) inhibition of B-cell receptor (BCR) stimulated CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payload. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity in vivo when multiple MOAs are enabled concurrently. ABBV-319 also showed potent anti-tumor activity across multiple B-cell lymphoma models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma treated with R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 for the treatment of B-cell malignancy in Phase I clinical trial (NCT05512390). Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Chewei (Anderson) Chang. ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-006.

Rare Drivers at Low Prevalence with High Cancer Effects in T-Cell and B-Cell Pediatric Acute Lymphoblastic Leukemia.

The genomic analyses of pediatric acute lymphoblastic leukemia (ALL) subtypes, particularly T-cell and B-cell lineages, have been pivotal in identifying potential therapeutic targets. Typical genomic analyses have directed attention toward the most commonly mutated genes. However, assessing the contribution of mutations to cancer phenotypes is crucial. Therefore, we estimated the cancer effects (scaled selection coefficients) for somatic substitutions in T-cell and B-cell cohorts, revealing key insights into mutation contributions. Cancer effects for well-known, frequently mutated genes like NRAS and KRAS in B-ALL were high, which underscores their importance as therapeutic targets. However, less frequently mutated genes IL7R, XBP1, and TOX also demonstrated high cancer effects, suggesting pivotal roles in the development of leukemia when present. In T-ALL, KRAS and NRAS are less frequently mutated than in B-ALL. However, their cancer effects when present are high in both subtypes. Mutations in PIK3R1 and RPL10 were not at high prevalence, yet exhibited some of the highest cancer effects in individual T-cell ALL patients. Even CDKN2A, with a low prevalence and relatively modest cancer effect, is potentially highly relevant for the epistatic effects that its mutated form exerts on other mutations. Prioritizing investigation into these moderately frequent but potentially high-impact targets not only presents novel personalized therapeutic opportunities but also enhances the understanding of disease mechanisms and advances precision therapeutics for pediatric ALL.

Combinatorial cellular therapy in pediatric solid tumors with natural killer (NK) and genetically engineered myeloid cells (GEMys).

2561 Background: Early human studies with Natural Killer (NK) cells have shown promise, harnessing their ability to infiltrate into tumors and be tumoricidal. However, these effects can be limited by adenosine, reactive oxygen species, and TGF-β in the highly immune suppressive tumor microenvironment (TME). TGF-β and downstream SMAD3 signaling in Natural Killer (NK) cells have been shown to cause decreased cytokine production, downregulation of activating cell surface receptors, and attenuated cytotoxic function against solid tumors. Exposure to TGF-β during cell culture generates expanded NK cells with increased resistance to TGF-β signaling, dubbed TGF-β imprinted NK cells. In vitrostudies have shown that these cells have increased cytokine production and cytotoxic function in the presence of TGF-β. However, their in vivo trafficking, modulation of the TME, and therapeutic efficacy remain understudied. Our lab has previously shown that when given to tumor-bearing mice, myeloid cells modified to produce IL-12 (IL-12-Genetically Engineered Myeloid cells, aka IL-12 GEMys) are effective at homing to tumor and metastatic sites and, via IL-12 secretion and TME modulation, increase NK cell presence and activation in these sites. They hold great promise as combination therapy, enhancing the effectiveness of other cellular therapies such as NK cell therapy. A current clinical trial exploring TGF-β Imprinted, Ex Vivo Expanded, Universal Donor NK Cell Infusions in relapsed sarcomas is underway (NCT05634369). Methods: In vitro experiments were conducted with the xCELLigence real-time cell analysis system. The human osteosarcoma cell line 143B and human rhabdomyosarcoma cell line RH-30 were evaluated for cell death after co-culture with healthy donor NK cells or TGF-β imprinted NK cells (E:T ratio of 5:1) in isolation and in combination with IL-12 GEMys (E:T ratio of 2:1). Results: TGF-β imprinted NK cells are significantly more effective at tumor cell killing of both 143B and RH30 cell lines in-vitro compared to regular expanded NK cells (WT NK cells), with &gt;90% decrease in cell index compared to 50-60%, respectively, after 48 hours of co-culture. IL-12 GEMys also significantly enhance the cytotoxic effects of WT NK cells, increasing cell killing to 70-80% compared to co-culture with untransduced GEMy controls. Conclusions: These initial in-vitro experiments provide insight into the promising effectiveness of NK-myeloid cell combination therapies in the treatment of pediatric sarcomas. Ongoing in vitroexperiments include cytokine quantification by ELISA and flow cytometric analysis of NK cell activation and proliferation markers. Our preliminary results provide rationale to continue studying the in vivo efficacy of TGF-β imprinted NK cell monotherapy and in combination with IL-12 GEMys in the treatment of tumor-bearing NSG mice.