Project description:We report that retention of intron 2 which affects expression of CD19 in CART-19 relapsed leukemia occurs in the context of full length CD19 transcript using Oxford Nanopore sequencing technology. By performing Direct RNA sequencing on Reh leukemia cell lines, we showed that intron 2 retention is functionally equivalent to nonsense mutations.
Project description:We developed a massively parallel reporter assay to determine the splicing effects of all mutations in the region comprising CD19 exons 1-3. Despite the great success of CART-19 (chimeric antigen receptor-armed autologous T-cells) immunotherapy to treat B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the targeted CD19 epitope. Since epitope loss can be caused by CD19 exon 2 mis-splicing, we set out to learn the regulatory code that controls CD19 alternative splicing.
Project description:We developed a massively parallel reporter assay to determine the splicing effects of all mutations in the region comprising CD19 exons 1-3. Despite the great success of CART-19 (chimeric antigen receptor-armed autologous T-cells) immunotherapy to treat B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the targeted CD19 epitope. Since epitope loss can be caused by CD19 exon 2 mis-splicing, we set out to learn the regulatory code that controls CD19 alternative splicing.
Project description:We developed a massively parallel reporter assay to determine the splicing effects of all mutations in the region comprising CD19 exons 1-3. Despite the great success of CART-19 (chimeric antigen receptor-armed autologous T-cells) immunotherapy to treat B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the targeted CD19 epitope. Since epitope loss can be caused by CD19 exon 2 mis-splicing, we set out to learn the regulatory code that controls CD19 alternative splicing.
Project description:The clinical success of chimeric antigen receptor (CAR) T-cell therapy for CD19+ B-cell malignancies may come at the expense of acute and chronic morbidities. Some patients suffer from significant, acute toxicities and those with persistent CAR T-cells require immunoglobulin therapy due to CAR-induced B-cell aplasia. Life-threatening effects include cytokine release syndrome, the exact etiology of which is unclear. To elucidate the underlying mechanisms of CAR-induced toxicities, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T-cells were adoptively transferred into mice whose normal B-cells express a hCD19 transgene at hemizygous levels. In contrast to homozygous mice, hemizygous mice have higher B cell frequencies, providing a greater target antigen load to drive CAR-T cell activation. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR-T cells had undetectable levels of tumor. However, recipients experienced acute B-cell aplasia, morbidities and mortality in an antigen- and dose-dependent manner. IL-6, INF-g, and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 blunted toxicity. This new model will prove useful in testing strategies designed to improve CD19-specific CAR T-cell therapy by reducing acute toxicities and reversing B-cell aplasia.
Project description:The clinical success of chimeric antigen receptor (CAR) T-cell therapy for CD19+ B-cell malignancies may come at the expense of acute and chronic morbidities. Some patients suffer from significant, acute toxicities and those with persistent CAR T-cells require immunoglobulin therapy due to CAR-induced B-cell aplasia. Life-threatening effects include cytokine release syndrome, the exact etiology of which is unclear. To elucidate the underlying mechanisms of CAR-induced toxicities, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T-cells were adoptively transferred into mice whose normal B-cells express a hCD19 transgene at hemizygous levels. In contrast to homozygous mice, hemizygous mice have higher B cell frequencies, providing a greater target antigen load to drive CAR-T cell activation. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR-T cells had undetectable levels of tumor. However, recipients experienced acute B-cell aplasia, morbidities and mortality in an antigen- and dose-dependent manner. IL-6, INF-g, and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 blunted toxicity. This new model will prove useful in testing strategies designed to improve CD19-specific CAR T-cell therapy by reducing acute toxicities and reversing B-cell aplasia.
Project description:The immune response to SARS-CoV-2 varies among patients. We used proteomic analysis to study CD19+ lymphocytes, which are important for the immune response. By comparing the proteomes of CD19+ cells from healthy individuals and COVID-19 patients, we discovered proteins and signaling pathways involved in the immune response during COVID-19. This knowledge improves our understanding of immune dysregulation in COVID-19 and can help develop targeted therapies. Our research also explores the role of CD19+ lymphocytes in COVID-19 and identifies potential biomarkers. Overall, our findings contribute to understanding COVID-19 immunology and inform future research efforts.