Single-cell sequencing of T cells harvested 4 hours after preparation by different methods
Ontology highlight
ABSTRACT: We successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with these PD1 knockout CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of T cells harvested 4 hours after preparation by different methods.
Project description:We successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with these PD1 knockout CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of T cells prepared by different methods.
Project description:We successfully developed a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with PD1 knockin CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of infusion products and T cells after administration.
Project description:CD19-directed chimeric antigen receptor (CAR) T-cell therapy has transformed outcomes for patients with relapsed/refractory large B-cell lymphoma (LBCL), yet the mechanisms underlying durable remission remain incompletely understood. While CAR T-cell persistence is associated with response, long-term remission can occur despite rapid CAR T clearance, suggesting the involvement of additional immune mechanisms. To investigate the role of the native T-cell repertoire in shaping response durability, we performed single-cell RNA and TCR sequencing (scRNA-seq/scTCR-seq) on longitudinal peripheral blood samples from LBCL patients treated with axicabtagene ciloleucel (axi-cel) in the ZUMA-1 trial. We compared immune landscapes and clonotypic dynamics among patients achieving durable remission (>1 year), those experiencing early relapse (<6 months), and those with refractory disease. Patients with long-term remission exhibited increased cytotoxic, proinflammatory, and proliferative native T-cell subsets, while early relapse was associated with immunoregulatory populations that may suppress T-cell activation. TCR profiling revealed robust clonotypic expansion of native cytotoxic T cells post-infusion in durable responders, with expansion patterns strongly predicting clinical outcomes. Notably, TCR screens did not identify known viral targets, suggesting tumor-specific immunity may mediate ongoing remission. These findings propose native T-cell clonotypic expansion as a key determinant of durable response to CAR T therapy and highlight its predictive potential for long-term clinical outcomes.
Project description:<p>Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (r/r) large B-cell lymphoma (LBCL) results in durable response in only a subset of patients. MYC overexpression in LBCL tumors is associated with poor response to treatment. We tested whether a MYC-driven polyamine signature, as a liquid biopsy, is predictive of response to anti-CD19 CAR-T therapy in patients with r/r LBCL. Elevated plasma acetylated polyamines were associated with non-durable response. Concordantly, increased expression of spermidine synthase, a key enzyme which regulates levels of acetylated spermidine, was prognostic for survival in r/r LBCL. A broad metabolite screen identified additional markers which resulted in a 6-marker panel (6MetP) consisting of acetylspermidine, diacetylspermidine and lysophospholipids which was validated in an independent set from another institution as predictive of non-durable response to CAR T therapy. A polyamine centric metabolomics liquid biopsy panel has predictive value for response to CAR-T therapy in r/r LBCL. </p>
Project description:<p>The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR; CTL019) has shown remarkable activity in patients with B acute lymphoblastic leukemia. Similar therapy can induce long-term remissions for relapsed/refractory chronic lymphocytic leukemia (CLL) patients, but in only a small subset of subjects. The determinants of response and resistance to CTL019 therapy of CLL are not fully understood. We employed next generation sequencing of RNA (RNA-seq) to identify predictive indicators of response to CTL019 treatment. We performed RNA-seq on leukapheresis and manufactured infusion product T cells from patients with heavily pre-treated and high-risk disease. To characterize potency, we also performed RNA-seq on the cellular infusion product after CAR-specific stimulation. Our findings indicate that durable remission in CLL is associated with gene expression signatures of early memory T cell differentiation (e.g., STAT3), while T cells from poorly- or non-responding patients exhibited elevated expression of key regulators of late memory as well as effector T cell differentiation, apoptosis, aerobic glycolysis, hypoxia and exhaustion. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to cellular therapies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products.</p>
Project description:BCMA targeting chimeric antigen receptor (CAR) T cell therapy have shown deep and durable responses in multiple myeloma. However, relapse following therapy is frequently observed, and mechanisms of resistance remain ill-defined. We performed single cell genomic characterization of longitudinal samples from a patient who relapsed after initial CAR T cell treatment with lack of response to retreatment.
Project description:Cellular evolution and molecular programs of chimeric antigen receptor-engineered (CAR)-T cells post-infusion are pivotal for developing better treatment strategies. Here, we constructed a high-precision single-cell transcriptional dynamic landscape of 7,578 CAR-T cells from 26 B-ALL patients and evaluated their long-term therapeutic efficacy. We demonstrated that the cytotoxic profile rather than memory property was a favorable biomarker for long-term remission. At the single-cell resolution, we uncovered the vast heterogeneity of CAR-T cells in vivo and identified eight CAR-T cell subtypes. Remarkably, the dominance of cytotoxic subtypes was coincident with long-term remission, while the emergence of subtypes with B-cell transcriptional features could be detected early and predict relapse. Furthermore, we defined transcriptional hallmarks of distinct CAR-T cell populations and revealed molecular changes along computationally-inferred cellular evolution of CAR-T cells in vivo . Collectively, these results illuminated intrinsic properties for durable response and provided molecular cues for improving CAR-T immunotherapy.
Project description:Significant response rates have been reported in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphomas (DLBCL), mantle cell lymphomas and follicular lymphomas. In contrast, in CTL019 trials, only 26% of CLL patients had durable antitumor responses with dramatic mechanisms of resistance to anti-CD19.CAR T-cell therapy. Low expression of programmed cell death protein 1 (PD1) has been identified as pre-treatment predictor of response in these trials, though the mechanisms responsible for a limited efficacy in CLL remain poorly understood. Additional studies have demonstrated that CD8+ anti-CAR T cells from patients with CLL exhibit impaired metabolic function compared to CD8+ T cells from healthy donors and that these features are associated with T-cell exhaustion and a reduction in T-cell activation and degranulation. Several T-cell defects have been observed in patients with CLL and are mainly related to impaired immune synapse (IS) formation, and these defects could ultimately impact on the CAR T-cell activation and expansion upon antigen encounter. Thus, the purpose of this study was to explore the possibility of using lenalidomide, an immunomodulatory agent that has induced significant, long-lasting responses in CLL patients, to enhance the therapeutic efficacy of CD23.CAR-redirected T cells.
Project description:Chimeric antigen receptor-T (CAR-T) therapy remains to be investigated in T-cell malignancies. CD7 is an ideal target for T-cell malignancies but is also expressed on normal T cells, which may cause CAR-T cell fratricide. Donor-derived anti-CD7 CAR-T cells using endoplasmic reticulum retention have shown efficacy in patients with T-cell acute lymphoblastic leukemia (ALL). Here we launched a phase I trial to explore differences between autologous and allogeneic anti-CD7 CAR-T therapies in T-cell ALL and lymphoma. Ten patients were treated and 5 received autologous CAR-T therapies. No dose-limiting toxicity or neurotoxicity was observed. Grade 1-2 cytokine release syndrome occurred in 7 patients, and grade 3 in 1 patient. Grade 1-2 graft-versus-host diseases were observed in 2 patients. Seven patients had bone marrow infiltration, and 100% of them achieved complete remission with negative minimal residual disease within one month. Two-fifths of patients achieved extramedullary or extranodular remission. The median follow-up was 6 (range, 2.7- 14) months and bridging transplantation was not administrated. Patients treated with allogeneic CAR-T cells had higher remission rate, less recurrence and more durable CAR-T survival than those receiving autologous products. Allogeneic CAR-T cells appeared to be a better option for patients with T-cell malignancies.