| Chimeric antigen receptor T cell(CAR-T)therapy has achieved impressive progress in patients with relapsed/refractory B cell acute lymphoblastic leukemia(R/R B-ALL),with high rates of complete remission(CR).However,the short-term remission and high recurrence rate after CAR-T therapy limit the clinical application of CAR-T therapy in R/R B-ALL.It remains a great challenge to improve the efficacy of CAR-T therapy,achieve durable remissions and reduce the relapse rate.Patients who achieved CR after CAR-T cell therapy eventually relapsed because of the antigen escape,poor persistence of CAR-T cells,T cell dysfunction/exhaustion and the immunosuppressive microenvironment.Considering that Interferon-α(IFN-α)exerts anti-leukemia function and multiple immunomodulatory effects,IFN-α is considered a potential drug for combination with CD 19/CD22 CAR-T therapy for R/R B-ALL.However,it is not clearly known whether IFN-α can improve the efficacy of CD19/CD22 CAR-T cell therapy and the mechanism by which IFN-α synergizes with CD 19/CD22 CAR-T cell therapy.Objectives1.We aimed to investigate whether IFN-α could enhance the anti-leukemic function of CD19/CD22 CAR-T cells and elucidate the mechanism by which IFN-α synergizes with CD19/CD22 CAR-T cell therapy.2.We aimed to investigate whether IFN-α combined with CD19/CD22 CAR-T cell therapy could activate endogenous T cells,promote T cell differentiation,improve the immunosuppressive microenvironment and enhance the anti-leukemic function of T cells.Methods1.Lentivirus encoding CD19/CD22 CAR was transduced into activated T cells.To investigate the synergistic effects of IFN-α combined with CD19/CD22 CAR-T cells,we assessed the cytotoxicity,cytokine production,CD 107a expression and phenotype of CD19/CD22 CAR-T cells.Moreover,we also evaluated the cytotoxicity,cytokine production,CD 107a expression and phenotype of CD19/CD22 CAR-T cells pretreated with IFN-α,to explore whether IFN-α could enhance the anti-leukemic function of CD19/CD22 CAR-T cells.To confirm the anti-leukemia activity of CD19/CD22 CAR-T combined with IFN-α in vivo,we developed a xenograft model of NALM-6 luciferase cells in NPSG mice.We performed RNA sequencing analysis to compare the difference of transcriptional profiles in the CD19/CD22 CAR-T cells that were treated with or without IFN-α.2.From September 2019 to September 2021,19 patients were screened and enrolled.All the enrolled patients had relapsed or refractory disease in response to their previous treatment.This clinical trial was designed to evaluate the safety and efficacy of IFN-αcombined with CD19/CD22 CAR-T cell therapy in patients with R/R B-ALL.We assessed the expansion and persistence of CD19/CD22 CAR-T cells and plasma cytokine levels.3.Peripheral blood(PB)samples were harvested on day before CAR-T cells infusion,before IFN-α treatment,one week after IFN-α treatment,and before allogeneic hematopoietic stem cell transplantation(allo-HSCT).Peripheral blood mononuclear cells(PBMCs)for each phase were isolated and subjected to Cytometry by Time-Of-Flight(CyTOF)analysis.For single-cell RNA sequencing(scRNA-Seq),samples were collected at three phases:before CAR-T cells infusion,before IFN-α treatment and before allo-HSCT.Results1.(1)The synergistic effect was significantly enhanced in the IFN-α at doses of 100 and 200 IU/mL combined with CD19/CD22 CAR-T cells group compared with CD19/CD22 CAR-T cells only group.(2)IFN-α at a dose of 200 IU/mL significantly enhanced the antileukemia activity of CD19/CD22 CAR-T cells.CD19/CD22 CAR-T cells treated with IFNa at a dose of 200 IU/mL exhibited stronger cytotoxicity and secreted higher levels of IL-2 and TNF-α.(3)IFN-α combined with CD19/CD22 CAR-T cell therapy activated untransduced T cells.(4)The CAR-T cells treated with IFN-α showed higher proportions of CAR-T cells with effector memory and effector memory CD45RA+phenotypes.T cells with central memory(TCM)and effector memory(TEM)were also increased.(5)In addition,we confirmed the synergistic effects of IFN-α combined with CD19/CD22 CAR-T cells in the NALM-6 B-ALL model.Compared to the CAR-T cell group,the mice in the combination group showed better survival.(6)Gene-expression analyses of CAR-T cells revealed significant transcriptional responses induced by IFN-α.IFN-α treated CAR-T cells were enriched in gene expression profiles involved in T cell activation,proliferation,memory,IFN-α pathway-related genes and immune synapse-related genes.2.(1)To assess the efficacy and safety of IFN-α combined with CD19/CD22 CAR-T cell therapy,we analyzed 19 patients with R/R B-ALL who were enrolled in the clinical trial between September 2019 and September 2021.(2)On day 28 after CAR-T infusion,all patients in both groups achieved CR or CR with incomplete hematologic recovery.Within 3-4 months after IFN-α combined with CD19/CD22 CAR-T therapy,9 patients proceeded to allo-HSCT.There were no significant differences in overall survival(OS)and leukemiafree survival(LFS)between two groups.(3)Generally,all patients were well tolerated and there were no CAR-T related deaths.Moreover,none of the patients suffered from more than grade 1 CRS after receiving IFN-α therapy.All adverse events were reversible and manageable.3.We performed CyTOF and scRNA-Seq to assess gene expression and cellular protein expression of immune cells in the PB during IFN-α combined with CD19/CD22 CAR-T cell therapy.(1)CyTOF analysis revealed that three CD8+TEM subpopulations were significantly increased after IFN-α combined with CD19/CD22 CAR-T cell therapy.(2)To better study the heterogeneity of CD8+T cells and CD4+T cells,we re-clustered CD8+T cells and CD4+T cells.Single-cell RNA sequencing identified 14 distinct clusters of CD8+T cells and 12 clusters of CD4+T cells.CD8+TEM cells and CD8+NKT cells were significantly increased during IFN-α combined with CD 19/CD22 CAR-T cell therapy.Compared with other clusters,the genes significantly upregulated in CD8+TEM cells were enriched in pathways that regulate immune response,T cell activation,immune effector process,leukocyte cell adhesion and antigen presentation.After the combination therapy,T cell memory and IFNa pathway related genes in CD8+TEM cells were up-regulated.(3)We used single-cell TCR sequencing to define clonotypes.After IFN-α combined with CD19/CD22 CAR-T cell therapy,expanded CD8+T cells mainly comprised CD8+effector T cells(cluster 01 and 07).Although before IFN-α combined with CD19/CD22 CAR-T cell therapy,expanded CD4+T cells mainly comprised regulatory T cells.After IFN-α combined with CD19/CD22 CAR-T cell therapy,expanded CD4+T cells mainly comprised CD4+TEM cells,CD4+NKT cells and CD4+effector T cells.Conclusions1.To our knowledge,it’s the first report to confirm the synergistic effects of IFN-αcombined with CD19/CD22 CAR-T cell therapy in vitro and vivo.IFN-α could enhance the anti-leukemic function of CAR-T cells by increasing cytotoxicity,cytokine production,promoting the maintenance of memory phenotype,effector function and up-regulating T cell activation,proliferation,memory,IFN-α pathway-related genes and immune synapse-related genes.2.Our study demonstrated that the combination treatment with IFN-α and CD19/CD22 CAR-T cell therapy was safe and effective for patients with R/R B-ALL.Moreover,IFN-αcombined with CD19/CD22 CAR-T cell therapy bridging to allo-HSCT could be a promising therapy for patients to achieve long-term remission and prolonged OS and LFS.3.IFN-α combined with CD19/CD22 CAR-T cell therapy can induce T cells to differentiate into TEM cells and enhance the effector function of T cells.IFN-α combined with CD19/CD22 CAR-T cell therapy inhibited the expansion of regulatory T cells,promoted the expansion of CD8+effector T cells,CD4+TEM cells,CD4+NKT cells and CD4+effector T cells,activated endogenous T cells and improved immunosuppressive microenvironment. |
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