Work

Mechanism of CRS: Critical Roles of IFN-γ, CD40L, and CD4 Cells.

Year: 2025

Type: article

Abstract: Despite remarkable clinical efficacy, Chimeric Antigen Receptor (CAR) T cell therapy in patients with hematologic malignancies is limited by immune-related toxicities including cytokine release syndrome (CRS) which occurs in more than 70% of patients. To identify genes and pathways critical for CRS, we generated a novel in vitro CRS assay in which human CD19 CAR T cells are incubated with luciferase expressing Ramos CD19+ target cells, and donor matched immature dendritic cells (iDC) that are previously primed with GM-CSF/IL-4. CART19 killing of targets is measured by BLI and CRS is assessed by measurement of IL-6 in the supernatant via ELISA . Two screens were performed. The first was a high throughput screen of >300 kinase inhibitors which identified two classes of kinase inhibitors , JAK1/2 and PI3Kgd inhibitors which had no impact on CART19 function but inhibited CRS. Neutralizing antibodies to IFN-γ or IFN-γR, or CRISPR gene editing of IFN-γ in CART19 or IFN-γR in iDCs had no effect on CART19 function but significantly reduced IL-6 production by ∼80-90%. These observations led to two clinical trials testing itacitinib (NCT04071366) and duvelisib (NCT05044039) as CRS prophylaxis after CART19 infusion. The second was a CRISPR/Cas9 KO screen in CD3/28 bead activated T cells focused on ∼200 genes expressed in activated T-cells, cocultured with THP1 cells (monocyte AML and source of IL-6). CD40L (among several others) was consistently identified as a lead candidate ( Fig 1 ). We compared the cytolytic activity and CRS of CD40L gene edited vs. wild type CART19 and found deletion of CD40L resulted in reduction of secreted IL-6 levels (80-90%) from iDCs while actually enhancing killing of target Ramos CD19+ cells ( Fig 2A ). We validated this by showing that neutralizing CD40L antibodies dramatically reduced IL-6 production without altering CART19 function ( Fig 2B ). To determine if there was a difference between CD4 and CD8 T-cells for CRS induction, we generated purified CD4 CART19 cells and CD8 CART19 cells and assessed IL-6 in our CRS assay. We found that although CD8 CART19 cells are the predominant cytotoxic cells in our assay, CD4 cells were the main driver of IL-6/CRS ( Fig 3A ). Surface CD40L was expressed rapidly and transiently after activation exclusively in CD4 CART19 (data not shown) and since CD40L can be cleaved from the cell surface, we measured secreted CD40L and found that CD4 CART19 cells secrete much higher levels of CD40L than CD8 CART19 ( Fig 3B ). Further, Lentiviral overexpression of CD40L in CD8 CART19 resulted in robust surface expression and secretion of CD40L as well as dramatic production of IL-6 by iDC (Data not shown). Together, our studies clearly implicate two major pathways that are necessary and sufficient for CRS: 1) IFN-γ/IFN-γR/JAK1/2 axis (both CD4 and CD8) and 2) CD40L/CD40 PI3Kg/d axis (exclusively CD4) and represent logical targets for future CRS mitigation strategies in vivo .

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.jtct.2025.01.009