Single-Cell Analysis of CAR-T Cell Activation Reveals A Mixed TH1/TH2 Response Independent of Differentiation

Abstract

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 + helper T (T H ) cells and CD8 + cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T H 1 and T H 2 signature cytokines, e.g. , interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3 . However, rather than conforming to stringent T H 1 or T H 2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T H 1/T H 2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T H 1/T H 2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma

“…Although cytotoxic CD8(+) T cells mediate direct tumor cell killing, CD4(+) T helper (TH) cells are also important in tumor cell eradication [ 47 ], as CD4(+) CAR T cells exert significant cytotoxicity comparable to CD8(+) CAR T cells [ 48 ]. According to recently published single-cell analysis data, both CD4(+) TH cells and CD8(+) cytotoxic T cells are equally effective in direct tumor cell killing, and their cytotoxicity is associated with both TH1 and TH2 cytokines, e.g., IFN-γ, TNF-α, IL-15, and IL-13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3 [ 49 , 50 ]. In addition, rather than stringent TH1 or TH2 subtypes, the predominant anti-tumor response is dependent on a highly mixed TH1/TH2 function in the same cell, suggesting the activation of BsAb-directed T cells or CAR T cells is a canonical process that leads to a mixed response combining both TH1 and TH2 cytokines together with GM-CSF [ 49 ].…”

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…37,[45][46][47][48][49] CD28 and 41BB co-stimulation in the context of CAR T cells has been extensively studied, including detailed phosphoproteomic and single-cell RNA sequencing (RNA-seq) analyses. [50][51][52] They activate different pathways within T cells, with CD28 signaling promoting glycolytic metabolism and an effector memory phenotype, in contrast to 41BB signaling, which induces oxidative metabolism and a central memory phenotype. 53 Depending on the number of co-stimulatory molecules included in the CAR signaling domain, CARs are either designated second (one domain) or third (two domains) generation.…”

Towards a Systems Immunology Approach to Unravel Responses to Cancer Immunotherapy

“…In a recent study, TCR-seq, integration site analysis, and scRNA-seq were used to profile anti-CD19 CAR-T cells before and after infusion, revealing that clonal diversity declines following infusion and that expanding clones have higher expression of proliferation and cytotoxicity genes (54). By analyzing the transcriptional and cytokine signatures (by means of scRNA-seq and single-cell multiplex cytokine secretion assay), together with live cell imaging of cytotoxic activity, Xhangolli et al demonstrated that anti-CD19 CAR-T cells display a highly mixed Th1/ Th2 function upon antigen-specific stimulation (55). Lymphodepleting chemotherapy is routinely administered prior to CAR-T cell infusion, and has been associated with improved in vivo cell expansion and persistence (56).…”