CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1

Abstract

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1–dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Precision-Cut Tumor Slices (PCTS) as an Ex Vivo Model in Immunotherapy Research

“…PCTS offer the opportunity to study the localization and function of the several immune cell types within the TME, and how the latter behave following treatment. With CD8+ cytotoxic and CD4+ helper T cells playing a central role in anti-tumor immunity, several PCTS-based studies have focused on understanding the localization and function of tumor infiltrating lymphocytes (TILs) in solid tumors, including lung, breast and melanoma [ 27 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ].…”

“…PCTS use in CAR-T cell research is still in its infancy. A 2021 study used EGFR-targeting CAR-T cells in co-cultures with either PCTS derived either from a BxPC3 EGFR-expressing pancreatic cancer PDX model or from PCTS derived from NSCLC and ccRC [ 39 ]. Confocal microscopy and dynamic imaging techniques were used to monitor CAR-T-cell migration and infiltration in the PCTS, whereas fluorescent calcium assay assessed CAR-T cell activation [ 39 ].…”

“…A 2021 study used EGFR-targeting CAR-T cells in co-cultures with either PCTS derived either from a BxPC3 EGFR-expressing pancreatic cancer PDX model or from PCTS derived from NSCLC and ccRC [ 39 ]. Confocal microscopy and dynamic imaging techniques were used to monitor CAR-T-cell migration and infiltration in the PCTS, whereas fluorescent calcium assay assessed CAR-T cell activation [ 39 ]. The results demonstrated CAR-T cell-specific activation that was dependent on the presence of EGFR-expressing tumor cells at the periphery of the tumor islets, which are initially the only ones permissive to CAR-T cell binding [ 39 ].…”

“…Confocal microscopy and dynamic imaging techniques were used to monitor CAR-T-cell migration and infiltration in the PCTS, whereas fluorescent calcium assay assessed CAR-T cell activation [ 39 ]. The results demonstrated CAR-T cell-specific activation that was dependent on the presence of EGFR-expressing tumor cells at the periphery of the tumor islets, which are initially the only ones permissive to CAR-T cell binding [ 39 ]. In response to EGFR CAR-T cell activation and IFN-γ secretion, the tumor cells upregulated the expression of the intercellular adhesion molecule-1 (ICAM-1), which in turn enabled the progressive entry of EGFR CAR-T cells from the periphery to the center of tumor islets [ 39 ].…”

“…The results demonstrated CAR-T cell-specific activation that was dependent on the presence of EGFR-expressing tumor cells at the periphery of the tumor islets, which are initially the only ones permissive to CAR-T cell binding [ 39 ]. In response to EGFR CAR-T cell activation and IFN-γ secretion, the tumor cells upregulated the expression of the intercellular adhesion molecule-1 (ICAM-1), which in turn enabled the progressive entry of EGFR CAR-T cells from the periphery to the center of tumor islets [ 39 ]. The importance of ICAM-1 expression by the tumor cells was further highlighted by the fact that EGFR-expressing tumor cells at the center of the tumor islets that weakly expressed ICAM-1 were not able to promote EGFR CAR-T cell activation “arrest” within the tumor islets [ 39 ].…”