IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways

J Cancer Res Clin Oncol. 2023 Aug;149(9):5547-5557. doi: 10.1007/s00432-022-04509-w. Epub 2022 Dec 6.

Abstract

Objective: To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells.

Background: High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma.

Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry.

Results: The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers.

Conclusion: A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

Keywords: CAR-T cells; CXCR3; GM-CSF; IL-2; MAPK; PI3K/AKT.

MeSH terms

  • Animals
  • CD28 Antigens / metabolism
  • Carcinoma, Hepatocellular* / metabolism
  • Carcinoma, Hepatocellular* / therapy
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology
  • Granulocytes
  • Humans
  • Interleukin-2 / pharmacology
  • Leukocytes, Mononuclear
  • Liver Neoplasms* / metabolism
  • Liver Neoplasms* / therapy
  • MAP Kinase Signaling System
  • Mice
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, CXCR3* / metabolism
  • Receptors, Chimeric Antigen* / metabolism
  • T-Lymphocytes / metabolism
  • Tumor Microenvironment

Substances

  • CD28 Antigens
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Interleukin-2
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Receptors, CXCR3
  • Receptors, Chimeric Antigen