Acute Conditioning of Antigen-Expanded CD8+ T Cells via the GSK3β-mTORC Axis Differentially Dictates Their Immediate and Distal Responses after Antigen Rechallenge

Abstract

CD8+ T cells protect against tumors and intracellular pathogens. The inflammatory cytokines IL-2, IL-15, and IL-7 are necessary for their expansion. However, elevated serum levels of these cytokines are often associated with cancer, poorer prognosis of cancer patients, and exhaustion of antigen-expanded CD8+ T cells. The impact of acute conditioning of antigen-expanded CD8+ T cells with these cytokines is unknown. Here, we generated antigen-expanded CD8+ T cells using dendritic cells and PC-3 cells. The cells were acutely (18–24 h) conditioned with IL-2 and either the GSK3β inhibitor TWS119, the mTORC1 inhibitor rapamycin, or the mTORC1/2 inhibitor Torin1, then their immediate and post-re-expansion (distal) cytokine responses after antigen rechallenge were evaluated. We found that acute IL-2 conditioning upregulated the immediate antigen-induced cytokine response of the tested cells. Following their re-expansion, however, the cells showed a decreased cytokine response. These IL-2 conditioning-mediated impacts were counteracted with TWS119 or rapamycin but not with Torin1. Our data revealed that the acute conditioning of antigen-expanded CD8+ T cells with IL-2 modulates the GSK3β-mTORC signaling axis. This modulation differentially affected the immediate and distal cytokine responses of the cells. The acute targeting of this signaling axis could, therefore, represent a novel strategy for the modulation of antigen-expanded CD8+ T cells.

Thapsigargin-Stimulated LAD2 Human Mast Cell Line Is a Potent Cellular Adjuvant for the Maturation of Monocyte-Derived Dendritic Cells for Adoptive Cellular Immunotherapy

“…Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient as described previously [ 61 ]. For iDC preparation in the serum-containing medium, the gradient-isolated PBMCs were fractioned into adherent and nonadherent cells as described previously [ 59 ].…”

“…The monocytes (adherent fraction) were differentiated into iDCs using fetal bovine serum-containing culture medium (KM medium; RPMI 1640 medium (Thermo Scientific), 10% heat-inactivated fetal bovine serum (HyClone, GE Healthcare Life Sciences, South Logan, UT, USA), 100 U/mL penicillin–streptomycin, 2 mM GlutaMax (Thermo Scientific)) supplemented with 1000 IU/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 IU/mL of IL-4 (Immunotools, Friesoythe, Germany). The cells were cultured as described [ 61 ]. For iDC preparation in the serum-free medium, the PBMC fractionation and monocyte differentiation into iDCs were performed in CellGro medium (CellGenix, Freiburg, Germany) supplemented with 2 mM GlutaMax, 100 U/mL penicillin–streptomycin, and the above-indicated cytokines.…”

“…For iDC preparation in the serum-free medium, the PBMC fractionation and monocyte differentiation into iDCs were performed in CellGro medium (CellGenix, Freiburg, Germany) supplemented with 2 mM GlutaMax, 100 U/mL penicillin–streptomycin, and the above-indicated cytokines. The cells were cultured as described [ 61 ].…”

LL-37 as a Powerful Molecular Tool for Boosting the Performance of Ex Vivo-Produced Human Dendritic Cells for Cancer Immunotherapy

“…Peripheral blood mononuclear cells (PBMCs) were prepared from the buffy coats of healthy blood donors using a density gradient separation as described previously [18]. PBMCs were then cryopreserved in the human-plasma-and DMSO-containing medium (RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA), 10% DMSO (Sigma-Aldrich, St. Louis, MO, USA), 10% human plasma serum (One Lambda, Canoga Park, CA, USA), 100 U/mL penicillin-streptomycin, 2 mM GlutaMax, 1 mM nonessential amino acid mix, and 1 mM sodium pyruvate (Thermo Scientific)).…”

SARS-CoV-2 spike glycoprotein-reactive T cells can be readily expanded from COVID-19 vaccinated donors

“…The REP was performed for the large-scale expansion of the peptide-enriched cell cultures [17]. As the feeder cells were used PBMCs isolated from buffycoats as described [30]. The buffy coats were obtained from the Institute of Hematology and Blood Transfusion in Prague and each donor provided signed written informed consent to participate in the study.…”