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  • Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Oncotarget (2016-12-16)
Latha B Pathangey, Dustin B McCurry, Sandra J Gendler, Ana L Dominguez, Jessica E Gorman, Girish Pathangey, Laurie A Mihalik, Yushe Dang, Mary L Disis, Peter A Cohen
ABSTRACT

Effective adoptive immunotherapy has proved elusive for many types of human cancer, often due to difficulties achieving robust expansion of natural tumor-specific T-cells from peripheral blood. We hypothesized that antigen-driven T-cell expansion might best be triggered in vitro by acute activation of innate immunity to mimic a life-threatening infection. Unfractionated peripheral blood mononuclear cells (PBMC) were subjected to a two-step culture, first synchronizing their exposure to exogenous antigens with aggressive surrogate activation of innate immunity, followed by γ-chain cytokine-modulated T-cell hyperexpansion. Step 1 exposure to GM-CSF plus paired Toll-like receptor agonists (resiquimod and LPS), stimulated abundant IL-12 and IL-23 secretion, as well as upregulated co-stimulatory molecules and CD11c expression within the myeloid (CD33+) subpopulation. Added synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65), were reliably presented to CD4+ T-cells and cross-presented to CD8+ T-cells. Both presentation and cross-presentation demonstrated proteasomal and Sec61 dependence that could bypass the endoplasmic reticulum. Step 2 exposure to exogenous IL-7 or IL-7+IL-2 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells with a predominant interferon-γ-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other γ-chain cytokines and/or combinations were initially proliferogenic, but followed by a contractile phase not observed with IL-7 or IL-7+IL-2. Regulatory T-cells were minimally propagated under these culture conditions. This mechanistically rational culture sequence, effective even for unvaccinated donors, enables rapid preparation of T-cells recognizing tumor-associated antigens expressed by the majority of human cancers, including pancreatic cancers, breast cancers and glioblastomas.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Fetal Bovine Serum, USA origin, sterile-filtered, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
Polyinosinic–polycytidylic acid sodium salt, TLR ligand tested
Sigma-Aldrich
IgG from human serum, reagent grade, ≥95% (SDS-PAGE), essentially salt-free, lyophilized powder
Sigma-Aldrich
Z-Leu-Leu-Leu-al, ≥90% (HPLC)
Sigma-Aldrich
Pseudomonas exotoxin A from Pseudomonas aeruginosa, lyophilized powder
Sigma-Aldrich
Lactacystin, ≥90% (HPLC)
Sigma-Aldrich
Resiquimod, ≥98% (HPLC)