Surrogate<i>in vitro</i>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

Abstract

// Latha B. Pathangey 1,* , Dustin B. McCurry 1,* , Sandra J. Gendler 1,2,3 , Ana L. Dominguez 1 , Jessica E. Gorman 1 , Girish Pathangey 1 , Laurie A. Mihalik 3 , Yushe Dang 4 , Mary L. Disis 4 and Peter A. Cohen 2,3 1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ, USA 2 Department of Immunology, Mayo Clinic, Scottsdale, AZ, USA 3 Department of Hematology and Oncology, Mayo Clinic, Scottsdale, AZ, USA 4 Tumor Vaccine Group, Center for Translational Medicine in Women&rsquo;s Health, University of Washington, Seattle, WA, USA * Shared first authorship Correspondence to: Peter A. Cohen, email: // Sandra J. Gendler, email: // Mary L. Disis, email: // Keywords : PBMC, TLR agonists, GM-CSF, IL-7, adoptive therapy, MUC1 Received : October 07, 2016 Accepted : November 23, 2016 Published : December 11, 2016 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 &gamma;-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 (&gt;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-&gamma;-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other &gamma;-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.