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1.
Oncotarget ; 8(7): 10785-10808, 2017 Feb 14.
Article in English | MEDLINE | ID: mdl-27974697

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.


Subject(s)
Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Interleukin-7/pharmacology , Mucin-1/immunology , Receptor, ErbB-2/immunology , Antigens, Neoplasm/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Cross-Priming/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Imidazoles/pharmacology , Immunity, Innate/drug effects , Immunity, Innate/immunology , Immunotherapy, Adoptive/methods , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-2/immunology , Interleukin-2/pharmacology , Interleukin-7/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mucin-1/metabolism , Neoplasms/immunology , Neoplasms/therapy , Peptides/immunology , Peptides/pharmacology , Receptor, ErbB-2/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism
2.
Clin Cancer Res ; 19(18): 5039-52, 2013 Sep 15.
Article in English | MEDLINE | ID: mdl-23873692

ABSTRACT

PURPOSE: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. EXPERIMENTAL DESIGN: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). RESULTS: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. CONCLUSION: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers.


Subject(s)
Colitis, Ulcerative/complications , Colonic Neoplasms/etiology , Crohn Disease/pathology , Mucin-1/physiology , Myeloid Cells/pathology , Animals , Azoxymethane/toxicity , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Bone Marrow Transplantation , Carcinogens/toxicity , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Crohn Disease/genetics , Crohn Disease/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Humans , Immunoenzyme Techniques , Mice , Mice, Knockout , Myeloid Cells/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
3.
Immunol Invest ; 41(6-7): 680-710, 2012.
Article in English | MEDLINE | ID: mdl-23017141

ABSTRACT

The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib's profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF's capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production.


Subject(s)
Indoles/therapeutic use , Myeloid Progenitor Cells/drug effects , Neoplasms/pathology , Neoplasms/therapy , Protein Kinase Inhibitors/therapeutic use , Pyrroles/therapeutic use , T-Lymphocytes/drug effects , Tumor Escape , Animals , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Immune Tolerance , Indoles/pharmacology , Mice , Myeloid Progenitor Cells/immunology , Myeloid Progenitor Cells/pathology , Neoplasms/immunology , Neoplasms/metabolism , Organ Specificity , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/immunology , Signal Transduction , Sunitinib , T-Lymphocytes/immunology , T-Lymphocytes/pathology
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