Exclusion of T Cells From Pancreatic Carcinomas in Mice Is Regulated by Ly6C(low) F4/80(+) Extratumoral Macrophages.

BACKGROUND & AIMS: Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor's immunosuppressive mechanisms, or both. METHODS: Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. RESULTS: Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells. CONCLUSIONS: Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.

Vaccination with immunotherapeutic Listeria monocytogenes induces IL-17(+) γδ T cells in a murine model for HPV associated cancer.

Interleukin 17 (IL-17) is produced during infection with Listeria monocytogenes and is also an important regulator of tumor development with both pro- and anti-tumorigenic effects. αß T cells and γδ T cells are among the principle producers of IL-17 in response to infection and other proinflammatory conditions. Listeria-based cancer immunotherapies induce IFNγ directed Th1 dependent tumor regression; however, the role of IL-17 in Listeria based immunotherapy has not been addressed. Therefore, we investigated the ability of attenuated Listeria-based immunotherapy to induce IL-17 producing cells in a model of cervical cancer and the potential impact that these cells have on anti-tumor vaccine efficacy. Here we show that vaccination of tumor bearing mice with Listeria vaccines resulted in elevated levels of intratumoral IL-17 and increased IL-17 production by γδ TCR+ cells, exclusively. IL-17 producing cells were lacking in tumors of γδ T-cell-deficient mice; however, the absence of γδ T cells, including IL-17+ γδ T cells, did not alter tumor progression or abrogate the efficacy of the Listeria-based vaccine indicating that αß T cells are key for clearance of the tumor. Th1 responses, known to be responsible for anti-tumor Listeria-based vaccine efficacy, appear to be sufficient for tumor regression in γδ T-cell-deficient mice. We conclude that the efficacy of Listeria-based vaccine does not rely on γδ T cells (or IL-17 produced by them) in a TC.1 tumor model; however, Listeria-based immunotherapy can be used to induce IL-17+ γδ T cells that are important for regression observed in alternative cancer models.

B Lymphocytes provide an infection niche for intracellular bacterium Brucella abortus.

BACKGROUND: Brucella spp. are intracellular bacteria that establish lifelong infections whose mechanisms of chronicity are poorly understood. Notably, B cells facilitate the establishment of the high infection plateau that persists for months. METHODS: We evaluated the contribution of murine B cells toward providing infection niches for Brucella by using flow cytometry and microscopy and by determining live bacterial counts associated with B cells both in vivo and in vitro. RESULTS: Herein we demonstrate that immunoglobulin M and complement-opsonized Brucella abortus infects and survives inside primary murine B cells protected from bactericidal effects of gentamicin. The entry was dependent on microfilaments for internalization and subsequently brucellae reside in a late endosomal/lysosomal compartment. Throughout the infection, 10% of colony-forming units from infected mice was associated with B cells, and these cells transferred disease to naive hosts. Furthermore, Brucella-positive cells were positive for transforming growth factor (TGF) ß1, and about 10% of such cells were B cells, similar to rates found for other intracellular pathogens that induce their hosts cells to produce TGF-ß1. CONCLUSIONS: To conclude, infected B cells contribute to chronic bacterial infections by providing an intracellular niche that may exert an immunoregulatory role. Although professional phagocytic cells harbor intracellular bacteria including Brucella, infection of lymphocytes by bacteria has not been previously appreciated.

Listeria and Salmonella bacterial vectors of tumor-associated antigens for cancer immunotherapy.

This review covers the use of the facultative intracellular bacteria, Listeriamonocytogenes and Salmonella enterica serovar typhimurium as delivery systems for tumor-associated antigens in tumor immunotherapy. Because of their ability to infect and survive in antigen presenting cells, these bacteria have been harnessed to deliver tumor antigens to the immune system both as bacterially expressed proteins and encoded on eukaryotic plasmids. They do this in the context of strong innate immunity, which provides the required stimulus to the immune response to break tolerance against those tumor-associated antigens that bear homology to self. Here we describe differences in the properties of these bacteria as vaccine vectors, a summary of the major therapies they have been applied to and their advancement towards the clinic.

Cancer immunotherapy using Listeria monocytogenes and listerial virulence factors.

Our laboratory is interested in how immunogenicity may be modulated in vivo in order to better design more effective immunotherapeutics against cancer. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic CTL epitopes. We have used this approach to target tumor antigens expressed on breast, melanoma and cervical cancer. We are also exploring the role of Listerial virulence factors in potentiating adaptive immune responses by activating innate immunity. Specifically, we are using these proteins as adjuvants for B cell lymphomas.

Promiscuity of MHC class Ib-restricted T cell responses.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.