title: Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines.
abstract: BACKGROUND
  The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-gamma or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site.
  
  
  METHODS
  These studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-gamma or TNF-alpha was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent.
  
  
  RESULTS
  We observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1alpha and MIP-1beta following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-gamma or TNF-alpha, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression.
  
  
  CONCLUSIONS
  These data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.
authors.affiliation: Department of Surgery, LMU Munich, Klinikum Grosshadern 81377 Munich, Germany. hauke.winter@med.uni-muenchen.de
authors.forename: Hauke
authors.initials: H
authors.lastname: Winter
authors.forename: Natasja K
authors.initials: NK
authors.lastname: van den Engel
authors.forename: Dominik
authors.initials: D
authors.lastname: Rüttinger
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schmidt
authors.forename: Matthias
authors.initials: M
authors.lastname: Schiller
authors.forename: Christian H
authors.initials: CH
authors.lastname: Poehlein
authors.forename: Florian
authors.initials: F
authors.lastname: Löhe
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Fox
authors.forename: Karl-Walter
authors.initials: KW
authors.lastname: Jauch
authors.forename: Rudolf A
authors.initials: RA
authors.lastname: Hatz
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Hu
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D002465:Cell Movement; D018925:Chemokines / Q000378:metabolism*; D002633:Chemotaxis; D005260:Female; D007167:Immunotherapy / Q000379:methods; D007371:Interferon-gamma / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008546:Melanoma, Experimental; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D009569:Nitric Oxide / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D018925:Chemokines; D009569:Nitric Oxide; D007371:Interferon-gamma