Tumor-specific T cells signal tumor destruction via the lymphotoxin β receptor

BACKGROUND: Previously, we reported that adoptively transferred perforin k/o (PKO), and IFN-γ k/o (GKO), or perforin/IFN-γ double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-α (LT-α) as a potential effector molecules of tumor-specific effector T cells. METHODS: Effector T cells were generated from tumor vaccine-draining lymph node (TVDLN) of wt, GKO, LT-α deficient (LKO), or PKO/GKO mice and tested for their ability to mediate regression of D5 pulmonary metastases in the presence or absence of LT-βR-Fc fusion protein or anti-IFN-γ antibody. Chemokine production by D5 tumor cells was determined by ELISA, RT-PCR and Chemotaxis assays. RESULTS: Stimulated effector T cells from wt, GKO, or PKO/GKO mice expressed ligands for LT-β receptor (LT-βR). D5 tumor cells were found to constitutively express the LT-βR. Administration of LT-βR-Fc fusion protein completely abrogated the therapeutic efficacy of GKO or PKO/GKO but not wt effector T cells (p < 0.05). Consistent with this observation, therapeutic efficacy of effector T cells deficient in LT-α, was greatly reduced when IFN-γ production was neutralized. While recombinant LT-α1β2 did not induce apoptosis of D5 tumor cells in vitro, it induced secretion of chemokines by D5 that promoted migration of macrophages. CONCLUSION: The contribution of LT-α expression by effector T cells to anti-tumor activity in vivo was not discernable when wt effector T cells were studied. However, the contribution of LT-β R signaling was identified for GKO or PKO/GKO effector T cells. Since LT-α does not directly induce killing of D5 tumor cells in vitro, but does stimulate D5 tumor cells to secrete chemokines, these data suggest a model where LT-α expression by tumor-specific effector T cells interacts via cross-linking of the LT-βR on tumor cells to induce secretion of chemokines that are chemotactic for macrophages. While the contribution of macrophages to tumor elimination in our system requires additional study, this model provides a possible explanation for the infiltration of inate effector cells that is seen coincident with tumor regression

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

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-1 alpha and MIP-1 beta 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. Conclusion: 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

Characterization of gastric adenocarcinoma cell lines established from CEA424/SV40 T antigen-transgenic mice with or without a human CEA transgene

BACKGROUND: Gastric carcinoma is one of the most frequent cancers worldwide. Patients with gastric cancer at an advanced disease stage have a poor prognosis, due to the limited efficacy of available therapies. Therefore, the development of new therapies, like immunotherapy for the treatment of gastric cancer is of utmost importance. Since the usability of existing preclinical models for the evaluation of immunotherapies for gastric adenocarcinomas is limited, the goal of the present study was to establish murine in vivo models which allow the stepwise improvement of immunotherapies for gastric cancer. METHODS: Since no murine gastric adenocarcinoma cell lines are available we established four cell lines (424GC, mGC3, mGC5, mGC8) from spontaneously developing tumors of CEA424/SV40 T antigen (CEA424/Tag) mice and three cell lines derived from double-transgenic offsprings of CEA424/Tag mice mated with human carcinoembryonic antigen (CEA)-transgenic (CEA424/Tag-CEA) mice (mGC2(CEA), mGC4(CEA), mGC11(CEA)). CEA424/Tag is a transgenic C57BL/6 mouse strain harboring the Tag under the control of a -424/-8 bp CEA gene promoter which leads to the development of invasive adenocarcinoma in the glandular stomach. Tumor cell lines established from CEA424/Tag-CEA mice express the well defined tumor antigen CEA under the control of its natural regulatory elements. RESULTS: The epithelial origin of the tumor cells was proven by morphological criteria including the presence of mucin within the cells and the expression of the cell adhesion molecules EpCAM and CEACAM1. All cell lines consistently express the transgenes CEA and/or Tag and MHC class I molecules leading to their susceptibility to lysis by Tag-specific CTL in vitro. Despite the presentation of CTL-epitopes derived from the transgene products the tumor cell lines were tumorigenic when grafted into C57BL/6, CEA424/Tag or CEA424/Tag-CEA-transgenic hosts and no significant differences in tumor take and tumor growth were observed in the different hosts. Although no spontaneous tumor rejection was observed, vaccination of C57BL/6 mice with lysates from gastric carcinoma cell lines protected C57BL/6 mice from tumor challenge, demonstrating the tumorigenicity of the tumor cell lines in nontransgenic mice of the H-2(b )haplotype. CONCLUSION: These tumor cell lines grafted in different syngeneic hosts should prove to be very useful to optimize immunotherapy regimens to be finally tested in transgenic animals developing primary gastric carcinomas

Combination immunotherapy and active-specific tumor cell vaccination augments anti-cancer immunity in a mouse model of gastric cancer

<p>Abstract</p> <p>Background</p> <p>Active-specific immunotherapy used as an adjuvant therapeutic strategy is rather unexplored for cancers with poorly characterized tumor antigens like gastric cancer. The aim of this study was to augment a therapeutic immune response to a low immunogenic tumor cell line derived from a spontaneous gastric tumor of a CEA424-SV40 large T antigen (CEA424-SV40 TAg) transgenic mouse.</p> <p>Methods</p> <p>Mice were treated with a lymphodepleting dose of cyclophosphamide prior to reconstitution with syngeneic spleen cells and vaccination with a whole tumor cell vaccine combined with GM-CSF (a treatment strategy abbreviated as LRAST). Anti-tumor activity to subcutaneous tumor challenge was examined in a prophylactic as well as a therapeutic setting and compared to corresponding controls.</p> <p>Results</p> <p>LRAST enhances tumor-specific T cell responses and efficiently inhibits growth of subsequent transplanted tumor cells. In addition, LRAST tended to slow down growth of established tumors. The improved anti-tumor immune response was accompanied by a transient decrease in the frequency and absolute number of CD4⁺CD25⁺FoxP3⁺T cells (Tregs).</p> <p>Conclusions</p> <p>Our data support the concept that whole tumor cell vaccination in a lymphodepleted and reconstituted host in combination with GM-CSF induces therapeutic tumor-specific T cells. However, the long-term efficacy of the treatment may be dampened by the recurrence of Tregs. Strategies to counteract suppressive immune mechanisms are required to further evaluate this therapeutic vaccination protocol.</p

Anti-Gr-1 Antibody Provides Short-Term Depletion of MDSC in Lymphodepleted Mice with Active-Specific Melanoma Therapy

Lymphodepletion, reconstitution and active-specific tumor cell vaccination (LRAST) enhances the induction of tumor-specific T cells in a murine melanoma model. Myeloid-derived suppressor cells (MDSC) may counteract the induction of tumor-reactive T cells and their therapeutic efficacy. Thus, the aim of the study was to evaluate a possible benefit of MDSC depletion using anti-Gr-1 antibodies (Ab) in combination with LRAST. Female C57BL/6 mice with 3 days established subcutaneous (s.c.) D5 melanoma were lymphodepleted with cyclophosphamide and reconstituted with naive splenocytes. Vaccination was performed with irradiated syngeneic mGM-CSF-secreting D5G6 melanoma cells. MDSC depletion was performed using anti-Gr-1 Ab (clone RB6-8C5). Induction of tumor-specific T cells derived from tumor vaccine draining lymph nodes (TVDLN) was evaluated by the amount of tumor-specific interferon (IFN)-γ release. LRAST combined with anti-Gr-1 mAb administration enhanced the induction of tumor-specific T cells in TVDLN capable of releasing IFN-γ in a tumor-specific manner. Additional anti-Gr-1 mAb administration in LRAST-treated mice delayed growth of D5 melanomas by two weeks. Furthermore, we elucidate the impact of anti-Gr-1-depleting antibodies on the memory T cell compartment. Our data indicate that standard of care treatment regimens against cancer can be improved by implementing agents, e.g., depleting antibodies, which target and eliminate MDSC

Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene-2

<p><b>Copyright information:</b></p><p>Taken from "Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene"</p><p>BMC Cancer 2006;6():57-57.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1421424.</p><p>Copyright © 2006 Nöckel et al; licensee BioMed Central Ltd.</p>clone cosCEA1[14] are shown as color coded boxes (light blue, leader; red, IgV-like domain; blue IgC-like domain; gray, transmembrane domain; white, 5' and 3'-untranslated region exons. Flanking vector sequences are indicated as black boxes. The location of the CEA minimal promoter present in the SV40 Tag gene transgene is indicated by dotted lines, the names of the transgenic lines are shown in the left margin. (B) Flow cytometry was performed by labeling of the indicated cells either with the CEA-specific mAb 26/3/13 (filled curves) or an isotype-matched antibody (open curves) followed by PE-labeled goat anti-mouse IgG antibodies. (C, D) For Western analysis, 10 μg of total protein from extracts of 424GC or mGC8 cells established from CEA424/Tag-transgenic mice and mGC2and mGC4cells from CEA424/Tag × CEA-transgenic mice were size separated by SDS polyacrylamide gel electrophoresis, transferred to a membrane and reacted with the CEA-specific mAb 26/3/13 (C) or hamster polyclonal anti-Tag antibodies (D). Extracts from Cos7L-CEA and Meth-A cells stably transfected with expression vectors encoding CEA or a Tag lacking a region with the nuclear localization signal (cTag) served as a positive control. The sizes of protein markers are indicated in the left margins

Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene-6

<p><b>Copyright information:</b></p><p>Taken from "Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene"</p><p>BMC Cancer 2006;6():57-57.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1421424.</p><p>Copyright © 2006 Nöckel et al; licensee BioMed Central Ltd.</p>ssively growing tumors without overt necrosis after transplantation and growth of the indicated cell lines for 35–40 days. mGC4cells were incubated with the serum of the indicated mice at different dilutions and bound primary antibodies were detected with a PE-conjugated anti-mouse antibody. (B, C) Three C57BL/6 mice each were injected subcutaneously three times at weekly intervals with 1 × 106 mGC8 cells killed by two freeze-thaw cycles. Two weeks after the last vaccination, the mice were challenged by injection with 1 × 106 (B) and 3 × 106 (C) live mGC8 cells, respectively (filled-in circles). As a control, tumor cells were injected in non-immunized mice (open circles). Tumor volumes were calculated as described in Material and Methods section. Results are shown as mean values +/- SD

Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene-7

<p><b>Copyright information:</b></p><p>Taken from "Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene"</p><p>BMC Cancer 2006;6():57-57.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1421424.</p><p>Copyright © 2006 Nöckel et al; licensee BioMed Central Ltd.</p>tumor cell lines in wild-type C57BL/6 mice, Tag- or Tag × CEA-transgenic mice. Depending on whether the proteins represent self or foreign antigens they can be regarded as TAA or TSA. In spontaneously developing autochthonous tumors both CEA and Tag represent TAA

Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene-3

<p><b>Copyright information:</b></p><p>Taken from "Characterization of gastric adenocarcinoma cell lines established from CEA424/-transgenic mice with or without a human transgene"</p><p>BMC Cancer 2006;6():57-57.</p><p>Published online 14 Mar 2006</p><p>PMCID:PMC1421424.</p><p>Copyright © 2006 Nöckel et al; licensee BioMed Central Ltd.</p>n vitro stimulation with Tag peptide-loaded RBL5 cells were incubated with irradiated 424GC, 424 fibroblasts, RBL5 and Tag T1, T2/3 or T4 peptide-pulsed RBL5 cells for 24 h and their IFNγ secretion into the culture media was determined by ELISA. Secretion of IFNγ by CTL stimulated with 424GC cells indicates that these cells present SV40Tag-specific peptides in an MHCI-restricted manner. (B) Coculture of 424GC and 424 fibroblasts were treated with 107 Tag-specific CTL in a petri dish for 48 h. Thereafter, non-adherent (dead) cells were removed. Left, coculture before CTL treatment; right, coculture after treatment. Arrows indicate the position of the tumor cells before addition of CTL