5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

BACKGROUND: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. METHODS: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. RESULTS: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. CONCLUSIONS: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

Colon-specific immune microenvironment regulates cancer progression versus rejection.

Immunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset of patients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissue-specific factors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironment shapes the immune response to CRC is needed to identify mechanisms of resistance to immunotherapies and guide the development of novel therapeutics. In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescence imaging and the immune signatures were assessed at a transcriptional level using NanoString and at a cellular level by flow cytometry. Despite initial tumor growth in all mice, only 25% to 35% of mice developed a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor. No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injected in non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumors exhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernible shortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting mice presented an early inflammatory response and an antitumor microenvironment enriched in CD8+ T cells. Taken together, our data demonstrate the role of the colon microenvironment in regulating the balance between anti or protumor immune responses. While emphasizing the relevance of the CRC orthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancer response to immunotherapy.

FAK alternative splice mRNA variants expression pattern in colorectal cancer.

The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine-kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients' outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0 , FAK28 and FAK6 ) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0 , the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis.

Macrocyclic lactones inhibit nasopharyngeal carcinoma cells proliferation through PAK1 inhibition and reduce in vivo tumor growth.

PURPOSE: The Epstein-Barr virus (EBV)-associated cancer nasopharyngeal carcinoma (NPC) is rare in Europe and North America but is a real public health problem in some regions of the world, such as southern Asia, North Africa, and for Inuit populations. Due to the anatomy and location of the nasopharynx, surgery is rarely used to treat primary NPC cancers. Treatment by radiotherapy, combined or not with chemotherapy, are efficient for primary tumors but often do not protect against fatal relapses or metastases. METHODS: Search for new therapeutic molecules through high content screening lead to the identification of Ivermectin (IVM) as a promising drug. IVM is a US Food and Drug Administration-approved macrocyclic lactone widely used as anthelmintic and insecticidal agent that has also shown protective effects against cancers. RESULTS: We show here that IVM has cytotoxic activity in vitro against NPC cells, in which it reduces MAPKs pathway activation through the inhibition PAK-1 activity. Moreover, all macrocyclic lactones tested and a PAK1 inhibitor are cytotoxic in vitro for EBV-positive and EBV-negative NPC tumor cells. We have also shown that IVM intraperitoneal repeated injections, at US Food and Drug Administration-approved doses, have no significant toxicity and decrease NPC subcutaneous tumors development in nude mice. CONCLUSION: Macrocyclic lactones appear as promising molecules against NPC targeting PAK-1 with no detectable adverse effect.

Fluorescence-guided surgery for cancer patients: a proof of concept study on human xenografts in mice and spontaneous tumors in pets.

Surgery is often the first treatment option for patients with cancer. Patient survival essentially depends on the completeness of tumor resection. This is a major challenge, particularly in cases of peritoneal carcinomatosis, where tumors are widely disseminated in the large peritoneal cavity. Any development to help surgeons visualize these residual cells would improve the completeness of the surgery. For non-disseminated tumors, imaging could be used to ensure that the tumor margins and the draining lymph nodes are free of tumor deposits. Near-infrared fluorescence imaging has been shown to be one of the most convenient imaging modalities. Our aim was to evaluate the efficacy of a near-infrared fluorescent probe targeting the αvß3 integrins (Angiostamp™) for intraoperative detection of tumors using the Fluobeam® device. We determined whether different human tumor nodules from various origins could be detected in xenograft mouse models using both cancer cell lines and patient-derived tumor cells. We found that xenografts could be imaged by fluorescent staining irrespective of their integrin expression levels. This suggests imaging of the associated angiogenesis of the tumor and a broader potential utilization of Angiostamp™. We therefore performed a veterinary clinical trial in cats and dogs with local tumors or with spontaneous disseminated peritoneal carcinomatosis. Our results demonstrate that the probe can specifically visualize both breast and ovarian nodules, and suggest that Angiostamp™ is a powerful fluorescent contrast agent that could be used in both human and veterinary clinical trials for intraoperative detection of tumors.

Regulatory properties of statins and rho gtpases prenylation inhibitiors to stimulate melanoma immunogenicity and promote anti-melanoma immune response.

Melanoma is a highly lethal cutaneous tumor, killing affected patients through development of multiple poorly immunogenic metastases. Suboptimal activation of immune system by melanoma cells is often due to molecular modifications occurring during tumor progression that prevent efficient recognition of melanoma cells by immune effectors. Statins are HMG-CoA reductase inhibitors, which block the mevalonate synthesis pathway, used by millions of people as hypocholesterolemic agents in cardiovascular and cerebrovascular diseases. They are also known to inhibit Rho GTPase activation and Rho dependent signaling pathways. Rho GTPases are regarded as molecular switches that regulate a wide spectrum of cellular functions and their dysfunction has been characterized in various oncogenic process notably in melanoma progression. Moreover, these molecules can modulate the immune response. Since 10 years we have demonstrated that Statins and other Rho GTPases inhibitors are critical regulators of molecules involved in adaptive and innate anti-melanoma immune response. In this review we summarize our major observations demonstrating that these pharmacological agents stimulate melanoma immunogenicity and suggest a potential use of these molecules to promote anti-melanoma immune response.

Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors.

Melanoma-expressed CD70 is involved in invasion and metastasis.

BACKGROUND: CD70 is a costimulatory molecule of the tumour necrosis factor family expressed in activated immune cells and some solid tumours. In lymphocytes CD70 triggers T cell-mediated cytotoxicity and mitogen-activated protein kinase phosphorylation. METHODS: We evaluated the expression of CD70 in biopsies and melanoma cell lines. Using melanoma cell lines positive or not for CD70, we analysed CD70 function on melanoma progression. RESULTS: We report CD70 expression in human melanoma cell lines and tumour cells from melanoma biopsies. This expression was observed in 95% of primary melanomas but only 37% of metastases. Both monomeric and trimeric forms of CD70 were detected in tumour cell membrane fractions, whereas cytoplasmic fractions contained almost exclusively monomeric CD70. In vitro and in vivo experiments demonstrated that CD70 expression inhibited melanoma cell migration, invasion and pulmonary metastasis implantation independently of the tumour immune microenvironment. Increasing the levels of the trimeric form of CD70 through monoclonal antibody binding led to an increase in CD70+ melanoma cell invasiveness through MAPK pathway activation, RhoE overexpression, ROCK1 and MYPT1 phosphorylation decrease, and stress fibres and focal adhesions disappearance. CONCLUSIONS: Our results describe a new non-immunological function of melanoma-expressed CD70, which involves melanoma invasiveness through MAPK pathway, RhoE and cytoskeletal modulation.

In vivo Effects in Melanoma of ROCK Inhibition-Induced FasL Overexpression.

Ectopic Fas-ligand (FasL) expression in tumor cells is responsible for both tumor escape through tumor counterattack of Fas-positive infiltrating lymphocytes and tumor rejection though inflammatory and immune responses. We have previously shown that RhoA GTPase and its effector ROCK negatively control FasL membrane expression in murine melanoma B16F10 cells. In this study, we found that B16F10 treatment with the ROCK inhibitor H1152 reduced melanoma development in vivo through FasL membrane overexpression. Although H1152 treatment did not reduce tumor growth in vitro, pretreatment of tumor cells with this inhibitor delayed tumor appearance, and slowed tumor growth in C57BL/6 immunocompetent mice. Thanks to the use of mice-bearing mutated Fas receptors (B6/lpr), we found that reduced tumor growth, observed in immunocompetent mice, was linked to FasL overexpression induced by H1152 treatment. Tumor growth analysis in immunosuppressed NUDE and IFN-γ-KO mice highlighted major roles for T lymphocytes and IFN-γ in the H1152-induced tumor growth reduction. Histological analyses of subcutaneous tumors, obtained from untreated versus H1152-treated B16F10 cells, showed that H1152 pretreatment induced a strong intratumoral infiltration of leukocytes. Cytofluorometric analysis showed that among these leukocytes, the number of activated CD8 lymphocytes was increased. Moreover, their antibody-induced depletion highlighted their main responsibility in tumor growth reduction. Subcutaneous tumor growth was also reduced by repeated intravenous injections of a clinical ROCK inhibitor, Fasudil. Finally, H1152-induced ROCK inhibition also reduced pulmonary metastasis implantation independently of T cell-mediated immune response. Altogether, our data suggest that ROCK inhibitors could become interesting pharmacological molecules for melanoma immunotherapy.

Blocking Tumor Necrosis Factor α Enhances CD8 T-cell-Dependent Immunity in Experimental Melanoma.

TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma.

Synthesis and Antiviral Activity Evaluation of Nitroporphyrins and Nitrocorroles as Potential Agents against Human Cytomegalovirus Infection.

Different nitroporphyrinoid derivatives were synthesized and studied as potential agents against human Cytomegalovirus. Interestingly, two nitrocorroles display strong activity against human Cytomegalovirus with IC 50 < 0.5 µM. These compounds also possess antiproliferative activities without detected in vivo toxicity. Therefore, nitrocorroles appear for the first time as potential active compounds that can be applied in human health.

Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments.

Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

Statins stimulate in vitro membrane FasL expression and lymphocyte apoptosis through RhoA/ROCK pathway in murine melanoma cells.

The capacity of FasL molecules expressed on melanoma cells to induce lymphocyte apoptosis contributes to either antitumor immune response or escape depending on their expression level. Little is known, however, about the mechanisms regulating FasL protein expression. Using the murine B16F10 melanoma model weakly positive for FasL, we demonstrated that in vitro treatment with statins, inhibitors of 3-hydroxy-3-methylgutaryl CoA reductase, enhances membrane FasL expression. C3 exotoxin and the geranylgeranyl transferase I inhibitor GGTI-298, but not the farnesyl transferase inhibitor FTI-277, mimic this effect. The capacity of GGTI-298 and C3 exotoxin to inhibit RhoA activity prompted us to investigate the implication of RhoA in FasL expression. Inhibition of RhoA expression by small interfering RNA (siRNA) increased membrane FasL expression, whereas overexpression of constitutively active RhoA following transfection of RhoAV14 plasmid decreased it. Moreover, the inhibition of a RhoA downstream effector p160ROCK also induced this FasL overexpression. We conclude that the RhoA/ROCK pathway negatively regulates membrane FasL expression in these melanoma cells. Furthermore, we have shown that B16F10 cells, through the RhoA/ROCK pathway, promote in vitro apoptosis of Fas-sensitive A20 lymphoma cells. Our results suggest that RhoA/ROCK inhibition could be an interesting target to control FasL expression and lymphocyte apoptosis induced by melanoma cells.

Coexpression of CD40L and CD70 by semiallogenic tumor cells induces anti-tumor immunity.

The immune system is potentially qualified to detect and eliminate tumor cells, but various mechanisms developed by tumor cells allow tumor escape. Strategies selected to promote antitumor responses have included genetic modifications of tumor cells to induce expression of costimulatory molecules. Moreover, alloantigens can also act as strong enhancers of the immune response. In this work, we have associated the expression of two costimulatory members of the TNF superfamily, CD40L and CD70 along with an allogenic MHC Class I (H-2K(d)) molecule expression on melanoma cells (B16F10, H-2(b)) to favor the antitumor immune response. B16F10 tumor growth slows significantly when CD40L and CD70 are coexpressed by tumor cells and the association with the allogenic molecule (H-2K(d)) enhances this effect. Growth kinetics of mock and CD40L-CD70-H-2K(d)-expressing B16F10 tumors in immunocompetent versus nu/nu and beige mice suggested that CD8(+) T lymphocytes and NK cells were involved in this antitumor immunity. A delay in mock tumor growth was observed when CD40L-CD70-H-2K(d)-expressing B16F10 cells and mock tumor cells were injected simultaneously and contralaterally. It was also shown that in vivo immunization of immunocompetent mice with CD40L-CD70-H-2K(d) B16F10 tumor cells improved the generation of cytotoxic lymphocytes against the wild-type melanoma cells expressing the syngenic MHC Class I molecule H-2K(b) (B16K1). These observations lay a path for new immunotherapeutic trials using semiallogenic fibroblasts expressing costimulatory molecules and tumor-associated antigens.

Induction of T-cell antitumor immunity and protection against tumor growth by secretion of soluble human CD70 molecules.

One of the strategies to promote an antitumor response is the genetic modification of tumor cells to induce expression of costimulatory molecules. We have tested the capacity of a soluble form of CD70 molecule (sCD70). After construction of a vector carrying the sCD70, we obtained stable sCD70-secreting TS/A tumor cells and allogenic MC57 fibroblasts. In all, 45% of wild-type (wt) tumors were rejected in immunocompetent mice when transfected sCD70-secreting cells were injected three times in the periphery of the wt tumors. Furthermore, the sCD70-secreting TS/A cells induced a protective memory against wt TS/A tumor growth: 70% of the wt tumors used for the challenge were rejected by mice, which had rejected tumors 45 days before in the presence of sCD70-secreting TS/A cells. It was also shown that in vitro mock TS/A tumor cell proliferation was inhibited by splenocytes harvested from mice injected with TS/A cells expressing CD70. Growth kinetics of wt TS/A tumors in immunocompetent versus nude mice suggested that T lymphocytes were implicated in the antitumor response, which was confirmed by membrane expression of specific markers. The data suggest that injection of genetically transfected cells secreting sCD70 in the periphery of wt TS/A tumors induces T-cell-mediated inhibition of tumor growth and builds up a protective antitumor memory.

Influence of histidine beta81 of HLA-DR101 on peptide binding and presentation to T-cell receptor.

HA(306-318) is an immunodominant peptide of the hemagglutinin of influenza virus that binds to most human leukocyte antigen (HLA-DR) alleles, while p18(73-85) is a HIV peptide characterized as a DR101 binding peptide. Our results demonstrate that crystal relaxation leads to the loss of a hydrogen bond between the beta81 histidine and the HA(306-318) peptide. This histidine is also involved in the binding of superantigens like SEA via a coordination of a zinc atom. To monitor the interaction of these peptides with this histidine of HLA-DR molecules, chemical modification, peptide binding on HLA-DR101 wild type and mutated molecules, and proliferation experiments were conducted, together with molecular simulation of HLA-DR/peptide molecular complexes. Our data suggest a different binding peptide pattern, depending of whether the peptide is HLA-DR101 allele specific or a shared one. Furthermore, tyrosine substitution at position beta81 does not affect either peptide binding or HA(306-318) clone-specific T-cell proliferation. On the contrary, the alanine substitution at position HLA-DR101 beta81 abrogated both peptide binding and T-cell proliferation. These results suggest that the histidine 81 on the DRbeta chain plays an important role in the HLA-DR peptide binding, more likely by polar interactions of the amino acid side chain ring with the peptide.

In vivo induction of antitumor immunity and protection against tumor growth by injection of CD154-expressing tumor cells.

As they should enhance tumor-specific antigen presentation by dendritic cells, tumor cell lines genetically modified to express CD154 molecules have been used in an attempt to induce protective antitumor immunity. Two murine models were used: the major histocompatibility complex (MHC) class I negative melanoma B16F10 and the MHC class I positive mammary adenocarcinoma TS/A. CD154 or mock-transfected B16F10 or TS/A cells were injected subcutaneously into H-2-compatible B6D2 mice. CD154 expression by tumor cells induced a complete rejection (in the TS/A model) or a striking reduction (in the B16F10 model) of modified tumors growth, but also a significant protection against the growth of mock tumor cells injected simultaneously, either mixed with the CD154-expressing tumor cells, or in the other flank of mice. Thirty days after CD154-expressing tumor rejection, splenic lymphocytes from surviving tumor-free mice were able to inhibit tumor proliferation in vitro and significant amounts of IFN-gamma were detected in the sera of these mice. Growth kinetics of mock and CD154-expressing tumors in immunocompetent versus nude mice suggest that T lymphocytes and natural killer cells responses are implicated in this antitumor immunity. The injection of CD154-expressing tumor cell induced an antitumor protective response, both locally and distant from the injection site. The effect was most pronounced in MHC class I expressing TS/A tumor model.