Connective tissue growth factor promotes fibrosis downstream of TGFbeta and IL-6 in chronic cardiac allograft rejection.

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFbeta, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFbeta transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGFbeta expression remained unchanged, indicating that IL-6 effects may potentiate TGFbeta-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFbeta and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.

Critical role for IL-6 in hypertrophy and fibrosis in chronic cardiac allograft rejection.

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.

Transplant acceptance following anti-CD4 versus anti-CD40L therapy: evidence for differential maintenance of graft-reactive T cells.

Inductive therapy with anti-CD4 or anti-CD40L monoclonal antibodies (mAb) leads to long-term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long-term cardiac allografts following inductive anti-CD4 or anti-CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti-CD4-treated mice (anti-CD4 cells) did not reject allografts while those receiving cells from anti-CD40L-treated mice (anti-CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti-CD40L cells mounted a Th1 response following transfer while anti-CD4 cells mounted a dominant Th2 response. Early (day 10) T-cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti-CD4 mAb. Thus, anti-CD40L therapy appears to result in graft-reactive T cells with a naïve phenotype while anti-CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti-CD40L mAb targets activated, but not memory, cells for removal or functional silencing.

Transforming growth factor beta-induced connective tissue growth factor and chronic allograft rejection.

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti-CD4 therapy and develop CR or anti-CD40L therapy and do not develop CR. Both TGFbeta and connective tissue growth factor (CTGF), which is induced by TGFbeta, were expressed in grafts with CR but were not expressed in grafts without CR. TGFbeta transfection of allografts in anti-CD40L-treated recipients resulted in CTGF expression and CR. However, TGFbeta transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFbeta alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFbeta induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

BRCA2 and homologous recombination.

Two recent papers provide new evidence relevant to the role of the breast cancer susceptibility gene BRCA2 in DNA repair. Moynahan et al provide genetic data indicating a requirement for BRCA2 in homology-dependent (recombinational) repair of DNA double-strand breaks. The second paper, by Davies et al, begins to address the mechanism through which BRCA2 makes its contribution to recombinational repair. BRCA2 appears to function in recombination via interactions with the major eukaryotic recombinase RAD51 [1,2,3]. We briefly review the context in which the two studies were carried out, we comment on the results presented, and we discuss models designed to account for the role of BRCA2 in RAD51-mediated repair.

Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA.

Dmc1 and Rad51 are eukaryotic RecA homologues that are involved in meiotic recombination. The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis. Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination. Here we report the purification of the Dmc1 protein from the budding yeast Saccharomyces cerevisiae and present basic characterization of its biochemical activity. The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA. Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative. Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor. In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP.

Nine novel conserved motifs in BRCA1 identified by the chicken orthologue.

The breast cancer susceptibility gene BRCA1 encodes a multifunctional protein that is mutated in many hereditary breast and ovarian cancers. We have cloned a homologue of the human BRCA1 gene from chicken; the gene encodes a 1749 amino acid protein that is 33% identical to human BRCA1. Phylogenetic analyses of the chicken and mammalian proteins indicate that the chicken gene is a bona fide BRCA1 orthologue, the first to be described from a non-mammal. Most of the chicken protein has diverged considerably from its mammalian orthologues, although the RING and BRCT repeat regions are highly conserved. This marked overall sequence divergence has allowed us to identify nine additional highly-conserved motifs (ranging from 8--56 amino acids in length) which are likely important for BRCA1 function.

Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis.

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions.

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.

DNA-liposome versus adenoviral mediated gene transfer of transforming growth factor beta1 in vascularized cardiac allografts: differential sensitivity of CD4+ and CD8+ T cells to transforming growth factor beta1.

We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.

Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes.

Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena. Here we report that Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated. Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation. Our findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes.

The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin.

Mutations in breast cancer tumor susceptibility genes, BRCA1 and BRCA2, predispose women to early onset breast cancer and other malignancies. The Brca genes are involved in multiple cellular processes in response to DNA damage including checkpoint activation, gene transcription, and DNA repair. Biochemical interaction with the recombinational repair protein Rad51 (Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435), as well as genetic evidence (Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518 and Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L., and Koller, B. H. (1999) Oncogene 18, 7900-7907), demonstrates that Brca1 is involved in recombinational repair of DNA double strand breaks. Using isogenic Brca1(+/+) and brca1(-/-) mouse embryonic stem (ES) cell lines, we investigated the role of Brca1 in the cellular response to two different categories of DNA damage: x-ray induced damage and cross-linking damage caused by the chemotherapeutic agent, cisplatinum. Immunoflourescence studies with normal and brca1(-/-) mutant mouse ES cell lines indicate that Brca1 promotes assembly of subnuclear Rad51 foci following both types of DNA damage. These foci are likely to be oligomeric complexes of Rad51 engaged in repair of DNA lesions or in processes that allow cells to tolerate such lesions during DNA replication. Clonogenic assays show that brca1(-/-) mutants are 5-fold more sensitive to cisplatinum compared with wild-type cells. Our studies suggest that Brca1 contributes to damage repair and/or tolerance by promoting assembly of Rad51. This function appears to be shared with Brca2.

Gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 prolongs cardiac allograft survival and inhibits donor-specific immunity.

Introducing immunomodulatory molecules into allografts by gene transfer may avoid the side-effects of systemic immunosuppression. vMIP-II and MC148 are two recently identified chemokine homologues encoded by human herpes virus 8 and Molluscum contagiosum, respectively, that have antagonistic activities against multiple different CC and CXC chemokine receptors. We hypothesized that introduction of these molecules into cardiac allografts may block leukocyte infiltration into the grafts and prolong survival. Vascularized and nonvascularized cardiac allografts in mice were performed and plasmid DNA encoding vMIP-II, MC148 and/or vIL-10 was transferred into the allograft at the time of transplantation. Gene transfer of either vMIP-II or MC148 into cardiac allografts markedly prolonged graft survival. Combining gene transfer of either one of these chemokine antagonists with vIL-10 gene transfer, which has a mechanistically different immunosuppressive action, further enhanced graft survival. vMIP-II and MC148 gene transfer both resulted in a marked decrease of donor-specific cytotoxic T lymphocytes (CTL) infiltrating the grafts and inhibited alloantibody production. These results demonstrate that plasmid-mediated gene transfer of virally encoded chemokine antagonists vMIP-II and MC148 can block donor-specific lymphocyte immunity within cardiac allografts and prolong graft survival. This is a new mechanistic approach to analyze, treat, and prevent graft rejection. Delivery of these or related molecules by gene transfer or conventional pharmacologic means may represent a novel therapeutic modality for alloactivation.

Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony-stimulating factor.

We performed a clinical study of five patients with melanoma to evaluate the immunobiological effects of retrovirally transduced autologous tumor cells given as a vaccine to prime draining lymph nodes. Patients were inoculated with both wild-type (WT) and GM-CSF gene-transduced tumor cells in different extremities. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were removed. There was an increased infiltration of dendritic cells (DCs) in the GM-CSF-secreting vaccine sites compared with the WT vaccine sites. This resulted in a greater number of cells harvested from the GM-CSF-VPLNs compared with the WT-VPLNs at a time when serum levels of GM-CSF were not detectable. Four of five patients proceeded to have the adoptive transfer of GM-CSF-VPLN cells secondarily activated and expanded ex vivo with anti-CD3 MAb and IL-2. One patient had a durable complete remission of metastatic tumor. Utilizing cytokine (IFN-gamma, GM-CSF, IL-10) release assays, GM-CSF-VPLN T cells manifested diverse responses when exposed to tumor antigen in vitro. In two of two patients, GM-CSF-VPLN T cell responses were different from those of matched WT-VPLN cells. This study documents measurable immunobiologic differences of GM-CSF-transduced tumor cells given as a vaccine compared with WT tumor cells. The complete tumor remission in one patient provides a rationale to pursue this approach further.

Tissue-specific consequences of the anti-adenoviral immune response: implications for cardiac transplants.

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding beta-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.

Xrcc3 is required for assembly of Rad51 complexes in vivo.

Rad51 is a member of a family of eukaryotic proteins related to the bacterial recombinational repair protein RecA. Rad51 protein localizes to multiple subnuclear foci in Chinese hamster ovary cells. Subnuclear Rad51 foci are induced by ionizing radiation or the DNA cross-linking agent cisplatin. Formation of these foci is likely to reflect assembly of a multimeric form of Rad51 that promotes DNA repair. Formation of damage-induced Rad51 foci does not occur in the Chinese hamster ovary cell line irs1SF, which is sensitive to DNA damaging agents. The Rad51 focus formation defect of irs1SF cells is corrected by a construct that encodes the repair protein Xrcc3. Xrcc3 is a human homolog of Rad51 previously isolated by virtue of its ability to correct the radiation sensitivity of irs1SF cells. Changes in the steady state level of Rad51 protein do not account for the irs1SF defect nor do they account for the appearance of foci following DNA damage. These results suggest that Xrcc3 is required for the assembly or stabilization of a multimeric form of Rad51 during DNA repair. Cell lines defective in two different components of DNA protein kinase formed Rad51 foci in response to damage, indicating DNA protein kinase is not required for damaged-induced mobilization of Rad51.

Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes.

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Inhibition of the alloantibody response by CD95 ligand.

We investigated the effect of Fas/APO1-ligand (CD95L) gene transfer on allogeneic immune responses in vivo. A colon carcinoma cell line from BALB/c mice, CT26, was stably transfected with a vector encoding mouse CD95L and was inoculated into C57BL/6 mice. CD95L expression markedly reduced allogeneic cytotoxic T lymphocyte and helper T lymphocyte activity directed toward CT26. Strikingly, expression of CD95L on these allogeneic tumors completely inhibited the generation of alloantibodies of both IgM and IgG subclasses. Thus, CD95L inhibited alloantibody production and conferred localized immune suppression through this mechanism. These results provide insight into the role of CD95L in regulating the alloantibody response and the generation of local immune responses.

Type 1 versus type 2 cytokine release by Vbeta T cell subpopulations determines in vivo antitumor reactivity: IL-10 mediates a suppressive role.

We have previously reported that CD8+ tumor-draining lymph node (TDLN) cells activated with anti-CD3 and IL-2-mediated tumor regression in adoptive immunotherapy. In this study, we examined the TCR Vbeta repertoire usage of TDLN cells with respect to cytokine release profiles and therapeutic efficacy in vivo. The majority of the whole population of TDLN cells after activation with anti-CD3 were composed of Vbeta3+, -5+, -7+, -8+, and -11+ cells. Enrichment of Vbeta subsets of TDLN cells by in vitro activation with anti-Vbeta mAb revealed Vbeta8+ cells released high amounts of IFN-gamma and granulocyte/macrophage-CSF (GM-CSF) with minimal amounts of IL-10 in response to tumor and mediated tumor regression in vivo. In contrast, enriched populations of Vbeta5+, Vbeta7+, and Vbeta11+ cells released low amounts of IFN-gamma and GM-CSF with high levels of IL-10 and had no in vivo antitumor reactivity. In vitro depletion of specific Vbeta subsets from the whole TDLN pool confirmed that the profile of cytokines released correlated with in vivo antitumor function. Therapeutic efficacy mediated by TDLN cells required the release of IFN-gamma and GM-CSF since in vivo neutralization of both cytokines inhibited tumor regression. The administration of anti-IL-10 mAb abrogated the suppressed antitumor response manifested by adoptively transferred TDLN cells, which elaborated increased levels of IL-10. Our study documents that type 1 cytokine release (i.e., IFN-gamma and GM-CSF) promotes in vivo tumor Ag recognition, in contrast to type 2 release (i.e., IL-10), which suppresses this interaction, and discriminates the functional activity of Vbeta subpopulations of effector cells.