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Abstract 130 Multiple myeloma (MM) bone marrow (BM) provides a homing microenvironment, which is important for tumor development, growth, proliferation, migration and chemoresistance. Our previous research has shown that macrophages (MΦ) are associated with malignant BM and confer MM cell chemoresistance in vitro. Based on these findings, we designed mechanistic studies to elucidate MΦ-mediated MM chemoresistance. MΦ protected MM cells from different chemotherapeutics, such as bortezomib, doxorubicin, dexamethasone, melphalan, or their combination-induced apoptosis. Cell-to-cell contact was crucial for such protection. Under a coculture condition, MΦ protected MM cells with wide ranging MM/MΦ ratio. Inhibition of phagocytosis did not affect the protection. An experiment examining MM patients' BM showed that MΦ was a dominant accessory cell population in patients' BM. Patients PBMC-derived MΦ or primary MΦ isolated from patient BM had protective activity. Further mechanistic study showed that surface molecules, such as P/E selectins (on MΦ) and their ligand PSGL-1 (on MM cells), were important for MΦ-mediated MM chemoresistance. Blockade antibodies against either P/E selectins or PSGL-1 significantly repressed MΦ-mediated drug resistance. MΦ had limited protection on PSGL-1-knocked down MM cells. Next, we examined intracellular signal transduction in MM cells, upon interaction with MΦs. Src family kinase and Erk1/2 kinase were activated in MM cells, after coculture with MΦs. Coculture also stimulated PSGL-1 and c-myc overexpression in MM cells. Inhibition of Src family kinase, Erk1/2 kinase, or c-myc by small molecule inhibitors impaired MΦ-mediated MM chemoresistance. The overexpression of PSGL-1, under coculture condition, could be repressed by IFN-α neutralization antibody. More importantly, coculture with MΦ could not stimulate Erk1/2 activation or c-myc overexpression in PSGL-1-knocked down MM cells, suggesting that Erk1/2 and c-myc were downstream of PSGL-1-initiated signal transduction. Finally, we established a NOD-SCID mouse model to test MΦ-mediated MM chemoresistance in vivo. MM tumors with MΦ infiltration grew significantly faster than MM, only, tumors and were more resistant to melphalan treatment. Overall, our data demonstrated a mechanism of MΦ-mediated chemoresistance, both in vitro and in vivo. Based on our findings, MΦ-targeted therapy in MM treatment may be beneficial to improve the effectiveness of MM chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 694 Osteoclasts (OCs), the responsible cells for bone resorption, are derived from monocytic precursor cells, with the stimulation of macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor κb ligand (RANKL). The formation and activity of OCs can be either promoted by activated T cell derived RANKL, IL-17, or suppressed by T cell derived IFN-γ, IL-10, and IL-4. On the other hand, OCs express MHC, and costimulatory molecules, secrete IL-10, TGF-β, TNF-α and IL-6 and can act as antigen presenting cells to activate T cells, which indicates that OCs can be considered as immune cells. However, the immune function of OCs is largely unknown, and whether activated T cells can regulate the immune function of OCs is also unclear. In this study, we investigated the effect of OCs on T cell responses, and the cross regulation between activated T cells and immune regulatory OCs. Results showed that autologous OCs could inhibit the proliferation of CD4+ T cells activated by allogeneic antigen, tetanus toxin, staphylococcal enterotoxin B, and anti-CD3/CD28 antibodies. The inhibitory rate range varied from 63% to 88%. To identify the mechanism of OC-mediated T cell suppression, we blocked or inhibited TGF-β, IL-10, (prostaglandin E2) PGE-2, and indoleamine 2,3-dioxygenase (IDO) with neutralizing antibodies or specific inhibitors during the coculture. Results showed that only 1-methyl-DL-tryptophan (1-MT, an IDO inhibitor) could rescue the T cell proliferation, which suggested that OCs mediated the T cell suppression through IDO. To confirm this result, we knocked down IDO expression in OCs with siRNA and found that T cell proliferation was restored completely. As normal OCs didn't express IDO, next we investigated which molecules induced IDO expression in OCs, when cocultured with activated T cells. Results showed that blocking IFN-γ and CD40 ligand (CD40L) could inhibit IDO expression in OCs and rescue the T cell proliferation, and recombinant IFN-γ and soluble CD40L could induce IDO expression in OCs, synergistically. In conclusion, our study identified that OCs can function as immune regulatory cells to suppress T cell proliferation through IDO, which is induced by activated T cell derived IFN-γ and CD40L. This study provides new insight into the reciprocal interaction between OCs and T cells and may be helpful to develop novel therapeutic strategies for diseases involved in both bone and immune systems, such as bone-invasive tumors and autoimmune arthritis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3040 Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of almost all patients with multiple myeloma (MM) and may be responsible for suppressed osteoblast formation. In our previous studies, we demonstrated that DKK-1 is a potent tumor-associated antigen in MM recognized by cytotoxic T lymphocytes (CTLs), which can effectively lyse autologous myeloma tumor cells in vitro (Qian et al., Blood 2007;110:1587-1594) and eradicate established patient-derived primary myeloma in SCID-hu mice upon adoptive transfer. To examine the potential of DKK1-based immunotherapy in MM, we investigated the efficacy of active vaccination with (murine) DKK1 DNA vaccine in a murine (MOPC-21) myeloma model. A plasmid DNA construct encoding defensin2-sFv was generated, and DKK1 full-length cDNA was cloned from DKK1-expressing mouse stromal cells by reverse transcription–polymerase chain reaction and genetically fused with defensin2 (DNA-vac). We first examined the ability of the vaccines to protect mice from developing myeloma. While 100% of mice vaccinated with vector control or PBS (10 mice for each group) developed tumors, 70% and 40% mice vaccinated with DNA-vac or DNA-vac with CpG (ODN 1826), respectively, developed tumors. These results clearly show that active vaccination with DKK1 DNA vaccines was able to protect mice from tumor challenge, and that combination with CpG was more effective than DNA vaccine alone. On day 90 after tumor challenge, all surviving tumor-free mice were rechallenged with the same myeloma cells and followed for tumor development. By day 180, All surviving mice that were vaccinated with DNA-vac or DNA-vac with CpG have no developed tumors, indicating that active vaccination with DNA vaccines induced strong tumor-specific memory immune responses to protect mice from tumor rechallenge. Next, we examined the therapeutic effects of DNA vaccines in our myeloma mouse model. DNA vaccine alone eradicated established myeloma in 1 out of 5 mice, while DNA vaccine plus CpG eradicated myeloma in 3 out of 5 mice bearing intermediate tumors (≥ 5 mm in diameter). These results indicate that DNA vaccine plus CpG was much more effective at eradicating established myeloma than DNA vaccine alone. Finally, the mechanisms of tumor protection induced by DNA vaccines were investigated. By depleting CD4+ or CD8+ T cells, we showed that CD8+ T cells are required for DNA-induced antimyeloma responses. In DNA-vaccinated mice, splenocytes contained increased numbers of DKK1-specific, IFN-g-secreting and proliferative T cells. The splenic CD8+ T cells exhibited enhanced cytotoxicity against myeloma cells. Furthermore, DKK1-specific CD8+ T cells were shown to be increased in DKK1-DNA vaccinated mice by using DKK1-peptide (P11, P15 and P210) tetramer staining. These results demonstrate the presence of myeloma-specific CTLs in vaccinated mice and show that DKK1 DNA vaccine can induce a potent CTL response capable of killing myeloma cells. Together, our study lays a basis for future clinical trials in MM by using DKK1 as a vaccine for all patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 3619 Poster Board III-555 Granulocyte colony-stimulating factor (G-CSF) controls neutrophil production in the bone marrow under steady state conditions and during demand-driven hematopoiesis occurring in response to infection. STAT3 is a principal signaling molecule activated by the G-CSF receptor (G-CSFR). We previously reported that STAT3 has an important role in demand-driven granulopoiesis, although its cellular and molecular mechanisms have been unclear. To address this, we investigated STAT3 function in emergency granulopoiesis stimulated by G-CSF administration or infection with Listeria monocytogenes, which is restrained by the G-CSF response pathway in vivo. Our results show that STAT3-deficiency renders hematopoietic stem cells and myeloid progenitors refractory to the proliferation-inducing effects of G-CSF or Listeria monocytogenes infection. STAT3-deficient myeloid progenitors have a cell autonomous defect in G-CSF-responsive cell cycle progression and undergo delayed granulocyte maturation relative to wild type cells. To define STAT3 target pathways in granulocytic progenitors, we investigated the expression of CCAAT enhancer binding protein (C/EBP) beta, a transcription factor that is necessary for G-CSF-driven emergency granulopoiesis. We found that STAT3 directly regulates G-CSF-responsive C/EBPbeta expression by binding to Cebpb promoter. Moreover, we show that STAT3 and C/EBPbeta co-regulate c-Myc during emergency granulopoiesis. These results place STAT3 as a crucial G-CSF-responsive signal transducer during demand-driven granulopoiesis, through its regulation of critical transcription factors in developing granulocytes. Disclosures: Zhang: Amgen: Research Funding. Nguyen-Jackson:Amgen: Research Funding. Watowich:Amgen, Inc: Research Funding.

Abstract 236 Neutrophil mobilization, the release of neutrophils from the bone marrow reserve into circulating blood, is important to increase peripheral neutrophil amounts during bacterial infections. Granulocyte colony-stimulating factor (G-CSF) and chemokines, such as MIP-2 (CXCL2), can induce neutrophil mobilization, but the mechanism(s) they employ remain unclear. The transcription factor STAT3 is the principal intracellular signaling molecule activated upon G-CSF-ligation of its receptor. Using a murine model with conditional STAT3 deletion in bone marrow, we demonstrated previously that acute G-CSF-responsive neutrophil mobilization and MIP-2-dependent neutrophil chemotaxis is mediated by STAT3. In this study, we show that STAT3 is also necessary for MIP-2-elicited neutrophil mobilization. STAT3 appears to function by regulating the amplitude of Raf/MEK/Erk activation by MIP-2, a signaling pathway that is important for MIP-2-mediated chemotaxis. In addition, we demonstrate that G-CSF stimulates the expression of the MIP-2 receptor via STAT3-dependent transcriptional activation of Il8rb. G-CSF treatment also induces changes in bone marrow chemokine expression levels in a STAT3-dependent manner, which may further affect neutrophil retention and release. Taken together, our study demonstrates that STAT3 regulates multiple aspects of chemokine and chemokine receptor expression and function within the bone marrow, indicating a central role in the neutrophil mobilization response. Disclosures: Nguyen-Jackson: Amgen, Inc: Research Funding. Panopoulos:Amgen, Inc: Research Funding. Zhang:Amgen, Inc: Research Funding. Li:Amgen, Inc: Research Funding. Watowich:Amgen, Inc: Research Funding.

Abstract 740 Bone destruction is common in osteolytic tumors such as multiple myeloma. Myeloma cells that reside in the bone cause osteolysis, which is characterized by severe bone pain, multiple fractures, and hypercalcemia in patients and thereby, compromises patient quality of life. While myeloma cells have been proposed to activate osteoclasts and/or inhibit osteoblasts leading to bone resorption, the mechanism underlying myeloma-induced osteolytic bone lesions is still poorly elucidated. As p38 mitogen-activated protein kinase (MAPK) is constitutively activated in myeloma, we hypothesized that p38 MAPK activation in myeloma cells might be responsible for myeloma-induced osteolytic bone lesions. Human myeloma cell lines ARP-1 and MM.1S caused bone lesions in SCID mice after intravenous injection. However, once p38 MAPK was knocked down by small hairpin RNAs (shRNAs) in these cell lines, myeloma was established but failed to cause bone lesions in SCID mice, whereas the wild-type or vector control myeloma cells established myeloma (with similar tumor burdens as myeloma cells with knocked-down p38 MAPK) and caused bone lesions in tibial bones, as measured by radiograph and peripheral quantitative m-computed tomography and further confirmed by histological examination. In vivo examination revealed that the number of bone surface-covering osteoclasts, osteoclast size, cellular nuclear numbers in per osteoclast, and levels of circulating collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker) were all reduced in mice injected with myeloma cells with knocked-down p38 MAPK as compared with controls. Consistently, we found that tumor p38 MAPK had active effects on in vitro osteoclast differentiation and bone resorption, and inhibited osteoblast differentiation and function, suggesting that constitutive activation of p38 MAPK in myeloma cells induces osteoclastogenesis. By functional studies and protein array analysis, we showed that monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) are upregulated downstream of p38 MAPK activation in myeloma cells and are responsible for enhanced osteoclastogenesis. Our results showed that p38 MAPK activation in myeloma cells upregulate MCP-1 and DKK-1 expression and production, which are released into the microenvironment. MCP-1 enhanced RANK expression on osteoclast precursors and DKK-1 increased RANKL secretion from stromal cells, all of which led to activation of NF-kB and MAPK signaling pathways in osteoclasts. In vivo blockade of MCP-1 and DKK-1 by specific antibodies significantly abrogated tumor p38 MAPK-induced osteoclast activation and bone lesions in established myeloma-SCID mice. Thus, our results have elucidated a novel mechanism that p38 MAPK activity in myeloma cells contributes to myeloma-induced osteolytic bone lesions. This study indicates that disruption of tumor p38 MAPK may be a new therapeutic approach to treat osteolytic bone lesions in myeloma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4804 Multiple myeloma is a B-cell malignancy characterized by the proliferation of plasma cells in the bone marrow. It is the second most common hematological malignancy and is still largely incurable. One of the major problems is that myeloma cells develop drug resistance upon interaction with bone marrow stromal cells. To better understand the importance of different stromal cell components in the bone marrow microenvironment, we examined the effects of macrophages on myeloma cell survival and myeloma cell response to chemotherapy. We report here that macrophages, in particular tumor-associated macrophages obtained by culturing macrophages with myeloma cell culture supernatants, are a protector of myeloma cells. Macrophages protected both myeloma cell lines and primary myeloma cells isolated from patients from spontaneous and chemotherapy drug-induced apoptosis via attenuating the activation of caspase-dependent apoptotic signaling. The protective effect was dependent on direct contact between macrophages and myeloma cells. Although tumor-associated macrophages secreted large amounts of IL-6, which is the most important survival factor for myeloma cells, our results showed that IL-6 neutralizing antibodies fail to significantly affect the protective effects of tumor-associated macrophages. The reduced numbers of apoptotic tumor cells in the cocultures were not the result of macrophage-uptake of apoptotic cells, because macrophages with or without the ability to phagocytose apoptotic cells provide similar protection to myeloma cells against chemotherapy-induced apoptosis. These findings are clinically relevant, because we examined bone marrow biopsies of patients by immunochemical analysis and found that CD68+ macrophages are heavily infiltrated in the bone marrow (tumor bed) of patients with myeloma but not control patients. Thus, our results indicate that macrophages are an important component of the bone marrow stromal cells and may contribute to myeloma cell survival and resistance to chemotherapeutic treatment in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 465 Immunotherapy may complement the current treatments for lymphomas. The lack of suitable shared lymphoma-associated antigens limits its applicability. Therefore, identification and utilization of novel and more potent tumor-associated antigens, particularly those shared among patients, are urgently needed to improve the efficacy of immunotherapy in the diseases. Recent studies have shown that Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by myeloma and other tumor cells, and is absent from normal tissues and organs except placenta and prostate. In the present study we demonstrated that DKK1 is also overexpressed in mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated HLA-A*0201+ DKK1-specific CTL lines and clones in vitro. These CTLs effectively lysed DKK1+/HLA-A*0201+ lymphoma cell lines Jeko-1 and Granta 519 cells, but not DKK1-/HLA-A*0201+ BJAB, RL and Mino cells nor DKK1+/HLA-A*020- CA46 and Daudi cells. Furthermore, the T-cell clones efficiently killed DKK1+/HLA-A*0201+ primary B-cell lymphoma cells from patients but not lymphoma cells from DKK1–/HLA-A*0201+ patients. HLA-ABC or HLA-A*0201 blocking mAbs significantly inhibited T cell-mediated cytotoxicity against peptide-pulsed T2 cells (P < .01, compared with medium control). No inhibitory effect was observed with mAb against HLA-DR and isotype control IgG. The results indicate that the cytotoxicity was attributed to MHC class I and more specifically, HLA-A*0201-restricted CD8+ CTLs. The CTLs did not kill DKK1–/HLA-A*0201+ DCs, B cells, or PBMCs, These results suggest that the CTLs recognized DKK1 peptides that are naturally processed and presented in the context of HLA-A*0201 molecules on lymphoma cells. To determine the in vivo antitumor activity, NOD-SCID and SCID-hu mice were used for lymphoma cell lines and primary lymphoma cells, respectively. Mice were treated with DKK1-specific CTLs after tumor established in NOD-SCID and SCID-hu mice. Control mice were treated with naïve CD8+ T cells or PBS alone. Tumor burden was measured according to levels of circulating human B2M, and survival rates were determined. Low levels (< 50 ng/ml) of circulating human B2M were detected in group treated DKK1-specific CTLs, while high levels (≥ 150 ng/ml) of circulating human B2M were detected in control mice. In SCID-hu model, X-ray examination showed that established tumors were eradicated in 60% mice treated with DKK1-specific CTLs, while large tumor burdens were found in all control mice. In NOD-SCID model, 40% of mice survived with the treatment of DKK1-specific CTLs. TUNEL assay further confirmed that tumor cells were lysed by DKK1-specific CTLs not naïve CD8+ T cells. These results indicate that DKK1-specific CTLs are able to eradicate established, patient-derived primary B- cell lymphoma in the hosts and adoptive transfer of DKK1-specific CTLs may be used for B-cell lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma (MM) remains fatal in the majority of patients with the disease, even though the application of new therapeutic agents has greatly advanced the treatment of the disease. There is an urgent need to develop novel therapeutic agents. We recently demonstrated that anti-β2-microglobulin monoclonal antibodies (anti-β2M mAbs) have remarkably strong apoptotic effects on myeloma in vitro and in SCID mice. However, whether the mAbs will be therapeutic and safe in treating myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined. In this study, we examined the antimyeloma activity and potential toxicity of the mAbs in a human-like situation. We developed and used a myeloma-HLA-A2-transgenic NOD/SCID mouse model. After myeloma establishment in NOD/SCID mice transgenic for HLA-A2 α-chain, we found that all tissues express human HLA-A2 and β2M, and detected high levels (2 mg/mL) of circulating human β2M. After one intraperitoneal (i.p.) injection of the mAbs (1 mg per mouse), the mAbs could be detected on the surface of tumors and murine organs. Furthermore, murine splenocytes were incubated with immobilized mAb W6/32 to crosslink MHC class I. Western blotting analysis showed that cross-linking human MHC class I triggers phosphorylation of Lyn, Syk, and PLC-γ2 in the treated cells, indicating that indeed human MHC class I are functional and able to transduce signals in murine cells. Thus, these findings demonstrate that the mouse model is suitable for our studies because myeloma-derived human β2M was highly elevated in the serum, and bound with the α-chain to form mature and functional human MHC class I on all tissues. By using the model, we first examined the therapeutic efficacy of the mAbs on established myeloma. Myeloma-bearing mice were i.p. injected with the mAbs (1 mg per mouse) twice a week for a total of 4 injections. The results showed that the mAbs effectively suppress myeloma growth, and activated caspase-9 and -3 and induced myeloma cell apoptosis in vivo. We also examined whether the mAbs damage human MHC class I-expressing normal cells and tissues. After treatment with the mAbs, murine organs were removed for histological examination. We found that although the mAbs were detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Furthermore, we elucidated the mechanisms underlying the selectivity of the mAbs in killing myeloma cells without damaging normal cells. By using the QuantiBRITE beads, we quantified the numbers of surface β2M and found that myeloma cells have approximately 265,918 surface β2M molecules whereas normal blood lymphocytes have approximately 79,521, indicating that myeloma cells express 3-fold more surface β2M than lymphocytes. Myeloma cells treated with specific siRNAs for human β2M expressed a similarly low level of β2M (91,723) and became resistant to mAb-induced apoptosis. Thus, our findings indicate that the mAbs may be safe and that the tissue-expressed and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.

Multiple myeloma is still an incurable plasma malignancy because patients are prone to quickly relapse after conventional treatment or even high-dose chemotherapy. At the present time, monoclonal antibodies (mAbs) are being successfully used to treat cancers. We recently discovered that anti-β2-microglobulin (β2M) mAbs (IgG1 isotype) induced myeloma cell apoptosis (Yang et al., Cancer Cell2006; 10:295–307). The mAbs bind, cross-link, and recruit surface β2M/MHC class I molecules on myeloma cells to lipid rafts where downstream signaling pathways are activated. Therefore, it is possible that enhancing the capacity of the mAbs to cross-link surface MHC class I could further improve the efficacy of mAb-induced myeloma cell apoptosis. To examine this hypothesis, we generated anti-β2M mAbs of IgM isotype with a pentameric structure. By using Annexin V and TUNEL assays, we showed that, compared with monomeric IgG mAbs, IgM anti-β2M mAbs exhibit stronger tumoricidal activity on all six myeloma cell lines and primary myeloma cells isolated from five myeloma patients in dose- and time-dependent manner. About 80% to 90% of myeloma cells were apoptotic when treated with IgM mAb at a concentration as low as 20 mg/mL in a 12-hour culture. Furthermore, IgM anti-β2M mAbs, which at the same dose had stronger therapeutic efficacy than IgG mAbs in vivo, had greatly reduced tumor burdens and retarded tumor growth in SCID mice. To examine whether the pentameric structure plays an important role in IgM mAb-mediated tumoricidal activity, IgM mAbs were treated with β-mercaptoethanol (2ME), a specific agent that breaks the J-chain of IgM antibody leading to irreversible disruption of the IgM pentameric structure. By using native gel electrophoresis, we confirmed that without 2ME treatment, IgM mAbs were pentamers with one band of 950 kDa. After treatment with 2ME, IgM mAbs displayed a strong band in 175 kDa (monomer), indicating that 2ME completely broke pentamers to monomers. Furthermore, we found that the addition of 2ME-pretreated IgM mAbs to cell culture induced much weaker myeloma cell apoptosis than pentameric IgM mAbs. By using Western blot analysis, we further showed that 2ME-pretreated IgM mAbs induce weaker JNK activation and caspase-9, -8, -3, and PARP cleavage. By using confocal microscopy, we showed that 2ME-pretreated IgM mAbs are much less efficient at recruiting β2M/MHC class I molecules into lipid rafts on myeloma cells, although 2ME-pretreated IgM mAbs bound well to myeloma cell surface. Thus, our findings indicate that enhancing cross-linking of surface β2M/MHC class I molecules may be a novel approach to improve the antimyeloma efficacy of the mAbs. This study also suggests that our IgM anti-β2M mAbs may be a better therapeutic agent for future clinical application.

H-2sA.SW and B10.S mice are susceptible and resistant to experimental autoimmune myocarditis (EAM), respectively. Our previous studies suggested that Eam1 on chromosome 1 is a non-MHC locus conferring susceptibility to cardiac myosin-induced (EAM) in A.SW mice. B10.A Eam1 congenic mice, carrying the Eam1 locus on the EAM-resistant B10.S background, develop EAM comparable to A.SW mice, suggesting that Eam1 plays a key role in the development of disease. We have previously reported that lymph node cells (LNC) of A.SW mice are more resistant to cyslophosphamide (Cy)-induced apoptosis in comparison to B10.S mice, and a similar apoptosis-resistant phenotype is found in NOD mice has been linked to the Idd5 locus overlapping with Eam1. Therefore, we hypothesized that the mechanisms by which Eam1 mediates the susceptibility to EAM could be due to its regulatory role in apoptosis. In the current study, we found that LNC from B10.A Eam1 congenic mice present an intermediate susceptibility to Cy-induced apoptosis, i.e. the percentage of apoptotic LNC with activated caspase 3, 8, 9 in congenic mice falls in between A.SW and B10.S mice. Interestingly, data suggest that A.SW mice have significantly lower levels of apoptotic LNC following immunization with cardiac myosin in CFA than B10.S mice. Our results therefore suggest that Eam1 partially down regulates LNC apoptosis and delays the removal of self-activated T cells, thereby providing an explanation for the role of Eam1 in the susceptibility to EAM. Supported in part by NIH grant HL077611