Supplementary Tables. Supplementary Table S1. Summary of cell lines used. Supplementary Table S2. Summary of Repeat-Dose Toxicity Study with LY2875358

Supplementary Methods and Supplementary Figure Legends

Supplementary Figures. Figure S1. Binding of LY2875358 to human and cynomolgus monkey MET-ECD and the blocking of HGF binding to MET-ECD by LY2875358 as determined by Biacore and ELISA assays Figure S2: Binding epitope regions of LY2875358 determined by H/D mass spectrometry were shown on the crystal structure of ternary complex MET-Sema-PSI with onartuzumab and HGFβ Figure S3. Effect of LY2875358 on HGF-induced cell migration compared to huOA-5D5. Figure S4. Effect of LY2875358 on pan-AKT phosphorylation in Caki-1, H441, and HepG2 cells. Figure S5. Effect of LY2875358 on Caki-1 cell proliferation. Figure S6. Effect of LY2875358 on HepG2 tubulogenesis. Figure S7. Effect of LY2875358 on staurosporin (STS) induced cell apoptosis. Figure S8. Effect of lysosome inhibitor leupeptin on LY2875358-induced MET localization to the lysosome. Figure S9. Effect of LY2875358 and Fab fragment of LY2875358 on HGF binding to MET, depletion of cell surface MET, and proliferation of MET amplified cell lines.

Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGFdependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity anddoes not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGFresponsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent andHGF-independent (METamplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059–70. 2014 AACR.

Abstract Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059–70. ©2014 AACR.

Abstract MET is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of MET can be induced by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by HGF-dependent autocrine or paracrine mechanisms. LY2875358 is a novel humanized bivalent MET antibody currently in phase I clinical testing (trial NCT01287546). LY2875358 has high neutralization and internalization activities against MET for inhibiting HGF-dependent and HGF-independent MET pathway activation and tumor growth. In HGF-dependent MET activation, LY2875358 blocks HGF binding to MET, HGF-induced MET phosphorylation and tumor growth both in cell culture and in mouse xenograft models, resembling activities of a humanized one-armed 5D5 MET antibody (monovalent antibody similar to Onartuzumab). In tumors with HGF-independent MET activation through MET gene amplification, LY2875358 induces internalization and degradation of MET, which results in decreased pMET and total MET, inhibition of cell proliferation and tumor growth in MKN45 and SNU5 gastric tumor lines and EBC-1 and H1993 NSCLC tumor lines. Moreover, LY2875358 enhances antitumor activity in combination with cisplatin or 5-FU in vitro and in vivo in MET amplified tumor cells. However, under the same ligand-independent conditions, the one-armed 5D5 antibody did not have anti-tumor activities. When HGF is added to tumor cells with high MET gene amplification, LY2875358 decreases cell proliferation, while the one-armed 5D5 antibody does not. In contrast to other bivalent MET antibodies, LY2875358 has no or otherwise negligible agonist activity and does not stimulate biological activities such as cell proliferation, scattering, invasion, tubulogenesis, apoptosis protection or angiogenesis in various HGF responsive cells. These findings indicate that LY2875358 has a different mechanism of action from the humanized one-armed 5D5 MET antibody. LY2875358 may be a promising therapy for treatment of patients whose tumors are driven by HGF-dependent and HGF-independent MET activation. Citation Format: Wei Zeng, Victoria Peek, Mark Wortinger, Jonathan Tetreault, Jinqi Xia, Jennifer Stephens, Kelly Credille, Darryl Ballard, Trish Brown-Augsburger, Jirong Lu, Chi-Kin Chow, Peter Vaillancourt, Ying Tang, Sau-Chi B. Yan, Ling Liu. LY2875358, a bivalent MET antibody with anti-tumor activity through blocking HGF as well as inducing degradation of MET, differentiates from a one-armed 5D5 MET antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2013-5465

c-Met is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). c-Met is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by ligand-dependent autocrine or paracrine mechanisms. Lung cancer is the leading cause of cancer death worldwide. Despite the successful development of EGFR- or EML4-ALK-targetd therapies, treatment options remain limited for patients with advanced lung cancer, making the identification of new therapeutic targets essential. c-Met expression was reported in 41-72% non-small cell lung cancer (NSCLC), amplification of c-Met occurs in 5-10 % of patients, and c-Met mutations have been detected in 8-13% of patients. We have developed a bi-valent c-Met antibody LY2875358 (LA480), which blocks ligand-dependent and ligand-independent c-Met activations. It is currently in clinical development. Here, we have demonstrated that LY2875358 alone or in combination with standard-of-care (SOC) affected cell proliferation, migration and signal transduction in NSCLC cells with c-Met gene amplification, mutations and overexpression. In vitro, LY2875358 induces wild type and mutant c-Met internalization and degradation. In vivo, LY2875358 alone shows a marked antitumor activity in Met amplification NSCLC xenograft models. The combination of LY2875358 with SOC chemotherapeutics treatments has better efficacy than either treatment alone, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2734. doi:1538-7445.AM2012-2734

cMet is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). cMet has been implicated in the initiation and progression of cancer due to the range of activities that cMet stimulates including proliferation, migration, morphogenesis, and survival. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation, or by ligand-dependent autocrine or paracrine mechanisms. Indeed, amplification of the c-Met gene, with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human cancers, including gastric, esophageal and non-small-cell lung carcinomas. It has been reported that ∼10-20% of gastric tumors have increased copy numbers of the MET gene and overexpression of c-Met significantly correlates with poor prognosis in gastric cancer. c-Met antibody LY2875358 treatment reduces proliferation of gastric cancer cell lines with ligand-independent activation of c-Met resulting from gene amplification. The ability of LY2875358 to internalize and deplete cell surface c-Met is implicated in its activity against ligand-independent driven gastric cell lines. Here, we demonstrate that the pre-clinical combination of c-Met antibody LY2875358 with gastric cancer standard-of-care treatment has better efficacy than either treatment alone, both in vitro and in vivo. These data suggest that LY2875358 in combination with standard-of-care may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2738. doi:1538-7445.AM2012-2738

Abstract Gastric cancer is common in Asia, and advanced disease is typically incurable. Development of effective treatments for gastric cancer is thus a high priority to address this highly unmet clinical need. c-Met gene amplification resulting in c-Met overexpression and pathway activation has been highly linked to tumor progression and metastasis. It has been reported that approximately 10-20% of gastric tumors have increased copy numbers of the MET gene. c-Met- amplified gastric cancer cell lines that are dependent on an activated c-Met pathway for cell growth have been reported to be highly sensitive to growth inhibition by small molecule inhibitors of c-Met. We report that LA480, a humanized monoclonal antibody specific for c-Met, can directly inhibit HGF-independent in vitro cell proliferation of three c-Met- amplified Asian gastric cell lines (MKN45, SNU-5, and NUGC-4), in a dose-dependent manner. After 48 hours of treatment with LA480, 60-70% percent inhibition of 3H-thymidine incorporation was observed. For one of these gastric lines (NUGC-4), sensitivity to LA480 increases with continuous passage of the culture. This increased sensitivity to LA480 over time appears to be associated with increased expression of cell surface c-Met and increased constitutive phospho-Met expression. In addition to inhibiting proliferation of c-Met- amplified Asian gastric lines, LA480 also directly inhibited proliferation of a c-Met- amplified gastric cell line (Hs 746T) from a Caucasian patient. These data suggest that targeting c-Met amplification with LA480 may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2430.

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4+ T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35–55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease.

Both IL-23- and IL-1-mediated signaling pathways play important roles in Th17 cell differentiation, cytokine production, and autoimmune diseases. The IL-1R-associated kinase 4 (IRAK4) is critical for IL-1/TLR signaling. We show here that inactivation of IRAK4 kinase in mice (IRAK4 KI) results in significant resistance to experimental autoimmune encephalomyelitis due to a reduction in infiltrating inflammatory cells into the CNS and reduced Ag-specific CD4(+) T cell-mediated IL-17 production. Adoptive transfer of myelin oligodendrocyte glycoprotein 35-55-specific IRAK4 KI Th17 cells failed to induce experimental autoimmune encephalomyelitis in either wild-type or IRAK4 KI recipient mice, indicating the lack of autoantigen-specific Th17 cell activities in the absence of IRAK4 kinase activity. Furthermore, the absence of IRAK4 kinase activity blocked induction of IL-23R expression, STAT3 activation by IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23R expression, Th17 differentiation, and autoimmune disease. The Journal of Immunology, 2009, 183: 568-577.

The present study characterized the induction of liver fibrosis and elaboration of biomarkers during progression of liver fibrosis induced by bile duct ligation (BDL) in rats. Following BDL, the severity of liver damage progressed in a time-dependent manner as assessed by an increase in bridging fibrosis, a hallmark of human liver fibrosis. Plasma protein C, albumin, and total protein levels were significantly decreased, whereas serum sICAM-1 and TIMP-1 levels were significantly increased along with fibrogenesis. Serum biochemical markers of liver function as well as circulating neutrophils, monocytes, RBCs, and platelets, significantly increased with progression of liver fibrosis. Inflammatory chemokines and cytokines were also significantly increased with progression of disease severity. The differential changes in GGT, HA, sICAM-1, neutrophils and several inflammatory chemokines suggest the involvement of inflammatory pathways in BDL-induced liver fibrosis. In summary, plasma protein C, along with several other noninvasive biochemical parameters, correlated significantly with severity of liver histopathology and may serve as potential biomarkers in liver fibrosis.

The present studies identified a novel DPP-4 resistant GLP-1 analogue LY548806 and characterized its pharmacological activity in a rat model of acute hyperglycemia. LY548806 was more than 20-fold less susceptible to DPP-4 cleavage and exhibited a 50-fold greater physical stability over native GLP-1, and was a full agonist with an EC50 of 10.6 ± 1.6 pM in vitro. In an acute hyperglycemia model, continuous infusion of LY548806 (0.1–10 μg/kg/hr) or insulin (0.01 U–0.1U/kg/hr) via the right jugular vein resulted in dose-dependent reduction of glucose levels with estimated ED50 values of 0.32 ± 0.11 ug and 0.08 ± 0.00 U respectively. In contrast to insulin, LY548806 did not produce hypoglycemia at higher dose levels. Continuous infusion of LY548806 (10 μg/kg/hr) or insulin (0.1 U/kg/hr) did not affect the plasma levels of the endogenous glucoregulatory hormones corticosterone, growth hormone, catecholamines, or insulin, indicating an insulin-independent effect of LY548806 in this model. LY548806 significantly lowered the plasma levels of free fatty acids (FFA) and glucagon, whereas insulin only reduced FFA levels. In conclusion, we have identified a stable GLP-1 analogue with improved solubility which effectively ameliorates acute hyperglycemia without the risk of hypoglycemia and may represent a novel therapeutic strategy for acute critical illness associated with acute hyperglycemia.

The present investigation was designed to identify potential biomarker(s) and assess the involvement of inflammatory pathway in dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Following DMN-treatment (10 mg/ml/kg, i.p., given three consecutive days each week for 4 weeks) body and liver weights were significantly decreased concurrent with increasing severity of liver damage assessed by bridging fibrosis, a histopathologic assessment and characteristic of human liver disease. Protein C along with albumin, C-reactive-protein (CRP), haptoglobin and total protein were significantly reduced and correlated with changes in liver histopathology. Biochemical markers of liver functions were significantly increased and correlated with changes in liver histopathology and plasma levels of protein C. Soluble intracellular-adhesion-molecule-1 (sICAM-1) levels were increased significantly but were poorly correlated with histopathology and protein C levels. Inflammatory chemokines and other analytes, monocyte-chemoattractant-protein-1 and 3 (MCP-1 and MCP-3), macrophage-colony-stimulating-factor (M-CSF) were significantly increased during the disease progression, whereas macrophage-derived-chemokine (MDC) and CRP were significantly suppressed. Circulating neutrophils and monocytes were also increased along with disease progression. The differential changes in sICAM-1, hyaluronic acid, gamma-glutamyltranspeptidase (GGT), neutrophil and other inflammatory chemokines suggest the involvement of inflammatory pathways in DMN-induced liver fibrosis. In conclusion, the progressive changes in protein C along with other noninvasive biochemical parameters whose levels were significantly correlated with disease progression may serve as biomarkers for pharmacological assessment of targeted therapy for liver fibrosis.

The effects of anesthetic agents, commonly used in animal models, on blood glucose levels in fed and fasted rats were investigated. In fed Sprague-Dawley rats, ketamine (100 mg/kg)/xylazine (10 mg/kg) (KX) produced acute hyperglycemia (blood glucose 178.4 ± 8.0 mg/dl) within 20 min. The baseline blood glucose levels (104.8 ± 5.7 mg/dl) reached maximum levels (291.7 ± 23.8 mg/dl) at 120 min. Ketamine alone did not elevate glucose levels in fed rats. Isoflurane also produced acute hyperglycemia similar to KX. Administration of pentobarbital sodium did not produce hyperglycemia in fed rats. In contrast, none of these anesthetic agents produced hyperglycemia in fasted rats. The acute hyperglycemic effect of KX in fed rats was associated with decreased plasma levels of insulin, adrenocorticotropic hormone (ACTH), and corticosterone and increased levels of glucagon and growth hormone (GH). The acute hyperglycemic response to KX was dose-dependently inhibited by the specific Α2-adrenergic receptor antagonist yohimbine (1–4 mg/kg). KX-induced changes of glucoregulatory hormone levels such as insulin, GH, ACTH, and corticosterone were significantly altered by yohimbine, whereas the glucagon levels remained unaffected. In conclusion, the present study indicates that both KX and isoflurane produce acute hyperglycemia in fed rats. The effect of KX is mediated by modulation of the glucoregulatory hormones through stimulation of Α2-adrenergic receptors. Pentobarbital sodium did not produce hyperglycemia in either fed or fasted rats. Based on these findings, it is suggested that caution needs to be taken when selecting anesthetic agents, and fed or fasted state of animals in studies of diabetic disease or other models where glucose and/or glucoregulatory hormone levels may influence outcome and thus interpretation. However, fed animals are of value when exploring the hyperglycemic response to anesthetic agents.

A rodent model of controlled acute hyperglycemia that is sensitive to glucose lowering agents insulin and GLP-1 analog (glucagon like peptide 1) has been developed. The studies show that anesthesia could be induced in fasted rats with ketamine (100 mg/kg) plus a low dose of xylazine (5 mg/kg) without inducing the acute hyperglycemia typically associated with these agents. Under these conditions continuous infusion of glucose (10 and 20%) via the jugular vein for 30 to 150 minutes induced hyperglycemia in a time-dependent fashion. Administration of “loading” boluses of glucose (0.2-0.6 ml of a 20% solution) prior to continuous infusion of 10% glucose produced more immediate and sustained hyperglycemia. Plasma levels of a variety of gluco-regulatory and stress hormones such as insulin, growth hormone (GH), glucagon, and corticosterone were determined. Only glucagon levels changed significantly during induction and maintenance of hyperglycemia. The infusion of insulin (0.1 unit/kg/hr) or GLP-1 analog (10 µg/kg/hr) effectively lowered blood glucose from its elevated levels. Insulin produced a significant increase in glucagon levels and GLP-1 analog produced a significant increase in insulin levels without any change in other gluco-regulatory and stress hormone levels. In conclusion, the present studies identified a novel approach for the induction of anesthesia and surgical manipulations without inducing hyperglycemia and further defined an approach for producing acute hyperglycemia in a controlled fashion in rodents. This model will be beneficial to study the influence of hyperglycemia in acute models of critical illness where hyperglycemia develops following the

A rodent model of controlled acute hyperglycemia that is sensitive to glucose-lowering agents insulin and glucagon-like peptide-1 (GLP-1) analog has been developed. The studies show that anesthesia could be induced in fasted rats with ketamine (100 mg/kg) plus a low dose of xylazine (5 mg/kg) without inducing the acute hyperglycemia typically associated with these agents. Under these conditions, continuous infusion of glucose (10 and 20%) via the jugular vein for 30 to 150 min induced hyperglycemia in a time-dependent fashion. Administration of “loading” boluses of glucose (0.2–0.6 ml of a 20% solution) prior to continuous infusion of 10% glucose produced more immediate and sustained hyperglycemia. Plasma levels of a variety of glucoregulatory and stress hormones such as insulin, growth hormone, glucagon, and corticosterone were determined. Only glucagon levels changed significantly during induction and maintenance of hyperglycemia. The infusion of insulin (0.1 U/kg/h) or GLP-1 analog (10 μg/kg/h) effectively lowered blood glucose from its elevated levels. Insulin produced a significant increase in glucagon levels, and GLP-1 analog produced a significant increase in insulin levels without any change in other glucoregulatory and stress hormone levels. In conclusion, the present studies identified a novel approach for the induction of anesthesia and surgical manipulations without inducing hyperglycemia and further defined an approach for producing acute hyperglycemia in a controlled fashion in rodents. This model will be beneficial to study the influence of hyperglycemia in acute models of critical illness where hyperglycemia develops following the precipitating event. This model was responsive to insulin and GLP-1 analog, both of which were effective in ameliorating hyperglycemia.

Background: Exposure to Aspergillus fumigatus allergens results in the sensitization and the development of allergic bronchopulmonary aspergillosis in susceptible individuals. Aspergillus antigen consists of a number of chemically diverse components and their cumulative or synergistic effect may result in disease. When mice were challenged with individual recombinant allergens, there was only reduced inflammation and immunological responses compared to the whole antigen. Various enzymes identified from A. fumigatus have been thought to cause airway damage. In the present study, we evaluated the effect of exposure to Asp f 13, an alkaline serine proteinase, and Asp f 2 in mice. Methods: BALB/c mice were challenged intranasally with Asp f 2 and Asp f 13 alone and in combination. The antibody response, pulmonary inflammation, and airway hyperreactivity were studied. Results: Results demonstrated no major difference in antibody response and airway responses among the different groups. The inflammatory responses in the lungs, however, showed marked differences in the various groups. Conclusion: In spite of the similar immunological responses in the different groups of mice studied, the results demonstrate enhanced inflammation in the lungs of mice exposed to a combination of both allergens. Allergens with proteinase activity have been found to be involved in airway inflammation and remodeling, which may also apply for Aspergillus-induced allergy.

A high proportion of peptide 1–11-specific T cells from H-2u (Vβ8+, H-2u) mice express the Vβ8 TCR chain. Peptide 89–101 is immunodominant for B10.RIII (Vβ8+, H-2r) mice; thus, it was of interest to determine whether Vβ8 TCR would be over-represented in a population of peptide 89–101-specific T cells of this strain. Second, it was asked whether MBP peptides other than 89–101 would induce EAE in these mice. Of 70 B10.RIII(71NS)/SnJ mice immunized with mouse myelin basic protein (MBP), 32 of 41 males (78%) and 11 of 29 females (38%) showed clinical signs of experimental allergic encephalomyelitis (EAE). All mice immunized with peptide 89–101 showed clinical signs. One of six mice immunized with peptide 91–103 showed clinical signs, and 9 of 16 mice, all males, responded with EAE when immunized with peptide 38–88. No clinical EAE was observed in mice immunized with peptide 43–67, 68–88, 55–74, 1–37 or 1–20. A peptide 89–101-specific T cell line was established. At the initial stimulation the line was 29% Vβ8+ versus 21% in normal controls, and the line did not transfer EAE adoptively. After five in vitro stimulations, the percentage of Vβ8+ T cells had increased to 54%, and the line was encephalitogenic. Encephalitogenicity was partially blocked by anti-Vβ8 monoclonal antibody. Thus, over-representation of Vβ8+ TCR by encephalitogenic peptide-specific T cells is not limited to peptide 1–11-specific T cells from H-2u mice. Immunization of RIIIA/J mice with MBP resulted in 100% showing clinical signs of EAE. In contrast, peptide 89–101 was ineffective in EAE induction in this strain, the MHC donor for the B10.RIII strain.