Methylprednisolone fails to attenuate lung injury in a mouse model of transfusion related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related morbidity and mortality. Anecdotally, TRALI patients have been treated with corticosteroids. However, evidence for its therapeutic rationale in TRALI is lacking. We determined the effects of corticosteroids on lung injury in a "two-hit" mouse model of antibody-mediated TRALI. STUDY DESIGN AND METHODS: BALB/c mice were primed with lipopolysaccharide, after which TRALI was induced by injecting major histocompatibility complex (MHC)-I antibody against H2K(d) . Mice infused with phosphate-buffered saline served as controls. Simultaneously, one group of TRALI mice was infused with methylprednisolone (MPS; 2 mg/kg). Mice were supported by mechanical ventilation for 2 hours, after which bronchoalveolar lavage fluid (BALF) and lung homogenate were obtained. Statistics were obtained by one-way analysis of variance or Kruskal-Wallis. RESULTS: Injection of MHC-I antibodies resulted in TRALI, indicated by pulmonary edema and increased BALF levels of protein and the proinflammatory mediators macrophage inflammatory protein-2, keratinocyte-derived chemokine, and interleukin (IL)-6. Administration of MPS did not affect the amount of edema nor pulmonary protein and chemokine levels. MPS reduced systemic inflammatory reaction as well as IL-6 levels in the BALF. CONCLUSION: In a two-hit model of antibody-mediated TRALI, MPS attenuated the IL-6 host response, but failed to prevent the development of lung injury.

Immunization with 60 kD Ro peptide produces different stages of preclinical autoimmunity in a Sjögren's syndrome model among multiple strains of inbred mice.

Sjögren's syndrome is a chronic illness manifested characteristically by immune injury to the salivary and lacrimal glands, resulting in dry mouth/eyes. Anti-Ro [Sjögren's syndrome antigen A (SSA)] and anti-La [Sjögren's syndrome antigen B (SSB)] autoantibodies are found frequently in Sjögren's subjects as well as in individuals who will go on to develop the disease. Immunization of BALB/c mice with Ro60 peptides results in epitope spreading with anti-Ro and anti-La along with lymphocyte infiltration of salivary glands similar to human Sjögren's. In addition, these animals have poor salivary function/low saliva volume. In this study, we examined whether Ro-peptide immunization produces a Sjögren's-like illness in other strains of mice. BALB/c, DBA-2, PL/J, SJL/J and C57BL/6 mice were immunized with Ro60 peptide-274. Sera from these mice were studied by immunoblot and enzyme-linked immunosorbent assay for autoantibodies. Timed salivary flow was determined after pharmacological stimulation, and salivary glands were examined pathologically. We found that SJL/J mice had no immune response to the peptide from Ro60, while C57BL/6 mice produced antibodies that bound the peptide but had no epitope spreading. PL/J mice had epitope spreading to other structures of Ro60 as well as to La, but like C57BL/6 and SJL/J had no salivary gland lymphocytic infiltration and no decrement of salivary function. DBA-2 and BALB/c mice had infiltration but only BALB/c had decreased salivary function. The immunological processes leading to a Sjögren's-like illness after Ro-peptide immunization were interrupted in a stepwise fashion in these differing mice strains. These data suggest that this is a model of preclinical disease with genetic control for epitope spreading, lymphocytic infiltration and glandular dysfunction.

CTLA4-Ig prevents alloantibody production and BMT rejection in response to platelet transfusions in mice.

BACKGROUND: Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion. STUDY DESIGN AND METHODS: We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody. RESULTS: CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection. CONCLUSIONS: These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions.

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

Exacerbation of autoimmune thyroiditis by a single low dose of whole-body irradiation in non-obese diabetic-H2h4 mice.

PURPOSE: To evaluate how irradiation affects thyroid autoimmunity in mouse models of Hashimoto's thyroiditis and Graves' hyperthyroidism. MATERIALS AND METHODS: Non-obese diabetic (NOD)-H2(h4) mice spontaneously develop anti-thyroglobulin (Tg) antibodies and thyroiditis when supplied with sodium iodine (NaI) in the drinking water. BALB/c mice develop anti-thyrotropin receptor (TSHR) antibodies and hyperthyroidism following immunization with adenovirus expressing TSHR (Ad-TSHR). Mice were irradiated as follows: A single whole-body irradiation with 0.05, 0.5 or 3 Gy one week before or after the beginning of NaI or immunization with Ad-TSHR, fractionated whole-body irradiations with 0.05 Gy twice a week or 0.5 Gy once a week from one week before NaI or Ad-TSHR immunization, or a single regional irradiation to the thyroid gland with 0.5 Gy one week before NaI. The effect of a single irradiation with 0.05, 0.5 or 3 Gy on splenocytes was also evaluated. RESULTS: A single whole-body irradiation with 0.5 Gy one week before NaI exacerbated thyroiditis and increased anti-Tg antibody titers in NOD-H2(h4) mice. In contrast, any irradiation protocols employed did not affect incidence of hyperthyroidism or anti-TSHR antibody titers in BALB/c mice. High-dose irradiation increased the relative ratios of effector T cells to regulatory T cells (an indication of enhanced immune status) but kills most of T cells. CONCLUSIONS: These results indicate that a single whole-body low-dose irradiation with 0.5 Gy exacerbates thyroiditis in NOD-H2(h4) mice, data consistent with some clinical evidence for increased incidence of thyroid autoimmunity by environmental irradiation.

Identification of MHC class I H-2 Kb/Db-restricted immunogenic peptides derived from retinal proteins.

PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation.

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

CD28 costimulation is critical for experimental allergic asthma in HLA-DQ8 transgenic mice.

The objective of this study was to investigate the contribution of the CD28 costimulatory molecules to allergen-induced primary and chronic inflammatory responses. To this end, we have developed and characterized a short ragweed allergen-induced asthma model involving sensitization of HLA-DQ transgenic mice followed by intranasal challenge with allergen. Forty-eight hours after primary challenge, sensitized DQ8 mice developed pulmonary eosinophilic inflammation, airway hyperreactivity, Th2 cytokines, and IgE/IgG1 Ab. This allergic inflammatory response was absent in H-2Abeta(0) and DQ8/CD28(0) mice. Secondary rechallenge with allergen 4 weeks later induced even greater inflammatory changes in the airways of DQ8 mice with eosinophils being the predominant inflammatory cells while only pulmonary lymphocytosis was observed in DQ8/CD28(0) mice. No inflammation was detected in H-2Abeta(0) mice. Proliferation and cytokine profile studies demonstrated that CD28 regulates T-cell activation and effector function. Therefore, CD28 is essential for the extrinsic asthma and can be a target for immunotherapy.

H-2 haplotype-dependent serum IL-12 production in tumor-bearing mice treated with various mycelial extracts.

IL-12 is considered to be one of the most important cytokines in anti-cancer therapy. We have demonstrated that substances derived from Basidiomycetes, such as active hexose-correlated compound (AHCC) and PSK induce the production of IL-12. In this study, the MHC dependency of IL-12 production induced by various mycelial extracts, PSK, AHCC and IL-X, was examined. During tumor-bearing, higher serum IL-12 levels were observed in H-2a and H-2b mice as compared to H-2d mice. Concerning the effect of genetic background of mice on response to mycelial extracts, AHCC administration enhanced the serum IL-12 level in H-2b mice but not in H-2d mice, while PSK administration increased the serum IL-12 level in H-2d mice but not in H-2b mice. IL-X, components derived from the same Basidiomycetes, also enhanced the serum IL-12 level in H-2b mice in the early stage of tumor like AHCC, and maintained serum IL-12 at a level higher than the normal value accompanying tumor growth, whereas AHCC did not restore the lowered serum IL-12 level accompanying tumor growth. These results showed that AHCC or IL-X is effective in a genetically Th1-dominant individual whereas PSK is effective in a genetically Th2-dominant individual or Th2-dominant status in advanced cancer patients. So we propose that the suitable combinations of various mycelial extracts may be effective methods of endogenous IL-12 induction for cancer patients of all stages, which is important as a cancer therapy that is relatively free from adverse reactions and which emphasizes the QOL in individual patients.

An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.

Targeting Janus kinase 3 to attenuate the severity of acute graft-versus-host disease across the major histocompatibility barrier in mice.

To prevent the development of acute graft-versus-host disease (GVHD) in lethally irradiated C57BL/6 (H-2b) recipient mice transplanted with bone marrow-splenocyte grafts from major histocompatibility complex (MHC) disparate BALB/c mice (H-2d), recipient mice were treated with the rationally designed JAK3 inhibitor WHI-P131 [4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline] (20 mg/kg, 3 times a day [tid]) daily from the day of bone marrow transplantation (BMT) until the end of the 85-day observation period. Total body irradiation (TBI)-conditioned, vehicle-treated control C57BL/6 mice (n = 38) receiving bone marrow-splenocyte grafts from BALB/c mice survived acute TBI toxicity, but they all developed histologically confirmed severe multi-organ GVHD and died after a median survival time of 37 days. WHI-P131 treatment (20 mg/kg intraperitoneally, tid) prolonged the median survival time of the BMT recipients to 56 days. The probability of survival at 2 months after BMT was 11% +/- 5% for vehicle-treated control mice (n = 38) and 41% +/- 9% for mice treated with WHI-P131 (n = 32) (P <.0001). Notably, the combination regimen WHI-P131 plus the standard anti-GVHD drug methotrexate (MTX) (10 mg/m2 per day) was more effective than WHI-P131 or MTX alone. More than half the C57BL/6 recipients receiving this most effective GVHD prophylaxis remained alive and healthy throughout the 85-day observation period, with a cumulative survival probability of 70% +/- 10%. Taken together, these results indicate that targeting JAK3 in alloreactive donor lymphocytes with a chemical inhibitor such as WHI-P131 may attenuate the severity of GVHD after BMT.

Strain-dependent antibody response induced by DNA immunization.

The antibody (Ab) response induced by DNA-based immunization was compared in various strains of inbred, H-2 congenic and outbred mice with different haplotypes of mouse major histocompatibility complex (H-2). Two different plasmid expression vectors encoding Aequorea victoria green fluorescent protein (GFP) or Escherichia coli, beta-galactosidase (beta-gal) were introduced into quadriceps muscle, and Ab production was examined using both enzyme-linked immunosorbent assay and immunoblot analysis. The beta-gal plasmid DNA immunization induced strong Ab production in all inbred, H-2 congenic and outbred strains at the early stages of immunization. By comparison with beta-gal peptide immunization, the degree of Ab response was H-2 haplotype-dependent. On the other hand, Ab production by GFP plasmid DNA immunization was observed in outbred strains, but not in some of the inbred and H-2 congenic strains. Also, outbred strains showed a high Ab response compared with other inbred and H-2 congenic strains by GFP peptide immunization. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of GFP or beta-gal transcripts at the DNA inoculation site in all the strains studied, even in inbred and H-2 congenic strains which showed no Ab production by GFP plasmid DNA immunization. These results indicate that the difference in Ab response induced by DNA immunization as well as by peptide immunization depends upon the H-2 haplotypes of host strains.

Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtusa) pollen, I. H-2 complex affects cross responsiveness to Cry j 1 and Cha o 1 at the T- and B-cell level in mice.

Common antigenicity among two purified Japanese cedar pollen allergens (Cry j 1 and Cry j 2) and one Japanese cypress pollen allergen (Cha o 1) was explored at the T-cell and B-cell level in mice of different H-2 haplotypes. Cry j 2 did not show any common antigenicity with Cry j 1 or Cha o 1. B10.S (H-2S) mice immunized with Cry j 1 or Cha o 1 generated T cells and antibodies reactive to both antigens, indicating the common antigenicity of these antigens. C57BL/6 (H-2b) mice were non-responders to Cry j 1. BALB/c (H-2d) mice immunized with Cry j 1 or Cha o 1 and C57BL/6 mice immunized with Cha o 1 generated T cells that were only reactive with the respective immunogen, but produced antibody reactive to both Cry j 1 and Cha o 1, indicating that Cry j 1 and Cha o 1 share their B-cell epitope but not their T-cell epitope. This finding may provide a clue for the clarification of the T-cell and B-cell epitopes of Cry j 1 and Cha o 1, even though the data are influenced by H-2 complex restriction in mice. Considering that H-2 complex restriction affects cross responsiveness to Cry j 1 and Cha o 1 at the T- and B-cell level in mice, we assessed the possible situation in humans exposed sequentially to Japanese cedar pollen and Japanese cypress pollen.

Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtusa) pollen, II. Determination of the cross-reacting T-cell epitope of cry j 1 and cha o 1 in mice.

We have previously detected common antigenicity between Cry j 1 and Cha o 1 in B10.S mice. B10.S mice immunized with Cry j 1- or Cha o 1-generated T cells and antibodies reactive to both allergens. In the present study, we investigated the cross-reacting and Cry j 1-specific T-cell epitopes in B10.S mice. Lymph node cells from B10. S mice immunized with Cry j 1 recognized Cry j 1 p111-130, p211-230, and p310-330 as well as Cha o 1 p209-228. The existence of the cross-reacting T-cell epitope in Cry j 1 and Cha o 1 was confirmed by the response of newly established p211-230-specific and Cha o 1 p209-228-specific T-cell lines. The minimum peptide sequence (p213-224) of the cross-reacting T-cell epitope was identical in Cry j 1 and Cha o 1. These findings clearly demonstrate that common antigenicity at the T-cell level between Japanese cedar and cypress pollen allergens was caused by the existence of an identitical-cell epitope in Cry j 1 and Cha o 1.

Induction of an antigen-specific CTL response by a conformationally biased agonist of human C5a anaphylatoxin as a molecular adjuvant.

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg). Groups of BALB/c mice (H-2d) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSWWTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admixed (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8+ CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR). The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway.

Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257-264 CD8+ epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD8+ T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca2+ and independent of vacuolar acidification. Moreover, inhibitors of the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation pathway is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconventional delivery of an exogenous Ag into the conventional cytosolic pathway.

Major histocompatibility complex and T cell receptor interaction of the P91A tum- peptide.

The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA700 proteasome regulatory complex. P91A stimulates a strong CD8+ T cell response when expressed on tumor cells or normal tissue and P91A-specific T cells express a restricted range of T cell receptors. Although it is a strong Ld-binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. Ld predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the Ld motif residue proline at position 2, residues 5-7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with Ld. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum+ peptide (QNRRALDL) to bind to Ld is addressed by molecular modeling.

Prevention of lethal graft-versus-host disease following allogeneic bone marrow transplantation in mice by short course administration of LF 08-0299.

We investigated the ability of LF 08--0299, a new immunosuppressive compound, to prevent murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). A short term LF 08--0299 treatment at optimal dosage protected more than 75% of recipient mice from lethal GVHD induced either across minor antigens alone or the full H2 barrier. Furthermore, LF 08--0299 still prevented lethal GVHD when treatment was delayed to 10 days post-BMT. Long-term LF 08--0299-treated survivors were free of clinical signs of GVHD, and histopathologic examination of liver, skin, and intestines was normal, demonstrating that recipient mice did not develop chronic GVHD. We assessed the immunocompetence of long-term surviving recipient mice. Results from MLR and CTL assays were weak whereas responses against unrelated H2 antigens were reduced but still preserved. Moreover, in vivo transfer experiments demonstrated that spleen cells from long-term survivors were unable to induce lethal GVHD in irradiated recipients of host origin, while spleen cells injected in irradiated recipients of a host-unrelated H2 were fully competent to induce a lethal GVHD. Together these results indicate that stable chimeric recipient mice were specifically tolerant to host antigens. We further showed that while LF 08--0299 can protect recipient mice from lethal GVHD, it also preserved a graft-versus-leukemia effect when mice were inoculated with P815 tumor cells. These data suggest that LF 08--0299 may be a novel pharmaceutical agent that would prevent GVHD in human unrelated bone marrow transplantation.

Murine experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein and Klebsiella pneumoniae 03 lipopolysaccharide (K03-LPS): a relation between H-2 haplotype and EAU induction.

The pathogenicity of interphotoreceptor retinoid-binding protein (IRBP) in the mouse and H-2 restriction of IRBP-induced experimental autoimmune uveoretinitis (EAU) was tested by repeated immunization using Klebsiella pneumoniae 03 lipopolysaccharide (K03-LPS) as an adjuvant. It was shown that IRBP had a greater capacity to induce EAU than S-antigen. Based on the incidence of EAU induction using B10 congenic mice and other strains, the susceptibility to EAU was, at least in part, controlled by the I-Ak haplotype of the H-2 subregion. The results also indicated that non-major histocompatibility complex (MHC) genes play some role in disease susceptibility.