H2 Class Research Articles

Development and Application of J3112: A/C Compressor Oil Separator Effectiveness Test Standard

<div class="section abstract"><section id="s_1" class="section"><h6 class="title"/><div class="htmlview paragraph">SAE Standard J3112 – “A/C Compressor Oil Separator Effectiveness Test Standard” - has been developed to provide an analytical means of determining if an a/c compressor with oil separator functionality meets the U.S. EPA criteria of a minimum 50% effectiveness in order to qualify for menu credits for greenhouse gas reduction, as specified in the U.S Government Code of Federal Regulations (CFR) Title 40, §86.1868-12. At the start of the EPA’s a/c efficiency credit program in 2017 OEMs could qualify for greenhouse gas menu credits without providing data on the effectiveness of the product being implemented, but EPA regulations required supporting data from 2020 MY through the end of the current published regulations in 2026 MY. J3112 was developed by the SAE Interior Climate Control MVAC Supplier Committee (TEVBES2E) in order to provide a standardized procedure for determining oil separator effectiveness in order to comply with the EPA requirements for 2020-2026 model years. This paper provides background on how J3112 was developed, and how it is used in order to provide documentation necessary to apply for Oil Separator credits with the EPA, in conjunction with SAE Recommended Practice J3174, “Recommended Practice for an Engineering Analysis for A/C Efficiency Credits”.</div></section><section id="s_2" class="section"><h2 class="title">Copyright Statement</h2><div class="htmlview paragraph">This paper puts into writing the material presented by the author in an on-demand online format for the SAE Thermal Management Systems Symposium on October 12-14, 2021. All figures contained in this paper are from the TMSS presentation, material which SAE holds the copyrights. Additionally, passages from SAE J3112 and J3174 are used in this paper, which is also SAE copyrighted material. Finally, any passages from the U.S. Government Code of Federal Regulations (CFR) included in this paper is public domain material.</div></section></div>

Optimal design of mixture experiments for general blending models

Mixture models of the Scheffé polynomial class are standard in several scientific fields. For these models there is a vast literature on the optimal design of experiments to provide good estimates of the parameters with the use of minimal resources. Contrarily, the optimal design of experiments for general blending models, extending the class of Becker, have not been systematically addressed. Nevertheless, there are practical examples where the models relating the response variables, the parameters and the factors including nonlinear blending effects fall into a general form. We propose a general formulation to find continuous and exact D– and A–optimal designs for general blending models. First, we consider designs to estimate the regression coefficients, and then extend the formulations to find locally optimal continuous designs for estimating both the coefficients and the power exponents. The treatment relies on converting the Optimal Experimental Design (OED) problem into an optimization problem of the Nonlinear Programming (or Mixed Integer Nonlinear Programming) class. We apply this approach to quadratic and special cubic general blending models of the H2 class of polynomials introduced by Becker, and to three examples of practical interest in combustion science and in the characterization of fuel properties.

Meta-populational demes constitute a reservoir for large MHC allele diversity in wild house mice (Mus musculus)

BackgroundThe MHC class I and II loci mediate the adaptive immune response and belong to the most polymorphic loci in vertebrate genomes. In fact, the number of different alleles in a given species is often so large that it remains a challenge to provide an evolutionary model that can fully account for this.ResultsWe provide here a general survey of MHC allele numbers in house mouse populations and two sub-species (M. m. domesticus and M. m. musculus) for H2 class I D and K, as well as class II A and E loci. Between 50 and 90% of the detected different sequences constitute new alleles, confirming that the discovery of new alleles is indeed far from complete. House mice live in separate demes with small effective population sizes, factors that were proposed to reduce, rather than enhance the possibility for the maintenance of many different alleles. To specifically investigate the occurrence of alleles within demes, we focused on the class II H2-Aa and H2-Eb exon 2 alleles in nine demes of M. m. domesticus from two different geographic regions. We find on the one hand a group of alleles that occur in different sampling regions and three quarters of these are also found in both sub-species. On the other hand, the larger group of different alleles (56%) occurs only in one of the regions and most of these (89%) only in single demes. We show that most of these region-specific alleles have apparently arisen through recombination and/or partial gene conversion from already existing alleles.ConclusionsDemes can act as sources of alleles that outnumber the set of alleles that are shared across the species range. These findings support the reservoir model proposed for human MHC diversity, which states that large pools of rare MHC allele variants are continuously generated by neutral mutational mechanisms. Given that these can become important in the defense against newly emerging pathogens, the reservoir model complements the selection based models for MHC diversity and explains why the exceptional diversity exists.

Various characteristics of Ni and Pt–Al2O3 nanocatalysts prepared by microwave method to be applied in some petrochemical processes

Alumina-supported metal nanocatalysts were prepared via the microwave method, by loading nano Ni particles (at 1, 3 and 5wt%) or nano Pt particles (at 0.3, 0.6 and 0.9wt%). Structural and adsorption features of the nano catalysts were revealed through XRD, DSC-DTA, TEM, H2-chemisorption and N2-physisorption. N2-adsorption–desorption isotherms of type IV were related typically to mesoporous materials with H2 class of hysteresis loops characterizing ink bottle type of pores. The well dispersed nano-sized metal particles were evidenced in the studied catalytic systems, exhibiting marked thermal stability up to 800°C. The catalytic performances of different catalyst samples were assessed during cyclohexane, normal hexane and ethanol conversions, using the micro-catalytic pulse technique at different operating conditions. The 5% Ni–γ–Al2O3 sample was found to be the most active in dehydration of ethanol to produce ethylene, as well as in n-hexane cracking. However, the 1% Ni–Al2O3 sample showed the highest dehydrogenation activity for selective production of benzene from cyclohexane. On the other hand, the 0.9% Pt–γ–Al2O3 sample exhibited the highest activity in the dehydration of ethanol and in the dehydrogenation of cyclohexane. The 0.3% Pt–γ–Al2O3 sample was the most active in the dehydrocyclization of normal hexane, as compared to the other catalyst samples under study.

NEAR-INFRARED H 2 AND CONTINUUM SURVEY OF EXTENDED GREEN OBJECTS. II. COMPLETE CENSUS FOR THE NORTHERN GALACTIC PLANE

We discuss 94 Extended Green Objects (EGOs) in the northern Galactic plane cataloged by Cyganowski et al, based on near-infrared narrowband H2 (2.122 {\mu}m and continuum observations from the United Kingdom Infrared Telescope. This data set is three times larger than our previous study, and is unbiased by preselection. As discussed in the previous paper, the morphologies of the 4.5 {\mu}m emission generally resemble those of the near-infrared continuum, but are different from those of the H2 emission. Of our sample, only 28% of EGOs with H2 emission show similar morphologies between 4.5 {\mu}m and H2 emission. These results suggest that the 4.5 {\mu}m emission mainly comes from scattered continuum from the embedded young stellar object (YSO), and partially from H2 emission. About half of EGOs are associated with H2 outflows, if the H2 outflow incompleteness is considered. The H2 outflow detection rate for EGOs with K-band detections (61%) is significantly higher than for those without K-band detections (36%). This difference may be due to the fact that both H2 and K-band emissions are associated with outflows, i.e., H2 emission and K-band continuum are associated with shocks and outflow cavities, respectively. We also compared the correlation between the H2 outflows and Class I 44 GHz methanol masers from literature. The methanol masers can be located upstream or downstream of the H2 outflows and some bright H2 spots or outflows are not associated with methanol masers, suggesting that methanol masers and H2 emission trace different excitation conditions.

Analysis of the performance of peptide predicting methods using MHC class I peptide sequencing in the C57BL/6 mouse (78.11)

Abstract MHC class I peptide-binding motifs can defined by sequencing of peptides naturally bound to MHC class I molecules. More recently MHC class I-peptide binding motifs have be defined on the basis of quantitative MHC-peptide binding assays in vitro using peptide libraries. The information on peptide binding motifs obtained using binding assays have been useful to develop numerous bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict peptides determined by sequencing of peptides eluted directly from MHC class I molecules. In this study we performed sequencing of endogenous peptides eluted from H2Kb and Db molecules expressed in the C57BL/6 mouse. Using data from 280 H2-class I-bound peptides we identified novel preferred anchor residues located in H2Kb and H2Db-associated peptides. When comparing the predicting affinity performance of 3 bioinformatics tools we found that between 60 to 95% of peptides eluted from H2Kb and H2Db molecules were correctly classified as binders by these 3 predicting algorithms, suggesting that these bioinformatics tools are reliable and efficient for prediction of naturally processed MHC class I ligands. These results expand our knowledge about H2 class I binding specificities and provide the identity of positions that can be used for better characterization of H2 class I peptide-binding motifs. Similar studies using sensitive methods for sequencing large numbers of peptides from other MHC molecules are needed to establish more precise motif information that can be used for the training of bioinformatics tool employed in the prediction of CD8 T-cell epitopes

Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction

Most major histocompatibility complex (MHC) class I-peptide-binding motifs are currently defined on the basis of quantitative in vitro MHC-peptide-binding assays. This information is used to develop bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict the binding of peptides determined by sequencing of naturally processed peptides eluted directly from MHC class I molecules. In this study, we performed large-scale sequencing of endogenous peptides eluted from H2K(b) and H2D(b) molecules expressed in spleens of C57BL/6 mice. Using sequence data from 281 peptides, we identified novel preferred anchor residues located in H2K(b) and H2D(b)-associated peptides that refine our knowledge of these H2 class I peptide-binding motifs. The analysis comparing the performance of three bioinformatics methods to predict the binding of these peptides, including artificial neural network, stabilized matrix method, and average relative binding, revealed that 61% to 94% of peptides eluted from H2K(b) and H2D(b) molecules were correctly classified as binders by the three algorithms. These results suggest that bioinformatics tools are reliable and efficient methods for binding prediction of naturally processed MHC class I ligands.

Specificity of amyloid precursor-like protein 2 interactions with MHC class I molecules

The ubiquitously expressed amyloid precursor-like protein 2 (APLP2) has been previously found to regulate cell surface expression of the major histocompatibility complex (MHC) class I molecule K(d) and bind strongly to K(d). In the study reported here, we demonstrated that APLP2 binds, in varied degrees, to several other mouse MHC class I allotypes and that the ability of APLP2 to affect cell surface expression of an MHC class I molecule is not limited to K(d). L(d), like K(d), was found associated with APLP2 in the Golgi, but K(d) was also associated with APLP2 within intracellular vesicular structures. We also investigated the effect of beta(2)m on APLP2/MHC interaction and found that human beta(2)m transfection increased the association of APLP2 with mouse MHC class I molecules, likely by affecting H2 class I heavy chain conformation. APLP2 was demonstrated to bind specifically to the conformation of L(d) having folded outer domains, consistent with our previous results with K(d) and indicating APLP2 interacts with the alpha1alpha2 region on each of these H2 class I molecules. Furthermore, we observed that binding to APLP2 involved the MHC alpha3/transmembrane/cytoplasmic region, suggesting that conserved as well as polymorphic regions of the H2 class I molecule may participate in interaction with APLP2. In summary, we demonstrated that APLP2's binding, co-localization pattern, and functional impact vary among H2 class I molecules and that APLP2/MHC association is influenced by multiple domains of the MHC class I heavy chain and by beta(2)m's effects on the conformation of the heavy chain.

Application of HLA Class II Transgenic Mice to Study Autoimmune Regulation

In the past decade, we participated in the increased use of HLA class II transgenic mice to delineate genetic control in autoimmune diseases. Our studies began with individual class II transgenes to determine permissiveness for experimental autoimmune thyroiditis (EAT), first in resistant strains and then in the absence of endogenous H2 class II molecules. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules serve as a determinant for EAT induction with either mouse thyroglobulin (mTg) or human thyroglobulin (hTg). This delineation enabled identification of pathogenic Tg peptides, based on DR3-binding motifs. HLA-DQ polymorphism was also detectable; hTg induced moderate EAT in DQ8(+), but not DQ6(+), mice. Coexpressing permissive and nonpermissive alleles, DR3(+) mice showed reduced EAT severity in the presence of DQ8, but not DQ6, DR2, or DR4. Determining the regulatory T cell (Treg) influences showed that Treg depletion increased thyroiditis incidence and severity without altering the major histocompatibility complex-based hierarchy in susceptibility. This increase after Treg depletion can also be observed in NaI-induced thyroiditis in DR3(+) mice, a means to study a major environmental factor in thyroid autoimmunity. DR3(+) mice were also immunized with human thyroid peroxidase cDNA, resulting in thyroiditis and an antibody (Ab) profile resembling patient antibodies (Abs). Similar immunization with human TSH receptor cDNA resulted in thyroid-stimulating Abs and elevated T(4) levels with moderate thyroiditis in some animals, suggesting a potential Graves' disease model that due to thyroid lesions is more complete than other models. Recently, Treg manipulation in cancer immunotherapy trials has triggered various autoimmune disorders. Thus, DR3(+) mice are being used to monitor a known risk factor for autoimmune thyroid disease in attempts to enhance tumor immunity.

Immunogenicity of a BCR-ABL e1a2 p190 Translocation in a Balb/c Mouse Model.

Intracellular processing of the breakpoint products of the bcr-abl fusion gene may generate novel peptides that, if capable of binding to HLA class I molecules, would be potential targets for a cytotoxic T lymphocyte (CTL) response. In humans, peptides derived from the e1a2 p190 breakpoint have generated peptide-specific MHC class II proliferative responses. Short peptides have shown binding to MHC class I molecules, although processing and presentation of endogenous p190 protein has not been shown. In a Kd Balb/c mouse model, we tested the hypothesis that single, key amino acids near the breakpoint could be the reason of lack of immunogenicity of the p190 breakpoint peptides. We synthesized a native peptide from the 190 breakpoint (AFHGDAEAL) as well as a synthetic mutated peptide (AYHGDAEAL), which showed excellent predicted binding on the BIMAS algorithm (1152 and 2880 respectively), although in vivo experiments did not show any specific CTL response. In order to assess if the lack of immunogenicity in vivo was due to the absence of binding to the MHC class I molecule rather than to poor TCR interactions, we designed a series of peptides where neutral amino acid, alanine, substitutions were introduced at different potential binding sites in the synthetic peptide: at position three (AYAGDAEAL), position four (AYHADAEAL), position five (AYHGAAEAL) and position seven (AYHGDAAAL). The binding of these altered peptides to H2 class I was assessed using a MHC stabilization assay on T2-Kd cells (TAP deficient cells). In spite of the good computer prediction for binding the MHC stabilization assay did not show evidence of binding of the native and synthetic peptides (<1.9 over the background). In contrast, alanine substitution in position five resulted in the best binding (3.5x over the background). Alanine substitutions in positions three, four and seven also showed improved binding to Kd molecules (2.6x, 3.1x and 3x over the background respectively). These results were confirmed by immunization in vivo with these altered peptides. Only the peptide with the alanine substitution in position five generated a immune response in a CD8 gamma-IFN Elispot assay. Since the altered peptides with the alanine substitutions in positions three, four and seven have shown binding to the Kd molecules, but not a CTL response, change of the aspartic acid in position five near to the fusion breakpoint allows appropriate presentation and recognition of the sequence by the TCR.

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single α or β subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the α polypeptide. Co-isolation with α chain requires the groove binding Ii-segment and depends on M 91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of α and β chains.

THE GENETICS OF THE PERSISTENT INFECTION AND DEMYELINATING DISEASE CAUSED BY THEILER'S VIRUS

Theiler's virus causes a persistent and demyelinating infection of the central nervous system of the mouse, which is one of the best animal models to study multiple sclerosis. This review focuses on the mechanism of persistence. The virus infects neurons for a few weeks and then shifts to white matter, where it persists in glial cells and macrophages. Oligodendrocytes are crucial host cells, as shown by the resistance to persistent infection of mice bearing myelin mutations. Two viral proteins, L and L*, contribute to persistence by interfering with host defenses. L, a small zinc-finger protein, restricts the production of interferon. L*, a unique example of a picornaviral protein translated from an overlapping open reading frame, facilitates the infection of macrophages. Susceptibility to persistent infection, which varies among inbred mouse strains, is multigenic. H2 class I genes have a major effect on susceptibility. Among several non-H2 susceptibility loci, Tmevp3 appears to regulate the expression of important cytokines.

Highly conserved pattern of recognition of influenza A wild-type and variant CD8 + CTL epitopes in HLA-A2 + humans and transgenic HLA-A2 +/H2 class I-deficient mice

As an in vivo model for studying human MHC (HLA) class I-restricted CTL responses to viral infection, we established a series of HLA Tg mice expressing HLA-A2, -B7 or -B27 human/mouse hybrid genes on a background deficient for H2 class I (Tg HLA hyb/H2 class I DKO). To determine whether CTL recognition of influenza A (flu) infection in Tg HLA-A2 hyb/H2 DKO mice is similar to HLA-A2 + humans, we compared the HLA-A2-restricted Tg mouse and human CD8 + T-cell responses to an immunodominant flu epitope (wild-type [WT] M1 58–66), as well as a variant of this peptide (var. M1 58–66). Similar to HLA-A2 + humans, our results show WT M1 58–66 is likely the dominant CTL epitope recognized in infected Tg HLA-A2 hyb/H2 DKO mice. Var. M1 58–66 was also recognized by WT peptide-reactive T cells from both HLA-A2 + humans and Tg mice, although slightly less efficiently than WT M1 58–66 in both cases. Reduced variant recognition was shown to be associated with reduced peptide/A2 binding, as well as a more limited repertoire of utilized TCR Vβ chains. The similar pattern of recognition and cross reaction observed here for the WT and variant M1 58–66 epitopes with HLA-A2 by human and Tg HLA mouse CTLs indicates that A2-dependent events of Ag processing, presentation and recognition are well-conserved between species. These findings demonstrate that this Tg HLA-A2/H2 DKO model will aid identification and development of epitopes as vaccines for numerous viral and tumor antigens for the HLA-A2 supertype.

PREDBALB/c: a system for the prediction of peptide binding to H2d molecules, a haplotype of the BALB/c mouse

PREDBALB/c is a computational system that predicts peptides binding to the major histocompatibility complex-2 (H2d) of the BALB/c mouse, an important laboratory model organism. The predictions include the complete set of H2d class I (H2-Kd, H2-Ld and H2-Dd) and class II (I-Ed and I-Ad) molecules. The prediction system utilizes quantitative matrices, which were rigorously validated using experimentally determined binders and non-binders and also by in vivo studies using viral proteins. The prediction performance of PREDBALB/c is of very high accuracy. To our knowledge, this is the first online server for the prediction of peptides binding to a complete set of major histocompatibility complex molecules in a model organism (H2d haplotype). PREDBALB/c is available at .

Strong memory CD8 + T cell responses against immunodominant and three new subdominant HLA-B27-restricted influenza A CTL epitopes following secondary infection of HLA-B27 transgenic mice

We previously showed that the known HLA-B27-restricted influenza A epitope identified from human studies, NP.383–391, was recognized by CTLs following influenza A infection of transgenic (Tg) HLA-B27/H2 class I-deficient (H2 DKO) mice. Here, we examined the kinetics of the primary NP.383–391-specific response in Tg HLA-B27/H2 DKO mice at the site of respiratory infection, along with the profile of additional influenza A epitopes recognized. While the temporal kinetics of the Tg HLA-B27/NP.383–391-specific CD8 + T cell response paralleled the H2-D b/NP.366–374-specific response of non-Tg H2 b mice, the magnitude was less. Using epitope prediction programs, we identified three novel B27-restricted influenza A epitopes, PB2.702–710, PB1.571–579, and PB2.368–376, recognized during both the primary and secondary response to infection. Although the secondary NP.383–391-specific response was dominant, PB1.571–579 and PB2.368–376 stimulated stronger proliferative expansion in memory T cells. Our results indicate a broader B27/influenza A CTL repertoire than previously known. Together with results for other HLA class I alleles, this information will become important in improving vaccine strategies for influenza A and other human pathogens.

Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. II. Deletion of Effector Cells by Fas-Fas Ligand Apoptosis

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8(+) T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8(+)1B2(+) effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8(+) T cells were from 2C mice, which lack Fas expression ((2CX lpr)F(2)), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8(+)1B2(+) effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the alpha3 domain of H2 class 1 on the effector cell.

HLA and H2 Class II Transgenic Mouse Models to Study Susceptibility and Protection in Autoimmune Thyroid Disease

Using single H2 and HLA class II transgenic mice, in the absence of endogenous H2 class II molecules, we have studied the permissiveness of class II molecules for experimental autoimmune thyroiditis (EAT). Resistant strains expressing susceptible class II molecules, H2A k or HLA-DR3, developed EAT, clearly demonstrating the importance of class II gene inheritance. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules were permissive for EAT induction with either mouse (m) or human (h) thyroglobulin (Tg). HLA-DQ polymorphism was also detectable, as hTg-induced EAT developed in DQ8+, but not DQ6+, mice. Class II gene interactions leading to reduced EAT severity were observed in H2 transgenic mice, when H2E transgene was expressed in H2A+ mice or H2A molecules were introduced into our novel H2A-E+ transgenic model. Similarly, in DR3+ mice, only the DQ8 transgene reduced EAT severity, depending on both background genes (C57BL/10 or NOD) and Tg species. Based on computer-predicted, class II-binding motifs, potential pathogenic Tg peptides, either unique to hTg (H2A-E+ model) or shared between mTg and hTg (HLA-DR3+ model), were identified. We have also developed a Graves' disease model by immunizing DR3+ mice with TSH receptor DNA. Thus, transgenic models are excellent tools to study human autoimmune thyroid diseases in the context of murine EAT.

Human MHC class I transgenic mice deficient for H2 class I expression facilitate identification and characterization of new HLA class I-restricted viral T cell epitopes.

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLA(nat)) (with human beta(2)-microglobulin) and as hybrid human/mouse (HLA(hyb)) molecules on the H2(b) background. To eliminate the influence of H2(b) class I, each HLA Tg strain was bred with a H2-K(b)/H2-D(b)-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLA(hyb) molecule and HLA-B27(nat)/human beta(2)-microglobulin led to peripheral CD8(+) T cell levels comparable with that for mice expressing a single H2-K(b) or H2-D(b) gene. Influenza A infection of Tg HLA-B27(hyb)/DKO generated a strong CD8(+) T cell response directed at the same peptide (flu nucleoprotein NP383-391) recognized by CTLs from flu-infected B27(+) humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7(hyb)/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418-426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.

The CD68 protein as a potential target for leukaemia-reactive CTL.

CD68, a haematopoietic differentiation marker of the monocyte-macrophage lineage, is expressed in various human malignancies including chronic and acute myeloid leukaemia (AML). While the majority of normal CD34(+) cells are negative for CD68 expression, CD34(+) cells from AML patients produce elevated amounts of this protein. The purpose of this study was to identify CTL epitopes in the human CD68 protein. Mouse CD68 was also analysed to search for epitopes that could be used in murine tumor model. Peptides binding to murine H2(b) class I molecules were identified and used to stimulate CTL responses from allogeneic donor mice to avoid immunological tolerance. High avidity CTL clones specific for three different peptide epitopes did not kill CD68-expressing murine target cells, indicating that endogenous antigen processing failed to produce sufficient amounts of these peptides. In contrast, allo-restricted human CTL specific for an HLA-A2-binding peptide of CD68 recognised not only picomolar concentrations of peptide, but also displayed low levels of killing against HLA-A2-positive K562 and THP-1 leukemia cell lines and blast cells from AML patients. These data suggest that human leukaemia cells express limited amounts of CD68-derived peptides, and that high avidity CTL capable of recognising sub-picomolar concentrations of peptides are required for efficient killing of leukaemia cells.

Transcription factor NF-IL6 (C/EBPbeta) activates the expression of the mouse MHC class I H2-Kb gene in response to TNF-alpha via the intragenic downstream regulatory element.

The 5'-enhancer-deleted genomic construct of the H2-K(b) gene, stably integrated into the genome of L(tk-) fibroblasts, retains full competence to be induced by tumor necrosis factor-alpha (TNF-alpha) treatment. The only defined regulatory region in this construct is the intragenic downstream regulatory element (H2DRE). Computational inspection uncovered two potential NF-IL6 (C/EBPbeta) binding motifs within the H2DRE. Chloramphenicol acetyltransferase (CAT) reporter gene assay revealed that NF-IL6 is able to elevate transcription from H2DRE. Moreover, transient transfection of an NF-IL6 expression vector increased both constitutive and TNF-alpha-induced mRNA levels of endogenous H2 class I genes, and transfection of an NF-IL6 dominant negative construct decreased the expression of endogenous H2 class I genes in a dose-dependent manner. Using the electrophoretic mobility shift assay (EMSA) and antibody supershift assay, we were able to qualify the two computationally identified NF-IL6 binding motifs as one high-affinity and one low-affinity binding site. We conclude that the H2-K(b) gene belongs to target genes of the NF-IL6 (C/EBPbeta) in the course of the cellular response to TNF-alpha, and we discuss some consequences of this conclusion in a general framework of inducible expression of the H2-K(b) gene.