Human Β2-microglobulin Research Articles

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: II. Application to the negative one-step affinity purification of human β2-microglobulin and Immunoglobulin G

The adsorption of two human proteins, β2-microglobulin and Immunoglobulin G, from uremic patient’s blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their “negative affinity” purification (purity estimated by silver nitrate SDS–PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.

Multiepitopic HLA-A*0201-Restricted Immune Response Against Hepatitis B Surface Antigen After DNA-Based Immunization

CTL together with anti-envelope Abs represent major effectors for viral clearance during hepatitis B virus (HBV) infection. The induction of strong cytotoxic and Ab responses against the envelope proteins after DNA-based immunization has been proposed as a promising therapeutic approach to mediate viral clearance in chronically infected patients. Here, we studied the CTL responses against previously described hepatitis B surface Ag (HBsAg)-HLA-A*0201-restricted epitopes after DNA-based immunization in HLA-A*0201 transgenic mice. The animal model used was Human Human D(b) (HHD) mice, which are deficient for mouse MHC class I molecules (beta(2)-microglobulin(-/-) D(b-/-)) and transgenic for a chimeric HLA-A*0201/D(b) molecule covalently bound to the human beta(2)-microglobulin (HHD(+/+)). Immunization of these mice with a DNA vector encoding the small and the middle HBV envelope proteins carrying HBsAg induced CTL responses against several epitopes in each animal. This study performed on a large number of animals described dominant epitopes with specific CTL induced in all animals and others with a weaker frequency of recognition. These results confirmed the relevance of the HHD transgenic mouse model in the assessment of vaccine constructs for human use. Moreover, genetic immunization of HLA-A2 transgenic mice generates IFN-gamma-secreting CD8(+) T lymphocytes specific for endogenously processed peptides and with recognition specificities similar to those described during self-limited infection in humans. This suggests that responses induced by DNA immunization could have the same immune potential as those developing during natural HBV infection in human patients.

Transgenic Mice Expressing Human HLA and CD8 Molecules Generate HLA-Restricted Measles Virus Cytotoxic T Lymphocytes of the Same Specificity as Humans with Natural Measles Virus Infection

Control of primary measles virus (MV) infection in humans and continued maintenance of immune memory that protects against reinfection are mediated primarily through the anti-MV T cell response, as judged by observations of children with defects in antibody formation but competency in making T cells. Further, the failure of T cell responses in those infected with MV most often leads to overwhelming infection. To better define and manipulate the elements involved in human T cell responses to MV, we analyzed the generation of HLA-restricted cytotoxic T lymphocytes (CTL) in a small animal model. Transgenic mice expressing the human class I MHC antigen HLA-B27 in conjunction with human CD8 molecules produced vigorous HLA-restricted CTL responses to MV antigens, paralleling those in MV infection of humans. In addition, such humanized mice generated human CD8 coreceptor-dependent HLA-B27-restricted CTL with the same specificity for recognition of MV fusion (F) peptide RRYPDAVYL as reported for humans during natural MV infection. Neither murine β2-microglobulin nor murine CD8 substituted adequately as coreceptors for the HLA-B27 heavy chain. By contrast, HLA-A2.1-restricted responses to measles could be generated in the absence of expression of human β2-microglobulin or CD8+ molecules in HLA-A2.1/Kb transgenic mice. Thus a small animal model is now available for studying strategies for optimizing human CD8+ T cell responses and for testing vaccines. This model offers the potential, when combined with the newly reported CD46 transgenic mouse model in which MV replicates in cells of the immune system, for uncoding the molecular mechanism of MV-induced immunosuppression.

Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide-2

Background/Purpose: Microinjection of a Fisher (F344) rat zygote with human HLA-B27 and β2-microglobulin genes induces spontaneous chronic gastrointestinal (GI) inflammation similar to lesions seen in patients with inflammatory bowel disease (IBD). This study was designed to evaluate the potential therapeutic benefit of GLP-2, an intestinal growth factor, in this transgenic rat model of IBD. Methods: Five F344 (control) and 10 HLA-B27 (on a F344 background) rats at 25 weeks of age were used. Rats were divided into the following 3 groups: group 1, F344 rats, no treatment (n = 5); group 2, HLA-B27, no treatment (n = 5); and group 3, HLA-B27, treated with a 14-day systemic infusion (via the jugular vein) of GLP-2 at 50 μg/kg/d (n = 5). After infusion, all rats underwent laparotomy, and the intestine from the ligament of Treitz to the rectum was harvested. Total mucosal damage (percent surface area) was measured using image analysis software (Sigmascan 2.0). Microscopic analysis was performed by a blinded reviewer and scored as follows: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation. Colonic mucosal total RNA was assayed for tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal standard, mRNA by reverse transcriptase polymerase chain reaction. Statistical analysis was performed using analysis of variance (ANOVA) and expressed as mean ± SEM. Results: Normal F344 rats did not show evidence of gross or histological lesions in the small or large intestine. GLP-2 reduced total mucosal damage from 9.0% ± 0.7% in group 2 to 0.9% ± 0.5% in group 3 (P <.01). The histological lesion score was reduced from 7.0 ± 0.6 in group 2 to 4.4 ± 0.8 in group 3 (P <.01). Furthermore, GLP-2 reduced the mean band intensity (MBI) of TNF-α (0.4 ± 0.04 in group 2 to 0 in group 3, P <.01) and IFN-γ (0.3 ± 0.02 in group 2 to 0 in group 3, P <.01). Conclusions: These data show for the first time that GLP-2 significantly reduces gross (90% decrease) and histological (40% decrease) lesions in this rat model of IBD. This is further supported by a significant decrease in gene expression of the inflammatory mediators TNF-α (100% decrease) and IFN-γ (100% decrease). These data suggest a potential therapeutic role for GLP-2 in IBD. J Pediatr Surg 35:847-851. Copyright © 2000 by W.B. Saunders Company.

Analysis of three HLA-A*3303 binding peptide anchors using an HLA-A*3303 stabilization assay.

The affinity of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues at position 2 (P2) (Ala, Ile, Leu, Val, Phe or Tyr) and the C-terminus (Arg) was analysed by a stabilization assay using RMA-S transfectants expressing HLA-A*3303 and human beta2-microglobulin. One hundred and nineteen of these peptides (51.3%) bound to HLA-A*3303, confirming that these residues are anchors for HLA-A*3303. Evaluation of P2 residues demonstrated that binding of peptides with Phe or Tyr at P2 is stronger than that of peptides with aliphatic hydrophobic residues at P2. This was confirmed by analysis of a panel of peptides mutated at P2. Analysis of the C-terminal mutant peptides showed that substitution of Lys for Arg had minimal influence on binding to HLA-A*3303. This implies that peptides carrying HLA-A*1101 anchor residues (Val, Ile, Phe or Tyr at P2 and Lys at the C-terminus) can bind to HLA-A*3303. However, such peptides showed lower binding for HLA-A*3303 than for HLA-A*1101. Thus, Arg at the C-terminus is much stronger anchor for HLA-A*3303 than Lys. The preference for Arg and Lys at the C-terminus by HLA-A*1101 and HLA-A*3303 respectively may be due to sequences of three residues (70, 97 and 114) forming the F-pocket of these HLA class I molecules. Statistical analysis of 232 peptides further showed a positive effect of negatively charged residues at P1 for peptide binding to HLA-A*3303. Thus, residues at P1, P2 and the C-terminus play an important role in peptide binding to HLA-A*3303.

Cell surface detection of HLA-E gene products with a specific monoclonal antibody

An HLA-E-specific monoclonal antibody (mAb) was obtained by immunization of human β2-microglobulin transgenic mice (M-TGM) with spleen cells from double transgenic mice expressing HLA-E molecules (EM-TGM). This mAb, designated V16, specifically recognizes in flow cytometry analysis the HLA-E expressing mouse cells, whereas it does not bind to mouse cells expressing various HLA class I molecules (HLA-A2, -A3, -A11, -A26, A29, -B7, -B27, -Cw3, -Cw7, and HLA-G). V16 mAb binds efficiently to human EBV-infected B lymphocytes, PHA blasts and PBL, thus establishing the surface expression of HLA-E in vivo on these cells.

Cell-Surface Expression and Alloantigenic Function of a Human Nonclassical Class I Molecule (HLA-E) in Transgenic Mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.

An scFv phage clone that binds to human β2-microglobulin

Using phage display technology, a novel mouse scFv phage clone D1 was generated from mice immunized with single-chain HLA-A2, the human MHC class I molecules. D1 recognized a determinant in β2-microglobulin. It reacted with β2-microglobulin associated with the heavy chain of HLA-A2, recombinant β2-microglobulin made in Escherichia coli or β2-microglobulin prepared from human urine. The heavy chain variable region of D1 belongs to gene family VII and the light chain variable region belongs to the kappa chain gene family XI according to the Kabat database.

Human serum β2-microglobulin levels: correlation with total serum IgG and placental IgG transfer in HIV-infected and non-HIV infected individuals

The acquisition of maternal immunoglobulin G (IgG) is fundamental to the immune defence of the neonate. The receptor responsible for IgG transfer across the human placenta has also been implicated in the maintenance of IgG levels in the circulation. β 2-microglobulin is part of the Fc receptor (FcR) that has recently been purified from the human placenta. In HIV infection, increasing serum levels of total IgG and β 2-microglobulin are observed as the disease progresses. Herein, we have investigated the correlation between β 2-microglobulin and total serum IgG levels in HIV-seropositive mothers and their term neonates (HIV group, n=37), as well as in HIV-seronegative mothers and their term neonates (control group, n=50). Serum maternal β 2-microglobulin was directly correlated with total serum IgG levels in HIV-infected mothers ( r=0.58; P=0.0002), but not in healthy HIV-seronegative mothers ( r=−0.20; P=0.16). Maternal serum β 2-microglobulin was also inversely correlated with placental antibody transfer of total IgG in mother-newborn pairs from the HIV group ( r=0.38; P=0.02), but not from the control group ( r=0.15, P=0.31). These results seem to indicate that, in HIV infection, elevated serum β 2-microglobulin levels could be involved in maintenance of abnormally high total serum IgG concentrations; by interfering with the binding of IgG to Fc receptors at the maternal-fetal interface, they might also reduce IgG transfer. By contrast, in normal non-HIV infected individuals, serum β 2-microglobulin levels do not appear implicated in regulation of these two phenomena.

Cell surface expression of HLA-E: interaction with human β2-microglobulin and allelic differences

The formation of a trimeric complex composed of MHC class I heavy chain, beta2-microglobulin (beta2m) and peptide ligand is a prerequisite for its efficient transport to the cell surface. We have previously demonstrated impaired intracellular transport of the human class Ib molecule HLA-E in mouse myeloma X63 cells cotransfected with the genes for HLA-E and human beta2m (hbeta2m), which is most likely attributable to inefficient intracellular peptide loading of the HLA-E molecule. Here we demonstrate that cell surface expression of HLA-E in mouse cells strictly depends on the coexpression of hbeta2m and that soluble empty complexes of HLA-E and hbeta2m display a low degree of thermostability. Both observations imply that low affinity interaction of HLA-E with beta2m accounts to a considerable extent for the observed low degree of peptide uptake in the endoplasmic reticulum. Moreover, we show that the only allelic variation present in the caucasoid population located at amino acid position 107 (Gly or Arg) greatly affects intracellular transport and cell surface expression upon transfection of the respective alleles into mouse cells. No obvious difference was found with regard to the sequence of the peptide ligand.

Arthritis in HLA-B27 Transgenic Animals

This review focuses on investigations of rats and mice transgenic for HLA-B27; these animals have been investigated for several years as potential models for the human spondyloarthropathies. Spontaneous multisystem disease occurs in rats with high expression of B27 and human β2-microglobulin (hβ2m). The disease is T-cell-dependent and is sensitive to both environmental and genetic manipulation. A spontaneous arthritis and enthesopathy has been observed by some investigators in nontransgenic mice which seems to be more prevalent in B27 transgenic mice. Peripheral arthritis has also been reported in B27 transgenic mice that lack mouse β2m. Potential insights from these animals into the pathogenesis of B27-related disease are discussed.

Functional comparison of bovine, murine, and human β2-microglobulin: interactions with murine MHC i molecules

Fetal calf serum is a well known source of bovine β2-microglobulin (β2m) which can exchange with endogenous β2m from, as well as promote peptide binding to, class I major histocompatibility (MHC I) molecules on cells cultured in vitro. Recombinant bovine β2m was expressed and purified for direct functional comparison to human and murine β2m for interactions with murine MHC I molecules H-2Kb, Db, Kd, Ld, and Dd. Bovine and human β2m were equivalent in stabilizing MHC I heavy chains and facilitating peptide loading, suggesting similar affinities for murine MHC I heavy chains. The activity of murine β2m was significantly weaker, consistent with previous work that demonstrated the lower affinity of murine human β2m for murine heavy chains compared to human β2m. Analysis of bovine β2m in fetal calf serum revealed ten-fold higher concentrations than in adult bovine serum, levels shown to significantly affect MHC I stability and peptide loading. The ramifications for the study of MHC I molecules from cells in culture and the evolutionary implications of the higher affinity interactions of human and bovine β2m are discussed.

A MURINE TRANSGENIC MODEL OF HUMAN CLASS I MHC-MEDIATED PROGRAMMED CELL DEATH

376 Human class I MHC molecules have recently been shown to mediate programmed cell death (PCD) signals via a Fas-independent pathway, suggesting that this pathway may, like Fas/Fas ligand, be useful for conferring immune privilege. The present study was conducted to develop a murine model for PCD mediated via human class I MHC molecules. Methods: B6 mice and B6 mice expressing both HLA B27 and human β2-microglobulin transgenes (Tg++ mice) were used. PCD was evaluated using viable cell recovery, ethidium bromide/acridine orange staining/fluorescent microscopy, and propidium iodide staining/FACS. Anti-human class I MHC mAbs used included W6/32 and 5H7(specific for an α3 domain monomorphic determinant). Results: Initial studies in mice expressing a single human class I MHC transgene did not demonstrate PCD induction, however, human class I MHC transgene expression was relatively low. Mice expressing HLA B27 and human β2 microglobulin transgenes, however, demonstrated higher human class I MHC expression and underwent PCD following treatment with 5H7 mAb. Splenocytes undergoing 5H7-induced PCD demonstrated classic features of PCD: cell shrinkage, cytoplasmic vacuolization, DNA condensation, and apoptotic body formation. Similar to the human studies of 5H7-induced PCD, studies in murine Tg++ cells demonstrated a greater sensitivity of B cells than T cells to PCD induction. The murine model was also similar to the human model in that class I MHC-mediated PCD induction was profoundly influenced by MHC receptor crosslinking and also dependent on the epitope specificity of the inducing anti-class I MHC mAb. Anti-CD3 mAb enhanced PCD induction by 5H7 mAb in both murine and human cells, however, IL-2 reversed 5H7-induced PCD only in murine cells. Conclusions: These studies indicate that 1) high level expression of human class I MHC in murine cells renders them susceptible to PCD induction via anti-human class I MHC mAb, 2) similar to human lymphoid cells, crosslinking and epitope specificity of the anti-class I MHC mAb markedly influence PCD induction, 3) anti-CD3 mAb enhances human class I MHC-mediated PCD in both human and murine cells, and 4) Tg++ mice provide a useful model for study of human class I MHC-mediated PCD.

A mutant human β2-microglobulin can be used to generate diverse multimeric class I peptide complexes as specific probes for T cell receptors

Antigen-specific receptors (TCR) on CD8 T lymphocytes form relatively short-lived complexes with their natural ligands: peptides in association with major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic heavy chain and a conserved light chain, β 2-microglobulin ( β 2-M). To produce soluble MHC-peptide complexes in a form that would bind more stably and could be used to identify, count, and isolate CD8 T cells having the appropriate TCR, we prepared multimeric MHC-peptide complexes. Our work builds on the assembly of recombinant MHC class I peptide complexes using a mutant human β 2-M chain (Tyr 67>Cys) which can form stable heterodimers with diverse MHC heavy chains. With biotin added to the SH group, the assembled MHC-peptide monomers formed multimers with avidin linked to a fluorochrome. The specific reactivity of the multimeric reagents with human and mouse cytotoxic T cells (CTL) is described. The present approach permits the production of class I multimers, without the necessity of genetic engineering each heavy chain, a significant advantage in view of the enormous polymorphism of MHC heavy chains. Because human β 2-M forms stable heterodimers with diverse class I heavy chains from various species (human and non human primates, mouse, etc.), this procedure is a general method for producing multimers of MHC-peptide complexes as T cell receptor-specific probes.

Ankylosing spondylitis, HLA B27, and beyond

Publication of the landmark Lancet paper showing the strong association between HLA B27 and ankylosing spondylitis 25 years ago 1 Brewerton DA Hart FD Nicholls A Caffrey M James DCO Sturrock RD Ankylosing spondylitis and HLA-27. Lancet. 1973; ii: 904-907 Summary Scopus (1410) Google Scholar stimulated an avalanche of research into HLA/disease associations. The subsequent weaker associations of HLA B27 with anterior uveitis, reactive arthritis, sacroiliitis, late-onset pauciarticular juvenile chronic arthritis in boys, and inflammatory bowel disease with axial joint involvement suggested a common genetic determinant for a group of disorders already linked as seronegative spondyloarthropathies by the clinical observations and family studies of the late Verna Wright, 2 Wright V Moll JMH Seronegative polyarthritis. North Holland Publishing Company, Amsterdam1976: 1-483 Google Scholar and others. Because reactive arthritis can be precipitated in B27-positive individuals by sexually acquired chlamydial infection or by enteric infections with Campylobacter, Clostridium, Salmonella, Shigella, and Yersinia spp, induction of disease by “arthritogenic” bacterial strains in genetically predisposed individuals has become a compelling idea for the pathogenesis of these disorders. This idea is supported by experiments in transgenic rats expressing HLA B27 and human β2-microglobulin. Such animals develop gut and joint inflammatory disease, 3 Hammer RE Maika SD Richardson JA Tang JP Taurog JD Spontaneous inflammatory disease in transgenic rats expressing HLA B27 and human β2m: an animal model of HLA B27 associated with human disorders. Cell. 1990; 63: 1099-1112 Summary Full Text PDF PubMed Scopus (814) Google Scholar which is mediated by normal gut bacteria, including Bacteroides species, 4 Rath HC Herfarth HH Ikeda JS et al. Normal luminal bacteria, especially bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA B27/human beta2 microglobulin transgenic rats. J Clin Invest. 1996; 98: 945-953 Crossref PubMed Scopus (677) Google Scholar and which does not occur when the animals are maintained in a germfree environment. 5 Taurog JD Richardson JA Croft JT et al. The germ free state prevents development of gut and joint inflammatory disease in HLA B27 transgenic rats. J Exp Med. 1994; 180: 2359-2364 Crossref PubMed Scopus (914) Google Scholar

Calnexin expression does not enhance the generation of MHC class I-peptide complexes

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K d , Kb , Kk , D d , Db , and Ld . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For Kb and K d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of Kb molecules bearing the immunodominant peptide from ovalbumin (OVA257 – 264 ). This was detected with a recently described monoclonal antibody specific for the Kb/peptide complex. Calnexin expression had no significant effect on the formation of Kb /peptide complexes generated from full-length OVA, cytosolic OVA257 – 264 , or endoplasmic reticulum-targeted OVA257 – 264 , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts.

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 degrees C) or by loading these cells with either exogenous human beta2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-gamma treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.

A New MHC Locus That Influences Class I Peptide Presentation

We have investigated the HLA-B27-restricted CTL response to HY minor histocompatibility antigens in rats and mice transgenic for HLA-B27 and human β2-microglobulin. A polymorphism was found at a locus within the H2 complex, producing two distinct but overlapping sets of B27-presented HY peptides. The locus, named Cim2, mapped between the K and Pb loci, and its product is therefore distinct from TAP, LMP, and tapasin. Identical findings in rats and mice, including identical HY peptide sequences and the failure of a rat Tap2A transgene to alter CTL recognition, suggest that a homologous locus with similar polymorphism exists in the rat. Cim2, or a closely linked locus, was found to exert a broad effect on peptide loading of both HLA-B27 and mouse class I alleles. The data thus establish a strong, previously unrecognized MHC-encoded influence on the class I antigen pathway.

HLA-A2.1–restricted Education and Cytolytic Activity of CD8+ T Lymphocytes from β2 Microglobulin (β2m) HLA-A2.1 Monochain Transgenic H-2Db β2m Double Knockout Mice

Three different HLA-A2.1 monochains were engineered in which either the human or mouse beta2-microglobulin (beta2m) is covalently linked to the NH2 terminus of the heavy chain by a 15- amino acid long peptide: HHH, entirely human, HHD, with the mouse H-2Db alpha3, transmembrane, and cytoplasmic domains, and MHD, homologous to HHD but linked to the mouse beta2mb. The cell surface expression and immunological capacities of the three monochains were compared with transfected cells, and the selected HHD construct was introduced by transgenesis in H-2Db-/- beta2m-/- double knockout mice. Expression of this monochain restores a sizable peripheral CD8(+) T cell repertoire essentially educated on the transgenic human molecule. Consequently, infected HHD, H-2Db-/- beta2m-/- mice generate only HLA-A2.1-restricted CD8(+) CTL responses against influenza A and vaccinia viruses. Interestingly, the CTL response to influenza A virus is mostly, if not exclusively, directed to the 58-66 matrix peptide which is the HLA-A2.1-restricted immunodominant epitope in humans. Such mice might constitute a versatile animal model for the study of HLA-A2.1-restricted CTL responses of vaccine interest.

Intestinal Inflammation and Barrier Function in HLA-B27/β2-Microglobulin Transgenic Rats

Background: Since intestinal inflammation is correlated with impaired barrier functions, transgenic HLA-B27/human β2-microglobulin rats that spontaneously develop intestinal inflammation were used to investigate whether onset of inflammation or impaired barrier function was the initial event. Methods: During the age period of 9–14 weeks, transgenic and non-transgenic (control) rats were gavaged weekly with the marker molecules, 51Cr-ethylenediaminetetraacetic acid, 1-deamino-8-D-arginine vasopressin, and albumin, which were quantified in blood or urine. Results: At 12 weeks of age the first signs of inflammation appeared with decreased body weight gain, decreased urine production, and onset of diarrhea. By 14–15 weeks of age all transgenic rats had developed intestinal inflammation, as confirmed by histology and increased myeloperoxidase content, whereas no inflammation was observed in controls. Intestinal passage of the markers did, however, not differ between transgenic and control rats over the studied period. Conclusions: The results suggest that intestinal inflammation precedes altered intestinal barrier function in this inflammation model.