Specificity and frequency of primary anti-HLA cytotoxic T lymphocytes in normal and HLA-B27.2-, HLA-B27.5-, and HLA-Cw3-transgenic mice. A transgenic model for MHC xenoantigen recognition

Previous studies have shown that the lymphocytes of naive mice produce a strong primary CTL responses in vitro to human MHC class I Ag presented by HLA-transgenic mouse (TGM) cells. A limiting dilution (LD) assay was used to analyze this xenoreactive CTL repertoire in mice. Frequencies of HLA class I-specific CTL precursors (CTLp) were estimated in naive normal and HLA-B27.2-, -B27.5- and HLA-Cw3-double TGM (i.e., mice expressing HLA and human beta 2-microglobulin (hu beta 2m]. The xenoreactive CTLp frequencies were compared to frequencies of CTLp to H-2 alloantigens estimated in naive normal mice. The results showed that the frequencies of HLA class I-specific CTLp are comparable with those of alloreactive CTLp. This overlap in CTLp frequencies suggests that HLA class I xenoantigens are recognized by primary mouse CTL as allelic variants of H-2K and H-2D. This was confirmed in split well analysis by the observation that the xenoreactive response was not restricted by self-MHC of the responding mouse. Thus, primary HLA class I-specific mouse CTL clones recognized their target Ag regardless of whether they were expressed on H-2-mismatched mouse cells or on human cells. The frequencies of HLA class I-specific CTLp in HLA-TGM were comparable to those in normal mice. We propose that MHC allo- and xenoreactive CTL responses are not caused by the activation of CTLp specific for self-MHC plus peptide but to the activation of CTLp recognizing MHC allo- and xenoantigens directly or as peptides presented by their native MHC molecules.     

HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human beta 2-microglobulin

A gene encoding the H chain of the human class I MHC Ag HLA-B27 was introduced into the germ lines of inbred C57BL/6 (B6) and non-inbred (B6 X SJL/J) F2 mice. By immunofluorescence and flow cytometry, the HLA-B27 gene product was expressed on lymphoid cells at levels comparable to the endogenous H-2b and H-2s class I MHC molecules. In both primary and secondary MLC between responder spleen cells from non-transgenic (B6 X SJL/J) F1 mice and transgenic stimulator cells, CTL were generated that specifically lysed mouse L cell (H-2k) or human B cell targets expressing HLA-B27, and this lysis thus appeared largely unrestricted by H-2. These results indicate that transgenic mice express a functional HLA-B27 gene product on cell surfaces in the absence of the human beta 2-microglobulin gene. These transgenic mice promise to be a valuable resource in the investigation of the unique role of HLA-B27 in inflammatory human disease.     

Cell surface expression and alloantigenic function of a human class I MHC heavy chain gene (HLA-B7) in transgenic mice

We have introduced the gene encoding the heavy chain of the human MHC class I Ag HLA-B7 into transgenic mice. The gene was shown to be expressed at both the RNA and protein level. Cell surface HLA-B7 was detected on whole spleen cells by immunoprecipitation and on purified T cells by flow cytometry (FACS). Normal mice immunized with H-2-syngeneic B7-transgenic spleen cells generated CTL capable of killing transgenic cells and B7-expressing human JY cells. Anti-HLA mAb blocked the killing of JY cells. These results indicate that the human class I Ag HLA-B7 can be expressed at the surface of transgenic spleen cells in the absence of human beta 2-microglobulin, and that a significant fraction exists in a form recognizable by nontransgenic CTL as a major histocompatibility Ag unrestricted by H-2.     

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.     

Cross-positive selection of thymocytes expressing a single TCR by multiple major histocompatibility complex molecules of both classes: implications for CD4+ versus CD8+ lineage commitment

This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8+ CTL clone. The Idhigh+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, Dq or Lq molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2q background, mature Idhigh+ T cells appeared among both CD4+ and CD8+ subsets in periphery, even in the absence of RAG-2 gene. When examined on beta2-microglobulin-/- background, CD4+, but not CD8+, Idhigh+ T cells developed, suggesting that maturation of CD8+ and CD4+ Idhigh+ cells was MHC class I (Dq/Lq) and class II (I-Aq) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4+/CD8+ lineage commitment are discussed.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

Competition between MHC class I alleles for cell surface expression alters CTL responses to influenza A virus

Mammalian cells express up to six different MHC class I alleles, many of which differ in terms of their interaction with components of the Ag presentation pathway and level of cell surface expression. However, it is often assumed in Ag presentation studies that class I alleles function independently of each other. We have compared cell surface expression levels and function of MHC class I molecules in F(1) hybrid mice with those in the homozygous parental strains. The level of cell surface expression of certain alleles in F(1) mice differed significantly from 50% of that found on the same cell type in the corresponding parental strain, suggesting allele-specific competition for cell surface expression, and not expression solely according to gene dosage. The strongest effect was observed in H-2(b) x H-2(k) F(1) mice, in which the H-2(b) class I molecules dominated over the H-2(k) class I molecules. The magnitude of H-2(k)-restricted CTL responses to influenza A virus infection was similar in the F(1) hybrid and parental H-2(k) mice. However, in H-2(k) mice expressing a K(b) transgene, cell surface levels of the endogenous class I molecules were down-regulated to a greater degree than in F(1) hybrid mice, and H-2(k)-restricted CTL responses against influenza A virus were greatly reduced, although the CTL repertoire was apparently present. Therefore, certain MHC class I molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response.     

Cutting edge: requirement of class I signal sequence-derived peptides for HLA-E recognition by a mouse cytotoxic T cell clone

The human nonclassical MHC class I molecule HLA-E has recently been shown to act as a major ligand for NK cell inhibitory receptors. Using HLA-E-expressing transgenic mice, we produced a cytotoxic T cell clone that specifically recognizes the HLA-E molecule. We report here that this T cell clone lyses HLA-E-transfected RMA-S target cells sensitized with synthetic class I signal sequence nonamers. Moreover, this T cell clone lyses human EBV-infected B lymphocytes, PHA blasts, and PBL, formally demonstrating the surface expression of HLA-E/class I signal-derived peptide complex on human cells. Furthermore, these data show that HLA-E complexed with class I signal sequence-derived peptides is not only a ligand for NK cell inhibitory receptors, but can also trigger cytotoxic T cells (CTL).     

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.     

Nonimmune thyroid destruction results from transgenic overexpression of an allogeneic major histocompatibility complex class I protein.

The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.     

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.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-microglobulin subunit

NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the alpha1, alpha2, alpha3, and beta2-microglobulin (beta2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the beta2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the beta2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine beta2m to human beta2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human beta2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of beta2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the alpha1 and alpha2 domains of the heavy chain.     

Phenotypical and functional characterization of the CD8+ T cell repertoire of HLA-A2.1 transgenic, H-2KbnullDbnull double knockout mice.

Homozygous HLA-A2.1 transgenic H-2KbnullDbnull double knockout (KO) mice were created. Their potential to develop HLA-A2. 1-restricted cytolytic responses was compared with that of their classical transgenic counterparts, which still express H-2Kb, Db molecules. On cell surfaces, both strains express similar amounts of chimeric (alpha 1 alpha 2 domains of human, alpha 3 cytoplasmic domains of mouse) HLA-A2.1 molecules in noncovalent association with mouse beta 2-microglobulin. Compared with mice that are totally deprived of histocompatibility class Ia molecules (H-2KbnullDbnull double KO), the expression of HLA-A2.1 in transgenic/double KO mice resulted in sizeable increase in the periphery of CD8+ T cells with a normally diversified TCR repertoire. A biased education in favor of HLA-A2.1, ascribable to the absence of H-2 class Ia molecules, was evidenced in these transgenic/double KO mice by their improved capacity to mount HLA-restricted cytolytic responses, regardless of whether they were virally infected or injected with synthetic epitopic peptide. HLA class I transgenic, H-2 class Ia KO mice should represent useful animal models for the preclinical evaluation of vaccine formulations aiming at the induction of HLA class I-restricted CTL responses.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.     

Expression of human HLA-B27 transgene alters susceptibility to murine Theiler's virus-induced demyelination

Infection of certain strains of mice with Theiler's murine encephalomyelitis virus results in persistence of virus and an immune-mediated primary demyelination in the central nervous system that resembles multiple sclerosis. Because susceptibility/resistance to demyelination in B10 congeneic mice maps strongly to class I MHC genes (D region) we tested whether expression of a human class I MHC gene (HLA-B27) would alter susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination. Transgenic HLA-B27 mice were found to co-express human and endogenous mouse class I MHC genes by flow microfluorimetry analysis of PBL. In the absence of the human transgene, H-2stf, or v mice but not H-2b mice had chronic demyelination and persistence of virus at 45 days after infection. No difference in degree of demyelination, meningeal inflammation, or virus persistence was seen between transgenic HLA-B27 and nontransgenic littermate mice of H-2f or H-2v haplotype. In contrast, H-2s (HLA-B27+) mice showed a dramatic decrease in extent of demyelination and number of virus-Ag+ cells in the spinal cord compared with H-2s (HLA-B27-) littermate mice. In addition, none of the eight H-2s mice homozygous for HLA-B27 gene had spinal cord lesions even though infectious virus was isolated chronically from their central nervous system. Expression of HLA-B27 transgene did not interfere with the resistance to demyelination normally observed in B10 (H-2b) mice. These experiments demonstrate that expression of a human class I MHC gene can modulate a virus-induced demyelinating disease process in the mouse.