Inhibition of allorecognition by an H-2Kb-derived peptide is evidence for a T-cell binding region on a major histocompatibility complex molecule

The class I and class II major histocompatibility complex (MHC) antigens are polymorphic cell-surface glycoproteins that present antigenic peptides to T lymphocytes in the generation of immune responses. While much is known about the recognition and processing of antigens, the nature of T-cell recognition sites on MHC molecules is poorly understood. Both structural and functional studies have suggested that the two major alpha-helical regions of the class I MHC molecule not only define the site for binding of antigenic peptide but also provide potential sites for interaction of the MHC molecule with the T-cell receptor. A peptide derived from one of these regions on the H-2Kb molecule, peptide Kb163-174, was previously shown to specifically inhibit the stimulation of an alloreactive T-cell hybridoma. To further investigate the role of this region in the recognition of H-2Kb, the effects of peptide Kb163-174 on allospecific T-cell lines and clones were studied. When peptide Kb163-174 was cocultured with either an H-2Kbm10 anti-H-2Kb cytotoxic T-lymphocyte (CTL) clone or a CTL line, this peptide inhibited lysis of H-2Kb targets. Pretreatment experiments showed that the blockade was due to interaction of the peptide with the effector T cells. Surprisingly, peptide Kb163-174 also inhibited lysis of H-2Kb targets by H-2Kbm1-, H-2Kbm3-, H-2Kbm6, and H-2Kbm8-anti-H-2Kb CTLs. These CTLs, which identify multiple antigenic sites on H-2Kb in the alpha 1 and alpha 2 domains, are not directed against amino acid residues 163-174 of H-2Kb. In addition, peptide Kb163-174 specifically blocked lysis of only H-2Kb and not H-2Ld targets by a single bulk CTL culture that was alloreactive on both H-2Kb and H-2Ld. These results indicate that peptide Kb163-174 interferes with T-cell receptor engagement of a contact site on the H-2Kb molecule. Thus, amino acid residues 163-174 define a site used by many alloreactive T cells to engage the H-2Kb molecule.     

Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro

Recombinant human tumor necrosis factor (TNF), purified to greater than 99% homogeneity, increases surface expression of class I major histocompatibility complex (MHC) antigens to a maximum of 9-fold on cultured human endothelial cells (HEC) and human dermal fibroblasts (HDF). The increase is concentration dependent (peak 20-100 units/ml) and time dependent (nearly maximal by 4 days); expression remains elevated in the continued presence of TNF and requires greater than 7 days to return to basal levels upon TNF withdrawal. The increase in surface expression appears to result from increases in steady-state mRNA levels for the class I antigens, although the increase in mRNA is proportionately greater than for surface expression. No surface expression of or mRNA for class II MHC antigens is detectable in either control or TNF-treated HEC or HDF. These effects are similar to those produced by leukocyte or fibroblast (type I) interferons (IFNs). The protein synthesis inhibitor cycloheximide (CHX), when added coincidentally with type I IFNs, leads to superinduction of mRNA for class I MHC antigens and, unexpectedly, leads to the appearance of mRNA for class II MHC antigens. CHX has no effect by itself upon mRNA levels for class I or class II MHC antigens, nor does it modulate the increases in mRNA produced by immune (type II) IFN. Most interesting, CHX blocks the increase in mRNA for class I MHC antigens induced by TNF. Thus TNF appears to act on MHC gene expression through a newly synthesized protein intermediate. Our results provide direct evidence that TNF can modulate gene expression in normal (untransformed) cell types and contribute to understanding the complex nature of MHC gene regulation. Finally, they suggest that TNF may act in vivo as an immunoregulatory molecule.     

Characterization of an incompletely assembled major histocompatibility class I molecule (H-2Kb) associated with unusually long peptides: implications for antigen processing and presentation.

We have identified two forms of a major histocompatibility complex (MHC) class I molecule, H-2Kb, distinguishable by specific antibodies through a study of a genetically engineered mouse cell line that overexpresses these molecules. One form, a complex associated with beta 2-microglobulin (native, beta 2m+ class I), is detectable by conformation-dependent antibodies. The other form, which remains after preclearing cell lysates of native class I, is only poorly, if at all, associated with beta 2-microglobulin (beta 2m- class I) and is detectable by an antiserum against the cytoplasmic tail region of H-2K molecules. Both forms are also present in normal cell lines. The affinity-purified native class I molecules bind short peptides (8 or 9 residues) and assemble tightly with beta 2-microglobulin. In striking contrast, the beta 2m- class I molecules bind peptides that are longer (> 15 residues) than those bound to native class I molecules. This finding is consistent with the recent evidence that peptides longer than 8-10 amino acid residues are transported into the endoplasmic reticulum and suggests the possibility of a control step for peptide presentation by MHC in which the incompletely processed peptides bind to the heavy chain and a selected fraction undergoes final processing and presentation on the cell surface.     

Molecular analysis of an EL4 cell line that expresses H-2Db but not H-2Kb or beta 2-microglobulin.

EL4/Mar is a variant cell line that expresses H-2Db but neither H-2Kb nor beta 2-microglobulin (beta 2m). Southern and RNA blot analysis and immunoprecipitation of metabolically labeled proteins established that the B2m gene(s), beta 2m mRNA, and beta 2m protein are normal in this cell line. Somatic cell hybridization showed that the defect in this cell line was in the synthesis of H-2Kb, and RNA blot analysis with an H-2Kb specific oligonucleotide established that the H-2Kb gene(s) in this cell line was not transcribed into a stable mRNA species. The apparent absence of beta 2m on the surface of this cell line suggests that there may be some feature of the H-2Db molecule that allows it to be expressed in the absence of detectable beta 2m.     

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.     

A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.

Genetically engineered or chemically purified soluble monovalent major histocompatibility complex (MHC) molecules, which have previously been used to study T cells, have not blocked cytotoxic T-cell responses. Here we describe a genetically engineered divalent class I MHC molecule which inhibits lysis of target cells by alloreactive cytotoxic T cells. This protein, H-2Kb/IgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, H-2Kb, and an immunoglobulin heavy chain polypeptide. The chimeric protein has serological and biochemical characteristics of both the MHC and IgG polypeptides. Nanomolar concentrations of H-2Kb/IgG inhibited lysis of H-2Kb-expressing target cells not only by alloreactive H-2Kb-specific T-cell clones but also by alloreactive H-2Kb-specific primary T-cell cultures. A direct binding assay showed high-affinity binding between the H-2Kb/IgG molecule and an H-2Kb-specific alloreactive T-cell clone. Unlabeled H-2Kb/IgG displaced 125I-labeled H-2Kb/IgG from T cells with an IC50 of 1.2 nM.     

Spontaneous H-2 mutants provide evidence that a copy mechanism analogous to gene conversion generates polymorphism in the major histocompatibility complex.

The analysis of H-2K products from spontaneously generated major histocompatibility complex (MHC) mutants and of the primary structure of other class I antigens suggests the genetic hypothesis that diversity in the MHC results from a copy mechanism analogous to gene conversion. The hypothesis was tested by making precise structural predictions about three partially characterized MHC mutants (bm1, bm3, and bm8). The predictions were based on consensus sequences among class I genes that differ from H-2Kb in the same region of the molecule as do the Kb mutants. In two cases (bm3 and bm8) we successfully predicted the correct amino acid substitution at positions known to be altered but for which the specific nature of the substitution had not been determined. In two additional cases (bm1 and bm8) we predicted and found both new mutation sites and the specific amino acid substitutions. The positions and identifications of the variant amino acids were determined by radiolabeled amino acid sequence analysis and DNA restriction endonuclease analysis. The interaction of MHC genes through a copy mechanism to generate diversity permits the introduction of multiple nucleotide base substitutions into class I sequences by a single genetic event. Such a mechanism may account in part for the large structural divergence among alleles of MHC loci and the high degree of MHC polymorphism among wild mice.     

Differential expression of MHC class II antigens in myelomonocytic leukemia cell lines

Major histocompatibility complex (MHC) class II antigens are discordantly expressed on hematopoietic progenitor cells. Their expression is linked to differential responsiveness of the cells to growth factors and inhibitors. We examined the expression of different MHC class II antigens in a panel of human myelomonocytic cell lines representing different stages of differentiation, by cytofluorographic analysis with monoclonal antibody (MoAb) and Northern blot analysis with specific cDNA probes. These analyses revealed discordant expression of different MHC class II antigens both in basal state and after gamma-IFN induction. Thus KG-1 myeloblast cells express all class II antigens (DR greater than DP greater than DQ) constitutively and their expression increased after gamma-IFN treatment. KG-1a, an immature blast variant of KG-1, does not express class II antigens, even after gamma-IFN treatment. THP-1, a monocytic cell line expresses DR but not DP or DQ under basal conditions. DP and DQ are, however, gamma-IFN inducible. The class II negative HL-60 promyelocytic cell line, expresses DR and DP but not DQ after gamma-IFN induction. In all the above cell lines, surface expression of class II antigens correlated with the levels of mRNA expression as determined with specific cDNA probes. In U-937, a monocytic cell line, no surface expression of class II MHC antigens was observed either with or without gamma-IFN, however, specific mRNA message was observed under basal conditions and was further increased with gamma-IFN, indicating a possible defect in assembly or transport of class II antigens. The patterns of class II MHC antigens in these leukemic cell lines may be a useful model to delineate molecular basis of discordant MHC class II expression during myelomonocytic differentiation.     

Direct demonstration of critical amino acid residues required for cytotoxic T-lymphocyte allorecognition of H-2 class I antigens

To identify critical amino acid residues recognized by alloreactive cytolytic T lymphocytes (CTL) generated between H-2Kb and H-2Kbm1, we have derived a series of cloned L-cell lines expressing the following mutant H-2Kb class I genes. Cell line L-KbTyr-Tyr expresses a mutant gene in which positions 155-156 of the Kb molecule have been changed from Arg-Leu to Tyr-Tyr, leaving position 152 unchanged. Cell line L-KbAla expresses the reciprocal mutant gene that has position 152 of the Kb molecule mutated from glutamic acid to alanine, leaving positions 155-156 unchanged. Electrophoretic mobilities of the mutant Kb molecules reflect only those changes predicted by the mutations. Mutant-specific (anti-Kbm1) and native-specific (anti-Kb) CTL lyse L-KbTyr-Tyr and L-KbAla target cells equally well. Unlabeled target inhibition of lysis revealed a pattern of recognition and inhibition that suggests that the amino acid differences between Kbm1 and Kb create at least two discrete determinants that can be recognized by different populations of CTL. The results suggest that these determinants consist, at least in part, of a linear amino acid sequence from which critical amino acid residues can be identified.     

Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms

Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.     

Major histocompatibility complex class I-specific and -restricted killing of beta 2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) recognize major histocompatibility complex (MHC) class I molecules, normally composed of a heavy chain, a beta 2-microglobulin (beta 2m), and peptide antigens. beta 2m is considered essential for the assembly and intracellular transport of MHC class I molecules as well as their peptide presentation to CTLs. Contrary to this dogma, we now report the generation of allospecific and restricted CD8+ and TCR alpha beta+ CTLs (where TCR is T-cell receptor) capable of killing beta 2m-deficient cells. Such CTLs were obtained by priming mice with live allogeneic beta 2m- spleen cells or mutant lymphoma cells producing MHC class I protein but no detectable beta 2m. Although both beta 2m- and beta 2m-expressing lymphoma cells were rejected in allogeneic mice, only the former were efficient inducers of CTLs recognizing beta 2m- cells. These CTLs were MHC class I (H-2Kb or Db)-specific and CD8-dependent and did not require serum as a source of external beta 2m in the culture. They could be induced across major and minor histocompatibility barriers. The H-2-restricted CTLs generated in the latter case failed to kill the antigen-processing-deficient target RMA-S cells. The results show that MHC class I heavy chains in beta 2m- cells can be transported to the cell surface and act as antigens or antigen-presenting molecules to allospecific and MHC-restricted CTLs.     

Ia+ murine epidermal Langerhans cells are deficient in surface expression of the class I major histocompatibility complex.

Murine epidermal Langerhans cells were analyzed with fluorescence microscopy and multicolor flow cytometry for the surface expression of major histocompatibility complex (MHC) class I and class II antigens. Langerhans cells of H-2k haplotype were identified in situ or in epidermal-cell suspensions by their surface expression of the MHC class II determinants I-Ak and I-Ek. More than 90% of class II-positive Langerhans cells in epidermal-cell suspensions expressed no or barely detectable amounts of MHC class I antigens. Quantitation by flow cytometry revealed that H-2k Langerhans cells expressed only 1.6-3.3% as much H-2Kk as did class II-negative keratinocytes in the same epidermal-cell suspensions. By fluorescence microscopy, class I MHC antigens were not detectable on Langerhans cells in situ when analyzed on sheets of intact epidermis. The deficient expression of class I MHC permitted highly purified Langerhans cell populations to be isolated from epidermal cell suspensions by treatment with anti-class I MHC monoclonal antibody and complement. It is likely that the uniquely low cell-surface expression of class I MHC antigen by Langerhans cells has relevance to both immune responses in the skin as well as to mechanisms of skin allograft rejection. In addition, it is conceivable that regulation of class I MHC expression on antigen-presenting cells in general is an important but hitherto unrecognized mechanism of immune regulation.     

Single-residue changes in class I major histocompatibility complex molecules stimulate responses to self peptides.

Single-residue changes were introduced into the murine major histocompatibility complex class I molecule H-2Kb at positions 65 and 69, which are predicted to point up from the alpha-helix of the alpha 1 domain and not into the peptide binding groove. Mutated and wild-type genes were transfected into the murine cell line P815 (H-2d). We present evidence that the changes did not affect the binding of three foreign peptides that are recognized by cytotoxic T lymphocytes (CTL) in association with H-2Kb. Additionally, the mutants provoked strong alloreactive responses in T cells from mice expressing unmutated H-2Kb. The alloreactive CTL were specific for self peptides, which could be extracted from wild-type H-2Kb molecules, recognized in the context of the mutant class I.     

Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.

To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.     

Altered growth of a human neuroendocrine carcinoma line after transfection of a major histocompatibility complex class I gene

The major histocompatibility complex (MHC) class I molecules are known to serve as recognition elements for cytotoxic T cells in mediating the rejection of transplanted tumors. We demonstrate that MHC molecules may have nonimmune functions in modulating tumor cell growth in addition to their classical role in antitumor immunity. A human neuroendocrine carcinoma cell line, COLO 320, with low levels of endogenous class I expression was transfected with the murine H-2Ld gene. Eleven independent stable clones were established, four containing only pRSV-neo and seven also containing varying copy numbers of the transfected Ld gene. The ability of the different clones to grow as colonies in soft agar correlated strongly with the relative amounts of Ld antigen expression (r = 0.89; P less than 0.001). There was a weaker correlation between increased clonogenic ability and higher levels of Ld mRNA (r = 0.67; P less than 0.05). There was no correlation between clonogenic ability and relative expression of amplified c-myc gene or of integrated pRSV-neo. Furthermore, in nude mice, Ld antigen expression was associated with increased formation of metastatic lung colonies 6 weeks after intravenous injection of 10(5) cells. These observations are consistent with the concept that MHC class I antigens may have a role in modulating the growth potential of certain tumor cells independent of their involvement in immune responses.