The conformational flexibility of class I H-2 molecules as revealed by anti-peptide antibodies specific for intracytoplasmic determinants: differential reactivity of beta 2-microglobulin "bound" and "free" H-2Kb heavy chains.

Immunoprecipitation experiments using anti-peptide antisera prepared against exon 6, exon 7 and exon 8-encoded intracytoplasmic regions of the H-2Kb gene product indicated that approximately 1/3 of the H-2Kb heavy chains in a cell surface-labelled glycoprotein fraction from EL-4 cells, or H-2b spleen cells, is not associated with beta 2-microglobulin (beta 2-m). This population of "free" H-2Kb heavy chains failed to react with alloantisera or monoclonal antibodies specific for conventional H-2Kb serological determinants, suggesting that significant conformational alterations were induced in the extracellular domains upon dissociation of beta 2-m. In addition, although antibodies to intracytoplasmic peptide 8 were able to react with both "free" and beta 2-m "bound" heavy chains, the determinants seen by anti-peptide 6 and anti-peptide 7 were only recognized in the "free" heavy chain. These data suggest that the conformational perturbation of the extracellular domains induced by beta 2-m dissociation can be "transmitted" to the intracytoplasmic region of the heavy chain. These results indicate the potential for a class I heavy chain-mediated transmembrane signalling event, and suggest that the "free" class I heavy chain might have a role to play in the major histocompatibility complex (MHC)-restricted presentation of T-cell determinants to cytotoxic T-lymphocytes.     

The CD8alphabeta co-receptor on double-positive thymocytes binds with differing affinities to the products of distinct class I MHC loci

The CD8 co-receptor is essential for TCR-dependent immune recognition and T cell development involving peptides bound to MHC class I (MHCI) molecules. The dominant interaction of CD8 alpha alpha and alpha beta co-receptors is with the alpha3 domain of an MHCI molecule. Whether this interaction is different for the products of various MHCI loci is currently unknown. Here we examine the interaction between H-2K(b) and H-2D(b), the two MHCI molecules in the C57BL / 6 mouse, and CD8 using H-2K(b) and H-2D(b) tetramers. The MHCI molecules bind to the CD8alpha beta co-receptor on double-positive thymocytes with different avidities (H-2K(b) > D(b)). The differences are linked to their respective alpha3 domains. Hence, an H-2D(b)K(b) tetramer comprising D(b)alpha1--alpha2 and K(b)alpha3 domains shows more binding than H-2D(b). We also quantitated the monomeric affinities of CD8alpha alpha and CD8alpha beta for H-2K(b) and H-2D(b). The H-2K(b) interaction with CD8alpha alpha and CD8alpha beta is stronger than that of H-2D(b). Given that T cell repertoire selection of DP thymocytes is a function of both TCR-pMHCI and CD8alpha beta-pMHCI avidities, these differences may explain the dominant role of H-2K(b) as compared to H-2D(b) in CD8 T cell development of C57BL / 6 mice. The influence of allelic and non-allelic alpha3 polymorphisms on thymic selection processes are discussed.     

Antigen recognition by the T cell receptor is enhanced by CD8 alpha-chain binding to the alpha 3 domain of MHC class I molecules, not by signaling via the cytoplasmic domain of CD8 alpha.

The binding specificities and function of mouse CD8 were studied using a CD4-CD8- allospecific T cell hybridoma, chimeric class I MHC molecules, and a CD8 alpha deletion mutant. By transfecting the mouse CD8 alpha gene into a IL-2 producing, H-2Kb specific hybridoma, IL-2 production was increased when L cells expressing Kb were used as stimulators. However, no increase in IL-2 was observed when a KbKbB7 hybrid molecule, composed of the alpha 1 and alpha 2 domains of H-2Kb, and the alpha 3 domain of HLA-B7, was used as a stimulator. Comparison between T cell hybridomas that expressed full-length CD8 alpha and a deletion mutant lacking part of the cytoplasmic domain revealed identical responsiveness for H-2Kb. The data suggest that the mouse CD8 alpha homodimer does not bind to the alpha 3 domain of HLA class I molecules and that CD8 alpha acts as a co-receptor with the TCR by binding the same MHC molecule for alloantigen recognition. Our data also provide evidence that CD8 alpha signal transduction through its cytoplasmic tail by association with p56lck is not an absolute requirement for antigen recognition by T cells.     

Immunopurification and insertion into liposomes of native and mutant H-2Kb: quantification by solid phase radioimmunoassay

To study the interaction between T cells and isolated H-2Kb, we developed protocols for the immunopurification of the molecule from monoclonal anti-H-2Kb immunoadsorbent columns and for its insertion in lipid vesicles. Patterns of reactivity of two anti-H-2Kb monoclonal antibodies (mAb) (20-8-4 and Y3) on H-2 recombinant and H-2Kb mutant mice indicated that mAb Y3 reacted with all six mutant forms of H-2Kb tested. Binding competition studies indicated that Y3 and 20-8-4 recognized distinct epitopes of H-2Kb. A solid phase radioimmunoassay was established using these two mAb to monitor H-2Kb activity in detergent containing cell lysates, after immunopurification, and after insertion into liposomes. About 70% of H-2Kb activity could be eluted from anti-H-2Kb-immunoadsorbents in the presence of 3 M NH4SCN and octyl glucoside (pH 7.4). A procedure of liposome formation combining gel dilution and dialysis yielded liposomes bearing H-2Kb molecules which could inhibit antibody plus complement cytolysis and could stimulate in vivo primed T cells to generate cytotoxic T lymphocytes in vitro. The present protocol can be extended to immunopurify and obtain H-2Kbm1 bearing liposomes.     

SEROLOGICAL AND IMMUNOCHEMICAL STUDIES OF H-2 ALLOSPECIFICITIES ON k36, A SYNGENEIC TUMOUR OF AKR

Expression of H-2 antigenic specificities on K36, a spontaneous leukaemia originating from AKR (H-2^k) mice, was studied by serology and immunochemistry. Two ascites lines of the tumour, as well as a tissue culture adjusted and cloned tumour line, were used in these studies with similar results being obtained. K36 expresses on its cell surface D-region encoded H-2K antigens but does not express K-region encoded H-2K alloantigens. It also expresses on its cell membrane, H-2 specificities of foreign haplotypes not present on normal AKR lymphoid cells. The molecular basis of the H-2D^d specificity on K36 (H-2^k was analysed by immunoprecipitation and polyacrylamide gel electrophoresis. The specificity was shown to be present on a glycoprotein of apparent molecular weight 45,000. However, antisera against the H-2D^d private specificity (H-2.4) precipitate additional glycoprotein of 45,000D and also 70,000D. In tryptic peptide maps of the isolated 45,000D fraction precipitated by anti-H-2.4 serum from radiolabelled K36 glycoprotein, all H-2D^d specific peptides were present in the same quantitative ratio. This is consistent with the structural identity of the foreign H-2D^d from the K36 tumour with normal H-2D^d and supports the hypothesis of a regulator system controlling the H-2 allelism. Under certain circumstances such a system could cause suppression of one and derepression of the other H-2 gene products.     

Sw-1近交系小鼠H-2单型的鉴定

采用一组标准H-2K、D及Ia定型血清,以微量补体依赖细胞毒试验方法,对Sw-1近交系小鼠作H-2单型的定型。初步确定Sw-1近交系小鼠其H-2单型为H-2~s,且带有Ia 4,8,11和/或16号抗原。     

Biochemical analysis of a public H-2 specificity revealed by an anomalous reaction of an alloantiserum with a chemically induced C57BL/10 fibrosarcoma

D-25 is a H-2 alloantiserum produced in (B10.D2 X C3H.NB) (H-2d X H-2p)F1 mice after immunization with B10.RIII(H-2r) cells, and which is known to recognize the H-2.25 public antigen on H-2k haplotypes. The "anomalous" reaction of D-25 with a partially purified deoxycholate-solubilized glycoprotein of B10-1 (H-2b) fibrosarcoma was studied with various biochemical techniques. The 125I-labelled precipitates were analysed both in one- and two-dimensional SDS-PAGE and by a partial proteolysis peptide mapping (Cleveland's mapping). The results indicate first that the D-25-related antigen was a genuine H-2 antigen normally associated with the B2-microglobulin, second that this antigen was borne by the Kb but not Db gene products, and finally that D-25 was able to precipitate the same antigen on normal H-2b spleen cells. We conclude that this serum recognized a normal H-2 specificity shared by H-2b and H-2r haplotypes and tentatively identified as the public antigen H-2.54 which for the first time could be assigned to the K but not to the D region of the H-2b haplotype.     

Allo-class I-reactive CD4-CD8- T cell hybridomas recognize the conformational change of class I molecule resulting from the exchange of the alpha 3 domain

H-2Kb-reactive, interleukin (IL) 2-producing T cell hybridomas possessing neither CD8 nor CD4 molecules were employed for the study of allorecognition on interspecies hybrid antigens by T cells in the absence of an influence of these accessory molecules. Both HTB176.10 and HTB177.2 T cell hybridomas reacted with KbKbB7 hybrid antigens as well as Kb antigens and they produced more IL2 in response to Kb antigens than KbKbB7 hybrid antigens. IL2 production of these T cell hybridomas was dependent on the surface expression level of Kb molecules on stimulators. Therefore, L cells expressing almost equivalent levels of Kb or hybrid antigens were selected for further functional studies by these T cell hybridomas. They apparently produced less IL2 in response not only to interspecies hybrid antigens but also to interspecies hybrid antigens in response to Kb antigens. These results indicated that T cell hybridomas recognized the conformational change of class I molecule resulting from the exchange of the alpha 3 domain via their T cell receptors.     

Identification of murine leukemia viral antigens detected on mouse cells by H-2 alloantisera.

H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system.     

Role of alpha3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs

CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the alpha3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule alpha3 domain in the activation of CD8(+) CTL, we have produced a soluble 227 mutant of H-2D(d), with a point mutation in the alpha3 domain (Glu227 --> Lys). 227 mutant class I-peptide complexes were not able to effectively activate H-2D(d)-restricted CD8 T cells in vitro, as measured by IFN-gamma production by an epitope-specific CD8(+) CTL line. However, the 227 mutant class I-peptide complexes in the presence of another MHC class I molecule (H-2K(b)) (that cannot present the peptide) with a normal alpha3 domain can induce the activation of CD8(+) CTL. Therefore, in order to activate CD8(+) CTL, the alpha3 domain of MHC class I does not have to be located on the same molecule with the alpha1 and alpha2 domains of MHC class I. A low-avidity CD8(+) CTL line was significantly less sensitive to stimulation by the 227 mutant class I-peptide complexes in the presence of the H-2K(b) molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the alpha3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR-MHC interaction.     

Microsequence analysis of Ia antigens from three strains of rats

Homologues of Ia antigens of the mouse are identified in three rat strains by partial N-terminal amino acid sequence analysis. Ia antigens of the rat were isolated by indirect immune precipitation using specific rat alloantisera. Rat Ia antigens consist of two components, alpha and beta, which have respective mol. wts. of approximately 35 000 and 28 000. Partial N-terminal sequence analysis of each of the alpha components of the H-1a, H-11 and H-1n haplotypes yields a single, apparently homogeneous sequence which is identical among the three haplotypes and is strikingly homologous to the alpha polypeptides of the I-E subregion of mouse and to the human polypeptide, p34. Partial N-terminal sequence analysis of the beta components shows that a mixture of polypeptides is present for each haplotype. There are differences in the beta sequences among the three haplotypes and potential homologies between the rat beta sequences and the sequences of the beta polypeptides of the I-A and I-E subregions of mouse. These obserations imply that the rat has at least two distinct groups of Ia molecules. The organization of genes encoding the Ia polypeptides in the major histocompatibility complex of the rat is discussed.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

Intra-H-2 Recombination in H-2b/H-2t1 Heterozygotes in the Mouse I. Four Cases of Crossing Over between the S and D Regions

An examination of 1009 backcross animals produced from H-2b/H-2t1 heterozygotes resulted in the detection of four cases of intra-H-2 recombination. Three of the recombinants received the K region from H-2b and the D region from the H-2t1 parenteral chromosome. The fourth recombinant received a K region from the H-2t1 parental chromosome and the D region from H-2b. Ss typing revealed that crossing over occurred between the S and D regions of the H-2 complex in all four cases. Three of the four recombinants were developed into inbred strains and assigned the haplotype designations H-2i8, H-2i9 and H-2at1.     

Influence of antigen density on degree of clonal deletion in T cell receptor transgenic mice.

The extent of peripheral clonal deletion of the T cell antigen receptor (TCR) from a CD8-dependent cytotoxic T lymphocyte of H-2k origin with alloreactivity for H-2Kb was measured in a TCR transgenic (Tg) model by use of a clonotype-specific mAb (anti-Ti mAb). Deletion varied depending on whether the TCR Tg was expressed in mice heterozygous (H-2k x b) or homozygous (H-2b x b) for the H-2Kb antigen. CD8 surface staining and functional analyses of peripheral T cells stimulated with polyclonal activators in bulk culture or by limiting dilution confirmed that in the H-2b x b mice few Ti+ cells were generated as measured by anti-Ti mAb-dependent target cell killing; while such cells could readily be detected in the H-2k x b mice. The latter maintained non-responsiveness to H-2Kb, as measured by cytolysis of H-2Kb-expressing tumor target cells, due to their down-regulation of surface CD8 expression. The question of whether the difference between H-2b x b and H-2k x b mice was due to the influence of positive selection imposed by the k haplotype in the H-2k x b hybrid, or to the lower antigen density in heterozygous than homozygous mice was addressed by analysis of H-2b x d Tg mice, H-2d being a non-selecting haplotype. Results obtained were similar for H-2b x d and H-2k x b Tg mice, suggesting that the density of the H-2Kb antigen may be one of the parameters controlling the extent of clonal deletion in the thymus.     

Preferred size of peptides that bind to H-2 Kb is sequence dependent.

The identification of naturally processed viral peptides reveals that major histocompatibility complex (MHC) class I epitopes are composed of nine or eight amino acid residues. Peptides eluted from H-2 Kb MHC class I molecules have been suggested, as a class, to be eight amino acid residues long. To assay for peptide-class I interactions, a stabilization assay described for surface labeled "empty" class I molecules was employed, but on biosynthetically labeled class I molecules. The Sendai virus nucleoprotein-derived octapeptide APGNYPAL does not bind and stabilize Kb molecules, whereas other octameric Kb-restricted peptides and the nonameric peptide FAPGNYPAL interact stably. We attribute the failure of Sendai octamer binding to the presence of proline in position two: replacement of proline renders the resulting octamers as efficient as FAPGNYPAL for binding and stabilization of H-2 Kb. Substitution of glycine in position three of APGNYPAL slightly improves its Kb stabilizing capacity. Iodination of the tyrosine residue significantly alters the binding properties of the nonamer peptide. We conclude that the length of epitopes as selected by the class I Kb molecule is influenced by their sequence and suggest that proper positioning of the NH2 terminus of peptides is essential for class I stabilizing properties. The ability to stabilize newly synthesized "empty" class I molecules with peptide argues against an involvement of beta 2 microglobulin exchange in the experiments described here.     

Enumeration of alloreactive helper T lymphocytes which cooperate with cytolytic T lymphocytes

The number of alloreactive helper T lymphocytes (HTL) which cooperate with cytolytic T lymphocytes have been quantified in a sensitive limit dilution system. Approximately 1 in 330 splenic T lymphocytes developed helper activity in response to alloantigen of a single haplotype. Most HTL appeared to cooperate nonspecifically with the cytolytic cells. A significant number of alloreactive HTL precursors recognized H-2 K or D alloantigen, but roughly 6-fold more HTL precursors responded to H-2 I, and roughly 20-fold more responded to non-H-2, presumably Mls, alloantigen. These results support the hypothesis that alloantigen encoded by the H-2 I and non-H-2 Mls regions preferentially stimulate helper T lymphocytes while alloantigens encoded by the H-2 K and D regions preferentially stimulate cytolytic T lymphocytes.     

A novel H-2s class I molecule expressed on a B-cell leukemia from SJL/J mice

Anti-H-2.33 [(B10.D2 X A)F1 anti-B10.A(5R)], which predominantly contains antibodies recognizing H-2Kb and IAb molecules, was found to be cytotoxic against DMLM 1678, a B-cell leukemia of SJL/J (H-2s) origin. The antiserum precipitated a typical class I (H-2-like) molecule from labeled tumor cell preparations as judged by molecular mass, papain susceptibility and association with beta 2-microglobulin. Sequential immunoprecipitation studies revealed that it was distinct from either H-2Ks or H-2Ds, the 2 molecules expressing the private antigens of the H-2s haplotype. Absorption analysis using congenic mice mapped the gene controlling the expression of the novel molecule telomeric to the S-region within the major histocompatibility complex.     

Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains.

We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice. The mouse anti Abs monoclonal antibody, KL304 in contrast reacts with the denatured Class II beta chains of H-2f, H-2j, H-2k and H-2s mice, but fails to bind H-2b, H-2d, H-2p or H-2q beta chain, suggesting residues 153-155 of this molecule to be critical for the epitope. Both KL295 and KL304 bind B10.WB (H-2j) which suggests a unique epitope in this strain of mice. Neither KL295 or KL304 react with native mouse class II cell surface molecules.