Cross-reactions between mouse Ia and human HLA-D/DR antigens analyzed with monoclonal alloantibodies

Two mouse monoclonal anti-I-E/Ck alloantibodies (H7-8.26 and H10-81.10) directed against 2 distinct determinants of the specificity Ia-7 and 1 anti-I-Ak alloantibody (H8-15.9) directed against a public determinant common to the I-A subregion products of the H-2k, H-2b, H-2d, H-2q, and H-2ja haplotypes identified cross-reactive determinants on lymphoid cells from various mammalian species, including rat, dog, pig, cow, hamster, and guinea pig. In man, these antibodies detected nonpolymorphic determinants of DR antigens on B cell-enriched peripheral blood lymphocytes from 50 unrelated individuals. These cross-reactive DR determinants were also detected on lymphoblastoid B cell lines, on PHA-activated peripheral T lymphocytes, and on allospecific cytolytic T cell clones, but not on various DR-negative human T leukemia cell lines. Two chains of 29,000 and 35,000 daltons m.w., corresponding to DR antigens, were precipitated by H7-8.26 and H8-15.9 antibodies from radiolabeled membrane extracts of Raji cells. Competitive binding experiments indicated that the 3 mouse anti-Iak antibodies identified 3 distinct cross-reactive determinants on human cells. The results indicate that: a) The cross-reactivity described between mouse I-E/C gene products (Ia-7) and human DR antigen(s) involves, in fact, several distinct and topologically distant determinants. b) At least 1 determinant cross-reacting with DR can be identified on I-Ak gene products. c) The intriguing genetic problem of mouse MHC allotypic determinant(s) being nonpolymorphic in man cannot be simply explained by the deletion of an I-E alpha chain in some strains of mice.     

The preferential cytolytic T lymphocyte response to immunodominant minor histocompatibility antigen peptides

C57BL/6 mice preferentially generate cytolytic T lymphocytes (CTL) to a limited number of immunodominant minor antigens and associated immunogenic peptides when primed with H2-matched Balb.B spleen cells despite multiple minor histocompatibility (H) antigen differences. We have examined the complexity of dominant H antigens recognized by these CTLs to estimate the number of peptides associated with single antigens. Peptides eluted from Kb molecules of lymphoblasts from Balb.B and CXB recombinant inbred (RI) strains were tested for sensitization of RMA-S cells for lysis by short-term C57BL/6 CTL lines specific for Balb.B and CXB strains. Anti-Balb.B CTLs recognized four Kb-bound peptides; subsets of these peptides were recognized by anti-CXB CTLs when tested with peptides from the respective CXB strains. Single peptides segregated independently among the CXB strains, confirming that single peptides were encoded by independently segregating alleles. These peptides were expressed in diverse inbred mouse strains and were recognized preferentially by C57BL/6 CTLs stimulated by different inbred mouse strains. This set of peptides was subclassified by their capacity to sensitize targets when presented in unfractionated mixtures of Kb-bound peptides. The peptide associated with the previously classified dominant CTT-2 antigen was the only peptide to strongly sensitize RMA-S cells for lysis under these conditions. These results suggest that dominant peptides have a wide strain distribution and may have a distinct advantage over dominated peptides in binding to class I molecules and/or in presentation to CTLs.     

T cell response to Borrelia garinii, Borrelia afzelii, and Borrelia japonica in various congenic mouse strains

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4+8- and Ia- T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8+ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Minor histocompatibility antigens

The existence of transplantation antigens, in addition to those encoded by genes in the MHC, has been known for over half a century. The molecular identification of these additional minor histocompatibility (H) antigens lagged behind that of their MHC counterparts, largely because minor H antigens are recognised by T cells and not by antibodies. In the past year, however, new minor H antigens have been identified at both the genetic and protein level and include Uty, a second novel gene encoding a male-specific epitope in mice, a novel autosomal gene encoding each of the H-13 alleles of mice, and a second male-specific epitope encoded by the SMCY gene.     

Histocompatibility-2 system in wild mice. II. H-2 haplotypes of t-bearing mice

As a first step in the study of the possible relationship between the T/t and H-2 complexes, the H-2 antigenic composition of the strains carrying factors t12, tw32, tw2, tw8, t1, t0, t6, tw1, tw71, tw73, tw12, tw5, tw75, and t38 was studied by using a battery of antisera containing antibodies against inbred-derived H-2 antigens. In addition, five t strains (t12, t6, tw5, tw1, and tw2) were selected for the production of antisera against the H-2 complexes carried by t chromosomes. Spleen, lymph node, and thymus cells from H-2b/t heterozygotes and tw2/tw2 homozygotes were injected into appropriate F1 hybrids between two inbred strains that carried the inbred-derived H-2 antigens of the donor. Four new H-2 antigens and one Ia antigen were uncovered and were assigned the symbols H-2.106 through H-2.109, and Ia.101, respectively. Three new H-2 haplotypes were also described, based upon the H-2 antigenic pattern of three t factors, t12, tw1, and tw5. These new haplotypes were given the symbols H-2t12, H-2tw1, and H-2tw5. When the t factors were grouped according to their H-2 haplotypes, their distribution, with certain exceptions, corresponded to the complementation groups. Thus, t chromosomes in the same complementation group carried similar, if not identical, H-2 haplotypes, despite the fact that these chromosomes were derived from widely separated geographic areas. Such an association between the t and H-2 complexes is most unusual in light of what is known of the polymorphism of H-2 haplotypes in wild mice populations. It suggests more than a casual relationship, at least at the population level, between the t and H-2 loci.     

A highly polymorphic microsatellite in the class II Eb gene allows tracing of major histocompatibility complex evolution in mouse

A hallmark of major histocompatibility complex (MHC) genes is their extraordinarily high level of polymorphism. Polymorphic residues on MHC molecules determine which peptide ligands they bind and present to effector T lymphocytes. Although the genetic mechanisms responsible for MHC polymorphism have been delineated, the timetable and the pathway of their diversification remain unclear. To trace MHC evolution, we have characterized a highly polymorphic microsatellite containing tandem repeats (TRs) of two tetranucleotide units, TGGA and GGCA, located at the 3' end of the second intron in the class II Eb gene of mouse. On the basis of length as well as sequence variations, 11 TR alleles were defined in 55 inbred mouse strains, which included MHC recombinant haplotypes and haplotypes derived from different subspecies of mouse. In this extensive sampling, a striking concordance was observed between the serologically identified class II proteins and the associated TR alleles. Examination of several strains carrying the same MHC haplotypes as well as strains carrying recombinant MHC haplotypes indicates that TR alleles are extremely stable. These observations suggest that TR polymorphism predates the separation of various subspecies of mouse. On the basis of sequence divergence, a genealogical tree has been constructed to depict evolution of the different TR alleles. Finally, evidence is presented that suggests this microsatellite polymorphism is generated by slipped-strand mispairing during DNA replication.     

Minors held by majors: the H13 minor histocompatibility locus defined as a peptide/MHC class I complex

The products of minor histocompatibility (H) loci are serious barriers to tissue transplantation even among major histocompatibility complex (MHC) identical individuals, frequently causing chronic graft rejection and graft versus host disease. Over 50 minor H loci map to mouse autosomal chromosomes but none are known at the molecular level. By expression cloning, we identified the H13 locus, a classical minor H locus first detected 30 years ago by the trait of graft rejection. The H13a allele is located on chromosome 2 and encodes a novel protein that yields the rare naturally processed nonapeptide SSVVGVWYL (SVL9) for presentation by the Db MHC class I molecule. The SVL9 peptide binds Db MHC despite the absence of the consensus binding motif, and a conservative methyl group substitution (Valine 4 <--> Isoleucine) explains why reciprocal T cell responses are elicited in H13a and H13b congenic strains.     

The conclusions from an analysis of 9 approaches to determining the value of KT/Vur

OBJECTIVES: The polymerase chain reaction (PCR)-based method was used to obtain and sequence three H-2K and three H-2D mouse complementary DNAs (cDNA) of class I major histocompatibility complex (MHC) molecules. METHODS: Messenger RNA was isolated from Conconavalin A-activated splenocytes of C57BL/10 (H-2b), C3H (H-2k), and Balb/c (H-2d) mice. We designed H-2K- and H-2D-specific primers as well as a common downstream primer based on previously published mouse class I MHC sequences. Using the PCR method and selective primers we isolated and sequenced H-2Kb and H-2Db cDNAs of C57BL/10, H-2Kk and H-2k cDNAs of C3H, as well as H-2Kd and H-2Dd cDNAs of Balb/c strains. RESULTS: Analysis of the nucleotide sequences documented similarity between our three H-2K cDNA sequences and all mouse MHC class I sequences available in the GenBank. Similarly, our three H-2D sequences were homologous with all mouse class I MHC sequences deposited in the GenBank. Our H-2K and H-2D sequences were also identical to numerous published sequences. CONCLUSIONS: Using these mouse cDNAs, we plan to determine the localization of polymorphic in vivo immunogenic amino acids in class I MHC H-2K and H-2D alloantigens.     

Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen

Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various inbred strains of mice to recombinant BCG expressing beta-galactosidase. These experiments demonstrated that BALB/c mice developed strong antibody responses against BCG expressing beta-galactosidase under the control of two different promoters. In contrast, C57BL/6, C3H, and CBA mice produced high anti-beta-galactosidase antibody titers only when immunized with recombinant BCG expressing beta-galactosidase under the control of the pblaF* promoter, which induced the production of high levels of this antigen. This difference in mouse responsiveness to recombinant BCG was not due to innate resistance to BCG infection, since similar immune responses were induced in Ity(r) and Ity(s) congenic strains of mice. In contrast, the analysis of anti-beta-galactosidase antibody responses of H-2 congenic mice in two different genetic backgrounds demonstrated that H-2 genes are involved in the immune responsiveness to beta-galactosidase delivered by recombinant BCG. Together, these results demonstrate that immune responses to an antigen delivered by recombinant BCG are under complex genetic influences which could play a crucial role in the efficiency of future recombinant BCG vaccines.     

Genetic control of immunity to Heligmosomoides polygyrus: presentation of promiscuous antigens to parasite-specific T cell hybridomas

The role of MHC class II in the presentation of Heligmosomoides polygyrus antigens has been investigated, using a number of T cell hybridomas produced in A and E positive and negative mice. By using fixed and irradiated antigen presenting cells (APC), further evidence has emerged, to support earlier data, that there can be differential processing requirements during the presentation of H. polygyrus antigens by A and E molecules. In concordance with these earlier observations, this work provides further evidence than individual T cells can respond to antigen when presented by more than one MHC molecule. Previously, this evidence has been restricted to individual MHC molecules of the same haplotype, but these data show that H. polygyrus produces antigens which can be presented by both syngeneic and allogeneic MHC molecules. These antigens do not appear to be synonymous with the previously described H. polygyrus superantigen, as presentation is restricted to specific MHC haplotypes. It is proposed that H. polygyrus may produce these antigenic molecules as part of its strategy to manipulate the host immune system.     

Expression of different members of the Ly-49 gene family defines distinct natural killer cell subsets and cell adhesion properties

The murine Ly-49 antigen belongs to a family of type II transmembrane molecules containing lectin-like domains. The original member of this family, Ly-49A, has been demonstrated to be expressed by a subpopulation of natural killer (NK) cells, bind certain class I major histocompatibility complexes (MHC), and act as a negative regulator of lytic activity. The expression patterns and functional activities of the other Ly-49s, however, is unknown. We extended the study of this family by isolating cDNAs encoding two new Ly-49 molecules. The reactivity of these and previously identified Ly-49 molecules with NK antibodies was tested in a COS cell expression system. YE1/32 and YE1/48 bound Ly-49A specifically, and 5E6 reacted only with Ly-49C. Three-color flow cytometric analysis demonstrated Ly-49A and Ly-49C expression defines complex, but distinct subsets within NK1.1+ cells. Some NK1.1-CD3+ as well as NK1.1-CD3- cells expressing Ly-49A or C were also detected. Analysis of MHC congenic strains of mice demonstrated that YE1/32+ and YE1/48+ NK cells are not deleted, as has been shown with the Ly-49A mAb A1. Furthermore, COS cells transfected with Ly-49A bound H-2d and H-2k cell lines, whereas Ly-49C transfectants bound H-2d, H-2k, H-2b, and H-2s. The antibodies 5E6 and 34-1-2S (anti-class I MHC) inhibited the binding of Ly-49C to an H-2s cell line. These results imply that the NK cell antigens Ly-49A and C bind to different repertoires of class I MHC molecules.     

Three cDNA clones encoding mouse transplantation antigens: homology to immunoglobulin genes

We constructed cDNA libraries from poly(A)+ RNA isolated from cell lines of two different inbred strains of mice, and screened the libraries with a cDNA clone encoding a human transplantation antigen. Three cDNA clones were identified, sequenced and found to encode amino acid sequences highly homologous to portions of a known mouse transplantation antigen. Comparison of the cDNA sequences of mouse transplantation antigens with the constant region domains of the mouse immunoglobulin mu gene reveals a striking homology, which suggests that the two genes share a common ancestor. Antibody genes undergo DNA rearrangement during B cell differentiation that are correlated with their expression. In contrast, DNA blots with these cDNA probes suggest that the genes for the transplantation antigens are not rearranged in the genomes of liver or embryo cells, which express these antigens, as compared with sperm cells, which do not express these antigens. In Bam Hl-digested liver DNAs from different inbred strains of mice, 10-15 bands of hybridization were found. Accordingly, the genes encoding the transplantation antigens appear to constitute a multigene family with similar gene numbers in different mice.     

Role of class-II major histocompatibility complex (MHC)-antigen-positive donor leukocytes in transfusion-induced alloimmunization to donor class-I MHC antigens

It has been shown that peripheral-blood mononuclear leukocytes (MNL) are responsible for transfusion-induced alloimmunization to donor major histocompatability complex (MHC) antigens. However, it is not known which subset of MNL is responsible for this immune response. Because elimination of class-II MHC antigen-positive passenger leukocytes effectively prolongs the survival of allografts, it has been hypothesized that class-II positive MNL are responsible for immunizing transfusion recipients to donor MHC antigens. To test this hypothesis, two different approaches were used. First, we compared the alloantigenicity of BALB/c mice (H-2(d)) peripheral blood MNL before and after depletion of class-II positive cells. CBA mice (H-2(k)) were used as transfusion recipients. Antibody development to donor class-I H-2 antigens was determined by flow cytometry and enzyme-linked immunoassay. After four weekly transfusions of MNL depleted for class-II positive cells, only 25% of recipient mice developed antibodies to donor H-2(d) antigens. In contrast, all mice transfused with control MNL became immunized. Second, we studied the alloantigenicity of peripheral MNL from C57BL/6 mice (H-2(b)) with homozygous deficiency of class-II MHC molecules in H-2 disparate recipient mice. After transfusions with class-II MHC molecule-deficient MNL, 0% of BALB/c, 40% of C57BR, and 25% of CBA-recipient mice developed antibodies to donor H-2(b) antigen. All control recipient mice were immunized. The antibody activities of the controls were also higher than those in the treatment group who became immunized. Thus, our study shows that class-II MHC antigen-positive MNL play a significant role in transfusion-induced alloimmunization to donor class-I MHC antigens. The results also support the hypothesis that direct antigen presentation by donor class-II positive MNL to the immune system of transfusion recipients is critical for the initiation of humoral immune response to donor MHC antigens.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus. This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis.     

Genetic regulation of the expression of H-2-K.5 antigen of erythroid cells by H-2K end

Difference in the amounts of H-2K.5 antigens present on erythrocytes was observed, using quantitative absorption method, among several B 10 congenic strains. However, no such variation was seen on the lymphocytes and lung cells. The variability in the amount of this antigen on the erythrocyte surface was primarily dependent on the haplotype of the H-2K end in the B 10 recombinant strains examined. This suggests that the regulator of H-2 expression on erythrocytes is in this region. Further genetic analysis confirmed that this regulation functions in the cis-position. Finally, the H-2K.5 antigenic activity was expressed on the reticulocytes of all strains tested, but appeared lower in the type 2 group indicating that the regulation begins early in erythropoietic differentiation and eventually results in a complete loss of detectable activity.     

An indirect radioimmunoassay for thymus leukemia (TL) antigens

An indirect radioimmunassay for thymus leukemia (TL)2 antigens has been developed and its specificity documented. The assay makes use of anti-TL antibodies produced in congenic mice (A-Tla(b)) and radioiodinated purified rabbit anti-mouse IgG. Using this assay, differences can be detected in the amounts of antigen expressed on thymocytes of the three known phenotypes (TL.1,2,3;TL.2;TL-) of inbred mouse strains. Significant differences are also detected in comparison of the thymocytes from homozygous TL.1,2,3 mice (A-Tla(a)) and heterozyhotes from Tla(a) and Tla(b) parents. Optimum conditions for the assay have been established. Its ability to detect antigens on glutaraldehyde-fixed cells and the binding of noncytolytic antibodies on both viable and fixed cells are documented. The assay has also been used to quantitate the changes in TL antigen expression on cells incubated in anti-TL antisera under conditions of antigenic modulation.     

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.     

Thyroid hormone control of thermogenesis and energy balance

An extensive analysis was made of receptor specificity and gene usage in the neutralising antibody (mAb) and Class II-restricted T cell responses to influenza haemagglutinin (HA) following natural infection of MHC (H-2(k) or H-2(d)) congenic mice with X31 virus (H3N2 subtype). Despite the diversity of available antigenic sites on the HA1 subunit, there was striking immunodominance in the mAb response as deduced by sequencing the HA genes of escape mutants and the corresponding antibody H and L chain gene rearrangements. Similarly, Class II restricted T cell responses of individual donors focused on a single antigenic site, or immunodominant peptide; and PCR sequence analysis of T cell receptor (alpha beta) gene usage indicated that T cell memory was derived from a single progenitor cell. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation.     

Major histocompatibility complex (MHC) class I KbDb -/- deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb -/- mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although beta2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb -/- mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by beta2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb -/- mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb -/- mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb -/- animals also have natural killer cells that retain their cytotoxic potential.