Interactions between MHC-encoded products and cloned T-cells. I. Fine specificity of induction of proliferation and lysis

To study the interactions between T cells and class I MHC products, we developed in vitro a T-cell line reactive to H-2K^b stimulating cells and derived T-cell clones from it. Although the T-cell line could proliferate in the absence of exogeneous T-cell growth factors when stimulated with H-2K^b spleen cells, each of the derived T-cell clones required both H-2K^b stimulating cells and an external source of T-cell growth factor for its propagation. Each of the T-cell clones was also cytolysic for H-2K^b target cells. Such T-cell clones allowed the comparison of the antigenic requirements for proliferation and cytolysis. By using H-2K ^b mutant mice, we found that while the original anti-H-2K^b T-cell line reacted with each of the six mutants tested, the individual T-cell clones could be distinguished in terms of their reactivity pattern. Similar fine specificity patterns were found when H-2K ^b mutant cells were used as stimulating or target cells for any given T-cell clone. Each of the three monoclonal H-2K^b-specific antibodies reacting with different epitopes of the H-2K^b molecule totally inhibited H-2K^b-induced proliferation and lysis by the T-cell clones. Further blocking studies involved use of Fab antibody fragments and definition of their reactivity on cells from the H-2K ^b mutants. We concluded that: (1) blocking with a monoclonal antibody does not prove identity of alloantigens recognized by the T-cells and the antibody; (2) a monoclonal antibody could either block or not block H-2K^b-CTL interactions depending on structural variations of the H-2K^b molecule not affecting the CTL-H-2K^b functional interaction; (3) blocking one type of H-2K^b-T-cell interaction (induction of proliferation) always affects the other type (cytolysis).Abbreviations used in this paper MHC major histocompatibility complex - CTL cytotoxic - T lymphocytes - Th T helper cells - PMA 4-phorbol 12-myristate 13-acetate - Con A Concanavalin A - LPS E. coli lipopolysaccharide - SCA Con A stimulated rat spleen-cells supernatant - SBD B6 anti-DBA/2 mixed lymphocyte culture supernatant - TCGF T-cell growth factors - IL-2 interleukin 2 - mAb monoclonal antibody - FCS fetal calf serum - PBS phosphate buffered saline - C complement     

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2K^d, H-2D^d and H-2L^d molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2L^d molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NFκB. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism.

Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2′-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.     

Recognition of Db and Kb gene products by influenza-specific cytotoxic T cells

A series of 16 H-2^b-restricted, A influenza virus-specific cytotoxic T-cell clones are described and characterized. One is K^b restricted, the others D^b restricted. The factors governing K^b or D^b restriction patterns seen in the mixed populations from which clones are derived are investigated. The K^b-restricted clone does not recognize K^b mutant bm1 and influenza and all 15 D^b-restricted clones do not recognize D^b mutant bm14 and A influenza virus; these results are discussed in the light of findings in a variety of other viral systems. Representative K^b- and D^b-restricted clones were used to assess the functional properties of cloned cosmids containing either K^b or D^b genes expressed in transformed L-cells (κ haplotype). The expression products of both cosmids functioned efficiently as mutually exclusive restriction elements for A influenza virus recognition.     

Interaction between MHC-encoded products and cloned T cells

The interaction between class I major histocompatibility complex (MHC) products and T cells was studied using H-2K^b-specific alloreactive T-cell lines and clones obtained by repeated in vitro stimulation with allogeneic cells. Induction of proliferation of these T cells appeared to involve two signals: the H-2K^b alloantigen and interleukins. Immunopurified liposome-inserted H-2K^b, which stimulates specific secondary in vitro cytotoxic T lymphocyte (CTL) responses, could not replace cell-associated H-2K^b in the stimulation of these T-cell lines, even in the presence of feeder cells and interleukins. When T-cell lines were initiated in vitro and repeatedly stimulated with H-2K^b liposomes and feeder cells, it was possible to obtain T cells that could proliferate in response to H-2K^b liposomes in the presence of feeder cells and interleukin-2-containing supernatants or on H-2K ^b -expressing cells. Only stimulation with cells permitted maintenance of these T cells in culture for more than 12 weeks. Analyses of cell surface markers and of patterns of inhibition of proliferation by monoclonal antibodies (mAb) of T-cell lines induced in vitro with cell- or liposome-associated H-2K^b indicated that T-cell stimulation by class I antigen can occur in at least two ways. In the first, the H-2K^b-induced proliferation of Lyt-1^- Lyt-2⁺ T4^- T cells is inhibited by H-2K^b- and by Lyt-2-specific mAb, but not by Ia or T4-specific mAb. In the second, both Lyt-2⁺ and T4⁺ T cells are involved and the H-2K^b-induced proliferation is inhibited by H-2K^b- and Lyt-2-specific mAb and by Ia- and T4-specific mAb.Abbreviations used in this paper Ab antibody - mAb monoclonal antibody - C complement - i.p. intraperitoneally - PBS phosphate-buffered saline - PBS-B-N PBS containing bovine serum albumin and NaN₃ - CTL cytotoxic T lymphocyte - Th T helper cell - MHC major histocompatibility complex - PMA 4-phorbol 12-myristate 13-acetate - SCA concanavalin A-stimulated rat spleen cell supernatant - SC16 EL4 clone 16 supernatant - IL-1 interleukin-1 - IL-2 interleukin-2 (T-cell growth factor) - FCS fetal calf serum - H-2K^b-lip. H-2K^b inserted in liposomes - C. E. cell equivalents     

Fine specificity and T-cell receptor β-chain gene rearrangements of five H-2Db-specific cytotoxic T-cell clones

A panel of cytotoxic T lymphocyte clones that recognize H-2^b target cells has been established. Six different clones were distinguished according to the following criteria. First, the fine specificity of the clones was determined by testing proliferation and cytotoxicity on target cells of recombinant mice. Clone 221 recognized H-2K^b, and five other clones recognized H-2D^b. Clone 433 distinguished itself from the other five D^b-specific clones by cross-reacting with an antigen on H-2^k cells. Second, the presence of an idiotypic determinant as defined by the 3179 clone-specific monoclonal antibodies was investigated in cytotoxicity inhibition experiments. One of the D^b-specific clones, 653, was inhibited by these antibodies and was therefore clearly different from the other D^b-specific clones. The third criterion involved the rearrangement pattern of the DNA coding for the chain of the T-cell receptor. Southern blot analysis showed that each clone had a unique pattern. Interestingly, clone 653 , which expresses the same idiotypic determinant as clone 3F9, had deleted the C 1 gene cluster, whereas this gene is functionally expressed in clone 3179.Abbreviations used in this paper C constant gene segment - Con A concanavalin A - CTLs cytotoxic T lymphocytes - D diversity gene segment - ³H-dThd tritiated thymidine - J joining gene segment - kb kilobase pairs - LPS lipopolysaccharide - MHC major histocompatibility complex - MLC mixed lymphocyte culture - SDS sodium dodecyl sulfate - V ariable gene segment     

Nonequivalence of Classical MHC Class I Loci in Ability to Direct Effective Antiviral Immunity

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K^b gene are highly susceptible to persisting Theiler''s virus infection within the CNS and subsequent demyelination, mice expressing the D^b transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K^b but encoding the peptide binding domain of D^b, develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K^bα1α2D^b gene (low) and D^b (high) in the CNS of infected mice mirror expression levels of their endogenous H-2^q counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.     

Coreceptor function of CD4 in response to the MHC class I molecule

The specificity of the T-cell receptor (TCR) and its interaction with coreceptors play a crucial role in T-cell passing through developmental checkpoints and, eventually, determine the efficiency of adaptive immunity. The genes for the α and β chains of TCR were cloned from T-cell hybridoma 1D1, which was obtained by fusion of BWZ.36CD8α cells with CD8⁺ memory cells specific for the H-2K^b MHC class I molecule. Retroviral transduction of the 1D1 TCR genes and the CD4 and CD8 coreceptor genes was used to obtain 4G4 thymoma variants that exposed the CD3/TCR complex together with CD4, CD8, or both of the coreceptors on their surface. Although the main function of CD4 is to stabilize the interaction of TCR with MHC class II molecules, CD4 was found to mediate the activation of transfected cells via TCR specific for the H-2K^b MHC class I molecule. Moreover, CD4 proved to dominate over CD8, since the response of CD4⁺CD8⁺ transfectants was suppressed by antibodies against CD4 and the A^b MHC class II molecule but not to CD8. The response of CD4⁺ transfectants was not due to a cross-reaction of 1D1 TCR with MHC class II molecules, because the transfectants did not respond to splenocytes of H-2^b knockout mice, which were defective in the assembly of the MHC class I molecule/β2 microglobulin/peptide complex and did not expose the complex on cell surface. The domination was not due to sequestration of p56^lck kinase, since CD4 devoid of the kinase-binding site was functional in 4G4 thymoma cells. The results were used to explain some features of intrathymic cell selection and assumed to provide an experimental basis for developing new methods of anticancer gene therapy.     

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K ^d , the only H-2^d class I responder allele for NP restriction. H-2 ^d /H-2 ^b F₁ mice (C57BL × DBA/2) primed by influenza infection lyse only H-2^d target cells treated with peptide 147–161 while H-2^b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by D^b restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K ^d and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.     

Identification of the Cloned Gene for the Murine Transplantation Antigen H-2Kb by Hybridization with Synthetic Oligonucleotides

The H-2K^b gene is a member of the large major histocompatibility complex class I gene family. Since many members of this family cross-hybridize with class I cDNA probes, the cloned H-2K^b gene was identified by hybridization with specific oligonucleotide probes. This clone was definitively shown to encode the H-2K^b polypeptide by partial DNA sequencing and by serological and tryptic peptide analyses of the expressed product.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

The complete amino acid sequence of the murine transplantation antigen H-2Db as deduced by molecular cloning

A mouse cDNA library derived from the EL4 cell line (b-haplotype) was screened with a probe containing a small part of the H-2K^b coding region. One of the clones isolated, pH203, encodes a protein whose deduced amino acid sequence is identical with the known sequence of H-2D^b in 141 of 141 positions available for comparison. The clone, therefore, is believed to code for the H-2D^b transplantation antigen. The cDNA insert of pH203 contains the coding region for residues 82 through the carboxy-terminus of H-2D^b, and includes 476 nucleotides of the 3-untranslated sequence. Comparison between the H-2D^b cDNA clone and a previously isolated H-2K^b cDNA clone shows homologies of 83% and 91% at the amino acid and nucleotide levels, respectively. Analysis of DNA sequences at the 3-coding and untranslated regions suggests that the mRNAs of H-2K^b and H-2D^b are spliced differently at their 3-coding ends.     

H-2 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

Primary structure of the H-2Db alloantigen

The complete amino acid sequence of the CNBr fragment comprising residues 229–284 of the murine major histocompatibility complex antigen H-2D^b has been determined using radiochemical methodology. The sequence was determined by N-terminal sequence analysis of the intact CNBr fragment and by sequence determinations of peptides derived from this fragment by trypsin and staphylococcal V8 protease cleavage. In addition to the amino acid assignments for H-2D^b, it was possible to assign the linkage position of the third N-linked glycosyl unit to the asparagine at residue 256. Additional amino acid sequence assignments have also been made for three other CNBr fragments that span residues 99–138, 139–228, and 308–331 of the H-2D^b molecule. The total protein sequence information available (222 of 338 residues) agrees in every comparable position with the protein sequence derived from the cDNA clone (pH203) isolated by Reyes and co-workers (1982b), which strongly suggests that this clone encodes H-2D^b. Combination of the protein sequence with that deduced from the cDNA clone provides the complete H-2D^b protein sequence. Comparison of this sequence with other available protein sequence information for murine class I molecules has revealed protein sequences that may be unique to either K or D region molecules.Abbreviations used in this paper HPLC high performance liquid chromatography - V8 Staphylococcus aureus V8 protease - MHC major histocompatibility complex     

Immunoselection of structural H-2Kb variants: Use of cloned cytolytic T cells to select for loss of a CTL-defined allodeterminant

Functional studies concerning the unique interaction between class I H-2 allodeterminants and cytolytic T lymphocyte (CTL) antigen receptors have benefitted from the development of H-2K^b mutant mouse strains and somatic H-2 variants selected with monoclonal antibody. Here, we describe the development of a novel approach to immunoselection of somatic H-2K^b variants employing a K^b-specific CTL clone as the negative selective agent. The rationale for this method is that the use of an alloreactive CTL clone as the selective agent should enable us to detect the emergence of structural K^b variants based on their loss of the relevant CTL-defined allodeterminant. Thus, these structural variants are well suited to an in-depth analysis of the functional relationship between H-2 antigens and receptor recognition by CTL. Using this approach, we successfully isolated two types of structural K^b variants, as well as numerous Kb-loss variants. The functional studies described in this paper indicate that these structural variants exhibit alterations in expression of both CTL-defined and serologically defined H-2K^b allodeterminants. The structural characterization of such variants should enable us to identify the precise amino acid residues responsible for the creation of the relevant CTL-defined K^b allodeterminants.     

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

Human MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K^b. The structure of MUC1-9 in complex with H-2K^b has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K^b complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide’s central region, partially characterized as a β-turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K^b.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

An H-2K gene of the t w32mutant at the T/t complex is a close parent of an H-2K qgene

Two recombinant mice have been recovered from the progeny of Ttf/t ^w32+ animals. They have lost the t^w32 lethality factor(s) and gained tufted, presumably from the T chromosome. Southern blot analysis of class I genes of these two new partial t ^PA027 and t ^PA286 haplotypes indicates that they have retained at least part of the major histocompatibility complex of the t ^w32 chromosome (H-2 haplotype H-2 ^w28). We have prepared a phage library of Eco RI-digested DNA from homozygous t ^PA027 animals. Upon screening the library with a cDNA probe specific for H-2K genes, we isolated a class I gene displaying all of the distinctive features of a genuine H-2K gene, and which could thus be defined as an H-2K ^w28 gene. The H-2K ^w28 gene is 92–95% homologous to H-2K ^band H-2K ^dgenes and differs significantly from the other class I genes sequenced so far. Homology with the H-2K ^bsequence reaches nearly 100% in the 3 part of the H-2K ^w28 gene. Moreover, the homology with an H-2K ^qcDNA sequence reaches 99.8%. Several hypotheses can account for the near identity of H-2K ^b, H-2K ^q,and H-2K ^w28 gene sequences: either recombination between H-2 ^w28 and H-2 ^band H-2 ^qsequences occurred before or at the.time the strain was established, or the class I genes of the t ^w32 chromosome and the H-2 ^band H-2 ^qgenes found in inbred strains of mice have separated from each other rather recently.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.