Anna Rocca

Localization of the conformational alteration of MHC molecules induced by the association of mouse class I heavy chain with a xenogeneic β2-microglobulin

Changes in the antigenicity of major histocompatibility complex (MHC) class I molecules resulting from the association of bovine beta 2-microglobulin (beta 2-m) with mouse class I heavy chains were investigated. Mice (H-2b) were immunized with syngeneic Concanavalin A (Con A) blasts induced in the presence of fetal calf serum (FCS) in conditions allowing exchange between mouse and bovine beta 2-microglobulin (beta 2-m). Spleen cells from hyperimmunized mice were fused with myeloma cells and two monoclonal antibodies which required for their reactivity the presence of FCS have been further studied. One of them (CAB 297) recognized a determinant of bovine beta 2-m which is present on free molecules in solution as well as when they are associated with either mouse or bovine class I heavy chains. In contrast, the second monoclonal antibody (CBB 70) did not react with free bovine beta 2-m molecules, nor with beta 2-m associated with bovine class I heavy chains. It did react with cells of some H-2 haplotypes (b, f, p and r) but only when their class I heavy chains are associated with bovine or with human beta 2-m. Therefore, expression of the CBB 70 defined antigenic determinant requires both xenogeneic beta 2-m and class I heavy chain of a given H-2 molecule. In order to precisely localize the antigenic determinant defined by this monoclonal antibody and therefore the region altered by the association of class I heavy chain with xenogeneic beta 2-m, we made use of exon shuffled class I molecules. The results indicate that changes induced by the association of bovine beta 2-m with H-2 class I heavy chain affect the conformation of the alpha 2 domain. These studies illustrate that MHC class I molecules exhibit a considerable conformational flexibility which could influence their ability to bind and present various peptides to the T-cell receptor.

Analysis of primary HLA-specific cytotoxic T cell response in graft-draining lymph nodes : a transgenic mouse model for in vivo recognition of human MHC antigens

In order to characterize primary anti-HLA cytotoxic T cells and especially those involved in graft rejection, we have utilized a transgenic mouse model. Mice (nontransgenic and HLA-transgenic) were grafted with spleen cells originating from H-2-matched transgenic mice expressing HLA-B27 molecules, and cells from graft-draining lymph nodes were tested in CML assay to investigate the primary in vivo induced CTL responses. The results showed that HLA-B27 molecules were able to raise strong primary xenogeneic CTL responses. Results from split-well analysis indicated that although recognition of HLA-B27 by primary CTL induced in nontransgenic recipients is predominantly unrestricted by H-2, a small fraction (ranging from 2% to 27%) of the primary in vivo induced CTL is able to recognize HLA-B27 in an H-2-restricted manner. HLA-specific H-2-restricted CTL had never so far been demonstrated in the primary T cell response. Thus the protocol used in our study for the generation of a primary CTL response seems to provide not only a more appropriate representation of cytotoxic T cells sensitized by a graft, but also to be a more sensitive approach than the usually used in vitro mixed lymphocyte culture.

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.

Immunization with Fibroblasts Transfected with a Cloned Retroviral DNA Induces H-2 Specific Antibodies In Syngeneic Recipients

Alloimmune sera induced by immunization between mice from H-2 congeneic strains are a potent tool for the detection of an extensive polymorphism of H-2 class I and class II molecules. In recent years a new category of H-2 class I specific antibodies has been described. These antibodies have been observed in mice prior to any intentional alloimmunization and are considered to be naturally occurring class I specific antibodies (Ivanyi et al. 1982; Lynes et al. 1984; Cerný-Provaznik et al. 1985 a, b, 1986). In other experiments H-2 specific antibodies have been induced by immunization with syngeneic cells. Various approaches have been applied: injection of syngeneic cells (Ivanyi et al. 1980; Capkova et al. 1983; Takahashi et al. 1987), syngeneic concanavalin A-induced blasts (Mierau and Cramer 1984), syngeneic tumor cells (Schmidt and Alonzo 1983; Philipps et al. 1985), and syngeneic Sendai virus-coated cells (Opolski et al. 1986; Kievits et al. 1987 a, b). The specificities of these H-2 class I specific antibodies were variable and ranged from narrow to widely cross-reacting serotypes. Autoreactivity was occasionally present but the antibodies were never exclusively or preferentially directed against self-H-2 antigens. The mechanism responsible for their induction is unknown.

Real and apparent H-2-specific antibodies induced by syngeneic immunization.

In an attempt to evaluate the consequences of the association of mouse class I heavy chains with xenogeneic (bovine) β2-m, we investigated the antibody response of mice (H-2 b , H-2 p , H-2 q , and H-2 s ) immunized with syngeneic Con A blasts cultured in the presence of fetal calf serum (FCS) in conditions allowing exchange between mouse and bovine β2-m. Almost all mice produced lymphocytotoxic antibodies. Such antibodies were not found in sera of mice innjected with FCS only. Spleen cells from hyperimmunized mice were fused with myeloma cells, and monoclonal antibodies (mAb) were isolated

Involvement of the H-2 Complex in Steroid Hormone Metabolism

We investigated DNA from mice with different H-2 haplotypes (b. d., f, j, k, p, g, r, s, u, v,). DNA was digested with five restriction enzymes (Eco RI, Taq I, Pvu II, Hind III, and Bgl II) and probed with human 21-OHase cDNA clone. Combined restriction fragment length polymorphism identify six different patterns of endonuclease fragment polymorphism of the murine 21-OHase region. Furthermore, we have investigated the serum level of two steroid hormones (progesterone and corticosterone) in the metabolic pathway involving the 21-OHase. Serum progesterone and corticosterone concentrations exhibit variations according to the H-2 haplotypes of the mice, suggesting that genetic factor(s) identical to or closely linked to the H-2 complex is involved in the control of their metabolism.

H-2 INFLUENCE ON THE PRODUCTION OF REAL BUT NOT OF APPARENT H-2-SPECIFIC ANTIBODIES INDUCED BY THE ASSOCIATION OF SYNGENEIC CLASS I HEAVY CHAINS WITH BOVINE β2-MICROGLOBULIN

Immunogenic properties of class I molecules resulting from the association of mouse class I heavy chains with a xenogeneic β2‐microglobulin (β2‐m) were investigated by studying the antibody response of mice of injections to syngeneic Con A lymphoblasts, induced in conditions allowing the replacement of endogenous β2‐m by exogenously added bovine (β2‐m provided by fetal calf serum (FCS‐Con A blasts). Lymphocyto‐toxic antibodies were regularly produced and according to their specificities they could be divided into two types: antibodies whose reactivity was (1) dependent on and (2) independent of the presence of bovine β2‐m on target cells. Although both types displayed an H‐2 dependent polymorphic reaction pattern, only antibodies recognizing class I molecules without bovine β2‐m can be considered as real H‐2‐specific antibodies. The others are only apparent H‐2‐specific antibodies: their polymorphic reaction pattern is dependent both on the presence of bovine β2‐m on the surface of target cells and on their H‐2 haplotype. A comparison of the antibody response of mice with various H‐2 haplotypes to injections of syngeneic FCS‐Con A blasts showed no significant difference in the induction of bovine β2‐m‐dependent antibodies (apparent H‐2‐specific) among the mice from all strains tested (H‐2b, H‐2p, H‐2q, and H‐2S). Unexpectedly, for most strains more than 60% of the immunized mice produced also β2‐m‐independent antibodies (real H‐2‐specific), with the exception of H‐2q mice, in which only 30% of sera were positive. The real H‐2‐specific antibody response is of two types: some mice (H‐2P and H‐2S) produced antibodies only reactive with allogeneic target cells whereas others (H‐2b and H‐2q) produced in addition antibodies that were reactive with syngeneic cells. Thus H‐2 appears to play an important role in the induction and specificity of the lymphocytotoxic H‐2‐specific antibodies induced upon immunization with cells expressing syngeneic class I heavy chains associated with bovine β2‐m.

Xenogeneic monoclonal HLA-specific antibodies defining mouse lymphocyte determinants encoded by the H-2 complex

H-2 antigenic determinants were defined by the use of polyclonal antisera or monoclonal antibodies coming from alloimmunized mice. When H-2-specific alloantisera were tested against human cells, a considerable amount of cross-reactivity was observed (Iva~kov~ et al. 1972, 1975). The intriguing fact is that such alloimmune Mhcspecific antibodies detect polymorphism of Mhc molecules in various species (reviewed in Iv~nyi 1981, Pla and Iv~nyi 1986). Another aspect of Mhc interspecies crossreaction is that not only allogeneic but also xenogeneic Mhc-specific antibodies detect Mhc polymorphism in third-party species (Brodsky et al. 1981, Chardon et al. 1983, Spooner and Ferrone 1984). Phylogenetically distant species share not only a general homology for the Mhc molecules but also some aspects of intraspecies polymorphism. Xenogeneic antibodies detect some kind of polymorphism when tested on a panel of targets of the immunized species itself. H-2 b mice immunized with human cells produced H-2Kor H-2D-specific (private or public) antibodies (Iv~inyi 1981). Serum antibodies against polymorphic mouse class I determinants were induced by immunization of A.TH mice with lymphocytes of chicken, frog, and fish (Shinohara et al. 1981). McMaster and Williams (1979) examined BALB/c rat class H-specific monoclonal antibodies against a panel of mouse spleen cells and found a polymorphic class II-specific reaction pattern. An A.TH human class II-specific monoclohal antibody detected an epitope related to, but not identical with, that defined by Ia.7-specific allogeneic monoclonal antibodies, while on human cells a monomorphic pattern of reactivity was observed (Falk et al. 1983). Thus, the intriguing nature of Mhc cross-reactions has emerged from serological studies. There are almost no similar data in other polymorphic systems. Immunoglobulin molecules are also homologous, but

Immunization with Fibroblasts Expressing Human β 2 -Microglobulin Induces H-2 Specific Antibodies in Syngeneic Recipients

It is now generally accepted that the expression of some antigenic determinants of major histocompatibility complex (MHC) class I molecules results from a complex interaction between a heavy chain, encoded by highly polymorphic loci within the MHC, and the β 2-microglobulin (β 2-m) encoded by a rather monomorphic non-MHC locus. In an attempt to evaluate the structural consequences of the association of murine class I heavy chains with xenogeneic β 2-m, we investigated the antibody response of the mice injected with syngeneic fibroblasts expressing human β 2-m.