NK and CTL recognition of a single chain H-2Dd molecule: distinct sites of H-2Dd interact with NK and TCR.

We generated transgenic mice expressing a single-chain beta2-microglobulin (beta2m)-H-2Dd. The cell-surface beta2m-H-2Dd molecule was expressed on a beta2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.

Rigidification of the alpha2 helix of an MHC class I molecule by a valine to proline mutation in position 165 does not prevent peptide-specific antigen presentation.

Although classical MHC class I glycoproteins bind peptide Ags for display at the cell surface, some MHC class I-related molecules such as the neonatal Fc receptor (FcRn) execute their function without binding peptide ligands. The three-dimensional structure of the FcRn suggested that a substitution of the conserved valine at position 165 of the alpha2 helix by proline contributed to a kink in the position of this helix relative to the alpha1 helix, and resulted in closing of the potential peptide-binding cleft. To test the contribution of proline 165 to the occlusion of the cleft and the binding of potential antigenic peptides, we introduced this mutation into the classical murine MHC class I molecule, H-2Dd, and characterized the ability of such a mutant to present peptide Ags to either a peptide-specific, H-2Dd-restricted T cell hybridoma (B4.2.3), or an allospecific, peptide-dependent, T cell hybridoma (3DT52.5.8). We show that the V165P mutation, expressed at the cell surface either in H-2Dd or in a single chain membrane version of H-2Dd, fails to eliminate recognition of the peptide/MHC complexes by two different T cells. Evaluation of a panel of synthetic substituted peptides suggests that subtle differences in the fine specificity of presentation can be discerned. Thus, the proline substitution at position 165 of FcRn and some other class I-like molecules is not the sole cause of the lack of peptide presentation.

Multiple point mutations in an endogenous retroviral gene confer high immunogenicity on a drug-treated murine tumor.

Exposure in vivo of murine L5178Y lymphoma cells to cytoreductive triazene derivatives leads to the generation of immunogenic variant lines expressing new transplantation Ags recognized by CTL. In one such clonal variant (clone D), at least one subset of T cell neoepitopes are provided by proteins previously shown by serology to be products of endogenous retroviral env sequences. We report here on characterization of PCR-amplified gp70 env genes in clone D. Relative to known gp70 sequences in parental cells and in current databases, one gp70 sequence presented four distinct nucleotide changes, two of which were apparently unique to clone D DNA and cDNA upon differential hybridization analysis. Transfection experiments with the entire gp70 gene or subgenic fragments encompassing a single putative mutation showed that products of the mutated env gene or fragments may confer immunogenicity in vivo and susceptibility in vitro to lysis by clone D-primed, H-2Kd- or H-2Ld-restricted CTL. By skin test assay of mice primed with either clone D or three mutated synthetic peptides, evidence was obtained that amino acid substitutions at the relevant positions of the gp70 protein may produce immunogenic T cell epitopes and that these epitopes are expressed in vivo by clone D. These data point to the role of mutated retroviral tumor peptides as rejection Ags in histocompatible hosts.

Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active beta 2-microglobulin domain.

As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2Dd class I MHC molecule (SC beta Ddm) which has beta 2-microglobulin (beta 2m) covalently linked to the amino terminus of a full-length H-2Dd heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2Dd-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SC beta Ddm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27 degrees or 37 degrees C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SC beta Ddm can be stably expressed in beta 2m-negative cells, showing that the single-chain molecule uses its own beta 2m domain to achieve correct folding and surface expression. Furthermore, the beta 2m domain of SC beta Ddm, unlike transfected free beta 2m, does not rescue surface expression of endogenous class I MHC in the beta 2m-negative cells. This strict cis activity of the beta 2m domain of SC beta Ddm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.

A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines.

Identification and immunogenic properties of an 80-kDa surface antigen on a drug-treated tumor variant: relationship to MuLV gp70.

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of L5178Y murine lymphoma cells with triazene derivatives. Most of these variants express new transplantation antigens which are not present on the original L5178Y tumor cells. In this study, a polyclonal syngeneic antiserum raised to one such variant (L5178Y/DTIC) was employed in immunoprecipitation studies of cell surface and metabolically labeled L5178Y/DTIC cells. One- and two-dimensional electrophoretic analyses of the immunoprecipitates detected a surface antigen of approximately 80 kDa. Additionally, a 45-kDa component was detected in the lysate of [35S]methionine-labeled cells. Anti-xenotropic MuLV gp70 serum precipitated material whose electrophoretic pattern was similar to that of the 80-kDa surface antigen. Sequential immunoprecipitation analysis revealed that the molecules reactive with the variant-specific antiserum were removed by the anti-xenotropic gp70 antibodies, whereas immunodepletion was only partial when the cell extract was first treated with the variant-specific antibodies. After Western blotting, the 80- and 45-kDa antigens precipitated by the variant-specific antibodies were injected intrasplenically into recipient mice. Only the animals sensitized with the 80-kDa antigen developed specific immunity to L5178Y/DTIC cells in that they displayed an increased frequency in CTL precursors (CTLp) to the variant cells. Sera from mice sensitized to the 80-kDa protein specifically inhibited the development of a primary CTL response to L5178Y/DTIC cells.

Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro.

The end-stage maturation of neutrophilic granulocyte precursor cells isolated from normal human bone marrow by Ficoll density centrifugation was studied in a liquid culture assay system used previously to study the maturation of guinea pig granulocyte precursors. Dialyzed normal human serum induced end-stage morphological maturation of human granulocyte precursors and this induction was proportional to a serum level of up to 5.0% in the assay medium. At serum concentrations greater than 5.0% a pronounced inhibition of maturation was observed. Passage of serum through a DEAE-Fractogel 650S column equilibrated with 0.01 M phosphate buffer (pH 7.0) resulted in the binding of the end-stage granulocyte maturation factor to the column. The activity eluted from the column in a fraction containing 17% of the starting serum protein that was inhibitor-free and was also capable of inducing the appearance of granulocyte alkaline phosphatase, a specific biochemical marker for granulocyte end-stage maturation. GMF is most likely a protein since it was destroyed by protease digestion. The data also indicate that neither purified human transferrin nor human recombinant granulocyte colony-stimulating factor can substitute for human serum GMF as a granulocyte end-stage maturation factor in this assay system. It was observed, however, that purified human transferrin greatly potentiated the effect of GMF suggesting that transferrin plays a supporting role in the end-stage maturation of human granulocytes in vitro. To our knowledge the evidence presented here indicates for the first time the existence of a neutrophilic granulocyte end-stage maturation factor in normal human serum.

Cell-mediated immunity to chemically xenogenized tumors--IV. Production of lymphokine activity by, and in response to, highly immunogenic cells.

To determine whether a novel pattern of lymphokine production might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a panel of murine tumors xenogenized by DTIC for production of soluble factors with lymphokine-like activity and induction of lymphokine release from naïve or specifically sensitized lymphocytes. In the L5178Y tumor system, a majority of xenogenized but not parental clones produced an IL-1-like factor, and this was associated, as a rule, with class II antigen expression and antigen-presenting ability. However, no such properties were exhibited by the xenogenized variants of P815 and L1210Ha cells, which nevertheless occasionally expressed other lymphokine (GM-CSF, IL-3) activities. On examining the ability of xenogenized and parental tumors to cause release of IL-1, IL-2, IL-3, IFN-gamma, TNF/LT and GM-CSF from T-cells, we found, as a rule, an increased lymphokine production when lymphocytes primed in vivo to a xenogenized tumor were restimulated in vitro with the same or parental cells.

Cell-mediated immunity to chemically xenogenized tumors. II. Evidence for accessory function and self-antigen presentation by a highly immunogenic tumor variant.

To determine whether antigen-presenting ability might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a murine lymphoma line xenogenized by a triazene derivative for expression of Ia antigens, ability to present soluble antigen in vitro, and production of factor(s) active in a mouse thymocyte assay. Results showed that Ia antigens, absent on nonimmunogenic parental L5178Y cells, were expressed on a xenogenized, highly immunogenic tumor variant (clone D), as detected by immunofluorescence. While the ability of parental cells to stimulate lymphocyte proliferation in vitro was lost on removal of Ia+ cells from the responder population, considerable augmentation of reactivity was observed upon depletion of Ia+ cells from the population of splenocytes responding to the xenogenized cells. Under these conditions, stimulation was blocked by anti-Ia antibodies, or an anti-L3T4 reagent or antibodies to the novel antigenic determinants induced by xenogenization. In addition, no stimulating activity was observed following exposure of clone D cells to glutaraldehyde or lysosomotropic agents such as chloroquine and ammonia. When the ability of clone D cells to present ovalbumin in vitro was assayed, it was found that the xenogenized cells could present the soluble antigen to specifically primed lymphocytes. Moreover, clone D cells could substitute for splenic adherent cells in the proliferative reaction of splenocytes to concanavalin A. Finally, when the supernate from clone D-cell culture pulsed with phorbol myristic acetate was tested in a mouse thymocyte assay, considerable IL-1-like activity was disclosed.

Cell-mediated immunity to chemically xenogenized tumors--III. Generation of monoclonal antibodies interfering with reactivity to novel antigens.

To develop monoclonal antibodies (MAbs) recognizing drug-mediated tumor antigens on a chemically xenogenized murine lymphoma, hybridomas were constructed with splenocytes from histocompatible mice hyperimmunized with L5178Y cells antigenically altered by triazene treatment in vivo (clone D, derived from a polyclonal L5178Y/DTIC subline). Screening of supernatants with parental and xenogenized cells showed that nine MAbs displayed exclusive or preferential reactivity with clone D cells as detected by immunofluorescence, and failed, as a rule, to bind normal or unrelated malignant cells of the same or different haplotype. Moreover, no reactivity was displayed to the triazene-xenogenized variants of antigenically unrelated tumors. All nine MAbs, however, were capable of binding a panel of L5178Y/DTIC clones in addition to clone D. When the ability of these antibodies to interfere with the development of cell-mediated immunity to clone D cells in vitro was tested, it was found that the proliferative reaction and generation of cytolytic activity by syngeneic lymphocytes were inhibited by addition of several MAbs to the tumor--lymphocyte co-cultures.

Cell-mediated immunity to chemically xenogenized tumors. I. Inhibition by specific antisera and H-2 association of the novel antigens.

T cell-mediated proliferative and cytotoxic responses occur in vitro to syngeneic tumor cells antigenically altered by mutagen treatment. One such xenogenized variant of the murine L5178Y lymphoma elicits IgG antibodies reactive with determinants on variant cells that are not expressed at detectable levels on parental or normal cells of the same H-2d haplotype and are also unrelated to public specificities of H-2b or H-2k histocompatibility antigens. In the present study we investigated the effect of those antibodies on development of cell-mediated responses in vitro to the xenogenized cells used for induction of the humoral response. The proliferative reaction, generation of cytolytic activity and target cell lysis were all inhibited by the anti-xenogenized tumor immune serum, whereas the corresponding reactions to the parental cells by syngeneic or allogeneic effector lymphocytes were not. In order to investigate the possible H-2 association of T cell-mediated responses to xenogenized cells, we also examined the effect on those reactions of antibodies specific for Class I or Class II products of the H-2d complex. The results obtained suggested a role for I-Ad molecules in the T cell proliferative response to the xenogenized cells, and also indicated a preferential association of the cytotoxic response with H-2Kd determinants.

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2.

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.

Transferrin induces maturation of neutrophil granulocyte precursors in vitro.

Previous studies showed that dialyzed serum from guinea pigs contained a factor which induces guinea pig granulocyte precursors to form mature neutrophil granulocytes in vitro. We show here that this maturation inducing activity of serum is associated primarily with transferrin. This activity is not species specific since both guinea pig and human transferrin serve equally well in this capacity. These findings raise the possibility that transferrin could serve as a physiological regulator of granulocyte maturation.

Search for class II major histocompatibility complex molecular involvement in the response of Lyt-2+ cytotoxic T lymphocyte precursors to alloantigen.

A possible requirement for class II major histocompatibility complex (Ia) molecules in the initial activation of cytotoxic T lymphocyte precursors (CTLp) for allocytotoxic responses was investigated. To avoid possible interaction with other alloreactive cell types, a highly purified population of Lyt-2+ splenocytes was used as a source of CTLp. In the light of preliminary results indicating that Lyt-2+ CTLp, even in the presence of interleukin 2 (IL2), could best be triggered into mature CTL in vitro by cells known to be Ia+, we examined whether an interaction of CTLp with Ia antigens (either on syngeneic accessory cells or on allogeneic stimulators) played a role in the development of allocytotoxicity. Results from experiments done with C57BL/6 Lyt-2+ splenocytes co-cultured with P815 stimulator cells and IL 2 showed that the early activation of CTLp was independent of Ia+ syngeneic accessory cells: (a) flow microfluorometry analysis of the responder population at the beginning or after 1 or 3 days of co-culture did not reveal the presence of Ia+ cells; (b) procedures for removal of residual Ia+ cells or of dendritic cells from the responder population before co-culture did not affect the development of cytotoxicity; (c) co-culture with monoclonal antibodies against syngeneic Iab antigens did not inhibit the CTLp activation. By comparing an Ia+ P815 tumor line with its Ia- clone as allogeneic stimulator cells, it was found that the CTLp activation was also independent of Ia alloantigen on the stimulator cells. The response against both the Ia+ and the Ia- stimulator cell types was not inhibited by monoclonal anti-L3T4 present in the co-culture, indicating that these responses were not affected by residual L3T4 helper cells.

Humoral response against murine lymphoma cells xenogenized by drug treatment in vivo.

Treatment of murine lymphomas with triazene derivatives may lead to the appearance of novel drug-mediated tumor antigens, a phenomenon known as chemical xenogenization. Such antigens, which are capable of eliciting specific transplantation resistance in histocompatible mice, have been previously detected by in vivo and in vitro cell-mediated immune responses. In the present report we address the question of the humoral antibody response to a chemically xenogenized lymphoma. Histocompatible mice were given several injections of live cells of the xenogenized tumor. Ten days after each immunization, pooled sera from different animals were analyzed for Ab content by means of flow microfluorometry analysis and CEL-ISA assay. The results reveal that antibodies of both IgG and IgM classes capable of binding the xenogenized tumor can already be detected after one single sensitization. However, the Ab titer gradually increases through subsequent immunizations, reaching a peak level after 3-4 injections at a time when most of the humoral response is made up of antibodies of IgG class. The specificity of the anti-xenogenized tumor hyperimmune sera was subsequently investigated by its reaction with the parental, non-xenogenized line and with normal tissue cells of the same or allogeneic haplotypes. The data obtained point out that cross-reactivity with the parental line could be completely removed by absorption of the hyperimmune sera on parental cells, which removed most of the IgM antibodies. Moreover, the presence of an excess of anti-parental antibodies on the xenogenized tumor cells does not prevent the subsequent binding of the hyperimmune absorbed serum, thus indicating that the novel determinant(s) recognized on xenogenized cells are not spatially related to those shared with the original parental tumor. In addition, the hyperimmune absorbed serum does not cross-react with normal hemopoietic or lymphoid cells of the same (H-2d) or allogeneic H-2b and H-2k haplotypes. Furthermore, no alien histocompatibility antigens of H-2b or H-2k haplotypes could be detected on the xenogenized tumor cell surface. Taken together, these data provide evidence that chemical xenogenization of a murine lymphoma leads to the appearance of novel determinant(s) detectable by specific antibodies.

A deletion in chromosome 1 in cells of a transplantable granulocytic leukemia (GL-13) in guinea pigs.

Chromosomes were examined in leukocytes from a unique transplantable granulocyte leukemia (GL-13) of strain 13 guinea pigs. The GL-13 leukemia has many similarities to human chronic myelogenous leukemia (CML). It has remained predominantly diploid after 90 transplant generations and is characterized cytogenetically by a terminal deletion of the long arm of one of the #1 pair of chromosomes. No other chromosomal abnormality was observed. These findings lend further support to the conclusion that the GL-13 leukemia may be a useful model for the study of human CML.

Detection of a common antigen on murine B cells and Lyt-2+ T cells by a rat monoclonal antibody, 14D10.

This paper reports the production of a rat monoclonal antibody, 14D10, which recognizes a determinant shared between B cells and Lyt-2+ T cells. Three subpopulations of spleen cells were defined based on the density of bound 14D10. They have been designated 14D10-SP (strongly positive), 14D10-WP (weakly positive), and 14D10-N (negative). Dual parameter immunofluorescence studies showed that 90% of 14D10-SP cells are IgM+ cells and more than 92% IgM+ cells are 14D10-SP. Of 14D10-WP cells, approximately 60% are Lyt-1+2+ T cells and less than 10% are IgM+ cells. However, most (greater than 90%) of Lyt-2+ T cells are reactive with 14D10. Staining of purified populations of Lyt-2+ and Lyt-2- T cells with fluoresceinated 14D10 also showed greater than 90% of Lyt-2+ T cells to be 14D10+, and fewer than 10% of Lyt-2- T cells are 14D10+. Mouse thymus contained very few (congruent to 5%) 14D10+ cells. Analysis of bone marrow cells shows almost all small cells to be 14D10+. Six Abelson virus-transformed cell lines all express the antigen recognized by 14D10.