Class I MHC-restricted cytotoxic responses to soluble protein antigen.
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Biomedical subjects
M J Bevan
Publications and source records attributed to M J Bevan.
Redirecting the cellular immune response.
A technology is described which combines the power of T lymphocytes in eliminating unwanted cells and causing beneficial inflammatory reactions with the great advantages of monoclonal antibodies. We show that heteroconjugate antibodies and hybrid antibodies, in which one of the component binding sites is anti-T-cell receptor and the other component binding is directed against any chosen target antigen, can focus T cells to act at the targeted site. We present experimental evidence for the in vitro efficacy of this system in viral infections and in the elimination of tumor cells. In an in vivo tumor model pilot experiments have been undertaken that resulted in a significant higher rate of survival of animals treated with hybrid antibodies compared to untreated animals.
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Introduction of soluble protein into the class I pathway of antigen processing and presentation.
In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphocytes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2Kb-restricted CTL specific for the OVA258-276 peptide. This synthetic peptide and CNBr fragments of OVA (242-285 and 242-273) were able to target H-2b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the Kb restricted determinant formed readily.
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Thymocyte subpopulations during early fetal development in the BALB/c mouse.
Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.
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Split tolerance induced by the intrathymic adoptive transfer of thymocyte stem cells.
The intrathymic transfer of semiallogeneic CD4/CD8 double-negative (DN) thymocyte stem cells into irradiated host mice resulted in a transient state of chimerism in adoptive host thymus, spleen, and lymph nodes. Host-derived T cells, isolated from the thymus and periphery of the chimeric mice, were found to be specifically nonresponsive to the MHC antigens of the semiallogeneic DN donor in cytotoxicity assays. This nonresponsiveness was not permanent, but persisted as long as appreciable numbers of Thy-1 alloantigen-positive progeny of the DN donor cells could be detected in the spleen and lymph nodes of adoptive host mice. FACS sorting of DN donor cells before intrathymic transfer indicated that nonresponsiveness could be induced by Thy-1+ cells and was therefore not attributable to contaminating thymic macrophages, dendritic cells, or B cells. When FACS-sorted Thy-1+ (bm5 x bm12)F1 DN cells were transferred intrathymically into C57BL/6 hosts, nonresponsiveness to DN donor MHC class I but not class II alloantigen (split tolerance) was observed. These experiments were repeated using FACS-sorted Thy-1+ DN donor cells that were semiallogeneic to the irradiated adoptive host at either MHC class I or class II locus with similar results. Limiting dilution analysis showed that host-derived CTL precursors were tolerant of DN donor MHC class I alloantigen and no evidence for the involvement of suppressor T cells was found. The data indicate that murine thymocytes themselves are capable of tolerizing to MHC class I but not class II alloantigen after intrathymic transfer. The implications for intrathymic T cell differentiation and maintenance of self tolerance are discussed.
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Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides.
Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.
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Thymocytes expressing CD8 differentiate into CD4+ cells following intrathymic injection.
Based on the cell surface expression of CD4 and CD8 molecules, murine thymocytes can be divided into four populations: these include CD4-, CD8- double-negative and CD4+, CD8+ double-positive subpopulations, both of which consist largely of immature cells, and the single positive, CD4+ or CD8+, subsets that contain functional helper or killer cells, respectively. The double-negative subset contains precursors of the other three populations and can reconstitute the thymus following intravenous or intrathymic transfer into irradiated hosts. In an attempt to establish the sequence of CD4 and CD8 expression during intrathymic development, we investigated the differentiation potential of highly purified CD8+ thymocytes by using intravenous or intrathymic adoptive transfer. Unlike the double-negative thymocyte subset, CD8+ cells did not have the ability to home to the thymus following intravenous transfer. However, when CD8+ thymocytes were injected directly into the thymus, they increased in number and gave rise to CD4+, CD8+ double-positive and CD4+ single-positive progeny. Furthermore, the rate of appearance of CD4+ cells from injected CD8+ precursors was faster than from the double-negative subset. Cells expressing a high surface density of CD8 convert to double-positive and CD4+ progeny without increasing in number, whereas CD8+ cells expressing a low surface density of the marker expand greatly and give rise to differentiated progeny. The results suggest that CD8 expression is an intermediate step on the differentiation pathway of mature CD4+ T cells.
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Cooperation between helper T cells and cytotoxic T lymphocyte precursors.
We have investigated the requirement for the presence of L3T4-positive T cells in the in vivo priming of Lyt-2-positive cytotoxic T-lymphocyte precursors. The antigens used were the male-specific antigen H-Y and autosomal minor histocompatibility antigens. In some experiments, responder mice were depleted of L3T4-positive cells by repeated intraperitoneal injections of the anti-L3T4 antibody, GK1.5. In other experiments, lymphoid cells from normal mice were fractionated in vitro by antibody-plus-complement treatment and the populations primed in irradiated adoptive hosts. In these antigen systems, depletion of L3T4-positive helper cells decreases the level of priming of cytotoxic T lymphocytes. With regeneration of the L3T4-positive subpopulation, the CTL response to antigen increases. To some extent, the reliance on L3T4-positive cells can be overcome by increasing the antigen dose. We conclude that in most physiological responses, L3T4-positive T cells enhance the cytotoxic T-cell response.
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Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and alloreactive cytotoxic T cells.
Cross-reactive recognition of alloantigen by "self + X"-reactive cytotoxic T lymphocytes (CTL) has been documented in a variety of systems. It has been shown previously that the H-2Kb-restricted CTL response of C57BL/6 (B6) mice to vesicular stomatitis virus (VSV) infection is partially cross-reactive on uninfected target cells expressing the H-2Kbm8 mutation. In this report, we describe the isolation and detailed characterization of such dual-reactive CTL. By employing EL4 tumor lines transfected with genes encoding various VSV proteins, we demonstrated that the majority of dual-reactive CTL recognize the internal N protein of VSV and are also reactive against uninfected bm8 targets. Although the response of normal B6 mice to bm8 stimulators shows no measurable cross-reactivity on VSV-infected targets, the response of VSV-primed B6 mice to bm8 stimulation is almost entirely cross-reactive, lysing VSV-B6 targets and uninfected bm8 targets roughly equally. Furthermore, about 70% of CTL clones isolated from such mice by bm8 stimulation are dual-reactive with respect to effector function. Analysis at the population and clonal levels with cold target competition and antibody blocking suggests that the bulk of dual-reactive CTL have a higher avidity for VSV-B6 targets than for bm8 targets. The extreme case of this is illustrated by a fraction of CTL clones, isolated and maintained on bm8 stimulators, which lyse VSV-B6 targets but do not lyse bm8 targets. One such CTL clone is shown to be specific for the bm8 antigen in proliferation assays. These results demonstrate that: the specificity of an alloreactive CTL response may be dramatically altered by previous antigenic encounters; and dual-reactive CTL display a significant difference in affinity of the CTL receptor-determinant interaction, depending on the target which is recognized.
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Expression and functional significance of the J11d marker on mouse thymocytes.
Subpopulations of thymocytes have been characterized phenotypically and functionally in relation to their expression of the marker defined by the monoclonal antibody J11d. Cortical-type L3T4+, Lyt-2+ thymocytes are all J11d+. Thymocytes that share the phenotype L3T4-, Lyt-2+ with peripheral Lyt-2+ T cells contain a J11d+ and a J11d- subset. These J11d- cells behave like peripheral Lyt-2+ T cells in two functional assays: they form clonal growth bursts in response to immobilized antibody against the T cell antigen receptor, and they act as precursors of alloreactive cytotoxic T cells. The J11d+ cells are inert in both of these assays. In contrast, L3T4+, Lyt-2- thymocytes do not contain a J11d+ subset.
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Hybrid antibody-mediated lysis of virus-infected cells.
Previously, cytotoxic T lymphocytes (CTL) had only been focused by hybrid antibodies to normal, cell-surface proteins and haptenated surface structures. In this report, we extend the application of this technology to mediate lysing of virus-infected cells by nonspecific CTL. Heteroconjugated antibodies between the anti-T cell antigen receptor antibody, F23.1, and monoclonal antibodies against either the hemagglutinin or nucleoprotein of the influenza virus PR/8 were constructed. We show in the present report that these bispecific constructs can target virus-infected cells for lysis according to the specificity of the virus-protein reactive monoclonal antibodies. Furthermore, even a virus protein that is only expressed in small quantities on the cell surface, such as nucleoprotein, can be exploited as target structure for heteroconjugated antibodies. These studies show that hybrid antibodies can focus a CTL response on virus-infected cells which might in the future be used to mount an immune response in situations without sufficient normal cellular defense.
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Mutual tolerization of histoincompatible lymphocytes.
T lymphocytes react strongly against foreign major histocompatibility complex encoded class I antigens by destroying incompatible tissue in vivo, and by generating cytotoxic T lymphocytes (CTL) in vitro. The absence of reactivity against self antigens may be due to clonal deletion of self-reactive T cells during their ontogeny in the thymus. The functional clonal deletion of mature T cells in the periphery was described recently: CTL recognizing antigen on other CTL are eliminated (Rammensee et al., Immunol. Today 1985. 6: 41). Hence, in a normal immune system only autoreactive cells would be eliminated. Here we show that injection of lymphocytes into class I-incompatible mice leads to abrogation of host anti-donor as well as donor anti-host reactivity, leaving a mixed population of host and donor T cells reactive against third-party antigens. The results demonstrate the existence of a peripheral failsafe mechanism for the elimination of autoaggressive CTL. Whether this failsafe mechanism is actually used under physiological conditions is a different question.
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Different classes of T lymphocytes have different mRNAs for the leukocyte-common antigen, T200.
The leukocyte common antigen, T200, is expressed on all white blood cells but not on other differentiated cells. Within the hematopoietic lineage, specific cell types display characteristic structural forms of the molecule on their surface. We show that murine cytotoxic T lymphocyte clones and helper T cell clones contain different size mRNA for this molecule, and that the early precursors of T200 glycoprotein made in the helper and cytotoxic T cells differ in Mr. Thus, in addition to differences in posttranslational modifications, it is highly likely that a difference in protein structure contributes to the distinct forms of T200 glycoprotein found on these functional T cell subsets.
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Activation of resting T lymphocytes by a monoclonal antibody directed against an allotypic determinant on the T cell receptor.
The murine monoclonal antibody F23.1 reacts with an allotypic determinant on the beta chain of the T cell receptor expressed by approximately 20% of T helper and cytotoxic T lymphocytes of most common mouse strains. This IgG2a antibody, either in soluble form or covalently coupled to Sepharose beads, can activate resting T cells from naive animals to proliferate. Interestingly, under all conditions of activation, the antibody can only induce proliferation if exogenous lymphokines in the form of Con A supernatant are provided. Thus, it is unlike most lectins and anti-T3 antibodies in this regard. Furthermore, under all conditions of culture, F23.1 activates preferentially the Lyt-2+ subset of T cells. This is the case even in the presence of accessory cells. Further evidence is provided that two soluble lymphokines, different from IL2, are required to initiate IL2-dependent growth and to allow the expression of lytic activity.
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T cell receptor expression and receptor-mediated induction of clonal growth in the developing mouse thymus. High surface beta-chain density is a requirement for functional maturity.
The ontogeny of T cell antigen receptor expression and function in the mouse thymus has been studied using a monoclonal antibody, F23.1, which recognizes a determinant on the beta chain of the receptor, and stains 25% of mature T cells and around 7-15% of adult thymocytes from most mouse strains. The same monoclonal antibody selectively activates Lyt-2+ peripheral T cells. Receptors are detectable by staining and fluorescence-activated cell sorter analysis from fetal day 17, and thereafter the overall frequency increases steadily towards adult levels. However, late fetal thymocytes express all of their antigen receptor beta chain at a very low level, visible by staining as a "shoulder" on the peak of negative cells. Thymocytes with high-density surface beta chain, visible by staining as a distinct peak, appear only after birth and are a prominent feature at neonatal day 4. In the late fetus, expression of beta chain can be detected on thymocytes with the "mature" L3T4-, Lyt-2+ phenotype. Despite this, F23.1-responsive precursors are not found in the fetal thymus, and appear in two waves, the first during day 1 of postnatal life and the second between days 3 and 4. These data suggest that high-density surface expression of T cell receptor beta chain occurs in parallel with functional maturation.
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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-2Kb 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-2Kb variants employing a Kb-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 Kb 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 Kb 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-2Kb 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 Kb allodeterminants.
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Hybrid hybridoma producing a bispecific monoclonal antibody that can focus effector T-cell activity.
Previous studies have shown that heteroconjugates of monoclonal antibodies in which one of the component antibodies is directed at the T-cell receptor and the other is directed against any chosen site can focus effector T cells to function at the targeted site. We report here the production of a hybrid hybridoma cell line, H1.10.1.6, which secretes large amounts of a bispecific hybrid antibody of the IgG2a class, that can focus T-cell activity. The parental hybridoma lines for the secondary fusion were F23.1, which secretes an antibody specific for an allotypic determinant on the T-cell receptor of most mouse strains, and 19E12, secreting an anti-Thy-1.1 antibody. The bispecific hybrid antibody was partially purified by hydroxylapatite chromatography and characterized by isoelectric focusing. It efficiently targets Thy-1.1-expressing tumor cells for lysis by F23.1 receptor-positive cytotoxic T-cell clones in vitro. Such hybrid antibodies produced by hybrid hybridoma cell lines may have application in the therapeutic targeting of tumors or sites of viral infections for attack by T cells.
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