Complex regulation of class II gene expression: analysis with class II mutant cell lines

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.     

T cell clones to two major T cell epitopes of myoglobin: effect of I-A/I-E restriction on epitope dominance

The murine T cell response to sperm whale myoglobin was analyzed for polyclonal and monoclonal T cells. For polyclonal T cells, the immunodominant epitope included residue Glu 109 when the antigen-presenting cells expressed I-Ad, whereas a Lys 140-containing epitope was immunodominant when the antigen-presenting cells expressed I-Ed only. Monoclonal T cells specific for each epitope were derived from a polyclonal line. T cell clones specific for the Glu 109 epitope were restricted to I-Ad, whereas the clones specific for the Lys 140 epitope were restricted to I-Ed. Thus, for an antigen that can be presented in association with either I-Ad or I-Ed, the immunodominance of particular epitopes depends strongly on the restriction element used. The immunodominance of each epitope-Ia combination may be due to a limited repertoire of T cells or selective presentation of epitope and Ia by accessory cells.     

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

The T cell receptor: the alpha and beta chains define idiotype, and antigen and MHC specificity

Three independent T cell hybridomas were isolated that have identical specificities for antigen and products of the major histocompatibility complex (MHC). All three react with the same clone-specific antireceptor antibody, and Southern blots show all three contain the same rearranged alpha and beta genes. Variants of one of these hybridomas, DO-11.10, were isolated that had lost the ability to respond to antigen plus MHC. These proved to have lost the DO-11.10-specific alpha or beta genes or both. Fusion of alpha-loss variants to beta-loss variants restored reactivity. These results indicate that the specific recognition of antigen plus MHC is determined solely by the alpha/beta-containing T cell receptor.     

Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones

We have analyzed the complexity of a unique tumor-specific transplantation antigen expressed by the murine ultraviolet light-induced fibrosarcoma 1591-RE. This tumor is highly immunogenic and is regularly rejected by normal mice. We have derived a cloned cytolytic T cell line showing a reactivity pattern representative of the cytolytic response of the host rejecting this regressor tumor. Using this T cell line (anti-A), variants of 1591-RE (1591-A-) were selected in vitro that had lost the same antigen as progressor variants of 1591-RE selected by the host in vivo. The in vitro derived variant was then used to generate a second T cell clone (anti-B) that recognized an antigen on the parental tumor that had been retained by the variants derived in vitro. Host-selected progressor variants were also found to have retained this antigen. By selecting for variants in vitro from the parental tumor with the anti-B T cell line, it was shown that the two different antigens (A and B) present on the parental tumor were lost independently of each other. Despite the independence of these two antigens, the host T cell response to the parental regressor tumor was invariably restricted to only the "immunodominant" A antigen.     

The T11 (CD2) molecule is functionally linked to the T3/Ti T cell receptor in the majority of T cells

Most mature human T lymphocytes express both the multichain T3 (CD3)/Ti T cell receptor for antigen (TCR), and the biochemically distinct 55-kDa T11 (CD2) glycoprotein. Stimulating the T11 molecule causes profound T cell proliferation and functional activation in vitro, but the relationship of T11-mediated activation to antigenic stimulation of T lymphocytes in vivo remains unknown. We now present evidence that T11 function is directly linked to TCR components in T3/Ti+ T11+ human T cells. First, we found that stimulating peripheral blood T cells with the mitogenic combination of anti-T11(2) cells with the mitogenic combination of anti-T11(2) plus anti-T11(3) monoclonal antibodies caused the phosphorylation of TCR T3 chains. The predominance of T3-gamma-phosphorylation that occurred in anti-T11(2) plus anti-T11(3)-treated T cells is similar to the pattern previously observed in antigen-stimulated T cell clones. Second, T11 function depended upon concurrent cell-surface expression of the TCR. Thus, when peripheral blood T cells were deprived of cell surface T3/Ti by anti-T3 modulation, anti-T11(2) plus anti-T11(3)-induced mitogenesis and transmembrane signal generation in the form of calcium mobilization were inhibited. The mechanism of TCR-T11 interdependence was investigated in a series of TCR-deficient variants of a T cell lymphoblastoid cell line. T3/Ti negative variants expressed cell surface T11, but anti-T11(2) plus anti-T11(3) failed to cause detectable calcium mobilization. The TCR-deficient variants also failed to express T11(3) activation epitopes after incubation with anti-T11(2) antibodies, suggesting that T11(3) expression is an essential and TCR-dependent intermediate in the T11 activation mechanism in these cells. Taken together, our results suggest that T11 function depends upon cell-surface expression of TCR in many T3/Ti+ T11+ T lymphocytes, and T11-mediated activation is intimately interconnected with TCR activation mechanisms. A model in which stimulating signals delivered via T11 may be a part of antigenic activation of T lymphocytes is presented.     

Antigen presenting ability of thymic macrophages and epithelial cells: evidence for defects in the antigen processing function of thymic epithelial cells

We compared the antigen presenting ability of cloned thymic macrophage and epithelial cell lines using T cell hybridomas with well-characterized activation requirements. A cloned thymic epithelial cell line (3D.1), preinduced with interferon-gamma (IFN-gamma) activated the T cell hybridoma 3DO-18.3 but not the T cell hybridoma DO-11.10. Analyses using preprocessed antigen suggest that the failure of 3D.1 to activate DO-11.10 is due to its inability to process chicken ovalbumin to produce a peptide recognized by the Ag:MHC T cell receptor of DO-11.10. The epithelial cell line 3D.1 was able to activate DO-11.10 if the superantigen staphylococcal enterotoxin B was used for activation instead of ovalbumin. These observations indicate that IFN-gamma-induced 3D.1 expresses sufficient I-Ad molecules to activate DO-11.10 but is unable to produce the peptide of ovalbumin recognized by DO-11.10. Furthermore, 3D.1 appears to be representative of nonmacrophage thymic stromal cells cultured in vitro, since heterogeneous cultures containing epithelial cells exhibited the same selective T cell activation characteristics. In contrast, thymic macrophage cell lines activated all T cells studied. These results suggest that there is a functional difference between the capacity of thymic epithelial cells and macrophages to process and present antigen to T cells.     

The effects of MHC gene dosage and allelic variation on T cell receptor selection

In a T cell receptor transgenic mouse model of thymic selection, the efficiency of selection of the transgenic alpha beta heterodimer is significantly enhanced in animals that express higher densities of the relevant major histocompatibility complex molecule (I-Ek/b). These results imply that there is a stochastic component to positive selection in the thymus. Allelic variants of the original selecting I-Ek molecule are either less efficient (E alpha k:E beta b) or incapable (E alpha k:E beta s and I-Ed) of mediating the selection of transgenic alpha beta + T cells. Two of these three I-E variants appear to differ from I-Ek in amino acid residues of the peptide binding site and not in residues capable of contacting the T cell receptor, suggesting that specific peptides, or conformations of peptides, play a role in positive selection. In contrast, mice transgenic for only the beta chain of this T cell receptor show selection for CD4+ T cells in the presence of all four I-E variants tested.     

Thy-1-negative and Ly-1-negative variants of T cells produce interleukin 2 in response to mitogens

IL 2 production by T cell variants, which lack the Thy-1 or Ly-1 surface glycoproteins, was studied. Cross-linking of the Thy-1 molecule resulted in IL 2 production by the EL4 thymoma and by a T cell hybridoma, suggesting that Thy-1 may play a role in T lymphocyte triggering. To further study the functional role of this molecule, Thy-1-negative variants were selected and analyzed for IL 2 production in response to phorbol-12-myristate-13-acetate (PMA) or to Con A. It was demonstrated that in spite of their failure to express Thy-1, the Thy-1-negative clones were capable of IL 2 production. These results indicated that although Thy-1 cross-linking triggers cell activation, a signal provided by Thy-1 is not indispensable for cell activation by mitogens. The T cell tumor line LBRM331A5 responds synergistically to IL 1 and PHA by releasing IL 2. It was demonstrated that anti Ly-1 monoclonal antibodies and PHA co-stimulated LBRM331A5 cells, as did IL 1 plus PHA. Thus, anti Ly-1 antibodies mimic the effect of IL 1, suggesting a role for Ly-1 antigen in T cell activation, perhaps by serving as an IL 1 receptor or as an associated molecule. To further study the functional role of Ly-1 and its relation to IL 1 receptor, Ly-1-negative variants of the LBRM331A5 cell line were selected and analyzed for IL 2 production in response to PHA plus IL 1. It was demonstrated that the Ly-1-negative clones were capable of IL 2 production as efficiently as Ly-1-positive clones. These results indicate that the Ly-1 and IL 1 receptor are distinct molecules, which are involved in different activation pathways.     

The major histocompatibility complex-restricted antigen receptor on T cells. IX. Role of accessory molecules in recognition of antigen plus isolated IA

Antibody inhibition studies were done to determine which molecules on the surface of the T cell hybridomas other than their receptors for antigen plus IAd were involved in interaction with antigen-presenting B cells, with artificial IAd membranes on glass beads, or with anti-receptor antibodies coupled to Sepharose beads. We found that T cell LFA-1 was only involved when B cells were used to present antigen plus IAd, whereas T cell L3T4 was involved in the response of T cells to antigen plus IAd either on cells or in artificial membranes, but not if anti-receptor antibodies were used to stimulate the T cells. From these results we concluded that LFA-1 may be involved in the recognition of a ligand on cells that was not present in artificial membranes, but that L3T4 might interact with a nonpolymorphic portion of class II molecules present in both intact antigen-presenting cells and the antigen-presenting artificial membranes.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

Comparison of structural requirements for interaction of the same peptide with I-Ek and I-Ed molecules in the activation of MHC class II-restricted T cells.

We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Immunoregulation of T-dependent responses by a cloned dendritic cell

We have described a cloned dendritic cell, Clone Den-1, which is a potent accessory cell for some T-dependent immune responses. Clone Den-1 activates T cells in both autologous and allogeneic mixed lymphocyte reactions, but cannot present soluble antigen to T cells that co-recognize nominal antigen plus I-Ab gene products. In addition, Clone Den-1 or factors produced by it can reconstitute plaque-forming cell responses by accessory cell-depleted B plus T cells to the T-dependent antigen sheep red blood cells. The relationship of this clone to heterogeneous populations of dendritic cells and other accessory cells is discussed.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Evidence for an involvement of T4+ cytotoxic T cells in tumor immunity

Cell-mediated immunity against cancer cells primarily involves class I major histocompatibility complex (MHC)-restricted cytotoxic T cells and natural killer (NK) cells. To investigate whether T4+ cytotoxic T cells also have a role in tumor-specific immunity, mice were immunized with a B cell lymphoma. T cell hybridomas were constructed from the immune spleen cells and analyzed for their cytotoxic ability against the immunizing lymphoma. A T4+, Lyt-1+ hybridoma cell line was developed (103L2) which specifically killed the immunizing tumor cells but not normal B cells or a range of other tumor cells of B or non-B origin. This cytotoxic hybridoma cell line differed from Lyt-2+ cytotoxic T lymphocyte cells and NK cells, commonly identified with cytotoxicity, in a number of important ways. First, the cells were class II MHC restricted; second, interleukin-2 was released from activated effector cells; and finally but most importantly, innocent nonparticipating bystander cells were also killed. The significance of this observation was that normal cells were protected, although a broad range of tumor cell types, including tumor antigen-negative mutants, were killed. It is therefore conceivable that T4+ cytotoxic T cells might play an important role in tumor immunity through the direct recognition and lysis of tumor cells while any tumor variants, arising due to antigen loss, would remain susceptible through the bystander killing effect and normal cells would remain unaffected. These results strongly suggest that tumor-reactive T4+ cytotoxic T cells belong to a new category of effector cells with an important role in tumor-specific immunity.     

Structural requirements for the interaction between peptide antigens and I-Ed molecules

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.     

Differentiation of an immature T cell line: a model of thymic positive selection.

Thymocyte differentiation is dependent upon recognition of major histocompatibility complex (MHC) molecules on thymic stroma, a process called positive selection. Here we describe an immature CD4+8+ T cell line derived from a TCR transgenic mouse that differentiates into CD4+8- cells in response to antigen and nonthymic antigen-presenting cells. When injected intrathymically, these cells differentiate in the absence of antigen. The ability of immature T cells to recognize MHC molecules in the absence of foreign antigen in the thymus can thus be attributed to a unique property of thymic antigen-presenting cells. These studies also demonstrate the phenotypic and functional changes associated with TCR-mediated T cell maturation and establish an in vitro model system of positive selection.