Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1(Δ/Δ) mutant mice

Background

Foxn1^Δ/Δ mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from an atypical progenitor population. In the current study, we tested the effects of these defects on thymic selection.

Methodology/Principal Findings

We used Foxn1^Δ/Δ; DO11 Tg and Foxn1^Δ/Δ; OT1 Tg mice as positive selection and Foxn1^Δ/Δ; MHCII I-E mice as negative selection models. We also used an in vivo system of antigen-specific reactivity to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection of both CD4 and CD8 SP thymocytes was reduced in Foxn1^Δ/Δ mutants compared to Foxn1^+/Δ control mice. These defects were associated with reduction of both MHC Class I and Class II expression, although the resulting peripheral T cells have a broad TCR Vβ repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output.

Conclusions/Significance

These data show that a primary defect in the thymic microenvironment can cause both direct defects in selection which can in turn cause indirect effects on the periphery, exacerbating functional defects in T cells.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Genetic control of the immune response to collagen. III. Coordinate restriction of cellular cooperation and antigen responsiveness by thymus-directed maturation

The H-2 restriction of T helper cells from thymus-reconstituted nude mice was examined. Hybrid athymic mice were bred from BALB/c.nu and C57BL/6.nu parental strains and reconstituted with fetal thymus tissue from either parental strain. T helper cells from these mice, immunized to SRBC, were restricted to cooperation with B cells of the thymic H-2 haplotype. These T helper cells were shown to have originated from the F1 host by functional sensitivity to antisera and complement. The H-2 restriction of thymus-reconstituted F1 nude mice was further investigated by examining expression of the Ir-collagen phenotype. Results showed that the level of antibody produced in response to type I calf collagen in thymus chimeras correlates with the H-2 haplotype (high responder or low responder) of the reconstituting thymus. These experiments indicate that the thymus environment of T cell maturation influences both the H-2 restriction and Ir-phenotype of a responding immune system.     

Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Transplantation of cultured thymic fragments. VI. H-2 recombinant donors

Athymic (nude) mice were transplanted with cultured thymic fragments from syngeneic, allogeneic, and partially allogeneic (recombinant) mice. Lymphocyte proliferation and cytotoxicity in vitro were measured to assess immunologic reconstitution. Transplanted nude mice were immunocompetent whether donor and recipient were disparate for class I, class II, or both H-2 gene types. Furthermore, allotolerance for thymic H-2 class I antigens was achieved independently of class II antigen allotolerance. Class I antigen tolerance was not broken during lymphocyte responses to unrelated alloantigens, ruling out insufficient help as the tolerance mechanism. Splenocytes, isolated from nude mice transplanted with fully allogeneic or syngeneic thymic fragments and stimulated in vitro with trinitrophenyl-modified cells, displayed H-2-restricted, hapten-specific cytotoxicity. Cytotoxic cells from allotolerant mice were restricted to either host or thymic H-2 antigens, depending on the stimulating cell haplotype. Response levels for thymic and host trinitrophenyl-modified cells were comparable. We have shown that allogeneic thymic epithelium transplanted into adult nude mice can induce allotolerance to class I and II H-2 antigens equally, and permits T lymphocyte interaction with cells bearing thymic donor or host H-2 antigens. Our results are consistent with a model wherein T lymphocyte self-receptors retain their genomic repertoire but can be selectively mutated or expanded by appropriate H-2 antigen presentation by the thymus.     

Differential effect of hybrid resistance on the localization of virus-immune effector T cells to spleen and brain

The hybrid resistance (Hr) effect operates in the lymphocytic choriomeningitis (LCM) in vivo transfer model to inhibit both the level of cytotoxicity T lymphocyte (CTL) generation in spleen and the induction of inflammation in cerebrospinal fluid (CSF). The effect is seen when LCM virus-immune T cells that are homozygous for H-2D ^b are injected into virus-infected, immunosuppressed recipients that are heterozygous for this allele, or into radiation chimeras that express an appropriate F₁ phenotype. Evidence that Hr to T -cell transfer is cell-dose-dependent and tends to diminish with age was found in both chimeric and normal F₁ mice. Inhibition of the capacity of injected T cells to cause meningitis is a more sensitive measure of Hr than is the further stimulation of CTL effectors in recipient lymphoid tissue. The injection of large numbers of H-2^b virus-immune T cells into (H-2 ^k X H-2 ^bF₁H-2 ^k) virus-infected recipients did not induce any cellular extravasation into CSF, though potent H-2^b-restricted CTL effectors were generated in recipient spleen. Evidence of minimal inflammatory process was found in one experiment where these chimeras were given a comparable dose of (H-2 ^b X H-2 ^d)F₁ immune spleen cells. Development of this Tcell-mediated immunopathological process depends essentially on the expression of the appropriate H-2 restriction element on radiation-resistant host cells which, in this case, presumably constitute part of the physiological barrier between blood and CSF.     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.     

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders^1-4. T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC⁵.Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events⁶. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes^7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY^cd4 model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice¹⁰.Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY^cd4 mouse model. While negative selection in HY^cd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.     

Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.     

Selection of the T-cell repertoire in transgenic mice expressing a transplantation antigen in distinct thymus subsets

Transgenic mice that expressed a transplantation antigen, H-2Kb, in an unusual tissue distribution have been developed. Gene-regulatory elements from the immunoglobulin heavy-chain locus (Emu enhancer and heavy chain promoter) were linked to the class I Kb gene and the construct microinjected into fertilized mouse eggs of a different haplotype. It was expected that such gene-regulatory elements would direct expression of the foreign class I molecules only to B and T lymphocytes. However, expression was also detected in a subset of thymus medullary epithelium. The Kb molecules expressed on this thymic subset were unable to positively select T cells for passage to the periphery. The mice were, however, tolerant of the cell types expressing the foreign Kb molecules and were also tolerant of Kb presented as skin grafts. These results suggest that not all components of thymic epithelium are involved in positive selection of T cells and that transplantation antigens expressed on non-dendritic cells can induce tolerance.     

Expression of subregion and haplotype preference for H-2Kk restricted cytotoxic T-cell responses in vivo and at the level of the precursor frequencies

Several strains of mice bearing the H-2K^k allele were found to generate in vivo strong CTL responses against TNP-haptenated syngeneic cells, while several other strains of mice were found to generate comparably weak or no responses. C3H × DBA/2)F1 mice (H-2^k × H-2^d) and A/J mice with the recombinant haplotype $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ generated CTL responses in vivo that were completely restricted toward the H-2^k haplotype or the K end of the $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ haplotype, respectively. The CTL activity of C3H × DBA/2)F1 and A/J mice against haptenated H-2^k targets was found to be more than 25-fold higher than the CTL activity on H-2^d targets. The CTL responses in vitro under macroculture conditions showed, on the other hand, only a 3- to 6-fold higher cytotoxic activity against the haptenated H-2^k targets as compared with haptenated allogeneic or H-2^d targets; and limiting dilution experiments in microcultures revealed that the CTL precursor frequencies were only 2- to 3-fold smaller for TNP-haptenated H-2^d or haptenated allogeneic targets than for haptenated H-2^k target cells. This indicated that sufficient numbers of H-2^d-restricted and allorestricted CTL precursors were actually present in these strains, but did not develop detectable cytotoxic activity in vivo. The exceptional property of the H-2^k haplotype is, therefore, only partly determined by a difference in the CTL precursor frequencies, and to the larger extent determined at the level of the activation of the CTL response.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

A genetic analysis of natural resistance to nonsyngeneic cells: The role of H-2

The genetic control of natural resistance in vivo to four natural killer (NK) cell-resistant H-2 homozygous lymphoid tumor cell lines was investigated by following the survival and organ distribution of cells prelabeled with radioactive iododeoxyuridine. Backcross mice derived from DBA/2J and CBA/J parents were injected with H-2 ^dtumor cells and tumor cell elimination was lowest in H-2 ^dhomozygotes. Natural killer cell activity was also reduced in mice with the H-2 ^dhaplotype, but no direct correlation between NK cell levels against YAC-1 or SL2-5 lymphoma cells and natural resistance in vivo was demonstrable. Analysis of 23 BXD recombinant inbred strains indicated that natural resistance to H-2 ^dtumors was restricted to H-2 ^bstrains. There was no direct association of NK cell activity with H-2 type in the BXD strains and NK cell levels did not correlate with tumor survival in vivo. By comparing natural resistance to H-2 ^dand H-2 ^btumors in DBA/2, C57BL/6, B6D2F₁, and B10.D2 mice we found that H-2 nonidentity between the tumor and the host, rather than the host H-2 haplotype, determined whether natural resistance occurred. Again, NK cell activity against YAC-1 cells was not predictive of tumor survival in these strains. These results provide genetic evidence that NK cells alone cannot account for natural resistance to H-2 nonidentical cells of hemopoietic origin.     

MHC epitopes of Ks and Dd restrict the same population of cytolytic T lymphocytes

When A. SW (H-2 ^s) mice are immunized with B10.S (H-2 ^S) epidermal cells, cytolytic T lymphocytes are evoked that efficiently lyse B10.D2 (H-2 ^d) as well as B10.S target cells. Immunogenetic analysis of this apparent H-2-unrestricted killing revealed that the most plausible explanation was a sharing of an H-2-restricting epitope by H-2K^S and H-2D^d molecules.     

Prolonged survival in mice with advanced tumors treated with syngeneic or allogeneic intra-bone marrow–bone marrow transplantation plus fetal thymus transplantation

Thymic function decreases in line with tumor progression in patients with cancer, resulting in immunodeficiency and a poor prognosis. In the present study, we attempted to restore thymic function by BALB/c (H-2^d) syngeneic (Syn), or B6 (H-2^b) allogeneic (Allo) bone marrow transplantation (BMT) using intra-bone marrow–bone marrow transplantation (IBM–BMT) plus Syn-, Allo- or C3H (H-2^k) 3rd-party fetal thymus transplantation (TT). Although the BALB/c mice with advanced tumors (Meth-A sarcoma; H-2^d, >4 cm²) treated with either Syn- or Allo-BMT alone showed a slight improvement in survival compared with non-treated controls, the mice treated with BMT + TT showed a longer survival. The mice treated with Allo-BMT + Allo-TT or 3rd-party TT showed the longest survival. Interestingly, although there was no difference in main tumor size among the BMT groups, lung metastasis was significantly inhibited by Allo-BMT + Allo-TT or 3rd-party TT. Numbers of CD4⁺ and CD8⁺ T cells, Con A response, and IFN-γ production increased significantly, whereas number of Gr-1⁺/CD11b⁺ myeloid suppressor cells and the percentage of FoxP3⁺ cells in CD4⁺ T cells significantly decreased in these mice. Furthermore, there was a positive correlation between survival days and the number of T cells or T cell function, while there was a negative correlation between survival days and lung metastasis, the number of Gr-1⁺/CD11b⁺ cells, or the percentage of FoxP3⁺ cells. These results suggest that BMT + TT, particularly Allo-BMT + Allo-TT or 3rd-party TT, is most effective in prolonging survival as a result of the restoration of T cell function in hosts with advanced tumors.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells

We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.