Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

In vitro induction of immunological tolerance

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.     

Analysis of the negative allogeneic effect using B cells and helper T cell lines as an indicator system: B cells identified as target cells for suppression

The target cells for H-2b T lymphocytes mediating a negative allogeneic effect in vitro were analyzed by using carrier-specific helper T cell lines of H-2b, H-2d, or F1 origin and hapten-primed T-depleted spleen cells also expressing one or both of these haplotypes. The helper T cell lines were shown to be carrier specific and H-2b or H-2d restricted. Most importantly, the lines derived from H-2b homozygous mice were devoid of alloreactivity against H-2d and vice versa. Titration of naive H-2b T lymphocytes to the indicator cultures resulted in suppression of the secondary anti-DNP response of the indicator cells whenever the B cells expressed H-2d antigens. The lack of suppression observed in mixtures in which only the helper T cell lines expressed H-2d antigens was not reversed by the increased addition of naive H-2bxd cells, indicating that an insufficient amount of H-2d antigens present on the low number of helper T cells used did not account for this finding. Moreover, the polyclonal plaque-forming cell responses of F1 spleen cells to LPS were also suppressed by naive parental T cells. From these findings it is concluded that the suppressor T cells directly recognize and inhibit allogeneic B cells without the involvement of helper T cells. In addition, it was shown that the suppression of secondary anti-hapten responses by naive allogeneic T cells is blocked by monoclonal anti-Lyt-2 antibody added at the onset of culture. Addition late in culture had no effect, pointing to a functional role of the Lyt-2-bearing structure at an early stage of the suppressive events resulting in the negative allogeneic effect.     

Regulation of a cytotoxic response toward alloantigens by T-amplifier and non-T-suppressor cells.

A system is described by which it is possible to generate in vitro non-specific T-amplifier and non-T-suppressor cells without accompanying formation of CTL. Such cells could be separated and tested for their capacity to regulate generation of CTL towards H-2 alloantigen. The experimental setup was as follows: M-locus or syngeneically activated spleen cells were fractionated on BSA-gradients and added to fresh, nylon wool purified (T) precursors. These cultures were stimulated during a second stage with H-2 alloantigen and lytic activities monitored with the chromium release assay. It was found that M-locus activated cells contained two separable subpopulations: 1) an anti-Thy 1 sensitive, non-adherent amplifier cell of intermediate density and 2) a blast cell which was insensitive to anti-Thy 1 as well as RaMIg treatment (i.e. a “null” cell). This cell was adherent, but non-phagocytosing and had strongly suppressive effects on the generation of CTL. It also suppressed—at least partially—the activity of fully differentiated CTL. In syngeneically stimulated cultures strongly enhancing T amplifier cells with blast cell characteristics were found.     

I-region restriction of alloantigen recognition: alloantigen expressed on the surface of a hybridoma cell must be presented in the context of self class II major histocompatibility complex determinants for recognition by a dual-reactive T-cell clone

A helper-T-lymphocyte clone, designated A10, proliferated in response to both hen egg ovalbumin (OVA) presented in the context of self I-Ak and to the alloantigen I-As. The alloantigen source could be provided by irradiated H-2s spleen cells and also by paraformaldehyde-fixed H-2s spleen cells. However, for fixed allogeneic spleen cells to stimulate proliferation of the cloned cells, it was necessary to add irradiated syngeneic I-Ak-bearing spleen cells, as fixed H-2s spleen cells added, by themselves, to A10 cells were nonstimulatory. We have extended these findings by generating a monoclonal hybridoma cell which expressed the I-As allodeterminant. Similar to our results with fixed allogeneic spleen cells, this source of alloantigen could stimulate A10 cells to proliferate only if irradiated syngeneic spleen cells were added to the cultures. These proliferative responses were effectively inhibited by anti-I-Ak monoclonal antibody (mAb) and by anti-I-As mAb. Furthermore, the response of A10 cells to the alloantigen-bearing hybridoma cells were also inhibited by the anti-L3T4 mAb GK1.5. Collectively, these data indicate that, in some situations, alloreactivity may be mediated by self class II major histocompatibility complex restriction of alloantigen-driven proliferation.     

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Alloantigen-induced T helper activity. I. Minimal genetic differences necessary to induce a positive allogeneic effect.

Addition of histoincompatible lymphocytes can influence the course of ongoing immune responses. Such allogeneic effects may either augment or diminish immune responses. We describe here the minimal genetic differences necessary to generate positive allogeneic effects (allohelp) in a humoral immune response. The antibody response to sheep erythrocytes of T cell-depleted mouse spleen cells was reconstituted by addition of syngeneic or allogeneic nylon wool column-passaged spleen T cells. T cells were pretreated with mitomycin C before culture to prevent development of allo-suppression and cytotoxic lymphocytes. Positive allogeneic effects were operationally defined as superior helper effects (to generate greater antibody forming cell responses) with T cells allogeneic rather than syngeneic to the responding B cells. Thus, addition of allogeneic T cells resulted in many more antibody forming cells than did equal numbers of syngeneic T cells, and fewer allogeneic than syngeneic T cells were necessary to generate comparable responses. With congenic, recombinant, and mutant mouse lines, genetic differences in the H-2 complex and those associated with Mls were each sufficient to provide positive allogeneic effects. With intra-H-2 recombinants, differences at either I or D were sufficient. A disparity at H-2K alone, as provided by the H-2 mutant B6.C-H-2ba against the parental line C57BL/6By, also induced helper effects. The significance of these results is discussed.     

Cellular requirements for production and release of the lymphocyte costimulator

The lymphocyte costimulator (CoS) is a lymphokine required for the activation of T cell responses to H-2 alloantigens or mitogen, CoS activity is found in the supernatant medium of Concanavalin A (Con A) stimulated spleen cells, In this paper we investigate the cellular requirements for CoS production by Con A-activated mouse spleen cells. Maximal lymphokine production in response to Con A depends on a co-operative interaction between T cells and a nylon wool-adherent cell present in the spleen of nude mice. T cells appear to be the major producers of CoS activity, doing so only in response to an initial inductive stimulus supplied by nude spleen cells. The inductive stimulus is found as a soluble factor in the supernatant of Con A-activated spleen cells, and can also be provided by stimulatory (S+), but not by non-stimulatory (S-), tumour cells H-2 identical with the responding T cells. The activation of lymphokine-producing T cells is thus a two-signal process, requiring both mitogen and an additional inductive signal. Once activated, homogeneous populations of T cells will release lymphokine in response to mitogen alone.     

Study of H-2 mutations in mice. VII. The defectiveness of the H-2ba mutant in the hemagglutination and graft vs host reactions]

Three mutants of the H-2K gene within the mouse major histocompatibility complex were studied. Antisera anti-H-2.5 + 39 were unable to agglutinate H-2ba and H-2bf mutant red blood cells. The same sera moderately agglutinated H-2b red blood cells. The H-2ba red blood cells did not absorb in vitro the activity from an anti-H-2.5 serum, while the H-2b cells did. Thus, these results indicate the existence of qualitative SD antigenic differences among H-2Kb derived mutants. In the proliferative graft versus host test the H-2ba spleen cells did not induce spleen enlargement in the H-2bd recipients, while pronounced reaction was recovered in bd-ba combination. The (baXb) F1 hybrid cells were as efficient as b/b homozygous cells. The data suggest that the H-2K gene product may be involved in the antigen recognition process by T cells.     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

The extent of self-MHC restriction of cytotoxic T cells in nude mice varies from mouse to mouse

There are conflicting results as to whether the response of athymic nude mice to TNP-modified self determinants is or is not H-2 restricted. We cultured spleen cells from 29 individual RNC (H-2k) nude mice with TNP-modified self determinants and tested the cultures for their ability to lyse TNP-modified self (RNC-TNP) and TNP-modified allogeneic (BALB/c-TNP) target cells. Each mouse was stimulated by two different protocols: either by the addition of TNP-modified irradiated nu/+ spleen cells or by TNP modification of the nude responder cells without addition of other cells. All mice could lyse RNC-TNP targets and about one-half could also lyse BALB/c-TNP targets, i.e., there was a 50:50 division between restricted and unrestricted responses. The magnitude of the response against RNC-TNP and whether the response was restricted were both independent of the method of stimulation. We conclude that H-2 restriction in these mice is imposed by an as yet unidentified environmental influence that can vary from one nude mouse to the next. The influence appears to act through negative selection because the modified self response is, if anything, higher in mice showing an unrestricted response.     

Stimulatory cells in the generation of T-cell-mediated cytotoxicity: Potent stimulating activity of a small radioresistant spleen cell population (RSCs)

The combined effects of irradiation followed by cultivation on a total spleen cell population in order to study the evolution of the stimulating potential in the in vitro generation of allogeneic cytotoxic T lymphocytes (CTLs) were tested. Results revealed that, after 3 days and up to at least 7 days of cultivating irradiated (1000 rad) spleen cells, the remaining living cells (radioresistant spleen cells or RSC) have the same potential to generate CTLs as irradiated noncultivated spleen cells. RSC can resist a 5000-rad irradiation and induce a primary cytotoxic response pattern similar to that of total spleen cells; they act in primary as well as in secondary cultures with optimal responder to RSC ratios of about 100, but are still stimulatory at MLC ratios up to 1000 or 5000. They are lysed by specific allogeneic CTLs and readily inhibit the specific lysis of H-2-identical labeled targets by CTLs. RSCs do not express unusual levels of H-2 or Ia antigens and do stimulate purified T cells. Alloantisera anti-H-2 are able to completely block the RSC-induced generation of CTL. This RSC population may prove to be a good model to study non-H-2- or H-2-associated, nonserologically detectable determinants interacting in the generation of T-cell-mediated cytotoxicity.     

Negative allogeneic effects in vitro. II. Mapping of histocompatibility differences leading to allosuppression.

When splenic T cells from normal mice recognize alloantigens on primed spleen cells there is a dramatic suppression of the secondary antihapten PFC response of the latter cells. From studies using intra-MHC recombinants it appears that differences at K or D alone can elicit allosuppression. There is sometimes suppression to differences in the I region. Even small differences in H-2K, as seen in the H-2b mutants, are sufficient to lead to such negative allogeneic effects. Thus, as far as has been determined, the specificity of allosuppresive T cells is indistinguishable from that of cytotoxic effector cells. Negative allogeneic effects and positive allogeneic effects different in the degree to which they influenced the primary and secondary responses. In our experiments no evidence for positive allogeneic effects was seen in the secondary response to TNP-carrier even with cells differing at the I region only. In contrast the primary response to SRBC was much enhanced by allogeneic cells and little suppression of that response was seen. It is suggested that the allosuppression is distinct from cytotoxicity and may be a profitable model for the study of suppressor T cells.     

Suppressors of the graft vs graft reaction in tolerance to alloantigens

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.     

AKR.H-2b lymphocytes inhibit the secondary in vitro cytotoxic T-lymphocyte response of primed responder cells to AKR/Gross murine leukemia virus-induced tumor cell stimulation.

We have previously shown that AKR.H-2b congenic mice, though carrying the responder H-2b major histocompatibility complex haplotype, are unable to generate secondary cytolytic T-lymphocyte (CTL) responses specific for AKR/Gross murine leukemia virus (MuLV). Our published work has shown that this nonresponsive state is specific and not due to clonal deletion or irreversible functional inactivation of antiviral CTL precursors. In the present study, an alternative mechanism based on the presence of inhibitory AKR.H-2b cells was examined. Irradiated or mitomycin C-treated AKR.H-2b spleen cells function as in vitro stimulator cells in the generation of C57BL/6 (B6) anti-AKR/Gross virus CTL, consistent with their expression of viral antigens. In contrast, untreated viable AKR.H-2b spleen cells functioned very poorly as stimulators in vitro. Viable AKR.H-2b spleen cells were also able to cause dramatic (up to > or = 25-fold) inhibition of antiviral CTL responses stimulated in vitro by standard AKR/Gross MuLV-induced tumor cells. This inhibition was specific: AKR.H-2b modulator spleen cells did not inhibit allogeneic major histocompatibility complex-specific CTL production, even when a concurrent antiviral CTL response in the same culture well was inhibited by the modulator cells. These results and those of experiments in which either semipermeable membranes were used to separate AKR.H-2b modulator spleen cells from AKR/Gross MuLV-primed responder cells or the direct transfer of supernatants from wells where inhibition was demonstrated to wells where there was antiviral CTL responsiveness argued against a role for soluble factors as the cause of the inhibition. Rather, the inhibition was dependent on direct contact of AKR.H-2b cells in a dose-dependent manner with the responder cell population. Inhibition was shown not to be due to the ability of AKR.H-2b cells to function as unlabeled competitive target cells. Exogenous interleukin-2 added at the onset of the in vitro CTL-generating cultures partially restored the antiviral response that was decreased by AKR.H-2b spleen cells. Positive and negative cell selection studies and the development of inhibitory cell lines indicated that B lymphocytes and both CD4- CD8+ and CD4+ CD8- T lymphocytes from AKR.H-2b mice could inhibit the generation of AKR/Gross virus-specific CTL in vitro. AKR.H-2b macrophages were shown not to be required to demonstrate AKR/Gross MuLV-specific inhibition, however, confirming that the inhibition by T-cell (or B-cell)-depleted spleen populations was dependent on the enriched B-cell (T-cell) population per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferative and cytotoxic immune functions in aging mice. II. Decreased generation of specific suppressor cells in alloreactive cultures

The ability of spleen cells from aged C57BL/6 mice to generate specific suppressor cells in mixed lymphocyte cultures (MLC) against allogeneic H-2 antigens was investigated. The suppressor cells from young and old mice were assayed in parallel for their ability to inhibit the proliferative response and the generation of cytotoxicity in fresh MLC. Suppressor cell generation was found to be significantly decreased in 41% of aged mice (23 to 28 mo) as compared to young controls (3-8 mo). The suppressor cells were H-2-specific, radiation-resistant (1000 R), and Thy-1+; they did not function by lysing the fresh stimulators or responder cells, or by absorbing the interleukin 2 in the fresh cultures. Suppression required very small numbers of cells to be effective. It was concluded that the effect of aging was less marked on specific suppressor cell generation than on generation of cytotoxic T cells in the MLC. However, a third type of response studied, the proliferative response, was affected earliest and most severely.     

T lymphocytes of young and aged rats. II. Functional defects and the role of interleukin-2

Splenic T lymphocytes of aged Lewis rats respond to Con A and PHA with diminished 3H-TdR uptake compared with splenic T lymphocytes of young Lewis rats. After immunization with allogeneic tumor cells, uptake of 3H-TdR in mixed lymphocyte-tumor cultures and T cell cytotoxicity against tumor target cells are significantly lower with spleen cells of aged rats compared with those of young rats. The culture of spleen cells of aged rats with Con A results in a diminished conversion of Ia-positive T cells from Ia-negative precursors compared with similar cultures of spleen cells of young rats. Spleen cells of both young and aged rats produce high amounts of IL-2 in response to Con A stimulation. "Old" T cells, however, bind relatively little IL-2, do not utilize it in culture, and do not respond to exogenous IL-2 with enhanced 3H-TdR uptake as do "young" T cells. In allogeneic MLTC, "old" T lymphocytes produce little IL-2 compared with "young" cells, and both "young" and "old" cells respond to exogenous IL-2 with enhanced 3H-TdR uptake and increased cytotoxic activity. The data suggest defects in the synthesis and/or recognition of IL-2 as well as defects in the regulation of Ia antigen expression may be responsible, in part, for the reduced T cell function in aged animals.     

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. II. Lack of antigenic specificity and immunogenetic requirements of allogeneic suppressive factor (ASF).

Mice were irradiated, infused with thymocytes and immunized with a variety of antigens, i.e., sheep or horse red blood cells (SRBC or HRBC), diphtheria toxoid (DT) or bovine gamma-globulin (BGG). The spleen cells (T.Spleen cells) were harvested 5 days later and cellfree extracts were prepared. The extracts contained an allogeneic suppressive factor (ASF) that was capable of inhibiting IgM antibody responses of allogeneic or semi-allogeneic unirradiated mice. ASF had to be injected within 24 hr of immunization to be effective and a single injection delayed, rather than abolished, the antibody response at the cellular level. However, daily injections of ASF resulted in persistent suppression of antibody response. ASF activity was antigen nonspecific, i.e., the antigen used to stimulate ASF production did not have to be the same as the antigen used to test for ASF activity. C3H T.Spleen extracts were even immunosuppressive when prepared by exposure to C3BF1 alloantigens only; such extracts suppressed antibody responses of C3BF1 and DBA/2 mice. C3H ASF was removed from extracts after incubation with C3BF1 spleen cells but not after incubation with C3H spleen cells. C3BF1 spleen cells which had been preincubated with C3H ASF were unable to generate antibody-forming cells upon transfer to irradiated C3BF1 host mice. This suggests that the ASF molecule may be or include receptors for alloantigens. The immunogenetic requirements for ASF activity were evaluated by injecting extracts from C3H, C57BL, C3BF and BALB/c T.Spleen cells into C3H, CBA, C57BL, BALB/c, DBA/2, A or C3H.A recipient mice. All extracts tested had ASF activity. However, all allogeneic recipients were not suppressed by the extract material. The suppressive activity of ASF seemed to require two (or more) antigenic differences between donors and recipients of extract material, an H-2K or I antigen difference and a second antigen difference, possibility Ig-1. In the limited numbers of strain combinations tested, T.Spleen extracts suppressed IgM antibody response only if exposed to H-2 and Ig-1 antigens, e.g., BALB/c (H-2d, Ig-1a) ASF suppressed A (H-2a, Ig-1e) but not C3H.A (H-2a, Ig-1a) or DBA/2 (H-2d, Ig-1c). Separate ASF molecules may react with separate antigens on the cell surface, i.e., with H-2 and gammaG2a. Alternatively, one ASF molecule may react with two structurally associated antigens. If the latter is correct, it is conceivable that the beta2-microglobulin which is non-covalently linked to the major component of H-2 molecules expresses allotypic antigens coded for by Ig-1 and beta2-microglobulin is one of the antigens recognized by ASF.     

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.