Functional differentiation of thymocytes induced by macrophages: cellular, humoral and genetic aspects

The cellular, humoral and genetical mechanisms of induction of T-effectors of the graft vs. host reaction (GHR) were studied in a double-cell culture of phagocytizing mononuclears with thymocytes. Experiments were carried out on mice of inbred and recombinant strains. The GHR intensity was estimated by the increase in the number of cells in the popliteal lymph node, regional with reference to the introduction of parental thymocytes into the F1 hybrid. For the induction of the GHR T-effectors from the immature population of thymocytes to be realized, a direct physical contact and identity by the H-2K locus of the major histocompatibility complex between the cooperating cells in culture are indispensable. An antiserum containing antibodies against the H-2K locus products prevents the induction. At the same time antibodies against antigens controlled by loci of I-region or the H-2D locus do not affect the accumulation of T-effectors. Contact interaction of phagocytizing mononuclears with thymocytes results in accumulation of a 65,000 D humoral factor in the culture medium. Incubation of the intact thymocytes with this factor ensures functional transformation of immature thymocytes to corresponding effector cells. For the humoral induction of T-effectors to be successfully realized, identity by the T-2K locus between the factor producents and intact thymocytes is indispensable, as well as in the conditions of direct intercellular interaction. It is suggested that H-2K specificity is incorporated into the factor structure.     

Control by genes of the major histocompatibility complex of macrophage-thymocyte interaction during induction of T-effectors of graft versus host reaction. II. Analysis at the level of humoral factor activity

The data are presented on the genetic restriction of interaction between humoral factors and intact thymocytes during the formation of T-cell effectors GVH from thymus cells. The source of humoral factors was a cultural medium from combined cultivation of macrophages and syngeneic thymocytes during 18 h. The intensity of the local GVH reaction was evaluated by the index of the lymph nodes enlargement. Genes of the H-2 system control interaction of mediators with thymocytes during the formation of T-cell effectors. H-2 region genes identity of supernatant generative cells and intact thymocytes was necessary for the effective induction of T-effectors GVH. I and D region differences did not affect the formation of T-cells inducing GVH.     

Control by major histocompatibility complex genes of macrophage interaction with thymocytes during induction of T-effectors of graft vs host reaction. I. Genetic control on the cellular level

H-2 complex control of interaction between macrophages and thymocytes during induction of T-cell effectors GVH was studied. Intensity of the local GVH reaction was evaluated by the index of the lymph nodes enlargement. Genes of the H-2 system control interaction of macrophages with thymocytes during the formation of T-cell effectors GVH. H-2K subregion genes identity of interacting cells was necessary and sufficient for the effective induction of T-effectors GVH. I and D region differences did not affect the formation of T-cells inducing GVH.     

The role of major histocompatibility complex genes in the reaction of macrophages and thymocytes during induction of T-effectors of the graft vs host reaction. III. Effect of antisera against H-2 complex antigens on the realization of the process

The data are presented on the influence of antisera to different products of the histocompatibility gene complex on the interaction between macrophages and thymocytes during the formation of T-cell GVH effectors. The intensity of the local GVH reaction was evaluated by the index of the lymph nodes enlargement. The close physical contact between cells was necessary for the induction of T-effectors GVH. Treatment of macrophages or thymocytes with anti-H-2 and anti-H-2K antibodies, prior to the beginning of combined incubation, or addition of these antisera to the cultural media suppressed the formation of T-cell inducing GVH. Anti-H-2 I and anti-H-2 D sera did not affect the formation of GVH T-effectors.     

Changes in the expression of the surface antigens of thymocytes during their cultivation with macrophages

The data on changes in expression of H-2 complex and Thy-1 antigens on cell surface of thymocytes resulting from their incubation with peritoneal macrophages has been presented. The process of joint cultivation of thymocytes with macrophages leads to significant decrease in number of cells with Thy-2-antigen and increase in that with H-2 complex antigens. An increase in H-2K+ cells in experimental thymocytes as compared to control ones was observed. No changes in H-2D expression was observed. A significant increase in Ia+ macrophages was observed after interaction with thymocytes as compared with intact mononuclear phagocytes.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Immunogenetic analysis of H-2 mutations. III. Genetic mapping and involvement in immune reactions of the H-2ka mutation.

Mutation M523 (H-2ka) occurred spontaneously in strain CBA/CaLacSto and was discovered during routine skin graft testing for genetic homogeneity. By linkage and complementation tests, the mutation was previously mapped in the K end of the H-2 complex. We demonstrate that the mutation occurred in the K region, without affecting the I region in the K end of the complex. The mutant antigens cause rejection of skin grafts, stimulate cells in mixed lymphocyte culture, and function as stimulators as well as targets in cell-mediated lymphocytotoxicity. Yet, they are serologically indistinguishable from the antigens of the original strain and do not induce formation of humoral antibodies upon immunization of the CBA strain. Together with the results obtained on testing of other H-2 mutants, the data strongly support the notion that classical H-2 antigens (i.e., products of the H-2K and H-2D loci) can function as lymphocyte-stimulating determinants, and that I-region differences are not required for the induction of strong cell-mediated lymphocytotoxicity.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

T100: a new murine cell surface glycoprotein detected by anti-Lyt-2.1 serum.

A cell surface glycoprotein (designated T100) of apparent m.w. 100,000 by SDS-PAGE under reducing and nonreducing conditions was precipitated from NP-40 extracts of surface radiolabeled thymocytes from a variety of inbred strains of mice by the standard noncongenic Lyt-2.1-typing serum. The inbred stain distribution, trypsin sensitivity on intact cells, and apparent m.w. of T100 suggest that it is different from Lyt-2.1. Inheritance and expression of T100 suggest that it is determined by an allele at a single locus, and testing of CXB recombinant inbred strains and B6.C minor histocompatibility congenic strains suggest that this locus is linked to H-25. Antiserum absorption experiments, two-stage cytotoxicity assays, and results of immunoprecipitations performed after prebinding antibody to radiolabeled thymocytes suggest that some T100 is accessible to antibody on the intact cell surface. However, for unknown reasons the number of cells required to absorb anti-T100 precipitating activity from antiserum was much higher than for removal of anti-Lyt-2.1 activity. A molecule with properties of T100 was also detected on lymph node cells and on the AKTB-1 lymphoma.     

Thymus hormones do not induce proliferative ability or cytolytic function in PNA+ cortical thymocytes

A variety of thymus hormone preparations, as well as drugs known to perturb cell differentiation, were tested for their ability to induce nonfunctional cortical thymocytes to become functional precursor cells. Murine cortical thymocytes, defined as the high peanut agglutinin (PNA) binding or as the low H-2K, major [86%] thymocyte subpopulation, were isolated by fluorescence-activated cell sorting. Their function was assessed in a high cloning efficiency, growth factor saturated, concanavalin A-stimulated limit-dilution culture system, determining the number of precursors of extended clones (PTL-p), or determining with a lectin-mediated tumor-lysis readout the number of precursors of cytolytic clones (CTL-p). The hormone preparations tested were crude or partially purified culture supernatants from thymus "epithelial" monolayers (TES), soluble extracts of thymic nonlymphoid tissue (STF), semipure thymus humoral factor (THF), and the pure peptides thymopoietin 32-36 (TP5) and "facteur thymique sérique" (FTS). These preparations were either added directly to the limit dilution cultures, or were first preincubated with the cells, which were then subjected to limit-dilution culture. In no case did the hormone preparations cause any increase in the level of PTL-p or CTL-p in the PNA+ or low H-2K thymocyte population, even though a conversion of only a few percent to functional cells could have been detected. Two possible explanations are considered. One is that the main function of these materials is to control post-thymic peripheral T cells, rather than to induce intrathymic differentiation. Another is that the typical cortical thymocyte is beyond the stage at which thymocytes can be induced by hormones, a view that is strengthened by the failure of either 5-azacytidine or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate to activate these cells. In this latter explanation the true intrathymic target of hormone action may be an earlier, and very minor, thymus subpopulation.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Influence of asparagine-linked oligosaccharides on tumor cell recognition in the mixed lymphocyte reaction

The ability of an Ia+ B cell lymphoma, AKTB-1b, to stimulate thymocytes in the allogeneic mixed lymphocyte reaction is dependent on its prior treatment with either swainsonine or deoxynojirimycin, two inhibitors of the processing of asparagine-linked oligosaccharides. In the absence of drug treatment, the tumor cells fail to stimulate thymocytes, whereas pretreatment of the tumor cells with either drug results in a five- to 10-fold increase in their ability to induce thymocyte proliferation. Drug-treated AKTB-1b stimulates thymocytes at levels comparable to those obtained with allogeneic splenocytes. In contrast, the untreated lymphoma does stimulate splenic lymphocytes, and pretreatment with either inhibitor only marginally increases the response. Genetic studies demonstrate that the thymocyte response is still H-2 locus restricted and can be blocked by monoclonal antibodies against two tumor cell major histocompatibility antigens, H-2K and I-A. Drug treatment does not change cell surface I-A expression, and H-2K levels are apparently decreased one-third by deoxynojirimycin but are not affected by swainsonine. To verify that the drug protocol used was capable of altering the glycoconjugates of membrane-associated proteins, the endo-beta-N-acetylglucosaminidase H (endo H)-sensitivity of immunopurified H-2K and I-A was analyzed by SDS-PAGE. These studies demonstrated that swainsonine treatment does result in cell surface expression of glycoconjugates with altered oligosaccharide moieties. Likewise, deoxynojirimycin treatment results in the cell surface expression of an I-A alpha polypeptide with altered oligosaccharide chains while only marginally affecting H-2K and not affecting the I-A beta chain. An intracellular form of the I-A beta chain sensitive to endo H digestion in the presence of deoxynojirimycin is not detectable at the cell surface. Neuraminidase-digested AKTB-1b are also capable of stimulating allogeneic thymocytes. These studies demonstrate that changes in the glycosylation state of the tumor cell can markedly influence its recognition by allogeneic lymphocytes, and further, that different T cell populations differ in their response to such changes.     

Genetic control of the humoral response to an H-2 public specificity.

The humoral response of mice to an H-2 public specificity, termed H-2. '28' was found to be under genetic control. The genes determining this specificity were mapped to both the H-2K and H-2D regions, suggesting possible structural homologies between the products determined by these two regions. Genetic analyses indicated that a single non-H-2-linked gene regulates the anti-H-2. '28' response. In a backcross study, no linkage was detected between this putative H-2. '28' Ir gene and either the V H region or the Ly-2 locus to which the K-light chain locus is linked. Thus, a regulatory rather than a structural genetic locus seems a more likely basis for differences in response to this antigen. Our data further indicate that control of the humoral response to H-2. '28' is determinant specific since responses of the same backcross mice to other K and D alloantigens were not found to be subject to the same control.     

Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanying the appearance of cytolytic and interleukin 2-producing cells

At the 14th day of gestation, embryonic thymocytes+ are large, functionally incompetent cells with H-2K+ Thy-1+ B14- Ly-2- L3T4- phenotype, some of which express TL antigen. Differentiation of these cells in organ culture is characterized by: 1) appearance of cells expressing Ly-2 and L3T4 molecules, first among the population of large cells, after 2 days of culture; 2) appearance of small H-2K- Thy-1+TL+B14+Ly-2+L3T4+ and H-2K-Thy-1+TL+B14-Ly-2+ L3T4+ cells between days 2 and 4; 3) accumulation of small H-2K- Thy-1+ TL+ B14- Ly-2+ L3T4+ (but not H-2K- Thy-1+ TL+ B14+ Ly-2+ L3T4+) cells until day 5 of culture, and their subsequent gradual disappearance which is paralleled by an increase of the proportion of medium-sized H-2K+ Thy-1+ TL- B14- cells with various Ly-2 L3T4 phenotypes; 4) appearance and subsequent accumulation of cytolytic and IL 2-producing cells between days 4 and 6. Comparison of these results with the data from similar in vivo studies shows that differentiation of organ-cultured thymocytes rather closely follows the in vivo development only during the first week of culture and shows significant deviations thereafter. Precursors of cytolytic cells and cytolytic effector cells as well as IL 2-producing cells are found among both Ly-2+ and Ly-2- populations of thymocytes, indicating that there is no clear association between Ly-2 phenotype and the ability to kill or to secrete IL 2.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions.

An in vitro assay was used for assessing the participation of various cell surface molecules and the efficacy of various cell types in the deletion of Ag-specific immature thymocytes. Thymocytes from mice expressing a transgenic TCR specific for the male Ag presented by the H-2Db class I MHC molecule were used as a target for deletion. In H-2d transgenic mice, cells bearing the transgenic TCR are not subjected to thymic selection as a consequence of the absence of the restricting H-2Db molecule but, nevertheless, express this TCR on the vast majority of immature CD4+8+ thymocytes. In this report we show that CD4+8+ thymocytes from H-2d TCR-transgenic mice are preferentially killed upon in vitro culture with male APC; DC were particularly effective in mediating in vitro deletion when compared with either B cells or T cells. Deletion of CD4+8+ thymocytes by DC was H-2b restricted and could be inhibited by mAb to either LFA-1 alpha or CD8. Partial inhibition was observed with mAb to ICAM-1, whereas mAb to CD4 and LFA-1 beta were without effect. These results are the first direct evidence of LFA-1 involvement in negative selection and provide further direct support for the participation of CD8/class I MHC interactions in this process. Like the requirements for deletion, activation of mature male-specific CD4-8+ T cells from female H-2b TCR-transgenic mice was also largely dependent on Ag presentation by DC and required both LFA-1/ICAM and CD8/class I MHC interactions; these results support the view that activation and deletion may represent maturation stage-dependent consequences of T cells encountering the same APC. Finally, our results also support the hypothesis that negative selection (deletion) does not require previous positive selection because deletion was observed under conditions where positive selection had not occurred.     

Bioassays for the detection of a cytokine that enhances the development of cytolytic T lymphocytes

Interleukin 2 (IL-2) is an important growth factor for cytolytic T lymphocytes (CTL). Other factors here termed cytolytic differentiation factor(s) (CDF) may be required for CTL responses, but it has been difficult to identify suitable bioassays. We report a polyclonal assay in which lytic activity develops after 2 days of culture with lectin and lymphokines. CDF is required for the development of Thy-1+, CD8+, CD4- CTL in this assay. The responsive T cells are thymocytes from certain strains of mice (A, Swiss NCS, B6.H-2k) or peripheral T cells cultured in the presence of high doses of hydrocortisone acetate. If these precautions are taken, little or no lytic activity develops in the presence of rIL-2 alone. CDF is present in the media of mitogen-stimulated mouse spleen or human blood mononuclear cells, but not in the conditioned media of a number of mouse and human cell lines. It is immunologically distinct from IL-2, but only acts in concert with IL-2 to induce CTL. A large panel of available mouse and human cytokines do not synergize with Il-2 in the bioassay. These include interferons, colony-stimulating factors, lymphotoxin/tumor necrosis factor, and IL-1, -3, and -4. Therefore a distinct CDF(s) seems essential for the induction of at least some CTL. The assays described here should be useful in purifying the molecule responsible for this biological activity.     

Thy-2: A murine thymocyte-brain alloantigen controlled by a gene linked to the major histocompatibility complex

We describe a new murine cell-surface alloantigen, provisionally designated Thy-2, which is expressed primarily on thymocytes and brain tissue. Although Thy-2 is also expressed at lower levels on bone-marrow and spleen cells, this antigen does not appear to be present on lymph-node, liver, or red blood cells. Immunoprecipitation of surface-labeled thymocyte extracts from a variety of inbred strains reveals this antigen to be a single polypeptide of 150 000 daltons. Quantitative membrane immunofluorescence demonstrates that Thy-2 is a minor cell-surface component which is present on the majority of thymocytes. Mice heterozygous at theThy-2 locus express approximately 50 percent as much antigen as positive homozygotes. Expression of the Thy-2 alloantigen is controlled by a single semidominant gene located approximately 3 cM to the right of theH-2K locus on chromosome 17.     

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.