Macrophages and the functional differentiation of thymocytes (a review of the authors' own data)

Cellular, humoral, and genetic mechanisms of induction of T-effectors of the transplant vs. host reaction (THR) have been studied in two-cell culture of phagocyte mononuclears and thymocytes. A direct physical contact and similarity of H-2K locus of major histocompatibility complex between the cooperating cells in culture is required for successful induction of T-effectors of THR. Contact interaction of macrophages with thymocytes leads to accumulation of a 67 KDa humoral factor in the culture medium. Incubation of intact thymocytes with this factor leads to functional transformation of immature thymocytes into corresponding effector cells. Similarity of H-2K locus of the factor producers and intact lymphocytes is also required for successful humoral induction of the T-effectors. The surface H-2K antigen is able to induce formation of THR t-effectors from non-reactive thymocytes. The H2-K-specific mediator, affinity-isolated from the supernatant of the macrophage-thymocyte culture can also cause this induction.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Sorption of a macrophage factor inducing functional thymocyte transformation on cells of organs of various sources

When the macrophages and thymocytes are jointly incubated, mononuclear phagocytes produce a humoral factor which induces the functional transformation of thymocytes promoting their differentiation into more mature cells. The factor was provisionally called FIT (factor of induction of thymocytes). The data are provided on adsorption of the factor under study on cells of different organ origin. Thymocytes adsorb the FIT in proportion with the cell concentration and time of interaction between the factor and thymocytes. The FIT was shown to be adsorbed only on the T-cells. It is suggested that the differential sorption of the FIT is due to the presence of the FIT receptors only on T-lymphocytes.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

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.     

Immune complex effects on murine macrophages. I. Immune complexes suppress interferon-gamma induction of Ia expression

We have studied the effects of immune complexes on the expression of macrophage surface proteins in vitro. Increased expression of the H-2 molecules I-A, I-E, and K on the macrophage membrane was induced by in vitro culture with crude lymphokine or interferon-gamma. Expression of all three of the molecules was additionally increased by stimulating the cultures with heat-killed Listeria monocytogenes. Addition of soluble immune complexes to the cultures did not have any effect on macrophage expression of these proteins. However, significant inhibition of lymphokine or interferon-gamma induction of I-A, I-E, and H-2K was observed when macrophages were cultured on plates to which immune complexes had been bound. This inhibition was dose dependent, required an immunoglobulin (Ig) molecule with an intact Fc portion, did not require the presence of T cells, and occurred in the presence of indomethacin. Complexes containing IgG1, IgG2a, IgG2b, and IgE, but not IgM or IgA, antibodies mediated the inhibitory effect.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Regulation of lymphocyte proliferation and differentiation by macrophages.

In this paper, we consider the role of a macrophage secretory product in promoting thymocyte differentiation, as well as a macrophage-immune T cell interaction that results in augmented secretion of lymphostimulatory factors. When cultured with the thymocyte-differentiating factor (TDF), thymocytes show a physiological increase in H-2D and K, decreased sensitivity to lysis with anti-TL and complement, and acquisition of responsiveness in the mixed lymphocyte culture. Development of the mature phenotype requires 2 to 3 days of culture and, once attained, is stable. The induced antigenic changes do not require cell division. The activity demonstrated by TDF, which is not attributable to interferon and cannot be replaced by 2-mercaptoethanol, is also displayed by normal thymic macrophages themselves. Enhanced secretion of TDF and of a distinct mitogenic protein follows the interaction of macrophages and immune T cells. This interaction is shown to require physical contact of the two cell types and is regulated by products of the I-A region of the major histocompatibility complex.