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.     

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.     

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.     

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.     

Enhancement of murine T cell I-J expression by limited proteolysis

I-J-encoded structures on peripheral T cells and thymocytes appear normally to be blocked or shielded by material that is susceptible to proteolysis. Limited proteolysis with trypsin, papain, pronase, or chymotrypsin increased the number of peripheral T cells and thymocytes lysed by anti-I-Jk serum and complement. Proteolysis did not induce I-Jk expression on B cells or on negative strain T cells. Increased lysis was enzyme concentration and time dependent and was not due to increased susceptibility of protease-treated cells to lysis by antibody plus complement; proteolysis rendered T cells and thymocytes less susceptible to lysis by anti-H-2Kk, anti-H-2Dd, and anti-Lyt-2 antibodies. Absorption experiments showed that I-Jk determinant density was increased in the protease-treated T cell population. The I-Jk determinants detected are proteins or glycoproteins; extended proteolysis removed these molecules from the T cell surface. Treatment of T cells or thymocytes with activated macrophage culture supernatant containing proteolytic activity produced a small but reproducible increase in I-Jk expression. Proteolysis of lymphocyte membranes, possibly mediated by macrophages, may have a role in cellular differentiation and immune activation.     

Neonatal infection with mouse thymic virus. Differential effects on T cells mediating the graft-versus-host reaction.

Neonatal infection with mouse thymic virus (TA), a murine herpes virus, produced extensive but temporary necrosis of the thymus which was maximal at 10 to 14 days of age. Studies of precursor and amplifier cells mediating graft-vs-host (GVH) reactivity of thymocytes, spleen cells (SC), and lymph node cells (LNC) of normal and TA-infected mice were made at 4 and 8 weeks of age. Infection with TA resulting in a profound reduction (70 to 80%) in the direct GVH reactivity of thymocytes at both ages; by comparison, the capacity of thymocytes to produce synergy when combined with normal LNC was normal at 8 weeks. Direct GVH reactivity of SC was depressed 90% 4 weeks after infection with TA but returned to near normal at 8 weeks. Direct GVH reactivity of LNC from TA-infected mice was normal at 4 and 8 weeks of age, but amplifier T cell activity in LNC was markedly depressed at 8 wekks. These results demonstrate that TA has highly selective effects upon subpopulations of T cells in thymus and lymph node.     

A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.     

Multiple CTL subsets generated by H-2.L locus products stimulation: evidence for an antigenic determinant private to H-2.Ld

Analysis of the fine specificity of CTL subpopulations raised by an H-2.L locus products stimulation (H-2dm2 anti-H-2d) was performed by absorption experiments by using monolayers of macrophages of H-2m, H-2q, H-2b, and H-2k haplotypes. The results show the existence of four CTL subsets. The pattern of reactivity of three of them could be correlated with that of antibodies present in H-2dm2 anti-H-2d antisera (anti-H-2.64, anti-H-2.65, and anti-H-2.Kk). The fourth CTL subset reacted with a specificity unique to H-2.Ld molecules (a private specificity?), absent on cells from H-2m, H-2q, H-2b, and H-2k haplotypes, and undescribed as yet by serologic methods. These data support the hypothesis that the H-2.L locus products are comparable in their antigenic properties to those of the H-2.K and H-2.D loci.     

Effect of the H-2 gene complex rates of fibroblast intercellular adhesion

The rate of collection of embryo fibroblast single cells by an embryo fibroblast monlayer was realted to the H-2 haplotype of the fibroblast monolayer. The rate was highest for the H-2s strains and lowest for the H-2k strains with all other strains examined being intermediate. As opposed to monolayers prepared from the A and C3H background animals, monolayers from B10 background mice only demonstrated an H-2 haplotype dependent rate differential after treatment with fetal calf serum or neuraminidase. The relationship that was seen between monolayer H-2 haplotype and rate of adhesion with embryonic monolayers was not observed with either congenic 3T3 cell lines or fibroblasts derived from adult tissues. It was further shown that the rate of single cell pick-up could be substantially reduced by incubating the monolayers with the appropriate polyspecific anti-H-2 antisera. The inhibition observed appeared to be directly related to anti-H-2 antibody binding and was not merely a function of ligand binding to the cell surface, as antisera directed against other fibroblast cell surface antigens did not significantly inhibit the adhesive rate. These results indicate a role for the H-2 gene complex in modulating fibroblast-fibroblast intercellular adhesion.     

Interaction between thymocytes and thymus-derived macrophages. I. Surface components participating in mutual recognition

Incubation of C57BL/6 thymus-derived macrophages (TDM phi) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDM phi formed rosettes with thymocytes after 6 hr of interaction at 4 degrees C. Rosette formation of the immature PNA+ thymocyte fraction was up to fivefold higher than that of PNA- and cortisone-resistant thymocytes. Pretreatment of PNA- thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 mM D-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-Ab mAb and its F(ab')2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.     

Modification of exogenous haemopoietic spleen colony formation in irradiated mice by antisera against mouse thymocytes and mouse brain.

Treatment of donor bone marrow in vitro with both anti-thymocyte serum (ATS) and anti-mouse brain serum (RAMBS) inhibits the formation of haemopoietic spleen colonies in irradiated and reconstituted mice. This activity of the antisera may be completely (ATS) or partly (RAMBS) eliminated by absorption with thymocytes. The effect of the antisera is complement-independent. Most likely it depends on the existence and functionality of phagocytes (macrophages) in the recipients.     

The role of Ia antigens in the activation of T cells by concanavalin A: an evidence for the species restriction between T cells and accessory cells

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.     

Specific interaction of phagocytosing mononuclears with T cells during formation of the thymocyte-induction factor

Data are provided on specific secretion by macrophages of a factor inducing the functional differentiation of thymocytes. The contact of mononuclear phagocytes with lymphocytes of T-cell type induces the formation of this transmitter. Interaction of macrophages with B-cells does not lead to the secretion of this monokine. Treatment of macrophages with nonspecific stimulators exerts no effect on the production of the factor.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts.

Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.     

Physical separation of "suppressor" from "helper" thymocytes.

Thymocytes from 1-month-old NZB/W mice were separated at unit gravity and then studied for helper or suppressor effects in a GVH assay. Thymocytes with a settling rate of 4 to 6 mm/hr suppressed but did not help. In contrast, help but no suppression characterized thymocytes settling at 6 to 10 mm/hr. Thus helper and suppressor thymocytes in this GVH assay were physically separated.     

Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells.

The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.     

Recombinant haplotype H-2 in mice of the congenic resistant strain B10.D1(R108)/Y

Recombinant H-2 haplotype of mouse strain B10.D1(R108)/Y (symbol R108) obtained in experiments with skin grafting in the course of developing the CR B10.D1/Y strain (strain DBA/LacY--the donor of H-2q) was studied. Strains with recombinant H-2 haplotypes a, h2, g1, i3, i5, i7, m, y1 were used. Alleles of different H-2 (K, I, D) regions were determined according to the presence or absence of genetic complementation in the F1 test with skin grafts. R108 recombinant was studied by serological methods with panel of anti-H-2 sera. Anti-H-2Kb (H-2.33) and anti-H-2Dq (H-2.30) monospecific antisera were used in microcytotoxicity test and in absorption experiments in vitro. It was concluded that crossing over between H-2b and H-2q chromosomes, which led to formation of recombinant H-2 haplotype of R108 mice, occurred at I region, between IA and IC subregions. The H-2 complex of R108 line has KbIAbIJ?IE?ICqSqDq alleles. bq1 symbol was proposed for the H-2 haplotype of B10.D1(R108)/Y strain.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. IV. Comparative analysis of the blocking effects of anti-Lyt-2 and anti-H-2 antibodies on allogeneic and PHA-mediated killing

Blocking of cell-mediated lympholysis (CML) by anti-Lyt-2 antibodies was compared with that by anti-H-2 antibodies which most likely inhibit CML by blocking antigen recognition by cytotoxic T lymphocytes (CTLs). Both antibodies were shown to inhibit the early Mg2+-dependent process of killing. Moreover, the anti-H-2-sensitive event was found to be reversible by the antibody as was the case with the anti-Lyt-2-sensitive event, suggesting that the two antibodies block the same event taking place during the Mg2+-dependent stage. Both types of antibody were also shown to be capable of inhibiting the phytohemagglutinin (PHA)-mediated non-specific killing activity of CTLs. However, in the case of anti-Lyt-2 antibodies, available monoclonal antibodies failed to inhibit PHA-mediated killing whereas conventional antisera did. The results thus suggest multiplicity and heterogeneity of Lyt-2 determinants or the existence of multiple products of Lyt-2-linked genes. In addition, an anti-H-2 antiserum also exerted a specific inhibitory effect on PHA-mediated killing. Thus there appears to be a general requirement for involvement of the Lyt-2 molecules on CTLs and major histocompatibility complex (MHC) products on the target cells. The implications of these observations are discussed.