Depressed lymphoproliferative response in mixed leukocyte reaction after Mis-locus alloimmunization

The proliferation of BALB/C lymphocytes preimmunized with Mis or H-2 incompatible cells in response to alloantigens was studied. The results show that preimmunization with Mis-incompatible spleen cells inhibits the lymphoproliferative response against alloantigens whereas preimmunization with H-2 incompatible spleen cells enhances it. It is suggested that Mis coded determinants activate suppressor mechanisms responsible for the unresponsiveness of these preimmunized lymphocytes against alloantigens.     

Macrophages in the mixed leukocyte culture reaction (MLC)

Two aspects of the mixed leukocyte culture reaction have been investigated. In the first study the capacity of two highly purified adherent cell populations, one consisting of neutrophils the other of macrophages, to support the reactivtiy of purified lymphocytes was compared. Activity was entirely confined to the latter preparation. In the second study, the ability of platelets, neutrophils, lymphocytes and macrophages to provoke a MLC reaction was studied. Macrophages were found to be 10 times as efficient in eliciting a reaction as an equal number of lymphocytes. Neutrophils and platelets appeared to be devoid of stimulating capacity. This lack of stimulation was shown to be due to a true deficiency in stimulating capacity rather than to an inhibition obtained by high concentrations of neutrophils and platelets. The two functions of the macrophage in MLC, its auxiliary helper role, and the elicitation of the reaction, may be dependent on one and the same mechanism.     

Activated B lymphocytes: stimulators of an augmented autologous mixed leukocyte reaction

The characteristics of the non-T cell(s) which stimulate T-lymphocyte proliferation in the autologous mixed leukocyte reaction (AMLR) have been at issue since this in vitro reaction was first described. Dendritic cells have been shown to be the most potent stimulator cells, but B cells, null cells, and macrophages have also been demonstrated to have the capacity to stimulate autologous T-cell proliferation. A cell preparation obtained from human peripheral blood was highly enriched for surface immunoglobulin-positive B cells. These cells were activated by brief culture with various B-cell mitogens and then compared to untreated B cells with regard to stimulatory activity in the AMLR. Mitogen-activated B cells were markedly augmented in their capacity to stimulate autologous T-cell proliferation when compared with untreated B cells. Fractionation of the B-cell preparation into high- and low-density subpopulations demonstrated that the high-density cells, enriched in resting B cells, had minimal stimulatory activity but could be activated to have increased AMLR-stimulatory capacity. Proliferation of the activated B lymphocytes was not required for the generation of the augmented AMLR. Response to both untreated and mitogen-activated B cells was a property of T4-positive T lymphocytes. The increase in stimulatory capacity was associated with a decrease in cell surface immunoglobulin, but no significant alteration in the percentage or fluorescence intensity of anti-Ia staining cells was detected. Activated B cells which are generated in vivo may acquire the capacity to generate T effector cells or factors important in the regulation of B-cell function.     

Human alveolar macrophages: HLA-DR-positive macrophages that are poor stimulators of a primary mixed leukocyte reaction

Previous studies demonstrated that alveolar macrophages (AM) from most normal human volunteers failed to stimulate the antigen-induced proliferation of peripheral blood T lymphocytes although greater than 90% of AM expressed HLA-DR antigens. The current studies establish that AM also fail to induce allogeneic peripheral blood mononuclear cells to proliferate in a mixed leukocyte reaction (MLR). Suppressive activity by AM was not an explanation for their failure to induce an MLR. Indirect immunofluorescence established the presence of both HLA-DR and DQ antigens on the majority of AM and the persistence of these antigens on cells in culture for up to 6 days, the period of time required to observe a maximal MLR. Metabolic labeling experiments also demonstrated that HLA-DR antigens were synthesized by AM. It was recently reported that AM secrete relatively small amounts of IL 1, an important ancillary signal provided by accessory cells to enhance the stimulation of lymphocyte proliferation. However, addition of optimal concentrations of IL 1 to cultures containing AM failed to enhance the MLR. Thus, there is at least one additional, but as yet undefined, requirement for an accessory cell to induce an optimal MLR besides the display of HLA-D region antigens and the secretion of IL 1. In contrast, AM were effective in specifically stimulating proliferation of alloreactive T cell lines, suggesting that at least some cell lines do not require this nonspecific undefined second signal. We speculate that although AM may not initiate primary immune responses in the lung, they may be important in maintaining immune-mediated inflammatory responses by specifically restimulating already activated T cells.     

HLA-B8 phenotype associated with an increased mixed leukocyte reaction

Peripheral blood leukocytes from normal volunteers and from patients with untreated Hodgkin's diseases have been assayed for the magnitude of their responses to pools of selected stimulating cells in a quantitative one-way mixed leukocyte reaction (MLR). Responses by leukocytes from individuals withHLA-B8 segregated above the median response regardless of whether the donors were healthy or were patients with Hodgkin's disease. These results suggest the presence of a gene (or genes) in the responding cells that has an effect on the magnitude of the MLR. This gene is in nonrandom association with the gene coding forB8 at theB region of theHLA complex. The observed linkage disequilibrium ofB8 with a gene controlling the proliferative response to alloantigens may be the basis for the increased frequency ofB8 in long-term survivors in carcinoma of the breast and Hodgkin's disease, and for the association ofB8 with several autoaggressive diseases.     

Capacity of B cells to function as stimulators of a primary mixed leukocyte reaction

The capacity of B cells to serve as stimulator cells for a primary mixed leukocyte reaction (MLR) was evaluated. Percoll-fractionated B cells were stimulated with lipopolysaccharide and dextran sulfate (L/D) or a B cell stimulatory factor (BSF-1)-containing culture supernatant, and then were fixed before being used as stimulator cells to more precisely define the state of activation associated with MLR stimulatory capacity. It was found that unstimulated B cells or B cells stimulated for 1 day with L/D or BSF-1 were incapable of initiating a primary MLR, whereas B cells incubated for 3 days in L/D were potent stimulators. The differential activity of 1 day L/D- and BSF-1-activated B cells compared with 3 day L/D-activated B cells was not related to the amount of the relevant MHC class I or class II alloantigens on these cell populations, because all three groups had large increments in MHC class II expression in the following order: BSF-1 greater than 3 day L/D greater than 1 day L/D, and had little difference in MHC class I expression. Also, all three populations were capable of stimulating both MHC class I- and class II-specific T cell hybrids. It was concluded that the capacity of 3 day L/D-activated cells to stimulate a primary MLR was due to the elaboration of necessary co-stimulator molecules. We evaluated whether interleukin 1 (IL 1) was the co-stimulator involved. That this was not the case was indicated by two findings. First, 3 day-activated L/D cells failed to express IL 1 activity as measured by a highly sensitive IL 1 assay that utilizes the T cell line D10.G4.1. Second, recombinant IL 1 added to MLR cultures containing 1 day L/D- or BSF-1 activated B cells failed to function as a co-stimulator. In contrast, the phorbol ester PMA was a potent co-stimulator in this system. We conclude from these experiments that appropriately activated B cells can function as stimulators of a primary MLR, and that they elaborate critical co-stimulator molecules, distinct from IL 1, that enable them to function in this regard.     

Suppression of the mixed leukocyte reaction by serum from polynucleotide-injected mice

Sera from mice which have been injected iv with either poly(A) X poly(U) or poly(I) X poly(C) 1 1/2 hr prior to bleeding were found to suppress the mixed lymphocyte reaction. This effect was reduced considerably by 18 hr. Characterization of the suppressive sera revealed it (a) was stable to heating at 56 degrees C for 1 hr and freezing at -20 degrees C for 1 month; (b) had a molecular weight greater than 30,000; (c) could be induced in sera from athymic nude mice; and (d) was present to a lower degree in sera from aging mice.     

Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin

The immunomodulating effects of heparin and natural and synthetic heparinoids (which are now undergoing clinical trials for the treatment of AIDS) on cellular immunity (DNA synthesis and cytotoxic responses of mouse lymphocytes to allogeneic cells and histocompatible tumors) were studied. The results showed that (1) high and low m.w. heparin enhanced mouse antitumor and antiallogeneic cell responses in vitro; (2) other sulfated heparinoids did not have this enhancing activity and some of them (including dextran sulfate) totally suppressed generation of cytotoxic cells; (3) these immunomodulating activities of heparin and heparinoids did not correlate with their anticoagulant effects, degree of sulfation, and mitogenic activity; (4) heparin did not increase the production of IL-2 and did not enhance the action of IL-2 on the cells in MLC, heparin also had no effect on the growth-promoting activity of IL-2 on cloned cytotoxic T cells; (5) heparin had a synergistic enhancing effect with IL-1 on the generation of cytotoxic cells in MLC; and (6) heparin abolished endothelial cell growth factor-induced suppression of cytotoxic response. The latter two effects by themselves, however, could not fully explain the entire immunoenhancing activity of heparin. These results indicate that heparin and heparinoids have multiple effects on the immune system and that some of them can enhance, whereas others can suppress cell-mediated responses.     

Cyclic nucleotide phosphodiesterase in vertebrates

The cellular concentration of cyclic nucleotides is largely dependent upon the activity of the enzymatic system responsible for their degradation: cyclic nucleotide phosphodiesterase. This enzymatic system thus plays a crucial role in the regulation of the multiple functions which are modulated by cyclic nucleotides in the organism. Many methodological problems, as well as the complexity of the phosphodiesterase system have long maintained a confusion in this field. Recent progresses (purification to homogeneity of some enzymatic forms, discovery of regulatory mechanisms, particularly) have brought a considerable evolution in the knowledge of the system. It is now well established that cyclic nucleotide phosphodiesterase exists under several isoenzymatic forms, the properties and distribution of which largely differ from a tissue to another. Some of these forms are relatively well characterized, while the representativity of others is still discussed. The significance of this multiplicity of isoenzymes, and their interrelationships are presently under study. A very interesting aspect in the study of this enzymatic system is that it is submitted to several physiological regulatory processes. Recent studies on this point suggest that phosphodiesterase might play a major role in the response of the organism to several hormones. These fundamental studies of phosphodiesterase system find a most interesting application in the pharmacological field. Indeed, numerous synthetic compounds which inhibit the enzyme present a strong pharmacological interest.     

Macrophage factors which enhance the mixed leukocyte reaction initiated by dendritic cells

Stimulator cells for the allogeneic MLR are dendritic cells but not macrophages (M phi). M phi, however, enhance the MLR initiated by relatively low doses of dendritic cells. The present report demonstrates that the enhancement of the MLR is mediated by two factors produced by M phi. One is IL-1, inasmuch as it has a Mr approximately 15 kDa, and both partially purified IL-1 and rIL-1 also enhance the MLR. The other has been identified as granulocyte-macrophage (GM)-CSF. It had a Mr approximately 25 kDa, and is reproduced by rGM-CSF. Moreover, the MLR-enhancing activity of both the 25-kDa molecule and rGM-CSF have been neutralized by anti-GM-CSF antiserum. Both IL-1 and GM-CSF have autonomous enhancing activity, but they collaborate with each other in enhancing the MLR. Both factors act on DC to augment their stimulatory activity for allogeneic T lymphocytes.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.