Modulation of allogeneic stimulation in man. I. Characterization of an in vitro induced suppressor macrophage population.

Cultured human peripheral blood mononuclear cells suppressed the allogeneic response of fresh autologous lymphocytes. This suppressor activity developed gradually over a period of one week. The cells primarily responsible for this effect were enriched by Ficoll density gradient centrifugation. It was found that the suppressor cell is a large, low density nylon wool adherent, radioresistant, phagocytic, and nonspecific esterase positive mononuclear cell. Moreover, these cells did not form E rosettes and were Fc positive. Electron microscopy confirmed that suppressor cells were macrophage like. Suppressor activity was not due to cytotoxicity, crowding, or steric hinderance by the cultured cells. The suppressor macrophage population did not appear to inhibit the allogeneic response via prostaglandin or arginase release, or interfere with the tritiated thymidine uptake by release of endogenous thymidine. The above system is viewed as an in vitro model of immune regulation by suppressor macrophages, in the context of allogeneic response.

Platelet HLA typing and cross-matching by a direct microcytotoxic assay. A new method employing carboxyfluoresceindiacetate-stained platelets.

Carboxyfluoresceindiacetate-labelled platelets can be used in direct microcytotoxic assays for the purpose of typing platelets for a number of antigenic determinants, such as HLA and beta-2 microglobulin. The assay when used in direct platelet cross-matching, where thrombocytopenic patient sera were screened, resulted in the uncovering of many extrapositive and weakly positive cross-matches not identified by the lymphocyte cross-match. The simplicity and rapidity of this new method makes it an attractive alternative for future use in research and clinical studies.

Use of monocytes in HLA-A, B, C and DR typings.

A simple method of improved serologic typing of monocytes for HLA-A, B, C and DR specificities is described. The method employs monocytes recovered from frozen samples of peripheral blood mononuclear cells; it chiefly involves pretreatment of monocytes with 0.01% iodoacetamide (IAA) prior to typing. The advantage of this method lies principally in the lowering of the background nonspecific cytotoxicities and false positive readings upon IAA addition to the monocyte preparations. Using this method monocytes can be typed for HLA-A, B, C determinants. Although the addition of IAA results in substantial typing improvements, we found the assignment of A, B, C specificities difficult due to the presence of extra positive reactions when monocytes were compared to T lymphocyte typings. probably due to the presence of DR or monocyte specific antibodies in the routinely used HLA antisera. This method proved to be most useful in DR typings where mono cytes in the presence of IAA were compared with autologous B cells in the absence of IAA. The differences in typings due to a decrease in false positive cytotoxic readings were significantly in favor of using IAA treated monocytes in DR typings (P < 0.0001). The use of IAA in the course of B cell or T cell typings bad no adverse consequences on either A, B, C or DR typings, respectively. Our results indicate a potential usefulness for the use of IAA in typing monocytes HLA determinants in general and for the DR determinants in particular.

Interaction with homologous erythrocytes of rat T cells which act as aggressors in the mixed lymphocyte reaction.

Substantial percentages of T-enriched spleen lymphocytes or thymocytes of inbred rats were found to form rosettes with the RBC of homologous strains. When an excess of RBC was used, essentially all of the rosette-forming subpopulation of lymphocytes was removed when the rosettes were separated by centrifugation. After depletion of the lymphocytes, reactive with RBC of one strain, most of the lymphocytes reactive with RBC of other strains could be recovered in the supernatant. A very large percentage of lymphocytes of the BN strain formed rosettes when a mixture of the RBC of five other strains was tested; the percentage was, however, somewhat lower than that predicted on the basis of complete additivity. Rosettes dissociated when warmed to 37 degrees C. The lymphocytes recovered were unable to form rosettes again. In nearly all instances, the subpopulation that formed rosettes with RBC of a given strain included essentially all of the lymphocytes that acted as aggressors against peripheral leukocytes or mytomycin C-treated thymocytes of that strain; the lymphocytes in the supernatant always retained activity as aggressors against the leukocytes of one or more other strains and retained their responsiveness to PHA. Lymphocytes recovered from rosettes, by warming to 37 degrees C, were highly reactive as aggressors in the MLR against the strain providing the RBC. Varying degrees of reactivity were noted against leukocytes of other strains.

Production of large amounts of antibodies, complement, and leukocytes in ascitic fluids of guinea pigs.

A method is described for the production in guinea pigs of large amounts of ascitic fluid containing non-specific IgG, antiprotein antibodies, complement, other serum proteins and leukocytes. The method is an adaptation of a procedure previously applied to mice. A major difference is the extended schedule of inoculations required for the induction of ascites in guinea pigs; a requirement for boosting with antigen intradermally while repeatedly inoculating adjuvant intraperitoneally; and the much larger quantities obtained. The average yield of ascitic fluid, when antigen was not used, was 113 ml per animal, and the average yield of IgG was 0.87 g. With antigen (keyhole limpet hemocyanin) the average yields were 143 ml and 1.6 g of antibody per guinea pig. Complement titers were 41 to 74% of those in serum. The number of leukocytes per ml of ascites ranged from 7 X 10(6) to 20 X 10(6). The method should be useful for the production of large amounts of leukocytes, antibodies and other serum proteins from a small colony of laboratory animals. In addition, cells can be obtained without the need to sacrifice the animal.

Heterogeneity of rabbit homologous skin sensitizing antibodies: IgE and a new subclass IgGa.

Homologous skin sensitizing antibodies in the rabbit can be differentiated by their requirement for complement in expressing the passive cutaneous anaphylactic reaction as complement dependent (CD) or complement independent (CI). CI antibodies are representative of the IgE class, while the CD antibodies belong to a proposed new subclass of IgG designated IgGa (a = anaphylaxis). The following are characteristics of CI (IgE) antibodies: elution at 0.05 M on DEAE at pH 8.1 migration ahead of 7S in ultracentrifugation, molecular weight 215,000 daltons by Sephadex G-200, fast gamma-electrophoretic mobility, isoelectric point, pI = 4.98 (5.68-4.58), 85-95% heat sensitivity, sensitivity to reduction plus alkylation and option at 0.01 M on DEAE, 7S in ultracentrifugation, MW 138,000 daltons, slow gamma-electrophoretic mobility pI = 6.90 (8.30-5.70), resistance to heating and to reduction plus alkylation and 1-day optimal SP. CI antibodies could be absorbed by anti-Fab, anti-epsilon, the homologous antigen and were not absorbed by anti-gamma, anti-alpha, or heterologous antigens. The CD antibodies were absorbed by anti-Fab, anti-gamma and not absorbed by anti-epsilon, anti-alpha, or heterologous antigens.