This chapter discusses the interaction of dextran-triggered murine splenic cell supernatants and allogeneic effect factor. In a study described in the chapter, the Marbrook method was used to immunize splenic cells in vitro. Balb/c splenic cells were cultured in Eagle's Minimal Essential Medium supplemented according to Mishell and Dutton with and without sheep red blood cells (SRBC). SRBC-specific plaque-forming cells of the IgM and IgG varieties were assayed on day 5 of immunization by the method of Cunningham and Szenberg. The detection of IgM and IgG PFCs was done according to Silver and Winn. B cells were separated from whole splenic cell populations on nylon-wool columns and the adherent subset was further treated with mouse anti-θ serum and guinea pig complement to eliminate T cells prior to their use for in vitro immunizations. Supernatants of splenic cells from saline-injected and dextran-injected mice were added to both splenic cell and B cell cultures 48 h after antigen. It was found that supernatants from splenic cells of dextran-injected mice caused a small but consistent increase in the number of IgM PFC. The number of IgG PFC was not significantly different from the control values.
This chapter presents an investigation of monocyte Fc receptor avidity and monocyte-mediated antibody-dependent cellular cytotoxicity (MMADCC) in man. Peripheral blood monocytes from normal subjects and patients with various immunodeficiency diseases, including the Wiskott–Aldrich syndrome (WAS) and intestinal lymphangiectasia (IL), were studied in an analysis presented in the chapter. In addition, pleural fluid macrophages were studied in one patient with IL. Mononuclear cells were incubated with 51Cr-labeled human blood group B erythrocytes in the presence of human anti-blood group B antiserum and the number of targets killed determined from the specific release of 5lCr into the medium. In this system, monocytes but not lymphocytes act as effector cells and the amount of killing observed correlates with the number of monocytes in the mononuclear cell effector population. The results showed that MMADCC requires the Fc portion of the antibody. However, as seen in the WAS patients, binding of the antibody-coated target to the monocyte is not in itself sufficient for lysis to occur, indicating that while antibody dependent cellular cytotoxicity requires an Fc receptor, the mere presence of a functional Fc receptor is not enough to trigger the cytotoxic event.
This chapter presents a few studies in which highly purified human peripheral lymphocytes were incubated in MEM containing 10% autologous serum, glutamine, and antibiotics, in the presence of optimal concentrations of phytohemagglutinin. Cultures were incubated at an initial cell concentration of 2 × 105 lymphocytes/ml, which is one log below the widely used concentration. Under these conditions, up to a fivefold increase in cell number was observed over a period of 7 days. DNA purified from cultures labeled with 5-bromodeoxyuridine between days 3 and 4 for 24 h, a period longer than one mean cycle time, was analyzed on cesium chloride equilibrium density gradients: the percentages of DNA in the heavy–heavy, heavy–light, and light–light regions were, respectively, 19, 56, and 25, providing direct evidence for replication of more than 50% of the DNA and reentry of some cells into a second proliferative cycle.
This chapter discusses the enhancement of weak MLC type reactions by hydrophilic polymers. In a study described in the chapter, BALB/c and C57BL/6 thymocytes were cultured for 4 days in Eagle's MEM + 10% FCS, with or without the X-irradiated lymphomas LSTRA and EL-4, respectively. By thymidine incorporation, no stimulation was observed with EL-4 in plain culture while occasionally a very small stimulation was observed with LSTRA. The effect of polyethyleneglycol M.W. 6000 (PEG-6) is much more pronounced. A titration curve of this polymer is shown in the chapter, and at the optimal concentration of 4–5%, PEG-6 causes an increase of 5- to 50-fold in the stimulation index. PEG-6 by itself has little effect on thymocytes in the absence of tumor cells. The above phenomenon can be followed microscopically as demonstrated in the chapter. Aggregates are induced by the polymer regardless of the presence of tumor cells. However, only where both isogenic tumor cells and 4% PEG-6 are present can blast cells be clearly seen.
Publisher Summary This chapter explores the functional mosaicism of the lymphocyte plasma membrane. Lymphocytes are activated to grow and divide when only a distinct proportion of plasma membrane-binding sites interacts with mitogens such as the lectin concanavalin A (ConA). To test whether binding sites responsible for cell activation are associated with specialized areas of the plasma membrane, calf thymocytes were disrupted by the nitrogen cavitation method and the microsomal membranes were isolated in a study described in the chapter. These vesicles, which are more than 90% derived from the plasma membrane, were subjected to affinity chromatography on ConA-sepharose. The results demonstrate that the plasma membrane of lymphocytes is not completely homogeneous. The areas of the plasma membrane that carry mitogen receptors, that is, high affinity binding sites, are distinct from the bulk membrane with regard to their enzymatic activities, suggesting a functional mosaicism in the vicinity of mitogen receptors.
This chapter discusses the receptors for IgG on human lymphocytes and relationship to each other of these receptors. Different methods that have been used to study a controversy as to which human lymphocyte subpopulations bear receptors for IgG (Fc-receptors) and the relationship to each other of these receptors. The issues that were examined were whether the receptors for IgG, as identified by different methods, are found on identical subpopulations of lymphocytes and also regarding the relationship of surface immunoglobulin to Fc-receptors. In a study described in the chapter, using several techniques, including depletion of lymphocyte subpopulations by gradient centrifugation, the interrelation of cells with Fc-receptors was evaluated. The results of examination of surface receptors on lymphocytes at the interface were essentially identical to baseline values. The results showed that Agg+ lymphocytes behaved in a manner similar to ShEA+ lymphocytes, depletion ShEA+ lymphocytes also depleted Agg+, HuEA+, and SIg+ lymphocytes, depletion of HuEA+ lymphocytes did not deplete Sig+ lymphocytes and the Sig+ lymphocytes remaining seemed also to be Agg+ and ShEA+, and during depletion experiments, lymphocytes that are AggSIg+ behaved in a manner similar to lymphocytes that were Agg+.
This chapter explores the role of H-2 CD antigens in secondary responses. Treatment of spleen cell suspension with UV light has been shown to selectively modify cell-surface antigens in a way that the cells are unable, after treatment, to elicit a proliferative response when used in a one-way MLC as stimulating cells. In an experiment described in the chapter, uv-treated cells syngeneic to the original stimulating cells were unable to generate any secondary cytotoxic response. However, an LD stimulus alone, presumably cross-reactive with LD antigens present on the original sensitizing cells, can restimulate a secondary cytotoxic response. When that LD stimulus is added to the CD stimulus in a three-cell experiment, a very good secondary cytotoxic response directed against that CD antigen is obtained, which is much higher than that generated by the LD stimulus alone. The potentiation seems to be specific; B10uv treated cells are unable to show the same phenomenon.
This chapter focuses on the stimulation of human lymphocytes, an activation by a sodium periodate (NaIO4)-induced growth factor. NaIO4 stimulates human peripheral blood lymphocytes to undergo DNA synthesis and cell division. Several distinct mechanisms for activation exist. Although lymphocyte growth factor (LGF) is evaluated in cultures of naive lymphocytes, it may contribute to late DNA synthesis in NaIO4-treated lymphocyte cultures. Such a component would be independent of the cell–cell contact required by the population that attains peak DNA synthesis within 48–72 h. Adherent cells influence the production of LGF and the response of autologous cells to LGF. The LGF activity could not be detected in the medium of NaIO4 stimulated depleted cultures, and LGF produced in adherent enriched cultures could not be assayed in depleted cultures.
This chapter presents an investigation of spontaneous cytotoxicity by human peripheral blood lymphocytes. It describes a few studies in which well-characterized subpopulations of human peripheral blood lymphocytes were prepared by a combination of immunoabsorbent techniques and were utilized as cytotoxic effectors. Peripheral blood mononuclear cells obtained from normal healthy volunteers by Ficoll–Hypaque gradient centrifugation were depleted of macrophages by iron carbonyl treatment, and the resultant macrophage-depleted lymphocyte population was separated into surface immunoglobulin (sIg)-negative and sIg-positive lymphocyte subpopulations utilizing Sephadex anti-Fab immunoabsorbent columns. Incubation of sIg-negative lymphocytes on antigen–antibody complexed plastic surfaces yielded a non-adherent sIg-negative, Fc-receptor-negative lymphocyte subpopulation. These effector cell populations were added to 51Cr-labeled Chang liver cell targets in a tissue culture medium containing 5% fetal calf serum, and following 18 h incubation, the %51Cr release from cultures containing effector cells and target cells was compared to the %51Cr release from target cells incubated in medium alone without added effector cells.
This chapter discusses the use and other aspects of a technique to extract Ia-like antigen from cultured human B lymphocytes and its expression on leukemic cells. The isolation procedures have required large quantities of starting material and many manipulations. The chapter presents a method whereby human B-lymphocyte Ia-like antigens can be isolated rapidly in sufficient yield that B-cell cultures or clinical specimens can be used as a starting material. By SDS-polyacrylamide gel electrophoresis, the isolated glycoprotein from the NC37 B-cell line had a molecular weight of 32 to 36,000 Da. Unabsorbed rabbit antisera to this membrane protein when assayed by indirect immunofluorescence showed preferential reactivity with B lymphocytes and minimal reaction with peripheral blood T cells, thymocytes, or other peripheral blood cells. When incubated with lymphocytes in the MLC, the antiserum suppressed thymidine incorporation. A membrane glycoprotein was obtained from human B lymphoblastoid cells and bore molecular, immunologic, and functional similarity to the murine Ia antigens. While these antigens are related predominantly to B-lymphocytes, their variable expression on leukemic cells in man indicates a broader role in hemopoietic cell differentiation.
Publisher Summary This chapter explores the effect of the ionophore A23187 upon lymphocyte calcium metabolism. The ionophore A23187 has been shown to induce lymphocyte proliferation in a calcium dependent manner, thus, lending support to the hypothesis that calcium is a critical intracellular signal in the initiation of lymphocyte transformation. A study described in the chapter investigated the effect of A23187 on human lymphocyte calcium metabolism in an effort to correlate changes in cellular calcium with initiation of transformation. Freshly isolated human peripheral lymphocytes suspended in MEM plus 10% AB serum were used in all experiments described in the chapter. Using the techniques of atomic absorption spectroscopy and 45 Ca uptake, it was determined that the mitogenic concentrations of A23187 result in a net increase in calcium content of human lymphocytes. However, the increased calcium was not retained, and over a period of many hours, the calcium content of A23187-treated lymphocytes declined to levels approaching control values. As the respiratory uncoupler FCCP was capable of blocking at least 95% of the A23187-induced 45 Ca uptake, it was concluded that most of the calcium accumulated in ionophore-treated lymphocytes was sequestered by the mitochondria.
Publisher Summary This chapter focuses on the nonspecific lymphocyte cytotoxicity for allogeneic target cells, also termed spontaneous cytotoxicity or background cytotoxicity reported by several investigators studying graft rejection and tumor immunology phenomena. Experiments were performed to determine whether this activation was the result of removal of some suppressor population during separation procedures, but reconstitution of the non-E-RFC with either the E-RFC (T cell) population or with unseparated mononuclear cells failed to reverse the cytotoxic activity. The mechanism of the nonspecific cytotoxicity has been investigated and appears to be mediated by release of a lymphotoxin-like moiety from the ER Sup-activated non-T cells.
This chapter discusses the intermediates of DNA replication and the rate of DNA elongation of in vivo proliferating thymocytes or lymphocytes stimulated in vitro by mitogens. In a study described in the chapter, in vivo proliferating thymocytes removed from thymuses of 3-week-old mice were immediately suspended at 37°C in culture medium, were pulsed by 3H-TdR for 2 mn, and were then chased in cold TdR for different periods. Washed pellets were lysed in alkali and were centrifuged on alkaline sucrose gradients. Newly synthesized DNA forms a homogeneous population of 5–10S fragments. From 2 mn up to 10 mn, the 3H-DNA shifted to a 40–45S band. This displacement operates by a direct translation from the 7S peak to the 45S one without intermediate steps. Thus, 5–10S and 45S fragments appeared as definite replication intermediates, which could be compared, respectively, to Okazaki fragments and replicons. DNA replication through in vitro stimulation was investigated by similar methods. Thymocytes stimulated by ConA and splenocytes of nude mice stimulated by LPS were compared. Thymocytes present usual intermediates of DNA replication and a usual rate of replication and elongation. Stimulation by ConA does not introduce any modification of these aspects of the DNA replication phenomenon.
This chapter presents a comparison of alveolar and peritoneal macrophages (MØ) as accessory cells in phytohemagglutinin (PHA)-induced proliferation of guinea pig lymphocytes. In a study described in the chapter, guinea pig tracheobronchial lymphnode lymphocytes were cultured alone and in the presence of alveolar or peritoneal cells from the same animal. PHA was added to the cultures and proliferation was measured by incorporation of tritiated thymidine at intervals over 160 h. The chapter presents a representative experiment from this series. The PHA response of lymphocytes with alveolar or peritoneal cells present was significantly greater than lymphocytes alone and with a peak response 24–48 h later. The PHA response of lymphocytes with alveolar MØ was significantly greater than with peritoneal MØ. Control experiments with mitomycin-treated alveolar and peritoneal cells indicated that these cells account for an insignificant amount of measured thymidine incorporation. The results suggest that MØ from different sources differ in their efficiency as accessory cells in PHA-induced lymphocyte proliferation with alveolar MØ being more efficient in this function.
Publisher Summary This chapter discusses some technical considerations for the separation of human peripheral blood lymphocyte subpopulations by rosetting with sheep erythrocyte. There are several points in the E-rosette forming cells (E-RFC) procedure where difficulties may be encountered. Best results are obtained when platlets are removed by aggregation with ADP prior to FH separation of peripheral blood mononuclear leukocytes (PBL) from whole blood. For optimal E-RFC formation, the sheep erythrocytes should be fresh (never older than two weeks) and from a sheep selected to form strong E rosettes. Fetal calf serum (FCS) employed in rosetting should be selected from a lot giving maximum numbers of E-RFC and should be absorbed with E if there is evidence of agglutinins. For rosette formation, PBL and E are usually pelleted together by centrifugation, and the optimum E:PBL ratio is approximately 40:1. A round-bottom centrifuge tube and the use of cold (4°C) media run down the tube's side will allow easier resuspension. The procedure for E-RFC formation was simplified by eliminating the centrifugation step. With this procedure, a higher E:PBL ratio of 80:1 and a final concentration of 40% fetal calf serum is used.
Publisher Summary This chapter highlights different aspects of the TYN–PLOP phenomenon in HLAD typing. The only way to type for HLA-D gene products has been the mixed lymphocyte reaction using homozygous typing cells. In the primed lymphocyte test (PLT), lymphocytes are specifically sensitized to one HLA-D determinant and then used as test reagents. This method is used to raise reagents that are specific for the unidentified HLA-D specificities. The non-HLA-D gene products might in some cases be capable of causing high stimulation, thus, camouflaging the HLA-D compatibility between a typing cell and a responder. More accurate estimations of gene frequencies can be obtained by PLT.
This chapter provides an overview of the examination results of isolation of tumor associated antigen(s) from Moloney virus induced leukemia. Several attempts have been made to isolate tumor associated antigen(s) from MBL-2, a Moloney virus induced ascitic lymphoma of C57BL/6 mice. Microcytotoxicity tests are performed using cytotoxic antiserum from C57BL/B6 MSV regressors. It is found that antigen(s) active in the inhibition of humoral cytotoxicity are released from tumor cells incubated with 3 M KCl for 1 h and reach maximum concentration after 4 h of incubation. Tumor-associated antigen(s) could also be recovered from the supernatant of the MBL-2 ascitic supernatant.
