Mitogen-induced Cellular Cytotoxicity Research Articles

Intra-epithelial lymphocytes: interferon-gamma production and suppressor/cytotoxic activities

Human intraepithelial lymphocytes (IEL) proliferate minimally in response to phytohaemagglutinin (PHA), but produce as much interleukin-2 (IL-2) as do peripheral blood lymphocytes (PBL). The addition of sheep erythrocytes during activation of IEL with PHA markedly augments both T cell functions. This study evaluates the ability of IEL to produce interferon-gamma (IFN-gamma) and to develop suppressor and cytotoxic activities when stimulated with mitogens in the presence or absence of sheep erythrocytes. PHA-activated IEL produced as much IFN-gamma as did PHA-activated peripheral blood CD8+ T lymphocytes. IEL activated by concanavalin A (Con A) demonstrated less suppressor activity directed against T cell proliferation than did Con A-activated peripheral blood CD8+ T lymphocytes. IEL generated less mitogen-induced cellular cytotoxicity and lymphokine-activated killer cell activity than did peripheral blood CD8+ T lymphocytes. The addition of sheep erythrocyte lysates during mitogen stimulation of IEL markedly enhanced their proliferation and lymphokine production but did not affect their suppressor or cytotoxic activities.

Defective natural killer cell activity in B-cell chronic lymphocytic leukaemia is associated with impaired release of natural killer cytotoxic factor(s) but not of tumour necrosis factor-alpha.

The mechanisms accounting for the impaired natural killer cell activity (NKa) in B-cell chronic lymphocytic leukaemia (B-CLL) were investigated in 34 B-CLL patients. We found that patients with B-CLL have indeed very low NKa which may be increased in the presence of recombinant human interferon-alpha or recombinant human interleukin-2. Patients had also very low mitogen-induced cellular cytotoxicity. Their absolute numbers of peripheral blood CD16+, CD57+, CD3+, and CD8+ cells were significantly increased. Patients' NK cells had a normal tumour cell binding capacity but failed to release sufficient amounts of soluble cytolytic molecules upon stimulation with K562 cells or activation with phytohaemagglutinin (PHA). However, B-CLL NK cells released tumour necrosis factor-alpha (TNF-alpha) following stimulation with PHA. We concluded that defective NKa in B-CLL patients is probably the result of an impairment in the production and/or release of soluble cytolytic mediators, but not of TNF-alpha by NK cells. Further studies on the production and release of other cytolytic molecules, such as perforin and granzymes, as well as studies on the possible inability of NK cells to activate the apoptotic mechanisms in the target cells are in progress in our laboratory.

Human mucosal cytotoxic effector cells

Human intestinal lamina propria mononuclear cells have been shown to mediate mitogen-induced cellular cytotoxicity, antibody-dependent cellular cytotoxicity, and lymphokine activated killer cell function. However, although natural killer cells have been demonstrated in the gut mucosa of rodents, recent reports found little or no spontaneous cytotoxic activity in the lamina propria of the human gut. Using the natural killer cell-related monoclonal antibody NKH-1, which has not previously been applied to studies of mucosal killer cell function, we have shown by immunofluorescence that 2%–3% of enzymatically dispersed lamina propria lymphocytes are NKH-1+. A “panning” technique was then used to enrich for the NKH-1+ cells. Panned cells were consistently ≥80% NKH-1+ by indirect immunofluorescence. Unlike their counterparts in the peripheral blood, the mucosal NKH-1+ cells were Leu-11−. Although unseparated lamina propria lymphocytes failed to exhibit natural killer activity against K562 targets in 4-h chromium release assays at effector to target ratios of up to 100:1, the NKH-1+ cells were cytolytically active at ratios of <5:1. Mucosal lymphocytes depleted of natural killer cells (NKH-1−) exhibited cytotoxic activity when cultured for 72 h with interleukin-2. The precursors of the lymphokine culture activated phenomenon were NKH-1−, Leu-11−, T4−, T3−, T11+, and T8+. Although lamina propria T3+ cells did not exhibit spontaneous or culture activated cytotoxicity, they were shown to exhibit nonspecific anti-CD3 (anti-T3)-induced T-cell cytotoxicity. In conclusion, (a) functional natural killer and lymphokine activated killer cells are both present in the human gut mucosa and represent distinct populations of cytotoxic cells. In addition, anti-CD3-induced cytotoxicity is a feature of mucosal T cells, (b) These mucosal killer cell subsets differ phenotypically from those previously described in the peripheral blood.

Cytotoxic lymphocytes in human intestinal mucosa.

Although cytotoxic lymphocytes have been well studied and characterized in the peripheral blood, the nature and function of these effector cells in human gut mucosa is still unclear. Previous studies have suggested that human intestinal lamina propria lymphocytes (LPL) exhibit mitogen induced cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) but that freshly isolated mucosal cells do not exhibit NK activity or exhibit it at very low levels (1–7). This is surprising since the intestinal mucosa is frequently affected by neoplastic, infectious and inflammatory diseases and is in apparent contrast with studies of the mucosal immune system in rodents where spontaneously cytotoxic cells are well represented (8,9). However, when isolated human colonic cells are cultured in the presence of interleukin-2 (IL-2) significant lytic activity is observed (lymphokine activated killers [LAK]) (10, 11).KeywordsMucosal Immune SystemCytotoxic LymphocyteLamina Propria LymphocyteHuman Colonic CellLamina Propria Mononuclear CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Langerhans cell-containing epidermal cells are not cytotoxic in antibody-Dependent - and mitogen-induced - cellular cytotoxicity

Langerhans cells (LC) constitute a small (2–6 %) subpopulation of mammalian epidermal cells (EC) and are the only EC that bear Fc-IgG receptors and that share several other cell-surface characteristics and functional capabilities with cells of the monocyte-macrophage series. Since Fc-IgG receptor-positive macrophages are potent effector cells in antibody-dependent cellular cytotoxicity (ADCC), we determined whether LC-containing EC populations are also able to mediate ADCC. In the ADCC assay employed, BALB/c LC-containing EC at different effector/target cell ratios consistently failed to lyse bovine red blood cell (BRBC) targets which were either trinitrophenyl(TNP)-modified or not modified and reacted with an appropriate antibody: a purified rabbit IgG anti-BRBC, or a mouse or rabbit anti-TNP antiserum. In addition, LCcontaining EC did not mediate mitogen-induced cellular cytotoxicity using purified phytohemagglutinin. BALB/c spleen cells, used as a positive control, gave substantial specific release in all experiments performed.

Dinitrochlorobenzene-induced colitis in the guinea-pig: studies of colonic lamina propria lymphocytes.

Dinitrochlorobenzene-induced colitis in guinea-pigs may be immunologically mediated: animals must be presensitised to dinitrochlorobenzene to develop colitis, sensitivity can be passively transferred by lymphocytes and the injury can be mitigated by immunosuppression. In this study, we examined lamina propria lymphocytes isolated from colons of animals with dinitrochlorobenzene-induced colitis, and appropriate controls. Lamina propria lymphocytes from colitis animals have a greater percentage of rabbit erythrocyte-rosetting cells (T cells) (20.1 +/- 3.0 vs 2.3 +/- 0.8, p less than .01) and a greater capacity to mediate mitogen-induced cellular cytotoxicity with phytohaemagglutinin than lamina propria lymphocytes from normal colon (% specific cytoxicity = 29.4 +/- 8.7 vs 5.0 +/- 4.5, P less than .005). There was no difference in the percentage of rosetting cells or cytotoxicity index of spleen or mesenteric lymph node lymphocytes between the colitis animals and controls. These data suggest that there are changes in the distribution and functional characteristics of lamina propria lymphocytes which correlate with mucosal cell injury in the dinitrochlorobenzene-colitis model.

Differential effects of ouabain on human cell-mediated cytotoxicity: II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10 −5 M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10 −9 M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.

Differential effects of ouabain on human cell-mediated cytotoxicity: I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed 51Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10 −4 M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations ( P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.

Fc receptors on human T lymphocytes: II. Cytotoxic capabilities of human Tγ, Tμ, B, and L cells

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC n−, EAC′+) did not mediate cytotoxicity. L cells (SRBC n−, EAC′−) mediated NC and ADCC but not MICC. T cells (SRBC n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.

The Effects of Cyclophosphamide on Mitogen-Induced and Antibody-Dependent Cellular Cytotoxicity in Guinea Pigs

Lymph node cells from normal guinea pigs, when stimulated with the non-specific mitogen PHA, were transformed to activated killer cells, capable of lysing 51Cr labeled mouse fibroblasts. Similarly, these lymphoid cells effected lysis of antibody coated chicken red cells in an antibody-dependent cellular cytotoxicity assay. Following cyclophosphamide, 150 mg/kg IP, the reactivity of an aliquot of lymph node cells to effect either cytotoxic reaction was not diminished. These results indicate that this immunosuppressant does not promote a selective decrease in either lymphoid effector. Rather, they are diminished in parallel with the generalized lympholysis resulting from drug administratioin. During the recovery phase, lymph node cells showed increased ability to lyse target cells, suggesting a rebound phase of heightened activity. Thymic cells from normal and cyclophosphamide treated animals were poor effectors in either cytotoxic assay. The addition of an equal number of thymocytes to lymph node cells resulted in decreased mitogen-induced cytotoxicity. Thymic inhibitory activity was mediated only by viable cells and the phenomenon did not represent an altered shift in PHA sensitivity. This suppressive activity persisted when thymic cells from cyclophosphamide treated-animals were employed, indicating that inhibitory cells were also not selectively depleted by this drug. In antibody-dependent cellular cytotoxicity assays, thymocytes from normal or cyclophosphamide-treated animals did not alter the cytotoxicity capacity of lymph node cells.

Cell-mediated cytotoxicity of desialylated human lymphocytes induced by a mitogenic mammalian liver protein

The ability of a purified rabbit liver membrane protein that selectively binds desialylated glycoproteins to induce desialylated human peripheral blood lymphocytes to mediate mitogen-induced cellular cytotoxicity has been determined. After short term exposure to purified hepatic binding protein, desialylated, but not intact, lymphocytes exhibited mitogen-induced cellular cytotoxicity against Chang target cells, which do not have exposed hepatic binding protein on their surface membrane. Furthermore, desialylated lymphocytes, in the absence of purified hepatic binding protein, reduced the proportion of isolated rabbit hepatocytes that adhered to plastic to a significantly greater extent than did intact lymphocytes, suggesting that the exposed hepatic binding protein demonstrated on the surface membrane of these target cells is capable of inducing mitogen-induced cellular cytotoxicity. The specific inhibition of this effect by asialo-orosomucoid, but not by intact orosomucoid, indicates that the site involved in the binding of asialo-glycoproteins to hepatic binding protein is probably also responsible for the induction of mitogen-induced cellular cytotoxicity. The demonstration that hepatic binding protein, a normal constituent of the surface membrane of mammalian hepatocytes, can induce mitogen-induced cellular cytotoxicity, suggests that mitogen-induced cellular cytotoxicity may be a mechanism of cellular injury in vivo.

Mitogen induced cellular cytotoxic activity as a monocyte function in human peripheral blood preparations

The effector cell population in man responsible for mitogen induced cellular cytotoxicity (MICC) of chicken erythrocytes was investigated using several separation techniques, including free flow electrophoresis. Electrophoresis produced substantial monocyte enrichment in some fractions with substantial depletion in others. MICC activity was seen to correlate with monocyte content in these fractions. Furthermore, removal of phagocytic cells with carbonyl iron and removal of adherent cells on plastic petri dishes depleted preparations of MICC activity. Thus it is suggested that under conditions described in this paper, the effector cell of the MICC assay in man appears to be a monocyte. This MICC effector cell was shown to be different from the effector cell in the natural killing (NK cells) of RPMI 6410 cells.

Cytotoxic Activities of Gut Mucosal Lymphoid Cells in Guinea Pigs

Populations of lymphoid cells were obtained from the gastrointestinal tract of guinea pigs and compared with splenic cells for effector cell activity in assays of spontaneous cell-mediated cytotoxicity (SCMC), mitogen-induced cellular cytotoxicity (MICC), and antibody-dependent cellular cytotoxicity (ADCC). Lymphoid cells were isolated from mesenteric lymph nodes (MLN), Peyer's patches (PP), and the small intestinal mucosa. Mucosal lymphoid cells were separated into a population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing lamina propria (LP) lymphocytes by a combination of physical and enzymatic methods. All populations of lymphoid cells from the gastrointestinal tract were capable of mediating MICC. Relative to spleen cells, IEL possessed more MICC effector cell activity whereas cells from MLN, PP, and LP possessed less activity. In contrast, SCMC and ADCC effector cell activities were present only in the IEL population and these cytotoxic activities were comparable to that of splenic cells. These results demonstrate that mucosal lymphoid cells possess cytotoxic effector cell activities that may contribute to local mucosal immunologic reactivity.

Lack of effector cell function in chronic lymphocytic leukemia, a B-cell malignancy, and the Sézary syndrome, a T-cell malignancy

In vitro reactivity of peripheral blood lymphocytes was examined from 13 patients with CLL and four patients with the Sézary syndrome. Lymphocyte proliferative responses to nonspecific mitogenic stimuli were generally depressed compared to normal controls. In vitro proliferative responses to specific antigenic stimuli were even more depressed. Lymphocyte effector functions which were independent of cellular division were also profoundly abnormal. Peripheral blood lymphocytes from both Sézary patients and CLL patients were incapable of mediating significant cytotoxicity toward 51Cr-labeled CRBC targets in either an antibody-dependent cellular cytotoxicity or a mitogen-induced cellular cytotoxicity model of nonspecific cytotoxicity. Similarly, cells from these patients were unable to produce normal quantities of lymphocyte-derived chemotactic factor when activated with either nonspecific mitogens or specific antigens. Thus malignant T cells and malignant B cells are incapable of performing many in vitro functions subserved by a broad range of normal lymphocytes.

Studies of complement receptors on cytotoxic effector cells in human peripheral blood

The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg −, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg−, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR.

The Effects of Pharmacologic Agents on Mitogen-Induced Cellular Cytotoxicity in Guinea Pigs

Using pharmacologic agents as probes, three aspects of the mitogen-induced cellular cytotoxicity reaction (MICC) were evaluated. These included the requirement for a concomitant lymphoid DNA synthetic response, the role of microtubules and microfilaments in effecting target cell lysis, and the potential inhibitory activity of hydrocortisone. Results, using both PHA and Con A stimulated guinea pig lymph node cells, indicate that this form of cytotoxicity occurs as a phenomenon independent of 3H-Tdr incorporation. Animals treated with cyclophosphamide showed cytotoxic reactions equivalent to untreated controls whereas mitogen-induced proliferative responses were greatly impaired. Further studies indicated that the in vitro exposure of lymph node cells to either mitomycin or cytosine arabinoside markedly reduced DNA synthesis but did not depress the ability of mitogen-stimulated lymphocytes to effect target cell lysis. The microtubular antagonists, vincristine, vinblastine and colchicine, inhibited cytotoxic reactions if the agents were present at the time that lymphocyte-target cell interaction was established. However, the antimicrofilament agent cytochalasin B was not inhibitory; this contrasts to this agent's ability to suppress antigen-induced cell-mediated cytotoxic reactions. In a third series of experiments, hydrocortisone showed a dose-related inhibition of MICC responses. The action of the steroid was apparent only if the drug was present at the onset of the culture; no suppression was observed if it was added after an initial one day incubation.

In vitro cell-mediated cytotoxicity in primary biliary cirrhosis and chronic hepatitis. Dysfunction of spontaneous cell-mediated cytotoxicity in primary biliary cirrhosis.

The in vitro cytotoxic function and target cell specificity of peripheral blood lymphocytes from selected patients with primary biliary cirrhosis and hepatitis B surface antigen-negative chronic hepatitis were investigated using 51Cr-labeled human Chang and EL-4 mouse sarcoma cell targets in assays of spontaneous cell-mediated cytotoxicity (SCMC) and mitogen-induced cellular cytotoxicity (MICC). In addition, antibody-dependent cellular cytotoxicity (ADCC) against Chang cells was assessed. At an effector-to-target cell ration of 100:1, the mean SCMC against Chang cells was much less in patients with primary biliary cirrhosis than that in either the controls (P less than 0.001) or the patients with chronic hepatitis (P less than 0.005) whereas the value for patients with chronic hepatitis did not differ significantly from that of the controls. The mean SCMC against EL-4 mouse sarcoma cells was also less in patients with primary biliary cirrhosis than in controls (P less than 0.005) whereas the value for chronic hepatitis was not significantly different from that of the controls or patients with primary biliary cirrhosis. In contrast, MICC against both targets and ADCC against Chang cells were similar for each group. Comparison of SCMC and MICC against both target cells, measured simultaneously, showed similar cytotoxic potenital against both target cells for each group. Effector cells capable of mediating cytotoxicity in each assay were defined by testing the cytotoxic function of lymphocyte subpopulations isolated from two representative patients with each disease using techniques of immunoabsorbent affinity chromatography and Fc receptor binding to antigen-antibody complexes. In both primary biliary cirrhosis and chronic hepatitis SCMC and ADCC were mediated by a subpopulation of lymphocytes which lack surface immunoglobulin (sIg-) and bear Fc receptors (Fc+). In contrast, MICC was mediated by sIg- cells which lack Fc receptors. Lymphocytes bearing sIg- were not cytotoxic in any assay. These results establish a difference in cytotoxic function in primary biliary cirrhosis and chronic hepatitis by defining the presence of a defect in spontaneous cytotoxic function of sIg-, Fc+ lymphocytes against Chang cells in primary biliary cirrhosis.

Mitogen Induced Cellular Cytotoxicity (MICC) in Multiple Myeloma

Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.

Immunoregulatory Properties of Fractions from Human Pregnancy Urine: Evidence That Human Chorionic Gonadotropin is Not Responsible

The immunologically privileged position of the histoin-compatible fetus and placenta is a striking example of a physiologic immunoregulatory mechanism. This study was designed to examine the effects of human chorionic gonadotropin (HCG) on the recognitive proliferative phase and the cytotoxic effector phase of in vitro cell-mediated immune responsiveness, since HCG has previously been reported to be immunosuppressive in vitro and in vivo. Commercial preparations of HCG were found to be potent inhibitors of lymphocyte proliferative responses to nonspecific mitogens like phytohemagglutinin (PHA), specific antigens such as streptolysin-O (SLO), and allogeneic cells as measured in the one-way mixed leukocyte response. Cytotoxic effector function of lymphocytes as measured by antibody-dependent cellular cytotoxicity (ADCC) and mitogen-induced cellular cytotoxicity were also markedly inhibited by these preparations. However, the 50% inhibitory concentration varied widely from lot to lot of these commercial materials. After dialysis, a portion of the inhibitory activity was lost from some but not all HCG lots. The dialysate from those lots with diminished activity was found to be immunosuppressive in vitro but contained no HCG detectable by radioimmunoassay. Following dialysis, the immunosuppressive activity of the various HCG lots remained variable and correlated poorly with values for HCG obtained by a double antibody radioimmunoassay. HCG preparations purified to a homogeneity sufficient for amino acid sequence were found to be only minimally immunosuppressive to the in vitro PHA response and had almost no effect on proliferative responses to antigens and allogeneic cells. These data do not support the concept of a primary immunoregulatory role for HCG, but they suggest that other uncharacterized compounds partially co-purified from pregnant urine along with HCG may have such immunoregulatory activity. Further characterization and identification of this immunoregulatory material(s) is essential, since it appears to have many of the properties of an ideal immunosuppressive compound: a) nontoxicity, b) ready reversibility, c) activity at very low concentration, and d) activity on a broad range of cellular immune functions.

Enrichment of the Murine Natural Killer (NK) and Mitogen Induced Cellular Cytotoxicity (MICC) Cells Using Preparative Free-Flow High Voltage Electrophoresis

A procedure using preparative free-flow high voltage electrophoresis is described for the fractionation of murine spleen and bone marrow cells so as to obtain cell subpopulations that are either enriched in or depleted of "natural killer" (NK) cells and "mitogen-induced cellular cytotoxicity" (MICC) effector cells. A nearly three fold enrichment in the NK and MICC activities of spleen cells was achieved. The enrichment in these cells could be further increased if the phagocytic cells were removed prior to electrophoresis. When bone marrow cells were fractionated a two and a half fold increase of NK activity, and a one and a half fold enrichment of MICC activity was achieved. In both cases, other fractions were nearly devoid of NK and MICC activity. The cell recovery after electrophoresis averages 70% of the cells applied, and at least 90% of these cells were viable. MICC and NK effector cells could not be separated to a useful extent electrophoretically but were found to be separable using Sephadex C-10 gel filtration columns. The MICC but not the NK cells were retained on these columns.