Immunomodulation of human leukocytes by staphylococcal enterotoxin A: augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.     

Isogeneic and allogeneic lymphocyte interactions may be controlled by cell surface immunoglobulin tropism.

A study of allogeneic MLC (mixed lymphocyte culture) and two types of isogeneic MLC has been conducted in which subsets of B-cells were serologically removed from pools prior to using the remainder as stimulator cells. Cellular division in the two types of ILC (isogeneic lymphocyte culture) was found to be triggered by lymphocytes with IgG₁ on their surfaces. In contrast, the stimulator cells in ALC (allogeneic lymphocyte culture) possessed membrane-bound IgG_2A and/or possibly IgG_2B. Splenic T-cells were incapable of stimulating replication of splenic or lymph nodal T-cells in the absence of B-cells. Splenic T-cell preparations served as weak stimulators of other allogeneic T-cells but only when B-cells, either isogeneic or allogeneic to the stimulator T-cells, were present. We propose that stimulation in the MLC occurs in two distinct steps. First, immunoglobulins on cell surfaces may function to bring appropriate subsets of cells together. Next, antigenic recognition occurs that results in blastogenesis. Furthermore, the tropism or attraction that certain immunoglobulins have for some cell types may determine which subsets of cells participate in allogeneic and which take part in isogeneic MLC.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Suppression of in vitro CML generation by alloactivated lymphocytes: analysis of antigen-nonspecific suppressive mechanisms

We have investigated the in vitro phenomena associated with antigen-nonspecific suppression of mixed lymphocyte culture (MLC) responses by allocativated lymphocytes. Using an experimental system that we described in a previous communication, we observed that a) the degree of suppressive activity generated by allocativation correlates directly with the intensity of proliferation observed during induction of suppressive activity, b) suppressive activity segregates exclusively with proliferating (lymphoblast) subpopulations of alloactivated lymphocytes, c) when suppressive cells are included in MLC, subsequent [3H]thymidine incorporation is enhanced and accelerated, rather than impaired, and d) a considerable proportion of the cells recovered from suppressed MLC appear to be the progeny of the suppressive population, and not the progeny of the MLC responder population. These data suggest that antigen-nonspecific suppression mediated by alloactivated lymphocytes has two related components: 1) cytokine preemption by suppressive (alloactivated) lymphocytes, and 2) MLC responder cell dilution by the progeny of suppressive lymphocytes. These data are consistent with the hypothesis that the antigen-nonspecific suppressive activity of alloactivated lymphocytes can reflect the coincidental ability of activated T cells to recognize and respond to mitogenic lymphokines in vitro. The data further explain why antigen-nonspecific suppression is difficult to reverse by addition of exogenous lymphokines to suppressed MLC.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Properties of reticulum cell sarcomas in SJL/J mice. III. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Experiments were performed to elucidate the mechanisms by which lymphocytes obtained from an M-antigen-incompatible strain reduce the specific mixed lymphocyte culture (MLC) response of lymphoid cell populations after injection into allogeneic recipients. Mice of strain CBA were injected with spleen cells from hybrids of the H-2-compatible, M-antigen-incompatible strain C3H. Normal C3H × CBA spleen cells increased the MLC reactivity of the host's lymphocytes during the first 1–3 days, and thereafter the response against C3H was drastically reduced. Mitomycin-treated or antibody-coated C3H × CBA cells rather enhanced the MLC responsiveness. Roughly similar results were obtained by injecting untreated H-2-incompatible C3H hybrid lymphocytes. Lymph node or spleen cell populations from CBA mice, injected with C3H × CBA cells up to 2 weeks earlier, were found to depress the MLC reactivity against C3H when transferred to new CBA hosts. The results indicate that injected cells had survived for 2 weeks in the host. On the other hand, H-2-incompatible C3H hybrid cells could not be detected even at day 3 after injection into CBA mice. The results also indicate that C3H hybrid lymphocytes have to be functionally intact and able to survive in the host for a relatively long period of time to be able to reduce the specific MLC response of the host's lymphocytes.     

Human immunodeficiency disease: impairment of cellular interactions leading to abnormal mediator production in mixed lymphocyte culture reaction.

Leukocyte migration inhibitory factor (LMIF) production in mixed lymphocyte culture (MLC) reactions is the result of cellular interactions based on two separate phenomena: the capacity of lymphocytes to stimulate in MLC, and the capacity of lymphocytes to respond in MLC. Puromycin-treated lymphocytes are capable of stimulating allogeneic cells for LMIF production, but are unable to respond with synthesis of LMIF (one-way MLC-LMIF test). We have studied the stimulating and responding capacity of lymphocytes from patients with different immunodeficiency syndromes in a one-way MLC-LMIF assay. Lymphocytes from patients known to have qualitative and quantitative defects of T cell or B cell functions (Hodgkin's disease, mycosis fungoides, thymoma, chronic lymphatic leukemia) were found to respond poorly as measured by mediator production although their stimulating fuction was frequently retained. Patients with advanced solid tumors often had both MLC-stimulating and responding functions depressed. There was no apparent correlation between mitogen response and MLC-induced LMIF responses or between MLC proliferative response (as measured by thymidine incorporation) and mediator production. Studying of stimulatory and responding capacity of lymphocytes in the MLC-LMIF assay provides a new tool for assessing immunocompetence and allows for in vitro evaluation of cellular interactions that may play an important role in vivo.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Clinical studies on cell-mediated immunity in patients with malignant disease

Summary In vitro mitogenic responses of lymphocytes of patients with advanced cancer of the stomach or lung were determined and the cells involved in the depressed responses of patients characterized. Proliferative responses to phytohemagglutinin and concanavalin A of lymphocyte rich mononuclear cells of the patients were impaired, but increased after in vitro unstimulated culture for seven days. Mitogenic responses were enhanced by depletion of monocytes using a Sephadex G-10 column, and further enhanced by removal of nylon wool nonadherent cells. Nylon wool nonadherent cells of the patients suppressed the mitogenic responses of autologous and allogeneic lymphocytes, but lost the suppressive activity during seven days' in vitro culture. Nonadherent cells of normal donors did not inhibit mitogenic responses. The results suggest that peripheral blood mononuclear cells of cancer patients may contain at least two populations of suppressor cells for mitogenic responses, monocytes and nylon wool nonadherent cells, which could be one of the causes of impaired mitogenic responses in these cancer patients.     

Functional significance of the Fc receptor on mixed lymphocyte culture-activated T blasts.

Fc receptor (FcR)-carrying blast cells were separated from nonFcR blast cells after priming in primary mixed lymphocyte culture (MLC) by erythrocyte-antibody rosetting and by 1-g velocity sedimentation. Both types of blast cells were cytotoxic to relevant allogeneic target cells in vitro. The FcR-positive and FcR-negative blasts were then plated on a feeder layer syngeneic to the primary MLC responder cells. After feeder layer culture both types of cells reverted into secondary small lymphocytes. When restimulated with the original stimulator cells, both types of secondary lymphocytes produced the relevant secondary cell-mediated lysis responses. Thus no functionally dissimilar subclasses of secondary T lymphocytes can be distinguished in the MLC-stimulated T-cell population on the basis of the FcR.     

Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Immunocompetence of the residual lymphocytes in frozen blood

Lymphocytes remaining in washed, previously frozen blood were isolated and their immunocompetence was studied on the basis of T- and B-cell markers, mitogenic responses, and mixed lymphocyte culture (MLC). The results showed that freeze-preservation of red cells considerably deteriorated immunocompetence of the residual lymphocytes in addition to preferential removal of polymorphonuclear cells. While approximately 60% of the residual lymphocytes excluded trypan blue dye, their E and EAC rosette formation decreased markedly to 6.4 and 5.2%, respectively.These lymphocytes showed weak proliferation to PHA and Con A stimulation, but failed to respond to alloantigens in MLC. In contrast, they retained stimulating activity to allogeneic lymphocytes in MLC. These observations suggest that some portion of the residual lymphocytes may remain viable enough to possess immunocompetence and recognizable immunogenicity.     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.     

Cytotoxic lymphocytes induced by soluble factors derived from the medium of leukocyte cultures.

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45 degrees C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered. The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.     

In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific

The effects of the arachidonic acid metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on the in vitro random migration of cloned murine T lymphocytes (derived from limiting dilution analysis of a C57BL/6 anti-DBA/2 mixed leukocyte culture) were examined. Experiments were also performed to study the effects of the cyclooxygenase inhibitor indomethacin on both random lymphocyte migration and lymphocyte migration in the presence of PGE2. The responses of cloned lymphocytes to PGE2 and LTB4 were compared with those of unsensitized lymph node lymphocytes. PGE2 at 100 ng/ml significantly inhibited (p less than 0.001) the in vitro migration of helper clones of T lymphocytes, but had no effect on random migration of cytotoxic T cells or helper independent cytotoxic (HIT) cloned cells. In contrast, LTB4 significantly (p less than 0.001) enhanced the random locomotion of helper, cytotoxic, and "HIT" cloned cells at 0.1 and 0.3 ng/ml. The effects of both PGE2 and LTB4 were found to be completely reversible by cell washing. Indomethacin (10(-7) M) did not alter random migration of any of the clones, and in particular, did not affect the inhibition of helper lymphocyte migration induced by PGE2. Unsensitized bulk lymph node lymphocyte migration was not affected by either PGE2 or LTB4. The results suggest that modulation of lymphocyte locomotor function by environmental stimuli may depend on cellular activation, and the locomotor responses of activated lymphocytes to arachidonic acid metabolites may be subset specific.     

Studies on cytotoxicity generated in human mixed lymphocyte cultures. III. Natural killer-like cytotoxicity mediated by human lymphocytes with receptors for IgM

Stimulation of human peripheral blood lymphocytes with allogeneic cells in mixed lymphocyte culture (MLC) results in increased NK-like cytotoxicity against K562 targets. The effector cells of this cytotoxicity were shown to include both Fcμ+ and Fcμ? cells, as shown by EAμ rosette separation and by combined rosette formation and single-cell analysis. Peak cytotoxic activity of Fcμ+ cells was found after 3 days of MLC stimulation. The Cytotoxicity against KS62 targets mediated by Fcμ+ cells could not be inhibited at all with alloantigen-bearing cells and could only be partially inhibited with another NK-sensitive target (MOLT- 4). This cytotoxicity could be generated from either Fcγ+ or FCγ? cells. These results indicate considerable heterogeneity of NK-like effectors and their precursors.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.