Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.     

Induction of interferon-gamma production by human natural killer cells stimulated by hydrogen peroxide

Interferon (IFN)-inducing activity of hydrogen peroxide in human peripheral mononuclear cells was investigated. Among the mononuclear cells, purified nonadherent cells produced IFN, but not B cells and monocytes. The maximal titer of IFN by purified nonadherent cells was observed after a 72-hr cultivation in the presence of 10(-2) mM H2O2 without affecting their viability. Furthermore, the purified nonadherent cells, but not the unpurified mononuclear cells, showed an augmented cytotoxicity to K562 when stimulated with hydrogen peroxide. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into the low and high density fractions for which natural killer (NK) cells and T cells were enriched, respectively. The NK-enriched low density fractions, but not the T cell-enriched high density fractions, showed IFN production by the stimulation of hydrogen peroxide. IFN production as well as large granular lymphocytes and HNK-1+, Leu-11+ cells of the NK-enriched fractions were abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1+) but not against T cells (OKT3) in the presence of complement. Moreover, hydrogen peroxide-inducing IFN production seems to be regulated by monocytes. The antiserum neutralizing IFN-alpha and IFN-beta failed to neutralize substantially IFN-produced NK cells. The treatment with either pH 2 or antiserum-neutralizing human IFN-gamma resulted in marked reduction, indicating that a major part of IFN was IFN-gamma. The purified nonadherent cells showed IFN production and augmented cytotoxicity when cultured separately from activated macrophages by opsonized zymosan; furthermore, both IFN production and enhancement of cytotoxicity were abrogated by catalase. These results suggest that both exogenous and endogenous hydrogen peroxide might be responsible for a part of immunoregulation.     

Functional characterization of T lymphocytes grown in semisolid agar

T lymphocyte colony forming cells (TL-CFC) grown in agar in the presence of PHA were assayed for their capacity to induce or suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells. This was measured by identifying cells containing intracytoplasmatic immunoglobulins by direct immunofluorescence. To validate the helper and suppressor system used in this paper, the inductive capacity of unfractionated T lymphocytes and their subpopulations bearing Fc-receptors for IgM (TM) and for IgG (TG) was measured. The unfractionated T cells and the TM fraction showed helper activity, whereas the TG cells expressed suppressor activity. The TL-CFC grown in agar in the presence of PHA manifested helper activity at low cell concentration. However, increasing the TL-CFC concentration finally caused suppression of B cell differentiation. The suppressor effect could be abolished by prior irradiation of the TL-CFC before seeding them in agar. These results indicate that T cells grown in agar have the functional capacity of T helper and T suppressor cells to induce and suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells.     

Modulation of human B cell immunoglobulin secretion by the C3b component of complement

The human C3b component of complement was found to inhibit the differentiation of human B lymphocytes into immunoglobulin-secreting cells in vitro. Pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses were inhibited by C3-coated zymosan particles and by purified human C3b. C3b inhibited the PWM-driven responses in a dose-dependent fashion, and it was necessary for C3b to be present in the early phases of the cultures. C3b acted directly on B cells rather than on helper T cells because it inhibited the PFC responses of MNC depleted of T cells and subsequently stimulated with a T cell-independent Epstein Barr virus mitogen. Furthermore, C3b failed to stimulate the generation of suppressor lymphocytes and/or monocytes that might have been responsible for the inhibition of B cell responses. Our results indicate that C3b or its fragments exert negative modulatory effects on human B lymphocyte responses.     

Asialo-GM1+ natural killer cells directly suppress antibody-producing B cells

Natural killer (NK) cells were tested for their ability to suppress antigen-induced antibody responses in vitro. Asialo-GM1+ (ASGM1+) cells were prepared from nylon-wool-nonadherent spleen cells obtained from normal mice. After depletion of Ig+, L3T4+ and Lyt-2+ cells, the ASGM1+-enriched cell population had high NK activity which was abrogated by treatment with anti-ASGM1 and C'. This NK-enriched ASGM1+ cell fraction significantly suppressed the generation of antibody-producing cells when added to in vitro immunization cultures of primed spleen cells. Treatment of the NK-enriched cell population with anti-ASGM1 and C' abrogated the ability of these cells to suppress antibody responses. In vitro antibody production by purified B cells was also suppressed in the presence of the NK-enriched cell population, although the kinetics of the suppression differed from that observed with unfractionated spleen cells. In addition, the NK-enriched cell population suppressed the proliferation of the B cell line WEHI-279.1. Suppression of WEHI-279.1 cells was abrogated when the NK-enriched cell population was treated with anti-ASGM1 and C'. These results suggest that normal NK cells suppress the generation of antibody-producing B cells and that this occurs, at least in part, through a direct regulation of the B cell.     

The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

Control of human B lymphocyte responsiveness: enhanced suppressor T cell activity after in vitro incubation.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) after a period of preincubation in vitro. When fresh PBM were co-cultured with preincubated PBM their response to PWM was inhibited, indicating that enhanced suppressor activity developed in the aged PBM concomitant with the loss of PWM responsiveness. Suppressor cell activity of aged PBM was present within the T lymphocyte population. The suppressor T cell inhibited PWM responsiveness of autologous and homologous PBM to an equivalent degree. The action of the suppressor cell was abrogated by inhibitors of DNA synthesis or by hydrocortisone. A suppressor T cell population with similar characteristics was found in freshly prepared PBM before in vitro incubation. Expansion of this suppressor T cell population during preincubation required cell division. There was no change in the functional capability of the helper T cell population as a result of similar in vitro culture. These observations indicate that a T cell population capable of suppressing PWM-induced generation of ISC can be selectively expanded by in vitro incubation of normal human PBM without additional mitogenic stimulation. Moreover, these data emphasize that control of B lymphocyte differentiation involves a critical interrelationship between T lymphocyte subpopulations exerting both positive and negative influences.     

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF)

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Suppression of B cell responses by natural killer cells is mediated through direct effects on T cells

We examined the ability of human natural killer (NK) cells to modulate T cell-dependent mitogen-induced B cell responses. Highly purified NK cells inhibited the polyclonal antibody responses of autologous pokeweed mitogen (PWM)-stimulated unfractionated mononuclear cells in a reverse hemolytic plaque-forming cell (PFC) assay. Investigation of the possible mechanism(s) of the suppressor activity of NK cells revealed that lysis of mitogen-stimulated cells was unlikely. Chromium-51 release cytotoxicity assays of PWM-stimulated mononuclear cells did not demonstrate lysis by NK cells. Additionally, the monoclonal antibody 13.3, which abrogates NK cell cytolysis, did not reverse NK cell-dependent suppression of PFC formation. The putative lytic molecule elaborated by NK cells, NK cytotoxic factor, did not suppress B cell responses, further supporting a nonlytic inhibitory mechanism. That NK cell-derived lymphokines such as IFN-alpha, IFN-gamma, or IL-2 were uninvolved in the down-regulation of B cells was corroborated by the failure of antibodies to these mediators to reverse the suppression. NK cells did not suppress PFC formation when T cells were replaced by supernatants from PWM-stimulated T cells; additionally, NK cells had no effect on the generation of these necessary T cell factors. However, the coculture of T cells with NK cells resulted in the induction of suppressor activity within the T cell population suggesting that this was the mechanism of NK cell-mediated suppression of B cell responses.     

Helper cell function of human fetal thymocytes

The role of human fetal thymocytes in the regulation of IgG and IgM production was examined. In pokeweed mitogen (PWM)-driven cocultures between thymocytes and normal adult peripheral blood mononuclear cells (PBMs) significantly enhanced secretion of both IgM (350% expected; P < 0.02) and IgG (450% expected; P < 0.001) was observed. In contrast, adult PBMs or adult T cells neither suppressed nor enhanced IgM or IgG production in coculture with allogeneic adult PBMs. Enhanced immunoglobulin secretion was not found when fetal thymocytes were cocultured with the T-cell-depleted fraction of adult PBMs. These results suggest that human fetal thymocytes (as early as 12 weeks gestational age) can provide helper cell function for both IgM and IgG secretion in PWM-driven cell cultures; however, a more mature T-cell effector is required for expression of this helper function.     

Activation and function of an autoreactive T cell clone with dual immunoregulatory activity

Previously it was demonstrated that the human autoreactive CD4+ T cell clone MTC-4 is bifunctional, having the capacity to augment differentiation of autologous B cells into Ig-secreting cells in the absence of PWM and the capacity to suppress such differentiation in the presence of PWM. In the present study it was shown that these two functions of MTC-4 are mediated by distinctly different mechanisms. In the presence of autologous class II MHC Ag, MTC-4 releases one or more non-MHC-restricted soluble factors which stimulate B cell differentiation. The helper factors are different from IL-2, and act on both resting (small) and activated (large) B cells. The suppressor function of MTC-4 cells is elicited when MTC-4 cells are co-cultured with autologous non-T cells preincubated with PWM for 4 h, but not with non-T cells preincubated with PWM for 24 h; thus, activated autologous non-T cells have a transient capacity to induce MTC-4 suppressor function. Induction of MTC-4 suppressor activity is not associated with increased proliferation of MTC-4 and is mediated by low numbers of these cells. Unlike helper function, MTC-4 suppression of Ig synthesis can occur late in B cell cultures, and MTC-4 suppresses Ig production by autologous B cells, but not by allogeneic B cells. Finally, in co-cultures with activated autologous non-T cells and allogeneic B cells, MTC-4 can simultaneously produce helper factors that augment Ig synthesis by allogeneic B cells and suppress Ig synthesis by autologous B cells. In summary, exposure of MTC-4 to autologous non-T cells causes release of non-MHC-restricted factors which augment Ig production by both resting and activated autologous B cells, whereas exposure of MTC-4 to recently activated B cells causes MTC-4 to express the additional function of directly suppressing Ig production by differentiated autologous B cells. Thus autoreactive T cells may be uniquely suited to regulate Ig production.     

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.     

Inhibition of human lymphocyte activation by wheat germ agglutinin: a model for saccharide-specific suppressor factors

The suppressive effect of wheat germ agglutinin (WGA) on lectin-stimulated blastogenesis and immunoglobulin production was studied. Addition of WGA at 10 micrograms/ml inhibited phytohemagglutinin (PHA)-, concanavalin-A (Con-A)-, and pokeweed mitogen (PWM)-induced mitogenic responses by 70-80%. PWM-driven immunoglobulin synthesis was suppressed by 45% with WGA. The inhibitory effects of WGA were not due to cell death or to interference with lectin binding at the cell surface. Inhibition was dependent on the presence of WGA in the cell culture during the first 24 hr of mitogen exposure and was observed in cultures of both monocyte-depleted peripheral blood mononuclear cells as well as T-cell-enriched populations. WGA-induced inhibition of blastogenesis was blocked by the addition of N-acetylglucosamine (GluNAc) which prevents WGA binding to the cell surface. WGA was found to mimic the suppressive effect of a soluble immune suppressor supernatant (SISS) derived from Con-A-activated mononuclear cell cultures. PHA responses were inhibited by 80 and 95% with SISS and WGA, respectively. The inhibition by both WGA and SISS was totally reversed with addition of GluNAc. Furthermore, WGA and SISS demonstrated competition for the same cell surface receptor site. WGA may therefore be useful as an in vitro model of a saccharide-specific, biologically relevant, soluble mediator for the investigation of mechanisms of immunologic suppression.     

Induction of suppressor cells from peripheral blood T cells by 15-hydroperoxyeicosatetraenoic acid (15-HPETE)

15-hydroperoxyeicosetetraenoic acid (15-HPETE), a lipoxygenase metabolite of arachidonic acid, inhibited polyclonal IgG and IgM production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells, whereas 15-hydroxyeicosetetraenoic acid (15-HETE) had little effect in this system. T cells preincubated for 18 hr with 15-HPETE caused substantial inhibition of IgG and IgM production of fresh, autologous B and T cells stimulated by PWM. The suppressive effect of the 15-HPETE-treated cells was lost if the cells were irradiated before the PWM culture, but not by treatment with mitomycin C. The suppressive effect was also lost if OKT8+ T cells were removed after, but not before, preincubation of the T cells with 15-HPETE. OKT8- T cells incubated with 15-HPETE for 18 hr showed a large increase in the percentage of cells staining with directly fluoresceinated Leu-2, another marker for suppressor cells. Thus, 15-HPETE induces functional and phenotypic suppressor cells from resting human peripheral blood T cells.     

Immunoregulatory human T lymphocytes triggered as a consequence of viral infection: clonal analysis of helper, suppressor inducer and suppressor effector cell populations

The regulatory functions of a series of human T cell clones specific for an autologous Epstein-Barr virus transformed B lymphoblastoid cell line were examined. Two T4+ T cell clones, termed AT4II and AT4IV, and one T8+ clone, AT8III, were maintained in culture for greater than or equal to 9 months and were characterized in detail. Both T4+ clones provided helper function for autologous B cell immunoglobulin production when added to unstimulated peripheral blood mononuclear cells. In addition, these same clones produced soluble inducer factors after specific antigenic stimulation. However, when AT4II, AT4IV and their subclones were tested on pokeweed mitogen stimulated peripheral blood mononuclear cells, it was found that AT4IV provided help for immunoglobulin production whereas AT4II cells were strongly suppressive. This suppression by AT4II was indirect and required the presence of fresh, autologous, unirradiated T8+ cells. In contrast, the T8+ AT8III clone markedly inhibited Ig production by autologous B cells in the absence of any additional T8+ cells from peripheral blood and produced a soluble suppressor factor upon specific antigenic triggering. Thus, after stimulation with autologous Epstein-Barr virus transformed cells, at least three discrete regulatory human T cell populations can be defined at the clonal level: helper, inducer of suppression and suppressor effector clones.