Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.     

Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.     

Regulatory effects of mast cells on lymphoid cells: the role of histamine type 1 receptors in the interaction between mast cells, helper T cells and natural suppressor cells

We have investigated the role of mast cells as modulators of lymphocyte function because the mast cells are concentrated in the areas of lymphoid storage; they are dependent upon T-cell growth factor for their proliferation; and they appear to be the principle if not sole storage site for histamine. We have tested the influence of mast cells on the proliferation of alloreactive cloned helper T cells, mixed leukocyte reactions, and the suppressive capacity of natural suppressor cells. We used an IL-3-dependent mast cell line that at high numbers (greater than 10(5)) suppressed and at low numbers (10(3) to 6 X 10(4)) augmented the proliferation of TH cells. Addition of histamine to cocultures enhanced the mast cell mediated proliferation of TH cells without directly affecting the helper cells. The action of histamine appeared to be mediated with H1 type receptors on these mast cells. Pretreatment of natural suppressor cells with supernatants from mast cell enhanced their suppressive capability. Here too, histamines enhanced suppression by the NS cell via histamine type 1 receptors on the natural suppressor cells. Our data suggest that mast cells may be a major modulator of the lymphoid cell immune function and demonstrate a role of histamine type 1 receptors in the interaction between mast cells, helper T cells, and natural suppressor cells.     

Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells

Many long term mouse Th clones express either the type 1 or type 2 Th cell (Th1 or Th2) cytokine secretion phenotype. In this report we present two lines of evidence for the existence of additional Th differentiation states. Lectin-stimulated spleen cells secreted moderate levels of IL-2 compared with long term Th1 clones, whereas the levels of other cytokines were more than 100-fold lower than those produced by either Th1 or Th2 clones. This suggests that many spleen cells produce substantial amounts of IL-2 but little or no IL-4, IL-5, IFN-gamma, IL-3, and granulocyte/macrophage-CSF. In contrast to long term Th clones, many short term alloreactive clones displayed cytokine secretion phenotypes intermediate between the Th1 and Th2 patterns. The proportion of recognizable Th1 and Th2 clones at early times in culture was greatly increased by immunization of the mice from which the responder and stimulator cells were derived; Brucella abortus immunization resulted in the isolation of exclusively Th1 clones, whereas infection with Nippostrongylus brasiliensis resulted in a strong trend toward the isolation of Th2 clones. The immunization of mice from which responder cells were derived strongly affected the type of Th clone obtained, whereas the source of stimulator cells had much less effect, suggesting that the commitment of Th cells to the Th1 or Th2 phenotypes occurred mainly in vivo. A model for the possible relationships of the various Th cells is presented.     

Response against single minor histocompatibility antigens. I. Functional and immunogenetic analysis of cloned cytolytic T cells

A clonal approach was used to investigate the cellular basis of a T cell response to single minor histocompatibility antigens (miHA). This analysis was performed by functional and immunogenetic characterization of a large number of clones derived from short-term mixed leukocyte culture (MLC) populations generated against the miHA, H-1.3. Forty-nine clones isolated from such MLC were specifically cytolytic for H-1.3-bearing, H-2Db-compatible target cells. Thirty-seven of the 49 cytolytic clones were driven to proliferate when stimulated by spleen cells bearing the H-1.3 alloantigen in the absence of added T cell-derived growth factor(s) (GF). The remaining 12 clones proliferated only when GF was added. A strong positive correlation was observed between antigen-induced proliferation and the production of interleukin 2 (IL 2) activity. A similar correlation was observed when comparing the ability of both antigen and concanavalin A to induce IL 2 activity from the clones. These data suggest that i) antigen-driven or helper T cell-independent cytolytic T cells (HITc) are frequent components of an MLC response to a single miHA, and ii) the ability of HITc to undergo antigen-driven proliferation is related to their ability to produce antigen-induced GF.     

Spleen cells from adult mice given total lymphoid irradiation (TLI) or from newborn mice have similar regulatory effects in the mixed leukocyte reaction (MLR). II. Generation of antigen-specific suppressor cells in the MLR after the addition of spleen cells from newborn mice

Spleen cells from newborn BALB/c mice were added to the mixed leukocyte reaction (MLR) between a variety of responder and stimulator cells. The newborn cells nonspecifically suppressed the uptake of (3H)-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. Suppressor cell activity fell rapidly during the first 4 days after birth, and could not be detected by day 20. Newborn spleen cells inhibited the generation of nonspecific suppressor cells during the MLR but did not inhibit the generation of antigen-specific suppressor cells. Thus, newborn spleen cells exhibit a pattern of regulation of the MLR similar to that reported previously for spleen cells from adult mice given total lymphoid irradiation (TLI). These regulatory interactions provide a model that explains the ease of induction of transplantation tolerance in vivo in newborn mice and in TLI-treated adult mice.     

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.     

Immunosuppressive lymphokine derived from natural suppressor cells

Cloned natural suppressor (NS) cells derived from spleens of total lymphoid irradiated BALB/c mice were incubated with the phorbol ester, PMA, and calcimycin for 4 h. After thorough washing, the induced NS cells were incubated in serum-free medium for 24 h and the supernatants were collected. The supernatants suppressed the MLR between normal adult responder and stimulator spleen cells. There was no Ag specificity or H-2 haplotype restriction of the MLR suppression. The supernatants did not inhibit [3H]thymidine incorporation per se, because they did not suppress mitogen stimulation of spleen cells. Protease digestion of the supernatants removed the suppressive activity, and dialysis studies indicated that the molecular size of the suppressive factor was larger than 50,000 Da and smaller than 100,000 Da. The suppressive activity was stable at 56 degrees C, pH 2, for 1 h. Thus, NS cell clones can be induced to secrete an immunosuppressive lymphokine, NS factor.     

Induction of interleukin 3 but not interleukin 2 or interferon production in the syngeneic mixed lymphocyte reaction

The syngeneic mixed lymphocyte reaction (SMLR) was assayed in the medium containing syngeneic normal mouse serum (NMS), by using nylon-adherent stimulator cells and nonadherent responder T cells, which were prepared from murine spleens in the absence of fetal calf serum (FCS) to avoid any sensitization to xenogeneic protein antigens. The responder cells in this SMLR, without definite background proliferation, generated specific proliferative response to the syngeneic stimulator cells in a dose-related fashion. The SMLR was accompanied by production of interleukin 3 (IL 3) but not interleukin 2 (IL 2) or interferon (IFN). No cytotoxicity against the syngeneic or allogeneic target cells was induced. Correlating with no production of IL 2 or IFN, no natural killer (NK) activity was detected. The proliferation was not inhibited by addition of specific antiserum for IFN-gamma. In contrast, proliferation in the responder cells when incubated with allogeneic stimulator cells was inhibited by anti-IFN-gamma serum and accompanied by production of IL 2 and IFN as well as IL 3, and by augmentation of NK activity and generation of cytotoxic T cells. Cell surface analysis revealed that the cells producing IL 3 in this SMLR system were Thy-1+ Lyt-1+2- helper T cells. Cells responding to the SMLR culture fluids with DNA replication were Thy-1-Lyt-1-2- asialo GM1- no-marker cells, which were the same as a population responsible for partially purified IL 3. On the other hand, when the responder cells were exposed to FCS before culture and assayed for SMLR in the FCS-free NMS medium, variable levels of IL 2 production were induced in response to the stimulator cells. The responder cells generated a high background DNA replication in the absence of syngeneic stimulators, suggesting that this IL 2 production may result from the stimulation of T cells by FCS as a foreign antigen. Overall, these results suggest that the SMLR may be a cellular interaction, in which non-T cells stimulate Lyt-1+2- helper T cells to produce IL 3 but not IL 2 or IFN. This IL 3 can, in turn, induce proliferation of IL 3 responding cells, which appear to be early precursors in lymphocyte differentiation, but no proliferative response or activation of IL 2- and IFN-dependent mature T cells or NK cells.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

Identification of a cord blood T cell subset of CD3+4-8-45R+ suppressing interleukin 2 production in the autologous mixed lymphocyte reaction and the mode of action of exogenous IL2 in the induction of IL2 production

As previously reported, the inability of cord blood T cells to produce IL2 in the autologous mixed lymphocyte reaction (AMLR) could be recovered by the treatment of stimulator non-T cells with interferon-gamma (IFN-gamma) and of the AMLR with exogenous IL2. In the present study, we showed that addition of untreated autologous cord blood T cells to the above-mentioned AMLR abrogated the IL2 production in a dose-dependent manner, suggesting active suppression by the untreated T cells because untreated cord blood T cells did not consume IL2. Suppressor activity was abrogated by the treatment of cord blood T cells with monoclonal anti-CD3 antibody plus complement or with monoclonal anti-CD45R (Leu 18) antibody, but not by the treatment with monoclonal anti-CD4 antibody and/or anti-CD8 antibody plus complement. These data showed that the cord blood suppressor T cells were CD3+4-8-45R+. This suppressor activity also disappeared by culturing with rIL2 for 8 hr. As the frequency of CD45R+ cord blood T cells was comparable to that of CD45R+ adult T cells and was minimally affected by the IL2 treatment, functional modulation of CD45R+ suppressor T cells by IL2 is suggested. Moreover, in spite of the inhibitory effect of anti-CD45R antibody on the suppressor activity, IL2 production was not induced merely by addition of anti-CD45R antibody directly to the responder cells in AMLR. Taken together, these data suggest the requirement of exogenous IL2 for IL2 production in that IL2-producing-precursor T cells themselves should be stimulated by IL2 in addition to the modulation of CD45R+ suppressor T cells by IL2.     

Role of accessory cell processing and presentation of shed H-2 alloantigens in allospecific cytotoxic T lymphocyte responses

The present study was undertaken to evaluate the role of accessory cell processing of MHC alloantigens in the initiation of primary allospecific CTL responses. To first determine whether antigen processing by accessory cells is involved in the initiation of allospecific CTL responses, accessory cells were retreated with the lysosomotropic drug chloroquine before their addition to culture. It was found that chloroquine pretreatment abrogated their ability to function as accessory cells only when they were of responder haplotype and had no effect when the accessory cells were of stimulator haplotype. Although accessory cells of either responder or stimulator haplotype can initiate allospecific CTL responses, we have previously demonstrated that they do so by activating distinct classes of T helper TH) cells. Indeed, the differential effects of chloroquine on accessory cells of responder or stimulator haplotypes were shown to reflect the fact that chloroquine pretreatment markedly impaired the ability of accessory cells to activate self-Ia-restricted TH cells, but had little effect on the ability of the same accessory cells to activate either allo-class I- or allo-class II-specific TH cells. We next examined the possibility that accessory cells of responder haplotype mediate alloresponses by acquiring and processing shed MHC alloantigens derived from the stimulator cell population. In these experiments, accessory cell-depleted stimulator cells were fixed with paraformaldehyde to inhibit shedding of their surface MHC alloantigens. It was observed that even though mixed stimulator cells were recognized normally by allospecific CTL precursors, they completely failed to stimulate CTL responses mediated by responder haplotype accessory cells, indicating that the function of such accessory cells is dependent upon their acquisition of shed MHC alloantigens. Taken together, the data presented in this report demonstrate that accessory cells of responder haplotype function in allospecific CTL responses by acquiring and processing shed class I MHC alloantigens, and by then presenting the processed alloantigens in association with self-Ia determinants to self-Ia-restricted TH cells. Thus, these data indicate that the self-Ia-restricted TH cells that are involved in allospecific CTL responses recognize processed class I alloantigens in association with self-Ia determinants.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.     

Frequent T4-positive cells with cytolytic activity in spleens of patients with Hodgkin's disease (a clonal analysis)

Purified T lymphocytes isolated from spleens of untreated patients with Hodgkin's disease (HD) were cloned by using a microculture system previously shown to allow clonal expansion of virtually all peripheral blood T lymphocytes. Cells were plated under limiting conditions with irradiated feeder cells and PHA. Interleukin 2 (IL 2)-containing supernatants were added 48 hr later. The phenotypic and functional characteristics of a total number of 221 clones derived from six different HD spleens were investigated and compared with those of 133 clones obtained from three spleens of otherwise healthy individuals who underwent posttraumatic splenectomy. The majority of T cell clones derived from HD spleens expressed the T4+ (helper/inducer) phenotype. However, further functional characterization showed that as much as 50% of these T4+ clones displayed cytolytic activity in a lectin-dependent lytic assay allowing detection of cytolytic cells of any specificity. In contrast, less than 10% T4+ clones derived from control spleens were cytolytic, as assessed by the same lectin-dependent lytic assay. The cytolytic potential of T4+ and T8+ clones established from spleens of patients with HD did not reflect the induction of lymphokine-activated killer cells, because only a minority of them displayed natural killer (NK) activity against NK-sensitive K562 and MOLT-4 cell lines. These findings indicate that T lymphocytes found in the spleens of patients with HD may represent, at least in part, the expansion of a subset present in small percentages among normal peripheral blood or spleen T lymphocytes, which is involved in a cytotoxic reaction.     

Ribavirin exerts differential effects on functions of cd4(+) Th1, th2, and regulatory T cell clones in hepatitis C

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10μg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that - in addition to its immunostimulatory effects on TH1 cells - ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.     

Cytolytic T lymphocyte clones that proliferate autonomously to specific alloantigenic stimulation. II. Relationship of the Lyt-2 molecular complex to cytolytic activity, proliferation, and lymphokine secretion

Previous analyses of the inhibitory effects of anti-Lyt-2 monoclonal antibodies (mAb) on cytolytic activity suggested that Lyt-2/3 antigens expressed on the surface of murine cytolytic T lymphocytes (CTL) are involved in antigen recognition. In the present study, we investigated the effects of anti-Lyt-2 mAb (in the absence of complement) on the functional activities of H-2K/D-specific Lyt-2+ CTL clones that proliferate to antigenic stimulation in the absence of helper T cells or added interleukin 2 (IL 2) and secrete lymphokines. For those clones that were inhibited in cytolysis by anti-Lyt-2 mAb, a parallel inhibition of antigen-dependent proliferation and lymphokine secretion (interferon, macrophage-activating factor) was observed. Inhibition of proliferation or lymphokine secretion could be overcome by the addition of IL 2 or lectin, respectively. Collectively, these results would strongly suggest that anti-Lyt-2 mAb were inhibiting CTL antigen recognition. Not all CTL clones, however, were inhibited in cytolysis by anti-Lyt-2 mAb, in which case proliferation and lymphokine secretion were similarly unaffected. This heterogeneity of Lyt-2+ CTL clones in their susceptibility to inhibition of cytolytic activity, proliferation, and lymphokine secretion by anti-Lyt-2 mAb is discussed in the context of a model proposing that Lyt-2/3 molecules function to stabilize the interaction between CTL receptors and the corresponding target/stimulating cell antigens. Such a stabilization may be required by CTL possessing few and/or low affinity receptors.     

Induction of CTL responses to alloantigens by a Db-specific T helper clone

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

Suppression of lymphocyte alloreactivity by early gestational human decidua. I. Characterization of suppressor cells and suppressor molecules

We examined the immunosuppressor role of the first trimester human decidua on lymphocyte alloreactivity in vitro in order to identify (1) the major cell classes in the decidua mediating the suppressor effect; (2) the stages in the lymphocyte alloreactive responses susceptible to the suppressor effects of the decidua; and (3) the precise nature of the suppressor molecules. Irradiated (2800 R), Ficoll-Paque-separated nucleated cells of the collagenase-dispersed early gestational (6.5-9.5 weeks menstrual age) decidua containing 70-94% typical decidual cells (identified on the basis of distinctive morphology and numerous cytoplasmic or surface markers) or their plastic-nonadherent fractions further enriched for decidual cells (approximately 96% pure) caused a strong dose-dependent suppression of the one way mixed lymphocyte reaction (MLR, i.e., proliferative response measured on Days 3, 4, or 5), when added at the onset of the mixed lymphocyte cultures (MLC). As few as 10(3) decidual cells caused a detectable inhibition of the MLR exhibited by 10(5)-1.5 X 10(5) responder lymphocytes. A smaller degree of suppression was noted with the plastic-adherent fractions of the early decidua (which retained all macrophages and granulocytes, but still included many decidual cells) or unfractionated cells of later gestational (10-13 weeks) decidua containing a higher incidence of leukocytes, granulocytes, and macrophages in particular, or the plastic-adherent fraction thereof, enriched for macrophages. Thus, decidual cells seem to represent an important suppressor cell class in the early gestational human decidua; however, suppression by decidual leukocytes, macrophages in particular, was also evident. The suppressor effect was unrelated to the major histocompatibility phenotype of the responder or the stimulator cells. It was not caused by cell crowding, since an equivalent number of irradiated K562 erythroleukemia cells had little effect on the MLR. The effect was exerted during both the initiation and the progression of the MLR. A delay in the addition of regulator cells progressively minimized the effect on the Day 4 MLR, but did not abolish it completely even when added as late as on Day 3. The major class of mediator molecules was identified as prostaglandins, primarily PGE2, on the basis of the following results: (1) the presence of indomethacin (10(-5) M) or varying dilutions of an anti-PGE2 antibody abrogated this suppression substantially or completely. (2) Addition of pure PGE2 (3 X 10(-7) to 1.1 X 10(-5) M), but not PGF2 alpha, reproduced a dose-dependent suppressor effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prethymic nylon wool-passed bone marrow cells, substituting for helper T cells, can augment the generation of cytotoxic T lymphocytes from their precursors.

Nylon wool-passed bone marrow (NW-BM) cells treated with anti-Thy.1 monoclonal antibody and complement were added to a mixed lymphocyte culture which contained a limiting number of lymph node cells, as responder cells, and a sufficient number of mitomycin-c-treated allogeneic spleen cells as stimulator cells. NW-BM cells of the same MHC haplotype as responder cells enhanced the generation of allo-specific cytotoxic T lymphocytes (CTL) not only at a relatively high dose (3 x 10(3) cells/well) of responder cells, but also at an extremely dilute dose (1 x 10(3) cells/well). NW-BM cells which had a third-party MHC haplotype, a haplotype different from both responder and stimulator cells, also enhanced the generation of CTL at relatively high doses, but not at low doses, of responder cells. NW-BM cells which had MHC haplotypes identical with those of responder cells induced CTL from helper T cell-depleted responder cells, but NW-BM cells which had the third-party haplotype did not. These results showed that the enhancing effects of NW-BM cells of the same MHC haplotype as responder cells might be due to a specific helper effect and the enhancing effect of NW-BM cells of the third-party haplotype might be due to a nonspecific filler effect, which only conditioned the cultured cells. It was also found that, to exhibit the helper effect, NW-BM cells had to possess MHC class II, but not MHC class I, molecules in common with CTL precursors. This study showed that in the induction of CTL, prethymic NW-BM cells had a capability comparable to that of mature helper T cells.