IL-1-induced prostacyclin production by cerebral vascular endothelial cells inhibits myelin basic protein-specific lymphocyte proliferation.

Addition of cerebral vascular endothelial cells (EC) to myelin basic protein (MBP) immune lymph node cells (LNC) cultured in the presence of MBP resulted in the inhibition of MBP-specific proliferative responses. Proliferation was not inhibited in cultures containing indomethacin (IM), suggesting a possible role for prostaglandins. Significant levels of 6-KPGF1 alpha, the stable hydrolysate product of PGI2, but not PGE2 were observed in culture (LNC + EC) supernatants but not in supernatants from cultures containing only LNC or EC. The levels of PGI2 release were proportional to the concentration of exogenous EC present in culture and synthesis of PGI2 could be blocked by IM. These results indicate the requirement for coculture in the generation PGI2. Additional experiments indicated that EC were required for the generation of PGI2 and that either macrophages (M phi), or recombinant murine IL-1 were able to replace LNC in cocultures with EC in order to generate PGI2. The ability of IL-1 to stimulate EC-derived PGI2 synthesis was dose dependent with maximal stimulation observed at 50 U/ml IL-1. The IL-1-induced production of PGI2 by EC as well as PGI2 production in cultures containing EC and either LNC or M phi was inhibited by treatment with anti-IL-1 antibody. These results indicate that EC are capable of inhibiting antigen-specific lymphocyte proliferation by producing PGI2, which can be induced by the lymphokine, IL-1.     

Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2)

The effect of rIL-6 on the growth and differentiation of highly purified human peripheral blood B cells was examined. IL-6 alone induced minimal incorporation of [3H]thymidine by unstimulated or Staphylococcus aureus (SA)-stimulated B cells and did not augment proliferation induced by SA and IL-2. Similarly, IL-6 alone did not support the generation of Ig-secreting cells (ISC) or induce the secretion of Ig by unstimulated or SA-stimulated B cells. However, IL-6 did augment the generation of ISC and the secretion of all isotypes of Ig induced by SA and IL-2. Maximal enhancement of B cell responsiveness by IL-6 required its presence from the initiation of culture. Delaying the addition of IL-6 to B cells stimulated with SA and IL-2 beyond 24 h diminished its effect on ISC generation. However, increased Ig production but not ISC generation was observed when IL-6 was added to B cells that had been preactivated for 48 h with SA and IL-2. This effect was most marked when the activated B cells were also stimulated with IL-2. IL-6 in combination with other cytokines such as IL-1 and IL-4 did not induce the secretion of Ig or generation of ISC in the absence of IL-2. Moreover, antibody to IL-6 did not inhibit the effect of IL-2 on the growth and differentiation of B cells stimulated with SA, but did inhibit the IL-6-induced augmentation of Ig secretion by B cells stimulated with SA and IL-2. IL-6 alone enhanced T cell dependent induction of B cell differentiation stimulated by PWM. Part of this enhancement was related to its capacity to increase the production of IL-2 in these cultures. These results indicate that IL-6 has several direct enhancing effects on the differentiation of B cells, all of which are at least in part dependent on the presence of IL-2. In addition, IL-6 can indirectly increase B cell differentiation by increasing IL-2 production by T cells.     

Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets--neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells--into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7- T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.     

Frequency of human B cells that differentiate in response to anti-CD3-activated T cells

Activation of T cells by mAb to the CD3 molecular complex induces the differentiation of many more Ig-secreting cells (ISC) from resting human B cells in bulk cultures than do other modes of polyclonal B cell activation. In the current experiments, a limiting dilution assay was used to demonstrate that this increase in ISC generation reflects an increased frequency of responding B cells. Highly purified B cells were cultured at densities of between 1000 cells and 0.5 cell per microwell with fresh, mitomycin C-treated T cells (T mito) or T cell clones stimulated by immobilized mAb to CD3. After 5 days in culture, the number of wells containing ISC was determined, and the frequency of responding B cells was calculated. The proportion of B cells responding to anti-CD3-stimulated T cells was very large (10.7 +/- 2.8%) and greatly surpassed that induced by other polyclonal activators. B cells cultured with anti-CD3-stimulated T cell clones responded better than did those cultured with T mito. The addition of exogenous IL-2 or IL-6 to cultures supported by activated T mito enhanced the frequency of responding B cells, whereas IL-4 did not increase the generation of ISC and inhibited the augmentation of B cell responses induced by IL-2. Supplementation of cultures with mitomycin C-treated B cells as accessory cells had less of an effect. The addition of both accessory cells and IL-2 markedly increased B cell responsiveness, with precursor frequencies of 60 to 80% noted. In some experiments, cultures were carried out for 7 to 14 days and supernatants were analyzed for IgM, IgG, and IgA secretion. B cells activated by anti-CD3-stimulated T cells produced all three Ig isotypes. When the classes of Ig produced by single B cells were examined, it was observed that the stimulation of individual B cell precursors led to the production of multiple Ig isotypes, suggesting that isotype switching occurs in these cultures. These results demonstrate that under optimum culture conditions, T cells stimulated with immobilized anti-CD3 can activate the majority of human peripheral blood B cells to produce Ig and induce isotype switching by many.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.     

Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.     

Independent regulation of B-cell inducing factor and IL-2 production by T lymphocytes, and direct and indirect promotion of immunoglobulin secretion by glucocorticosteroid

The conditions for induction of B-cell inducing factor (BIF) by human peripheral blood T cells was investigated. BIF was assayed by induction of immunoglobulin secreting cells (ISC) in peripheral blood B (non-T) cells stimulated with Staphylococcus aureus bacteria strain Cowan I (Sac), and in the IgM cell line SKW6.4. Maximum BIF production occurred with high concentrations of the T-cell mitogens phytohemagglutinin, concanavalin A, and PWM. Dexamethasone (Dex) also induced BIF production in T cells at 10(-5) to 10(-7) M. At 10(-5) and 10(-6) M Dex, the T-cell supernatants had to be dialyzed before testing because Dex alone stimulated variable levels of ISC in both test B-cell assays. Dex did not enhance BIF production by T cells that were optimally stimulated by lectin. BIF levels were maximum by Day 2 of T-cell cultures and remained high at Days 3 and 4. In contrast, IL-2 reached a peak at Day 1 and declined drastically by Day 4. We previously showed that IL-2 at less than 100 U/ml did not induce ISC in B cells and did not alter ISC induction by BIF. Dex did not induce IL-2 production and inhibited IL-2 production induced by Con A, in contrast to the promoting effects of Dex on BIF production, providing further evidence for the independence of BIF and IL-2 production and B-cell stimulation.     

Interleukin 2 induction of antigen-nonspecific suppressor cells

Although Interleukin 2 (IL-2) is essential to the generation of immune responses it may also be important as a regulator of these same responses, as both primary and secondary anti-SRBC responses are greatly diminished when IL-2 is included in culture. IL-2 must be present within the first 24 hr of culture to affect maximum suppression. This inhibition is mediated by suppressor cells which are expanded by pulsing spleen cells with IL-2 for 48-72 hr. Their development is not antigen dependent and their action is antigen nonspecific. Suppressor cell activity can be generated from either naive or primed animals which are equally effective in inhibiting primary or secondary anti-SRBC responses. Suppressor cells can be propagated for long periods of time in T-cell growth factor-containing medium. These long-term cultured cells retain the ability to inhibit various immune responses such as mitogen- and alloantigen-induced proliferation, the generation of cytotoxic T lymphocytes and humoral responses. These cells suppress these responses by absorbing IL-2, as demonstrated by their ability to remove IL-2 upon incubation at 4 degrees C, and the reversal of suppression by the addition of supraoptimal amounts of IL-2.     

Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

Comparative analysis of splenic cell proliferation induced by interleukin 3 and by syngeneic accessory cells (syngeneic mixed leukocyte reaction): evidence that autoreactive T-cell functioning instructs hematopoietic phenomena

Murine syngeneic mixed leukocyte reaction (SMLR) was studied under totally autologous culture conditions using syngeneic normal mouse serum in the culture. SMLR was detected in splenic, but not in lymph node, nonadherent responding cell populations (NWNAC). In the absence of stimulator, accessory cells (AC), IL3-containing fluids also induced splenic, but not lymph node, NWNAC growth. SMLR-derived supernatants contained IL3, but not IL2, activity, and production of this IL3 activity could be prevented by adding anti-CD4 mAbs to SMLR cultures. Precursor frequencies of both SMLR and IL3 splenic responses were very low and similar, and there was a synergism between IL3 and AC in induction of NWNAC growth. Growth of responding NWNAC was further enhanced by T-cell depletion with anti-Thy1 mAb and complement. Lack of T-cell proliferation in the SMLR was confirmed by BUdR and light protection experiments. Autoradiographs indicated that the same cell type grew in both SMLR and IL3-induced NWNAC cultures. Besides blast cells, cells with the appearance of immature monocytes with 3H-labeled nuclei were found in both kinds of culture. No labeled lymphocytes could be found. Both SMLR and IL3-induced NWNAC cultures contained expanded numbers of M-CSF-responsive monocyte precursors. On the other hand, SMLR- but not IL3-induced cultures contained expanded numbers of IL3-responsive, immature precursors capable of giving rise to large colonies of monocytic-like cells. Although IL2 could not be detected in SMLR supernatants, both cell growth and IL3 production could be blocked with anti-IL2 receptor and anti-IL2 mAbs. Exogenous IL2, on the other hand, enhanced both cell growth and IL3 production in the SMLR. These results indicate that, under totally autologous conditions, CD4+ autoreactive T-cells do not proliferate in the SMLR, but rather instruct the growth of splenic hematopoietic precursors capable of differentiating along the monocytic lineage. Autoreactive T-cell activation in the SMLR seems to involve minimal IL2 production, which is critically necessary for triggering IL3 production in a markedly amplified manner. These results suggest a link between normal regulation of hematopoiesis and MHC-restricted, autoreactive T-cell activation.     

Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.     

DNA vaccines encoding interleukin-8 and RANTES enhance antigen-specific Th1-type CD4(+) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes. However, no data currently exist in an infectious challenge model system. We analyzed the modulatory effects of selected chemokines (interleukin-8 [IL-8], gamma interferon-inducible protein 10 [IP-10], RANTES, monocyte chemotactic protein 1 [MCP-1], and macrophage inflammatory protein 1 alpha [MIP-1 alpha]) on immune phenotype and protection against lethal challenge with herpes simplex virus type 2 (HSV-2). We observed that coinjection with IL-8 and RANTES plasmid DNAs dramatically enhanced antigen-specific Th1 type cellular immune responses and protection from lethal HSV-2 challenge. This enhanced protection appears to be mediated by CD4(+) T cells, as determined by in vitro and in vivo T-cell subset deletion. Thus, IL-8 and RANTES cDNAs used as DNA vaccine adjuvants drive antigen-specific Th1 type CD4(+) T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1 alpha increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.     

Histamine acts directly on human T cells to inhibit interleukin-2 and interferon-gamma production

Histamine acts directly on human T cells to inhibit lymphokine production without the involvement of accessory cells. Histamine inhibits the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by purified human peripheral T cells activated in the presence of either intact monocytes or metabolically inactive fixed Raji and U698 cells as accessory cells. Purified T cells do not respond more than marginally to staphylococcal enterotoxin A (SEA) or phytohemagglutinin (PHA) in the absence of accessory cells. However, activation by the phorbol ester PMA in conjunction with either PHA or the calcium ionophore A23187 induces large amounts of IFN-gamma and IL-2. Histamine suppresses the lymphokine production in these pure T-cell cultures to a similar extent as in monocyte-containing cultures. Histamine is also shown to suppress DNA synthesis by purified T cells cultivated at a low cell density, eliminating any possible involvement of small numbers of contaminating accessory cells. In vitro preactivated T cells are shown to retain their capacity to respond to histamine when stimulated by PMA and A23187 or by mitogen in the presence of Raji cells. The conclusion that histamine acts directly on T cells and does not require accessory cells to induce suppression is further confirmed by the demonstration that IL-2 production by the human T-cell leukemia line Jurkat was significantly suppressed by histamine in a H-2 receptor-restricted manner.     

Regulation of human T-cell production of interleukin 2 by Leu 11 (CD16) positive large granular lymphocytes

Peripheral blood large granular lymphocytes (LGL) expressing Leu 11 (CD16) antigen with potent natural killer cytotoxicity inhibited soluble antigen-induced T-cell production of interleukin 2 (IL-2). Depletion of Leu 11-reactive cells from T-cells doubled IL-2 activity (P less than 0.05). Leu 11-enriched cells did not express high affinity IL-2 receptors nor did they deplete IL-2 activity from culture media. Upon addition in low ratios to Leu 11-depleted cells, Leu 11-enriched fractions increased antigen-induced IL-2 production three-fold (P less than 0.05), whereas at higher ratios IL-2 production was suppressed P less than 0.01. Additionally, adherent monocytes were increasingly accessory when added in graded numbers to Leu 11-depleted but not T-cell cultures. In the presence of small numbers (5%) of Leu 11-enriched cells, however, monocytes down-regulated IL-2 production of Leu 11-depleted cell cultures. Thus Leu 11-reactive lymphocytes have noncytotoxic functions and may play a major role in immunoregulation.