Identification of a factor(s) from cloned murine natural suppressor cells that inhibits IL-2 secretion during antigen-driven T cell activation.

Crude supernatants were obtained from cloned murine natural suppressor (NS) cells activated in vitro with phorbol-myristate-acetate (PMA) and calcium ionophore (A23187). Supernatants suppressed IL-2 production in the mixed leukocyte reaction (MLR) with BALB/c spleen cells, but no reduction was observed in the response to PHA, Con A, or anti-CD3 antibody. Suppressive activity was partially purified by DEAE ion exchange chromatography, and inhibited the antigen-presenting function of the macrophage line 1G18-LA in an assay system with the ovalbumin-specific T cell hybridoma, 3DO-18.3. In addition, the antigen-presenting function of the A20 B cell line was inhibited in an assay with a sperm whale myoglobin (SpWMb)-specific T cell hybridoma (8.2.1d.H1a). Results with blocking antibodies suggest that this factor appears to be a unique cytokine.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.     

Effect of T cell activation on lymphocyte-endothelial cell adherence and the role of VLA-4 in the rat.

Lymphocyte migration from the blood is in part controlled by lymphocyte surface adhesion molecules, such as VLA-4 and LFA-1. Small lymph node lymphocytes from rats adhere poorly to rat microvascular endothelial cells (EC), while lymphoblasts from antigen-challenged lymph nodes have an enhanced adherence to EC and preferentially migrate to inflamed tissues. This lymphoblast adherence is partially inhibited by anti-VLA-4. The effects of in vitro activation of lymph node lymphocytes on lymphocyte-EC adhesion were examined. In vitro stimulation of T cells with concanavalin A or calcium ionophore and phorbol myristate acetate (PMA) for 1-4 hr caused a marked (7- to 12-fold) increase in lymphocyte adhesion to unstimulated and IFN-gamma- or LPS-treated EC. This adhesion was partially inhibited by anti-VLA-4, was not associated with increased VLA-4 expression, was partially inhibited at 4 degrees C, and was virtually eliminated at 4 degrees C with anti-VLA-4. Anti-CD3 or IL-2 stimulation of T cells also enhanced lymphocyte adhesion but required 2-3 days of culture. This adhesion was not inhibited by anti-VLA-4 and was almost totally inhibited at 4 degrees C, suggesting a primarily LFA-1-mediated adhesion. In conclusion, stimulation of T cells with Con A or calcium ionophore plus PMA caused a rapid enhancement of lymphocyte-EC adhesion mediated in part through VLA-4, while stimulation of T cells with anti-CD3 or IL-2 enhanced lymphocyte adhesion apparently independent of VLA-4.     

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

Analysis of protein phosphorylation patterns reveals unanticipated complexity in T lymphocyte activation pathways.

Protein kinases are considered likely to play important roles in the still dimly understood process by which mitogens induce resting T lymphocytes to enter the cell cycle. Using two-dimensional electrophoretic analysis of lysates from orthophosphate-labeled cells, we have compared patterns of phosphorylation in freshly isolated murine splenic T cells exposed to three mitogenic agents: antibody to the epsilon-chain of the TCR CD3 complex, the plant lectin Con A, and a mixture of PMA and ionomycin, which together bypass the signal transduction apparatus to activate intracellular pathways. Of 14 phosphoproteins found whose level of phosphorylation was increased (at least fivefold) by anti-CD3 epsilon antibody, 13 also responded to the mixture of PMA and ionomycin. Surprisingly, however, only 5 of these 14 also responded strongly to Con A exposure. We also identified two substrates that were phosphorylated in response to Con A but not to anti-CD3. Phosphorylation patterns were also studied in T cells exposed to either PMA or ionomycin alone, to gain further insight into the role of protein kinase C and calcium-dependent events in the activation process. Of 16 phosphoproteins that responded to mixtures of PMA and ionomycin, 4 were shown to require the ionomycin signal, 2 to require the PMA signal, and 3 others to respond only when both activators were present; the other 7 responded to either agonist added alone. In addition, we found two PMA-sensitive phosphoproteins in which phosphorylation was inhibited by ionomycin induced calcium signals. Finally, we identified several phosphoproteins which show differential responsiveness in CD4+ and CD8+ T cells. Classification of kinase substrates based on their differential susceptibility to these stimuli should provide new insights into the mode of action of agents and diseases that affect T cell activation.     

Evidence for the involvement of three distinct signals in the induction of IL-2 gene expression in human T lymphocytes

The regulation of IL-2 gene expression during T cell activation and proliferation has been investigated in primary cultures of purified human peripheral blood T cells. Prior results indicated that stimulation of T cells by anti-CD28 mAb plus PMA could induce IL-2 expression and T cell proliferation that was entirely resistant to cyclosporine. The present studies examined whether CD28 augments IL-2 expression by a unique pathway or merely acts at a point common to CD3-induced proliferation but distal to the effects of cyclosporine. The induction of maximal IL-2 gene expression required three signals provided by phorbol ester, calcium ionophore, and anti-CD28 mAb. Stimulation of cells by optimal amounts of calcium ionophore and PMA induced IL-2 mRNA that was completely suppressed by cyclosporine. The addition of anti-CD28 to T cells stimulated with PMA plus calcium ionophore induced a 5- to 100-fold increase in IL-2 gene expression and secretion that was resistant to cyclosporine. The CD28 signal was able to increase steady state IL-2 mRNA levels even in cells treated with maximally tolerated amounts of calcium ionophore and PMA. The three-signal requirement did not reflect differential regulation of lymphokine gene expression between the CD4 and CD8 T cell subsets or differences in the kinetics of IL-2 mRNA expression. The signal provided by CD28 is distinct from that of CD3 because although anti-CD28 plus PMA-induced proliferation is resistant to cyclosporine, anti-CD3 or anti-CD3 plus PMA-induced IL-2 expression is sensitive. Thus, these studies show that three biochemically distinct signals are required for maximal IL-2 gene expression. Furthermore, these studies suggest that lymphokine production in T cells is not controlled by an "on/off" switch, but rather, that CD28 regulates a distinct intracellular pathway which modulates the level of IL-2 production on a per cell basis. The observation that CD28 stimulation results in IL-2 concentrations that exceed 1000 U/m1 in tissue culture supernatants suggests that a role in vivo for CD28 might be to amplify immune responses initiated by the CD3/T cell receptor complex. Finally, the observation that CD28 interacts with the signals provided by PMA and calcium ionophore shows that the function of CD28 is not merely to act as a scaffold to stabilize or enhance signalling through the CD3/TCR complex.     

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.     

Hyaluronan synthesis is required for IL-2-mediated T cell proliferation

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.     

Evidence for phosphatidylinositol-linked forms of human and avian fibronectin receptors

Phosphatidylinositol (PI)-linked forms of surface molecules have been hypothesized to mediate the initial stages of cell adhesion or signal transduction. We report evidence for the occurrence of a functional PI-linked subset of cell surface fibronectin receptors (FNR). Treatment of human MG63 osteosarcoma cells or primary chicken embryo fibroblasts (CEF) with PI-specific phospholipase C (PI-PLC) reduced cell surface FNR expression by 30% as detected by immunofluorescence. PI-PLC treatment of cell membranes purified from [35S]methionine-labeled CEF or MG63 cells led to a similar loss of membrane-associated immunoprecipitable FNR from the pelleted membranes, while such treatment led to the appearance of FNR in the supernatant of treated MG63 membranes. Biosynthetic labeling of CEF FNR with [3H]palmitate and [3H]ethanolamine demonstrated the acylation and putative PI linkage of avian FNR subunits. PI-PLC treatment of CEF and MG63 cells also reduced fibronectin-specific adhesion in a short-term in vitro assay, suggesting that the avian and human FNR occur in PI-linked isoforms which appear to contribute to cell adhesion to fibronectin.     

Immunodeficiency of aging: restorative effects of phorbol ester combined with calcium ionophore

Current models for lectin-induced T cell proliferation suggest that activation of protein kinase C (PK-C) and elevation of cytoplasmic Ca2+ may both play important roles in the earliest phases of signal transduction. To learn more about the relative inability of T cells from old mice to proliferate in response to mitogenic stimuli, we attempted to stimulate T cells by the synergistic effects of a PK-C activator, phorbol myristate acetate (PMA), and the calcium ionophore ionomycin. T cells from young mice respond as well to optimal combinations of these agents as they do to the strong polyclonal activator Con A, but T cells from old mice respond much better to PMA plus ionomycin than they do to Con A. This result suggests that an inability to transduce the signal supplied by extracellular ligands into the intracellular signals represented by Ca2+ and PK-C activators may underlie the age-associated loss of T cell reactivity. We also found evidence for a second defect in old T cells related to their response to elevated intracellular Ca2+: old T cells, compared with young, required higher levels of ionomycin for maximal proliferation.     

Expression of prohibitins on the surface of activated T cells

Prohibitins (prohibitin-1 and -2) comprise a family of highly conserved proteins that are mainly localized to mitochondria. Recent studies showed that prohibitins are up-regulated upon T cell activation and play an essential role in maintaining mitochondrial homeostasis. In the present study, we found that a considerable proportion of prohibitin-1 and -2 induced in response to T cell activation was expressed on the surface of activated T cells. When mouse and human T cells were stimulated with PMA and ionomycin, prohibitins expressed on the cell surface were increased significantly, peaking at 48 h after stimulation. Stimulation of mouse T cells with anti-CD3 and anti-CD28 antibodies also remarkably induced the cell surface expression of prohibitins. Their expression on the cell surface was also detected in T cell leukemia cells such as Jurkat cells. In Jurkat cells, prohibitin-1 and -2 were co-localized with CD3 on the cell surface, and anti-CD3 antibody-induced signaling, the MAP kinase cascade, was inhibited on treatment with protein A magnetic beads co-conjugated with anti-CD3 antibody and anti-prohibitin-1 or anti-prohibitin-2 antibody. These results suggest that prohibitins expressed on the surface of activated T cells are involved in the T cell receptor-mediated signaling cascade.     

T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice.

The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.     

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

Comparison of mitogenic responses of young and old rhesus monkey T cells to lectins and interleukins 2 and 4

Purified T cells from rhesus monkeys, like human T cells, do not show a significant mitogenic response to lectins or PMA, but when combined with PMA or accessory cells, PHA and Con A induce a vigorous mitogenic response. This response is strongly impaired in purified T cells from old rhesus monkeys compared to young T cells, from 56 to 72%, and parallels results obtained with T cell preparations containing accessory cells. Likewise, purified T cells do not respond to interleukin 2 (IL-2) or IL-4, but in the presence of PMA, a significant mitogenic response occurs in the young but not the old T cells. This response is augmented by accessory cells, but is still very deficient in the old T cells. These results show that the IL-2 independent activation of T cells triggered by IL-4, like the conventional IL-2 activation, is age impaired. The deficient response to IL-2 implies an age-related deficiency in IL-2 receptor as well in aged rhesus T cells, and may account for the less effective response of the old cells to calcium ionophore (+PMA) activation. The use of purified T cells in these studies obviate the influence of accessory cells, and thus simplify interpretation.     

The role of protein kinase A anchoring via the RII alpha regulatory subunit in the murine immune system

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.