Evidence that genistein, a protein-tyrosine kinase inhibitor, inhibits CD28 monoclonal-antibody-stimulated human T cell proliferation.

In the present investigation, we compared the immunosuppressive effects of genistein and CsA on anti-CD28 stimulated human T cell proliferation, IL-2 production, and IL-2R expression. Genistein, an isoflavanoid compound, is a specific protein tyrosine kinase inhibitor and inhibited the PMA plus anti-CD28 stimulated T cell proliferation. In contrast, proliferation of T cells stimulated with PMA plus anti-CD28 is resistant to the inhibitory effects of CsA. Similar results were obtained with IL-2 synthesis and IL-2R expression. PHA plus anti-CD28 or PMA plus anti-CD28-induced IL-2 synthesis was inhibited by genistein, and CsA, though it inhibited the PHA plus PMA-stimulated IL-2 synthesis, failed to have any effect on PMA plus anti-CD28-induced IL-2 synthesis. Genistein at the concentration that inhibited T cell proliferation and IL-2 synthesis also showed significant inhibitory effects on PMA plus anti-CD28 stimulated IL-2R expression while CsA had no effect on IL-2R from these cultures. Our data suggest that genistein is a powerful immunosuppressive agent, with no toxic effects on T cells, and has the potential for use in the prophylaxis and treatment of allograft rejection. Since genistein blocks the CsA-resistant pathway of T cell proliferation, the combined usage of these two agents may provide better immunosuppressive effect and a lesser degree of CsA-induced nephrotoxicity.     

Activation-Induced Expression of Cell Surface CD28 on Mouse T Lymphocytes is Inhibited by Cyclosporine A

T-cell activation requires both T-cell receptor signaling and a costimulatory signal provided by CD28 which enhances and prolongs interleukin-2 (IL-2) production. To determine the effect of cyclosporine A (CsA) on constitutive and activation-induced CD28 expression, mouse T cells were exposed to CsA (0.1 microM) in the absence or presence of anti-CD3 monoclonal antibody (mAb). CD28 expression was then determined by flow cytometry. CsA treatment prevented activation-induced CD28 expression but did not affect constitutive CD28 expression. Inhibition of inducible CD28 expression by CsA was not rapidly reversible, requiring 48h of restimulation in the absence of CsA for CD28 expression to return to control levels. T cells activated in the presence of combined anti-IL-2 and anti-CD25 mAb (both 10 microg/mL) also exhibited reduced CD28 expression, suggesting that activation-induced CD28 expression is, at least in part, an IL-2-dependent process. However, the inhibitory effect of CsA on activation-induced CD28 expression was maintained in the presence of exogenous IL-2 (250 U/mL). We conclude that CsA, by inhibiting activation-induced expression of costimulatory CD28 molecules by T lymphocytes, may interfere with the ability of CD28 to provide an optimal costimulatory signal for sustained IL-2 production following T-cell activation.     

Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. I. Inhibition of expression of alloantigen-activated suppressor cells, as well as induction of alloreactivity

The effect of FK506 on in vitro human lymphocyte responses was assessed in comparison with cyclosporine. FK506 suppressed, in a dose-dependent fashion, the lymphocyte response to stimulation with PHA and with alloantigens in primary mixed lymphocyte reactions at a 70-100-fold lower concentration than CsA--namely, 50% inhibition (IC50) was obtained with 8.6 nM FK506 and with 750 nM CsA in the PHA response, and with 0.21 nM FK506 and with 20 nM CsA in MLR. Allocytolytic T lymphocyte induction was also inhibited by FK506, whereas the ability of CTL to lyse targets was not affected by the agent, indicating that FK506 did not affect the recognition and binding of alloantigen by CTL. FK506 inhibited, in a dose-dependent fashion, both IL-2 receptor and transferrin receptor expression on the alloactivated lymphocytes--whereas this agent inhibited only incompletely both expression of both receptors on lymphocytes stimulated with PHA. Lymphocytes from primary MLR cultured in the presence of FK506 were tested for suppressor cell activity on day 8 of culture. FK506 did not allow for the expression of alloantigen-activated suppressor cells when used in a dose sufficient to inhibit CTL generation.     

Discodermolide--a new, marine-derived immunosuppressive compound. I. In vitro studies.

The in vitro immunosuppressive properties of a novel, marine-derived compound, discodermolide, are reported here. Discodermolide suppressed the proliferative responses of splenocytes in the murine two-way mixed lymphocyte reaction (MLR) and concanavalin A stimulated cultures, with IC50 values of 0.24 microM and 0.19 microM, respectively. There was no evidence of cytotoxicity for murine splenocytes at concentrations of discodermolide as high as 1.26 microM. Similarly, discodermolide suppressed the proliferative responses of human peripheral blood leukocytes (PBL) in the two-way MLR, and Con A and phytohemagglutinin mitogenesis. The IC50 values were 5.65 microM, 28.02 microM, and 30.12 microM for the MLR, Con A, and PHA mitogenic responses, respectively. There was no evidence of cytotoxicity toward human PBL at discodermolide concentrations as high as 80.64 microM. Discodermolide was equally effective, compared with cyclosporine, in suppressing the PMA-ionomycin induced proliferation of purified, murine T cells, with IC50 values of 9.0 nM and 14.0 nM for discodermolide and CsA, respectively. The production of IL-2 by PMA-ionomycin stimulated T cells was not inhibited by discodermolide; however, the percentage of IL-2 receptor-bearing cells as measured by immunofluorescence with 7D4 antibody, specific for the 55-kDa chain (p55) comprising the murine IL-2 receptor, was reduced. The expression of a similar chain comprising the human IL-2 receptor (Tac antigen, p55) by PHA or Con-A-stimulated PBL was similarly suppressed by discodermolide. The precise mechanism of action of discodermolide remains to be elucidated.     

环孢素A对T细胞活化CD28通路的影响

Ｔ细胞需两种以上刺激信号才能充分活化。环孢素Ａ通过阻断ＣＤ３经通路而产生免疫抑制效应，本文研究ＣｓＡ对ＣＤ２８活化通路的影响。研究发现以抗ＣＤ２单抗和抗ＣＤ２８单抗共刺激后，淋巴细胞增殖比以ＣＤ３单抗单独刺激时显著增强，且对ＣｓＡ不敏感，ＩＤ５０较单独刺激时高４－５倍，研究表明．：ＣＤ２８通路能提供共刺激信号，使ＴＸ以分活化增殖，并且这一活化通路能对ＣｓＡ产生抵抗 。     

Effect of interleukin 2 on the immunosuppressive action of cyclosporine

The influence of exogenous interleukin 2 (IL-2) on the immunosuppressive effect of cyclosporine in the mixed lymphocyte response (MLR) was examined. Results show that addition of exogenous IL-2 to a MLR containing graded doses of CsA (0.01-2.5 micrograms/ml) restored a normal proliferative response to alloantigens. In contrast, the effect of exogenous IL-2 on the induction of cytotoxic lymphocytes in primary MLR in the presence of CsA was variable. At the highest doses of CsA (0.5-2.5 micrograms/ml), no cytotoxic T cell activity could be detected, regardless of the presence of exogenous IL-2. However, at a lower dose of CsA (0.1 microgram/ml) that routinely resulted in the total inhibition of cytotoxic T cell induction, addition of exogenous IL-2 resulted in significant levels of detectable cytotoxic T cell activity. The effect of time-sequential addition of CsA or CsA-plus-exogenous-IL-2 on the proliferative and CML responses in MLR was also examined. Results show that addition of CsA to ongoing primary MLR cultures within the first 48-96 hr of culture results in the significant inhibition of the proliferative and CML response in MLR. Addition of CsA-plus-exogenous-IL-2 to ongoing cultures resulted in no significant inhibition of the proliferative response. In contrast, addition of CsA-plus-exogenous-IL-2 within the first 4 hr of culture did not overcome the immunosuppressive effect of CsA. At 18 hr of culture addition of CsA resulted in complete suppression of the CML response, whereas the addition of CsA-plus-IL-2 resulted in significant levels of cytotoxicity. Thereafter addition of CsA-plus-IL-2 resulted in enhanced levels of cytotoxic T cell activity compared with cultures receiving CsA alone. Taken together, our results suggest that: (1) exogenous IL-2 can overcome the immunosuppressive effect of CsA on the proliferative response in MLR to alloantigens; (2) at high levels of CsA, IL-2 cannot overcome the immunosuppressive effect of CsA on the induction of cytotoxic T-lymphocytes; (3) there are doses of CsA at least in vitro, that allow for the activation of the cytotoxic T cell, presumably with the acquisition of a receptor for IL-2 but without the clonal amplification due to inhibition of IL-2 production; and (4) time-sequential studies revealed that the development of responsiveness to IL-2 by the precursor cytotoxic T cell occurs 4-18 hr after exposure to the stimulating alloantigen with clonal expansion if IL-2 is present.     

Inhibition of in vitro immunoglobulin production by rapamycin.

Like FK506, rapamycin, a structural analog of FK506, is a strong immunosuppressant. The immunosuppressive effect of Rapa in in vitro IgG, IgM, and IgA production by human lymphocytes was examined in this study. To inhibit spontaneous or pokeweed mitogen-stimulated production of Ig by human peripheral blood lymphocytes, about one thousandfold lower concentrations of Rapa (IC50 = 0.3 nM-2 nM) were required than of cyclosporine (IC50 = 0.3 microM-2 microM). T cells were the direct targets of Rapa, because preincubation of T cells with Rapa abolished the T cells helper effect to T-dependent Ig production. Rapa also had direct suppressive effect on B cells, since Rapa suppressed IgG production by pure B cells stimulated with IL2 and Staphylococcus aureus Cowan I. Kinetic studies measuring IgG production and cell proliferation revealed that Rapa acted at the activation stage of T and B cells. Exogenous IL2 substantially reversed the inhibitory effect of CsA but not that of Rapa in Ig production. This study is the first report on the strong suppressive effect of Rapa on human humoral immune response with a quantitative comparison with that of CsA. The underlying mechanisms are also explored. The results indicate the potential usefulness of this drug in treatment of presensitized transplantation patients, with whom cytotoxic Ab is a major obstacle to a successful transplantation.     

西罗莫司和环孢素A对Th17细胞和调节性T淋巴细胞分化影响的比较

目的 比较西罗莫司(SRL)和环孢素A(CsA)对体外诱导CD4+T淋巴细胞向Th17和调节性T淋巴细胞分化的影响.方法 使用抗CD3和抗CD28单克隆抗体(简称抗CD3单抗和抗CD28单抗)刺激CD4+T淋巴细胞,并在培养体系中分别加入转化生长因子β(TGF-β);TGF-β+白细胞介素6(IL-6);TGF-β+IL-6+SRL;TGF-β+IL-6+CsA.72 h后用流式细胞术检测细胞内Foxp3和IL-17的表达水平,观察CD4+T淋巴细胞的分化情况及SRL和CsA对CD4+T淋巴细胞体外分化的影响.结果 在经抗CD3单抗和抗CD28单抗刺激后,TGF-β可诱导Foxp3+细胞(调节性T淋巴细胞,Treg)的增殖与分化,而TGF-β+ID6可诱导Th17细胞的增殖与分化.在培养体系中加人SRL和CsA,均可使Th17细胞的增殖与分化减少;SRL可促进Treg的增殖与分化,而CsA抑制Treg的增殖与分化.结论 SRL可以促进CD4+T淋巴细胞向Treg增殖与分化,而抑制Th17细胞的增殖与分化;CsA既抑制Treg的增殖与分化,又抑制Th17细胞的增殖与分化.     

A comparison of the inhibitory effects of immunosuppressive agents cyclosporine, tetranactin, and didemnin B on human T cell responses in vitro.

The agents cyclosporine, tetranactin (TN), and didemnin B (DB) were compared for their ability to inhibit proliferative human T cell responses in vitro, using anti-CD3, PHA, alloantigen, or tetanus toxoid as stimuli and using monocytes or Langerhans cells as antigen-presenting cells/accessory cells (APC/AC). We found that all three agents suppressed T cell activation in a dose-dependent fashion, irrespective of the stimulus of APC/AC type used. Both T cells and APC/AC were affected by the drugs. DB appeared to be the most potent suppressive drug (IC50 = 1-4 ng/ml), whereas CsA and TN exerted approximately similar potency (IC50 = 50-60 ng/ml). Remarkably however, DB was toxic at a concentration of 10 ng/ml, which is quite close to the inhibition-inducing dose. No toxicity was observed with CsA and TN at doses up to 5000 ng/ml. The agents TN and DB could interrupt ongoing T cell responses and could block responsiveness to exogenous recombinant IL-2. Expression of IL-2 receptors was slightly inhibited by all three drugs. Expression of MHC class II molecule HLA-D and of adhesion molecules LFA-1, LFA-3, and ICAM-1 was clearly reduced by DB, giving an explanation for the observed inhibition of cluster formation between T cells and APC/AC. Except for a slight reduction of LFA-3 by TN, CsA and TN did not affect the expression of any of these cell surface markers or the formation of clusters. Differences in the effects of CsA, TN, and DB on immune responses in vitro and on the phenotype of T cells and APC/AC suggest that these immunosuppressive drugs have different inhibitory mechanisms.     

An in-vitro screening assay for the detection of inhibitors of proinflammatory cytokine synthesis: a useful tool for the development of new antiarthritic and disease modifying drugs

OBJECTIVE: This work targets the development of a new tool to help develop new anticytokine drugs that prevent or reduce the progression of arthritic diseases. The specific aim of our study was to establish a fast and reliable in vitro screening assay of cytokine synthesis inhibitors (TNFalpha, IL-1beta) which shows better correlation with enzyme assays than previously reported in vitro assays. The test system should be able to detect p38-MAP kinase inhibitors. MATERIAL AND METHODS: Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood. Cells were adjusted at 1 x 10(6) cells/ml. PBMCs were stimulated with lipopolysaccharide (LPS; E. coli serotype 026:B6: 1 microg/ml) in the presence of test compound (10(-5)-10(-8)M) for 4h at 37 degrees C in a 5% CO(2)-incubator. Induced TNFalpha and IL-1beta protein were measured by ELISA. RESULTS: The following are representative examples of inhibitors which effect cytokine synthesis. Corticoid Dexamethasone inhibits IL-1beta and TNFalpha synthesis at IC(50) of 38 nM and 25 nM, respectively. ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC(50) of 0.34 microM for IL-1beta production and 0.26 microM for TNFalpha synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1beta- and TNF-alpha-synthesis (IC(50)sof 0.052 microM and 0.46 microM) in the same degree as p38-MAP kinase activity (IC(50): 0.34 microM). Same results could be shown for SB 210313, which had same efficacy on IL-1beta and TNFalpha biosynthesis (IC(50)'s: 1.88 microM and 1.01 microM) and on p38-MAP kinase (IC(50): 6.85 microM). Also for SB 202190 this correlation in inhibition of IL-1beta and TNFalpha synthesis (IC(50)'s: 0.055 microM and 1.01 microM) and p38-MAP kinase inhibition (IC(50): 0.088 microM) could be shown. CONCLUSION: This study shows the screening assay using PBMCs stimulated with LPS for IL-1beta and TNFalpha synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1beta and TNFalpha synthesis which is mainly mediated by p38-MAP Kinase. These assay allows fast detection of IL-1beta and TNFalpha synthesis inhibitors with different modes of action, including p38-MAP kinase inhibitors. The results obtained with our in-vitro screening assay show good correlation with results from enzyme assays.     

抗CD3与抗CD28单克隆抗体共刺激对淋巴细胞免疫活化基因mRNA表达的影响

目的　观察ＣＤ２８ 共刺激信号对淋巴细胞免疫活化基因表达的影响及环孢素Ａ（ＣｓＡ）对它们的抑制作用。方法　分离健康人外周血单个核细胞, 分别给予抗ＣＤ３ 单克隆抗体（ 抗ＣＤ３ｍＡｂ） 单刺激或抗ＣＤ３ ｍＡｂ＋ 抗ＣＤ２８ 单克隆抗体（抗ＣＤ２８ ｍＡｂ）共刺激培养; 加或不加入ＣｓＡ。于培养后６ ｈ 和２４ ｈ 收集细胞。以逆转录聚合酶链反应（ＲＴＰＣＲ） 检测βＡｃｔｉｎ、ＣＤ４０Ｌ、ｂｃｌｘＬ、ＴＧＦβ１ 、Ｆａｓ、ＦａｓＬ、穿孔素和颗粒酶ＢｍＲＮＡ 表达水平。结果　ＣＤ２８ 共刺激信号能显著增强免疫活化基因ｍＲＮＡ 的表达;ＣｓＡ 对ＦａｓＬ和颗粒酶Ｂ的ｍ ＲＮＡ表达能产生抑制, 而对共刺激后其余免疫活化基因的表达无明显抑制。结论　ＣＤ２８ 共刺激信号通过提高多种Ｔ 淋巴细胞相关的免疫活化基因的表达水平, 而使免疫反应的活化、增殖和效应过程均得到增强;ＣｓＡ 对这一过程并非完全无效, 仍可能具有调节作用。     

Inhibition of interleukin 2 receptor expression in normal human T cells by cyclosporine. Demonstration at the mRNA, protein, and functional levels.

In view of the importance of the IL-2 receptors in the expression of antiallograft immunity and the currently existing controversy regarding the effect of CsA on the induction of IL-2 receptors, we explored the effect of cyclosporine on the induction of interleukin-2 receptor alpha and beta in normal human T cells. The effect of CsA on the induction of IL-2 receptors was examined at the levels of mRNA expression (with the aid of the polymerase chain reaction), protein (by SDS-PAGE analysis of chemically crosslinked 125I-IL-2 membrane protein complexes and by FACS), and function (by Scatchard analysis of 125I-IL-2 binding to T cells). The T cells were signaled with sn-1,2-dioctanoylglycerol and ionomycin or with crosslinked anti-CD3 and anti-CD2 mAbs. Our experimental design revealed that (A) CsA inhibits the induction of IL-2 receptor alpha and beta in normal human T cells, (B) the inhibitory activity is realized by a direct effect on T cells, and (C) the inhibitory activity is detectable at the pretranslational level--CsA significantly reduced the induction of mRNA encoding IL-2 receptor alpha and IL-2 receptor beta. These observations together persuasively demonstrate the ability of CsA to interrupt the emergence of IL-2 receptors on the surface of normal human T cells.     

Synergistic inhibitory activities of interleukin-10 and dexamethasone on human CD4+ T cells

BACKGROUND: We have previously demonstrated that interleukin (IL)-10 synergizes with dexamethasone (Dex) in inhibiting proliferation of human T cells, stimulated in an antigen-presenting cell (APC)-dependent manner. Because IL-10 effectively inhibits APC accessory functions, the synergism could have been a result of its effect on APC. We then investigated the effects of Dex and IL-10 on T-cell subpopulations, stimulated in an APC-independent manner. METHODS: CD4 and CD8 T cells were stimulated with anti-CD3, with or without Dex and IL-10, alone or in combination. Proliferation, glucocorticoid (GC) receptor binding, anti-CD3-induced tyrosine phosphorylation, IL-2 production, and expression of IL-2 receptor alpha, beta, and gamma chains were evaluated. The pharmacologic interactions were analyzed using the isobole method. RESULTS: IL-10 synergized with Dex in inhibiting CD4 but not CD8 T-cell proliferation. The synergism was not associated with modifications of GC receptor number or affinity, nor with modifications of anti-CD3-induced tyrosine phosphorylation. IL-10 synergized with Dex in inhibiting IL-2 production and increased Dex inhibitory effect on the expression of the IL-2 receptor alpha chain, which is up-regulated by CD3 stimulation and IL-2. Only Dex inhibited the beta and gamma chain expression, which, interestingly, is not up-regulated by IL-2. IL-2, as well as IL-7 and IL-15, reversed the effects of IL-10 but not those of Dex. CONCLUSIONS: IL-10 synergizes with Dex in inhibiting CD4 T-cell proliferation. Its synergizing effect is mediated by the inhibition of IL-2 production. Dex exerts additional activities, such as the inhibition of beta and gamma chain expression. Therefore, IL-10 could be useful for the enhancement of GC-based immunosuppressive therapies.     

Mechanisms of total lymphoid irradiation-induced immunosuppression. II. Failure of con A-stimulated splenocytes from TLI-treated mice to express IL-2 and IL-2 receptor RNA

Total lymphoid irradiation is a radiotherapy procedure used as an alternative immunosuppressive regimen in organ transplantation. Following TLI mature lymphocytes are depleted, and splenocytes do not proliferate to mitogens, produce IL-2, or express IL-2 receptors. We now show that mitogen stimulated splenocytes from TLI-treated mice do not secrete IL-2 protein by an IL-2 ELISA assay. Northern blot analysis and RNase protection assays reveal that TLI splenocytes do not make IL-2 RNA or IL-2 receptor RNA following mitogen stimulation. TLI splenocytes produce at least 1000 times less IL-2 RNA after Con A stimulation than normal splenocytes. TLI therapy resembles anti-CD4 therapy and CsA in that each results in an IL-2-"depleted" state.     

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.     

Differential regulation by dexamethasone and cyclosporine of human T cells activated by various stimuli.

Calcium ionophores such as ionomycin (IONO), CD3 antibody (CD3), or CD28 antibody (CD28) have been shown to stimulate T cells in a quite different fashion. However, each stimulator induces full activation of resting T cells in the presence of phorbol myristate acetate. Human T cells were activated with PMA + CD3, PMA + IONO, or PMA + CD28 and the inhibitory effects of dexamethasone (DEX) and cyclosporine were examined on [3H]-TdR incorporation, IL-2 production, and IL-2 receptor expression. Three inhibition patterns emerged: PMA + CD3 stimulation was DEX-sensitive and CsA-sensitive, PMA + IONO stimulation was CsA-sensitive but DEX-resistant, PMA + CD28 stimulation was DEX-sensitive but CsA-resistant. Although the degree of inhibition by DEX and CsA was different in [3H] TdR incorporation, IL-2 production, and IL-2 receptor expression assays, the inhibitory pattern of these drugs was similar in each of the assays, indicating that human T cell activation is differentially regulated by DEX and CsA depending on the stimulator.     

The immunosuppressive mechanism of total lymphoid irradiation. I. The effect on IL-2 production and IL-2 receptor expression

Total lymphoid irradiation is an effective immunosuppressive therapy used to prepare patients for organ transplantation and to treat several autoimmune diseases. We have used the mouse model to investigate the mechanism of TLI-induced immunosuppression. Mice were irradiated with 250-rad daily fractions to a total dose of 3500 rads. Splenocytes from control and TLI-treated mice were analyzed for IL-2 production, IL-2 receptor expression after mitogen stimulation, and percent L3T4 positive cells. At two weeks following the completion of TLI, IL-2 production from whole spleen populations had decreased 90% compared with controls (TLI mean IL-2 units = 25 +/- 9.0, control mean = 277 +/- 104). IL-2 receptor expression on splenocytes following Con A stimulation was decreased 80% compared with controls (TLI mean percentage of IL-2 receptor expressing cells = 16.2 +/- 4.1, control mean = 82.0 +/- 7.5). L3T4+ cells that expressed IL-2 receptor were also decreased following TLI (TLI mean = 4.4 +/- 1.4, control mean = 22.0 +/- 3.8), as were proliferative responses to Con A and PHA. Addition of recombinant mouse IL-2 did not restore IL-2 receptor expression or proliferative responses. Whole TLI-treated splenocytes did not suppress IL-2 production or proliferative responses of normal splenocytes. These immunologic abnormalities recovered over time, and by 8 weeks post TLI IL-2 production, IL-2 receptor expression, L3T4+ cell numbers and proliferative responses had returned toward normal. These results suggest that TLI therapy transiently depletes IL-2 producing, IL-2 receptor expressing, and mitogen responsive lymphocytes. The immunosuppression is not mediated through a suppressor cell and is independent of IL-2 production.     

Enhancement by cyclosporin A of taxol-induced apoptosis of human urinary bladder cancer cells

Taxol is a microtubule-stabilizing agent which induces apoptosis in various cancer cells. In this study, we found that T24 cells derived from high grade human urinary bladder cancer were relatively resistant to taxol and that the IC50 value determined by a colorimetric WST-1 assay was 406.0 nM. Interestingly, cyclosporin A (CsA), an immunosuppressive drug, dramatically enhanced sensitivity to taxol, and the IC50 value was decreased to 47.5 nM in the presence of 1 microM CsA. KK47 cells derived from low grade human urinary bladder cancer showed high sensitivity to taxol with an IC50 value of 78.8 nM which decreased to 14.4 nM in the presence of 1 microM CsA. FK506, another immunosuppressive drug, also enhanced sensitivity to taxol. Furthermore, a concomitant loss of calcineurin activity was observed after the treatment of both cell lines with both CsA and FK506. Taxol induced apoptosis of the cells, as assessed by Hoechst 33258 staining and by the measurement of caspase 3 activity. Immunoblot analysis with an antibody against Bcl-2 phosphorylated at serine 70 demonstrated that taxol induced the phosphorylation of Bcl-2 with its enhancement in the presence of CsA. In addition, treatment of the cells with CsA significantly decreased the expression of Bcl-2 at both the protein and mRNA levels. These results suggest that the enhancement of taxol-induced apoptosis by immunosuppressive drugs is at least partly due to the inhibition of calcineurin activity and the loss of the antiapoptotic function of Bcl-2 via the enhancement of phosphorylation and the reduction of expression.     

Cyclosporine effect on anti-CD3 monoclonal antibody-stimulated mitogenesis, phorbol ester comitogenesis, and PGE2 production

Human peripheral blood mononuclear cells (H-PBMC) from 10 healthy donors were stimulated to proliferate with phytohemagglutinin lectin (PHA), anti-CD3 monoclonal antibody (mAb), and anti-CD3 mAb plus phorbol 12, myristate 13 acetate (TPA), a protein kinase C (PKC) agonist. Anti-CD3 mAb-mediated mitogenesis was 35-75% of that observed with PHA. When TPA was added to a dose of mAb that by itself did not cause mitogenesis, proliferation equal to 50-90% of the maximally mitogenic dose occurred. TPA did not enhance proliferation with maximally mitogenic doses of antibody. Dimethyl-prostaglandin E2, dibutyryl cyclic AMP, and forskolin (an adenyl cyclase agonist) inhibited PHA, anti-CD3, and anti-CD3/PMA-mediated mitogenesis. Cyclosporine (CSA) inhibited anti-CD3 and anti-CD3/TPA mitogenesis in a dose-dependent fashion. While CSA inhibited anti-CD3 and anti-CD3/TPA mitogenic signals, it did not affect PGE2 production by anti-CD3 mAb-stimulated H-PBMC. In the presence of CSA, PGE2 production in PHA-stimulated H-PBMC was increased. PGE2 inhibits lymphocyte proliferation via a cyclic AMP-mediated mechanism and may enhance maturation of suppressor cells. CSA inhibits anti-CD3 mAb and anti-CD3/TPA proliferative signals in H-PBMC yet has no effect or may even enhance production of suppressive PGE2. The maturation of antigen-specific suppressor cells elicited by CSA may involve active down-regulation of CD3 receptor and PKC-dependent events while PGE2 production continues.