T-lymphocyte apoptosis is increased by non-interleukin-2-dependent induction in human mixed lymphocyte cultures

INTRODUCTION: Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. METHODS: Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (10(6) cells/mL) were cocultured with equal numbers of responder cells. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. RESULTS: Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. CONCLUSIONS: Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.     

Analysis of cloned T cell function. II. Differential blockade of various cloned T cell functions by cyclosporine

Cyclosporine has profound suppressive effects on selected in vitro functions of cloned T lymphocytes. Cyclosporine inhibits the antigen-induced proliferation of the helper T cell clone 12-11. The effective dose required to reduce this response by 50% (ED50) is 28 ng/ml. In contrast, the proliferation of clone 12-11 induced by exogenous growth factors in secondary mixed lymphocyte culture supernatant (2 degrees MLC SN), is relatively insensitive to cyclosporine (ED50 = 4600 ng/ml). Furthermore, cyclosporine abrogates both antigen-induced and mitogen-induced secretion of lymphokines by clone 12-11, indicating that cloned helper T cell function is sensitive to cyclosporine even when interactions between specific alloantigens and their cell surface receptors are bypassed with mitogen. The suppressive effect of cyclosporine is not limited to helper T cell clones. The cytolytic T lymphocyte (CTL) clone 5MD2-2 is also sensitive to cyclosporine. Again, cyclosporine (100 ng/ml) blocks the antigen-driven, but not the exogenous lymphokine-driven, component of clone 5MD2-2 proliferation. This suppression does not result from the occlusion of antigen receptors or from antigen deformation by cyclosporine, because clone 5MD2-2 remains capable of antigen-specific cytolysis in the presence of cyclosporine concentrations that can suppress its proliferation. Finally, the ability of clone 5MD2-2 to remove IL-2 activity from culture media, a function that is significantly enhanced by contact with specific alloantigen, is not influenced by suppressive cyclosporine concentrations.     

Differential effects of interleukin-2 blockade on apoptosis in naïve and activated human lymphocytes

BACKGROUND: Certain transplantation immunosuppressive strategies are primarily based on the interruption of interleukin (IL)-2 signaling by calcineurin inhibition or anti-IL-2 receptor-antibody blockade. However, recent evidence suggests that IL-2 is necessary for peripheral deletion of allograft-specific lymphocytes. STUDY DESIGN: In this study, we examined the apoptotic effects of the calcineurin inhibitor, cyclosporine A, the chimeric anti-interleukin-2 receptor monoclonal antibody, basiliximab, and the rabbit antihuman thymocyte preparation Thymoglobulin (rATG) on phytohemagglutinin-activated human lymphocyte models designed to simulate initial exposure to the graft or ongoing rejection of the graft. RESULTS: We found that rATG increases Fas expression, decreases Bcl-2 expression, and induces early apoptosis in na?ve lymphocytes. However, rATG has more of a necrotic effect on activated lymphocytes. Basiliximab and cyclosporine had little effect on apoptosis, but did alter Bcl-2 and Fas expression. CONCLUSIONS: Compared with IL-2 pathway inhibitors, rATG increases lymphocyte apoptosis, probably via Bcl-2 pathway inhibition. Because apoptosis is required for the development of graft tolerance, induction strategies that use IL-2-independent pathways may be advantageous.     

Rapamycin impairs antigen uptake of human dendritic cells

BACKGROUND: Rapamycin is a recently introduced immunosuppressive agent. Its effect on lymphocytes has been extensively studied. Whether it can also modulate dendritic cell (DC) function is unknown. METHODS: The effect of rapamycin on differentiation, antigen uptake, and the immunostimulatory capacity of human DC was examined. DC were derived from monocytes upon culture with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor in the presence or absence of rapamycin (0.1-100 ng/mL). Surface phenotype and antigen uptake capacity of DC were assessed by flow cytometry. Immunostimulatory capacity was measured by mixed lymphocyte culture. RESULTS: Rapamycin reduced DC recovery and increased DC apoptosis. DC differentiated in the presence of rapamycin (rapa-DC) had increased expression of CD1a, CD1b, and CD1c and decreased expression of MHC I, MHC II, CD80, CD86, and CD40. Antigen uptake receptor expression (mannose receptor, CD32, CD91, CD46) was decreased, and receptor-mediated endocytosis of fluorescein isothiocyanate-dextran was markedly impaired in rapa-DC, as were fluid phase endocytosis of Lucipher Yellow and phagocytic activity of bacteria and dead or apoptotic cells. CD40 ligand-induced production of both IL-12 and IL-10 was reduced in rapa-DC, and allogeneic T lymphocyte responses were moderately impaired when rapa-DC were used as stimulator cells. Neither cyclosporine nor FK506 affected DC function. However, the effects of rapamycin on DC could be completely inhibited by a 10-fold excess of FK506 but not by up to 100-fold excess of cyclosporine. CONCLUSION: Rapamycin has a unique and profound inhibitory effect on DC function, which seems to be at least in part mediated by the FKBP immunophilins.     

Rapamycin does not induce anergy but inhibits expansion and differentiation of alloreactive human T cells

BACKGROUND: Studies in mice have shown that rapamycin inhibits cell cycle progression and promotes the development of clonal anergy. We here addressed the question if rapamycin can induce anergy of human T cells and studied the effects of rapamycin on activation, proliferation and expression of cytotoxic effector molecules of alloresponsive T cells in mixed lymphocyte cultures. METHODS: Peripheral blood mononuclear cells from healthy individuals were labeled with CFSE to monitor subsequent cell divisions. Cells were cocultured with allogeneic irradiated cells in the presence or absence of rapamycin. Flowcytometric analysis was performed after staining for surface CD4, CD8, and CD25 and for intracellular perforin, granzyme B, active caspase-3, and TGF-beta. Bio-Plex cytokine assay was done to measure the secretion of IL-2, IL-4, IL-10, and IFN-gamma. RESULTS: Addition of rapamycin at a final concentration of 10 ng/ml strongly decreased precursor frequencies of alloreactive CD4+ and CD8+ T cells. However, when these cells were washed and subsequently specifically restimulated in the absence of rapamycin, the proliferative capacity appeared normal. Next to lowering precursor frequencies, rapamycin also inhibited T cell expansion by inducing apoptosis in divided alloreactive CD4+ and CD8+ T cells. Rapamycin did not interfere with the formation of CD25brightCD4+ T cells during allogeneic stimulation and did not inhibit their suppressive function. Furthermore, the drug decreased production of effector molecules perforin and granzyme B by alloreactive T cells and diminished alloreactive cytotoxicity. CONCLUSION: Our data show that rapamycin strongly inhibits proliferation and effector functions of alloreactive T cells in vitro, but does not induce alloantigen specific nonresponsiveness.     

大面积烧伤脓毒症患者T淋巴细胞免疫功能的改变及临床意义

目的 了解大面积烧伤脓毒症患者T淋巴细胞免疫功能的变化，探讨其与脓毒症的关系。方法 选择59例烧伤面积≥30％TBSA的患者，分为脓毒症组43例和非脓毒症组16例。采集两组患者伤后1、3、5、7、14、21、28d的外周静脉血，检测T淋巴细胞增殖能力和白细胞介素2（IL-2）的分泌水平，并行相关性分析；通过流式细胞仪检测CD3^＋／CD4^＋ T淋巴细胞的百分率及其凋亡率，并行相关性分析。结果 与非脓毒症组比较，脓毒症组患者伤后l、14、2l、28dT淋巴细胞增殖能力和IL-2的分泌水平均显著下降（P〈0．05或P〈0.01），两指标呈显著正相关（r=0．82，P〈0．01）。伤后1、5、14、21、28d，脓毒症组患者CD3^＋／CD4^＋T淋巴细胞百分率明显低于非脓毒症组，而其凋亡率呈相反趋势（P〈0.05或P〈0．01），两指标呈显著负相关（r=-0.66，P〈0．05）。结论 大面积烧伤脓毒症患者T淋巴细胞免疫功能持续处于抑制状态，T淋巴细胞凋亡参与了脓毒症细胞免疫紊乱的病理生理过程。     

New insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs

OBJECTIVE: To determine the precise in vivo interaction between T-cell costimulatory blockade and conventional immunosuppression in transplantation. SUMMARY BACKGROUND DATA: Blocking B7 or CD154 T-cell costimulatory activation pathways prevents allograft rejection in small and large animal transplant models and is considered a promising strategy for clinical organ transplantation. METHODS: A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD154 or CTLA4Ig monotherapy and conventional immunosuppressive drugs in promoting long-term graft acceptance. The frequency of alloreactive T cell was measured by ELISPOT. Chronic rejection was examined by histology. RESULTS: Cyclosporine, tacrolimus, and anti-IL-2R monoclonal antibody therapy abrogated the effect of a single-dose protocol of anti-CD154 therapy. In contrast, rapamycin acted synergistically with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose schedule of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the effect was not affected by the concomitant use of any immunosuppressive drugs. CONCLUSIONS: The widespread view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection.     

Rapamycin enhances the number of alloantigen-induced human CD103+CD8+ regulatory T cells in vitro

BACKGROUND: Regulatory T cells (T(reg) cells) may be operational in both the induction and maintenance of transplantation tolerance. We recently showed that alloantigen-induced CD103+ CD8+ T cells strongly suppressed T-cell proliferation in mixed lymphocyte culture (MLC) via a contact-dependent mechanism. CD103 directs T lymphocytes to their ligand E-cadherin, which is expressed on renal tubular epithelial cells, and CD103+ CD8+ T cells have been described to be present in late renal allograft rejection. METHODS: We studied the influence of prednisolone, cyclosporin, tacrolimus, CD25 monoclonal antibodies, rapamycin, and mycophenolate mofetil (MMF) on the development and functional activity of alloantigen-activated CD103+ CD8+ T cells in MLC. RESULTS: Calcineurin inhibitors, MMF, and CD25mAb did not influence the number of CD103 expressing CD8+ T cells. In contrast, corticosteroids diminished CD103 expression on alloactivated CD8+ T cells, which appeared to be caused by their inhibitory action on myeloid dendritic cells. Addition of rapamycin to allocultures led to an increased percentage of CD103+ CD8+ alloreactive T cells. Moreover, in the presence of rapamycin, these cells tended to show higher suppressive capacity. CONCLUSIONS: Alloreactive CD103+ CD8+ T(reg) cells may expand and exert their suppressive function during immunosuppressive treatment with rapamycin. These data are relevant in the design of immunosuppressive drug regimens intended to induce and/or maintain transplantation tolerance.     

Correlation of in vitro CD4+ T helper cell function with clinical graft status in immunosuppressed kidney transplant recipients.

We recently identified three distinct T helper pathways which contribute to interleukin-2 (IL-2) production by human peripheral blood lymphocytes following stimulation with HLA alloantigens. In two of these pathways, CD4+ T helper cells respond to alloantigen using either self antigen-presenting cells (sAPC)* or allogeneic antigen-presenting cells (aAPC). A third pathway involves CD8+ T helper cells using aAPC. Previous in vitro studies have shown that the T helper pathway dependent on CD4+ T helper cells and sAPC (CD4-sAPC) is the most susceptible to suppression by cyclosporine. In the present study, we measured alloantigen-stimulated IL-2 production by PBL from 42 kidney transplant recipients to characterize the strength of the three T helper-APC pathways. In 58% of patients, a loss of the CD4-sAPC pathway was identified and was correlated with cyclosporine treatment. However, several patients not receiving cyclosporine also exhibited a similar loss of T helper cell function, suggesting that cyclosporine is not the only factor involved. Of 27 patients exhibiting depressed CD4-sAPC function, none had evidence of ongoing/recent graft rejection. In contrast, of 11 patients with no defects in the three pathways of in vitro T helper cell function, 6 had evidence of chronic graft rejection. Of considerable interest are the data obtained from a separate group of 4 patients who had episodes of acute rejection during the study. In each case, at the time of the rejection episode, all exhibited an intact CD4-sAPC pathway. However, samples tested prior to the rejection episode or after successful treatment of the rejection episode showed a depressed CD4-sAPC pathway. These results suggest that depression of the CD4-sAPC pathway represents adequate immunosuppression for graft retention and that patients not exhibiting such suppression are at increased risk for both acute and chronic graft rejection. These data may have relevance for diagnosis and/or prediction of graft rejection and may provide an in vitro method of monitoring the functional degree of immunosuppression in transplant recipients.     

The effect of cyclosporine on ornithine decarboxylase induction with mitogens, antigens, and lymphokines

Ornithine decarboxylase (ODC) is the initial enzyme in polyamine synthesis. An increase in ODC activity is associated with increased RNA, DNA, and protein synthesis. We have used the induction of ODC by mitogens and alloantigens in human peripheral blood lymphocytes as an intracellular marker of protein synthesis and lymphocyte activation. The immunosuppressive agent cyclosporine was found to inhibit both the mitogen and alloantigen stimulated induction of ODC in lymphocytes in a manner that parallels inhibition of subsequent 3H-thymidine incorporation. When purified T lymphocytes were stimulated with mitogen alone, minimal ODC activity was detected. The addition of 5% monocytes, human Interleukin-1 (IL-1), or T cell growth factor (IL-2) enhanced mitogen-induced ODC activity in T lymphocytes 4-10-fold. Cyclosporine inhibited the induction of ODC when T lymphocytes were combined with monocytes or growth factors. We conclude that (1) the induction of ODC in human lymphocytes by mitogen and alloantigen is inhibited in the presence of cyclosporine; (2) the induction of ODC activity in purified T lymphocytes requires the presence of both mitogen and monocytes or their products; (3) IL-1 and IL-2 can supplement for monocytes and augment the phytohemagglutinin induction of ODC in T lymphocytes; and (4) cyclosporine inhibits ODC induction in T lymphocytes stimulated with mitogen in the added presence of monocytes, IL-1, or IL-2. The inhibition of ODC induction and polyamine synthesis by cyclosporine adds insight into its mode of action on the mechanisms involved in early T cell activation.     

CD8+ lymphocytes augment chronic rejection in a MHC class II mismatched model

Chronic rejection, or cardiac allograft vasculopathy (CAV), remains the leading cause of late death in heart transplant recipients. The precise role and contributions of T lymphocyte subsets to CAV development remains unknown. METHODS: Donor hearts from B6.C-H2bm12 mice were transplanted into T lymphocyte subset knockout recipients and T lymphocyte-reconstituted nude recipients. No immunosuppression was used. Intimal proliferation was measured morphometrically. In vitro studies were performed to analyze the donor-specific activation status of recipient CD8+ lymphocytes by examining cellular proliferation, interleukin-2 secretion, and interleukin-2Ralpha expression. Intracellular cytokine staining assay was performed to determine both the profile and source of intragraft cytokines. RESULTS: Hearts transplanted into wild-type recipients developed severe CAV by 24 days. Intimal lesions were absent in the hearts that were transplanted into nude and CD4-/- knockout mice (containing CD8+ lymphocytes). In contrast, the donor hearts in CD8-/- knockout recipients (containing CD4+ lymphocytes) developed CAV, but significantly less than in wildtype. Adoptive transfer of T lymphocyte subset populations into nude recipients confirmed that CAV was absolutely contingent on CD4+ lymphocytes, and that CD8+ lymphocytes played an additive role in intimal lesion progression in the presence of CD4+ lymphocytes. Although CD8+ lymphocytes alone did not cause CAV in vivo, we demonstrated that MHC class II disparate alloantigens activated CD8+ lymphocytes both in vivo and in vitro. Finally, both CD4+ and CD8+ lymphocytes contributed to the intragraft IL-2 and IFN-gamma production. CONCLUSIONS: In this MHC class II mismatched murine model, CAV is a T lymphocyte dependent event, and absolutely contingent on the presence of CD4+ lymphocytes. Furthermore, CD8+ lymphocytes (1) are activated by MHC class II disparate antigens and (2) play a significant role in the progression of lesion development. Finally, both CD4+ and CD8+ lymphocytes contribute to CAV development via secretion of IFN-gamma, a known mediator of CAV in this model.     

急性胰腺炎病人T淋巴细胞亚群改变与差异性凋亡

目的 动态观察急性胰腺炎(AP)病人T淋巴细胞亚群的变化及凋亡情况.方法 应用流式细胞仪三色荧光分析对44例轻型急性胰腺炎(MAP)病人和32例重症急性胰腺炎(SAP)病人发病第1、3、7天外周血T淋巴细胞CD4+、CD8+两个亚群各自的比例变化与凋亡情况进行动态监测.结果 与MAP组及健康对照组相比,SAP组病人在整个观察期内CD4+T细胞明显减少,CD4+/CD8+比值明显下降;同时发现CD4+T细胞的凋亡率明显增加,与上述二者的变化呈显著负相关.MAP组上述各指标呈一过性改变.而CD8+T细胞凋亡率在各组间及组内不同时点均无显著差别.结论 AP病人,特别是SAP病人病程中T淋巴细胞CD4+、CD8+两个亚群发生差异性凋亡,从而导致严重而持久的细胞免疫功能抑制,这可能是SAP病程中后期容易发生严重感染的重要原因.     

Interleukin-10 and apoptotic death of circulating lymphocytes in surgical/anesthesia trauma

OBJECTIVE: To examine the relationship between circulating interleukin-10 (IL-10) and the occurrence of lymphocyte apoptosis after surgical/anesthesia trauma. METHODS: Data were collected prospectively on 18 adult patients undergoing elective major surgery. Blood sampling for assessment of lymphocyte apoptosis and IL-10 levels was performed on the day before surgery (t(0)) and at 24 and 96 hours after operation (t(1) and t(2), respectively). After lymphocyte isolation, quantification of apoptosis was made by staining apoptotic cells with 7-amino-actinomycin D. Plasma IL-10 concentrations were measured using enzyme-linked immunosorbent assay. RESULTS: A significantly increased frequency of apoptotic CD4(+) and CD8(+) cells (p < 0.05) was observed at t1 measurement (8.10% +/- 0.58% and 12.21% +/- 1.47% for CD4(+) and CD8(+), respectively) compared with preoperative values (1.53% +/- 0.38% and 1.32% +/- 0.45% for CD4(+) and CD8(+), respectively). Plasma IL-10 levels showed a significant elevation at both t(1) and t(2) times, peaking at t(1). At t(1), IL-10 levels were correlated with the frequency of CD4(+) and CD8(+) apoptotic lymphocytes (r = 0.78, p = 0.0005 for IL-10 vs. apoptotic CD4(+); r = 0.71, p = 0.003 for IL-10 vs. apoptotic CD8(+)). CONCLUSION: Surgical trauma is associated with a significant but transient increase in lymphocyte commitment to apoptosis and IL-10 production. The exact relationship linking the overproduction of IL-10 with lymphocyte apoptosis after a surgical operation is still elusive and requires further investigation.     

Effect of cyclosporine on T lymphocyte development. Relationship to syngeneic graft-versus-host disease

This report investigates the effects of cyclosporine on the reconstitution of T lymphocytes after syngeneic bone marrow transplantation and its role in the development of a novel T cell-mediated autoimmune disease, syngeneic graft versus host disease. We analyzed the effect of CsA treatment on T lymphocyte differentiation during reconstitution after bone marrow transplantation and correlated the maturation of CD4+ and CD8+ T cell subsets with the onset of syngeneic GVHD. Administration of CsA following syngeneic bone marrow transplantation leads to a developmental arrest of mature CD4+ and CD8+ T lymphocytes in the thymus and a marked reduction in cells expressing the alpha beta T cell receptor. The reduction of CD4+ and CD8+ T cell subsets is also reflected in the peripheral lymphoid compartment with an altered CD4/CD8 ratio. Functional assessment of the cells revealed that CD8+ cells respond normally to mitogenic signalling whereas CD4+ cells exhibit marginal proliferative responses. Both subsets of T lymphocytes respond to syngeneic B lymphoblasts, comparable to the response of T lymphocytes from non-CsA-treated syngeneic BMT recipients, suggesting that autoreactive cells are produced despite CsA treatment. Following discontinuation of CsA, T cell differentiation in the thymus is rapidly restored to normal. However, concurrent with the onset of syngeneic GVHD, a compensatory insurgence of CD4+ T helper cells is observed.     

Effect of FK506 treatment on allocytolytic T lymphocyte induction in vivo: differential effects of FK506 on L3T4+ and Ly2+ T cells.

Although FK506 has been widely investigated as a potent suppressor of organ allograft rejection in animals, little is known about the effect of FK506 on T cell responses to allografts in vivo. In the present study, we have studied the effect of FK506 on the induction of allocytolytic T lymphocyte using mice primed with alloantigens and treated with FK506. FK506 suppressed the CTL induction of spleen cells and peritoneal exudate cells (PEC) in a dose-dependent manner. Time-course kinetic studies indicated that the CTL activity was markedly dependent on the time of administration of FK506 to the mice. Lymphocytes from these FK506-treated animals were found to be reactivated upon exposure to the same alloantigens in a secondary mixed lymphocyte culture (MLC). Furthermore, FK506 was shown to have a differential effect on the activation of helper (L3T4+) and cytotoxic (Ly2+) T cell subpopulations. L3T4+ T cells from the mice primed with alloantigens and treated with FK506 had normal helper activity in the generation of CTL in MLC, whereas Ly2+ T cells from these mice were profoundly suppressed CTL activity upon reexposure to the same alloantigens in a secondary MLC. Exogenous IL-2 or L3T4+ T cells could overcome the immunosuppressive effect of FK506 on the CTL induction of Ly2+ T cells in a secondary MLC. Finally, we have demonstrated that this FK506 effect appeared to be antigen nonspecific since Ly2+ T cells from alloprimed FK506-treated mice failed to induce CTL against the third-party alloantigens as well as the same alloantigens in a secondary MLC.     

IFN-γ Decreases CTL Generation by Limiting IL-2 Production: A Feedback Loop Controlling Effector Cell Production

IFN-γ is produced by cytotoxic T lymphocytes (CTL) but can also decrease CTL generation. We used IFN-γ-R1-deficient (GRKO) and IFN-γ-deficient (GKO) mice to study the effects of IFN-γ in MLC on the generation of CTL activity and CTL number, IL-2 production and cell proliferation. CTL activity was increased in MLC when GRKO responders or GKO stimulators and responders were used, compared to wild-type (WT) MLC. The number of cells displaying the CTL phenotype (CD3⁺, CD8⁺, CD25⁺) was also increased, accompanied by increased IL-2 production and proliferation. Combinations of WT or GRKO CD4+ T cells with WT or GRKO CD8+ T cells as responders showed that IFN-γ mostly affects CD4+ T cells to limit CTL generation. Intracellular staining indicated that IL-2 production was largely by CD4+ T cells. Moreover, addition of IL-2 to WT responders mimicked GKO CTL generation and activity, whereas neutralizing IL-2 decreased CTL activity in GRKO and WT responders. Thus IFN-γ reduces CTL generation in alloimmune responses largely by limiting proliferation of IL-2 producing CD4+ T cells. This creates a feedback loop in which effectors produce IFN-γ that limits IL-2 production which in turn limits CTL generation.     

Stimulated response of peripheral lymphocytes may distinguish cyclosporine effect in renal transplant recipients receiving a cyclosporine+rapamycin regimen

BACKGROUND: Clinically, cyclosporine (CSA, Neoral) is titrated to concentrations, and not to pharmacological effect. METHODS: Intracellular interleukin- (IL) 2 was measured in phorbol myristic acid-ionomycin-stimulated peripheral lymphocytes by flow cytometry, after isolation from 14 renal transplant recipients receiving CSA+prednisone, and double-blind rapamycin (rapamycin:placebo=4:1). RESULTS: The proportion (%) of CD4+IL-2+ lymphocytes corresponding to CSA levels (mean+/-SD ng/ml) measured preoperatively (TO=O), and on postoperative day 8, before (356+/-63), and 2 hr after the morning dose (Cmax=1567+/-669), decreased from 39+/-16 to 15+/-8 and 3+/-1.6, respectively. Reciprocally, unresponsive lymphocytes (%CD4+IL-2-) increased with increasing CSA levels and predicted an EC50 of 249 ng/ml (CSA concentration at which CD4+IL-2- cells increased by 50% over baseline) in an Emax pharmacodynamic model. CONCLUSIONS: Clinically, the pharmacological effect of CSA is quantifiable, and lies in the upper end of the predicted range. In our Neoral-treated sample population, Cmax was associated with the least variable "cyclosporine effect." Such information could potentially individualize immunosuppression, and lead to rational dosing strategies.     

Immunoregulation and apoptosis induction of nitric oxide in the human mixed lymphocytes culture

Nitric oxide (NO) is a multifunctional molecule in a variety of physiologic and pathologic processes. Its precise effect on human T lymphocyte responses against alloantigens are not yet fully known, although it has been reported that NO is antiproliferative and can cause apoptosis in several cell types. To address these issues, we analyzed the effects of an NO donor on mixed lymphocyte cultures (MLC) and on apoptosis induction in T lymphocytes activated with alloantigens. NOC 18 was used as an NO donor. The MLC was performed with human peripheral blood mononuclear cells isolated from healthy volunteers. Cell division and interleukin (IL)-2 production were measured with CFSE labeling and an EIA kit, respectively. After cells were incubated with NOC 18 for 24 hours, DNA fragmentation was assessed using the diphenylamine assay. Pre-culture of cells with NOC 18 for 24 hours resulted in significant inhibition of cell proliferation and IL-2 production in MLC. NOC 18 induced DNA fragmentation of cells harvested from an MLC following 7 days of the culture, in a dose-dependent manner, whereas it never exerted any influence on DNA fragmentation of freshly isolated cells. A chemical NO donor, NOC-18, may have immunosuppressive ability when treatment of responder cells occurs before the beginning of the MLC and may induce apoptosis of alloantigen-activated T lymphocytes.     

儿童肾病综合征T细胞亚群检测的临床意义

目的：探讨儿童原发性肾病综合征（NS）T细胞亚群检测的临床意义。方法：对25例NS活动期和缓解期患儿应用流式细胞仪检测T2细胞亚群的变化。结果：NS活动期组CD3^ ,CD4^ ,CD4^ /CD8^ 比值，NK[CD(16 56)]^ 细胞均明显低于缓解期组（P＜0．01）和对照组（P＜0．01）。结论：说明NS细胞免疫功能减低，T细胞亚群检测可作为肾病综合征活动指标之一。