The distinct effects of FK506 on the activation, proliferation, and differentiation of human B lymphocytes.

We examined the effect of FK506 on the activation, proliferation and differentiation of human B lymphocytes in vitro. FK506 inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan strain I (SAC) and phorbol myristate acetate (PMA) in a dose-dependent manner. Inhibition of cell proliferation by FK506 was caused by a selective block of G0 to G1 phase transition leading to cell arrest. In addition, the proliferative response of in vivo-activated B cells and lymphokine-driven B cell proliferation were also found to be sensitive to FK506. Interestingly, FK506 did not affect the expression of activation antigens such as CD23, IL-2 receptor (CD25), and transferrin receptor (CD71). Finally, FK506 had little effect on B cell antibody generation in a T cell-independent system. Conversely, FK506 suppressed neither proliferation nor immunoglobulin secretion in a human B lymphoblastoid cell line. These results indicate that FK506 has discrete effects on the different stages of the B cell maturation.     

他克莫司对肝移植受者外周血T淋巴细胞亚群及共刺激分子表达的影响

目的 探讨他克莫司（FK506）对肝移植受者外周血T淋巴细胞亚群及其表面共刺激分子表达的影响。方法 采用荧光标记单克隆抗体和流式细胞技术，测定术后采用FK506治疗的肝移植受者（FK506治疗组）在用FK506后1、2、3、4周时的外周血T淋巴细胞亚群及其表面共刺激分子CD28、CD152和ICOS分子的表达情况，以健康志愿者（健康对照组）和患终末期肝脏疾病拟行肝移植者（肝病对照组）为对照。结果 CD3^＋T淋巴细胞在各组间的差异均无统计学意义（P〉0．05）。经FK506治疗后，肝移植患者的CD4^＋T淋巴细胞逐渐减少，CD8^＋T淋巴细胞逐渐增加，并恢复至健康对照组水平（P〉0．05）。FK506治疗组T淋巴细胞亚群表面CD28分子和ICOS分子表达逐渐下降，并明显低于健康对照组（P〈0．05），而CD152分子表达增加，且明显高于健康对照组（P〈0．05）；其ICOS分子表达水平的下降晚于CD28分子，CD4^＋CD28^＋T淋巴细胞、CD8^＋CD28^＋T淋巴细胞和CD4^＋ICOS^＋T淋巴细胞均呈现相近的变化规律。结论 FK506能迅速纠正移植受者T淋巴细胞亚群紊乱，并抑制正性共刺激分子CD28和ICOS的表达，促进负性共刺激分子CD152的表达。     

Plant alkaloid tetrandrine and its analog block CD28-costimulated activities of human peripheral blood T cells: potential immunosuppressants in transplantation immunology

BACKGROUND: T lymphocyte activation mediated by CD28 costimulation plays a critical role in graft rejection. Plant alkaloid tetrandrine, purified from a Chinese antirheumatic herb, is a potent immunosuppressant. Here, we examined its effects on several CD28-costimulated T-cell activities. In addition, such effects were readily compared with the effects of three tetrandrine analogs. METHODS: T lymphocytes were purified from whole blood by negative selection. The stimuli that mimic CD28 costimulation included both anti-CD3 + anti-CD28 monoclonal antibody and PMA+anti-CD28 monoclonal antibody. The determination of CD28-costimulated cell proliferation was performed by tritium uptake, cytokine production by ELISA, cell surface interleukin 2Ra and CD69 expression by flow cytometry, and mixed leukocyte reaction by tritium uptake. Drug cytotoxicity was determined by trypan blue exclusion, propidium iodide staining, and MTT colorimetric assays. RESULTS: Tetrandrine inhibited CD28-costimulated T-cell proliferation and cytokine production through a mechanism different from that of cyclosporine. In addition, tetrandrine down-regulated both T helper 1 and T helper 2 cytokine production in CD4+ and CD8+ T-cell subpopulations. By examining cytokine production and T-cell activation marker expression, we further demonstrated that, among tetrandrine and its analogs tested, dauricine was the most potent suppressor of CD28-costimulated T-cell activities. Furthermore, the different immunosuppressive activities of these compounds were not associated with their cytotoxic capacities. Finally, the unparalleled inhibitory potency of dauricine on both mixed leukocyte reaction and CD28-costimulated T-cell proliferation suggests that dauricine preferentially targeted CD28-costimulated T-cell activities. CONCLUSIONS: This is the first report to show that tetrandrine and its analogs potently inhibited both PMA+CD28-costimulated and CD3 + CD28-costimulated activation of human peripheral blood T cells. Based upon their structural similarity and different immunosuppressive potency, these in vitro data also provide very useful information for further identification and development of more potent and less toxic immunosuppressants to achieve transplantation success.     

Effect of FK506 on donor T-cell functions that are responsible for graft-versus-host disease and graft-versus-leukemia effect

BACKGROUND: FK506 is a potent immunosuppressive agent that is used in human graft-versus-host disease (GvHD) prevention. However, the precise mechanisms for GvHD prevention and the effect on graft-versus-leukemia (GvL) activity are unknown. This study was undertaken to determine the effect of FK506, given at clinically relevant doses, on donor T-cell functions responsible for GvHD and GvL activity. METHODS: The effect of FK506 on GvHD prevention and GvL activity was investigated using a murine model of allogeneic bone-marrow transplantation in which mice were injected with a P815 leukemic cell line. The regulatory role of FK506 on donor T cells was tested by analysis of donor T-cell expansions in the spleen and donor anti-host T-cell proliferative and cytotoxic responses. mRNA expression of type 1 T helper (Th1), Fas ligand (L), and granzyme B were also evaluated in target organs of GvHD. RESULTS: FK506 significantly prolonged the survival of GvHD mice when given at the trough level of 17.6 ng/mL, whereas it also blocked GvL effect in P815-injected GvHD mice. FK506 reduced the expansion of donor CD8+ and, to a lesser extent, CD4+ T cells in the spleen and inhibited donor anti-host T-cell proliferative and cytotoxic responses. It also inhibited the induction of Th1, FasL, and granzyme B mRNA expression in target organs of GvHD. CONCLUSIONS: FK506 inhibits both GvHD and GvL activity when given at clinical doses by inhibiting donor T-cell expansion, donor anti-host T-cell reactivity, and Th1 immune responses.     

Cyclosporine preserves the anergic state of human T cells induced by costimulation blockade in vitro

BACKGROUND: Costimulation blockade based tolerance-inducing therapies might be disrupted by adjunct conventional immunosuppressive drug use. In the current study, we evaluated the compatibility of various immunosuppressive agents on costimulation blockade-based immunosuppression and T-cell anergy induction of human alloreactive T-cells in vitro. T-cell anergy is crucial in transplantation tolerance. METHODS: T cell anergy was induced in human mixed lymphocyte cultures in vitro, by monoclonal antibodies directed against the costimulatory ligands CD40 and CD86. The effect of coadministration of conventional immunosuppressive drugs (CsA, rapamycin or FK506) on the inhibitory potential of costimulation blockade and the induction and maintenance of T cell anergy was analyzed. RESULTS: We found that monoclonal antibodies against CD40 and CD86 and the simultaneous use of conventional immunosuppressive drugs resulted in strong immunosuppression of proliferation and cytokine production. Rapamycin, in contrast to FK506 and CsA, facilitated T-cell apoptosis. However, drug cotreatment prevented costimulation blockade induced T-cell anergy. Induction of human T-cell anergy in vitro required approximately 5 days of culture. Coadministration of drugs at day 5 after the start of mAb treatment, when anergy was established, did not increase the immunosuppressive effect of mAb treatment. But interestingly, in the majority of experiments, in contrast to rapamycin and FK506, CsA did not affect the anergic state when given after T-cell anergy induction. Moreover, the cell death facilitating potential of rapamycin vanished when used later after T-cell activation. CONCLUSIONS: Timing and choice of conventional drug are crucial in the success of costimulation blockade-based tolerance induction therapies.     

In vivo pharmacokinetic and pharmacodynamic evaluation of the malononitrilamide FK778 in non-human primates

The malononitrilamide FK778 is a leflunomide analogue with a shorter half-life than leflunomide. Groups of cynomolgus monkeys were treated orally with various doses of FK778 once daily for 7 days: group A, 10 mg/kg ( n=4); group B, 5 mg/kg ( n=3); and group C, one single loading dose of 20 mg/kg followed by 5 mg/kg once daily ( n=2). Trough plasma concentration of FK778 was measured by HPLC. Lymphocyte proliferation and expression of T-cell activation surface antigens were assessed by flow cytometry. In group A, trough plasma concentration of FK778 reached steady state at 48 h. After 7 days, lymphocyte proliferation was 23+/-7.4% (mean +/- SEM) and expression of CD71, CD25, CD11a and CD95 on T cells was less than 50% of pre-treatment baseline values. In group B, trough plasma levels of FK778 did not reach steady state, but dropped to near-zero levels after 3 days and on day 7 and lymphocyte proliferation and T-cell surface antigen expression were not different from pre-treatment baseline values. In group C, FK778 trough levels did not reach steady state, but drug exposure was evident over the entire period of treatment, and on day 7, lymphocyte proliferation was 11.4+/-8.6% of pre-treatment baseline values. We conclude that FK778 inhibits lymphocyte proliferation and expression of T-cell activation antigens in vivo in non-human primates after 1 week of treatment. These effects are related to the total drug exposure over the time of treatment. At doses lower than 10 mg/kg daily, FK778 is cleared from the circulation between the dosing intervals, thus failing to exert its inhibitory effects on immune functions.     

Acute influence of FK506 on T-lymphocyte populations of peripheral blood and spleen in rats

BACKGROUNDS: Tacrolimus (FK506) is an immunosuppressive agent used in clinical transplantation. We studied the acute impact of FK506 on T-lymphocyte populations of peripheral blood and spleen in rats. METHODS: The animals injected with FK506 (2 mg/kg) subcutaneously were sacrificed at 12, 24, or 48 hours after administration. CD3(+), CD4(+) and CD8(+) T-cell populations in peripheral blood and spleen were analyzed by FACS. RESULTS: After treatment with FK506, the proportion of CD4(+) cells significantly increased in the spleen at 12 hours, decreasing to control levels at 24 and 48 hours. There was a slight increase in the CD4(+) subpopulation in the peripheral blood at 12 and 24 hours and a significant increase at 48 hours. The CD8(+) T-cell subpopulations in peripheral blood and spleen were stable during FK506 administration. CONCLUSIONS: These results indicated that FK506 increased CD4(+) spleen lymphocyte activation in a short time, inducing a subsequent increase in CD4(+) cells subsets in peripheral blood.     

Human T cell responses to human and porcine endothelial cells are highly sensitive to cyclosporin A and FK506 in vitro

BACKGROUND: Human T cells proliferate in response to both human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) via the second signals LFA-3/CD2 and B7-2 (CD86), respectively. Previous studies have shown that stimulation of T cells via CD28 or phorbol myristate acetate (PMA) activation is highly resistant to inhibition by cyclosporine A (CsA) and tacrolimus (FK506), as is the response of T cells to phytohemmaglutinin in the presence of endothelial cells. We have investigated the inhibitory effects of CsA and FK506 on the direct response of human CD4+ T cells to HUVEC and PAEC and the effect of adding B7-1 transfectants. METHODS: T cell proliferation, interleukin-2 release bioassays and a multiple cytokine bioassay employing the TF-1 cell line were used as indicators of T cell responses to HUVEC and PAEC either in the presence or absence of CsA and FK506. In some experiments, B7-1 transfectants were also added. RESULTS: Proliferative responses and interleukin-2 release were highly sensitive to CsA, the ID50 being significantly less for HUVEC (6.5 ng/ml) than PAEC (15 ng/ml). The ID50 of CsA for the mixed lymphocyte response (MLR) was similar to PAEC (18.6 ng/ml), all these values being significantly less than the T cell activation by phytohemmaglutinin (PHA) (227 ng/ml). Addition of B7-1 transfectants significantly increased interleukin-2 production by T cells/HUVEC and resistance to CsA was greatly increased to an ID50 of > 1000 ng/ml. In contrast, addition of B7-1 transfectants to T cells/PAEC had no effect either on T cell proliferation, IL-2 production, or CsA resistance. Similar results were obtained with FK506. Using the TF-1 cell line, it was determined that cytokines other than IL-2 are released during CD4+ T cell/EC interactions, with similar sensitivity to CsA and FK506. CONCLUSIONS: It is concluded that both allogeneic and xenogeneic T cell/endothelial responses should be inhibited by therapeutic levels of CsA in vivo, assuming the absence of trans-stimulation by B7 molecules.     

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.     

Potential for improved therapeutic index of FK506 in liposomal formulation demonstrated in a mouse cardiac allograft model.

BACKGROUND: FK506 is a potent immunosuppressant that has improved clinical outcomes in kidney and liver transplantation both as a primary and as a rescue immunosuppressive agent. Despite these benefits, the potential value of FK506 is limited by toxic side effects that result in a narrow therapeutic index. By encapsulating the active drug within liposomes (LipoFK506), a new formulation has been developed that might improve this therapeutic index. METHODS: The biodistribution of tritiated-FK506 administered i.v. showed that the drug remained associated with the liposomal carrier in vivo, and that its tissue distribution was increased in heart and spleen compared to nonliposomal FK506. The immunosuppressive efficacy of lipoFK506 compared with conventional FK506 formulation was tested in vivo. CBA (H2k) mice were engrafted with BALB/c (H2d) mouse hearts with daily immunosuppression using either 1 mg/kg FK506, or 1 mg/kg LipoFK506, from day 0 to 14. RESULTS: At day 7 the blood trough level of FK506 in the FK506 group was 10-fold higher (25 microg/L) than that in the LipoFK506 group. In both groups the median heart allograft survival was similar at around 26 days. The possibility that FK506, or LipoFK506, might influence antibody-mediated tolerogenesis was addressed in the same model: neither formulation prevented tolerance induction by CD4 and CD8 blockade. CONCLUSION: LipoFK506 is a novel formulation of FK506 that is efficacious at low blood trough FK506 levels. This property has a direct potential benefit for clinical organ transplantation.     

Triptolide is a potent suppressant of C3, CD40 and B7h expression in activated human proximal tubular epithelial cells

BACKGROUND: Previous studies have shown that triptolide possesses potent immunosuppressive and anti-inflammatory properties. Increasing recognition of the importance of the proximal tubular epithelial cells (PTEC) in renal disease and renal transplantation raises the question of whether triptolide suppresses the pro-inflammatory activity of PTEC. METHODS: Cultured human PTEC were exposed to tumor necrosis factor-alpha (TNF-alpha) and immunosuppressant (triptolide or CsA or FK506) for 24 hours, followed by RT-PCR, ELISA, flow cytometry and Western blotting analysis for complement C3, CD40, B7h expression. RESULTS: TNF-alpha up-regulated C3, CD40 and B7h production by PTEC. This up-regulation was inhibited by all three immunosuppressants with different intensity. Firstly, triptolide (4 to 8 ng/mL), CsA (4000 to 6000 ng/mL) and FK506 (2000 ng/mL) inhibited up-regulation of C3 mRNA, but CsA and FK506 had less of an effect than triptolide. Secondly, triptolide (4 to 8 ng/mL) completely inhibited C3 expression at both mRNA and protein levels. In contrast, CsA and FK506 had only slight effects on C3 expression at the protein level. Thirdly, triptolide (4 to 8 ng/mL), CsA (500 to 2500 ng/mL) and FK506 (1250 ng/mL) inhibited up-regulation of CD40 and B7h mRNA, the effect on B7h and CD40 mRNA expression by CsA and FK506 being greater than that on C3 mRNA expression. CONCLUSION: Triptolide effectively inhibited up-regulation of C3, CD40 and B7h on PTEC. Triptolide was more effective than CsA and FK506 at inhibiting C3 expression. This suggests that triptolide, at non-cytotoxic concentrations, has the potential to reduce the inflammatory and immunostimulatory properties of PTEC, in addition to any of the previously reported actions on T cell or B cell function.     

Use of mixed lymphocyte reaction to identify subimmunosuppressive FK-506 levels in mice

The immunosuppressive agent FK-506 has a well-described neuroregenerative effect that is mediated by a mechanism independent of calcineurin inhibition. FK-506 levels that fall below the threshold for immunosuppression could therefore potentially enhance nerve regeneration while minimizing toxicity. The purpose of this study was to characterize the dose-dependent effects of FK506 on T-cell proliferation, and establish a subimmunosuppressive dosing regimen for FK-506 in mice. Forty BALB/cJ mice were randomized to four groups corresponding to 0, 0.25, 0.5, or 1.0 mg/kg/day doses of FK-506. Ten days postoperatively, animals were sacrificed, and mixed lymphocyte reaction assays were performed to quantify the immune response to nerve allografts. Mice receiving 0.25 and 0.5 mg/kg/day of FK-506 exhibited a robust T-cell proliferation response, with stimulation indices approaching those of untreated animals. Mice treated with 1.0 mg/kg/day of FK-506 demonstrated significantly decreased T-cell proliferation. These results establish 0.5 mg/kg/day as an upper limit for subimmunosuppressive FK-506 administration.     

Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities

BACKGROUND: We describe immunomodulatory effects of FK734, a humanized version of a mouse anti-human CD28 mAb (clone TN228), in vitro and in a chimeric human-mouse model of allograft rejection. METHODS: Cytokine production and proliferation were assessed in a mixed lymphocyte reaction containing FK734, human T cells, and endothelial cells or monocytes. FK734 was also administered to SCID mice engrafted with human skin and adoptively transferred with human peripheral blood mononuclear cells allogeneic to the skin graft. RESULTS: In vitro, FK734 enhanced secretion of interleukin-2 and interferon-gamma as well as proliferation of CD4+ and CD8+ T cells stimulated by allogeneic human leukocyte antigen (HLA)-DR+ human umbilical vein endothelial cells (which lack B7 molecules and FcgammaRs) or by blood monocytes (which express low levels of B7 molecules and FcgammaRs) compared with control mAb, but these effects were significantly smaller than those provided by mAb 28.2, a stimulatory mouse anti-human CD28 mAb, at comparable concentrations. However, FK734 generally inhibited cytokine secretion and T cell proliferation in cocultures with human umbilical vein endothelial cells transduced to express CD86. In vivo using SCID/beige mice bearing human skin with adoptively transferred peripheral blood mononuclear cells, administration of FK734 protected human endothelial cell-lined microvessels, significantly but incompletely reducing endothelial cell injury and T cell infiltration into the graft one or two weeks later. CONCLUSIONS: FK734 is a partial agonist of CD28 signaling that can reduce human T cell alloresponses in the presence of strong costimulation by B7 molecules in vitro and can reduce T cell-mediated skin allograft rejection in vivo.     

Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model

The effects of FK506, and TAK-779, antagonists of CCR5 and CXCR3, were investigated using a rat intestinal transplantation model. Small intestines from DA rats were heterotopically transplanted into LEW rats. The recipients were treated with FK506 (1 mg/kg/day, day 0-5) and TAK-779 (10 mg/kg/day, day 0-10). Graft survival and immunological responses to these materials were estimated by mixed lymphocyte reactions and IFN-?? production. The expression of chemokine receptors on lymphocytes was also examined. The average duration of survival was 7.0 ± 0.3, 12.0 ± 1.0, 9.8 ± 0.5 and 18.0 ± 1.5 days in the allogeneic, FK506, TAK-779 and the two-drug combined groups, respectively. Cell proliferative responses and IFN-?? production were suppressed to a significant extent in the FK506 group compared with the TAK-779 group. In addition, the two-drug combination showed a tendency for stronger suppression than FK506 alone, correlated with in vivo and histopathological data. The numbers of both CD4⁺ and CD8⁺ cells were significantly suppressed in the blood of the recipients of both the FK506 and the TAK-779 groups, and in Peyer's patches of the graft of the TAK-779 group, but the FK506 group was not, as evidenced by FACS analysis. In addition, double-staining of graft-infiltrating lymphocytes showed a significant reduction in lymphocyte numbers, expressing CCR5 and CXCR3 in the TAK-779 group, but not evident in the FK506 group, compared to the allogeneic group.While FK506 suppresses cell proliferation and effecter function, it has less effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effects of a combined therapy with TAK-779 could represent a novel treatment for intestinal transplantation.     

Regulatory effect of FK506 on CD152 and PD-1 in the liver allorecipients

AIM: We dynamically observed the expression of CD152 and PD-1 on T-cell surfaces in peripheral blood of liver allorecipients to explore the regulatory effect of FK506 on negative costimulatory molecules. METHODS: We evaluated 20 liver allorecipients, 19 end-stage liver disease patients, and 20 healthy volunteers for FK506 concentrations measured by an enzyme-multiplied immunoassay technique and flow cytometry to determine T-cell subsets as well as CD152 and PD-1 expression. RESULTS: After liver transplantation, the frequency of CD4+ T cells gradually decreased to significantly lower level than those in the disease controls (P < .05). CD8+ T cells in each treatment group were obviously higher than those in the disease controls (P < .05). The expression of CD152 on CD4+ and CD8+ T cells was greater than those in healthy controls (P < .05); and at 2 and 4 weeks, higher than those in disease controls (P < .05). The expression of PD-1 on CD4+ T cells from the 2 weeks after treatment was significantly greater than that in healthy controls (P < .05), and that on CD8+ T cells increased obviously from the 4 weeks compared with disease controls (P < .05). CONCLUSION: FK506 up-regulated the expression of CD152 and PD-1 on the T-cell surface inhibiting proliferation and activation of effector T cells, favoring the survival of allorecipients.     

Clinical value of the flow cytometric method for measuring lymphocyte subset activation: spontaneous activation of T-cell subpopulations is associated with acute GvHD

Thymidine ((3)H-TdR) incorporation remains the most commonly used method to quantifying T-cell proliferation. This method, however, does not provide information about specific lymphocyte subpopulations responding to different stimuli. In our study, we modified previously described nonradioactive flow-cytometric T-cell activation assay measuring the expression of a CD69+ antigen on T-cell subsets and applied it to analysis of lymphocyte subsets activation/proliferation in children after allogeneic hematopoietic cell transplantation (HCT). We compared the percentage of spontaneously activated lymphocyte subpopulations (background) and the percentage of PHA-P, PWM, and SEB-stimulated cell subsets from two groups of patients: group 1, children with Graft versus Host Disease (GvHD) and group 2, children without any signs of GvHD at the time of analysis. High rate of spontaneous T-cell subset activation was found in group 1 with CD3+CD8+Ts cells being the most affected cell population. High background activation of Th and B cells correlated with the occurrence of autoimmune phenomena posttransplant. Rapid quantification of CD69+ expression on unstimulated and stimulated T-cell subsets proved to be a valuable method for monitoring children after allogeneic HCT. High proportion of activated, unstimulated Ts cells observed in the GvHD group may underline the critical role of CD3+CD8+ cells in the pathogenesis of GvHD. Thus in future immunosuppressive therapy may be adjusted according to the proportion of activated Ts cells.     

CD154 is expressed during treatment with calcineurin inhibitors after organ transplantation

BACKGROUND: CD154 (CD40 ligand) monoclonal antibody prevents allograft rejection in rodents and monkeys. Inasmuch as calcineurin inhibitors (CI) inhibit CD154 expression by pharmacologic agents in vitro, we investigated whether CD154 is also inhibited by CI in vivo and in vitro during allogeneic stimulation. METHODS: CD154 expression was determined by immunohistochemistry and flow cytometry in human lymph nodes and spleen sections from rhesus monkeys with or without CI treatment. The effect of CI on induction of CD154 expression was studied by stimulating lymphocytes with phorbol 12-myristate 13-acetate (PMA) and ionomycin or with allogeneic monocyte-derived mature dendritic cells. RESULTS: Lymph nodes from patients with or without CI cyclosporine (CsA) or FK506 (FK) treatment showed comparable CD154 expression, which was present on the cell surface of T cells. CD154-expressing cells were also present in spleens from monkeys treated with CsA in comparable numbers to those in the nontreated group. Moreover, in several liver transplant rejection biopsies taken during CI therapy CD154-expressing cells were observed. In vitro, CsA and FK strongly inhibited induction of CD154 expression on peripheral blood mononuclear cells by pharmacologic stimuli. Maximum inhibition was found at 50 ng/ml CsA and 20 ng/ml FK. CD154 expression induced by dendritic cells in peripheral blood mononuclear cells or spleen cells was also almost completely inhibited by CsA. CONCLUSION: Although CI strongly suppressed pharmacologic and allogeneic induction of CD154 expression on T cells in vitro at concentrations at approximately clinical trough levels, CD154 is prominently expressed during CI therapy in lymphoid tissue and (sporadically) in liver allografts. This suggests that the CD154-CD40 pathway remains functional during CI therapy, which may contribute to allograft rejections in the clinical setting.     

Cytokine gene expression by alloactivated cells in SCID mice

OBJECTIVES: The severe combined immune deficient (SCID) mouse provides a neutral environment to study human immune responses. We therefore tested human gene expression of Interleukin (IL) 2, 4 and 10, interferon gamma (IFNgamma); transforming growth factor beta 1 (TGFbeta1); and CD40 ligand (CD40L) in splenic extracts of SCID mice after engraftment of PBLs from two persons (direct MLR) or one person plus allopeptides (indirect MLR) in the presence or absence of cyclosporin A (CsA) or FK506. METHODS: Cytokine gene expression was detected by RT and quantitative (for IFN-gamma, TGFbeta1 and CD40L) PCR. All cells, allopeptides, CsA (25 mg/kg/day for 7 days) or FK 506 (0.5 mg/kg/day for 7 days) were administered intraperitoneally (IP). RESULTS: In both direct and indirect MLR the numbers of SCID mice expressing the human cytokine genes varied between 33% for IL4 and 100% for IL10, IFN-gamma, TGFbeta1, and CD40L. There was significant interpersonal variation in levels of gene expression. Concomitant CsA or FK506 administration for 7 days did not abrogate early or late (1 week after discontinuation of CsA or FK506) cytokine gene expression in either the direct or indirect MLR, but paradoxically enhanced levels of IFN-gamma, TGFbeta1 and CD40L gene expression in some experiments. CONCLUSIONS: The results explain late rejection after rapid calcineurin inhibitor withdrawal or reduction, and illustrate the potential use of SCID mice as a surrogate model to study graft outcome by determination levels of gene expression and sensitivity to immunosuppressive agents in the in vivo alloresponse.     

Suppression of primary T-cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490

BACKGROUND: Tyrphostin AG490 has recently been shown to block interleukin (IL)-2 receptor gamma-chain-associated Janus kinase 3. Here, we analyzed the effect of AG490 on T-cell alloresponses in vitro. METHODS: For the evaluation of T-cell activation, DNA synthesis, surface marker expression, cytokine secretion, intracellular calcium mobilization, early protein tyrosine phosphorylation, and apoptosis were measured. RESULTS: AG490 effectively inhibited T-cell proliferation in human mixed lymphocyte culture (MLC) even when added 4 days after culture initiation. Inhibition of IL-2-dependent proliferation in T-cell blasts and the incapability of IL-2 or IL-15 to restore proliferation in AG490-treated MLC suggests interference with cytokine receptor signaling. T-cell receptor-triggered early protein tyrosine phosphorylation, calcium mobilization, up-regulation of CD69, and initial CD25 expression were not affected. Interestingly, AG490 substantially inhibited production of IL-2 and interferon-gamma in T cells stimulated with alloantigen or via CD3 and CD28. In CD28-independent activation models (e.g., stimulation with phorbol myristate acetate plus ionomycin), however, cytokine secretion was not inhibited. Pretreatment of primary MLC with AG490 resulted in substantial down-regulation of secondary responses to cells from the original donor as opposed to third-party cells or phytohemagglutinin. Unresponsiveness was induced also in T cells stimulated with CD3 monoclonal antibody. Induction of apoptosis in polyclonally activated T cells and the incapability of IL-2 to reverse specific hyporesponsiveness, suggest programmed cell death as an important mechanism underlying antigen-specific down-regulation of alloresponses. CONCLUSIONS: We demonstrate that AG490 blocks different manifestations of T-cell activation. This and its ability to induce alloantigen-specific hyporesponsiveness point to a potential use for interfering with alloreactivities in vivo.     

Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers

BACKGROUND: FK778, a malononitrilamide analog of leflunomide, is currently being investigated for use in clinical transplantation. METHODS: Whole blood from cynomolgus monkeys (n=4) and healthy human volunteers (n=4) was incubated with different concentrations of FK778 and stimulated with mitogens in culture medium. Lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow-cytometric analysis of expression of proliferating cell nuclear antigen on cells in S/G(2)M phase. Flow cytometry was also used to assess expression of T and B lymphocyte activation surface antigens and production of intracellular cytokines by T cells. RESULTS: Not only lymphocyte proliferation, but also expression of various T cell surface antigens (CD25, CD11a, CD95, CD154) was suppressed by FK778. Fifty percent effective concentration values for the different immune functions were lower in human blood than in blood from cynomolgus monkeys. CONCLUSIONS: FK778 inhibits multiple immune functions. Their flow cytometric evaluation can be used to assess the effects of the drug in vivo.