Dendritic cells genetically engineered with adenoviral vector encoding dnIKK2 induce the formation of potent CD4+ T-regulatory cells

BACKGROUND: Immature dendritic cells (DC), characterized by low expression of both major histocompatibility complex class II antigens and co-stimulatory molecules, can be instrumental in the induction of peripheral tolerance. Because nuclear factor (NF)-kappa B is central to DC maturation, the authors engineered DC with an adenoviral vector (Adv) encoding for a kinase-defective dominant negative form of IKK2 (dnIKK2) to block NF-kappa B activation and inhibit DC maturation. METHODS: DC were obtained by culturing bone marrow from Brown Norway (BN) rats with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 11 days. To block NF-kappa B activation, at day 9, cells were transfected with AdV-dnIKK2. At day 11, cells were used as stimulators in primary mixed leukocyte reaction (MLR) with naive Lewis rat lymphocytes as responders. CD4+ T cells were purified from primary MLR and tested in secondary MLR with allogeneic mature DC and in co-culture MLR with naive lymphocytes. The tolerogenic potential of dnIKK2-DC was evaluated in vivo in a model of rat kidney allotransplantation. RESULTS: DnIKK2-DC were immature and lacked any allostimulatory activity. T cells preexposed to allogeneic dnIKK2-DC were hyporesponsive to a secondary stimulation with mature DC and acquired potent regulatory properties, inhibiting naive T-cell proliferation toward allogeneic stimuli. Pretransplant infusion of allogeneic donor dnIKK2-DC prolonged the survival of a kidney allograft from the same allogeneic donor, without the need for immunosuppressive therapy. CONCLUSIONS: Allogeneic DC, rendered immature by dnIKK2 transfection, induce in vitro differentiation of naive T cells into CD4+ T-regulatory cells, effective at low ratios with target cells, rendering them applicable for cellular therapy of immune-mediated abnormalities and for preventing transplant rejection.     

Down regulation of CD45 expression on CD4 T cells during acute renal allograft rejection: Evidence of a decline in T suppressor/inducer activity

Acute rejection is associated with the activation of helper and cytotoxic cells. A shifting balance between the suppressor/inducer CD45+ CD4+ and T helper/inducer (CD4+CD45–) cells may be responsible for the transition from quiescence to overt rejection. We examined the kinetics of CD45 expression on CD4+ T cells in renal allograft recipients from pretransplant values to acute rejection and after reversal of rejection, searching for a shift in balance between helper/inducer and suppressor/inducer cell subsets. Using two color flow cytometry, the peripheral blood levels of CD4+, CD4+CD45– [T helper/inducer (Thi)], CD4+CD45+ [T suppressor/inducer (Tsi)], CD3+, and CD8+ T cells subsets and their interrelationships, were determined in 49 patients prior to transplantation, and in 10 of them, during acute rejection and after its reversal. Results were analyzed and compared to data obtained from 10 healthy blood donors. Acute rejection was associated with a significant decline in CD45+ CD4+ expression compared to quiescent phase (22% ± 3.7%vs. 26.5% ± 3.2%, p = 0.05) and controls (29.5% ± 6.2%, p = 0.01). No difference was observed compared to pretransplant levels (19.9% ± 3.2%, p = ns). CD45–/CD45+ (Thi/Tsi) ratio was lowest during quiescence (0.75) compared to rejection (0.97, p = 0.05), in controls (0.98, p = 0.05) and pretransplant values (1.4, p = 0.01). Acute rejection was characterized by higher Thi/CD8+ and lower Tsi/CD8+ ratio (103 and 88 respectively, p = 0.045), compared to clinical quiescence (104 and 116 respectively, p = 0.039). These data suggest that acute rejection is associated with down regulation of CD4+CD45+ suppressor/inducer subset. This shift may account for the transition from quiescence to overt rejection, concurring with reports on CD4+CD45 regulatory function.     

Generation of CD4-positive suppressor T cells from mixed lymphocyte cultures in the presence of interleukin 2 receptor antibody TU69. An in vitro model for transplantation tolerance induction

The specificity of a novel monoclonal antibody (moAB), TU69, directed to the interleukin 2 receptor (IL-2R) was verified by sequential immunoprecipitation with anti-Tac. TU69 cross-competed with anti-Tac in binding analyses. When TU69 was added during the sensitization of normal peripheral blood mononuclear cells (PBMC) to allogeneic HLA-class I or -class II mismatched stimulator PBMC, alloproliferative responses and specific cytotoxicity were no longer detectable and the generation of natural killer (NK)-like effector cells was partially inhibited. Remarkably, however, the generation of CD4+ nonspecific suppressor T cells in such mixed lymphocyte cultures (MLC) was not inhibited--but, in contrast, was strongly enhanced in the presence of TU69. These suppressor cells inhibited unrelated allospecific responses in vitro to background levels even at a ratio of 50:1 responder:irradiated suppressor T cell lines. Such a potent experimental suppressor system suggests a possible application of TU69 for in vivo tolerance induction after transplantation, by down-regulating allospecific effector cells and allowing the generation of tolerance to graft antigens.     

人CD8+CD28-抑制性T淋巴细胞的体外扩增及其抗原特异性调节作用

目的 探讨在体外大量扩增CD8+CD28-抑制性T淋巴细胞(Ts细胞)的方法,并检验其免疫调节作用.方法 分离健康志愿者全血中CD8+T淋巴细胞,在含不同细胞因子和异体抗原提呈细胞(APC)的培养条件下进行体外扩增.应用流式细胞术对扩增过程中CD28 -细胞亚群的比例进行监测.分为3组进行混合淋巴细胞培养,反应细胞均为CD4+T淋巴细胞:B-APc组以来源于原致敏供者的APC作为刺激细胞,I-APC组以HLA-A、B、DR全错配的无关供者的APC作为刺激细胞,Dynabeads组以包被有抗CD3和CD28单克隆抗体的免疫微球Dynabeads作为刺激细胞;扩增后的Ts细胞作为第三方调节细胞加入混合淋巴细胞培养中,测定其对CD4+T淋巴细胞增殖的抑制作用.结果 在含白细胞介素2(IL-2)+ IL-7+ IL-15的培养条件下,CD8+T淋巴细胞培养后CD8+CD28-T淋巴细胞亚群的比例最高(P＜0.05).B-APC组不加入Ts细胞时,增殖的CD4+T淋巴细胞比例为66.7％,当加入的Ts细胞与反应细胞的比例分别为0.5∶1、0.1∶1和0.02∶1时,增殖的CD4+T淋巴细胞比例分别为16.5％、34.1％和62.6％.Ts细胞对CD4+T淋巴细胞的增殖有明显抑制作用,而在I-APC组和Dynabeads组中,此抑制作用不明显.结论 应用含IL-2+ IL-7+ IL-15的培养条件联合异体APC刺激可以在体外大量扩增Ts细胞,扩增所得Ts细胞在体外对供者CD4+T淋巴细胞的增殖有明显抗原特异性抑制作用.     

Differentiation of human alloreactive CD4+ and CD8+ T cells in vitro

BACKGROUND: Recent studies have revealed that, during viral infection, ordered phenotypic and functional changes occur in human antigen-specific T cells. We analyzed whether a similar differentiation program is induced after alloantigen stimulation in vitro. METHODS: Peripheral blood mononuclear cells and purified CD4(+)CD45RA+, CD4(+)CD45RO+, and CD8+ T cells from healthy individuals were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were co-cultured with allogeneic irradiated cells. Flow cytometric analysis was performed on days 3, 5, and 7 of culture using surface CD45RA, CD27, CD28, CCR7, and intracellular perforin and granzyme B markers in relation to CFSE dilution. RESULTS: Based on the decrease in CFSE fluorescence, both CD4+ and CD8+ T cells showed an early and vigorous response to allogeneic stimulation. Loss of CD45RA expression and up-regulation of CD27 and CD28 costimulatory molecules was an early event occurring in the first generations of dividing cells. Differentiation at later stages of proliferation was characterized by loss of CCR7 homing receptor expression, more pronounced in CD4+ than in CD8+ T cells, indicating the decreased ability of these cells to traffic to secondary lymphoid organs. Production of the cytotoxic effector molecules perforin and granzyme B increased with the number of cell divisions. CONCLUSIONS: Our data thus show that short-term phenotypic and functional changes of alloreactive T cells follow the differentiation pattern seen in the early stages of an antiviral immune response.     

Persistent expansion of CD4+ effector memory T cells in Wegener's granulomatosis

In order to test the hypothesis that Wegener's granulomatosis (WG) is associated with an ongoing immune effector response, even in remission, we examined the distribution of peripheral naive and memory T-lymphocytes in this disease, and analyzed the function-related phenotypes of the memory T-cell population. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from WG-patients in remission (R-WG, n=40), active WG-patients (A-WG, n=17), and age-matched healthy controls (HCs, n=21). Expression of CD4, CD8, CD45RO, CCR7, interleukin (IL)-18Ralpha, ST2L, and FoxP3 were determined by four-color flow cytometric analysis. CD45RO and CCR7 were used for distinction between naive and memory T cells, IL-18Ralpha, ST2L, and FoxP3 for the assessment of Type1, Type2, and regulatory T-cells, respectively. In R-WG, the CD4+CD45RO+CCR7- effector memory T-cell subpopulation (TEM) was relatively increased, whereas the CD4+CD45RO-CCR7+ naive T-cell population (TNaive) was decreased as compared to HC. The distribution of naive and memory CD8+T cells did not differ between R-WG, A-WG, and HC, nor did CD4+CD45RO+CCR7+ central memory T cells (TCM). In contrast to HC, the percentage of CD4+TNaive cells in R-WG correlated negatively with age, whereas CD4+TEM cells showed a positive correlation. In R-WG, a skewing towards Type2 T cells was observed in CD4+TEM cells. No differences were detected in FoxP3+CD4+TEM cells between R-WG and A-WG, whereas the FoxP3-CD4+TEM cells were increased in R-WG and decreased in A-WG as compared to HC. Collectively, peripheral blood homeostasis of CD4+T cells is disturbed in R-WG with the persistent expansion of non-regulatory CD4+TEM cells. These cells might be involved in relapse and may constitute a target for therapy.     

Inhibition of alloresponsive naive and memory T cells by CD7 and CD25 antibodies and by cyclosporine.

Human naive and memory T cells can be isolated from each other by their CD45RA and CD45RO expression, respectively. This enables the assessment of their differential sensitivities to immunosuppressive agents for the first time. We have investigated the ability of cyclosporine or CD7 and CD25 antibodies to selectively block alloantigen stimulated naive and memory T cells in vitro. CD7 antibodies blocked the proliferation of naive (P less than 0.025) but not memory T cells in a primary MLR. CD25 antibody inhibited both naive and memory subsets but a significantly greater effect was found on the memory T cells (P less than 0.005). The constitutive CD7 and CD25 antigen expression on resting naive or memory T cells was related to the inhibitory activities of these antibodies on both subsets. Accordingly, naive T cells expressed more CD7 antigen than memory cells while memory T cells displayed low levels of CD25 antigen that was absent from naive populations before activation. Cyclosporine, like CD25 antibody, inhibited both subsets in a primary MLR but had a greater effect on memory cells (P less than 0.02). Memory T cells, therefore, are more dependent than naive cells on IL-2 for proliferation. There was great individual variation in the ability of CsA to block the MLR. The simultaneous addition of CD25 or CD7 antibody together with CsA, however, enhanced the MLR inhibition as the effects of all three were additive. This suggested interference by these agents at different points during T cell activation. Thus, in CsA sensitive individuals, one-tenth of the optimal CsA concentration together with CD25 antibody maintained maximum immunosuppression in vitro. These results demonstrate the possibility of using CD7 and CD25 antibodies for selective inhibition of naive or memory T cells and also the possibility of augmenting the inhibition of and reducing the CsA concentration required for clinically effective immunosuppression.     

Renal allograft rejection in CD4+ T cell-reconstituted athymic nude rats. The origin of CD4+ and CD8+ graft-infiltrating cells

PVG-rnu/rnu nude rats reject a fully allogeneic DA renal allograft after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many CD8+ leukocytes within the graft. In order to clearly establish the provenance of these CD8+ cells infiltrating rejecting kidney allografts, nude recipients (PVG-RT7a) were injected with CD4+ T cells from the PVG-RT7b congenic strain bearing an allotypic variant of the leukocyte-common antigen. Dual fluorescence and immunohistochemistry demonstrated that approximately 75% of the total infiltrate was host-derived; the donor-derived RT7b population was almost entirely (92-99%) CD4+, CD5+, CD3+, and alpha beta TCR+. At least 97% of the CD8+ cells were of nude origin. There was no evidence of donor-derived CD8+ cells or of a CD4+8+ double-staining population. Unexpectedly, nearly half of the alpha beta TCR+ cells from the grafts were of nude origin.     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

Patterns of T cell-accessory cell interaction in the generation of primary alloresponses in the pig.

Partially inbred, MHC-homozygous miniature swine provide a unique model for the study of organ transplantation and the induction of tolerance in large animals. Models of both vascularized solid organ transplantation and bone marrow transplantation have previously been established. The availability of monoclonal antibodies reactive with porcine leukocyte subset antigens now makes possible studies of the cellular immunology in this species, affording the opportunity to examine mechanisms of transplant tolerance and graft rejection in increasing detail. Using such antibodies and peripheral blood leukocytes from pigs of recombinant MHC haplotypes, we have examined porcine T cell-accessory cell interactions in vitro with attention to T cell subsets and the class of MHC alloantigen stimulation. Primary allospecific MLR and CML cultures were studied after depletion of accessory cells from responder and/or stimulator populations. Although class II MHC antigens were expressed on the majority of porcine T cells before and after depletion, these cells were insufficient for antigen presentation, since there was an absolute requirement for ACs in the generation of primary alloresponses. Proliferative and CTL alloresponses could be generated provided that ACs of either stimulator or responder type were present. Selective depletion of CD4+ T cells from the responder population demonstrated: (a) that the interaction mediated by self ACs was CD4-dependent; (b) that two pathways exist for interaction involving allogeneic ACs; and (c) that the interaction involving allogeneic class II is CD4-dependent, while that with allogeneic class I is not.     

Human CD8 responder cells can be directly activated to proliferate and to produce IL-2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells

Abstract: In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4₊ and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL-2, but not by IL-lα, IL-lβ, or IL-6. Production of IL-2 was not detected in the xenostimulated CD8+ responder cells, nor could IL-2 production be restored by the addition of IL-lα, IL-1β, or IL-6. The presence of human CD4₊ responder cells was crucial both for a xenoproliferative response and for IL-2 synthesis. However, when the expression of the IL-2 receptor (CD25) on the CD8₊ T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8₊ T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL-2 reconstituted the cytotoxicity by purified CD8₊ T cells; however, IL-lα, IL-lβ, or IL-6 did not.
Our results show that purified human CD4₊ T cells respond directly against pig PBMCs, whereas purified CD8₊ T cells do not. Furthermore, responsiveness in CD8₊ T cells is completely restored by the addition of human IL-2.     

Suppressor function of umbilical cord blood-derived CD4+CD25+ T-regulatory cells exposed to graft-versus-host disease drugs

BACKGROUND: This study examines the effects of the most commonly used graft-versus-host disease (GVHD) prophylactic drugs on inducing apoptosis and suppressor cell function of human umbilical cord blood (UCB) CD4+25+ Treg and CD4+25- cells. METHODS: Cyclosporin A (CSA), methylprednisolone (MP), methotrexate (MTX), and mycophenolic acid (MPA) were added to the final 6 days of expansion cultures of Treg or CD4+25- T-cells isolated from the same donor and each concurrently cultured under the same conditions. Cell viability was measured for CD4+25+ as compared to CD4+25- T-cells and Treg function was assessed. The effects of these immunosuppressive drugs, Treg cells, or both also were tested in a primary allogeneic mixed lymphocyte response (MLR) response. RESULTS: The cell viability percentages were lower for CD4+25- cells than for Treg cells when MP, MTX, or MPA was added for the last 6 days of an expansion culture. Under these interleukin (IL)-2 based expansion conditions, CSA had no effect. The addition of any of the four GVHD prophylactic agents to the expansion phase of culture did not reduce the MLR suppressive capacity of Treg cells. Overall MLR suppression was increased when Treg cells were added along with CSA and MP to a primary MLR culture, whereas MTX modestly reduced Treg suppression. CONCLUSION: These data indicate a general resistance of expanded UCB Treg cells to GVHD immune suppressive agents and support trials to test UCB Treg infusions under the cover of GVHD prophylactic drugs in hematopoietic cell transplantation.     

Expansion of CD25+CD4+ regulatory T cells by natural mature dendritic cells

Because of the anergy of CD25+CD4+ regulatory T cells, it is unclear how the number of these regulatory T cells is sustained and expanded in normal physiologic circumstances. In the present study, we examined the effect of natural allogeneic mature dendritic cells (DCs) on the proliferation and function of CD25+CD4+ T cells. Our data showed that natural allogeneic mature DCs stimulated CD25+CD4+ T-cell growth vigorously, whereas immature DCs had little effect on the proliferation of CD25+CD4+ T cells. After expansion by mature DCs, CD25+CD4+ T cells maintained their expression of Foxp3 and suppressed the proliferation of CD25- CD4+ T cells similar to freshly isolated CD25+CD4+ T cells. Our results introduce a potentially critical role played by natural allogeneic mature DCs, which exist in normal physiologic circumstances, in controlling CD25+CD4+ regulatory T-cell expansion and function.     

Afferent and efferent pathways in T cell responses to MHC class I+, II-hepatocytes

We have previously reported that purified hepatocytes stimulate significant in vitro allospecific cytotoxicity when cocultured with naive responder splenocytes in the mixed lymphocyte hepatocyte culture (MLHC). In this report we examined the expression of MHC antigens on the surface of hepatocytes, the phenotypic lymphocyte subset(s) that respond(s) to allogeneic hepatocytes, and the phenotype of allospecific cytolytic effectors generated in MLHC. Hepatocytes expressed MHC class I but not MHC class II antigens by immunofluorescent microscopy and fluorescence activated cell sorting. The lack of MHC class II on the surface of hepatocytes was also indirectly supported by the inability of hepatocytes to stimulate proliferation of a class II-directed allospecific helper T cell clone. The generation of allospecific cytotoxicity in MLHC required the participation of L3T4+, Ly2- T cells and L3T4-, Ly2+ T cells in the naive responder splenocyte population since depletion of these subsets with mAb and complement abrogated the development of allo-CTLs. Furthermore, adherent accessory cells in the naive responder splenocyte population appeared to play a role in the generation of allospecific cytotoxicity in MLHC since depletion of this population by plastic adherence and passage through a Sephadex G10 column resulted in significantly reduced allospecific cytotoxicity. Depletion of day 5 allosensitized cells of Ly2+ but not L3T4+ T cells by mAb and complement eliminated allospecific cytotoxicity--indicating that cytolytic effectors generated in MLHC appear to be L3T4-, Ly2+ T cells.     

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

Activation and Maturation of Alloreactive CD4-Independent, CD8+ Cytolytic T Cells

The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.     

利用RNA干扰诱导产生的CD8+CD28-抑制性T淋巴细胞的免疫学特性

目的 探讨通过RNA干扰技术诱导产生的CD8+ CD28-抑制性T淋巴细胞(Ts细胞)的免疫学特性.方法 取SD大鼠骨髓,培养分离树突状细胞(DC),设计、合成主要组织相容性复合物(MHC)Ⅰ类小片段干扰RNA(siRNA),以MHC Ⅰ siRNA转染DC.先以Wistar大鼠肠系膜淋巴组织液刺激转染MHCI siRNA的DC,然后将DC与从SD大鼠脾脏分离得到的CD8+T淋巴细胞共同培养,通过磁珠法分离出Ts细胞.分别在由SD大鼠脾脏淋巴细胞(反应细胞)和Wistar大鼠肠系膜淋巴组织细胞(刺激细胞)组成的混合淋巴细胞培养体系中加入数量不等的Ts细胞,检测反应细胞增殖情况;分别以Wistar大鼠肠系膜淋巴组织细胞和卵白蛋白(OVA)刺激SD大鼠脾脏淋巴细胞,然后再按不同比例加入Ts细胞,检测各组脾脏淋巴细胞的增殖情况;在由SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入可溶性重组白细胞介素2(rrIL-2),观察IL-2对Ts细胞功能的影响;采用实时定量聚合酶链反应(PCR)测定Ts细胞中转化生长因子β(TGF-β和γ干扰素(IFN-γ)mRNA的表达,流式细胞仪和实时PCR检测Ts细胞上CD25分子的表达.结果 Ts细胞对SD大鼠脾脏淋巴细胞和Wistar大鼠肠系膜淋巴组织细胞之问的混合淋巴细胞反应(MLR)具有抑制作用,但对于SD大鼠脾脏淋巴细胞和OVA之间的MLR则无抑制作用.在SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入rrIL-2后,SD大鼠脾脏细胞的增殖并无明显增加(P＞0.05).与CD8+CD28+T淋巴细胞和CD8+ T淋巴细胞比较,Ts细胞的TGF-β和IFN-γ mRNA的表达量明显升高(P＜0.01,P＜0.05),而CD25的表达量明显降低(P＜0.05).结论 采用经MHC I siRNA干扰的DC能够诱导CD8+T淋巴细胞产生CD8+ CD28-Ts细胞;Ts细胞在体外具有免疫抑制特性,其免疫抑制作用不被外源性IL-2所逆转,且其免疫调节作用具有抗原特异性.     

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. III. Suppression of graft-versus-host disease in murine allogeneic bone marrow transplantation by the PG27 extract

BACKGROUND: PG27 is an active fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii Hook f. We tested PG27 in murine allogeneic bone marrow transplantation (BMT) and investigated the mechanism of graft-versus-host disease (GVHD) suppression. METHODS: Recipients in the C57BL/6 --> BDF1 murine BMT model received oral or intraperitoneal PG27. RESULTS: Fourteen days of PG27 given orally or intraperitoneally prevented GVHD development and produced extended disease-free survival (more than 300 days) for many animals. PG490-88, a semisynthetic derivative of PG490 (triptolide, present in PG27), was also efficacious. PG27 reduced day 7 splenic allospecific cytotoxic T lymphocyte levels by more than 99% compared with vehicle-treated mice. Compared with normals, spleens from control allogeneic BMT mice displayed significantly reduced mononuclear cell content, an increased percentage of CD8+ cells, fewer CD4+ cells, and more activated ([interleukin-2 receptor+], IL-2R+) CD8+ T cells. PG27 increased mononuclear cell recovery, and significantly reduced the day-14 percentages of CD3+ and IL-2R+ cells in allogeneic BMT mice, producing results similar to those for syngeneic BMT mice. PG27 significantly increased concanavalin A-stimulated in vitro IL-4 production by day-14 splenocytes, with a 7- to 8-fold higher level than that produced by control cells. CONCLUSIONS: PG27 treatment for only 14 days prevented GVHD induction and development and produced long-term survival. PG27 largely normalized splenic T lymphocyte subsets, reduced allospecific cytotoxic T lymphocyte activity, and increased IL-4 production capability. PG27 may suppress GVHD by the induction of anergy and a deviation away from a proinflammatory phenotype, which could be reflected in the increased potential for IL-4 production.