Selective immunosuppression with anti-interleukin 2 receptor-targeted therapy: helper and suppressor cell activity in rat recipients of cardiac allografts

(LEW X BN)F1 cardiac allografts are rejected within 8 days in unmodified LEW rats. ART18, a mouse anti-rat IgG1 monoclonal antibody which binds specifically in vitro to the interleukin 2 receptor (IL 2R) molecule expressed primarily on activated T cells, prolongs allograft survival in a dose-dependent fashion to ca. 3 weeks (p less than 0.001) after being administered for 10 days after transplantation. This effect was related to the specificity of the antibody for IL 2R, as therapy with ART62 (a monoclonal antibody recognizing MHC class I antigen but not binding the rat IL 2R) was ineffectual. Suppressor activity was detected in spleen cells of ART18-treated grafted hosts: in vivo, splenic T suppressor/cytotoxic fraction adoptively transferred into normal LEW improved donor-specific but not third-party test graft survival (17 days, vs. 8 days, respectively, p less than 0.001); in vitro, mixed lymphocyte reaction was profoundly but nonspecifically inhibited (less than 5% of test mixed lymphocyte reaction, p less than 0.001 as compared to acutely rejecting controls). In contrast, splenic T helper (Th) cells from ART18-treated hosts were functionally depressed, as noted by their passive transfer into immunologically anergic B recipients of cardiac allografts (rejection in ca. 40 days, vs. ca. 13 days after transfer of Th from specifically sensitized rats). ART18 treatment also resulted in diminished elaboration of IL 2 as compared to normal (p less than 0.005) or acutely rejecting hosts (p less than 0.001); however, a remarkable increase in the production of IL 3 occurred (p less than 0.001). These results demonstrate that IL 2R-targeted therapy of immunocompetent graft recipients produces a selective immune defect in which donor-specific T suppressor cells are spared, but Th cells attenuated or destroyed. Decreased elaboration of IL2 concomitantly augments the release of IL 3, a lymphokine which might play a role in suppressor effect in vivo. In addition, IL 2R-targeted therapy of the immunodeficient graft recipients abrogates the capacity of alloactivated T cells to re-establish acute immune responsiveness.     

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.     

环孢菌素A抑制小鼠同种心脏移植急性排斥反应中Lymphotactin表达的研究

目的：研究小鼠同种心脏移植急性排斥反应中淋巴细胞趋化因子（lptn）的表达情况及环孢菌素A（cyclosporine A, CsA）的抑制作用。 方法： 改良Banff评分系统判断同种小鼠移植心急性排斥反应程度，RT-PCR检测移植心组织内淋巴细胞趋化因子表达水平，ELISA方法检测心脏移植小鼠脾细胞活化T细胞核因子(NFAT)活性。 结果： C57BL/6-Balb/c急性排斥组小鼠移植术3 d后脾脏显著增大。术后第5、7 d移植心肌间淋巴细胞浸润程度评分分别为2.667±0.577和2.333±0.577。C57BL/6-Balb/c+CsA组小鼠移植术后脾脏肿大明显减轻，术后第5、7 d心肌间淋巴细胞浸润程度评分分别为1.000±0.000和1.333±0.577。急性排斥组和CsA处理组小鼠移植心脏在术后第5 d和第7 d都可检测到Lptn mRNA阳性表达，但CsA处理组Lptn mRNA的表达明显弱于急性排斥组。治疗剂量的CsA可以完全抑制NFATc1活性。 结论： Lptn在早期移植免疫事件中具有重要的作用，CsA仅能部分抑制Lptn mRNA的表达。活化T细胞Lptn的表达调控存在NFAT以外的途径。     

Dual effects of the alloresponse by Th1 and Th2 cells on acute and chronic rejection of allotransplants

The contribution of direct and indirect alloresponses by CD4⁺ Th1 and Th2 cells in acute and chronic rejection of allogeneic transplants remains unclear. In the present study, we addressed this question using a transplant model in a single MHC class I‐disparate donor–recipient mouse combination. BALB/c‐dm2 (dm2) mutant mice do not express MHC class I L^d molecules and reject acutely L^d+ skin grafts from BALB/c mice. In contrast, BALB/c hearts placed in dm2 mice are permanently accepted in the absence of chronic allograft vasculopathy. In this model, CD4⁺ T cells are activated following recognition of a donor MHC class I determinant, L^d 61–80, presented by MHC Class II A^d molecules on donor and recipient APC. Pre‐transplantation of recipients with L^d 61–80 peptide emulsified in complete Freund's adjuvant induced a Th1 response, which accelerated the rejection of skin allografts, but it had no effect on cardiac transplants. In contrast, induction of a Th2 response to the same peptide abrogated the CD8⁺ cytotoxic T cells response and markedly delayed the rejection of skin allografts while it induced de novo chronic rejection of heart transplants. This shows that Th2 cells activated via indirect allorecognition can exert dual effects on acute and chronic rejection of allogeneic transplants.     

Telomere shortening and cellular senescence in a model of chronic renal allograft rejection

Cellular senescence has been suggested to play a role in the deterioration of renal graft function and has been linked to telomere shortening. We have investigated markers of cellular senescence in the F344 to LEW rat model of chronic renal transplant rejection. Syngeneic and LEW to F344 transplants were used as controls. Substantial telomere shortening was observed in all transplants, including allogeneic and syngeneic grafts from day 7 post-transplant onwards. Ischemia of native F344 kidneys was already sufficient to induce telomere shortening. It is known that shortened telomeres can activate cell cycle regulators, such as p21 and p16. Accordingly, all cases showed a transient p21 increase, with a maximum at day 7 and a sustained expression of p16. Importantly, senescence-associated beta-galactosidase staining, a cytological marker for senescence, was only observed in tubular epithelial cells of chronically rejecting F344 allografts from day 30 post-transplantation onwards. Long-term surviving LEW allografts or syngeneic F344 grafts were negative for senescence-associated beta-galactosidase. In conclusion, ischemia during transplantation results in telomere shortening and subsequent activation of p21 and p16, whereas senescence-associated beta-galactosidase staining is only present in chronically rejecting kidney grafts.     

Beneficial effect of amrinone on murine cardiac allograft survival.

Amrinone is a non-glycoside positive inotropic agent with an inhibitory effect on a cyclic adenosine monophosphate (AMP) phosphodiesterase isoenzyme. In the present study, we examined the immunosuppressive action of amrinone, since several other cyclic AMP-elevating agents have been shown to suppress T lymphocyte activation. First, the in vivo effects of amrinone were investigated. Oral amrinone treatment, at 40 mg/kg per day, significantly prolonged median cardiac allograft survival compared with non-treated controls (22.0 days versus 10.5 days, P < 0.01) when DBA/2 mouse hearts (H-2d) were heterotopically transplanted into C57B1/6 mice (H-2b). Histopathological examination showed that there was less prominent cellular infiltration in the amrinone-treated than in the non-treated allografts. Plasma amrinone concentrations of mice after a single oral dose of 40 mg/kg were within the range of clinical relevance. To clarify the mechanism of action, in vitro studies were done. The generation of specific cytotoxic T lymphocytes after mixed lymphocyte culture was significantly suppressed by addition of amrinone to the culture medium at 5 micrograms/ml. The production of IL-2 and the interferon-gamma during mixed lymphocyte culture was also suppressed by amrinone at 5 micrograms/ml. However, the level of intracellular cyclic AMP in mouse splenic lymphocytes was not affected significantly by the same dose of amrinone. In conclusion, amrinone has immunosuppressive actions at the therapeutic doses, and it may be a beneficial agent for therapy against acute cardiac allograft rejection.     

Role of RANTES in experimental cardiac allograft rejection

The host response to alloantigen results in upregulation of Class II MHC antigens and associated cytokine production (especially IL-2 and interferon-gamma (IFN-gamma)) as well as lymphocytic infiltration and cellular activation which leads to graft damage/destruction. RANTES (Regulated upon Activation, Normal T-cell Expressed and presumably Secreted) is a member of the beta subfamily (CC) of chemokines and has been shown to function as a lymphocyte chemoattractant. We now describe the requirement for RANTES in cardiac heterotopic allograft (brown Norway into Lewis rats) rejection in rats. By Northern blot analysis, mRNA could be detected in allografts at 6 and 8 days post-transplantation but not in isogenic (Lewis) grafts. RANTES protein could be demonstrated by Western blot analysis in homogenates from allografts at day 8 but not at day 0 and could not be identified in isogenic cardiac transplants. By immunostaining, RANTES protein was present in mononuclear cells of allografts at day 6 but was absent in the isogenic transplants. When rats were treated with anti-RANTES serum, there was a significant delay in rejection time (cessation of beating) of the allografts. These data demonstrate that expression of RANTES in rat cardiac allografts is linked to the rejection phenomenon.     

卵巢癌患者一线化疗后Tc1/Tc2亚群漂移及免疫应答功能动态变化的研究

目的 研究晚期卵巢癌患者经一线化疗后体内Tc1/Tc2免疫细胞亚群漂移及免疫应答功能的动态变化,以期明确化疗后机体是否存在抗肿瘤免疫抑制暂时逆转的"窗口期".方法 选取术后行紫杉醇联合卡铂化疗的晚期卵巢上皮癌患者共13例为患者组,健康女性13例作为对照组.采集化疗前(S_0)、化疗后5～7 d(S_1)、化疗后12～14 d(S_2)和化疗后25～28 d(S_3)外周血,流式细胞术检测患者外周血中Tc1/Tc2免疫细胞亚群的动态变化,同时采用体外自体肿瘤细胞裂解物脉冲淋巴细胞共培养的方法检测不同时间点CD8~+细胞IFN-γ的分泌功能.结果 晚期卵巢癌患者化疗前Tc1比例、Tc1/Te2细胞比值较对照组显著降低,而Tc2比例则显著升高.化疗后Tc1细胞比例在S_2期较S_0期显著增高,Tc2细胞比例在化疗后无显著变化,Tc1/Tc2细胞比值在S_2期显著增高.当体外给予自身肿瘤细胞裂解物刺激诱导培养后,分泌IFN-γ的CD8~+细胞比例在S_2期达到最高.结论 卵巢癌患者于一线化疗后机体Tc1/Tc2亚群动态漂移,其细胞比值在S_2期显著升高;S_2期亦是CD8~+细胞免疫应答达到最高峰的时间点.化疗有可能通过机体免疫重建诱生了增强的抗肿瘤免疫应答,从而使机体抗肿瘤免疫抑制得到暂时逆转.免疫重建期可能是免疫治疗实施的最佳窗口期.     

Lymphotactin: a key regulator of lymphocyte trafficking during acute graft rejection.

The attraction of leucocytes to allografts is essential for rejection. The process is controlled by chemokines. In order to clarify the role of lymphotactin (a cytokine that represents a novel branch of the chemokine superfamily) in regulating leucocyte trafficking during graft rejection, we used rat renal transplantation models to examine its gene expression and the distribution of lymphotactin-expressing cells in renal grafts. Lymphotactin mRNA was upregulated strongly in acutely rejecting renal allografts. The mRNA was undetectable in isografts, chronically rejecting renal allografts or normal kidney. Once lymphotactin was expressed, large numbers of infiltrating lymphocytes were seen. Moreover extended studies demonstrated that in cultured rat spleen cells the expression of lymphotactin mRNA was markedly induced by phytohaemagglutinin (PHA) or phorbol myristate acetate (PMA), and such induction was inhibited by the immunosuppressive drugs FK506 and cyclosporin. Collectively, these observations provide new evidence demonstrating that lymphotactin is a key regulator of lymphocyte motility and adhesiveness during acute allograft rejection. FK506 and cyclosporin inhibition of lymphotactin expression is likely to represent an important molecular mechanism of the action of the drugs.     

Local immunosuppressive therapy with monoclonal anti-T cell antibody on renal allograft survival in the rat.

Considerable interest in the experimental and clinical use of MoAbs as potential therapeutic agents in allograft rejection has been generated by the recent reports of striking prolongation. In this study we investigated the efficacy of the local administration of MoAb OX-19 which is directed to the rat CD5 equivalent, through the renal artery using a rat kidney transplant model, in order to develop a potent method for modifying rejection while minimizing the systemic side effects. Untreated Lewis rats (LEW, RT-1(1)) rejected Brown-Norway rat (BN, RT-1n) kidney at 7.8 +/- 0.2 days (n = 10). Mean survival time (MST) of recipients treated with OX-19 (75 micrograms/kg per day) as single bolus injections via the dorsal penile vein for 7 days was 7.0 +/- 0.2 days (n = 5, NS). LEW hosts receiving OX-19 (75 micrograms/kg per day) continuously for 7 days via a femoral vein by using an osmotic minipump (IV-treated group) showed a slight prolongation of graft survival (MST = 8.8 +/- 0.9 days, n = 5), but this was not statistically significant. On the other hand, local continuous intrarenal arterial infusion of OX-19 (75 micrograms/kg per day) for 7 days (RA-treated group) significantly prolonged the graft survivals (MST = 16.8 +/- 1.3 days, n = 8, P < 0.01). Histological examination of MoAb-treated LEW hosts on day 6 post-grafting revealed that kidney grafts from RA-treated hosts showed a slight tubular necrosis, but reduced mononuclear cell infiltration, whereas kidney grafts from IV-treated hosts displayed a severe mononuclear cell infiltration around the artery with interstitial oedema. Moreover, the local intrarenal administration of OX-19, even when the dose is delayed until day 4 after renal grafting, has a therapeutic effect for on-going acute allograft rejection (MST = 11.4 +/- 0.8 days, n = 8) compared with administration of OX-19 intravenously from day 4 after grafting (MST = 7.6 +/- 0.2 days, n = 5, P < 0.01) or with no treatment (MST = 7.8 +/- 0.2 days, P < 0.01). The phenotype of graft infiltrating cells (GIC) was investigated on day 6 post-grafting. There was a significantly lower percentage of cells positive for OX-19, OX-8, OX-26 (transferrin receptor), and OX-39 (IL-2 receptor) in the RA group than in the IV group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autoantibodies to human melanocyte-specific protein Pmel17 in the sera of vitiligo patients: a sensitive and quantitative radioimmunoassay (RIA)

Lethally irradiated LEW rats reconstituted with syngeneic bone marrow and given CsA for a 4-week period develop a graft-versus-host-like disease upon withdrawal of CsA. This T cell-mediated autoimmune disease is referred to as CsA-induced autoimmunity (CsA-AI). CsA-AI-susceptible LEW rats and resistant BN rats differ greatly in the composition of their peripheral T cell compartment. To dissect the role of MHC and non-MHC genes in the development of peripheral T cell subsets in combination with susceptibility to CsA-AI the respective MHC congenic strains (LEW-1N and BN-1L) were examined for their T cell subsets and for their ability to develop CsA-AI. In this study we show that the Th1/Th2-like cell ratio as well as susceptibility to CsA-AI are under control of the non-MHC genes. This suggests that the Th1/Th2-like cell ratio is a critical determinant for development of CsA-AI. Alternatively, resistance can be attributed to lack of target organ susceptibility due to the absence of the target autoantigen in resistant rat strains. This interpretation is rejected, since both BN as well as BN-1L rats consistently develop the characteristic macroscopic and microscopic signs of CsA-AI upon adoptive transfer with autoreactive LEW-1N and LEW T cells, respectively. Therefore, it can be concluded that the non-MHC genes encode for immune deviation and thereby determine susceptibility or resistance to CsA-AI.     

Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Expression of allograft endothelial VCAM-1 can be manipulated with antagonist of IFN-alpha or IL-4 and is not required for allograft rejection.

This report provides evidence to support the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and IL-4 promote the expression of new endothelial surface molecules in rejecting murine heterotopic cardiac allografts. The microvascular endothelia of these cardiac allografts all develop strong reactivity with the monoclonal antibodies (mAbs) YN1.1/74 (anti-ICAM-1), M/K-2 (anti-VCAM-1) and MECA-32 (undefined molecule) within 3 to 5 days of graft implantation. Daily treatment of the allograft recipients with pentoxifylline (PTX), soluble TNF receptor (TNFR:Fc), anti-interleukin-4 (IL-4) mAb (11B11), or soluble IL-4 receptor, each abrogate the expression of endothelial VCAM-1 and reduce the endothelial reactivity with the mAbs YN1.1/74 and MECA-32 to levels found in cardiac isografts. This is accompanied by a reduction, but not an elimination, of interstitial leukocytic infiltration. Despite this, cardiac allograft recipients treated with PTX or the mAb 11B11 rejected allografts at the same rate as untreated allograft recipients, ie, within 10 to 12 days after graft implantation. These rejected grafts contained mRNAs for TNF-alpha and IL-4, as well as for all other cytokines that have been associated with rejecting allografts. This suggests that endothelial VCAM-1 expression, which is characteristic of rejection, is not an essential element of the rejection process. Interestingly, the grafts from the PTX-treated recipients continued to display rare, isolated VCAM-1 positive cells in the interstitium, which may be dendritic cells. In general, these studies demonstrate a role for IL-4 and TNF-alpha in the alterations of vascular endothelial phenotype observed in rejecting cardiac allografts. They also demonstrate that endothelial VCAM-1 expression is not essential for the allograft rejection process, and that the role of VCAM-1 in this process may be more subtle than was initially suspected.     

Homozygosity at the major histocompatibility complex is required for optimal immunogenicity of bone marrow cell allografts in irradiated rats

Hemopoietic histocompatibility ( Hh ) Genes associated with the H-2 region control the antigenicity of hemopoietic cell grafts in the mouse. We have tested for similar genes in rats. Wistar Furth (WF, RTI ) or Lewis (LEW RTI ¹) bone marrow cell grafts did not proliferate in spleens of lethally irradiated (WFxLEW) Fl hybrid rats as assessed by measuring the incorporation of 5-iodo-2'deoxyuridine—¹²⁵I (IUdR) into recipient spleens 5 days after transplantation. In contrast, (WFxLEW)Fl hybrid marrow cells grew well in both WF and LEW parental strain hosts. (WFxDA)Fl or (WFxLEW)Fl hybrid rats were backcrossed to WF parental strain rats to produce progeny, either homozygous, or heterozygous for the MHC. The RTl type of 46 individual backcross progeny was determined using a 5 day mixed-lymphocyte reaction (MLR). Correlation between RTl type and growth of marrow grafts of individual backcross rats was determined bt using each rat as a bone marrow donor for irradiated LEW hosts. Marrow grafts from rats heterozygous for RTl were accepted in all 25 cases, whereas, grafts from 19 of 21 homozygous donors were rejected by the LEW hosts. Thus, homozygosity, for Hh determinants in or near the RTl region appears to be necessary for optimal immunogenicity of bone marrow allografts.     

Type 1 CD8^＋ T Cells are Superior to Type 2 CD8^＋ T Cells in Tumor Immunotherapy due to Their Efficient Cytotoxicity, Prolonged Survival and Type 1 Immune Modulation

CD8^＋ cytotoxic T （Tc） cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8^＋ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8^＋ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin （OVA）-pulsed dendritic cell （DCovA）-activated CD8^＋ type 1 Tc （Tcl） cells secreting IFN-T, and CD8^＋ type 2 Tc （Tc2） cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor （TCR） transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tcl and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8^＋ Tc2, Tcl cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8^＋ Tcl and Tc2 cells skewed the phenotype of CD4^＋ T cells toward Thl and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8^＋ Tcl and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8^＋ T cells for adoptive T cell therapy of cancer.     

尖锐湿疣患者外周血Thl/Th2及Tc1/Tc2亚群的检测及意义

目的 探讨辅助性T淋巴细胞(Th细胞)和细胞毒性T淋巴细胞(Tc细胞)的极化情况在尖锐湿疣(CA)发病机理中的作用及其与疾病复发的关系.方法 采用三色荧光抗体染色流式细胞术检测细胞内细胞因子的方法 ,检测30例CA患者和20名健康对照者外周血CD3+CD8-/IFN-γ+(Th1)、CD3+CD8-/IL4+(Th2)、CD3+CD8+/IFN-γ+(Tc1)和CD3+CD8+,IL-4+(Tc2)细胞比例.结果 与健康对照组比较,CA患者外周血中Thl细胞含量显著减少(P<0.01),Tc1细胞含量、Th1/Th2比值、Tc1/Tc2比值均减少(P<0.05);其中15例复发CA患者的Th1/Th2比值较正常对照显著减少(P<0.01),而Te1/Te2比值与健康对照组比较差异无统计学意义.结论 CA患者外周血Th1和Tc1细胞数减少,而Th2和Tc2细胞相对占优势,复发的CA患者这种失衡更趋严重.CA患者机体免疫状态可能存在Th1→Th2、Tc1→Tc2方向的漂移,这种漂移可能是人乳头瘤病毒(HPV)不易被有效清除或CA反复发生的一种免疫机制.     

CD4+ Th1 cells promote CD8+ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

The cooperative role of CD4⁺ helper T (Th) cells has been reported for CD8⁺ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4⁺ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8⁺ T cell proliferation and activation. In this study, we used CD4⁺ Th1 and CD8⁺ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4⁺ Th1 cell''s help effects on active CD8⁺ Tc1 cells and the molecular mechanisms involved in CD8⁺ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4⁺ Th1 cells with acquired pMHC I by OVA-pulsed DC (DC_OVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8⁺ Tc1 cells in vitro, and promoting CD8⁺ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8⁺ Tc1 with CD4⁺ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4⁺ Th1’s help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8⁺ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.     

人同种异体抗原诱导的T细胞克隆在体外的建立及鉴定

目的：在体外建立长期培养的人T细胞克隆。方法：以^60Co照射的无关PBMC作为饲养细胞，在条件培养液中以有限稀释法对经单向混合淋巴细胞反应活化的T细胞进行克隆化。用免疫荧光染色和流式细胞术分析鉴定所获克隆。结果：获得的16株克隆中，除1株外，均为αβT细胞。15株αβT细胞克隆中，有9株为Th(3株Th0、6株Th2)，6株为Tc(5株Tc0，1株Tc2)。结论：建立了15株稳定的T细胞克隆并进行了鉴定，为Tc1和Tc2亚群标记和功能的研究打下了基础。     

The regulatory T cell effector molecule fibrinogen‐like protein 2 is necessary for the development of rapamycin‐induced tolerance to fully MHC‐mismatched murine cardiac allografts

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long‐term immunosuppression. To investigate mechanisms of rapamycin‐induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC‐mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4⁺ and CD8⁺ immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin‐induced, donor‐specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen‐like protein 2 (FGL2). Depletion of Treg cells with anti‐CD25 (PC61) and treatment with anti‐FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co‐expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual‐staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg‐associated genes (foxp3, lag3, tgf‐β and fgl2) had increased expression and pro‐inflammatory genes (ifn‐γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin‐induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.