转hCTLA4Ig树突状细胞诱导T细胞免疫耐受的实验研究

目的　通过逆转录病毒载体将人CTLA4Ig转染DCs ,探讨转人CTLA4Ig(hCTLA4Ig)树突状细胞 (DCsRev)诱导T细胞免疫耐受的可能性。方法　通过重组逆转录病毒将目的基因hCTLA4Ig转染到大鼠骨髓来源的DCs中 ,通过流式细胞检测目的基因hCTLA4Ig表达及DCs表面分子的改变 ;通过混合淋巴细胞反应 (MLR)检测DCsRev抑制T细胞免疫反应的能力。 结果　重组逆转录病毒转染DCs的最大效率为 91 2 5 % ;在功能上 ,DCsRev不但丧失了刺激MLR的能力 ,并且能够强烈抑制MLR中反应T细胞的增殖 ,而且抑制率与加入DCsRev的数量和DCsRev预处理反应T细胞的时间长短有关。具体来说 ,DCsRev数量在 10 3 ～ 10 4之间时 ,抑制率与剂量呈正相关 ,最高为 71 96%。而当DCsRev数量达到 5× 10 4抑制率下降为 5 9 2 %。在 12～ 48h之间 ,随着预处理时间的延长 ,抑制率却不断下降 ,预处理 12h抑制率最高 ,为 99 6%。但不做预处理 ,在反应开始时同时加入DCsRev ,则抑制率明显降低 ,仅为 5 9 2 %。对腹腔注射DCsRev大鼠脾T淋巴细胞体外分析表明 ,DCsRev也能在动物体内诱导耐受 ,但这种免疫耐受状态不能维持终身。结论　通过逆转录病毒载体将人CTLA4Ig转染DCs,不但DCs表面CD86分子被CTLA4Ig有效的封闭 ,并且能够诱导抗原特异性T细胞的免疫耐受     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

腺病毒载体介导CTLA4Ig基因治疗小鼠变应性鼻炎

目的 检测CTLA4Ig-重组腺病毒载体（Ad-CTLA4Ig）在变应性鼻炎治疗中的作用。方法 采用卵清蛋白（OVA）致敏和激发诱导小鼠变应性鼻炎。实验组在OVA激发前30min予以Ad-CTA4Ig腹腔注射。未转CTLA4IgcDNA的腺病毒载体（Adv）作对照。比较各组动物鼻部症状和鼻粘膜形态学改变，并采用ELISA法测定血清中OVA－特异性IgE水平。结果 CTLA4Ig重组腺病毒实验组小鼠鼻部症状和鼻粘膜病理改变不明显，并且血清中OVA－特异性IgE水平明显低于对照组（P＜0.05）。结论 Ad-CTLA4Ig可防治小鼠应变性鼻炎的发生，提示CTLA4Ig-重组腺病毒载体有可能运用于临床变应性鼻炎的治疗。     

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.     

Regulated activity of the IgH intron enhancer (E{micro}) in the T lymphocyte lineage

The activity of the IgH (E_µ) enhancer in the T lymphocytelineage has been investigated using both transgenic mice andtransfection studies. Thymocyte fractionation experiments indicatethat a transgene consisting of the bacterial chloramphenicolacetyl transferase (CAT) gene, linked to Eµ and the SV40early promoter (E_µ–CAT), is expressed only in thymocyteswith a mature medullary phenotype and not in immature cells.Transfection of this same construct into two thymoma cell linesrepresenting different stages of thymocyte development mimicsthe pattern of activity observed in vivo. Further transfectionexperiments suggest that this pattern of expression might beattributed to the differential activity of the E2E3 and octanucleotidemotifs of E_µ during development. In contrast, an Ig transgene(linked to E_µ and an Ig V promoter) is expressed in themajority of thymocytes. We envisage that the different patternsof expression of the two transgenes reflect interactions betweentheir respective promoters and the factors which are bound toE_µ at different stages of thymocyte development. Althoughdiffering in their pattern of expression within the thymus,the two transgenes share the property of extinction in peripheralT lymphocytes. These results indicate that the expression ofE_µ-linked transgenes in the thymus cannot simply be explainedby activation of the enhancer in a lymphoid progenitor cellprior to B/T lineage divergence. Rather, the enhancer (or componentsof it) must be independently activated (and inactivated) duringT lymphocyte development. Furthermore, this activity is consistentwith the developmental timing of Ig D_H–J_H rearrangementsin these cells.     

Signal transduction in human B cells initiated via Ig{beta} ligation

Ig and Igß heterodimers are non-covalently associatedwith Ig to compose the antigen receptor complexes on B cells.The demonstration that different sets of tyrosine kinases bindto the cytoplasmic tails of Ig and Igß suggests thatIg and Igß may activate distinct second messengerpathways. In this study, we examined the effects of mAbs againstan exposed epitope of human Igß on pre-B and B celltriggering. Cross-linkage of Igß on B cells leadsto activation of tyrosine kinases, hydrolysis of phosphatidylinositides,and elevation of intracellular Ca²⁺, effects qualitatively identicalto those of anti-_µ mAbs. Our observations thus indicatethat cross-linking of Igß does not segregate signaltransduction pathways connected with the cytoplasmic talls ofIg and Igß. Ig ligation has been reported to be moreeffective in triggering pre-B than B cells, whereas our resultsindicated that Igß ligation is more efficient in triggeringB than pre-B cells. In addition to their activation properties,the anti-Igß mAbs effectively modulated B cell receptorcomplexes and blocked terminal differentiation of all plasmacell isotypes. The findings support the idea that anti-Igßcould serve as a universal B cell immunosuppressant.     

Apoptosis of ileal Peyer's patch B cells is increased by glucocorticoids or anti-immunoglobulin antibodies

The ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (<5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24h, 60 ± 1 % of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose-dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2–2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 ± 6%, compared with 2.4 ± 0.1 % in untreated lambs. Anti-immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti-Ig (PIg47A), DNA fragmentation was 74 ± 2 % and 75 ± 3 %, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca²⁺, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti-Ig. We speculate that the apoptosis of ileal PP B cells in situ may be modulated by glucocorticoids and by the cross-linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self-reactive B cells during the expansion of the Ig repertoire in the ileal PP.     

Partial restoration of B cell development in Jak-3(-/-) mice achieved by co-expression of IgH and E(mu)-myc transgenes

Jak-3 is a non-receptor tyrosine kinase that plays an important role in coordinating signals received through a wide range of cytokine receptors, including the IL-7 receptor (IL-7R). Jak-3-deficient mice have a profound block in B cell development at the pro-to-pre-B cell transition and have very few peripheral B cells. This block has been postulated to reflect the inability of Jak-3(-/-) pro-B cells to respond to IL-7. Here we demonstrate that B cell development can be partially restored in Jak-3-deficient mice when they are bred to mice carrying both a rearranged Ig heavy chain (IgH/Igmu) transgene and a c-myc transgene expressed in the B cell lineage. Jak-3(-/-) mice expressing both of these transgenes exhibit significant increases in the number of B cells in the bone marrow and, to a lesser extent, in the spleen. However, very few rescued B cells were detectable in mice greater than 4 months of age. To determine whether resident hyperactivated Jak-3(-/-) peripheral T cells are responsible for the elimination of the rescued B cells in older mice, we bred IgH transgenic (Igmu Tg)/myc Tg/Jak-3(-/-) mice to T cell-deficient (TCRalpha(-/-)) mice. Data from these experiments suggest that the paucity of B cells in older Jak-3(-/-) mice is largely attributable to the lack of Jak-3 in the B cells themselves. Thus, Jak-3 seems to play several important roles in B cells: during development, to enable cell division, Ig gene rearrangement and cell differentiation, and in mature cells, to promote B cell survival in the periphery.     

The effect of phenytoin in vitro on normal human mononuclear cells and on human lymphoblastoid B cell lines of different Ig isotype specificities

The anticonvulsant drug phenytoin, in less than cytotoxic concentrations, caused significant reductions in Ig secretion by unstimulated or EBV-stimulated normal MNC, as measured by PFC or secretion of Ig into the culture medium. Isotype-specific LBL varied in their sensitivity, the secretion of IgA (1 line) and IgG (3 lines) being reduced by phenytoin near therapeutic concentrations, whereas that of IgM (1 line) was resistant. Six-day exposure of MNC to phenytoin caused no selective depletion of or enrichment for B cells, monocytes or T cell subsets. The results suggest that the reduction in serum Ig levels reported in phenytoin-treated epileptic patients is, at least in part, due to a direct effect of the drug on the B lymphocyte. However, among EBV-activated normal MNC, those secreting IgA were no more sensitive to the drug than those secreting IgG or IgM, and other factors may, therefore, operate to cause the preferential reduction in serum IgA in phenytoin-treated patients.