Protochordate concordant xenotransplantation settings reveal outbreaks of donor cells and divergent life span traits

If fulminate rejection in allogeneic and xenogeneic engraftments is not an evolutionary relict feature, then any treatment that ablates the host surveillance's effector arms capabilities and eliminates graft vs. host reactivity should induce donor chimerism in transplant settings. We demonstrate here marked proliferative response of Botryllus (Urochordata) blood cells months following their infusions (2×10⁴–10⁵ blood cells per host) into the concordant xenogeneic environment of irradiated Botrylloides soma. The state of infused cells was followed by Botryllus specific microsatellite alleles on DNA samples from host zooids and vascular system. Increased growth rates and life spans of engrafted hosts in some cases, and sudden chimerical death following the outbreak of donor cells in others, indicate a ‘double-edged sword’ expression of concurrent evolutionary selected mechanisms. This DES phenomenon in immunity underlies divergent stem cell competition phenomena in multicellular organisms, leading in mammals, to cases of autoimmune diseases vis-à-vis long-lasting microchimerism events following an iatrogenic transplantation.     

巢式序列特异性引物PCR方法检测HLA单倍体相合供受者间母胎微嵌合的临床应用研究

目的探索临床应用巢式序列特异性引物聚合酶链式反应（PCR-SSP）方法检测HLA单倍体相合供受者间母胎微嵌合状态的可行性。方法选取25对拟行HLA单倍体相合造血干细胞移植治疗的实体肿瘤患者及其供者,供受者为母子关系15例,父子关系10例。采集供受者外周血提取基因组DNA,采用巢式PCR-SSP方法检测患者外周血中供者来源的HLA-DRB1位点,并计算母胎微嵌合阳性率。随机选取经巢式PCR-SSP证实嵌合阳性和阴性且供受者性别不同的患者各4例,采用荧光原位杂交（FISH）技术进行重复检测,并与巢式PCR-SSP方法的检测阳性率进行比较,同时采用噻唑蓝法检测这8例患者分别与其亲缘供者和HLA完全不相合无关第3人之间的混合淋巴细胞增殖反应（MLR）,并计算增殖指数（SI）。结果巢式PCR-SSP方法灵敏度可高达0.001%,并具有较好的特异性。巢式PCR-SSP检测显示,在母子移植关系患者中母胎微嵌合阳性检出率为40%（6/15）,而在父子移植关系患者中母胎微嵌合阳性检出率为0。巢式PCR-SSP方法的检测灵敏度与FISH技术相比明显增高,其检测阳性率分别为50%（4/8）和12.5%（1/8）。MLR检测显示,嵌合阳性患者对其亲缘供者和无关第3人外周血单个核细胞（PBMC）的SI分别为（0.949±0.023）、（1.320±0.095）,嵌合阴性患者对其亲缘供者和无关第3人PBMC的SI分别为（1.133±0.036）、（1.245±0.069）;嵌合阳性患者对其亲缘供者PBMC的增殖反应强度与嵌合阴性患者相比明显降低（P=0.001）,并且也显著低于其对无关第3人PBMC的增殖反应强度（P=0.003）。结论巢式PCR-SSP方法灵敏度高、特异性好,适用于临床HLA单倍体相合供受者间母胎微嵌合状态的快速检测。     

Transplantation of hematopoietic stem cells for induction of unresponsiveness to organ allografts

Although it has been recognized since the early days of Owen and Medawar that engraftment of donor stem cells, induced in utero spontaneously or intentionally neonatally, results in life-long unresponsiveness to donor alloantigens. However, successful induction of transplantation tolerance in adult life still represents an unsolved problem. Engraftment of donor stem cells using conventional modalities involves intensive myeloablative or lymphoablative immunosuppression, which is associated with toxicity and mortality and such methods are not suitable for organ allograft recipients. In this chapter, we present an innovative approach for induction of donor-specific unresponsiveness to bone marrow and organ allografts without myeloablative conditioning. Our methods is based on cyclophosphamide-induced, alloantigen-primed lymphocyte depletion. Cyclophosphamide is administered 1 day following infusion of donor hematopoietic cells, thus eliminating predominantly host T lymphocytes reacting against donor cell challenge, and resulting in relative unresponsiveness to donor alloantigens. Subsequently, life-long tolerance to fully mismatched donor skin allografts can be accomplished by a second infusion of stem cells from the same donor, with donor T cells displacing residual alloreactive host cells that may have escaped deletion. Taken together, we believe that induction of true permanent and specific tolerance to organ allografts using donor hematopoietic cells could become a clinical reality in the foreseeable future.     

异基因外周血干细胞移植后淋巴细胞和粒细胞嵌合体的动态改变

目的：探讨异基因外周血干细胞移植(allo-PBSCT)后T细胞、粒细胞嵌合体的动态改变及临床价值。方法：将多重PCR扩增短串联重复序列(STR—PCR)的法医试剂进行条件优化，间隔短时间抽取9例恶性血液病患者(5例清髓性PBSCT，4例非清髓性PBSCT)的外周血样，STR—PCR定量分析T细胞和粒细胞的嵌合体，并观察其对移植后应用免疫抑制剂的指导作用。结果：STR—PCR定量分析嵌合体的敏感性为5％，并具高度可重复性。清髓性PBSCT后10d( 10d)，5／5例患者的粒细胞迅速演变为完全供者嵌合体(CDC)， 14d，4，／5例患者的T细胞获得CDC。非清髓性PBSCT， 7～ 14d，供者T细胞信号的植入速度快于粒细胞；随后，供者粒细胞的比例突然增加，并迅速获得CDC，T细胞的植入却渐缓慢，最后，T细胞取得CDC的时间迟于粒细胞。依据供者T细胞信号的植入程度，及时调整非清髓性PBSCT后环孢素A(CsA)的用量，移植早期T细胞即获CDC，随访2～16个月，T细胞和粒细胞均呈稳定的供者植入状态。结论：供者T细胞的完全植入迟于粒细胞．动态监测T细胞嵌合体，可能有助于免疫抑制剂的调整。     

Chimerism and tetragametic chimerism in humans: implications in autoimmunity, allorecognition and tolerance

The presence of cells or tissues from two individuals, chimeras, or the presence of cells and tissues that include the gonads, tetragametic chimerism can be detected by the analysis of cytogenetics and analysis of polymorphic genetic markers, using patterns of pedigree inheritance. These methodologies include determination of sex chromosomes, major histocompatibility complex (MHC) polymorphisms and panels of short tandem repeats (STRs) that include mitochondrial DNA markers. Studies routinely involve cases of temporal chimerism in blood transfusion, or following allotransplantation to measure the outcome of the organ, lymphopoietic tissues or bone marrow grafts. Demonstration of persistent chimerism is usually discovered in cases of inter-sexuality due to fusion of fraternal twins or in cases of fusion of embryos with demonstrable allogeneic monoclonality of blood which, excluded maternity or paternity when blood alone is used as the source of DNA. In single pregnancies it is possible to produce two kinds of microchimerism: feto-maternal and materno-fetal, but in cases of fraternal twin pregnancies it is possible to identify three different kinds which are related to cases of vanishing twins that can be identified during pregnancy by imaging procedures; (1) hematopoietic, (2) gonadal, and (3) freemartins when the twins have different sex and the individual born is a female with either gonadal or both gonadal and hematopoietic tissues. Fraternal twin pregnancies can also produce fusion of embryos. Such cases could be of different sex presenting with inter-sexuality or in same sex twins. One of such cases, the best studied, showed evidence of chimerism and tetragametism. In this regard, the case was studied because of disputed maternity of two of her three children. All tissues studied, except for the blood, demonstrated four genetic components but only two in her blood of four possible showed allogeneic monoclonality consistent with the interpretation that her blood originated from one hematopoietic stem cell. Also, microchimerism, due to traffic of cells via materno-fetal or feto-maternal has been prompted by reports of their potential association with the development of autoimmune disorders including systemic lupus erythematosus (SLE) and systemic sclerosis, and in allotransplantation. In addition, their relevance of chimerism in the positive and negative selection of T cells in the thymus has not been addressed. T lymphocytes play a central role in controlling the acquired immune response and furthermore serve as crucial effector cells through antigen specific cytotoxic activity and the production of soluble mediators. Central tolerance is established by the repertoire selection of immature T lymphocytes in the thymus, avoiding the generation of autoreactive T cells. Expression of chimeric antigens in the thymus could modify the generation of specific T cell clones in chimeric subjects and these mechanisms could be important in the induction of central tolerance against foreign antigens important in allo-transplantation. In this review, we discuss the genetics of chimerism and tetragametism and its potential role in thymic selection and the relevance in allotransplantation and autoimmune disorders. This review is dedicated to the memory of Robert A. Good, MD, PhD, an outstanding physician and scientist, one discoverer of the functions of the Thymus in immunobiology and the pioneer of human bone marrow allotransplantation. Presented at the First Robert A Good Society Symposium, St. Petersburg, FL 2006.     

Non-myeloablative mixed chimerism approaches and tolerance, a split decision

Stable mixed chimerism has been considered the most robust tolerance strategy. However, rejection of solid donor tissues by chimeras has been observed, a state termed split tolerance. Since new non-myeloablative mixed chimerism approaches are being actively pursued, we sought to determine whether they lead to full tolerance or split tolerance and to define the mechanisms involved. Fully mismatched mixed chimeras generated by induction with various lymphocyte-depleting antibodies along with either low-dose irradiation or busulfan and temporary sirolimus, maintained stable mixed chimerism but nevertheless rejected donor skin grafts. Generation of stable mixed chimerism using antibody targeting CD40L, but not depleting antibodies to CD4 and CD8, could prevent split tolerance when skin grafts were given together with donor bone marrow. Minor antigen matching abrogated the ability of effector T cells to reject donor skin grafts. A CFSE killing assay indicated that chimeras were both directly and indirectly tolerant of donor hematopoietic cell antigens, suggesting that minor mismatches triggered a tissue-specific response. Thus, split tolerance due to tissue-restricted polymorphic antigens prevents full tolerance in a number of non-myeloablative mixed chimerism protocols and a 'tolerizing' agent is required to overcome split tolerance. A model of the requirements for split tolerance is presented.