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。     

Impact of fetal-maternal tolerance in hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is known to cure various hematological disorders; however, its widespread use is limited due to a lack of histocompatible donors. Reciprocal cell traffic between the mother and fetus during pregnancy gives rise to postpartum fetal-maternal lymphohematopoietic microchimerism, which is frequently detected in the blood or tissue of healthy individuals. Studies in clinical and experimental transplantation provide evidence that exposure to non-inherited maternal antigens (NIMAs) during pregnancy may result in long-lasting fetomaternal microchimerism and tolerance induction. Studies of HLA-mismatched HSCT have suggested a relatively lower incidence of severe graft-versus-host disease (GVHD) after transplantation from a NIMA-mismatched donor. Studies using a mouse model have also demonstrated a “child-to-mother” bone marrow transplantation from an NIMA-exposed donor to reduce the morbidity and mortality of GVHD in an antigen-specific manner while preserving the graft-versus-leukemia effects and favoring the immune reconstitution, thus resulting in a marked improvement in outcome after HSCT. Prospective clinical studies are therefore warranted to confirm these beneficial effects of fetal-maternal tolerance in allogeneic HSCT.     

Possible roles and determinants of microchimerism in autoimmune and other disorders

Microchimerism is the presence of a low level of non-host stem cells or their progeny in an individual. The most common source of microchimerism is pregnancy. During pregnancy, bi-directional trafficking of hematopoietic cells occurs through the placenta and these microchimeric cells persist for decades after childbirth. A possible role of microchimerism in the pathogenesis of some (systemic sclerosis, systemic lupus erythematosus, primary biliary cirrhosis, autoimmune thyroid diseases and juvenile myositis) but not all autoimmune diseases has been suggested by recent studies. Contradictory reports exist regarding HLA allelic associations with persistent T lymphocyte microchimerism. Although much of the focus of past studies has been on microchimerism in the effector arm of the immune system, increasing evidence suggests that microchimeric cells may differentiate into many lineages in different tissues raising additional possible roles for these cells. The possibility of microchimerism in many organs should induce an exploration of how persistent mixtures of cells of different genetic backgrounds throughout the body may influence diverse physiologic processes during life. In the present review, we discuss possible influencing factors and roles of all forms of microchimerism in autoimmune and non-autoimmune diseases. A better understanding of the immune mechanisms, along with the identification of environmental and genetic risk factors, is crucial for further deciphering the many possible implications of maternal-fetal and fetal-maternal cell trafficking in health and disease.     

Fetal microchimerism and autoimmune disease

Microchimerism is defined by the presence of circulating cells, bi-directionally transferred from one genetically distinct individual to another. The acquisition and persistence of fetal cell microchimerism, small numbers of genetically disparate cells from the fetus in the mother, is now a well-recognized consequence of normal pregnancy. Some of the autoimmune diseases that show a predilection for women in their child-bearing years and beyond are linked to fetal microchimerism from previous pregnancies. Microchimerism has been investigated in different autoimmune disorders, such as systemic sclerosis, systemic lupus erythematosus, autoimmune thyroid diseases, and primary biliary cirrhosis. Recent data have demonstrated the promising role of microchimeric cells in the maternal response to tissue injuries by differentiating into many lineages. Therefore, further understanding of fetal-maternal microchimerism may help in anticipating its implications in disease as well as in more general women's health issues.     

Communication between human mast cells and CD4+ T cells through antigen‐dependent interactions

Mast cells (MCs) are immune cells residing in tissues where pathogens are first encountered. It has been indicated that MCs might also be involved in setting the outcome of T‐cell responses. However, little is known about the capacity of human MCs to express MHC class II and/or to capture and present antigens to CD4⁺ T cells. To study the T‐cell stimulatory potential of human MCs, CD34⁺ stem cell derived MCs were generated. These cells expressed HLA‐DR when stimulated with IFN‐γ, and, importantly, presented peptide and protein for activation of antigen‐specific CD4⁺ T cells. The interplay between MC and T cell led to increased HLA‐DR expression on MCs. MCs were present in close proximity to T cells in tonsil and expressed HLA‐DR and CD80, indicating their ability to present antigens to CD4⁺ T cells in T‐cell areas of human LNs. Our data show that MCs can present native antigens to human CD4⁺ T cells and that HLA‐DR expressing MCs are present in tonsil tissue, indicating that human MCs can directly activate T cells and provide a rationale to study the potential of MCs to prime and/or skew human T‐cell responses.     

Human leucocyte antigen‐defined microchimerism early post‐transplant does not predict for stable lung allograft function

Microchimerism is the presence of foreign cells in an individual below 1% of total cells, which can occur in the setting of solid organ transplantation. This study quantitated donor‐derived cellular subsets longitudinally in human leucocyte antigen (HLA)‐mismatched lung transplant recipients (LTR) during the first post‐operative year and evaluated the pattern of peripheral microchimerism with clinical outcomes. Peripheral blood mononuclear cells (PBMC) isolated from non‐HLA‐B44 LTR who received HLA‐B44 allografts were sorted flow cytometrically into three cellular subsets. Real‐time quantitative polymerase chain reaction (q–PCR) demonstrated that donor‐derived HLA‐B44 microchimerism is a common phenomenon, observed in 61% of patients. The level of donor‐derived cells varied across time and between LTR with frequencies of 38% in the B cells/monocytes subset, 56% in the T/NK cells subset and 11% in the dendritic cells (DC) subset. Observations highlighted that microchimerism was not necessarily associated with favourable clinical outcomes in the first year post‐lung transplantation.     

In situ characterization of the cell-surface antigens of the mononuclear cell infiltrate and bile duct epithelium in primary biliary cirrhosis

To analyze the tissue distribution of mononuclear cells and HLA antigens in primary biliary cirrhosis, we studied liver biopsies of 12 patients at different stages of the disease, using the avidin-biotin-peroxidase technique and monoclonal antibodies directed against T and B lymphocytes, T-cell subsets, macrophages, NK/K cells, dendritic cells, and HLA class I and II antigens. To evaluate the proportion of activated T cells we used anti-interleukin-2-receptor antibodies and a double-staining technique for T cells and class II HLA antigens. In all biopsies activated T cells predominated in the portal areas and around the damaged bile ducts. T4 cells almost always outnumbered T8 cells. While B cells, NK/K cells, and dendritic cells were always scarce, macrophages constituted about 30% of the cellular infiltrate. Biliary epithelium, which normally expresses HLA class I antigens, displayed mainly HLA class II antigens. The predominance of T4 cells around the bile ducts, which express class II antigens, suggests that class II-restricted T4 lymphocytes may mediate liver damage in primary biliary cirrhosis.     

Proteogenomic discovery of cancer antigens: Neoantigens and beyond

Host T cells infiltrate the cancer lesion and contribute to patient survival. T cells recognize antigen peptides displayed by the cancer cell human leukocyte antigen (HLA) system. Cancer antigens constitute an essential element of T‐cell discrimination and play an indispensable role in anti‐cancer responses. HLA ligandome analysis directly and comprehensively detects the peptides that are naturally presented by HLA of given cells, leading to discovery of cancer antigens. A proteogenomic approach, which combines conventional proteomics with genomic information, has further deciphered the landscape of the cancer HLA ligandome. Neoantigens that arise from somatic mutations are arguably the major type of peptides patient T cells recognize. Moreover, cancer cells present peptides derived from alleged noncoding regions, which also elicit T‐cell responses thereby serving as cancer antigens. The diversity of newly discovered antigen sources implies that T cells are capable of sensing a variety of genomic aberrations in cancer.     

Detection of microchimerism by minor histocompatibility antigen HA-1 allele-specific nested polymerase chain reaction.

Minor histocompatibility antigens (mHags) can induce T-cell reactivities with important consequences for the graft-versus-leukemia effect and the development of graft-versus-host disease in HLA-matched stem cell transplantation settings. Recently, mHag-specific T cells were also demonstrated in multiparous woman and in solid organ transplant recipients. Microchimeric cells have been detected in the latter settings. To study whether microchimerism is instrumental in the induction and/or maintenance of mHag T cells, we developed an HA-1 allele-specific nested polymerase chain reaction. To optimize and validate the reliability of this method at different levels of microchimerism, serial dilutions of HA-1(H) cells titrated into HA-1(R) cells were tested. We demonstrated that the HA-1(H) allele can be reliably and consistently detected at concentrations as low as 1:10(5) without losing specificity. The developed HA-1-specific nested polymerase chain reaction is an important tool that facilitates the detection of HA-1 microchimerism in various clinical specimens and that promotes investigation of the effects of microchimerism on induction of mHag-specific T cells in the various settings of immunization.     

Expression of HLA class I/II antigens and T cell immune response in human neuroendocrine tumors of the pancreas

Peptide presentation by HLA class I and II antigens regulates specific antigen recognition by T cells. The present study aimed to investigate T cell infiltration and its relation to HLA antigen expression in pancreatic neuroendocrine tumors. Fresh tissue samples were collected from five insulinomas and six other neuroendocrine tumors (one gastrinoma, one glucagonoma, two carcinoid, and two neuroendocrine carcinomas). Normal pancreatic and splenic tissue samples were used as controls. Investigation of infiltrating lymphocyte populations, as well as staining of HLA class I and II antigens, were performed by standard immunohistochemistry. The majority of investigated tumors demonstrated an intratumoral infiltration by CD3+, CD4+ and CD8+ T cells that was significantly higher than in normal pancreatic islets. Only a minority of tumor-infiltrating T cells showed the CD45RO+ phenotype. The expression of HLA class I antigen was altered in 10 of 11 tumors. A loss of beta-2microglobulin represented the most frequent type of alteration to HLA class I expression, although the total loss of HLA class I was found in only one case of neuroendocrine carcinoma. HLA class II molecules were expressed by endothelial and lymphoid cells and not by tumor cells. In conclusion most neuroendocrine pancreatic tumors induce a T cell mediated immune response resulting in an intratumoral infiltration with CD3+, CD4+ and CD8+ T cells. Loss of beta-2microglobulin is a frequent alteration in these tumors, which may influence the normal function of the HLA class I antigen complex. In contrast to malignant tumors of the exocrine pancreas, expression of HLA class II was absent in neuendocrine pancreatic tumor cells.     

Distribution of HLA class 1 antigens in normal human tissue and in mammary cancer.

With a monoclonal antibody which reacts with all HLA class 1 antigens it was found that these antigens are not uniformly distributed in all nucleated cells. Rather HLA class 1 antigens are restricted in their distribution to lymphoid cells, endothelial cells of small vessels, and certain epithelia including mammary duct cells. These antigens were not detected on hepatocytes, specialised cells of the central nervous system, or on the tumour cells of 8 out of 17 human mammary cancers. Given the hypothesis that T cells only respond to foreign antigens on cells which share a common major histocompatibility antigen, these results imply that the T cell responses to viral infections of hepatocytes--for example, hepatitis B virus and the CNS--for example, subacute sclerosing encephalitis, are mediated through an antigen system other than HLA class 1. The absence of HLA class 1 antigen on many mammary cancer cells may be of prognostic significance if T cell modulation of tumour growth is mediated through this class of antigens.     

HLA-A匹配的人胃癌细胞系诱生胃癌特异性T淋巴细胞

目的 用ＨＬＡ－Ａ匹配的人胃癌细胞系诱导对胃癌细胞具有特异性杀伤作用的Ｔ淋巴细胞。方法 分离胃癌患者及正常人外周血淋巴细胞（ＰＢＬ）,用ＨＬＡⅠ类型别已知、经照射的异体胃癌细胞系刺激,进行筛选性的混合淋巴细胞肿瘤细胞培养（ＭＬＴＣ）,初步获得ＨＬＡ－Ａ可能匹配的淋巴细胞供体,血清法测定其ＨＬＡ－Ａ、Ｂ分子确认匹配后,进一步进行ＭＬＴＣ,＾３Ｈ掺入法评估其增殖能力,直接免疫荧光法分析其细胞表型,＾５     

Antigen-Presenting Cells in Human Decidual Tissue

ABSTRACT: The responses of peripheral blood human T lymphocytes supported by decidual antigen-presenting cells (DAPCs) to a variety of immunogenic stimuli were studied and compared to those of T cells supported by peripheral blood antigen-presenting cells (PAPCs). Antigen-presenting cells were isolated from early normal decidual tissue or peripheral blood by elution with ethylenediamine tetraacetic acid of cells that after Ficoll-Paque separation bear receptors for and have bound to fibronectin. DAPCs pulsed with soluble or particulate antigens induced proliferation of T cells with an efficiency equivalent to PAPCs. Decidual tissue APCs also showed the ability to stimulate auto- and alloreactivity. Treatment with anti-human lymphocyte antigen (HLA) class II antibody and ultraviolet radiation resulted in substantial inhibition of the accessory cell function of DAPCs as well as of PAPCs. Bromodeoxyuridine and light treatment of alloreactive T cells generated in vitro was used to demonstrate that DAPCs primed with a synthetic polypeptide antigen (T,G)-A-L can stimulate only HLA class II-compatible T lymphocytes.     

Divergent effects of thyroid HLA-antigens on T and natural killer (NK) cell cytotoxicity.

We have used non-autoimmune non-neoplastic human thyroid cells to explore the role of surface class I and DR antigens on these cells' sensitivity towards T and Natural Killer (NK) cell cytotoxicity. Non-treated thyrocytes expressed class I but no DR antigens. Following incubation with gamma-interferon (gamma-IFN) class I antigens were markedly elevated and DR expression was induced. Whereas non-treated thyrocytes were minimally lysed by sensitized T cells, they served as appropriate targets for NK cells. Following incubation with gamma-IFN, the thyroid cells became highly sensitive to T cell lysis, with no significant reduction in their vulnerability to NK cell killing. The addition of monoclonal anti class I or DR antigens, or brief acid treatment which specifically eliminates class I molecules, inhibited T cell cytotoxicity but enhanced the sensitivity to lysis by NK cells. Thus, the presence of HLA antigens on the same thyroid cells have an opposite effect on two major cytotoxi mechanisms. Our findings are relevant within the context of recent suggestions of intervening with target HLA antigens for the management of autoimmune and malignant diseases.     

Expression of class I and class II markers on populations of leukemic cells

The study of class I and class II antigen expression on leukemic cells brought the following conclusions: most of the leukemic cells show a slower number of class I antigenic sites than normal peripheral blood lymphocytes (PBL) but, in most cases, this does not hinder HLA typing; contrarily to normal PBL, leukemic cells seem to carry "non HLA" antigens (and/or non classical HLA antigens) which are probably responsible of the false positive reactions frequently observed at the time of HLA typing; most of the leukemic cell types express DR antigens (except those belonging to the T lineage) but DQ antigen expression (and in some cases MT antigen expression) varies depending on the cell type studied: well defined on mature B hemopathies, DQ expression is often lower than DR expression on acute leukemic cell types.     

Interleukin 2 production by alloantigen-stimulated CD4+ and CD8+ human T cell subsets: frequency of HLA class I or class II-reactive precursor cells and clonal specificity of activated T cells

A recently developed limiting dilution (LD) method was used to analyze the frequency and specificity of IL2-producing cells within alloantigen-stimulated human CD4+ and CD8+ T cell subsets. Cell sorter-separated CD4+ and CD8+ responder cells were cocultured under LD conditions with HLA class I and/or class II different Epstein Barr virus (EBV)-transformed lymphoblastoid cells line (LCL) stimulator cells in the absence of additional factors. After 3 days, IL2 in cell-free culture supernatants was measured by a colorimetric assay on IL2-dependent murine CTLL cells. Under these conditions, one out of 200-500 CD4+ and one out of 300 to 1000 C8+ T cells produced IL2 when stimulated by HLA class I and class II disparate LCL. By using selected responder and stimulator cells differing only in HLA class I (A, B, C) or class II (DR) antigens, it was found that CD4+ T cells produced IL2 in response to HLA class II antigens, while CD8+ T cells produced IL2 in response to HLA class I antigens. Surprisingly, high frequencies of IL2-secreting CD4+ T cells were noted in certain HLA-DR-identical responder-stimulator combinations. To investigate whether HLA class II antigens other than DR (i.e., DQ or DP) activate CD4+ cells to IL2 secretion, we analyzed a set of HLA-A,B,C and -DR,DQ-identical responder-stimulator cells which differed only in DP antigens. In several of these instances, we measured high frequencies (f = 1/1000 to 1/2000) of HLA-DP-reactive CD4+ IL2 producers, while the frequencies in LD cultures stimulated with autologous LCL were low (f = 1/10,000 to 1/30,000). The specificity of alloantigen-activated IL2-secreting T cells was assayed by restimulation with the original or HLA-mismatched third-party LCLs. CD4+ responder cells could be efficiently and specifically restimulated to IL2 production after a resting period of 3 to 4 days, while CD8+ cells were refractory to restimulation under these conditions. Together these data demonstrate that: 1) CD4+ and CD8+ cells are stimulated to IL2 production by HLA class II and class I antigens, respectively; 2) alloantigen-activated CD4+ IL2 producers are highly specific for stimulating HLA antigens as shown by a split culture and restimulation approach; and 3) significant numbers of CD4+ IL2-producing T cells can be activated by selected HLA-DR-identical, DP-different stimulator cells.     

Microchimerism in Human Health and Disease

During pregnancy some cells traffic between the fetus and the mother. Recent investigative work indicates a low level of fetal cells commonly persists in the maternal circulation for years, or even indefinitely, after pregnancy has been completed. The term microchimerism refers to one individual harboring DNA or cells at a low level that derive from another individual. Chronic graft-versus-host disease (cGvHD) shares similarities with some autoimmune diseases and is an iatrogenic form of chimerism, occurring as a complication of hematopoietic stem cell transplantation. The HLA genes of the donor and the host are known to be of central importance to the development of cGvHD. When also considered in light of the female predilection to autoimmunity, these series of observations led to the hypothesis that microchimerism and HLA genes of host and non-host cells are involved in some autoimmune diseases. The hypothesis can also apply to men, children, and women who have not been pregnant because there are other sources of microchimerism. Persistent microchimerism can follow a blood transfusion, or can occur from transfer between twins in utereo. Additionally, maternal cells have recently been found to persist in her immune competent progeny. A number of studies have investigated a potential role of microchimerism in human diseases including systemic sclerosis (SSc), primary biliary cirrhosis (PBC), Sjögren's syndrome, polymorphic eruption of pregnancy, myositis, and thyroid disease. While some studies lend support to the concept that microchimerism is involved in the pathogenesis of selected autoimmune diseases, studies also indicate microchimerism is not uncommon in other human conditions and in healthy individuals.     

Presentation of soluble antigen to human T cells by products of multiple HLA-linked loci: analysis of antigen presentation by a panel of cloned, autologous, HLA-mutant Epstein-Barr virus-transformed lymphoblastoid cell lines

Epstein-Barr virus-transformed human B lymphoblastoid cell lines (EBV-LCL) can present soluble antigens to antigen-primed T lymphocytes. In this study, we used HLA antigen-loss mutants of an EBV-LCL line (LCL 721) to demonstrate that the presentation of a soluble antigen from Candida albicans (CAN) by EBV-LCL to primed T cells can be restricted by multiple HLA determinants. Haplotype-deletion mutants that contained only the maternal or only the paternal HLA-haplotype were used to demonstrate the preferential role of autologous HLA antigens in presenting soluble antigens to Candida-primed T cells from the donor of LCL-721, and to T cells from her mother and father. Immunoselected mutants of LCL-721 showing a variety of distinct phenotypes that are deficient in HLA-DR, DQ, or DP antigen expression were tested as antigen-presenting cells. The antigen-presenting ability of these class II deficient EBV-LCL variants weakened with progressive loss of class II HLA determinants expressed on the cell surface. Our study, therefore, provides evidence for multiple HLA restriction determinants, including HLA-DR, DQ, and DP. Furthermore, LCL lacking all HLA-DR, DQ, and DP expression because of homozygous deletion of these MHC class II genes still presented CAN and Tetanus toxid (TET), although to a much lesser degree than presented by LCL-721. This suggests that determinants other than DR, DQ, and DP which are expressed on these EBV-LCL may also function as restriction elements for the proliferative T-cell response to soluble antigens.     

Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.