同种异基因骨髓细胞移植诱导嵌合体小鼠生成

采用C57BL/J和BALB/c两种小鼠进行骨髓细胞移植。移植前受体小鼠经 6 0Gy6 0 Coγ射线照射。用细胞染色法和流式细胞术分析了射线对小鼠淋巴细胞的损伤强度和照射后移植了骨髓细胞的小鼠在不同时间内的淋巴细胞亚群变化。结果表明 ,(1 ) 6 0Gyγ射线照射受体鼠 ,能杀伤 95 5 %的外周淋巴细胞 ,但是不影响自身H 2Kb 分子在淋巴细胞表面表达的百分数。残留细胞中CD3+ T细胞、NK1 1 + 淋巴细胞、CD3+ /NK1 1 + 淋巴细胞的阳性率分别由照射前的 40 44%增加到45 30 %、 1 1 3 %增加到 37 41 %和 1 0 7%增加到 5 86 % (P≤ 0 0 1 ) ;(2 )小鼠的淋巴细胞数量从照射后 30d开始恢复 ,到1 90d达到正常值 ;(3 )照射后进行同种异基因骨髓细胞移植后的第 60天 ,受体淋巴细胞表面自身H 2Kb 抗原表达下降 ,CD3+ /NK1 1 + 淋巴细胞百分数增加。随H 2Kb 的抗原表达的恢复 ,CD3+ /NK1 1 + 淋巴细胞百分数下降 ;(4)在细胞移植的第 1 90天 ,受体小鼠 (黑色 )表型呈现出供体鼠 (白色 )颜色特征。在嵌合体小鼠外周淋巴细胞中 ,有 2 5 %为供体H 2Kd 阳性细胞 ,CD3+ /NK1 1 + 淋巴细胞百分数为 1 88% ,明显高于正常值 (P≤ 0 0 1 )。以上结果表明 ,6 0Gy6 0 Coγ射线照射能诱导免疫耐受 ,其耐受性的形成可能与最初保     

Dynamics of Cytotoxic T Lymphocyte Precursors in Vivo Assessed by Change in the Radiation Sensitivity

The dynamics of cytotoxic T lymphocyte precursors (CTL-p) in mice injected with allogeneic spleen cells (SC) was studied with special reference to changes in their radiation sensitivity. Whole-body 400 rad X-ray irradiation of allo-SC-primed and unprimed mice virtually abolished the capacity of their SC to proliferate and to generate CTL in primary or secondary mixed leucocyte culture (MLC). However, the impaired ability of SC to generate CTL in the primary MLC was restored by interleukin 2 (IL-2). This showed that helper cells whose activity was replaceable with IL-2 (IL-2-producing cells) were functionally more radiation-sensitive than CTL-p in unprimed mice. In contrast, the radiation-impaired activity in secondary MLC was not restored by IL-2, suggesting that memory CTL-p in allo-SC-primed mice were unexpectedly sensitive to radiation. The D37 values determined from the percentage of residual CTL-p activity of SC in bulk cultures 1 day after irradiation were 525 rad for virgin CTL-p and 75 rad for memory CTL-p. Further studies demonstrated that the radiation-sensitive memory CTL-p were generated from relatively radiation-resistant precursors, largely independent of radiation-sensitive IL-2-producing cells and of cellular proliferation. The mean frequency of CTL-p in SC measured by limiting dilution assay was not significantly increased by the priming. This supports our conclusion that the development of the memory CTL-p activity in allo-SC-primed mice did not depend on clonal expansion. Whole-body 400 rad-irradiation reduced the frequency of CTL-p in SC from unprimed mice to 1/2-1/3 and that in SC from allo-SC-primed mice to 1/8-1/15. This supports the view that the majority of radiation-resistant virgin CTL-p functionally mature to radiation-sensitive memory CTL-p without cellular proliferation in allo-SC-primed mice.     

Lymphocyte Chimerism After Bone Marrow Transplantation

Specific HLA antibodies were used to eliminate donor and recipient cells, respectively, from lymphocyte suspensions prepared from the blood of a child who had been transplanted with bone marrow from an HLA-A- and HLA-B-incompatible, HLA-D-compatible donor. About 70% of the lymphocytes were of donor HLA type, the remaining of recipient type. The phytohemagglutinin-responsive lymphocytes were exclusively limited to the lymphocyte population carrying donor-type HLA antigens. Membrane immunofluorescence investigations of the donor and recipient populations showed a low percentage of IgM-positive lymphocytes in the donor population and an extremely high proportion of IgM-positive lymphocytes in the recipient population. About 90% of the donor lymphocytes were T cells, as judged by their capacity to form rosettes between sheep erythrocytes and T lymphocytes; no cells in the recipient cell population expressed this ability.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Immunodeficiency after Allogeneic Bone Marrow Transplantation in Man

This study was undertaken to clarify the mechanism behind the severely decreased lymphocyte proliferative response upon stimulation with mitogens and antigens seen after allogeneic bone marrow transplantation (BMT) in man. We investigated eight BMT patients and eight controls and found that the proliferative response of patient cells was reduced both when the cells were stimulated with phytohaemagglutinin (PHA) and when they were stimulated with a combination of phorbol myristate acetate (PMA), which is an activator of protein kinase C (PKC), and the calcium ionophore A23187, which irreversibly opens for calcium transport into the cell (median relative responses were 41 and 37%, respectively). However, the PHA-induced increase in the concentration of intracellular free calcium in post-BMT cells was not significantly different from the values found in control cells and the expression of interleukin 2 (IL-2) receptors (CD25) was only slightly decreased. However, the production of IL-2 was severely decreased in patient cells after stimulation with A23187/PMA (median 3541 units), although it was higher than in PHA-stimulated control cells (median 354 units). These results show that a direct activation of PKC by PMA combined with an increase in intracellular free calcium by A23187 cannot overcome the lymphocyte proliferation deficiency in cells from patients after allogeneic BMT. The data suggests that the defect is affecting the diacylglycerol pathway considerably more than the inositol triphosphate pathway.     

T Cell Repertoire Evolution after Allogeneic Bone Marrow Transplantation: An Organizational Perspective

High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-β sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-β sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-β VJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution.     

Patient Perspectives Regarding Allogeneic Bone Marrow Transplantation in Myelofibrosis

Hematopoietic stem cell transplantation (HCT) is a curative treatment for patients with myelofibrosis (MF); however, many HCT-eligible patients decline this potentially life-saving procedure. The reasons behind this decision are not clear. We sought to survey patients with MF to understand their perspective on HCT. A 63-question survey was posted on myeloproliferative neoplasm patient advocacy websites. A total of 129 patients with MF responded to the survey. Among these patients, 49 (41%) were referred for HCT, and 41(32%) attended the transplantation consult. Of the patients who attended the transplantation consult, 24 (59%) did not plan on going on to HCT, and 16 (41%) intended to proceed with HCT. Reasons for the decision to not undergo transplantation included the desire to not be ill, desire to not spend time in the hospital, and concerns about overall quality of life. Specifically, concerns related to financial impact and the risk of graft-versus-host disease (GVHD) were expressed. Patients who decided to proceed with HCT felt that this would extend their survival and allow them to be around family for longer. This is the first survey to investigate patient perceptions regarding HCT for MF. Less than one-half of the patients were referred for HCT, and of those, less than one-half planned on proceeding with the transplantation, suggesting that many patients do not receive this life-saving procedure. Further exploration of the basis of patients’ reluctance to proceed with HCT is warranted.     

Cytotoxic T Lymphocyte Recognition of HLA-G in Mice

Several features of HLA-G’s sequence and expression pattern distinguish HLA-G from its classical counterparts. These features, including HLA-G’s limited polymorphism and its expression at the maternal-fetal interface, have been used as a basis for suggesting a distinct functional role for this nonclassical class I HLA molecule. On the other hand, published data do demonstrate that HLA-G has much in common with its classical counterparts. It associates with β2-microglobulin and cytosolic peptides, it binds to CD8, and its presence can inhibit NK-cell-mediated lysis of HLA-G-bearing target cells. To develop a model in which HLA-G’s function could be more thoroughly studied, we produced several HLA-G-expressing transgenic mouse strains. We report here the results of skin graft experiments which show that nontransgenic mice reject HLA-G-expressing transgenic murine skin as foreign and that this rejection is associated with the presence in the recipient of lymphocytes capable of specifically lysing HLA-G-expressing cells. In addition, experiments are described which demonstrate that HLA-G transgenic mice recognize HLA-G as a “self” molecule. Together the reported data demonstrate that HLA-G is capable of stimulating an HLA-G-restricted CTL response, that HLA-G molecules can serve as target molecules in lytic interactions with CTLs, and that HLA-G is involved in education of the lymphocytic repertoire of HLA-G transgenic mice.     

Effect of Cyclosporin A on the Generation of Cytotoxic T Lymphocytes in Mouse Mixed Lymphocyte Culture

Cyclosporin A inhibited at equal concentrations both the proliferative response and the generation of cytotoxic T lymphocytes in one-way mouse spleen cell mixed lymphocyte culture. The 50% inhibitory concentration was in all experiments 10(-2) to 10(-1) microgram/ml. The inhibition was directly proportional to how early the drug was added to the culture: a complete inhibition of both responses was obtained if the drug was added on day 3, and a partial inhibition if added on day 4 of culture. Cytological analysis of the cultured cells demonstrated that resting lymphocytes were not damaged at 100-1000-fold concentrations of the drug giving complete inhibition of the blastogenic response. The results suggest that cyclosporin A is most effective if present throughout the induction phase of the immune response.     

Late Mortality after Allogeneic Bone Marrow Transplantation in Childhood for Bone Marrow Failure Syndromes and Severe Aplastic Anemia

Children with bone marrow failure syndromes and severe aplastic anemia (SAA) are treated with allogeneic blood or marrow transplantation (BMT). However, there is a paucity of studies examining late mortality risk after allogeneic BMT performed in childhood for bone marrow failure syndromes and SAA and evaluating how this risk differs between these diseases. We investigated cause-specific late mortality in 2-year survivors of allogeneic BMT for bone marrow failure syndromes and SAA performed before age 22years between 1974 and 2010 at 2 US transplantation centers. Vital status information was collected from medical records, the National Death Index, and Accurint databases. Overall survival was calculated using Kaplan-Meier techniques. The standardized mortality ratio (SMR) was calculated using age- sex-, and calendar-specific mortality rates from the Centers for Disease Control and Prevention. Among the 2-year survivors of bone marrow failure syndromes (n?=?120) and SAA (n?=?147), there were 15 and 19 deaths, respectively, yielding an overall survival of 86.4% for bone marrow failure syndromes and 93.1% for SAA at 15years post-BMT. Compared with the general population, patients with bone marrow failure syndromes were at a higher risk for premature death (SMR, 22.7; 95% CI, 13.1 to 36.2) compared with those with SAA (SMR, 4.5; 95% CI, 2.8 to 7.0) (P < .0001). The elevated relative risk persisted at ≥15years after BMT for both diseases. The hazard of all-cause late mortality was 2.9-fold (95% CI, 1.1 to 7.3) higher in patients with bone marrow failure syndromes compared with those with SAA. The high late mortality risk in recipients of allogeneic BMT in childhood for bone marrow failure syndromes calls for intensified life-long follow-up.     

Frequency of Cytotoxic T Lymphocyte Precursors to Herpes Simplex Virus Type 1 as Determined by Limiting Dilution Analysis

The conditions for establishing a limiting dilution assay to measure cytotoxic T lymphocyte precursors (CTL-P) against herpes simplex virus type 1 (HSV-1) were determined. Analysis by Poisson statistics demonstrated that the estimated frequency of HSV-1-reactive cells in the spleens of normal mice was less than 1/250,000. In contrast, mice immunized previously with infectious HSV-1 demonstrated a CTL-P frequency between 1/3,500 and 1/15,670. The generation of a maximum cytotoxic T lymphocyte response required that mice be primed in vivo with infectious virus. Immunization with inactivated virus either failed to elicit detectable CTL-P frequencies or gave frequencies markedly less than those induced with infectious virus. To obtain positive cultures, the responder cell population had to be exposed to stimulator splenocytes expressing viral antigens. Normal splenocytes without virus or normal splenocytes with T cell growth factor did not result in significant cytotoxicity. Split culture analysis comparing cytotoxicity against syngeneic and allogeneic virus-infected targets provided evidence for specificity, H-2 restriction, and the T cell nature of the CTL-P. It was determined that precursors were eliminated by treatment with anti-Thy 1, Lyt 2.1, or Lyt 1.1, indicating the CTL-P were Lyt 1(+)2(+) cells. Cytotoxicity was reduced after treatment of the responders with anti-Lyt 2 plus complement, which gave further evidence of the T cell nature of the cytotoxic T lymphocytes. These experiments demonstrated the feasibility of using the limiting dilution approach as a highly sensitive and quantitative means to measure the cell-mediated immune response to HSV-1 antigens.     

Listeriosis in Recipients of Allogeneic Bone Marrow Transplants from Unrelated Donors

 Two cases of listeriosis in patients submitted to matched unrelated donor bone marrow transplantation are reported. The patients developed listerial septicemia and listerial septicemia with meningitis and encephalitis 39 and 29 days after transplantation, respectively. Including the present two cases, 19 Listeria monocytogenes infections in related and unrelated donor allogeneic bone marrow transplant recipients have been reported to date. Infection occurred earlier in unrelated donor transplant recipients. Listeriosis is a rare complication in allogeneic bone marrow transplant recipients; however, the widespread practice of performing transplants from a donor-alternative to a human leukocyte antigen-compatible sibling and, in this setting, the need for intensified immunosuppression may predict an increasing and earlier occurrence of listeriosis.     

Involvement of MHC-Linked Hemopoietic-Histocompatibility Genes in Allogeneic Bone Marrow Transplantation in Mice

Genes controlling resistance of irradiated mice of allogeneic hemopoietic cells were mapped within, or closely linked to the D region of MHC and were designated Hemopoietic-histocompatibility genes (Hha). Hhp genes responsible for resistance to parenteral hemopoietic cells had also previously been detected on the D-end of MHC. Hh genes are regarded as determinants of cell surface antigens (Hh antigens) phenotypically expressed, in contrast to Histocompatibility antigens (H antigens) only on blood-forming and leukemic cells. The inheritance of Hh genes is not codominant, unlike thformed on peripheral blood lymphocytes and platelets. Sixteen Tunisian families were typed with 37 patients and 108 relatives. Genetic transmission of the disease of the HLA system seemed to be independent in this study. Comparison of HLA gene frequencies (unrelated) parents of patients and a control population showed no difference, proving that there is no clear association in population between deleterious XP genes and a particular HLA gene. However, an excess of identical HLA among pairs of diseased siblings would suggest that the disease is polymorphic and a form of the XP could be linked to HLA.     

Clonally Expanded CD8+ T cells in Allogeneic Bone Marrow Transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with haematologic malignancies. Several lines of evidence demonstrate that donor T cells are involved in the antitumour effects observed after BMT. Thus, patients receiving T-cell-depleted BMT have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated BMT, and patients experiencing graft-versus-host disease (GVHD) have a lower risk of disease relapse than patients who do not experience GVHD. Although the importance of donor T cells for the curative action of BMT has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. In a recently initiated project, we have conducted a longitudinal study of T-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. Peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (MNCs) were isolated and cryopreserved. CD8⁺ T cells were isolated from the MNCs by use of immunomagnetic beads or FACS and analysed for the presence of clonally expanded cells by T-cell receptor clonotype mapping based on RT-PCR and denaturing gradient gel electrophoresis (DGGE). Using this gel-based methodology, clonally expanded T cells were monitored after transplant and compared to the clinical data of the patients. The preliminary results demonstrates the presence of clonally expanded CD8⁺ T cells at all time points analysed. Furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. The appearance of newly emerged clonotypes which coincided with clinical GVHD could indicate a role for these T cells in the pathogenesis of GVHD.     

Separation and Functional Analysis of Subpopulations of T Cells in the Bone Marrow of the Mouse

Functional capacities of T-cell precursors have been evaluated by cell culture experiments after fractionation of bone marrow cells from normal and nude mice by 1 g velocity sedimentation. The results indicate that the T-cell precursors in the bone marrow are cycling cells, that T-cell precursors in the bone marrow of nude mice have more limited potentials than those of normal mice, and that both Thy-1.2-positive and Thy-1.2-negative precursors are stimulated by T-cell activators.