Thymus-independent expansion of T lymphocytes in children after allogeneic bone marrow transplantation

The contribution of the thymus-dependent pathway and thymus-independent pathways for T cell regeneration after BMT in children is still unclear. We analyzed the kinetics of T cell regenerative pathways after allogeneic BMT. The number of CD4+CD45RA+ T cells, a thymus-dependent population, was very low until 3 months after BMT. The numbers of CD28- T cells and CD8+ T cells expressing CD8alpha/alpha homodimer (CD8alpha/alpha+ T cells), a thymus-independent population, increased shortly after BMT, beyond the levels of healthy children in some patients. The numbers of Vgamma9+Vdelta2+ and Valpha24+ T cells, which represent populations of extrathymic development, were less than 200/microl during the 6 months after BMT. There was a significant inverse correlation between the percentages of CD4+CD45RA+ and CD28-T cells at 1 month, and a positive correlation between the percentages of CD28- and CD8alpha/alpha+ T cells at 2 and 3 months after BMT. The mean age at BMT was higher in patients with a high level of CD8alpha/alpha+ T cells than in those without an increase in these cells, suggesting the influence of thymic function on the regenerative pathways. These results suggest that the thymus-independent pathway is the dominant source of T cells even in children shortly after allogeneic BMT.     

Graft-versus-leukaemia following allogeneic bone marrow transplantation: emergence of cytotoxic T lymphocytes reacting to host leukaemia cells.

Cytotoxic T lymphocyte precursor (CTLp) frequency assays were examined in patients with chronic myeloid leukaemia (CML) following bone marrow transplantation (BMT) using recipient lymphocytes or CML cells as targets in a 51Cr release cytotoxicity assay. Eighteen patients were studied; 11 received marrow from a fully HLA A, B and DR matched sibling donor, and six from matched unrelated donors or a partially matched sibling (one patient). Two of the unrelated donor transplant recipients received marrow depleted of T lymphocytes, and the remainder received unmanipulated marrow and cyclosporin with or without methotrexate as prophylaxis against graft-versus-host disease (GVHD). Donor cells tested before BMT did not generate CTL against the patients' leukaemia, but up to 9 months after BMT a low frequency of CTLp directed against the patients' CML cells (Lk-CTLp) was detected in all patients. The Lk-CTLp frequency was significantly lower than the frequency of CTLp directed against the recipients' PHA transformed pretransplant lymphocytes (Ly-CTLp) (p less than 0.05). Lk-CTLp showed MHC restricted cytotoxicity and did not demonstrate cytotoxicity in an NK assay. The Lk-CTLp frequency correlated with both GVHD severity and relapse: severe GVHD was only seen with Lk-CTLp frequencies greater than 1:400,000, while leukaemic relapse was only observed in two patients with Lk-CTLp frequencies less than 1:400,000. These results show that a low frequency of alloreactive cells of presumed donor origin with cytotoxic potential against residual leukaemia normally circulate after BMT. Their relationship with the graft-versus-leukaemia phenomenon and their cross-reaction with GVHD reacting cells remain to be determined.     

Cocaine-contaminated allogeneic bone marrow transplantation.

Should a person with history of drug addiction be categorically denied as a bone marrow donor? The answer to the question is controversial. We report a case of allogeneic bone marrow transplantation for refractory acute myeloid leukemia preceded by essential thrombocythemia. The donor used cocaine and marijuana the night before the bone marrow harvest.     

Analysis of T lymphocytes after bone marrow transplantation using monoclonal antibodies

Using monoclonal antibodies to cell surface antigens and fluorescent cell sorter analysis, we studied peripheral blood lymphocyte subsets after bone marrow transplantation (BMT). In 13 patients studied 3 mo or more after BMT, the ratio of T-cell subsets defined by antibodies OKT4 and OKT8 was reversed (OKT4/OK%8 = 0.7 +/- 0.3) in comparison to normal volunteers or bone marrow donors (ratio OKT4/OKT8 = 1.7 +/- 0.4) (p less than 0.001). This reversed ratio persisted for up to 3 yr after BMT. In contrast to a previous report, presence of an abnormal ratio of T-cell subsets did not correlate with clinically significant graft- versus-host disease (GVHD). In agreement with a previous study, (26% +/- 8%; less than 4% in normals (p less than 0.001) and antibody OKT10 reactive cells (39% +/- 20% versus 10% +/- 4%) (p less than 0.01), suggesting in vivo activation. However, their PBL did not react with antibody B3/25 (antitransferrin receptor), a marker found on normal PBL after in vitro activation by mitogens (BMT patients less than 5%; normal PBL T cells plus PHA 45% +/- 11%). These results demonstrate that BMT patients have: (A) an abnormal ratio of T-cell subsets in the presence or absence of clinically significant GVDH disease so that these measurements were not useful in monitoring patients; (B) an increased number of T cells with cell surface phenotype (OKT8+, Ia+, OKT10+, B3/25-) that is distinct from normals but similar to patients with infectious mononucleosis or acquired hypogammaglobulinemia.     

TCR gamma/delta positive lymphocytes after allogeneic bone marrow transplantation.

Peripheral gamma/delta+ T cells were studied in patients following allogeneic bone marrow transplantation (BMT) by indirect immunofluorescence utilizing two monoclonal antibodies (G1 and A13) able to recognize the two major subpopulations (V delta 2+ and V delta 1+, respectively) of these cells. We found that the relative percentage of 'total' (gamma/delta+ T lymphocytes) (V delta 2 + V delta 1 positive cells), and particularly of G1+ (V delta 2+) cells, in CD3+ lymphocytes was higher in transplanted patients, and especially in those presenting with acute graft-versus-host disease (aGVHD), than in normal controls. This finding was confirmed by the analysis of the V delta 2+/V delta 1+ cell ratio which was again significantly higher in patients with aGVHD as compared to controls. Similarly, the absolute number of 'total' gamma/delta+ and V delta 2+ cells was also significantly increased in patients with aGVHD. TCR gamma/delta+ T cells increased as a function of time after BMT reaching a plateau value at about day 60 post-BMT. When patients were stratified for the presence or absence of aGVHD this correlation was maintained only for patients with aGVHD. Finally, most V delta 2+ cells expressed surface T cell activation markers such as CD25 (IL-2 receptor) and DR (MHC class II) antigens. Our results suggest a possible involvement of gamma/delta+ T cells and particularly of V delta 2+ cells in the clinical and immunological events (aGVHD) occurring after allogeneic BMT.     

Conditioning regimens for allogeneic bone marrow transplantation.

Conditioning regimens for transplantation are important in determining transplant outcome. This review focuses on transplantation in aplastic anemia and leukemia using marrow from HLA-identical siblings. Results of conditioning with newer regimens such as busulfan plus cyclophosphamide and etoposide plus total body irradiation are reviewed and compared to results achieved with cyclophosphamide and total body irradiation. The potential for improved results using recent innovations such as dose adjustment of busulfan, agents which may decrease transplant-related toxicity, and directed radiation are discussed.     

多种免疫抑制剂联合应用于单倍体骨髓移植

目的：观察多种免疫抑制剂联合使用后半相合未去T细胞骨髓移植术后患者免疫功能的恢复情况。方法：38例白血病患者接受HLA2～3个位点不匹配的亲缘骨髓移植，用ATG、CD25抗体、CSA、MTX、MMF预防移植物抗宿主病(GVHD)，定期对受者在移植术后1、3、6、12、18个月的外周血淋巴细胞亚群和血清免疫球蛋白进行检测。结果：移植术后1个月患者淋巴细胞各亚群均明显下降，CD4^ T最明显，CD4^ T细胞绝对数在 6月达200个／μ，导致CD4／CD8持续倒置，在移植后18个月才逐渐恢复。CD3^ 、CD8^ 、CD19^ B细胞计数在BMT后的6～12个月基本恢复。因常规输注丙种球蛋白BMT后IgG一直正常，IgM和IgA分别在移植后6～9个月和18个月恢复。结论：单倍体相合骨髓移植后1年患者免疫功能处于低下状态，CD4^ T和IgA恢复最慢，CD4^ 细胞百分比和绝对值持续的低下，CD4／CD8持续倒置是移植后感染高发的原因，该期间要适当应用免疫支持治疗及环境保护。     

Graft-versus-leukemia effect using mixed allogeneic bone marrow transplantation

Previous studies have demonstrated that T cell-depleted (TCD) syngeneic marrow protects against graft-versus-host disease (GVHD) when given along with an allogeneic lymphocyte plus bone marrow (BM) inoculum to lethally irradiated mice. In spite of this anti-GVHD effect, TCD syngeneic marrow is ultimately eliminated by non-TCD allogeneic marrow, permitting complete allogeneic reconstitution. These observations suggested that allogeneic BM might also eliminate host-type leukemic cells in a model in which TCD syngeneic marrow is co-administered to provide protection from GVHD. In the present studies, we describe the establishment of a new model using the EL4 leukemia/lymphoma. Lethally irradiated B10 (H-2b) mice were given a lethal dose of EL4 cells (H-2b) along with syngeneic marrow or a mixture of TCD syngeneic plus non-TCD allogeneic (B10.D2, H-2d) marrow. Non-TCD allogeneic marrow, in contrast to TCD or unmanipulated syngeneic marrow, delayed or prevented mortality from the otherwise lethal EL4 inoculum, without producing clinically apparent GVHD. The anti-leukemic effect of allogeneic marrow alone was not attenuated by the co-administration of TCD syngeneic marrow, and such animals repopulated as completely allogeneic chimeras. Similar anti-leukemic effects of mixed marrow inocula in a haploidentical strain combination, and an anti-leukemic effect against established tumor were also demonstrated. This model may have the potential to increase the safety of clinical bone marrow transplantation across greater HLA disparities, while permitting utilization of the anti-leukemic and alloengraftment-promoting effects of T cells in allogeneic marrow inocula.     

Depletion of T cells from bone marrow for allogeneic transplantation: method for treatment of bone marrow in bulk

T lymphocytes were depleted from donor marrow for 23 patients undergoing allogeneic bone marrow transplantation using an anti-T-cell antibody, CT-2, and complement. The methodology is described in detail for in vitro depletion of large quantities of bone marrow. The extent of T-lymphocyte depletion using various T-cell markers, the percent of marrow lost in the processing and quantity of antibody, and complement needed are presented. These techniques for in vitro T-lymphocyte depletion were reproducible and did result in an average final yield of 47% of the harvested donor marrow.     

Selective depletion of CD8+ T lymphocytes for prevention of graft-versus-host disease after allogeneic bone marrow transplantation

The effects of selectively depleting CD8+ cells from donor bone marrow were assessed in 36 patients receiving transplantation from an HLA-identical sibling as treatment for leukemia. Donor bone marrow underwent ex vivo treatment using anti-Leu-2 monoclonal antibody and complement. Patients received cyclosporine post-transplant for 6 months. Thirty-three patients had initial engraftment. Three failed to have hematologic recovery, and one patient with initial engraftment had late graft failure. The actuarial incidence of grade greater than or equal to 2 acute graft-versus-host disease was 28% +/- 18% and was usually confined to the skin. Of 33 patients with engraftment, 32 were complete chimeras and one had mixed chimerism. The tempo of hematologic and immunologic recovery was comparable with that reported with transplantation of unmodified bone marrow, although CD4+ and CD8+ T cells recovered at comparable rates. The actuarial rate of leukemia relapse was 11% +/- 10%, occurring in three patients with acute leukemia but in none of 13 patients transplanted for chronic myelogenous leukemia. Actuarial survival was 57% +/- 17% at 2 years. These data indicate that after transplantation of marrow depleted of CD8+ cells, engraftment with prompt hematologic and immunologic recovery generally occurs, with a relatively low rate of acute graft-versus-host disease. Graft failure remains a problem despite retention of CD4+ cells within the donor marrow. The lack of leukemia relapse in patients with chronic myelogenous leukemia suggests retention of a graft-versus-leukemia effect, at least for this malignancy.     

T cell regeneration after allogeneic and autologous bone marrow transplantation

Venous blood T cell phenotypes were analysed with monoclonal antibodies after 11 allogeneic and 17 autologous bone marrow transplants. In seven cases studied in the early regenerative period, cells with a thymocyte phenotype were present in the blood. In the large majority of patients treated with both allografts and autografts there was an imbalance of phenotypic 'helper' and 'suppressor' T cell subsets with initially a relative and later an absolute increase of 'suppressor' T cells. This imbalance was still present at over 250 d in eight out nine cases. Suppressor T cells bearing HLA-Dr antigens were abundant in one case of fatal GVHD but not in another, and were also increased following two autografts. It is concluded that T cell phenotyping is not of diagnostic value in sick patients following bone marrow transplantation when graft-versus-host disease is suspected.     

HTLV-I-associated myelopathy following allogeneic bone marrow transplantation.

A 39-year-old polytransfused patient with aplastic anemia acquired transfusion-associated HTLV-I infection shortly before transplantation. The patient underwent allogeneic bone marrow transplantation and developed HTLV-I associated myelopathy 3 years later. Clinical abnormalities and a host of atypical findings are presented in the context of previous reports describing uncommon features of the disease.     

Thrombotic microangiopathy following allogeneic bone marrow transplantation.

Thrombotic microangiopathy is one of the complications of bone marrow transplantation and is related to other complications such as graft-versus-host disease, veno-occlusive disease, diffuse alveolar hemorrhage, and cytomegalovirus infection. Thrombotic microangiopathy occurred in three out of 12 patients who underwent allogeneic bone marrow transplantation over the past 1 year at our department. We compared the changes in cytokines and other molecules between patients with and without microangiopathy from before conditioning to the early post-transplantation period. All three patients with microangiopathy showed a significant increase of interleukin-12 at the time of leukocyte recovery after transplantation (two-way layout analysis of variance; P < 0.05), while none of the patients without microangiopathy showed an increase of interleukin-12. No significant differences were found between the two groups with respect to the other cytokines and molecules that were tested. These findings suggested that thrombotic microangiopathy might be predicted at an early stage after bone marrow transplantation by detecting an increase of interleukin-12 at the time of leukocyte recovery. The possibility that thrombotic microangiopathy is related to inflammation or autoimmunity was also suggested.     

Capillary leak syndrome associated with elevated IL-2 serum levels after allogeneic bone marrow transplantation

Summary The pathophysiological mechanisms involved in the development of a spontaneous systemic capillary leak syndrome (CLS) are unknown. In contrast, CLS is a well-known side effect of high-dose interleukin-2 (IL-2) therapy in solid tumors. We report on a patient who developed CLS with high serum levels of endogenous IL-2 under immunosuppressive therapy for chronic graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation (BMT). Generalized edema persisted for 10 weeks. The condition resolved after antibiotic therapy of a septic shock with hemolyzing streptococci group A. Thus, a latent infection may alter cytokine homeostasis and may cause CLS in BMT patients.     

Clinical trial depleting T lymphocytes from donor marrow for matched and mismatched allogeneic bone marrow transplants

Nine patients received T-lymphocyte-depleted histocompatible bone marrow and 28 patients received T-lymphocyte-depleted histoincompatible bone marrow. Eight of nine patients receiving matched bone marrow quickly engrafted without severe graft-versus-host disease (GvHD). None of the eight patients received anti-GvHD prophylaxis medications. Two of these eight patients are currently alive. Nonengraftment and severe GvHD were problems seen in some of the patients given the histoincompatible bone marrow. Additional cytarabine pretransplant permitted engraftment in those patients undergoing histoincompatible transplants for treatment of malignancy, and prednisone and cyclosporine posttransplant reduced the incidence of acute GvHD in those given T-lymphocyte-depleted grafts. Seven of these 28 patients are currently alive. T-lymphocyte-depleted marrow can reduce the occurrence or prevent severe acute GvHD, especially when combined with additional prednisone and cyclosporine; however, the impact on relapse patterns and survival remains to be determined. The occurrence of nonengraftment and treatment-related lymphomas are formidable problems to overcome.