MHC antigen expression in human first trimester spinal cord with implications for clinical transplantation procedures.

We report human leukocyte antigen (HLA) class I expression in 5-17% and class II in 0-9% of first trimester human spinal cord cells. After 8 days in culture with gamma-interferon, >87% of the spinal cord cells expressed HLA class II. However, mixed cultures of adult human peripheral lymphocytes and immature human spinal cord cells, showed no induction of lymphocyte proliferation prior to or after gamma-interferon exposure in culture. In conclusion, we report non-immunogenic expression of HLA antigens in the human first trimester spinal cord.

Long-term follow-up of a randomized trial comparing T cell depletion with a combination of methotrexate and cyclosporine in adult leukemic marrow transplant recipients.

Forty-eight adult leukemic recipients of HLA-identical sibling marrow were randomized to T cell depletion using anti-CD8 and anti-CD6 antibodies plus complement (n = 28) or prophylaxis with methotrexate (MTX) and cyclosporine (CsA) (n = 25). Patient characteristics were comparable in the two groups. The median observation time was 5 1/2 years. Transfusions, infections, and acute GVHD did not differ between the groups. Chronic GVHD occurred in 52% of patients receiving T cell-depleted marrow and 23% of those receiving MTX + CsA (P = 0.06). Overall probability of relapse was similar in both groups and actuarial leukemia-free survivals at 5 years were 39% and 35% in the two groups, respectively. Among patients with chronic myeloid leukemia (CML), leukemia-free survival at 5 years was 25% in patients receiving T cell-depleted marrow compared with 51% in those given MTX + CsA (P = 0.09). In patients with acute leukemia the probability of relapse was 24% in the group receiving T cell-depleted marrow compared with 73% in those treated with MTX + CsA (P = 0.06). Leukemia-free survival was 55% and 21% in the two groups, respectively (NS). CML patients tended to have a poorer prognosis and those with acute-leukemia better outcome with T cell depletion than with combined MTX + CsA. It is concluded that T cell depletion is unsuitable for patients with CML, but may be considered in patients with acute leukemia.

Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies.

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

TNF alpha levels are increased during bone marrow transplantation conditioning in patients who develop acute GVHD.

TNF alpha levels were determined by ELISA in serum from 112 BMT patients during pre-transplant conditioning. Patients who developed post-transplant complications had significantly higher TNF alpha levels than those without complications (mean 620 pg/ml vs 440 pg/ml, P = 0.04). In particular this effect is associated with patients who developed grade II-IV acute GVHD (mean 960 pg/ml, P < 0.001) and chronic GVHD (mean 724 pg/ml, P = 0.001). High TNF alpha levels were the only statistically significant risk factor for acute GVHD. IL-1 beta and IL-6 levels were not correlated with TNF alpha levels or posttransplantation complications. In multivariate analysis of chronic GVHD, patient age > 17 years and CMV disease were the only statistically significant risk factors. Relapse was associated with low levels of TNF alpha during conditioning (mean 318 pg/ml, P = 0.02). In multivariate analysis, high risk disease was the only factor that correlated with relapse. Low risk patients had significantly higher levels than high risk patients (551 vs 377, P= 0.04). CML and MDS patients had higher TNF alpha levels than acute leukemia patients. There was no difference in TNF alpha levels between patients conditioned with BU/CY and CY/TBI. We conclude that determination of TNF alpha levels during conditioning may be useful in the prediction of acute GVHD.

Cryopreservation of fetal stem cells.

Fetal liver cells may be transplanted, in utero, to treat disorders affecting the hematopoietic system. Conventional immunological methods were employed to determine whether pooled cryopreserved cells could be a suitable basis for a tissue bank. We found that cryopreservation did not adversely change the population, viability or immunological capacity of human fetal liver cells and therefore, pooled and cryopreserved fetal stem cells may be suitable for transplantation.

Immunological capacity of human fetal liver cells.

The aim of this study was to evaluate immunological characteristics of human fetal liver (FL) cells, fresh and cryopreserved, 7-12 weeks post-conception. With monoclonal antibodies, HLA-associated determinants were demonstrated on FL. Although serological HLA determination of A, B, C and class II antigens was not possible, genomic HLA class II typing using RFLP technique or PCR amplification with sequence-specific primers was feasible. MLC induced only minor responses. Exposure to standard mitogens and polyclonal B cell activators did not stimulate DNA synthesis or antibody production. ABO antigens were expressed and determined. The apparent low immunological capacity of FL cells may reduce the risk of rejection and graft-versus-host disease when such cells are used in transplantation.

Effects of cryopreservation on subsets of fetal liver cells.

Human fetal livers from 6 to 13 weeks postconception were analysed before and after cryopreservation. The percentages of cell subsets, detected by MoAbs, did not change significantly after cryopreservation. Compared with BM, fetal liver contained significantly smaller subsets of cells identified by MoAbs, with two exceptions. Fetal liver contained a mean of 47% M5 positive cells versus 31% in BM, and there was no difference in the numbers of CD34+ cells. The colony-forming capacity was studied: 53 colonies grew from 10(5) cells from fresh fetal liver compared with 51 colonies from cryopreserved cells. For fresh BM the corresponding value was 88 per 10(5) cells. Incubation time for fetal stem cells was 17-18 days while the corresponding time for BM cells was 8-10 days.

Prevention of graft-versus-host disease with T cell depletion or cyclosporin and methotrexate. A randomized trial in adult leukemic marrow recipients.

In adult leukemic marrow recipients of HLA identical sibling marrow, 23 patients were randomized to T cell depletion and 25 received cyclosporin (CSA) and four doses of methotrexate (MTX) to prevent graft-versus-host disease (GVHD). Anti-CD8 and anti-CD6 antibodies plus complement depleted 95.3 +/- 5.8% (mean +/- SE) CD3 cells. All patients engrafted except one who died early. Patients receiving T cell-depleted marrow had a faster time to 0.2 x 10(9) WBC/l (p less than 0.001), but required more erythrocyte units (p = 0.03). Platelet transfusions, infections and time in hospital did not differ. The incidence of grade II-III acute GVHD was 23% following T cell depletion and 12% for those receiving CSA + MTX. Chronic GVHD occurred in 51% and 23% in the two groups, respectively (p = 0.06). Recipients of T cell-depleted marrow who developed grade I-III acute GVHD received more T cells compared to those without acute GVHD (p = 0.02). The major cause of death in both groups was relapse, the cumulative incidence of which, at 4 years, was 39% in the recipients of T cell-depleted marrow and 54% in the CSA + MTX group. The 3-year actuarial leukemia-free survival was 42% and 44% in the two respective groups.

Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation.

The use of hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy, although this approach has provided heterogeneous results. In this paper we have undertaken molecular, phenotypic and functional studies aimed at identifying the presence of fully competent T lymphocytes in samples of fetal livers and cord blood. We found mature VDJ TCR beta chain transcripts in fetal liver cells taken from 7 to 16 weeks of gestation and a similar pattern was detected in cord blood cells sampled from 13.5 to 20.5 weeks of gestation. A Vbeta8 gene sequence comparable to that detected in adult PBMC was found in fetal liver samples at 9 or 17 weeks gestation. PreTalpha message was detected in all samples and its expression decreased in fetal blood samples with increasing gestational age while Calpha message appeared at 9.4 weeks and its expression increased during gestational age. T cell clones obtained from fetal liver cells showed a mature TCR alphabeta+, CD8+ phenotype and displayed strong alloreactivity against allo-MHC class I molecules. The presence of alloreactive T lymphocytes may explain the failure to engraft in fetuses older than 13 to 16 weeks and may provide insights into fetal liver transplantation. Bone Marrow Transplantation (2000) 25, 135-141.

Polyclonal antibody secretion during acute graft-versus-host disease.

Spontaneous plaque-forming cells (S-PFC) were followed in 67 bone marrow transplantation (BMT) recipients and 41 controls. Patients with no acute graft-versus-host disease (GVHD) had decreased IgA and IgM S-PFC up to 7 weeks after BMT compared with controls. Patients with acute GVHD had increased IgG, IgA, and IgM PFC compared with controls and patients without GVHD during the first 4 weeks after BMT. The maximum number of S-PFC increased with increasing severity of acute GVHD. However, at diagnosis of GVHD there was no difference in S-PFC in patients who resolved their GVHD or in those who developed more severe GVHD. After 6 weeks, patients with acute GVHD had significantly decreased IgA and IgM S-PFC compared with normal. No major changes in S-PFC were induced during various infections. However, a patient who developed urticaria had a dramatic increase in S-PFC. Patients studied more than a year after BMT had reduced IgM S-PFC compared with controls. It is concluded that S-PFC are reduced after BMT, but markedly enhanced during acute GVHD.

Rapid detection of cytomegalovirus antigen on alveolar cells in bronchoalveolar fluid from transplant patients with cytomegalovirus pneumonia.

Cytomegalovirus-specific mouse monoclonal antibodies and immunomagnetic beads conjugated with sheep anti-mouse immunoglobulin G were used to screen for cytomegalovirus (CMV) antigen expressed on the surface of alveolar cells in bronchoalveolar lavage (BAL) fluid from 23 transplant recipients with interstitial pneumonia. The beads formed rosettes around the cells when CMV antigen was present on the cell surface. Results could be evaluated by light microscopy within 2 hours of receiving the specimen. Cells in BAL fluid obtained from 15 immunocompetent individuals served as controls. Cytomegalovirus antigen was expressed on the surface of the alveolar cells from 12 transplant recipients and detected by this method as rosette formation on 1-8% of the cells. CMV was isolated from culture of cells in BAL fluid and blood from all these patients except 1, while intracellular CMV antigen was detected by monoclonal antibodies and immunofluorescence in 3 only. Serological changes, indicating an active CMV infection, were present in 11/12 patients. One patient with a CMV pneumonia, later confirmed by autopsy, failed to respond with any antibody titer. No rosette formation occurred on alveolar cells from any of the 15 immunocompetent controls. Thus, the method studied appears to be a valuable complement to other diagnostic methods for CMV pneumonia in transplant recipients. When correlated with the isolation of CMV from culture, the sensitivity was 100% and specificity 92%.