Serotherapy with thymoglobulin and alemtuzumab differentially influences frequency and function of natural killer cells after allogeneic stem cell transplantation.

Although thymoglobulin and alemtuzumab are frequently used in hematopoietic stem cell transplantation (HSCT), little is known of their effects on NK cells, which mediate important functions in post-transplantation immunology. In the present study, we determined NK cell death in vitro using propidium iodide and Annexin V. The NK cell activity in 34 patients at day +30 after allogeneic HSCT was assessed using the CD107a assay. Alemtuzumab and thymoglobulin were similarly very potent in inducing NK cell death in vitro. Even in low concentrations (<1 microg/ml) the antibodies induced apoptosis and necrosis in a relevant percentage of NK cells (>30%). However, the number of tumor reactive (CD107a+) NK cells was 13.16 per mul and 1.15 per microl (mean) in patients receiving T-cell depletion with 6 mg/kg thymoglobulin and in patients receiving 100 mg alemtuzumab, respectively (P=0.02). Although thymoglobulin and alemtuzumab are equally NK cell toxic in vitro, the recovery of NK cell frequency and anti-tumor reactivity is reduced in recipients of alemtuzumab. Our findings can be explained by a longer half-life of alemtuzumab as compared to active thymoglobulin under therapeutic conditions. Prolonged immunosuppression with increased risk of infections and tumor relapse are a potential threat to patients undergoing HCST and receiving alemtuzumab as T-cell depletion.

Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan.

Radioimmunotherapy (RIT) of relapsed lymphoma is gaining increasing importance. Especially the commercially available anti-CD20 antibody 90Y-ibritumomab tiuxetan is currently under investigation in various trials including dose escalation and autologous hematopoietic progenitor cell support. It is not clear, however, whether the implementation of this radiolabeled antibody into another treatment option for relapsed or poor risk lymphoma patients-allogeneic hematopoietic cell transplantation-interferes with or delays successful engraftment. This study reports encouraging results with 2 relapsed lymphoma patients (1 transformed marginal zone lymphoma and 1 mantle cell lymphoma) who underwent allogeneic hematopoietic cell transplantation from HLA-matched donors. The conditioning regimen consisted of Rituximab 250 mg m(-2) on days -21 and -14, 0.4 mCi kg(-1) body weight 90Y-ibritumomab tiuxetan on day -14 and fludarabine (30 mg m(-2)) plus cyclophosphamide (500 mg m(-2)) on days -7 to -3. The data demonstrate that engraftment is fast and reliable with leukocytes >1 x 10(9) L(-1) on day 12 and platelets >50 x 10(9) L(-1) on day 10. Thus, the incorporation of radioimmunotherapy into allogeneic transplant protocols combines established modalities with proven anti-lymphoma activity and, hence, offers an attractive new therapeutic option for relapsed lymphoma patients.

A model for the detection of clonality in marked hematopoietic stem cells.

The semirandom location of retroviral integration in the target cell genome introduces a marker in the form of a fusion sequence composed of a genomic and a proviral part that is unique for each transduced cell and its clonal progeny. High-sensitivity detection of these fusion sequences would allow the tracking of clonal contributions of individual, marked hematopoietic progenitor, and stem cells in vivo. Clone detection by Southern blot has helped to analyze models of oligoclonal repopulation but is limited in sensitivity and specificity. Inverse PCR (Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414-2419) can demonstrate the clonal identity by sequencing but does not permit simultaneous detection of multiple clones. In an efficiently transduced rhesus macaque model (Tisdale et al., Blood 92: 2681-2687; Wu et al., Mol. Ther. 1: 285-293) Kim et al. (Blood 96: 1-8) have identified more than 40 insertion sequences from marrow CFU by inverse PCR. However, no previous study has been able to directly analyze the number of clones active in vivo. Here we demonstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal contributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integration sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked hematopoiesis in small and large animals as well as in human gene transfer.