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Bone marrow transplantation (BMT) is the treatment of choice in children affected by primary immunodeficiency (PID). Because only 10-15% of affected children have a familial HLA-identical donor alternative therapeutic options are BMT from a matched unrelated donor or an haploidentical BMT. In our experience only 40% of these children find a donor within the International Registry. Therefore, the remaining 50% children affected by PID are candidates for haploidentical BMT. Unfortunately, in PID other than sever-combined immunodeficiency (SCID), low engraftment rates have been reported because of minimal residual immunity. In order to enhance engraftment rate in haploidentical BMT in PID we suggest a protocol with addition of donor peripheral stem cells after mobilization with granulocyte colony-stimulating factor (G-CSF) (16 micrograms/kg for 5 days) and bone marrow cells. This procedure increases the cell load, which allows intensification of the conditioning regimen for induction of faster engraftment. The separation of CD34+ cells from leukapheresis products was achieved in the first 6 patients by the Isolex 300 system (Baxter) with a CD34+ cell purity range of 80-95% and in another three patients by the Clinimacs System (Miltenyi). The peripheral blood stem cells were cryopreserved until BMT, 15 days after G-CSF stimulation when the bone marrow was harvested, processed and T-cell depleted with Campath 1-M in the first 6 cases while the Clinimacs System was used in the remaining cases and no T-cell depletion was required. We included 9 patients in the study protocol: SCID (4), Omenn's syndrome (3), LAD (1) and CID (1). The mean value of peripheral CD34+ cells infused was 13.42 x 10(6)/kg and the mean CD3+ cells number was 0.385 x 10(5)/kg; the mean value of BM CD34+ cells infused was 10.62 x 10(6)/kg and the mean CD3+ cell number was 2.39 x 10(5)/kg. The mean number of infused CFU was 8.1 x 10(5)/kg for PBSC and 3.59 x 10(5)/kg for BM. The 9 patients achieved more than 0.5 x 10(9) peripheral blood neutrophils/L at a mean of 14.6 days (range: 6-22 days). One patient affected by SCID showed complete chimerism, but he died after BMT of systemic CMV infection; the other 8 patients are alive and well and 4 of them show complete chimerism in all cell lines. Split chimerism was documented in 2 SCID cases (CD3+ lymphocytes were of donor origin, monocytes were autologous and granulocytes were mainly autologous); 1 patient affected by Omenn's syndrome received 3 transplants (1 from the mother and 2 from the father, T-cells alone and bone marrow) and achieved engraftment with complete chimerism after the third transplant; the patient affected by LAD also received 3 transplants (2 bone marrow infusions and 1 PBSC infusion) achieving complete chimerism after the third one. In conclusion, the engraftment achieved in all treated patients, and the acceptable conditioning-related toxicity suggest that this approach could be successfully applied to children affected by PID and candidates for haploidentical BMT.

BACKGROUND AND METHODS. The outcome of mismatched bone marrow transplantation is still severely hampered by graft versus host disease (GVHD) and graft rejection. Procedures for the recognition and selective elimination of T cells are still unsatisfactory due to the increased incidence of graft failure and late rejection. Lymphocyte proliferation and generation of cytotoxic T cells (CTL) in response to allogeneic cells are considered good in vitro correlates of GVHD and have been used in the present study to asses the capacity of two drugs (vincristine, VCR, and methylprednisolone, MP) to affect the T cells involved in these reactions. RESULTS AND CONCLUSION. Treatment in vitro with VCR and MP has been shown to inhibit the functional capacity of peripheral blood lymphocytes to proliferate (mean reduction 95.8%) and to generate CTL in response to haploidentical stimulator cells. Responsiveness to antigens and mitogens was affected to a minor extent (mean reduction 40% and 65.5%, respectively), and the method allowed recovery of hemopoietic precursors. The results suggest that treatment of donor bone marrow with VCR and MP is worth studying as a new approach to the prevention of GVHD in haploidentical bone marrow transplantation.