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Objective: We have evaluated T-cell reconstitution and reactivity in patients receiving nonmyeloablative haploidentical hematopoietic cell transplantation (HCT) protocols involving an anti-CD2 monoclonal antibody (MEDI 507) to treat chemorefractory hematopoietic malignancies., Methods: Three cohorts of four patients each and one cohort of six patients received one of four Medi-507-based regimens, all of which included cyclophosphamide, thymic irradiation, and a short posttransplantation course of cyclosporine., Results: Following marked T-cell depletion, initially recovering CD4 and CD8 T cells were mainly memory-type cells. A high percentage of CD4 T cells expressed high levels of CD25 in recipients of all protocols, except the only protocol to include fludarabine, early post-HCT. CD25 expression varied inversely with T-cell concentrations in blood. CD25(high) CD4 T cells expressed Foxp3 and cytotoxic T-lymphocyte-associated protein 4, indicating that they were regulatory T cells (Treg)., Conclusions: Fludarabine treatment prevents Treg enrichment after haploidentical nonmyeloablative stem cell transplantation, presumably by depleting recipient Tregs. In vitro analyses of allorecognition were consistent with a cytokine-mediated rejection process in one case and in another provided proof of principle that mixed chimerism achieved without graft-vs-host disease induces donor- and recipient-specific tolerance. More reliable achievement of this outcome could provide a promising strategy for organ allograft tolerance induction.

Numerous studies have demonstrated an association of posttransplant HLA antibodies with decreased long-term graft survival. The presence of C4d deposition in these cases supports the hypothesis that antibody and complement deposition are involved in the pathogenesis of graft failure. Development of HLA antibodies may predate the clinical manifestation of chronic rejection (CR). However, frequency of donor-specific antibody is low when all patients are screened regardless of their graft function, and it may be more valuable to look for antibody only in patients with mild dysfunction. Effective treatment for CR has not been identified, although increased immunosuppression has been shown to decrease antibody levels and stabilize graft function. Many patients have been identified with good graft function despite the presence of circulating donor-specific HLA antibody. Additional studies focusing on the mechanism behind the apparent protection from the detrimental effects of antibody in such patients are needed.

HLA antibodies can develop for many different reasons and are often present in the serum of patients awaiting renal transplantation. These antibodies can limit the patient's ability to find a compatible donor and are a risk factor for poor graft outcome. Detection of HLA antibodies is important for identifying compatible donors, assessing the pretransplantation risk for rejection, and diagnosing humoral rejection posttransplantation. The results of antibody screens are also useful when interpreting crossmatch results. Assays used for detecting panel-reactive and donor-specific HLA antibodies, including cytotoxicity, flow cytometry, and solid phase assays, are described in this review.

We sought evidence for non-MHC antibody-mediated rejection in renal allografts by a systematic study of rejected HLA-identical sibling renal allografts. Among 162 recipients of HLA-identical, ABO-compatible sibling donor kidneys transplanted at the Massachusetts General Hospital from 1964 to 2005, we identified 15 grafts that were lost from rejection and two additional grafts with reversible acute rejection, which provided 30 samples for study. All samples were stained for C4d by immunofluorescence in frozen tissue (n = 7) or by immunohistochemistry in paraffin embedded tissues (n = 10). We found that two of 17 grafts had positive C4d staining of peritubular capillaries. Histology revealed acute antibody-mediated rejection in one and acute cellular rejection type 1 in the other. Both grafts were matched at HLA-A, B, and C loci and had a nonreactive mixed lymphocyte response. Genotyping and serological analysis were not available. Compared with a published series, C4d+ irreversible rejection was more common in HLA nonidentical than HLA-identical grafts (75% vs 6.7%, respectively, P < .002). We conclude that antibody-mediated rejection, presumably due to non-MHC antigens other than ABO-blood groups does occur, but infrequently. This may account for some of the HLA antibody negative cases that develop antibody-mediated rejection.

Objective: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism., Patients and Methods: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease., Results: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras., Conclusion: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.