Effects of activating NK cell receptor expression and NK cell reconstitution on the outcomes of unrelated donor hematopoietic cell transplantation for hematologic malignancies.

Inhibitory NK cell receptors are recognized as important determinants of NK cell activity in hematopoietic cell transplantation (HCT). The role of activating receptors and their acquisition after HCT is less certain. Therefore, we comprehensively evaluated both inhibitory and activating receptors in 59 patients receiving unrelated donor HCT. NK cell numbers normalized quickly relative to B and T cells; however, the expression of both inhibitory and activating isoforms of killer immunoglobulin-like receptors (KIRs) was delayed. Most NK cells expressed an immature phenotype during the first 6 months post-HCT; however, we found high expression of activating NKp46 and NKp44 natural cytotoxicity receptors (NCRs), and cytotoxicity was preserved. Early reconstituting NK cells from unmanipulated grafts showed lower cytotoxicity than those from T-cell-depleted grafts. Differences in NK cell reconstitution had significant effects on clinical outcomes. Patients whose NK cells reconstituted earlier had better survival and lower relapse rates. The best survival group was recipients who possessed HLA-C2 but their donor lacked the cognate-activating KIR2DS1. Collectively, our data underscore the clinical relevance of reconstituting NK cells and their activating KIRs and NCRs. In addition to NK cell quantification and genotyping, comprehensive assessment of NK cell functions and phenotypes, including activating receptors, is essential.

Inhibitory KIR-HLA receptor-ligand mismatch in autologous haematopoietic stem cell transplantation for solid tumour and lymphoma.

Genes that encode killer Ig-like receptors (KIRs) and their HLA class I ligands segregate independently; thus, some individuals may express an inhibitory KIR gene but not its cognate ligand. We hypothesised that these patients with KIR-HLA receptor-ligand mismatch have a low risk of relapse after an autologous haematopoietic stem cell transplantation (HCT). Sixteen consecutive patients with lymphoma or solid tumour were enrolled onto a prospective study. They received high-dose busulphan and melphalan followed by autologous CD133(+) HCT. We found that 8 of the 16 patients experienced disease progression after autologous HCT, including 5 of the 6 patients (83%) with no inhibitory KIR-HLA mismatch and 3 of the 6 patients (50%) with 1 mismatched pair; none of the 4 (0%) patients with 2 mismatched pairs experienced disease progression. Survival analyses showed that inhibitory KIR-HLA mismatch was the only significant prognostic factor (P=0.01). The potential applicability of the receptor-ligand mismatch model to autologous HCTs and to patients with lymphoma or solid tumour is clinically significant because of the prevalence of the HCT procedure.

Purification of human natural killer cells using a clinical-scale immunomagnetic method.

BACKGROUND: Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS: Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS: The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION: We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

Gamma delta T lymphocyte regeneration after T lymphocyte-depleted bone marrow transplantation from mismatched family members or matched unrelated donors.

The recovery of gamma delta T lymphocytes was studied in 31 recipients of T cell-depleted allogeneic bone marrow (BMT) to determine if the dynamics of reconstitution could be related to graft-versus-host disease (GVHD) or other complications of marrow transplantation. Two distinct patterns of regeneration were apparent. In 12 patients, there was a progressive rise in both the percentage and the absolute number of peripheral blood gamma delta T cells over the first year post-transplantation, but these increases never breached levels found in 14 healthy donors. Each of the 19 remaining patients had abnormally high proportions and numbers of gamma delta T cells on at least two occasions following transplantation. The clinical factor that best explained these observations was the frequency of intercurrent infections. Of 19 patients with abnormally increased percentages and numbers of gamma delta T lymphocytes, 18 had one or more episodes of confirmed viral or fungal infection, contrasted with only two of 12 in the comparison group (P < 0.001). There was no significant association of gamma delta T cell recovery patterns with the presence of GVHD (P = 0.33). We conclude that the recovery of gamma delta T lymphocytes after marrow transplantation may vary. Supranormal levels of this T cell subset are associated with infection and may contribute significantly to cellular immune defenses against fungal or viral disease.

Production of genetically modified Epstein-Barr virus-specific cytotoxic T cells for adoptive transfer to patients at high risk of EBV-associated lymphoproliferative disease.

EBV-induced lymphoproliferative disease (EBV-LPD) is a disorder most commonly associated with the immunocompromise that follows allogeneic organ transplantation. In patients receiving T cell-depleted bone marrow from HLA-mismatched or HLA-matched unrelated donors, the incidence of EBV-LPD is particularly high, ranging from 5 to 30%. Administration of EBV-specific cytotoxic T lymphocytes may be one means of preventing and treating this disease. We now describe a method that allows the routine and timely preparation of large numbers of such cells to allow their safe administration to bone marrow transplant recipients. We also describe how these cells may be genetically marked before infusion, to determine their fate and disposition in vivo.

Early identification of Epstein-Barr virus-associated post-transplantation lymphoproliferative disease.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is a common, usually fatal, complication developing after transplantation of bone marrow from HLA-mismatched or HLA-matched unrelated donors. Prompted by recent reports of successful treatment of EBV-LPD, we investigated methods which could result in early identification of patients at high risk for this disorder, thus improving the likelihood of successful therapeutic interventions. Both the outgrowth of transformed B lymphocytes ex vivo (100% correlation) and the detection of EBV DNA by a PCR method (80% correlation) showed statistically significant association with the histopathological diagnosis of EBV-LPD. Because these abnormalities can be detected prior to the onset of clinical disease. It should now be possible to use a combination of the methods described here to identify patients at high risk of developing EBV-LPD, thus enabling early therapeutic intervention.

Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.

The contribution of infused bone marrow cells to long-term haemopoietic recovery in patients undergoing autologous bone marrow transplantation is unknown. Such information would help to clarify the role of this procedure in cancer therapy and would aid in the development of strategies to reduce the risk of subsequent aplasia. By transferring a neomycin resistance marker gene into the marrow cells of 20 patients before transplantation, we were able to trace the pattern of haemopoietic reconstitution postinfusion. The marker gene was present and expressed in all haemopoietic lineages in vivo in 15 of 18 evaluable patients at 1 month post-transplantation, in 8 of 9 patients at 6 months, and in 5 of 5 at 1 year. The marker has remained detectable for up to 18 months--the duration of our study. Our findings indicate that harvested bone marrow consistently contributes to long-term multilineage recovery of haemopoiesis after autologous marrow transplantation in cancer patients. These results provide a rationale for the continued exploration of more ablative preparative regimens with single or sequential autologous marrow transplants.