Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

Haploidentical stem cell transplantation (haploSCT) offers an alternative treatment option for advanced leukemia patients lacking a HLA-compatible donor. Transfer of NK cells represents a promising therapeutic option in combination with SCT, as NK cells can promote graft versus leukemia with low risk of GVH disease. In this study, we show results from a phase I/II trial in which 24 acute myeloid leukemia patients underwent haploSCT in combination with early transfer of unmodified NK cells and observed a promising 2-year overall survival rate of 37%. By performing immunomonitoring and subsequent principal component analysis, we tracked donor NK-cell dynamics in the patients and distinguished between NK cells reconstituting from CD34(+) precursors, giving rise over time to a continuum of multiple differentiation stages, and adoptively transferred NK cells. Transferred NK cells displayed a mature phenotype and proliferated in vivo during the early days after haploSCT even in the absence of exogenous IL-2 administration. Moreover, we identified the NK-cell phenotype associated with in vivo expansion. Thus, our study indicates a promising path for adoptive transfer of unmodified NK cells in the treatment of high-risk acute myeloid leukemia.

The type of ATG matters -- natural killer cells are influenced differentially by Thymoglobulin, Lymphoglobulin and ATG-Fresenius.

Although ATG is frequently used in hematopoietic stem cell transplantation and solid organ transplantation, little is known on its effects on NK cells, which mediate important functions in post-transplantation immunology. We incubated peripheral blood lymphocytes of healthy donors with Thymoglobulin, Lymphoglobulin or ATG-Fresenius. Cell death and apoptosis of NK cells and T cells were determined by flow cytometry using propidium iodide and Annexin V. As expected, there were no significant differences between the different ATGs regarding their T cell toxicity. Surprisingly, we found profound differences between the different ATGs regarding their impact on NK cells: In clinically relevant concentrations Lymphoglobulin had less toxic effects on NK cells as compared to Thymoglobulin or ATG-Fresenius: the median percentages of apoptotic or necrotic NK cells in response to 1 mug/ml Lymphoglobulin, ATG-Fresenius and Thymoglobulin were 2%, 35% and 38%, respectively (p<0.001). This is the first report of differential effects of different ATGs on NK cells. Lymphoglobulin appears to be superior to Thymoglobulin or ATG Fresenius regarding the preservation of NK cell mediated immunity. Randomized trials addressing the impact of different ATGs on lymphocyte subpopulations in the clinical setting are urgently warranted.

A novel method to quantify and characterize leukemia-reactive natural killer cells in patients undergoing allogeneic hematopoietic stem cell transplantation following conventional or reduced-dose conditioning.

The antitumor activity of natural killer (NK) cells has recently been shown to be assessable at a single-cell level via flow cytometric detection of CD107a. We used this novel method to prospectively quantify and characterize tumor-reactive NK cells in patients undergoing myeloablative or nonmyeloablative conditioning and allogeneic hematopoietic stem cell transplantation (HSCT). Degranulation of NK cells in the peripheral blood of 34 patients after HSCT (day +30, day +90) was determined by evaluating CD107a expression after coincubation of the cells with tumor targets. The percentage of degranulating NK cells was higher after nonmyeloablative conditioning than after myeloablative conditioning (P<.001), indicating a higher activation state and increased antitumor activity of NK cells after nonmyeloablative conditioning. We were able to analyze NK cell subsets separately and found that CD56bright NK cells following HSCT are functionally different from CD56bright NK cells in healthy donors, as indicated by a high percentage of degranulating NK cells in response to tumor targets in patients after HSCT. The CD107a assay is a new and feasible method to quantify and characterize tumor-reactive NK cells after HSCT. Using this method, we found that NK cells had high antitumor cytotoxic activity after nonmyeloablative conditioning, which may contribute to the effectiveness of nonmyeloablative conditioning.

The anti-lymphoma effect of antibody-mediated immunotherapy is based on an increased degranulation of peripheral blood natural killer (NK) cells.

BACKGROUND: In patients treated with rituximab and alemtuzumab for lymphomas or CLL, antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action. Therefore, assessment of ADCC is mandatory to understand the complex mechanisms leading to the anti-lymphoma effects of monoclonal antibodies (mAb). Due to methodical difficulties, little is yet known about the relevant cell subpopulations and effector mechanisms leading to tumor lysis in ADCC. METHODS: We used a novel flow cytometric assay that detects CD107a as a marker for NK-cell degranulation to characterize and quantify peripheral blood natural killer (NK) cells mediating ADCC in vitro and in vivo. RESULTS: We observed specific and dose-dependent NK-cell activation after administration of rituximab and alemtuzumab. The number of degranulating NK cells was closely related to the concentration of mAb and the effector:target ratio. We were able to quantify and characterize the peripheral blood NK cells mediating ADCC. The majority of degranulating NK cells had the phenotype: CD56(dim), CD69(+), NKG2D(+), NKp30(-), NKp46(-), and CD94(-). Furthermore, we found that the CD107a assay can also visualize ADCC under clinical conditions as we observed increased numbers of NK cells degranulating in response to CD20(+) lymphoma cell lines in patients with non-Hodgkin's lymphoma treated with rituximab. CONCLUSIONS: We were able to quantify and characterize NK cells mediating ADCC with a new and feasible method. The CD107a assay may be useful for predicting treatment responses of individual patients and may help find the optimal dosage and timing for treatment with mAb.

Allogeneic hematopoietic cell transplantation (HCT) following reduced-intensity conditioning in patients with acute leukemias.

The majority of patients with acute leukemia enter complete remission following induction therapy, but relapse despite consolidation and maintenance chemotherapy. Allogeneic hematopoietic cell transplantation (HCT) is the most effective consolidation therapy but unfortunately associated with high transplant-related mortality (TRM). In order to decrease TRM but still apply a graft-versus-tumor effect, allogeneic HCT protocols with reduced-intensity conditioning were developed and more than 5000 HCT, of which 1500 for acute leukemia, performed. Detailed information is available on more than 400 patients with acute leukemia. The results, summarized in this article, confirm that reduced-intensity preparative regimens lead to full donor chimerism and to generation of graft-versus-leukemia (GvL) effects with curative potential in older patients (>60 years). Prospective-controlled clinical trials are needed in younger patients to compare results of HCT after reduced-intensity conditioning to those of HCT with conventional conditioning.

Mesenchymal stem cells remain host-derived independent of the source of the stem-cell graft and conditioning regimen used.

BACKGROUND: Human bone marrow contains hematopoietic stem cells and stroma cells known as mesenchymal stem cells (MSC). MSC are cells with the morphological features of fibroblasts, which, in addition to their nursing function for hematopoietic stem cells, retain the ability to differentiate into cartilage, bone, fat, muscle, and tendon and have an important immunmodulatory function. To understand in more detail hematopoietic engraftment and immune modulation after hematopoietic cell transplantation, we investigated the ability of donor MSC to engraft after hematopoietic cell transplantation in dependency to the conditioning regimen (myeloablative vs. reduced intensity) and source of the graft (bone marrow vs. peripheral blood). METHODS: Bone marrow MSC of 12 patients were analyzed, a median of 23.4 (range 0.9-137.8) months after human leukocyte antigen matched but gender mismatched bone marrow transplantation after myeloablative conditioning (n=4) or peripheral blood cell transplantation after myeloablative (n=4) or reduced intensity conditioning (n=4). MSC were characterized by morphology, positivity for CD 105+, CD73+, CD 44+, and CD 90+, and by their capacity to differentiate into adipocytic and osteogenic cells. Recipient and donor origins were determined by fluorescent in situ hybridization for sex chromosomes. RESULTS: While overall blood and bone marrow chimerism was 100% donor type, MSC remained in all patients of recipient origin (>96%). There was no difference between patients receiving bone marrow and peripheral blood grafts, nor was any difference observed between patients receiving full intensity in comparison with reduced intensity conditioning. CONCLUSIONS: We conclude that MSC remain of host type irrespective of the conditioning regimen and graft source.

Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation.

Allogeneic hematopoietic stem cell transplantation (SCT) regimens incorporating the lymphocytotoxic CD52 antibody alemtuzumab demonstrate efficient engraftment and reduced graft-versus-host disease (GVHD). However, these protocols substantially impair posttransplantation antiviral and antitumor immunity. To accelerate immune reconstitution after alemtuzumab-based reduced-intensity SCT, we administered prophylactic CD8-depleted donor lymphocyte infusions (DLIs) starting on days 60 and 120 after transplantation. DLIs were processed in an immunomagnetic good manufacturing practice depletion procedure resulting in a 2.5- to 6-log reduction in CD8 T cells. Of 23 high-risk patients with hematologic malignancies, 11 received a total of 21 CD8-depleted DLIs. Five patients developed transient grade I acute GVHD following transfer. Only 2 patients with HLA-C-mismatched donors showed grade II and III acute GVHD and subsequently progressed to limited chronic GVHD. Following DLIs, 4 patients with declining hematopoietic donor chimerism converted to full chimeras. A 2.1-fold median increase of circulating CD4 T cells was observed within 2 weeks after infusion. Non-DLI patients did not show a comparable rise in CD4 counts. Four patients demonstrated enhanced frequencies of cytomegalovirus-specific CD4 and CD8 T cells following transfer. Our results suggest that prophylactic CD8-depleted DLIs accelerate immune reconstitution after lymphodepleted HLA-matched SCT and carry a low risk of inducing severe GVHD.

The response of autologous T cells to a human melanoma is dominated by mutated neoantigens.

Our understanding of pathways leading to antitumor immunity may depend on an undistorted knowledge of the primary antigenic targets of patients' autologous T cell responses. In the melanoma model derived from patient DT, we applied cryopreserved short-term autologous mixed lymphocyte-tumor cell cultures (MLTCs) in combination with an IFN-gamma enzyme-linked immunospot (ELISPOT) assay to cDNA expression screening. We identified three previously unknown peptides processed from melanosomal proteins tyrosinase (presented by HLA-A(*)2601 and -B(*)3801) and gp100 (presented by HLA-B(*)07021) and five neoantigens generated by somatic point mutations in the patient's melanoma. The mutations were found in the genes SIRT2, GPNMB, SNRP116, SNRPD1, and RBAF600. Peptides containing the mutated residues were presented by HLA-A(*)03011, -B(*)07021, and -B(*)3801. Mutation-induced functional impairment was so far demonstrated for SIRT2. Within MLTC responder populations that were independently expanded from the patient's peripheral blood lymphocytes of different years, T cells against mutated epitopes clearly predominated. These results document a high degree of individuality for the cellular antitumor response and support the need for individualizing the monitoring and therapeutic approaches to the primary targets of the autologous T cell response, which may finally lead to a more effective cancer immunotherapy.