ALBERT

Description: Background The prophylactic use of systemic antifungal agents reduces morbidity and mortality after allogeneic hemopoietic stem cell transplantation (HSCT). However, the efficacy of this approach in pts receiving intensive chemotherapy or autologous HSCT is yet unproven. This trial was designed to evaluate the efficacy of L-AmB prophylaxis in high-risk neutropenic pts. Methods: 231 pts with hematological malignancies and expected neutropenia (N) of more than 10 days (d) following intensive chemotherapy or autologous HSCT were enrolled, 219 pts became neutropenic and were randomized to receive either 50 mg L-AmB i.v. every second d (arm A) or no systemic antifungal prophylaxis (arm B). Treatment with L-AmB started 1–3 d prior onset of N and was continued until neutrophil recovery, breakthrough IFI, intolerable toxicity or death. The level of significance was 0.05 (two-sided) for all statistical analyses. Calculations were performed using commercially (SPSSWIN 12.0) software, the calculations for GEE were performed using the software MAREG. Results Pt. characteristics: Eligible pts 219; arm A: 110; arm B: 109. Reasons for exclusion were: Absence of N (8), infection prior N (3) and pts decision (1). Baseline characteristics were balanced for age (mean 53.8 years), underlying disease (119 AML, 27 ALL, 64 NHL, 9 other), duration of N (mean 14.8 D) and treatment modality (primary 149, secondary 42, transplant 28). Primary endpoint: The incidence of proven and probable IFI was 5 of 110 pts (4.6%) in arm A and 22 of 109 pts (20.2%) in arm B (p = 0.001, RR = 2.9, CI 1.3 – 6.5). Key secondary endpoints: Pneumonia of unknown origin occurred in 6 pts (5.5%) vs. 28 pts (25.7%) (p 〈 0.001), the incidence of possible, probable and proven IFI was 11 pts (10.2%) vs. 42 pts (39.6%) (p 〈 0.001), systemic antifungals were used in 24 pts (22%) vs. 64 pts (59%) (p 〈 0.001), and death occurred in 4 pts (3.7%) vs. 9 pts (8.2%) (p = 0.16) in arm A vs. arm B. Toxicity: No grade 3 or 4 toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not different between the treatment groups. Conclusion: Intermittent application of low dose L-AmB is save. The significant lower incidence of IFI in pts treated with L-AmB prophylaxis supports its use in prolonged N.

Description: Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p

Description: Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T], B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T]). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498], B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Description: Abstract 1335 Poster Board I-357 Alloreactive T cells are crucial for graft-versus-host-disease (GVHD) pathophysiology, and we hypothesized that controlling their trafficking can ameliorate GVHD. P-selectin is a dimeric glycoprotein found on most inflamed endothelium, which interacts with multiple lectin-type molecules on leukocytes, including T cells. We used murine allogenienc BMT models to study GVHD and found that P-selectin−/− recipients exhibited significantly less GVHD mortality and morbidity, as well as decreased GVHD of the skin, liver and small bowels. However, WT and P-selectin−/− allo-BMT recipients had comparable large bowel GVHD. This decrease in target organ and systemic GVHD was associated with diminished infiltration of alloactivated T cells into the Peyer's Patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLO) on day 14 and day 35 post-transplant. However, donor alloreactive T cells in WT and P-selectin−/− allo-BMT recipients had similar alloactivation and apoptosis, and donor alloactivated T cells from WT and P-selectin−/− allo-BMT recipients with GVHD showed similar proliferation in vitro in a mixed leukocyte reaction, suggesting that the inflammatory environment in WT and P-selectin−/− recipients was comparable. Finally, non-transplanted P-selectin−/− mice, and P-selectin−/− mice which had received the allo-BMT conditioning regimen but not a donor graft, had similar cellularity in the majority of tissues examined as corresponding WT controls. This suggests that the differential cellularity of donor alloactivated T cells in WT and P-selectin−/− allo-BMT recipients with GVHD is probably largely dependent on trafficking and tissue infiltration during inflammation. Since P-selectin glycoprotein ligand 1 (PSGL1) is the best-described P-selectin ligand, and all leukocytes constitutively bear high levels of membrane PSGL1, we next hypothesized that PSGL1−/− donor alloreactive T cells would be defective in trafficking into GVHD target organs, and that PSGL1−/− donor T cells would cause decreased target organ damage, systemic GVHD, and mortality. However, allo-BMT recipients of WT and PSGL1−/− donor T cells had comparable survival and clinical GVHD scores, and further analyses on day 14 post-transplant revealed that recipients of WT and PSGL1−/− donor T cells also had similar numbers of donor alloactivated T cells in the spleen, liver, mesenteric and peripheral lymph nodes, and Peyer's Patches. Additionally, WT and PSGL1−/− donor T cells had comparable proliferation as measured by CFSE dilution, and comparable alloactivation in vivo as determined by levels of CD25, CD44, and CD62L, suggesting similar T cell function. As PSGL1−/− and WT donor T cells appeared to have equal functionality and accumulated in GVHD target tissues and lymphoid tissues in a similar fashion, we asked whether PSGL1−/− T cells might display other P-selectin ligands. Flow cytometric analyses of T cells from non-transplanted PSGL1−/− mice, and analyses of PSGL1−/− alloactivated T cells on day 14 after allo-BMT, revealed that these cells displayed substantial levels of cell-surface P-selectin ligands as defined by positive staining with recombinant P-selectin-IgG-Fc fusion protein at levels similar to those found on WT T cells, suggesting that although absence of P-selectin on host tissues may ameliorate GVHD, multiple donor leukocyte P-selectin ligands interact meaningfully with P-selectin. Our studies suggest that P-selectin may be required for trafficking into inflamed tissues but not SLO, and that donor T cells may utilize multiple P-selectin ligands apart from PSGL1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable target for GVHD prophylaxis or treatment. Disclosures No relevant conflicts of interest to declare.

Description: Background: In vivo T cell depletion with ATG or Alemtuzumab is effective to reduce the incidence of graft-versus host disease (GVHD) caused by alloreactive T cells. However, there is also a potential impact of these substances on the function of natural killer (NK) cells who are the predominant cells in peripheral blood in the early phase after hematopoietic stem cell transplantation (HSCT) and mediate beneficial graft-versus-tumor activity. Using a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation, we investigated the effect of T cell depletion with ATG and Alemtuzumab on NK cell function in the early phase after HSCT. Methods: PBMCs of 34 patients (pts) at day +30 after allogeneic HSCT and of 16 healthy donors were coincubated at 37°C for 3 h with the NK sensitive cell line HL60. In each tube, containing 400μl effector/target suspension (2x106 cells), 20μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. After the first 1 h 10μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3) for flow cytometry. The percentage of CD107a expressing NK cells was assessed and the absolute number of degranulating NK cells/μl was calculated. Results: Treatment Characteristics: Fourteen pts received ATG, ten pts were treated with Alemtuzumab and ten patients did not receive T cell depletion. The source of donor was: MRD 12 and MUD 22. NK cell count: The median NK cell count was: 250/μl in healthy individuals, 250/μl in pts without T cell depletion, 400/μl in pts with ATG and 100/μl in pts receiving Alemtuzumab (p

Description: Background In a recently reported randomized trial (ASH meeting 2005, oral presentation #1310) low-dose intravenous L-AmB reduced the incidence of IFI (20.2% vs. 4.6%, p

Description: Introduction: Allogeneic stem cell transplantation (alloSCT) has become an integral part in the therapy of patients with malignancies of the lympho-hematopoietic system. One of the main reasons for treatment failure after alloSCT is relapse of the underlying disease, which, in the majority of cases, is associated with a poor prognosis. Adoptive immunotherapy by the use of donor lymphocyte infusions (DLI) was shown to be effective in this setting. However, the conditions and the optimal timing of DLI administration for prophylaxis or treatment of (impending) relapse remains controversial. Patients and Methods: We retrospectively analyzed 160 consecutive patients (median age: 48 (range: 17-69) years) who received DLI after previous alloSCT performed at our center between 1998 and 2014. Indications for alloSCT were: acute myeloid leukemia (AML) (N=68), acute lymphoblastic leukemia (ALL) (N=49), myelodysplastic syndrome/myeloproliferative neoplasia (MDS/MPN) (N=26), or myeloma/lymphoma (N=17). The disease risk index (DRI) was low (N=1), intermediate (N=101), high (N=43), or very high (N=6) (unknown: N=9). Comorbidities, as specified by the hematopoietic cell transplantation-specific comorbidity index (HCT-CI), were low (N=38), intermediate (N=79), or high (N=38) (unknown: N=5). In N=71 patients a 10/10 human leukocyte antigen (HLA) matched-related donor was chosen, whereas N=89 patients were transplanted from an unrelated donor, either matched (N=73) or mismatched (N=16). Conditioning was either myeloablative (MAC) (N=71) or reduced-intensity (RIC) (N=89). The median interval from alloSCT to first DLI was 7.1 (range: 1.0-93.2) months. Indication for DLI was prophylactic (e.g. high-risk of relapse or active disease at the time of alloSCT) (N=28), pre-emptive (e.g. persistent or increasing mixed chimerism/molecular relapse) (N=86), or hematologic relapse (N=46). Pre-treatment before DLI was none/cessation of immunosuppression (N=129), lymphodepleting chemotherapy (N=16), or other (N=15). The median number of DLI units given was 2 (range: 1 - 6) and the median cumulative CD3+ cell dose/kg body weight given was 1.1 x 10E7 (range: 5.0 - 16.0 x 10E7). Results: The median follow-up of all patients from day of alloSCT was 37.3 (range: 3.0 - 202.6) months, whereas the median follow-up from day of first DLI administration was 21.2 (range: 0.3 - 200.5) months. Overall survival (OS) of the entire cohort at 1, 3, and 5 years after alloSCT was 80.5%, 63.8%, and 57.7%. Calculated from the day of first DLI OS at the same time points was 68.8%, 61.0%, and 55.8%. At five years after alloSCT OS in the group of patients with AML or ALL was significantly lower as compared to patients with MDS/MPN or myeloma/lymphoma, i.e. 52.0% versus 66.2% (p=0.043). Furthermore, OS in the group of patients receiving pre-emptive DLI was virtually identical to patients who received prophylactic DLI, i.e. 70.4% versus 69.8% at 5 years. In contrast, patients with hematologic relapse prior to DLI had an inferior outcome, i.e. an OS of 23.3% at 5 years. In addition to indication for DLI administration, i.e. prophylactic or pre-emptive versus therapeutic, the occurrence of chronic graft-versus-host disease (cGvHD) was the strongest predictor for outcome, i.e. long-term survival. Conclusions: Taken together, our data indicate that adoptive immunotherapy by the use of DLI is capable of inducing long-term remissions in patients after alloSCT. As pre-emptive and prophylactic treatment yielded virtually identical results, latter may be reserved for selected patients with (very) unfavorable disease characteristics, e.g. AML or ALL with active disease at the time of transplant. The optimal type of pre-treatment needs to be determined by investigating larger patient cohorts. Disclosures No relevant conflicts of interest to declare.

Description: Angiogenesis and inflammation are two closely related processes and inhibition of angiogenesis can ameliorate inflammatory diseases by reducing the recruitment of tissue infiltrating leukocytes. However, there is limited evidence on initial mechanisms of both processes and it is unknown if angiogenesis contributes to initiation of inflammation or is a mere consequence of inflammation. Recent research showed that graft-versus-host disease (GVHD) is associated to angiogenesis making the endothelium a potential new therapeutic target during allogeneic stem cell transplantation (bone marrow transplantation, allo-BMT). We studied kinetics and quantity of angiogenesis and leukocyte infiltration in murine acute GVHD models (LP/J→B6, B6→Balb/c, B6→BDF and 129S2/Sv→B6) by assessing CD31, CD4, CD8, CD11b by immune staining and FACS analysis. In GVHD target organs colon, skin and liver, we found significant increased vascular density and endothelial cell (EC) number but no leukocyte infiltration as early as day+2 after BMT (Fig. 1a,b). Leukocyte infiltration occurred secondary to angiogenesis at day+7 or later. No angiogenesis and inflammation occurred in the non-acute GVHD target organs joints, cardiac and skeletal muscle (Fig. 1c) suggesting a previously unrecognized role of the endothelium in GVHD organ tropism. Similar analyses in a standard model for inflammatory bowel disease, the 3% dextran sulfate sodium-induced (DSS) colitis, confirmed that angiogenesis preceded leukocyte infiltration in colon (d+1 vs. d+3) implicating that the significance of our results surpasses the field of transplantation biology. We aimed at identifying pathways that are relevant for initiation of angiogenesis during GVHD and first investigated the Vegfa/VEGFR1+2 axis. Based on our previous observation demonstrating a reduction of GVHD due to inhibition of neovascularization by anti-VE-cadherin antibodies (Penack et al, JNCI 2010), we performed therapeutic interventions with monoclonal blocking antibodies. Anti-VEGFR1+2 (DC101+MFI, Imclone) and anti-Vegfa (B20-4.1.1, Genentech) did not lead to GVHD reduction (Fig. 1d). Furthermore, we found no consistent upregulation of Vegfa and VEGFR2 expression levels in GVHD target organs during initiation of GVHD or later time points (Fig. 1e) suggesting that the Vefga/VEGFR2 pathway is not a major mechanism for initiation of angiogenesis in GVHD target organs. We next assessed known endothelial activation signs and performed qPCR analysis of adhesion molecules Icam1, Vcam1, E- and P-selectin (Fif. 1f) as well as immune staining of MHC-II (Fig. 1g). We confirmed previous knowledge that endothelial cells upregulate adhesion molecules and MHC-II during established GVHD. In sharp contrast, adhesion molecules and MHC-II were downregulated or not changed during the initiation of angiogenesis at day+2. This suggests that alternative pathways are important during initial angiogenesis in early GVHD. To identify alternative pathways during initial angiogenesis in an unbiased approach, we performed microarray analyses of FACS-sorted colon ECs (Fig. 2a) and LC-MS/MS proteome analyses of MACS-isolated liver ECs (Fig. 2c) at day+2 after allo-BMT vs. syn-BMT. Strikingly, we detected substantial metabolic and cytoskeletal changes in ECs of allo-BMT recipients (Fig. 2b,d). Recently, metabolic processes have been shown to be associated with cytoskeletal remodeling during EC migration and angiogenesis (Uebelhoer et al., J Vasc Res 2016). Accordingly, measuring single-cell mechanical characteristics with Real-Time deformabiliy cytometry (Otto et al., Nature Methods 2015) revealed profoundly higher deformation (D = 0.0547 ± 0.0011) and overall softening of ECs from allo-BMT recipients (Fig. 2e) indicating to enhanced EC migration and proliferation. We demonstrate that angiogenesis initiates GVHD in target organs and is restricted to target organs. Our findings implicate that endothelial biology plays a major role in GVHD organ tropism and disease development. We revealed novel genes and proteins regulating migration and proliferation of EC in initial angiogenesis during GVHD. Our study amends the knowledge on the interplay between the vasculature and inflammation opening a new window to develop therapeutic strategies targeting the endothelium. Disclosures No relevant conflicts of interest to declare.

Description: Endothelial dysfunction has been shown to be associated with severe complications and increased NRM after alloSCT. Endothelial risk markers, such as angiopoetin-2 (ANG2) serum levels and thrombomodulin single nucleotide polymorphisms can be used for pre-transplant prediction of endothelium-related complications after alloSCT (Blood 2011;118:1685; J Clin Oncol. The aim of the present study was to develop a tool for prediction of endothelial dysfunction prior to alloSCT that can be easily used in clinical practice. For this purpose, we focused on routine parameters which are used to diagnose transplant-associated thrombotic microangiopathy (TMA), which is an endothelial complication associated with high NRM. Based on the fact that TMA is defined by high creatinine, high lactate dehydrogenase (LDH), low thrombocyte counts, schistocytes, and loss of haptoglobin, we hypothesized that the simplified formula termed 'Endothelial Activation and Stress Index (EASIX)' might be valuable for predicting TMA, NRM and overall survival (OS) after alloSCT. Design: The capacity of pre-transplant EASIX ("EASIX-pre") obtained directly prior to conditioning for alloSCT for predicting TMA was tested retrospectively in 771 consecutive adult patients undergoing alloSCT in Heidelberg between 2001 and 2013 (training cohort) using cause-specific Cox regression analysis. The correlation of EASIX-pre with pre-transplant ANG2 and suppressor of tumorigenicity-2 (ST2) serum levels was assessed by Kruskal-Wallis test / Pearson correlation. The prognostic strength of EASIX-pre for NRM, time to relapse (TTR) and OS was calculated in the training cohort and in three independent validation cohorts (Berlin adults n=386, Seattle adults n=450 and Cincinnati children n=247) by calculating the prediction error (integrated Brier score), concordance index, and calibration index. Different intensities of conditioning (MAC+RIC+non-MAC) as well as all donor types and all degrees of HLA-matching were included. Hazard ratios (HR) were estimated to illustrate the effect of a two-fold change in EASIX-pre. Results: In the training cohort, EASIX-pre was a significant risk factor for TMA in univariable (HR=1.24, p=0.03) and in multivariable models including age, disease score, ATG, donor sex, recipient sex, graft source, diagnosis and statin intake as covariates (HR 1.28, p=0.02). EASIX-pre correlated with pre-transplant serum levels of the endothelium-related markers ANG2 and ST2. Increasing EASIX-pre was significantly associated with increasing NRM (uni: HR 1.22, p

Description: Background: Recent data suggest that NK cell mediated antibody dependent cellular cytotoxicity (ADCC) is a major mechanism of action of the anti-CD20 monoclonal antibody (mAb) rituximab and the anti-CD52 mAb alemtuzumab, which are frequently applied in patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. However, the exact mechanisms leading to NK cell activation are not completely understood and the cytotoxic subpopulation of peripheral blood NK cells mediating ADCC remains to be defined. In order to quantify and characterize the NK cells mediating ADCC, we used a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation. Methods: PBMCs from healthy individuals were coincubated at 37°C for 3 h with different human leukemia and lymphoma cell lines. In each tube, containing 200μl effector/target suspension (4x105 cells), 15μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. To assess antibody dependent cellular cytotoxicity (ADCC) saturating concentrations (10μg/ml) of rituximab or alemtuzumab were used. After the first 1 h 5μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3, NKG2D, CD69, CD94, NKp30, NK46) for flow cytometry. NK cell-mediated cytotoxicity (specific lysis) was analyzed by flow cytometric detection of propidium iodide uptake. Results: After coincubation with NK sensitive K562 cells up to 6% of CD56+ cells expressed CD107a, indicating that a subpopulation of NK cells releases cytotoxic granules after contact with these target cells. In contrast, coincubation with NK-resistant leukemia cells (ML2, EHEB, DAUDI, RAJI, AM0-1, YT-1) was not followed by an increased surface expression of CD107a. However, when rituximab was added to CD20+ lymphoma or leukemia cells (EHEB, DAUDI, RAJI) we observed that up to 15% of NK cells expressed CD107a after coincubation. In contrast no increased CD107a surface expression was observed when rituximab was added to the CD20− cell lines AMO-0 and YT-1, which excludes unspecific NK cell activation. When alemtuzumab was added to the CD52+ cell lines AMO-1, DAUDI, EHEB, RAJI and YT-1, surface expression of CD107a on NK cells was increased considerably. The majority of degranulating NK cells had the phenotype: CD56dim, 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. The number of degranulating NK cells was closely related to the concentration of rituximab and the effector:target ratio, showing a maximum at a ratio of 1:1 and concentrations above 5μg/ml. CD107a surface expression and specific lysis demonstrated a strong positive correlation (r2 = 0.99), confirming that NK cell cytotoxicity can be assessed by this method. Conclusion: The CD107a assay represents a promising new method not only for assessment of natural cytotoxicity on a single cell level but also for determination of ADCC in vitro and in patients treated with mAb. In clinical practice, it may help to find optimal doses and time schedules for the treatment with different mAbs.