Natural Killer Cell Recovery Research Articles

Natural Killer (NK) Cell Recovery After Bone Marrow Transplantation in Mice: Early Emergence of a Novel Ly49+ Stage in Differentiation Independent of MHC Haplotype.

Abstract 4477NK cells play critical roles in resistance to viral infections and in cancer. NK cells exist as subpopulations of cells that bear inhibitory and/or activating receptors to MHC. In mice, these are the Ly49+ subsets which have been shown to play a role in the rejection of bone marrow allografts as well as resistance to CMV. These Ly49 receptors also play a central role in the “education”, “arming” or “licensing” of NK cells. NK cell recovery after bone marrow transplantation is thought to proceed quickly but the acquisition of the Ly49 receptors has been incompletely characterized. NK cell differentiation has for the most part been characterized through in vitro models. We therefore examined the recovery of NK cells after syngeneic BMT in mice and assessed the effects of MHC on the recovery of the Ly49+ subsets using congenic recipients. NK cells were rapidly detected in mice as early as 14 days after BMT. Interestingly, the dominant population observed had a mature NK cell phenotype based on the expression of CD94, CD43, CD11b, etc. However, the majority of NK cells had a unique expression of Ly49G2 single positive NK cells with median fluorescence intensity (MFI) approximately twice as high as on NK cells from normal animals. This phenotype was not observed in untreated normal mice and occurred in B6 and B10 (H2b) as well as B10.D2 (H2d) strain mice showing independence from MHC. When these cells were sorted and assessed for cytotoxic activity, the cells had comparable killing efficiency as NK cells from untreated mice. Over time, NK cells with normal Ly49 subset distribution and phenotype were observed with the disappearance of this earlier subset. However, total splenic NK cell numbers remained significantly reduced in the BMT recipients as long as 120 days after BMT and despite normal recovery and numbers of T cells. These results demonstrate the existence of novel stage(s) in NK cell differentiation that occur independently of MHC and that can be characterized by sole expression of Ly49G2 at high levels. However, deficits in NK cell numbers persist in mice for extended periods of time. These findings suggest that NK cell recovery may occur in waves with some inhibitory receptors programmed to repopulate first and that surface expression intensity also needs to be assessed. Furthermore, the long-term deficits in total numbers suggest that overall activity may be compromised for extended periods clinically post-BMT. Disclosures:No relevant conflicts of interest to declare.

Results of a Phase II Study of Haploidentical Hematopoietic Cell Transplantation (HHCT) in Adults Using Reduced Intensity Conditioning and CD3/CD19-Depleted Grafts: Clinical Outcome and Immune Reconstitution.

Abstract 1203Poster Board I-225HHCT using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion is of low toxicity and enables fast engraftment even with grafts of low CD34 content. Immune reconstitution may be improved compared to graft CD34 selection due to the cotransplantation of facilitating cells, CD34- progenitors, dendritic cells and natural killer (NK)-cells. A prospective multicenter phase II study of HHCT using CD3/CD19-depleted grafts after RIC with fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) was initiated. No post grafting immunosuppression was applied if the graft contained <5×104 CD3+ cells/kg. 60 adults with median age of 45 years (range, 19-61) have been enrolled from 2002 to 2009. Diagnoses were AML (n=37), ALL (n=8), NHL (n=6), myeloma (n=4), CML (n=3), CLL (n=1) and MDS (n=1). Patients were “high risk” because of refractory disease (n=6), cytogenetics (n=5), chemosensitive relapse (n=26) or relapse after prior HCT (allo=16, auto=6, both=1). At HHCT, 30 patients were in CR and 30 in PR. Grafts contained median of 6.8×106 (range, 3.5-22) CD34+ cells/kg, 4.0×104 (range, 0.9-44) CD3+ T cells/kg and 2.8×107 (range, 0.00-37.3) CD56+ cells/kg. Engraftment was rapid with median of 12 days to >500 granulocytes/μL (range, 9-50) and 11 days to >20.000 platelets/μL (range, 7-38). Incidence of grade II-IV acute GVHD and chronic GvHD (cGvHD) was 47% and 15%, respectively. Non-relapse-mortality on day 100 was 25% and 44% for the whole study period. Current overall survival (OS) is 18 of 60 patients (30%) with median follow-up of 525 days (range, 21-1542) of patients alive resulting in a Kaplan-Meier estimate 1-year OS of 41% and 2-year OS of 24%. Kaplan-Meier estimate 1-year OS was 40 % in AML, 38 % in ALL and 67% in NHL patients. No positive impact of KIR-mismatch on survival even in the subgroup of patients with AML was observed. Patients with cGVHD had a trend toward better survival with 56% vs. 39% (p=0.09). 14 patients died because of infections. Detailed immune reconstitution was analyzed in 24 patients. NK-cell engraftment was fast with median of 248 CD16+56+CD3- cells/μl (range, 1-886) on day 20. Increased natural cytotoxicity receptors (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells in the first 3 months. T-cells regenerated delayed with median of 191 CD3+ cells/μl (range, 38-799) on day 100. A slow reconstitution of CD8+ and CD4+ T-cells with median of 66 CD8+ (range, 8-170) vs. 70 CD4+ cells/μl (range, 12-301) on day 100 and 157 (range, 32-379) vs. 181 cells/μl (range, 19-980) on day 400 was observed. The subset of memory T-cells regenerated faster compared to naïve T-cells with median of 25 CD4+45RA+ (range, 4-109) vs. 80 CD4+45RO+ cells/μl (range, 0 to 255) and 46 (range 7-191) vs. 164 cells/μl (range, 66-323) on days 80 and 250, respectively. T-cell repertoire was skewed with oligoclonal T-cell expansion to day 100 and normalization after day 200. B-cell reconstitution was slow with median of 8 (range, 0-407) CD19+20+ cells/μl on day 100. 6 of these 24 patients received donor-lymphocyte-infusions for relapse or mixed chimerism resulting in acceleration of immune recovery in T- and NK-cells. In conclusion, HHCT using RIC and CD3/CD19 depleted grafts is of low toxicity and allows fast engraftment even with low doses of CD34 cells. Overall survival seems promising in a high risk patient cohort. Recovery of NK cells occurs early and fast. KIR receptor expression remains low in the first 3 months. T- and B-cell reconstitution is delayed but seems faster compared to published data after CD34 selection. To evaluate the treatment protocol earlier during the course of disease a new study has just been initiated. Disclosures:Off Label Use: The use of Fludarabine, Thiotepa, Melphalan and OKT-3 in the conditioning is off-label-use.

Impact of Early Lymphocyte Recovery On Transplant Outcome After Cord Blood Transplantation.

Abstract 3386Poster Board III-274 Background:Previous studies have shown that rapid recovery of the absolute lymphocyte count (ALC) is associated with improved transplant outcomes after related and unrelated donor allogeneic stem cell transplantation (allo-SCT). Natural killer (NK) cells are the first lymphocytes to recover after allo-SCT and comprise the majority of peripheral blood lymphocytes after transplantation. Rapid recovery of NK cells at 1 month post allo-SCT has been associated with rapid neutrophil and platelet engraftment and decreased non-relapse mortality. Studies have shown that prolonged T-cell lymphopenia and compensatory expansion of B and NK cells occurs early after cord blood transplantation (CBT). However, no consistent link has been reported between lymphocyte/NK cell recovery and transplant outcome after CBT. Methods:Records from 40 CBT patients at our institution (3/2006-6/2009) were reviewed to determine the impact of lymphocyte recovery on transplant outcome in an IRB-approved study. The ALC was calculated based on the total white cell count and automated differential. Common transplant outcome variables (graft failure, acute and chronic graft versus host disease [GVHD], relapse, non-relapse mortality [NRM] and survival) were studied in relation with ALC at 1, 2, 3 months post-CBT. ALC was analyzed as a continuous variable and also as a dichotomous variable with two distinct groups based on transplant associated outcomes. Results:The majority of patients (n=33, 83%) received CBT for hematological malignancies. The median age was 22 years (range: 0.6-64), and 24 (60%) were male. Disease status at transplantation included 29 (73%) high risk and 8 (20%) standard risk hematological malignancies. Conditioning consisted of myeloablative regimens in 28 (70%) (TBI=21, non-TBI=7). Fifty percent of patients received thymoglobulin with their conditioning regimen. All patients received calcineurin inhibitors and mycophenolate for GVHD prophylaxis. Nineteen patients (48%) received two cord blood units. The median nucleated cell dose was 4.1 × 10 7 cells/kg (range, 2.2-27.3). Five (13%) patients experienced graft failure. Acute GVHD (grade II-IV) occurred in 30 (75%) patients; chronic GVHD occurred in 15 of 31 patients (48%) surviving beyond 100 days. Six (15%) experienced relapse and 9 (23%) died from non-relapse causes. At the time of analysis, 26 patients (65%) were alive with a median follow-up for surviving patients of 422 days (range 51-1256). Patients with ALC<100/μl at 1 month post-CBT showed increased graft failure (4 of 10 vs. 1 of 29; p=0.011). Patients with ALC>150/μl at 1 month post-CBT had decreased NRM (0 vs. 37%±10%, p=0.011) and improved survival (46%± 32% vs. 41%±13%, p=0.013) (Figure 1). There was no impact of ALC at 1, 2, or 3 months post-CBT on relapse or acute or chronic GVHD. There was no relationship between age, type of conditioning regimen, disease risk, number of cord units (one vs. two) or nucleated cell dose on ALC recovery. Conclusion:Our results indicate that ALC 1 month post-CBT is a surrogate marker for engraftment, and that low ALC (<150/μl) identifies an “at-risk” population of patients after CBT. These results point to the importance of developing patient-specific strategies to improve outcomes in those who fail to achieve satisfactory lymphocyte counts in the first month post-CBT. A limitation of this study is the small sample size, and validation in larger prospective multicenter CBT studies is warranted. [Display omitted] Disclosures:Jagasia:Genzyme: Research Funding.

Characterization of Early Natural Killer Cell Reconstitution Following Autologous Transplantation in Multiple Myeloma.

Abstract 4641 BackgroundDespite the advent of novel therapies, multiple myeloma (MM) remains incurable and high-dose chemotherapy (HDC) with autologous stem cell transplantation (SCT) remains an important therapy for this incurable malignancy. Prior research has suggested that early lymphocyte immune reconstitution following HDC/SCT may be associated with improved outcomes; however, little is known regarding the biologic mechanisms underlying this phenomenon. A potent natural killer (NK) cell versus MM effect has also been demonstrated by our group and others previously, and NK cells are the first lymphocyte subset to recover following HDC/SCT. Herein, we characterize the nature of this recovery to suggest that the early post-transplant period may be an especially favorable environment in which to augment the NK cell versus MM effect to improve outcomes for patients with MM undergoing HDC/SCT. MethodsWith informed consent and under institutional review board approval, peripheral blood samples from patients with multiple myeloma undergoing HDC/SCT were collected on the day of first peripheral white cell count > 1500/uL post- HDC/SCT. All patients received intravenous melphalan (200mg/m2) as the preparative regimen. Mononuclear cells were isolated and, by flow cytometry, CD56(+),CD3(-) NK cells were comprehensively immunophenotyped for expression of inhibitory and activating ligands. NK cell activation and cytotoxicity were assessed by ELISPOT and chromium release assays. Clinical outcome was assessed as a function of early NK cell recovery. Results11 patients (median age 57, 8 male, 3 female) with a median of 1 prior regimen to transplant (range 1-9) have been assessed to date. 1 patient was mobilized with AMD-3100, 3 had cyclophosphamide mobilizations and 7 were mobilized with neupogen alone. The average CD34(+) cell dose received was 5.22 (+/- SD 2.39). The median day of peripheral white cell recovery was day +12 (range 11-18). The median absolute lymphocyte count on sample procurement day was 410 (range 340 – 820), of which an average of 37% (range 13 – 70) were NK cells. Although total white count on procurement day correlated with CD34(+) cell dose (p = 0.02), neither absolute lymphocyte count or NK cell count correlated significantly with CD34(+) cell dose and mobilization strategy did not affect NK cell count. NK cell reconstitution correlated with best response to HDC/SCT (p=0.02), the average NK cell count for patients ultimately achieving CR was 389 (+/- 131) as compared to those achieving PR or less (171 +/- 101). Interestingly, nearly half of reconstituting NK cells (46% +/- SD 20) expressed CD117 (c-kit receptor), a marker shown previously to be associated with earlier stages of NK cell development. Inhibitory KIR (CD158a on 36% +/-17 and CD158b on 36% +/- 24) and NKG2A (76% +/- 10) expression also seemed restricted to the CD117(+) NK cells. This population of NK cells also displayed NKG2D (25% +/- 22) and CD16 (73% +/-8). Reconstituting NK cells were capable of interferon gamma production against MM tumor targets similar to NK cells isolated from healthy donors (E:T = 20:1, 956 +/- 98 spots/well versus 896 +/-85, p = n/s). Granzyme B production against MM tumor targets also compared to normal controls (E:T = 20:1, 547 +/- 66 versus 573 +/- 50, p= n/s). Moreover, reconstituting NK cells showed enhanced cytotoxicity against MM tumor targets when pre-treated with an inhibitory KIR blocking antibody (IPH2101/1-7F9) similar to healthy donor NK cells. DiscussionTaken together, we provide quantitative and qualitative data to suggest that early NK cell recovery post-SCT may contribute to outcome in patients with MM. This may be an optimal period to implement immunomodulatory strategies to augment NK cell function to improve outcomes for patients with MM undergoing HDC/SCT. Reconstituting NK cells, although exhibiting a relatively unique immunophenotype compared to mature NK cells in healthy, normal donors, appear to retain properties of activation and cytotoxicity. Given the favorable effector:target ratio in vivo following SCT, these results support further research into immunotherapeutic manipulation of the reconstituting NK cell clone to improve efficacy of HDC/SCT in patients with MM. Disclosures:Andre:Innate Pharma: Employment. Squiban:Innate Pharma: Employment. Romagne:Innate Pharma: Employment.

5-Aza-2'-Deoxycytidine Has a Biphasic Effect On Ex Vivo Expanded Natural Killer Cells.

Abstract 2664Poster Board II-640Adoptive transfer of activated autologous or allogeneic natural killer (NK) cells has emerged as a safe and potentially efficacious immunotherapy for cancer. Alloreactive HLA haploidentical NK cells in the HSCT setting have been reported to enhance engraftment, reduce GVHD and prevent relapse of leukemia. The main restrictions to developing NK cells for immunotherapy are the limited quantity of cells available for adoptive transfer and the modulation of NK cell receptors and/or NK cell ligands as a recognized mechanism of immune escape in oncogenesis. To address the first hurdle, we have developed an efficient method to expand CD3−CD56+ primary NK cells in vitro using K562 artificial APCs expressing membrane-bound IL-21. Altering the balance of activating and inhibitory signals with immunosensitizing drugs is an attractive approach to addressing the second hurdle of NK cell therapy. DNA methylation inhibitors such as 5-Aza-2'-deoxycytidine and 5-azacytidine are being tested for their activity in AML, and have been shown to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct immunologic effect of these hypomethylating agents on NK cell function, reconstitution, and phenotype.Using the calcein release assay, we investigated NK cell survival and lytic function against the NK cell-sensitive HLAnull 721.221 cells after exposing expanded NK cells to 5-Aza-2'-deoxycytidine. Human peripheral blood NK cells that had been expanded ex vivo in our laboratory were incubated with a wide range of 5-Aza-2'-deoxycytidine concentrations for 3 days in the presence of 50 IU/mL IL-2. Cell viability was tested every 24 h.We found that 5-Aza-2'-deoxycytidine at higher concentrations resulted in reduced NK cell viability, but at lower concentrations inhibited NK cell killing. At these lower doses, NK cell inhibition was not associated with loss of viability, resulting in an overall U-shaped dose response curve to 5-Aza-2'-deoxycytidine with maximum inhibition in the 0.3 – 2.5 micromolar range.This biphasic response to 5-Aza-2'-deoxycytidine matches that previously described for malignant cells in which reduced proliferation at higher concentrations resulted in reduced demethylation inhibition (Qin et al, Clin Cancer Res. 2007 Jul 15;13(14):4225–32). To assess the role of demethylation in NK cells in a more dynamic setting, we are investigating 5-Aza-2'-deoxycytidine effects on NK cell proliferation and phenotype during ex vivo expansion, and assessing NK cell recovery, phenotype, and function in a prospective cross-over trial of patients receiving 5-azacytidine post HSCT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Improving the outcome of cord blood transplantation: use of mobilized HSC and other cells from third party donors

We developed the strategy of umbilical cord blood transplants (UCBT) with co-infusion of a limited number of highly purified mobilized haematopoietic stem cells (MHSC) from a human leucocyte antigen (HLA) unrestricted third party donor (TPD). Short post-transplant periods of neutropenia were usually observed in adults with haematological neoplasms receiving UCBT with a relatively low cell content and 0-3 HLA mismatches after myeloablative conditioning. This resulted from an early and initially predominant engraftment of the TPD-MHSC. After a variable period of double complete TPD + UCB chimerism, final full UCB chimerism was achieved (cumulative incidence >90%) within 100 d. Early recovery of the circulating neutrophils resulting from the 'bridge transplant' of the TPD-MHSC reduced the incidence of serious neutropenia-related infections, also facilitating the use of drugs with myelosuppressive side effects to combat other infections. The observed incidence of graft-versus-host disease and relapses was low, with overall and disease-free survival curves comparable to those of HLA identical sibling transplants. Post-transplant recovery of natural killer cells occurred soon after the transplant and B cells recovered around 6 months, but T-cell recovery took more than 1 year. Available data show that T cell recovery derives from UCB-HSC through thymic differentiation and that cytomegalovirus (CMV)-specific lymphocytes develop following CMV reactivations.

Day 15 Interleukin 15 Levels Affect Natural Killer Cell Recovery After Autologous Stem Cell Transplantation

Recently, we reported in a prospective study (Porrata et al. Biology of Blood & Marrow Transplantation 2008;14(7):807–16) that the absolute number of natural killer (NK) cells at day 15 post-autologous stem cell transplantation (ASCT) affects survival. Factors affecting NK cell recovery at day 15 post-ASCT are not completely understood. Interleukin-15 (IL-15) is a cytokine associated with NK cell differentiation and homeostasis. Thus, we studied the role of day 15 IL-15 on NK cell recovery post-ASCT. From February 2002 to February 2007, 50 non-Hodgkin lymphoma (NHL) patients enrolled in the study. We identified higher levels of day 15 IL-15 compared with normal controls and pre-ASCT IL-15 levels [normal IL-15 levels: median = 0 pg/ml (range: 0–3.37 pg/ml); pre-ASCT IL-15 levels: median = 0.09 pg/ml (range: 0–229.42 pg/ml); and day 15 IL-15: median = 5.48 pg/ml (range: 0–36.94), p < 0.0001)]. ROC curve and AUC analysis showed that day 15 IL-15 was a significant marker for NK cell recovery (AUC = 0.87, p < 0.0001). The best cutoff level was 9.5 pg/ml. Groups (≤9.5 or >9.5 pg/ml) were balanced in regard to histology (p = 0.48); gender (p = 0.8); stage (p = 0.7); LDH (p = 0.5); disease status prior to ASCT (p = 0.9); performance status (P = 0.1); and extra nodal involvement (p = 0.4). We identified an inverse correlation between day 15 IL-15 and day 15 NK cell recovery (r = −0.5, p< 0.0001). Patients with a day15 IL-15 ≤ 9.5 pg/ml experienced higher numbers of NK cells compared with those with a day 15 IL-15 > 9.5 pg/ml [median 142 cells/μl (range: 10–416 cells/μl) versus median 57.5 cells/μl (range: 28–999 cells/μl), p < 0.001, respectively]. Using logistic regression models, univariately day 15 IL-15 was a predictor for NK cell recovery (p < 0.0003) and multivariately day 15 IL-15 remained a predictor for NK cell recovery (p < 0.0004). To our knowledge, this is the first study showing an association between day 15 IL-15 and NK cell recovery in ASCT. We showed an inverse relationship between day 15 IL-15 and NK cell numbers consistent with a homeostatic association. The data presented here suggest that day 15 NK cell recovery might be driven by the cytokine milieu post-ASCT. Recently, we reported in a prospective study (Porrata et al. Biology of Blood & Marrow Transplantation 2008;14(7):807–16) that the absolute number of natural killer (NK) cells at day 15 post-autologous stem cell transplantation (ASCT) affects survival. Factors affecting NK cell recovery at day 15 post-ASCT are not completely understood. Interleukin-15 (IL-15) is a cytokine associated with NK cell differentiation and homeostasis. Thus, we studied the role of day 15 IL-15 on NK cell recovery post-ASCT. From February 2002 to February 2007, 50 non-Hodgkin lymphoma (NHL) patients enrolled in the study. We identified higher levels of day 15 IL-15 compared with normal controls and pre-ASCT IL-15 levels [normal IL-15 levels: median = 0 pg/ml (range: 0–3.37 pg/ml); pre-ASCT IL-15 levels: median = 0.09 pg/ml (range: 0–229.42 pg/ml); and day 15 IL-15: median = 5.48 pg/ml (range: 0–36.94), p < 0.0001)]. ROC curve and AUC analysis showed that day 15 IL-15 was a significant marker for NK cell recovery (AUC = 0.87, p < 0.0001). The best cutoff level was 9.5 pg/ml. Groups (≤9.5 or >9.5 pg/ml) were balanced in regard to histology (p = 0.48); gender (p = 0.8); stage (p = 0.7); LDH (p = 0.5); disease status prior to ASCT (p = 0.9); performance status (P = 0.1); and extra nodal involvement (p = 0.4). We identified an inverse correlation between day 15 IL-15 and day 15 NK cell recovery (r = −0.5, p< 0.0001). Patients with a day15 IL-15 ≤ 9.5 pg/ml experienced higher numbers of NK cells compared with those with a day 15 IL-15 > 9.5 pg/ml [median 142 cells/μl (range: 10–416 cells/μl) versus median 57.5 cells/μl (range: 28–999 cells/μl), p < 0.001, respectively]. Using logistic regression models, univariately day 15 IL-15 was a predictor for NK cell recovery (p < 0.0003) and multivariately day 15 IL-15 remained a predictor for NK cell recovery (p < 0.0004). To our knowledge, this is the first study showing an association between day 15 IL-15 and NK cell recovery in ASCT. We showed an inverse relationship between day 15 IL-15 and NK cell numbers consistent with a homeostatic association. The data presented here suggest that day 15 NK cell recovery might be driven by the cytokine milieu post-ASCT.

Rapid NKG2D Reconstitution on NK Cells Possibly Plays a Major Role in the Development of Graft-Versus-Leukemia Effect in the Early Period after Unrelated Cord Blood Transplantation.

Background: Cord blood transplantation (CBT) is being increasingly used for treatment of hematologic malignancies due to its potent anti-leukemic effects and the low incidence of graft-versus-host disease (GVHD). Recent study has shown that recovery of natural killer (NK) cells is prompt in the blood of CBT recipients both in numbers and functions during the first two months, suggesting that NK cells play an important role in the GVL effect in CBT. NKG2D is an activating receptor expressed by NK cells, which recognizes human MHC class I chain related A and B (MICA/B) and UL16-binding protein 1–4 (ULBP1-4) on target cells, and regulates NK cell activity. However, NKG2D reconstitution following CBT is presently unclear. In this study, we investigated the dynamic expression of NKG2D and other receptors on CD3−CD56+ NK cells in the early period after CBT.Material and methods: The study panel comprised 21 leukemia patients receiving allogeneic hematopoietic cell transplants. Of these, 10 received unrelated CBT and 11 received allogeneic transplants with peripheral blood (PB) combined with bone marrow (BM). To examine the recovery of NKG2D, and other receptors expressed by NK cells, flow cytometry was used to evaluate samples obtained from the recipients on day of clinical engraftment, 30, 60, and 90, respectively following transplantation.Results: Recovery of NK cells was prompt in both transplantation groups (CBT and PB combining with BM), which is consistent with the previous reports. Interestingly, there are significant differences in NKG2D reconstitution between CBT and PB combining with BM transplantation in early stage after transplantation. CBT recipients showed significantly increased expression of NKG2D compared to PB combining with BM transplantation recipients on day of clinical engraftment (79.58±8.71 VS 36.7±14.33 P<0.03 ) and day 30 after transplantation ( 80.03±13.49 VS 56.8±18.21 p<0.035 ). However there was no differences for NKG2D expression between the CBT and PB combined with BM transplantation groups on day 60(82.55±9.10 VS 82.3±28.64 p<0.01 )and on day 90 (85.82±18.21 VS 90.3±27.89 p<0.01), respectively. The inhibitory NK receptor NKG2A reconstitution occurred at the early period, and the expression was not significantly changed during the first three months in CBT, although the level was lower than the other transplantation group. The reconstitution of other inhibitory NK receptors (CD94, CD158a and CD158b) and activating receptor (NKp46) was not found statistically significant differences between the two transplantation groups. T and B cells reconstitution started gradually, and remained at low level at the end of investigation period in CBT recipients.Conclusion: NK cells contribute to GVL effects after transplantation maybe due to rapid NKG2D reconstitution. Rapid NKG2D reconstitution on NK cells in CBT recipients than blood combined bone marrow transplantation suggests that CBT appears superior to blood combined bone marrow transplantation in the treatment of leukemia patients.

Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL.Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry.Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets.Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

Temporal, quantitative, and functional characteristics of single-KIR–positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation

AbstractIn this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34+ selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR+ NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56bright/CD56dim NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56bright, and NKG2A and CD62L up-regulation on CD56dim, suggest sequential CD56bright-to-CD56dim NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR+ NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR+ NK cells selected from an alloreactive donor.

Effects of the NK Cell Recovery on Outcomes of Unmanipulated Haploidentical Blood and Marrow Transplantation for Patients with Hematologic Malignancies

The goal of this study was to investigate the association of natural killer (NK) cell recovery with clinical outcomes after unmanipulated haploidentical blood and marrow transplantation. We sequentially monitored the reconstitution kinetics of circulating NK cells, CD56 bright and CD56 dim, in 43 patients by flow cytometry, and the functionality recovery of cytokine or cytotoxicity of NK cells by flow cytometry or lactate dehydrogenase release assay after transplantation. Reconstitution of NK cells was rapid but accompanied by skewing of cell subsets mainly in CD56 bright, which recovered earlier. Linear regression analysis demonstrated that dose of CD34 + cells in the allografts was inversely correlated with the ratio of T/NK cells (β = −0.506, P = .003) and CD56 dim/CD56 bright cell (β = −.403, P = .018) by day 14 after hematopoietic stem cell transplantation (HSCT), and the dose of CD3 + T cells in the allografts was also inversely correlated with the ratio CD56 dim/CD56 bright cells by day 14 after HSCT (β = −0.474, P = .005). Moreover, the dose of CD56 dim NK cells in the allograft was positively associated with the day 14 CD56 brigh NK cells (β = 0.494, P = .032) and inversely correlated with the day 14 ratio of CD56 dim/CD56 bright cells (β = −0.617, P = .005). Compared with nonacute graft-versus-host disease (GVHD) patients, patients with acute GVHD (aGVHD) had a higher level of NK subsets during week 2 posttransplantation. Cox regression analysis revealed that the patients with more CD56 bright NK cells in the recovery stage had a higher survival rate (hazard risk [HR], 0.406; P = .017) and the patients with a higher ratio of T/NK (>1.0) had a higher chance of getting aGVHD (HR, 3.436; P = .059) and chronic GVHD (HR, 3.925; P = .028). Our results suggest that the recovery of NK cells is and can be used as an indicator to predicate the clinical outcomes after unmanipulated haploidentical transplantation.

Effect of cyclosporin A on interleukin-15-activated umbilical cord blood natural killer cell function

Interleukin (IL)-15-activated natural killer (NK) cells may provide a graft-versus-leukemia (GvL) effect post-umbilical cord blood (CB) transplantation. The effect of cyclosporin A (CsA), a calcineurin-inhibitor used for prophylaxis of graft-versus-host disease (GvHD), on IL-15-mediated activation, cytotoxic function and target-induced apoptosis of CB NK cells, was examined in comparison with adult peripheral blood (APB) NK cells. CsA was added to anti-CD3+/-IL-15-stimulated CB and APB mononuclear cells (MNC) for a 5-day incubation. CD3- CD56+ NK cell recovery was determined by flow cytometric analysis. Magnetic bead-purified CB and APB NK cells were stimulated with IL-15 for 18 h under the influence of CsA. NK activation (CD69), K562 cytotoxicity and NK-K562 interactions (CD54, perforin and annexin-V expression 4 h following contact with K562 cells) were assessed by flow cytometry. CsA decreased CD3- CD56+ NK cell recovery in anti-CD3-stimulated CB MNC 5-day cultures, an effect that could be counteracted by IL-15; comparable effects were observed with APB. Short-term (18-h) experiments revealed that CsA down-regulated K562 cytotoxicity of IL-15-activated (P=0.018) but not resting (P=0.268) purified CB NK cells. IL-15-induced CB NK CD69 expression showed increased CsA sensitivity over APB (P=0.012). CsA down-regulated K562 cell-induced CD54 (P=0.028) but not perforin (P=0.416) expression of IL-15-activated CB NK cells. Target-induced apoptosis of IL-15-activated CB (P=0.043) but not APB (P=0.144) NK cells was decreased by CsA. We have demonstrated differential CsA sensitivity of IL-15-activated CB and APB NK cells. These results may be used to improve the design of IL-15-activated NK cell adoptive immunotherapy in cancer patients receiving CsA post-CB transplantation.

Recovery of the NK Cells and Its Intending Clinical Roles after Unmanipulated Haploidentical Blood and Marrow Transplantation.

Objectives: The goal of this study was to investigate the association of natural killer (NK) cell recovery with clinical outcomes after unmanipulated haploidentical blood and marrow transplantation.Methods: We sequentially monitored the reconstitution kinetics of circulating NK cells, CD56bright and CD56dim, in 43 patients by flow cytometry, and the functionality recovery of cytokine or cytotoxicity of NK cells by flow cytometry or LDH release assay after transplantation.Results: Reconstitution of NK cells was rapid but accompanied by skewing of cell subsets mainly in CD56bright, which had a greater growing speed. Linear regression analysis demonstrated that dose of CD34+ cells in the allografts was inversely correlated with the ratio of T/NK cells (β=−0.506, P=0.003) and CD56dim/CD56bright cell (β=−0.403, P=0.018) by day 14 after HSCT, and dose of CD3+ T cells in the allografts was also inversely correlated with the ratio CD56dim/CD56bright cells by day 14 after HSCT (β=−0.474, P=0.005). Compared with non-acute graft-versus-host disease (aGVHD) patients, patients with aGVHD had a higher level of NK subsets during week 2 post-transplantation. Cox regression analysis revealed that the patients with more CD56bright NK cells in the recovery stage had a higher survival rate (hazard risk [HR], 0.353; p=0.019) and the patients with higher ratio of T/NK (>1.0) had a higher chance of getting aGVHD (HR, 3.889; p=0.034) and cGVHD (HR, 3.925; p=0.028).Conclusions: Our results suggest that the recovery of NK cells is and can be used as an indicator to predicate the clinical outcomes after unmanipulated haploidentical transplantation. [Display omitted]

106-P: Interaction of HLA KIR matching/mismatching with natural killer cell recovery in T cell depleted HLA identical stem cell transplantation

106-P: Interaction of HLA KIR matching/mismatching with natural killer cell recovery in T cell depleted HLA identical stem cell transplantation

Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia

Natural killer (NK) cells are the first lymphocytes to recover after allogeneic stem cell transplantation (SCT) and can exert powerful graft-versus-leukemia (GVL) effects determining transplant outcome. Conditions governing NK cell alloreactivity and the role of NK recovery in sibling SCT are not well defined. NK cells on day 30 post-transplant (NK30) were measured in 54 SCT recipients with leukemia and donor and recipient killer immunoglobulin-like receptor (KIR) genotype determined. In univariate analysis, donor KIR genes 2DL5A, 2DS1, 3DS1 (positive in 46%) and higher numbers of inhibitory donor KIR correlated with higher NK30 counts and were associated with improved transplant outcome. NK30 counts also correlated directly with the transplant CD34 cell dose and inversely with the CD3+ cell dose. In multivariate analysis, the NK30 emerged as the single independent determinant of transplant outcome. Patients with NK30 >150/microl had less relapse (HR 18.3, P=0.039), acute graft-versus-host disease (HR 3.2, P=0.03), non-relapse mortality (HR 10.7, P=0.028) and improved survival (HR 11.4, P=0.03). Results suggest that T cell-depleted SCT might be improved and the GVL effect enhanced by selecting donors with favorable KIR genotype, and by optimizing CD34 and CD3 doses.

Good manufacturing practices production of natural killer cells for immunotherapy: a six‐year single‐institution experience

Natural killer (NK) cells, a subset of lymphocytes and part of the innate immune system, play a crucial role in defense against cancer and viral infection. Herein is a report on the experience of clinical-scale, good manufacturing practices (GMPs) production of NK cells to treat advanced cancer. Two types of NK cell enrichments were performed on nonmobilized peripheral blood mononuclear cell apheresis collections with a cell selection system (CliniMACS, Miltenyi): CD3 cell depletion to enrich for NK cells and CD3 cell depletion followed by CD56 cell selection to obtain a more pure NK cell product. After overnight incubation with interleukin-2 (IL-2), cells were washed, resuspended in 5 percent human serum albumin, and then released for infusion. A total of 70 NK cell therapy products have been manufactured for patient infusion since 2000. For the CD3 cell-depleted NK cell products, the mean purity, recovery, and viability were 38, 79, and 86 percent, respectively. For the CD3 cell-depleted/CD56 cell-enriched NK cell products, the mean purity, recovery, and viability were 90, 19, and 85 percent, respectively. Gram stain, sterility, and endotoxin testing were all within acceptable limits for established lot release. Compared to the resting processed cells, IL-2 activation significantly increased the function of cells in cytotoxicity assays. Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.

Total Lymphocyte and Natural Killer (NK) Cell Count Day 30 Post-Transplant Strongly Predict Transplant Outcome after T Cell Depleted Allogeneic Stem Cell Transplantation.

One hundred and fifty-seven patients with leukemia (80 CML, 48 AML/MDS, 29 ALL) received a T cell depleted myeloablative allogeneic stem cell transplantation (SCT) from an HLA-matched sibling between 09/1993–09/2005. Conditioning consisted of TBI (12–13.6 Gy) + cyclophosphamide (96) or cyclophosphamide and fludarabine (61). The stem cell source was G-CSF mobilized peripheral blood stem cell (PBSC) in 129 and bone marrow in 28 patients. T cell dose with graft ranged from 0.2 – 2 × 105/kg CD3+ cells. GVHD prophylaxis was with low dose cyclosporine (level 100–200 ng/ml) in 103 and standard dose in 54. Patients without ≥ grade II acute GVHD received 1–2 donor lymphocyte infusion of 107 CD3+ cells/kg between days 45 and 100. Absolute lymphocytes on day 30 (LC30) was available in 154 patients (3 patients died before day 30) and 54 day +30 post-transplant samples were available for lymphocyte subset analysis. Median lymphocyte count on day +30 (LC30) was 400/μl, (range 10–3295) and 150/μl, (range 6–1005) for CD56+, CD16+ CD3− NK cells (NK30). Statistical analysis was performed on SPSS14 software. Median age of the group was 34 years (range 10–56). 78 patients had standard risk (SR) disease in first remission or first chronic phase of CML. The remaining 79 had high risk (HR) disease. At the time of analysis 85 patients are (51.5±4%) are alive with median follow-up of surviving patients 1392 days (range 147–4208). Only 9 patients (3 above median LC30) developed aGVHD before day+30. Patients with ≥ median LC30 had significantly better transplant outcome: survival 71±5 vs. 36 ±5.5%, p<0.0001; DFS 71±5 vs. 31±5 %, p<0.0001; NRM 10±3.5 vs. 38 ±6%, p<0.0001; relapse 22±5 vs. 51±7.5%, p=0.004; ≥ II aGVHD 34 ±5 vs. 51±6%, p=0.05. In multivariate analysis only disease risk and LC30 emerged as independent prognostic factors: LC30 above 400/μl was associated with improved survival (RR 4.3), DFS (RR 4.5), less relapse (RR 10.3), NRM (RR 3.3) and aGVHD (RR 2.3). LC30 impacted on outcome of both HR and SR disease groups (Figure). LC30 and NK30 were highly correlated (r2– 0.45, p<0.0001) and NK30 above 150/μl was also associated with improved transplant outcome: In multivariate analysis of this subset of 54 patients, SR disease and NK30 emerged as the only independent factors with better outcome for NK30 >150/μl: higher survival (RR 3), and DFS (RR 3), less relapse (RR 4.8), less NRM (RR 3) and less aGVHD (RR 5.3). This study does not define whether LC30 is a surrogate for NK cell count or whether both are a surrogate for some other undetected prognostic factor. However the inverse relationship between NK count and aGVHD suggests an NK-mediated effect through elimination of host antigen presenting cells as has been described in mismatched SCT but also in HLA identical SCT by Cook et al ( Blood 2004, 103, 1521) Prospective studies to correlate transplant outcome with NK cell recovery and function after HLA identical SCT are indicated. [Display omitted]

Clinical-Scale Combined CD4/CD8 Depletion for Donor Innate Lymphocyte Infusion (DILI).

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection procedures holds significant promise for application in adoptive immunotherapy protocols. In vitro and in vivo studies demonstrated that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells and γδ T cells have potent anti-tumor activity without inducing Graft-versus-Host disease (GvHD). However, current clinical-scale approaches to reduce the risk of GvHD usually use T-cell depletion (such as CD34+ selection or CD3+ depletion). However, both procedures also deplete γδ T cells which may be advantageous in mediating Graft-versus tumor effects and augmenting innate immune response against infections. Here we present a new method for depletion of T cells with potential GvHD reactivity and simultaneously enrichment of innate lymphocytes (NK cells and γδ T cells) from standard leukapheresis products by using a single-step immunomagnetic protocol which efficiently depletes CD4+ and CD8+ αβ T cells under good manufacturing conditions (GMP). Efficiency of CD4+ and CD8+ T cell depletion from (unstimulated) leukapheresis products (n=6) containing up to 2.0 × 1010 white blood cells, was demonstrated by 4-color flow cytometric analysis (mean log depletion of CD4+ cells: 4.12 (3.17–4.9); mean log depletion of CD8+ cells: 3.77 (2.97–4.54)). In addition to efficient depletion of CD4+ and CD8+ cells, immunomagnetic CD4/CD8 depletion resulted in enrichment of NK cells and γδ T cells (mean NK cell recovery: 38%(19–72), mean γδ T cell cell recovery: 50%(34–79)). In vitro assays of the final product demonstrated that NK cells and γδ T cells preserved their proliferative and cytotoxic capacity. We conclude that simultaneous depletion of CD4+ and CD8+ cells is feasible and can be performed in large scale under GMP conditions with sufficient depletion efficacy of αβ T cells and recovery of functionally intact innate effector lymphocytes (NK cells and γδ T cells) for potential use in adoptive immunotherapy studies.

Early Reconstitution of Natural Killer Cells Following Non-Myeloablative Hematopoietic Stem Cell Transplantation Predicts the Development of Acute Graft-Versus-Host Disease.

Natural killer (NK) cells are among the first lymphocytes to recover following allogeneic hematopoietic stem cell transplantation (HSCT). Their levels may reflect both homeostatic and inflammatory processes. In this study we analyzed the early reconstitution of NK cells in fifty-one patients with hematologic malignancies after a reduced-intensity regimen consisting of fludarabine and cyclophosphamide and its relationship to the development of acute graft-versus-host disease (aGVHD) and cytomegalovirus (CMV) reactivation.NK cells were severely depleted by the fludarabine-cyclophosphamide conditioning regimen. On the day of the transplant, the NK levels had been reduced in all patients to 0 cells/microliter. Subsequent NK cell recovery was rapid. By two weeks post-transplant NK cells had recovered to median levels comparable those in the original hosts (median= 119 cells/microliter; range, 0–816) and by four weeks increased to a median of 161 cell/microliter (range, 6–701; p< 0.0001) as compared to pre-transplant levels. By the second month post-transplant the median levels decreased and were maintained at pre-transplant levels.CMV antigenemia developed in 23 of the 51 patients at a median of 30 days post transplant (range, 20–213 days). The median NK cell reconstitution at 2 weeks in the patients who subsequently developed CMV antigenemia was 158 cells/microliter (range, 2–816) and was higher than that among patients without CMV antigenemia (median 71 NK cells/microliter, range 10–228;Wilcoxon rank sum test p = 0.05).Grade I to III aGVHD developed in 31 out of 51 patients (median time = 27 days; range, 11–92 days). The NK cell levels in the patients who developed aGVHD were significantly higher at two weeks post-transplant (median NK = 193 cells/microliter; range, 0–816) than those in the patients who did not (median NK = 66 cells/microliter; range 2–215) (p = 0.005 by Wilcoxon rank sum test). Furthermore, based on a Kaplan-Meier analysis of time to aGVHD as a function of the NK levels at two weeks post-transplant, patients who had NK cell counts greater than 195 cells/microliter at two weeks post transplant had a significantly higher probability of developing aGVHD (p = 0.0036 by log-rank test) Figure 1.These results suggest that the early NK cell reconstitution may serve as a biomarker of transplant outcomes, especially in predicting the development of acute GVHD. [Display omitted]

Rapid Helper T-Cell Recovery at 3 Months Correlates to Successful Transplant Outcomes after Allogeneic Stem Cell Transplantation.

Background: The current study attempted to evaluate the role of a simple quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes, including overall survival (OS), non-relapse mortality (NRM), and opportunistic infections, after allogeneic stem cell transplantation (SCT).Methods: A total of 69 patients receiving an allogeneic SCT were included. The disease entities were as follows: AML 42, ALL 5, CML 15, NHL 5, and high-risk MDS 2. The peripheral lymphocyte subset counts, such as CD3+ T-cells, CD3+4+ helper T-cells, CD3+8+ cytotoxic T-cells, CD19+ B-cells, and CD56+ natural killer (NK) cells, were measured 3, 6, and 12 months post-transplant.Results: The CD19+ B-cell reconstitution was slow, while a rapid CD56+ NK cell recovery was noted. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (p<0.0001), NRM (p=0.0007), and opportunistic infections (p=0.0108) when stratifying patients with cut-off value of 200×106/L CD4+ helper T-cells. A rapid CD4+ helper T-cell recovery was also independently associated with a higher CD4+ helper T-cell transplant dose (p=0.006) and donor type (p<0.001) in a regression analysis. An early CD4+ helper T-cell recovery at 3 months was associated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate survival analysis, a combination of a higher CD34+ cell dose and rapid recovery of CD4+ helper T-cells at 3 months was found to a have favorable prognosis in terms of OS (p=0.001, hazard ratio [HR] 3.653) and NRM (p=0.005, HR 4.836), yet not relapse.Conclusion: A rapid recovery of the CD4+ helper T-cell count above 200×106/L at 3 months seemed to correlate with a faster immune reconstitution and predict a successful transplant outcome. [Display omitted]