Alloreactive NK Cells Research Articles

Haploidentical Peripheral Blood Stem Cell Transplantation with Post-Transplantation Cyclophosphamide in Primary Refractory Acute Myeloid Leukemia

In a pilot study, 75 patients with Primary Refractory (PRef) AML without matched family donors were offered post-transplantation cyclophosphamide (PTCY) based haploidentical peripheral blood stem cell (PBSC) transplantation. Twenty-seven patients (36%) opted for haploidentical transplantation with or without further chemotherapy. There was no difference in the patient or disease characteristics amongst patients undergoing transplantation or not. The conditioning regimen comprised of FluCyMel (n=5), FluBuMel (n=17) and FluTreoTBI (n=5). MMF was tapered between days 14 and 21 posttransplant in the absence of GVHD and cyclosporine A was tapered between days 60 and 90. The progression free survival at a median follow-up of 25 months was 36.6% and 0% in the transplant and the non-transplant group (p=0.0001). Prompt engraftment was noted at a median of 14 days irrespective of disease status or conditioning regimens. Cumulative incidences of acute graft-versus-host disease (GVHD) and chronic GVHD were 26.6% and 8% respectively. The overall incidence of infections remained low, with CMV reactivation and invasive aspergillosis occurring in 9 and 2 patients respectively. CMV disease was diagnosed in 2 patients. Non-relapse mortality at 1 year was 16.7%. The incidence of disease progression was 54%. Factors positively impacting progression free survival were < 15% marrow blasts at transplant and a Natural Killer Cell Ligand Mismatch (NKLMM) donor. NKLMM, Haplotype or B scores had no impact on CMV infection or GVHD. However, Bx Haplotype was associated with lower NRM (5%, 95%CI-1-9) compared to 48.6% (95%CI 28.2-69.0) in AA Haplotype (p=0.01). Disease status did not impact the overall survival (p=0.11) in the HSCT cohort. In fact, NKLMM donors with B haplotype had the greatest impact on overall survival in both the HSCT cohort (71.4%, 95%CI 54.3-88.5%) compared to 20% (95%CI 8.8-31.2, p=0.01) in those without the same. Our data suggests PTCY based Haploidentical PBSC transplantation is feasible in patients with PRef AML and donor NKLMM might improve progression free survival, provided the conditioning protocol and the post-grafting therapy offer the optimum platform for alloreactivity of NK cells. DisclosuresNo relevant conflicts of interest to declare.

In vitro generation of allo-reactive human NK cells from umblillical cord blood CD34+ progenitor cells

Compare to autologous NK cells, allogeneic NK cells have more anti-tumor cytotoxicity without GVHD. Adoptive transfer of allo-reactive NK cell can be ideally used combined with other immunotherapy. However, in vivo persistency of transferred NK cells in the recipients are still an obstacle in NK cell-based immunotherapy. As most of NK cells purified from peripheral blood of donors are fully differentiated, their short lifespan might be a reason. In this study, we developed a new method of “young” human NK cell generation which can be used for immune cell therapy. CD34+ hematopoietic precursor/stem cells were purified from umbilical cord blood and differentiated into NK cells in vitro. Stem cell expansion and lineage commitment can be modulated by stromal cells and exogenous cytokines. Most of CD34+ cells express receptors for IL-3, Flt3-L, and SCF, while a small subset expressed IL-7Rα. Without feeder cells, CD34+ cells were initially expanded with exogenous cytokines (IL-3, Flt3-L, IL-15, SCF, and IL-7) but most of cells were differentiated into myeloid cells. When the precursor cells were co-cultured with feeder cells (murine embryonic liver stromal cell line EL08-1D2), CD56+ NK cells were successfully generated after 3 weeks (x20,000 fold expansion). Interestingly, as use of feeder cells was necessary for NK cell differentiation during early days (D0 to 5), feeder cell interaction may have a role on the proliferation of early lymphoid precursors or myeloid/lymphoid fate decision of hematopoietic stem cells. When we used EL08-1D2 feeder cells transduced with human Notch ligand DLL4, most of hematopoietic precursors were differentiated into lymphoid lineage cells. Some of generated CD56+ cells were non-cytotoxic group3 innate lymphoid cells (ILC3s), and ILC3s do not express most of NK cytotoxic receptors (NKG2D, NKp30, NKp46, 2B4, CD94, NKG2A, DNAM-1, and CD16). Cytotoxic NK cells can be discriminated from ILC3s based on CD11a/CD18 (LFA-1) expression, and this was confirmed using CD107a assay and IFN-γ staining. While ILC3s require SCF and IL-7 for their generation, NK cells can be successfully generated with IL-15 and Flt-3L only. As small populations of the generated NK cells express KIRs which are essential for allogeneic cytotoxicity, we epigenetically induced KIR expression using a demethylating agent 5-aza-2′-deoxycytidine during NK cell differentiation. When 5-aza-2′-deoxycytidine was treated on immature NK cells (D14 to 21), it efficiently induced KIR expression while it did not on fully differentiated NK cells (at D21). In conclusion, this new method can be used for novel NK cell based immunotherapy.

Haploidentical hematopoietic transplantation from KIR ligand–mismatched donors with activating KIRs reduces nonrelapse mortality

AbstractBecause activating killer cell immunoglobulinlike receptors (KIRs) are heterogeneously expressed in the population, we investigated the role of donor activating KIRs in haploidentical hematopoietic transplants for acute leukemia. Transplants were grouped according to presence vs absence of KIR-ligand mismatches in the graft-vs-host direction (ie, of donor-vs-recipient natural killer [NK]-cell alloreactivity). In the absence of donor-vs-recipient NK-cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplant pairs with donor-vs-recipient NK-cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection related (KIR2DS1 present vs absent: hazard ratio [HR], 0.25; P = .01; KIR3DS1 present vs absent: HR, 0.18; P = .006), and better event-free survival (KIR2DS1 present vs absent: HR, 0.31; P = .011; KIR3DS1 present vs absent: HR, 0.30; P = .008). Transplantation from donors with KIR2DS1 and/or KIR3DS1 was also associated with a 50% reduction in infection rate (P = .003). In vitro analyses showed that KIR2DS1 binding to its HLA-C2 ligand upregulated inflammatory cytokine production by alloreactive NK cells in response to infectious challenges. Because ∼40% of donors able to exert donor-vs-recipient NK-cell alloreactivity carry KIR2DS1 and/or KIR3DS1, searching for them may become a feasible, additional criterion in donor selection.

NK cells with KIR2DS2 immunogenotype have a functional activation advantage to efficiently kill glioblastoma and prolong animal survival.

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig-like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2(+) NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2(-) immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2(-) NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell-mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2(+) donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56(+)CD16(+) NK cells from a KIR2DS2(+) donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2(-) NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.

Association of Donor Killer Immunoglobulin-like Receptor (KIR) Genotype with Outcome after HLA-Matched Related Donor Hematopoietic Cell Transplantation for AML

Although outcomes after allogeneic hematopoietic cell transplantation (alloHCT) for AML have improved, this has mainly been attributed to a reduction in transplant-related mortality rather than reduced leukemia relapse. NK cell alloreactivity is regulated by inhibitory and activating signals mediated through cell-surface receptors including the killer immunoglobulin-like receptors (KIRs). Group A and B KIR haplotypes have distinct centromeric (Cen) and telomeric (Tel) gene-content motifs and donor Cen group B KIR haplotypes have been reported to be associated with decreased relapse and improved survival in AML patients undergoing unrelated donor alloHCT. We hypothesized that donor KIR genotype may also be predictive of outcomes after matched related donor (MRD) alloHCT. We evaluated 93 AML patients in CR1/CR2 who underwent T-cell replete alloHCT using HLA- matched related donors at our institution from 1/2000-3/2013. Sixty-six had myeloablative conditioning (MAC) that that was busulfan/cyclophosphamide-based and 27 had reduced-intensity conditioning (RIC) with fludarabine/total body irradiation or busulfan/fludarabine. Donors were KIR genotyped to assign haplotypes A/A vs. B/X and the distinctive Cen and Tel gene-content motifs of group A and B KIR haplotypes according to the presence or absence of one or more B haplotype-defining KIR genes. KIR B–content score for each KIR genotype was defined as the number of Cen and Tel gene-content motifs containing B haplotype–defining genes (range, 0-4). As compared to those with haplotypes B/X (n=40; B content scores of 1-4) those with haplotype A/A (n=25; B content score of 0) undergoing MAC had significantly lower 100-day, 6-, 12- and 24-month non-relapse mortality (NRM) (8% vs. 0%, 13% vs. 0%, 15% vs. 0%, 25% vs. 0%, respectively, Figure 1) which was confirmed on multivariable analysis (HR 9.19, p=0.03). There were no differences between these groups regarding patient and transplant-related characteristics, or for acute or chronic GVHD, relapse, or survival. The causes of death in the group with haplotypes B/X were most commonly attributed to infection and then GVHD. However, within the group with B/X haplotypes, the B motif content score (1-4) was not associated with significant differences in NRM (HR 0.79, p=0.56). No difference in outcomes was observed for those undergoing RIC. The number of donor activating KIR genes (2SD1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1) was then assessed. As compared to those with 3-6 activating KIR genes (n=20) those with 0-2 (n=41) undergoing MAC had significantly lower 100-day, 6-, 12- and 24-month non-relapse mortality (NRM) (15% vs. 0%, 15% vs. 5%, 15% vs. 5%, 29% vs. 8%, respectively, Figure 2) which was confirmed on multivariable analysis (HR 4.07, p=0.01). There were no differences in other post-transplant outcomes when comparing these groups or when considering those undergoing RIC. An increase of 1 donor activating KIR also was highly associated with NRM (HR 1.37, p=0.008). Overall, these results suggest that in the MRD MAC alloHCT setting donor KIR genotype may be predictive of increased NRM risk, particularly for those with B/X haplotypes and greater numbers donor activating KIRs. No comparable effects were observed in the RIC setting. Future strategies to further enhance immune reconstitution post-transplant may be appropriate to pursue for these higher risk patients. These results may have potential implications to improve donor selection for those AML patients with multiple HLA-matched related donors and need to be validated in larger cohorts. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Role of Alloresponsive Ly49+NK Cells in Rat Islet Allograft Failure in the Presence and Absence of Cytomegalovirus

There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2(+) NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2(+) NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2(+) NK cells and accelerated islet graft failure. The Ly49i2(+) NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1(+) NK cells infiltrated the grafts, but nonalloreactive NKR-P1B(+) cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.

NK Cell KIR Ligand Mismatches Influence Engraftment Following Combined Haploidentical and Umbilical Cord Blood (UCB) Transplantation In Patients With Severe Aplastic Anemia (SAA)

BackgroundAllogeneic hematopoietic stem cell transplant (HSCT) is curative for patients (pts) with severe aplastic anemia (SAA). For SAA pts who lack HLA-identical donors, we explored a HSCT approach that co-infuses PBSCs enriched for CD34+ cells from a haplo-identical relative combined with a single umbilical cord blood unit (UCB). Although most pts undergoing this approach had early haplo-myeloid engraftment that was eventually supplanted by the UCB unit, engraftment patterns were highly variable; in some pts full cord myeloid chimerism was delayed greater than one year, while others had loss of cord engraftment with sustained full haplo-donor myeloid chimerism. Here, we investigated the impact of various patient, UCB, and haplo-donor characteristics, as well as NK cell KIR ligand mismatches between graft sources, on engraftment kinetics following haplo-cord HSCT. MethodsPts with SAA or SAA evolved to MDS unresponsive to immunosuppressive therapy with severe neutropenia (ANC<500), who lacked an HLA-matched donor and had an available haploidentical family member, and had at least one ≥ 4/6 matched UCB unit with a minimum TNC dose ≥1.5x107 cells/kg were eligible for HSCT. Pts were conditioned with cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine-ATG (160 mg/kg) and 200 cGy of total body irradiation. On Day 0, pts received a CD34 selected (Miltenyi CliniMacs) G-CSF mobilized haplo-donor allograft combined with a single UCB. Tacrolimus and MMF were given for GVHD prophylaxis. Results16 pts (median age: 18.9 yrs, range: 4.5-27.9) including 13 SAA and 3 with SAA evolved to MDS underwent haplo-cord transplant. Ten pts received a 4/6, and six pts received a 5/6 HLA-matched UCB unit. A median 3.6x107 TNC/kg (range: 1.96-6.93) from the UCB unit, and 3.3x106CD34+ cells/kg (range: 3.0-4.1) from the haplo-donor were transplanted. All 16 pts had sustained engraftment with 15/15 pts evaluable past day 100 having transfusion independence. At a median follow-up of 570 days (range: 55-1826), 14/16 pts survive for an overall survival rate of 81.8%. Two pts died at day 414 and 402 post-HSCT from viral related complications (CMV pneumonitis and limbic encephalopathy). Neutrophil and platelet recovery occurred at a median 10 (range: 9-22), and 21 (range: 10-213) days post-HSCT, respectively. By post-HSCT day 11, 15/16 pts had neutrophil recovery. Cord myeloid engraftment (cord ANC>500, calculated from chimerism data) occurred in 13/16 pts at a median 42 days. 3/16 did not achieve a cord ANC>500 but had sustained haplo-donor engraftment. The cumulative incidence of acute grade II-IV GvHD was 38.1%. Engraftment profiles were highly variable among pts; 12 achieved full cord chimerism in all cell lineages, 2 remained mixed haplo-cord chimeras, and 2 failed to have UCB engraftment but had sustained 100% haplo-donor myeloid chimerism. Higher degrees of HLA matching (out of 10 alleles) between recipient and UCB unit were associated with faster rates of full cord engraftment (p=0.006) and a higher probability of complete loss of haplo-donor chimerism (p=0.018). KIR ligand incompatibility in the haplo vs. cord direction (defined as the presence of a KIR ligand in the haplo-donor graft that is absent in the UCB unit at HLA epitopes Bw4, HLA-C Group 1 & 2, HLA-A3, and HLA-A11) negatively impacted cord myeloid engraftment. 5/5 (100%) pts who failed to achieve full cord myeloid chimerism by post-HSCT day 400 had haplo vs. cord KIR ligand incompatibility. Moreover, both pts who failed to have UCB engraftment and had sustained haplo-donor chimerism had haplo vs. cord KIR ligand incompatibility. In contrast, only 3/11 (27%) pts who achieved full cord myeloid chimerism post-HSCT by day 231 had haplo vs. cord KIR ligand incompatibility (p=0.026). KIR ligand incompatibility in the cord vs. haplo direction showed no significant effect on haplo-donor myeloid engraftment. ConclusionThese results show that haplo-cord HSCT is an effective treatment option for pts with SAA who lack an HLA-matched donor. Further, these results suggest that NK cell alloreactivity, occurring as a consequence of KIR ligand mismatch between the two graft sources, may have a negative impact on cord engraftment when haplo vs. cord KIR ligand incompatibility is present. In summary, this study highlights a novel factor that should be considered during graft selection for haplo-cord transplantation. Disclosures:No relevant conflicts of interest to declare.

HLA-Haploidentical Stem Cell Transplantation with Treg and Tcon Adoptive Immunotherapy promotes a Strong Graft-Versus-Leukemia Effect

In HLA-haploidentical stem cell transplantation we showed adoptive immunotherapy with naturally occurring T regulatory cells (nTregs) followed by conventional T cells (Tcons) prevented acute and chronic graft-versus-host-disease (GvHD), favoured lymphoid reconstitution and immunity against pathogens (Di Ianni et al., Blood 2011). One major concern is whether a graft-versus-leukemia (GvL) effect is maintained since FoxP3+Tregs can also suppress immune response against tumour. However, we showed in immunodeficient NSG mice engrafted with human myeloid and lymphoblastic acute leukemia cells (7x106 iv), Treg (3x106)+Tcon(3x106) infusion rescued mice from leukemia death in the absence of GvHD. In contrast, controls died of leukemia (when not treated or infused with Tregs only) or of GvHD (when infused with Tcons only).Since September 2008, 45 consecutive patients (21 male; 24 female; median age 40, range 20-59) with high-risk acute leukemia ( 33 AML, 12 ALL) were transplanted. The conditioning regimen included TBI, thiotepa, fludarabine and cyclophosphamide (25 patients in the 1st clinical trial) or alemtuzumab/ATG (20 patients in the 2nd clinical trial). On day -4, patients received an infusion of freshly isolated donor Tregs (mean 2.5/kg ±1; Foxp3 81.01%±16.47; Helios/FoxP3 54%±8.4; CD127 11.72%±7.653) followed on day 0 by a megadose of purified CD34+ cells (9.7x106/kg ± 3.1) and Tcons (mean 1.1 /kg ±0.6; CD3 90.72%± 9.6; CD4 57.77% ± 8.85; CD8 31.21% ± 8.59). No post-transplant GvHD prophylaxis was given. 43 of the 45 patients achieved primary, sustained full-donor type engraftment. Only 6/43 evaluable patients developed ≥ grade II acute GvHD (3 alive, treated and now off-therapy). At a median follow-up of 28 months (range 4-58) no patient has developed chronic GvHD. CD4 and CD8 counts reached 100/µL medianly on days 40 (range 25-150) and 45 (range 18-100) and 200/µL on days 55 (range 45-160) and 60 (range 50-140). In KIR-ligand mismatched transplants (Ruggeri et al., Science 2002; Ruggeri et al., Blood 2007), donor versus recipient alloreactive NK cells were also preserved. At a median follow-up of 46 months (range 7-58) 23/43 patients are alive and disease free with a probability of DFS of 0.56. The cumulative incidence of TRM is 0.37. The cumulative incidence of relapse was 0.06 as only 2 AML patients transplanted from non-NK alloreactive donors have relapsed to date. This incidence is extremely low considering the high-risk patient population (9 with FLT-3/ITD; 8 with complex karyotypes; 4 with t(9:22); 2 PIF; 1 secondary AML; 1CR after second-line induction; 1 with CNS and skin localization at diagnosis; 19 ≥ 2CR). In conclusion, adoptive immunotherapy with Tregs and Tcons exerts a powerful GVT activity in both AML and ALL patients in the absence of GvHD. Disclosures:No relevant conflicts of interest to declare.

At the Bench: Preclinical rationale for exploiting NK cells and γδ T lymphocytes for the treatment of high-risk leukemias

NK cells and γδ T lymphocytes display potent cytolytic activity against leukemias and CMV-infected cells and are thus, promising immune effector cells in the context of allo-HSCT. NK cells express HLA class I-specific inhibitory receptors and preferentially kill HLA class I(low) tumors or virus-infected cells. Killing occurs upon engagement of activating NKRs with ligands that are up-regulated on tumors and infected cells. A similar activating receptor/ligand interaction strategy is used by γδ T cells, which in addition, use their TCRs for recognition of phosphorylated antigens and still largely undefined ligands on tumor cells. In the haploidentical allo-HSCT setting, alloreactive NK cells, derived from donor HSCs, can exert potent antileukemia activity and kill residual patient DCs and T cells, thus preventing GvHD and graft rejection. However, generation of KIR(+) alloreactive NK cells from HSCs requires many weeks, during which leukemia relapses, and life-threatening infections may occur. Importantly, mature NK cells and γδ T cells can control certain infectious agents efficiently, in particular, limit CMV reactivation, and infusion of such donor cells at the time of HSCT has been implemented. Development of novel, cell-based immunotherapies, allowing improved trafficking and better targeting, will endow NK cells and γδ T lymphocytes with enhanced anti-tumor activity, also making them key reagents for therapies against solid tumors. The clinical aspects of using NK cells and γδ T lymphocytes against hematological malignancies, including the allo-HSCT context, are reviewed in the related side-by-side paper by Locatelli and colleagues [1].

At the Bedside: Innate immunity as an immunotherapy tool for hematological malignancies

The identification of an anti-tumor effect displayed by cells of innate immunity has opened new scenarios, not only in the field of allo-HSCT but also for nontransplanted patients with hematological malignancies or solid tumors. Donor-derived NK cells have been shown to contribute to the eradication of malignant cells after allo-HSCT, when recipients lack ligands for their inhibitory receptors. These alloreactive donor NK cells can also kill recipient APCs and CTLs, thus preventing the occurrence of GvHD and graft rejection. The role of activating receptors on the capacity of NK cells to kill leukemia targets has become evident in the last years. The adoptive infusion of ex vivo-activated NK cells has been investigated recently in Phase I/II trials on patients with hematological malignancies and solid tumors, with promising results. γδ T lymphocytes are also able to display anti-tumor activity-this providing the biological rationale for Phase I/II trials in lymphoproliferative disorders and solid tumors. Aminobisphosphonates are clinically available compounds able to boost γδ T cell function. As γδ T cells do not cause GvHD, they could also be transduced with tumor-associated chimeric antigen receptors and safely infused in allo-HSCT recipients. Basic aspects of innate immunity relevant to the field will be covered by a companion review article.

The EFIS‐EJI Ruggero Ceppellini Advanced School of Immunology Innate Immunity 2012: From Evolution to Revolution

The EFIS‐EJI Ruggero Ceppellini Advanced School of Immunology Innate Immunity 2012: From Evolution to Revolution

NK Cells Display Poor Anti Lymphoma and AML Function At Early Times Following Allogenic HLA-Identical SCT for Leukemia and Lymphomas.

AbstractAbstract 3045The functional role of NK cells in leukemia control as well as their role against viruses have prompted numerous studies analyzing NK cell function in allogeneic haematopoietic stem cell transplantation (HSCT) in the recent years. However, most studies refer to unrelated donor, HLA- match or mismatch HSCT and little is known about matched sibling HSCT, which is a more common setting.Here, we have studied the reconstitution of NK cell in 27 HLA-identical patients. Patients were sampled at early (<2months), intermediate (<4months) and late (>4 months) time points after engraftment of G-mobilized peripheral blood of geno identical siblings. Reduced intensity conditioning (fludarabin 4d+Busulfan 2d) was given to all patients. Post-graft immunosuppression consisted of cyclosporine (CSA). Percentage and numbers of NK cells was monitored together with the relevant markers including CD56, CD16, KIR, NKG2A, CD57 and NKG2C. Functional studies were performed by stimulation with the reference cell line K562 and when available the autologous leukemia. The functional assays evalauted both degranulation (CD107a+b) and cytokines (IFng, TNFa).Similarly to previous studies, a large percentage of CD56bright NK cells (up to 90%, median 40% of NK cells) was observed during the first months, a percentage that tended to resume to normal after late time points (> 5 months). Remaining CD56dim NK cells mostly expressed NKG2A. CD57 expression was low at the first time points and increased with time to reach healthy controls by late time points.Overall, these data showed that up to 6 months developing NK cells display a very immature phenotype with high CD56bright frequencies and high NKG2A expression with low frequencies of CD57. KIR expression was monitored by means of KIR2DL1/S1, KIR2DL2/S2/L3 and KIR3DL1 staining. Surprisingly, KIR expression was close to that of healthy controls, but we observed a higher frequencies of NK cells expressing no or only one KIR, which is consistent with an immature phenotype of NK cells. Next, we analyzed the functional capacities of developing NK cells. Degranulation after interaction with a classical, HLA-negative NK target cell, K562, was comparable to that of healthy control NK cells. Similarly, developing NK cells were fully capable to produce MIP1b after interaction with a target cell. By contrast, stimulation by K562 resulted in a lower production of pro-inflammatory cytokines such as IFNg and TNFa whereas chemokine production (MIPb) was conserved. These results are in line with other obtained in unrelated HSCT.Finally, the normal cytotoxic potential of developing NK cells prompted us to check whether these cells had an anti-tumor potential. For this purpose, we tested the function of the patients NK cells post-transplantation against the aulogous or allogeneic blast (AML, CLL) or cell line (Follicular lymphoma). We analyzed the degranulation capacities of developing NK cells against leukemic cells of the same histology as the original pathology of the patients. A patients treated by HSCT for a follicular lymphoma was tested against RL, a follicular lymphoma cell line. Although, degranulation against K562 was normal (control), these NK cells failed to degranulate against RL, even in the presence of anti HLA blocking antibodies. Similarly, in transplanted patients for B-CLL, NK cell failed to recognize and degranulate against allogeneic primary leukemic B cells. Comparable to healthy controls, only the use of Rituximab allowed the recognition and killing of B-CLL cells. Finally, we tested the reactivity of developing NK cells against autologous AML blast, taken from the patients at diagnosis. Although some degree of reactivity could be observed in some patients, overall degranulation as well as cytokine production was low compared to reactivity against K562. Altogether these data showed that although reconstitution of NK cells after genoidentical allogeneic HSCT was rapid, functionality of NK cells was partially impaired as we observed a dichotomy between degranulation capacities and cytokine production potential. These in vitro data suggest that at the early times post transplantation the immunotherapeutic impact of HSCT does not only rely on the alloreactivity of NK cells from the donors in HLA-matched, sibling. Their role in combination with T cells as well as their anti-viral and GvH function deserve further studies. Disclosures:No relevant conflicts of interest to declare.

In Vitro Skewing of the Human KIR Repertoire Leads to Enhanced NK Cell Alloreactivity

In Vitro Skewing of the Human KIR Repertoire Leads to Enhanced NK Cell Alloreactivity

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant

This review focuses on advances in the field of natural killer (NK) cell alloreactivity as a form of immunotherapy in the setting of allogeneic haematopoietic transplantation. Recent research has focused on mechanisms underlying the acquisition of function by NK cells after haematopoietic transplantation, on the therapeutic impact of NK-cell alloreactivity in various forms of transplantation and in adoptive immunotherapy. Finally, studies have highlighted the role of NK-cell responses in viral infections after transplantation. Donor-versus-recipient NK-cell alloreactivity is established as a key therapeutic element in human leukocyte antigen haplotype-mismatched haematopoietic transplants in adult acute myeloid leukaemia (AML) and paediatric acute lymphoblastic leukaemia (ALL). NK-cell allotherapy for leukaemia is deployed through stem cell transplantation (and ensuing NK-cell reconstitution) across killer-cell immunoglobulin-like receptor ligand mismatches. Donor-derived NK cells were also reported to respond to cytomegalovirus by acquiring features that are reminiscent of the specificity and memory of adaptive (T-cell) immune responses. As NK cells are the earliest immune cells to recover after transplant, this observation suggests they may contribute to controlling viral reactivation early after transplant.

KIR-Ligand Incompatibility Is Not Associated with Relapse Reduction After Double Umbilical Cord Blood Transplantation,

Abstract 4150Natural killer (NK) cell alloreactivity has been implicated in graft-versus-host (GVH), graft-versus-leukemia (GVL), and host-versus-graft reactions following hematopoietic stem cell transplantation (HSCT). Though the basic mechanisms of NK cell alloreactivity are well described, comparatively little is known about its clinical effects. In umbilical cord blood (UCB) transplantation, both stem cells and donor-derived T-cell numbers are limited compared to other transplant modalities, so NK cell effects may be more pronounced. NK cell-mediated cytotoxicity is mediated by a balance of activating and inhibitory cell-cell contacts, the most important of which entails engagement of MHC-I on target cells by inhibitory killer immunoglobulin-like receptors (KIR) on NK cells. This interaction inhibits NK cell cytotoxicity, whereas the absence of MHC-I on target cells stimulates cytotoxicity, thus providing a mechanism for immune surveillance against pathologic states in which MHC-I is down-regulated such as cancer and viral infection. Applied to the scenario of HSCT, based on the specificity of various KIR for MHC-I encoded by particular HLA-B and HLA-C alleles, GVH NK cell alloreactivity can be predicted from the HLA genotypes of donors and recipients. The clinical impact of predicted GVH NK cell alloreactivity, termed KIR ligand incompatibility, has been investigated retrospectively in various transplant settings (Verheyden 2008), including UCB transplantation (Brunstein 2009, Willemze 2010), with inconsistent results regarding its association with relapse, GVH disease incidence, and survival. Methods: We stratified a cohort of 80 patients who underwent double umbilical cord blood (UCB) transplantation according to the presence or absence of KIR ligand incompatibility in the graft-versus-host direction. The median age was 48 years. Patients had a mix of myeloid and lymphoid malignancies, the most common of which were AML (31%) and non-Hodgkin lymphoma (18%). 59% of patients received non-myeloablative conditioning. Results: We found no association between KIR ligand incompatibility and neutrophil (p=0.30) or platelet (p=0.95) engraftment, incidence of acute (p=0.70) or chronic (p=0.44) GVHD, relapse (p=0.67), or progression-free (p=0.73) and overall (p=0.71) survival. Graft rejection, defined as neutrophil engraftment with donor-predominant chimerism followed by loss of donor chimerism, occurred in six patients in the cohort, all but one of whom received KIR-ligand-incompatible UCB units (p=0.08). Of these six patients, the only one who received a KIR-ligand-compatible transplant received a far less immunosuppressive conditioning regimen than the rest of the cohort due to comorbidities. When this patient was retrospectively excluded from the analysis, the association between graft rejection and KIR ligand incompatibility was statistically significant (p=0.01). An analysis accounting for the dominance of one cord unit and its associated KIR incompatibility was not performed due to the concordance of KIR ligand compatibility/incompatibility among transplanted cord unit pairs. Conclusions: These results suggest a role for NK cell alloreactivity in mediating graft rejection in double UCB transplantation but no effect on relapse or survival. These findings warrant further evaluation in larger cohorts. Disclosures:No relevant conflicts of interest to declare.

Significance of Missing Inhibitory KIR Ligands in Nonmyeloablative, HLA-Haploidentical (Haplo) BMT with Posttransplantation High-Dose Cyclophosphamide (PT/Cy)

Abstract 840Significance of Missing Inhibitory KIR Ligands in Nonmyeloablative, HLA-Haploidentical (Haplo) BMT with Posttransplantation High-Dose Cyclophosphamide (PT/Cy).Yvette L. Kasamon1, Leo Luznik1, Mary S. Leffell1, Hua-Ling Tsai1, Heather J. Symons1, Javier Bolaños-Meade1, Gary Rosner1, Lawrence E. Morris2, Pamela A. Crilley3, Richard J. Jones1 and Ephraim J. Fuchs1, (1)Johns Hopkins University, Baltimore, MD, (2)Northside Blood and Marrow Transplant Program, Atlanta, GA, (3)Hahnemann University Hospital, Philadelphia, PA Introduction:NK cells can influence haplo BMT outcomes including risks of relapse and GVHD. Our group previously reported that, in allogeneic BMT for hematologic malignancies that incorporates PT/Cy, donor-recipient iKIR (inhibitory killer-cell immunoglobulin-like receptor) gene mismatches and having a KIR haplotype B donor were associated with improved outcomes (BBMT 2010;16:533). Because the reported impact of NK cell alloreactivity models in haplo BMT has been variable and some models are relevant to donor selection, we expanded our analysis of iKIR ligand status in this transplantation platform. Patients and methods: Outcomes of 212 uniformly treated patients (pts) enrolled on two similar clinical trials of related-donor, haplo BMT were retrospectively analyzed. Planned treatment consisted of fludarabine (30 mg/m2 IV on days −6 to −2), Cy (14.5 mg/kg IV on days −6 and −5), total body irradiation (200 cGy on day −1), and non-T-cell depleted bone marrow infusion. GVHD prophylaxis consisted of high-dose Cy (50 mg/kg IV on days 3 and 4), mycophenolate mofetil on days 5–35, and tacrolimus on days 5–180 without taper, with filgrastim begun on day 5. All pts (median age 51, range 1–73) had poor-risk hematologic malignancies; 60 (28%) had prior BMT. Diagnoses were Hodgkin lymphoma (31 pts), NHL (69), CLL (21), multiple myeloma (6), acute leukemia or lymphoblastic lymphoma (62), MDS (9), CML (9), CMML (4), PV (1). Missing ligands (ML; defined as absence in the recipient of one or more HLA ligands for iKIRs) and donor/recipient iKIR ligand incompatibility (LI; defined as presence of an iKIR ligand in the donor that is absent in the recipient) were determined using high-resolution HLA typing of class I alleles. For study purposes, HLA-A*2301, A*2402, and A*3201 were included in the Bw4 serologic group and effects attributable to HLA-A3 and A11 ligand groups were excluded. Results: With a median 2.9 year follow-up (range, 0.3–7 years) in pts without events, the actuarial 2-year progression-free survival (PFS) was 34%. On competing-risk analysis, cumulative incidences of grade II–IV acute GVHD and chronic GVHD were 28% and 14%, respectively; 1-year cumulative incidences of relapse and nonrelapse mortality (NRM) were 42% and 14%. Baseline characteristics in pts with ML (157 pts, of whom 76 had LI) and without ML were similar. On univariate analysis, pts with LI had no significant difference in PFS (figure A), grade II–IV acute GVHD, relapse or NRM compared to those without LI. In contrast, as compared to no ML, the presence of ML was associated with a statistically significantly improved PFS on univariate analysis (hazard ratio [HR] = 0.68, p = 0.03; figure B). This association was not identified in our previous analysis, potentially due to underpowering or inclusion of pts receiving one dose of PT/Cy. No statistically significant difference was detected according to the presence or absence of ML in the cumulative incidences of acute GVHD, relapse, or NRM, although presence of ML was associated with a tendency toward lower NRM risk (HR = 0.61, p = 0.17). On multivariate analysis, the presence of ML was found to be independently associated with a significant improvement in PFS (HR = 0.66, p = 0.03). This multivariate model also confirmed our previous observation (BBMT 2010;16:482) that greater donor-recipient HLA disparity was not detrimental to PFS (HR = 0.57, p = 0.04 for 3–4 antigen mismatches versus fewer at HLA-A, B, C, and DRB1 combined). We are currently investigating the impact of KIR haplotypes in this setting with the goal of optimizing donor selection. Conclusion: The presence of ML may be beneficial in haplo BMT with postgrafting immunosuppression that includes high-dose Cy. Further studies are needed to confirm and define the mechanisms of this effect. Our findings do not support selection of donors on the basis of LI in this transplantation platform. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cell Docking, Movement and Cell-Cell Interactions of Heterogeneous Cell Suspensions in a Cell Manipulation Microdevice

This study demonstrates a novel cell manipulation microdevice for cell docking, culturing, cell-cell contact and interaction by microfluidic manipulation of heterogeneous cell suspensions. Heterogeneous cell suspensions include disparate blood cells of natural killer cells and leukemia cancer cells for immune cell transplantation therapy. However, NK cell alloreactivity from different healthy donors present various recovery response levels. Little is still known about the interactions and cytotoxicity effects between donor NK cells and recipient cancer cells. The cell-based micro device first showed the capability of cell docking, movement, contact and cell-cell interaction with respect to cell cytotoxicity of NK cells against cancer cells. With various flow tests for live cell loading, flow rates of 10 μL/h were chosen for injection in the central and side flows such that both types of suspension cells could be gently docked at the gap structure in a reaction zone. The trapping number of particles and cells was linearly proportional to the gap length. Finally, the cytotoxicity of around 40% was found to be similar in the case of dilute cells and a large cell population. As a result, the cell manipulation microdevice has been validated for live suspensions of natural killer and cancer cells, and exhibited the capability to measure the cytotoxicity of dilute cell suspensions.

Tregs combined with mature donor T cells hasten immune reconstitution without triggering GvHD in HLA haploidentical transplantation

Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in preclinical studies, the present phase I/II clinical trial evaluated the impact of early infusion of donor CD4/CD25+ Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34+ cells. Twenty-eight patients (median age 41, range 21-60) were enrolled from September 2008 onwards; 22 had AML (10 in CR1 at high risk, 10 in ≥CR2 and 2 in relapse), 5 had ALL (4 in CR1; 1 in relapse) and 1 had high grade NHL in relapse. Conditioning was: 8 Gy single fraction TBI, thiotepa (4 mg/kg × 2), fludarabine (40 mg/m2 × 5), cyclophosphamide (35 mg/kg × 2). All patients received immunoselected Tregs (CliniMACS, Miltenyi Biotec) (23/28 2 × 106/kg bw; 5/28 4 × 106/kg bw) and 4 days later positively immunoselected CD34+ cells (median 8.2 × 106/kg bw, range 5.0-19.1) together with Tcons (4/28 0.5 × 106/kg bw; 17/28 1 × 106/kg bw; 5/28 2 × 106/kg bw; 2/28 did not receive Tcons). CD4/CD25+ Tregs (purity 92.7 ± 2.1) consisted of 33.6% ± 13.1 CD25high; 58.1% ± 6.6 CD25int; 5.8% ± 2.5 CD25low; 65.7% ± 11.8 FoxP3; 17.4% ± 7.2 CD127 (mean ± SD). No GvHD prophylaxis was administered. 26/28 patients engrafted. No GvHD developed in 24/26 patients, 2 developed ≥ grade II GvHD. Ten patients died (3 VOD, 2 fungal pneumonia, 1 bacterial sepsis, 1 CNS aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 MOF). CD4 and CD8 counts reached, respectively, 50/μL medianly on days 34 (range 19-63 days) and 24 (range 15-87); 100/μL medianly on days 47 (range 28-100 days) and 34 (range 19-95); 200/μL on days 70 (range 41-146 days) and 61 (range 21-95). A wide T-cell repertoire developed rapidly with high frequencies of specific CD4+ and CD8+ for opportunistic pathogens. Episodes of CMV reactivation were significantly fewer than after our standard haplo transplants. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved. In conclusion, in the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution.

Role of alloreactive KIR2DS1+ NK cells in haploidentical hematopoietic stem cell transplantation

In allo-HSCT, donor-derived, "alloreactive" NK cells have been shown to play a crucial role in the treatment of acute leukemia, contributing to eradication of leukemic blasts (GvL effect) and to clearance of residual recipient DCs and T lymphocytes (thus, preventing GvHD and graft rejection, respectively). Such alloreactive NK cells do not express CD94/NKG2A but express inhibitory KIRs, specific for HLA class I allotypes, present in the donor but lacking in the recipient. This review is focused on the role of the activating KIR2DS1 receptor (specific for the C2-epitope of HLA-C) in haplo-HSCT. Recent data indicate that KIR2DS1 expression in HSC donors may represent a remarkable advantage in alloreactive NK responses. This is a result of a substantial increase in the NK-mediated capability to kill, not only recipients' leukemic cells but also DCs and T cell blasts. The beneficial effects mediated by alloreactive KIR2DS1(+) NK cells may occur after de novo expression of CCR7 upon interaction with allogeneic, KIR ligand-mismatched CCR7(+) cells. As a consequence, they can be redirected to LNs, where they can prevent priming of donor T cells and induction of GvHD. Finally, KIR2DS1 expression may also significantly amplify the size of the alloreactive NK cell subset by switching a subset of "not alloreactive" NK cells into potent alloreactive cells.