Cytolytic Activity of NK Cell Clones Against Acute Childhood Precursor

ORIGINAL ARTICLE Cytolytic activity of NK cell clones against acute childhood precursor-B-cell leukaemia is inﬂuenced by HLA class I expression on blasts and the differential KIR phenotype of NK clones

T Feuchtinger1, M Pfeiffer1, A Pfafﬂe1, H-M Teltschik1, D Wernet2, M Schumm1, R Lotﬁ2, R Handgretinger1 and P Lang1

1Department of Paediatric Haematology/Oncology, University Children’s Hospital, Tu¨bingen, Germany and 2Institute of Transfusion Medicine, Eberhard Karls University, Tu¨bingen, Germany

Relapse after allo-SCT in patients with acute leukaemia Introduction remains a major problem. A beneficial impact of alloreactive natural killer (NK) cells has been reported Stem cell transplantation from matched or partially for myeloid malignancies, but has been questionable for matched unrelated donors as well as mismatched related B-lineage ALL. We analysed lysis of primary paediatric donors has become an established procedure for the precursor-B-ALL blasts by 285 NK cell clones to treatment of children with high risk and relapsed leukae- investigate whether HLA class I expression on the blasts mia.1–3 GVHD-related mortality has been reduced signifi- and phenotypic killer cell Ig-like receptor (KIR) expres- cantly by T-cell depletion, but relapse still remains the sion on NK cells affect the lytic activity against ALL major problem after successful transplantation.4,5 In blasts. Precursor-B-ALL blasts with low HLA-I expres- contrast to myeloid malignancies, controversial effects of sion were lysed by a majority (79%) of NK cell clones, T-cell depletion on relapse rate in ALL have been whereas those with high HLA-I expression showed low reported.1,3,6,7 As normal numbers of NK cells reconstitute susceptibility to NK clones independent of their KIR from donor progenitors within a few weeks after T-cell expression patterns. NK cell activity against susceptible depleted SCT, NK cells are possible effectors for a GVL blasts was regulated by differential surface expression of reaction in the early phase post transplant and may be used the three major KIRs (CD158a, CD158b, CD158e). NK for immunotherapy.8 NK cell alloreactivity was found to be clones with none of these three KIRs or a single KIR that associated with a reduced risk of relapse in AML after recognized no ligand, were not inhibited by the targets and HLA haplotype-mismatched haematopoietic SCT.9,10 exerted higher lysis (P ¼ o0.0005) in comparison to NK Although NK cell-related GVL effects were expected in clones expressing KIRs with a ligand on the ALL blasts. patients with B-lineage ALL, nodifference in the clinical In conclusion, the quantity of HLA-I expression on outcome was seen between patients with alloreactive NK precursor-B-ALL blast regulates overall NK cell suscept- cells and those patients without.10 This was attributed to ibility; in case of reduced HLA expression, differential the fact that B-lineage-ALL cells were not killed by NK surface expression of KIRs affects NK cell alloreactivity cells. However, recent reports indicate a different suscept- against those blasts. ibility of childhood ALL and adult ALL.11 In this study, we Bone Marrow Transplantation (2009) 43, 875–881; show that a relevant number of NK cell clones exert doi:10.1038/bmt.2008.398; published online 19 January 2009 cytotoxic activity against primary childhood precursor-B- Keywords: NK cells; ALL; childhood; KIR; HLA ALL blasts and we investigate factors of the observed functional heterogeneity. The heterogeneous activity of NK cell populations arises from the differential cell surface expression of various inhibitory and activating receptors.12 The alloreactions of human NK cell clones have been defined mainly by the activity of inhibitory receptors with specificity for HLA class I determinants.13,14 The killer cell Ig-like receptor (KIR) family includes inhibitory receptors for polymorphic determinants of HLA-A (KIR3DL2), Correspondence: Dr T Feuchtinger, Department of Paediatric HLA-B (KIR3DL1/CD158e) and HLA-C (KIR2DL1/ Haematology/Oncology, University Children’s Hospital, Eberhard Karls CD158a, KIR2DL2, KIR2DL3/CD158b).15 In the absence University, Hoppe Seyler Strasse 1, Tu¨ bingen D 72076, Germany. of the cognate ligand, NK cells kill allogeneic target cells. The first two authors contributed equally to this work. Because alloreactive killing depends mainly on absent E-mail: [email protected] Received 6 June 2008; revised 10 October 2008; accepted 10 October inhibitory signals from HLA-I ligands, control of allo- 2008; published online 19 January 2009 reactive NK cell activity has been described as ‘missing NK clone cytotoxicity vs childhood ALL blasts T Feuchtinger et al 876 self’.16 Despite the complexity of NK cell receptors for cells were labelled with the fluorescence enhancing ligand HLA-I molecules, the most prevalent, and dominant, BATDA (Wallac Oy, Turku, Finland) for 30 min (LCL patterns of alloreactivity are those caused by two groups cells), or 60 min (leukaemic blasts) at 37 1C. After five of HLA-C epitopes, termed as HLA-Cw3-related (ligated washing steps, the target cell suspension was adjusted to toCD158b) and HLA-Cw4-related alleles (ligated to 2 Â 105 cells/ml and seeded intomicroplates(5000 target CD158a). Deficiency of the inhibitory signal due to cells/well). Effector to target (E:T) ratio was 5:1. The assays incompatibility to non-self HLA-I or reduced expression were continued as published.20 Specific lysis was calculated of HLA-I results in activation of NK cells. In particular, as follows: % specific lysis ¼ (experimental releaseÀsponta- malignant cells with reduced HLA-I expression, including neous release)/(maximum releaseÀspontaneous re- solid tumours, lymphomas and leukaemias, are supposed to lease) Â 100. Statistical analysis of differences in specific be valid targets for lysis by NK cells.17–19 In this study, we lysis were analysed by paired or unpaired t-tests using analysed NK cell clones for antileukaemic activity against Graph Pad Prism software. w2-test for KIR distribution was primary precursor-B-ALL blasts and investigated the carried out using sigma-stat software. relevance of phenotypic KIR expression for specific lytic alloreactivity against B-lymphoid leukaemic blasts. Re- duced HLA-I expression on ALL blasts conditioned good Phenotypic analysis NK susceptibility and enabled analysis of the broad Flow cytometry was performed using saturating concentra- variability in effector function. tions of the following: directly labelled antibodies CD56, CD158a, CD158b, CD158e (all purchased from Becton Dickinson, Heidelberg, Germany), NKG2A (purchased Materials and methods from Beckman Coulter, Kreefeld, Germany). Data acquisition was performed on a FACS Calibur using Cell quest Preparation of NK cell clones and target cells software (both from Becton Dickinson). A four-colour Natural killer cell clones from four healthy donors with FACS analysis was performed with CD158a, CD158b, either HLA-Cw4-related (n ¼ 2) or HLA-Cw3-related HLA CD158e and CD56 todetect NK cloneswith single alleles (n ¼ 2; see Table 1) were screened for alloreactive expression of KIRs and to evaluate KIR expression. The killing of primary, cryopreserved, precursor-B-ALL blasts primary leukaemic blasts were obtained from BM biopsies from BM of childhood leukaemias. PBMCs depleted of T at the time of diagnosis. Blasts showed high expression of cells by negative immuno-magnetic selection with anti-CD3 CD10, CD19, CD34 (490% of BM cells). Lymphocyte moAb (MACS microbeads, Miltenyi-Biotec, Bergisch- function-associated Ag 1a (CD11a) was negative. Gladbach, Germany) were plated at a concentration of 10 CD45RA/RO (all from Becton Dickinson), HLA-I (Dako- cells per well in 96-well microtiter plates, activated with Cytomation, Hamburg, Germany) and HLA-E (Abcam, PHA, and cultured with IL-2 and irradiated feeder cells Cambridge, MA, USA) expression was reduced when 9 þ compared with normal B-cells (Figure 1). For quantitative as described. CD3 cells were o0.1% of viable cells after CD3 depletion. Clones with detectable CD3 þ T cells were analysis of HLA-I expression, leukaemic blasts were excluded from the analysis. Cloned NK cells were used as incubated with a saturated concentration of unconjugated effectors in cytotoxicity assays. Target cells were cryopre- primary antibodies W6/32 (anti-HLA-ABC, Dako) and served, primary, childhood B-lineage leukaemic blasts. The isotypic control IgG1 (Immunotech, Marseille, France) for 1 study was authorized by the ethical institutional review 45 min at 4 C. Cells were washed twice and then incubated 1 board. Patients and healthy donors gave informed consent. with FITC-F(ab’)2 conjugates (Dako) for 45 min at 4 C. Set-up and calibration beads coated with known amounts of mouse antibodies (QIFIKIT, Dako) were stained with Cytotoxicity assay the same secondary Ab for the same time. Analysis was Cytolytic activity of NK cells was tested in a 2 h BATDA performed according to the manufacturer’s instructions: 0 0 00 00 [bis (acetoxymethyl) 2,2 :6 ,2 -terpyridine-6,6 -dicarboxy- set-up beads were used tooptimizethe instrument settings, late)] europium release assay. Cryopreserved primary calibration beads were used for construction of the precursor-B ALL blasts were used as target cells. Target calibration curve (mean fluorescence intensity against Ab- binding capacity). Ab-binding capacity of the leukaemic Table 1 HLA genotype of healthy donors and primary precursor- blasts was calculated by interpolation on the calibration B-ALL blasts curve. One Ab-binding site was assumed per one surface HLA type Corresponding KIR HLA-I molecule. The linear range of the calibration beads reaches from 0 to 606 000 and therefore all results were in Healthy donors this range. Donor 1 B35 B51 Cw0401 Cw1510 CD158a/CD158e Donor 2 B07 B58 Cw0302 Cw0702 CD158b/CD158e Donor 3 B51 B62 Cw4 CD158a/CD158e Donor 4 B44 Cw0304 Cw0702 CD158b/CD158e Results Precursor-B-ALL blasts Patient 1 B40 B51 Cw0304 Cw1402 CD158b/CD158e HLA-I and HLA-E expression on precursor-B-ALL blasts Patient 2 B08 B62 Cw3 Cw7 CD158b control NK cell susceptibility Patient 3 B51 B55 Cw3 Cw14 CD158b/CD158e Natural killer cell susceptibility in relation to HLA-I Patient 4 B41 B15 Cw0304 Cw1701 CD158a/CD158b expression was screened with 285 NK clones from healthy

Bone Marrow Transplantation NK clone cytotoxicity vs childhood ALL blasts T Feuchtinger et al 877 HLA-1 expression donors and primary ALL blasts from four paediatric patients (twowith reduced and twowith high HLA-I Control 150 expression; Figure 1). Mean lysis of blasts with reduced HLA-I was 46.6±24% s.d. compared with 12.0±9% s.d.

120 in blasts with high HLA-I expression (P ¼ o0.001). In Figure 2 is shown the distribution of the functional activity

90 of the NK clones. Only 4 out of 125 NK clones showed a higher lysis HLAhigh leukaemic blasts. Expression of HLA-I (ligand for KIR) and HLA-E (ligand for NKG2A) on these 60 target cells was analysed by ﬂow cytometry, to conﬁrm the possibility of a receptor–ligand interaction between tested 30 NK cell clones and primary B-ALL blasts. HLA-I expression was as follows: 85991 and 135458 HLA epitopes 0 per cell on the two leukaemias with low HLA-I expression 100 101 102 103 104 and 356427 and 587986 HLA epitopes per cell on the two 140 low leukaemias with high HLA-I expression (Figure 2). Normal HLA blasts B cells express a mean of 231064±49929 (n ¼ 10) HLA-I 120 epitopes per cell. A study of different childhood B-lineage

100 leukaemias has recently shown a reduced HLA-I expression in two-thirds of cases.11 Mean HLA-E expression on the 80 two leukaemias with low HLA-I expression was 890 epitopes per cell and 1665 on the leukaemias with high 60 HLA-I expression.

40 NK clones could lyse primary precursor-B-ALL blasts 20 The broad spectrum of NK cell function was tested against

0 NK susceptible precursor-B-ALL blasts and compared 100 101 102 103 104 with K562 (Figure 3). More than 79% of tested NK clones (n ¼ 98) effectively killed precursor-B-ALL blasts (440% 70 HLAhigh blasts specific lysis; see Figure 3a). Mean specific lysis was 54.2% (±20% s.d.). To exclude functional inactivity, positive 60 controls were performed with the erythroblastoid cell line 50 K562 in each clone. The mean specific lysis of K562 was 104±11% s.d. (Figure 3b), showing a constantly high lytic 40 activity against this standard NK target. These results

30 confirm that generally hyporesponsive NK cell clones were not among the clones and the differences in the lysis of 20 leukaemia cells (Figure 3a) could be attributed to target inhibition. Additional blocking of HLA-I with a MoAb 10 (W6/32) on the target cells significantly (P ¼ 0.001) 0 enhanced the specific lysis (n ¼ 5; data not shown). 100 101 102 103 104

20 Healthy B-cells Phenotype of NK cell clones Natural killer cell clones were further analysed for relevant factors, which could influence the variable cytotoxic 15 activity against precursor-B-ALL blasts. The various patterns of predominantly inhibitory receptors on NK clones were analysed by four-colour flow cytometry. This 10 method could show differential expression of the Ig-like receptors CD158a, CD158b and CD158e on a single cell basis. One-third of NK clones expressed none of these three 5 KIR and were termed CD158aÀ/bÀ/eÀ, another third of NK clones expressed one of these three KIR and were termed single KIR þ . The remaining third expressed 2–3 0 100 101 102 103 104 KIRs out of CD158a/b/e. Ninety percent of the tested clones were NKG2A þ . The occurrence of CD158aÀ or Figure 1 Expression of HLA-I on primary precursor-B-ALL blasts and CD158b single þ NK clones correlated with the donor healthy B cells: expression of HLA class I is shown for two representative ALL þ blasts. Standardized beads allow an estimation of HLA epitopes per cell using the HLA type: HLA-Cw3 donors had significantly more NK geometrical mean in flow cytometry. HLAlow blasts had 85991 HLA epitopes per clones expressing the specific receptor CD158b for their cell, HLAhigh blasts 356 427 and normal B cells 274 364 HLA epitopes per cell. own ligands (42 CD158b single þ out of 160 clones) than

Bone Marrow Transplantation NK clone cytotoxicity vs childhood ALL blasts T Feuchtinger et al 878 HLA expression on blasts: Low High

100 90 80 No. of 70 tested NK 60 clones: 50 n = 98 40 n = 62 30 20 n = 62 10 n = 63 Specific lysis by NK clones [%] 0 12 3 4 Primary childhood pre B-ALL blasts [patient No.] Figure 2 Natural killer (NK) cell lysis of four primary ALL blasts according to HLA class I expression: NK cell activity was tested against primary precursor-B-ALL blasts from four paediatric patients. Two of them with high and two with low HLA class I expression. NK cell susceptibility was signiﬁcantly higher in blasts with reduced HLA class I expression (P ¼ o0.001). Therefore, further study of effector-related variability in NK cell lysis was studied with HLAlow blasts from patient no. 1 and 2.

NK clones with CD158a (12 CD158a single þ out of 160 100 NK clones; P ¼ o0.004), which should not be inhibited by their own HLA type. Conversely, HLA-Cw4 þ donors 80 produced signiﬁcantly more NK clones with single KIR expression of CD158a (20 CD158a single þ out of 103 NK 60 clones) than clones with single KIR expression of CD158b (10 CD158b single þ out of 103 NK clones, P ¼ o0.001). 40 Specific lysis of

preB-ALL blasts [%] NK activity against primary ALL blasts is affected by 20 differential KIR expression Precursor-B-ALL blasts with low HLA expression (HLAlow 0 blasts; patients 1 and 2) showed a variable susceptibility to 0 102030405060708090100 NK clone lysis and this variability could be attributed to the KIR phenotype of the NK clones (Figures 4a and b). NK cells expressing none of the three major KIRs 100 (CD158aÀ/bÀ/eÀ clones) efﬁciently lysed HLAlow blasts, irrespective of the donor HLA type (mean lysis exerted by 80 NK clones from Cw3 þ donors: 60%, mean lysis exerted by NK clones from Cw4 þ donors: 62%; see Figures 4a and b). 60 These CD158aÀ/bÀ/eÀ clones exerted signiﬁcantly higher lysis of HLAlow blasts than clones expressing one single

of K562 [%] 40 Specific lysis KIR (CD158b) that can recognize ligands on the target cells. NK clones expressing one single KIR (CD158a) that 20 does not bind to HLA ligands on the target cells (HLA- Cw3) were not inhibited in their cytolytic function and 0 À À À 0 102030405060708090100 reached specific lyses similar tothat ofCD158a /b /e À À À No. of NK cell clones clones. Thus, CD158a /b /e clones and single positive clones with receptor–ligand mismatch appeared to be the Figure 3 Majority of natural killer (NK) clones lyse precursor-B-ALL most effective, whereas clones with a possible receptor– blasts expressing reduced HLA class I levels. Alloreactive activity of 98 NK cell clones from healthy donors were screened with two target cells: HLA- ligand engagement exerted a lower activity. The response Cw3 þ and -Cw4À precursor-B-ALL blasts (HLAlow blasts) from patient pattern of the described NK cell sub-populations was no. 1 (a) and the erythroblastoid cell line K562 (b) as a positive control for independent of the HLA-Cw match between NK cells and NK cell activity to exclude functionally inactive clones. The majority of all blasts (Figures 4a and b). Furthermore, in both groups tested NK clones effectively lysed precursor-B-ALL blasts (440% specific lysis). The constantly high lysis of K562 (MHC-I negative cell line) confirm (panels a and b) a significant difference was observed that NK clones were not generally hyporesponsive NK and the differences between clones with a possible KIR ligation and clones in the lysis of leukaemia cells (a) could be attributed to target inhibition. without a possible KIR ligation. This difference in cytolytic

Bone Marrow Transplantation NK clone cytotoxicity vs childhood ALL blasts T Feuchtinger et al 879 PreB-ALL with HLAlow expression PreB-ALL with HLAhigh expression

P=<0.0001

70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 with E:T HLA-CW match - + + + - + + + Specific lysis of blasts [%] - /e - /e - /b - /b CD158a CD158b CD158e CD158a CD158b CD158e >1 inh. KIR >1 inh. KIR CD158a CD158a

P=0.0005

70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 - + + + - + + +

Specific lysis of blasts [%] - /e - /e with E:T HLA-CW mismatch - /b - /b KIR CD158a CD158b CD158e CD158a CD158b CD158e >1 inh. KIR >1 inh. CD158a CD158a KIR expression on NK cell clones (n=284)

Panel A: HLA-Cw3+ -Cw4– NK cells vs HLA-Cw3+ -Cw4– leukaemia cells Panel B: HLA-Cw3– -Cw4+ NK cells vs HLA-Cw3+ -Cw4– leukaemia cells Figure 4 Lysis of precursor-B-ALL blasts by natural killer (NK) cell clones is related to the quantity of HLA class I on ALL blasts and the differential KIR phenotype on NK clones. Cytolytic NK function is tested in HLAlow blasts (a and b) and in HLAhigh blasts (c and d) from children with precursor-B ALL. The lytic activity of the NK clones is shown on the x axes, according to the KIR phenotype. NK cell/blast combinations are shown with identical HLA-C type (a and c) and with mismatched HLA-C type (b and d). In case of reduced HLA class I expression, NK cell susceptibility is increased to a variable degree. This variability depends on the differential KIR expression. (a) Shows the cytotoxic activity of HLA-Cw3 þ and -Cw4À NK cell clones from healthy donors against HLA-Cw3 þ and -Cw4À ALL blasts from two children (patient nos. 1 and 2), according to the KIR expression on NK cells. (b)Shows the cytolytic activity of HLA-Cw4 þ and -Cw3À NK cell clones from healthy donors against the ALL blasts from the same two children, according to the KIR expression on NK cells. NK clones without any of the three major KIRs or NK clones expressing one single KIR (CD158a) that is not engaged by its ligand on the target cells (HLA-Cw3) are not inhibited in their cytolytic function in comparison with NK clones expressing one or more inhibitory KIR (CD158b, 158e) with a cognate ligand on the targets. Although MoAbs against CD158 a/b/e could not distinguish between activatory and inhibitory isoforms of KIR and CD158b detects KIR2DL2 and KIR2DL3, the difference reached statistical significance determined by unpaired t-tests between mean values±s.e.m. activity between NK clones with inhibiting ligand and those malignancies after haematopoietic SCT in adults. Recently, without was 20% (P ¼ o0.0001 within HLA Cw3 þ NK in a limited number of paediatric patients with lymphatic clones and P ¼ 0.0005 within HLA Cw4 þ NK clones; leukaemia, a lower relapse rate after mismatched trans- Figures 4a and b). The HLA-C types of the donors were plantation was attributed to NK cell alloreactivity:21 NK not predictive, as similar lyses were obtained with both cell alloreactivity correlated to KIR-ligand mismatch Cw3 þ and Cw4 þ donors. The NK lysis of ALL blasts with within the HLA-I alleles of donor and recipient.10,22 In high HLA expression (HLAhigh blasts, patients 3 and 4) was particular, three groups of KIRs and their ligands have not affected by the KIR expression (Figures 4c and d). The been considered to play a predominant role in this model: specific lysis remained below 20% and no significant Cw3-related HLA C alleles (recognized by CD158b), Cw4- difference could be seen between clones with different related HLA C alleles (recognized by CD158a) and HLA KIR expression. Bw4 alleles (recognized by CD158e). The phenotypical detection of KIR expression may enable the selection of viable NK cell populations for potential immunotherapy, Discussion compared with genotyping of KIR expression. However, the MoAbs could not distinguish between inhibitory and Natural killer cell-mediated alloreactive GVL reactions activating isotypes of the same receptor and anti-CD158b have been shown to reduce relapse rates of myeloid could even bind to KIR2DL2 and KIR2DL3. Despite the

Bone Marrow Transplantation NK clone cytotoxicity vs childhood ALL blasts T Feuchtinger et al 880 fact that phenotypical characterization of KIR expression ligand.31 However, initially hyporesponsive NK cells could not distinguish between inhibitory and activating showed inducible effector function after stimulation with isotypes, engagement of the majority of these KIR will cytokines.28,29 As NK cell clones are stimulated with high result in an inhibition of the cytotoxic activity in the vast dose IL-2, the observed antileukaemic reactivity of our NK majority of NK cells.10,23–26 On the basis of these cell clones with inappropriate inhibitory receptors, may not assumptions, the effect of alloreactive NK cells on relapse be contradictory to these observations. Clinical IL-2 rate after haploidentical transplantation was predictable by application post-SCT might be considered an option to the KIR ligand mismatch. In contrast, B-cell lineage activate such donor-derived cells in vivo. Furthermore, the malignancies in adults have not been considered a relevant distinction of NK population according to three KIR target for NK cell-mediated antileukaemic effects.9 In this receptors remains a simplified view of the complex study, we focus on the effector function of NK cells against mechanisms controlling NK cell activity. A large number the most common paediatric malignancy—acute lympho- of activating and/or inhibitory receptors and potential blastic precursor-B-cell leukaemia of infancy and child- ligands have been identified in the past years, and the hood. We show in this study that (1) childhood primary clones called CD158aÀ/bÀ/eÀ or KIR single positive NK precursor-B-ALL blasts with reduced HLA-I expression cells in fact express additional receptors and triggering were lysed by a relevant proportion of NK cell clones from surface molecules that have not been taken into healthy donors, whereas blasts with high HLA-I expression consideration. were mainly resistant toNK-mediated lysis. This indicates In summary, we show that certain paediatric precursor- a relevance of HLA expression for NK cell-mediated lysis. B-ALL blasts can be lysed by NK clones, depending (2) In case of HLAlow blasts, an additional effect appeared predominantly on the intensity of HLA-I expression. The to be relevant: NK clones expressing at least one KIR, differential surface expression of inappropriate KIR, which binds toHLA alleles ofthe target, exerted positively affected the lytic activity against those primary significantly lower lysis than NK clones without a childhood B-lineage ALL blasts. Clinical studies may corresponding KIR. Hence, NK clones with high lytic answer the question, whether these NK cell-mediated activity were characterized by the lack of the three major effects exist in vivo after allogeneic mismatched transplan- KIRs or by a KIR, which is not engaged by the HLA-I tation in paediatric leukaemia. alleles of the target. The intensity of HLA expression on precursor-B-ALL targets appears to be a predominant factor for NK cell susceptibility. Precursor-B-ALL blasts Acknowledgements with increased HLA-I expression (as compared with healthy B cells) are mainly resistant and block NK We thank A Barbarin-Dorner, L Koschnik for excellent cell clones, whereas reduced HLA expression allows technical assistance, A Velardi, L Ruggeri for helpful advice certain degrees of NK lysis. Only in this case, incompat- and D Martin for reviewing the manuscript. LCL with defined ibility or lack of the corresponding KIR on NK clones HLA-C expression was kindly provided by A Velardi, Perugia, improved lysis. This phenomenon was independent from Italy. This study was supported by a grant from the Deutsche the HLA-C types of NK cells and ALL blasts and Forschungsgemeinschaft (DFG) Grant no. 2001.048.1 (PL) and independent of an HLA-C match or mismatch between SFB685. NK cells and ALL blasts, as CD158aÀ/bÀ/eÀ or single Declaration of commercial interest: All authors have no positive clones were found in both Cw3 and Cw4 þ donors financial or commercial and personal interest or relation- (Figure 4, upper panel compared with lower panel). In ships to disclose with other people or organizations that the case of CD158aÀ/bÀ/eÀ NK cells, cytolytic function of could inappropriately influence or bias their work. those cells is expected to be regulated by NKG2A–HLA-E interaction. In our blasts with reduced HLA-I expression, additional decrease of HLA-E expression was detected as References compared with healthy B cells. This may explain why CD158aÀ/bÀ/eÀ NK cells were not inhibited through 1 Green A, Clarke E, Hunt L, Canterbury A, Lankester A, Hale G et al. Children with acute lymphoblastic leukemia who NKG2A. receive T-cell-depleted HLA mismatched marrow allografts The existence of NK cells with an incomplete pattern of from unrelated donors have an increased incidence of primary À À À KIR expression (CD158a /b /e or single positive) was not graft failure but a similar overall transplant outcome. Blood only shown in the somewhat arbitrary situation of NK cell 1999; 94: 2236–2246. cloning but also after clinical transplantation of highly 2 Godder KT, Hazlett LJ, Abhyankar SH, Chiang KY, purified CD34 þ progenitors. NK cells derived from those Christiansen NP, Bridges KD et al. Partially mismatched progenitors acquired a specific pattern of KIR expression related-donor bone marrow transplantation for pediatric step by step within the first 3 months.21 During this period patients with acute leukemia: younger donors and absence of of receptor acquisition post-allogeneic SCT, subsets of NK peripheral blasts improve outcome. 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