Human CD80/IL2 lentivirus-transduced acute myeloid leukaemia ...

research paper

Human CD80/IL2 lentivirus-transduced acute myeloid leukaemia (AML) cells promote natural killer (NK) cell activation and cytolytic activity: implications for a phase I clinical study

Wendy Ingram, Lucas Chan, Hayrettin Guven, David Darling, Shahram Kordasti, Nicola Hardwick, Linda Barber, Ghulam J. Mufti and Farzin Farzaneh Department of Haematological Medicine, King’s College London, London, UK

Received 8 January 2009; accepted for publication 10 March 2009 Correspondence: Professor G. J. Mufti, Department of Haematological Medicine, King’s College London, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK. E-mail: [email protected]

Summary Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/ IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46Æ6% to 90Æ4%) and allogeneic AML cells (from 11Æ8% to 20Æ1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0Æ4% to 22Æ5%). These studies demonstrated that vaccination of patients with CD80/ IL2-transduced AML cells could provide a powerful strategy for T/NK cellmediated stimulation of anti-leukaemic immunological responses. Keywords: acute myeloid leukaemia, lentivirus, CD80, IL2, NK cells.

Natural killer (NK) cells represent innate effector cells that help provide defence against pathogens as well as tumours. The cytolytic activity of NK cells is governed by a complex balance of activation and inhibitory receptor signalling which, when disrupted, may impair effective anti-tumour immune responses (Biassoni et al, 2001; Bryceson et al, 2006). Acute myeloid leukaemia (AML) encompasses a heterogeneous group of clonal hematopoietic stem cell disorders that are associated with impaired NK cell activity (Costello et al, 2002; Jaffe et al, 2001; Tajima et al, 1996). The natural cytotoxicity receptors (NCRs) NKp30, NKp44 and NKp46 are highly NK cell-specific, non-human leucocyte antigen (HLA) restricted activating molecules and are important in the cytolytic response against leukaemia (Cantoni et al, 1999; Costello et al, 2002; Fauriat et al, 2007; Moretta et al, 2001; Pende et al, 1999; Pessino et al, 1998; Sivori et al, 1997). Dysregulation of the

activating receptor NKG2D and its ligands have also been described in AML, (Nowbakht et al, 2005; Pende et al, 2005; Salih et al, 2003) whereas the role of other activating receptors, such as DNAX accessory molecule 1 (DNAM-1, CD226) and CD244 (2B4), are unclear. Strategies to promote NK-mediated cytotoxicity against AML are the subject of ongoing studies. Interleukin 2 (IL2) therapy has been used in clinical trials for the induction of NK cell activation, but this results in adverse events and toxicity, particularly from capillary leak syndrome (Benyunes et al, 1993; Boughton & Simpson, 1999; Farag et al, 2002a; Lim et al, 1992; Maraninchi et al, 1998; Margolin et al, 1999; Meloni et al, 1996). Haploidentical donor transplantation exploits NK cell alloreactivity as a result of killer immunoglobulin-like receptor (KIR) mismatching and aims to enhance the graft versus leukaemia (GvL) response following transplantation

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 145, 749–760

First published online 20 April 2009 doi:10.1111/j.1365-2141.2009.07684.x

W. Ingram et al (Farag et al, 2002b). The effect of KIR incompatibility on outcome is currently under debate as the initial improved outcome reported by Ruggeri et al has not been a consistent finding (Farag et al, 2006; Hsu et al, 2006; Ruggeri et al, 2002, 2007; Schaffer et al, 2004). We and others have previously shown that modification of tumour cells to express the co-stimulatory molecule CD80 in combination with a stimulatory cytokine, such as IL2, promotes T cell activation and induces T cell-mediated rejection of the tumour in murine models (Emtage et al, 1998; Gaken et al, 1997; Stripecke et al, 2000). Furthermore, efficient transduction of AML blasts can be achieved using a self-inactivating lentiviral vector encoding CD80 (B7Æ1) and IL2 (Chan et al, 2005). The primary aim of this strategy is to induce T cell activation. This is based on the knowledge that AML cells express high levels of major histocompatibility complex Class I and II and frequently CD86 (B7Æ2), but invariably lack expression of the co-stimulatory molecule CD80 (Vollmer et al, 2003; Whiteway et al, 2003). Transduction of AML cells with CD80 could therefore enable them to act as antigen presenting cells. Indeed, we have previously reported enhanced T cell proliferation with a T-helper cell type 1 (Th1) cytokine profile following in-vitro culture with CD80/ IL2 lentivirus-transduced AML cells (Chan et al, 2005). However, the effect of CD80/IL2 modification of AML cells on NK cell activation and cytotoxicity has not been previously studied. Importantly, both IL2 and CD80 may have a role in NK cell activation. Expression of CD80 on tumour cells has been shown to render the tumour more susceptible to NK-mediated lysis (Wilson et al, 1999). In addition, NK cells may also express CD28 providing a potential mechanism for CD80 stimulation (Galea-Lauri et al, 1999). Hence, the advantage of this strategy is the possible stimulation of both T and NK cell cytotoxicity, thereby maximising the potential anti-leukaemia immune responses. The present study examined the effect of stimulation by CD80/IL2 lentivirus-transduced cells on allogeneic and autologous NK cell activation. Furthermore, the cytolytic activity of stimulated NK cells was examined against K562 and primary AML cell targets.

World Health Organisation classification of AML was confirmed by immunophenotyping, cytogenetics and molecular analysis at King’s College Hospital, London (Jaffe et al, 2001). The diagnoses were as follows: AML without maturation n = 3, AML with maturation n = 3, acute promyelocytic leukaemia n = 2, acute myelomonocytic leukaemia n = 4, AML with multilineage dysplasia n = 4. The median age of AML patients was 51 years (range 24–74 years) and was 39 years (range 31–79 years) for healthy donors.

Monoclonal antibodies and flow cytometry The percentage (%), absolute number (using trypan blue dead cell exclusion) and phenotypic characterization of NK cells (CD3)CD56+) from both healthy donors and AML patients was assessed on days 0 and 7 of culture. Surface staining with fluorochrome-conjugated monoclonal antibodies (mAbs) from BD Bioscience against the following antigens was performed: CD3 (UCHT1), CD8 (SK1), CD56 (B159), CD16 (3G8), CD69 (L78), HLA-DR (L243), CD107a (H4A3), CD244/2B4 (2-69), KIR2DL1 (CD158a) (HP-3E4), KIR2DL2/3 (CD158b) (CH-L), KIR3DL1 (CD158e/NKB1) (DX9), DNAM-1/CD226 (DX11), NKG2D (1D11), NKp30 (p30-15), NKp46 (9E2). Also analysed were CD25 (BC-96) eBioscience, NKG2A (13 144) and NKG2C (134 591) R&D Systems, NKp44 (2Æ29) Miltenyi Biotec, CD85j/ILT-2 (HP-F1) Beckman Coulter and CD28 (YTH913Æ12) Serotec. The mean fluorescence intensity (MFI) for each NK cell receptor was recorded on days 0 and 7 of co-culture. The change in receptor density (MFI ratio) was calculated using the MFIday 7/MFIday 0; values >1 represented an increase in relative receptor density. There was no change in the MFI of the isotype-matched control between day 0 and day 7 of culture. Surface staining of cell lines and AML blasts was performed prior to lentivirus transduction with fluorochrome-conjugated mAb against CD80 (L307Æ4) BD Bioscience and HLA-class I (W6/32) Dako. Flow cytometry was performed on the FACSCantoII (BD Biosciences) and data analysed using flowjo software (Tree Star Inc, version 7.2.4).

Lentiviral-mediated gene transduction Materials and methods Acquisition of patient and donor material Peripheral blood (PB) and/or bone marrow (BM) was obtained from 16 AML patients at presentation and PB from eight patients in complete remission (CR). PB from 12 healthy donors was used as controls. The study was approved by King’s College Hospital research ethics committee and all patients and donors gave written informed consent in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) and AML blasts were isolated from blood or BM by density gradient centrifugation (Histopaque; Sigma, St Louis, MO, USA). The diagnosis and 750

Production and titration of lentiviral vectors was performed as previously described (Chan et al, 2005). AML blasts or leukaemia cell lines (U937, HL60) were thawed and plated at a cell density of 5 · 105)1 · 106/ml in X-VIVO 15 (Cambrex Bio Science). AML blasts were supplemented with recombinant human stem cell factor (rhSCF, 20 ng/ml) and recombinant human interleukin 3 (rhIL3, 10 ng/ml) (Peprotec EC) for 24 h prior to lentiviral-mediated gene transfer. Aliquots of lentivirus were thawed and used at a multiplicity of infection of 10, supplemented with 10 lg/ml diethylaminoethyl cellulose dextran (Amersham Pharmacia, Buckinghamshire, UK). The cell were washed 24 h following lentiviral infection, then cultured for a further 48 h prior to analysis of CD80


CD80/IL2-transduced AML Cells Stimulate NK Cell Activity (fluorescent-activated cell sorting) transgene expression and IL2 (DuoSet enzyme-linked immunosorbent assay; R&D systems) secretion.

Cell culture Primary AML blasts or either U937 or HL60 cell lines were used as stimulator cells. Unmodified or lentivirus-transduced stimulators were washed, re-suspended in fresh X-VIVO 15 and c-irradiated at 25–50 Gy for primary AML cells or cell lines respectively. PBMCs from healthy donors or AML patients were used as responder cells and cultured at a stimulator: responder ratio of 1:2. As a comparator for cultures stimulated with CD80/IL2 lentivirus-transduced cells, rhIL2 (Peprotec EC) at either low dose (100 IU/ml) or high dose (1000 IU/ml) was added to responder cells on day 1 of culture. K562, Raji, U937 and HL60 cell lines were maintained in culture in RPMI 1640 medium supplemented with 10% foetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin prior to use in cell culture assays.

Cytotoxicity assays

(KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A, CD85j) were analysed in 16 AML patients at presentation and compared with 12 healthy donor controls. A significantly lower percentage (P = 0Æ02) and absolute number (P = 0Æ004) of NK cells was detected at presentation. The percentage and MFI of the activation receptors DNAM-1 (P < 0Æ001), NKG2D (P < 0Æ001) and CD16 (P < 0Æ001) were significantly lower in AML patients (Fig 1). No significant differences were detected in the expression of NKp30, NKp46, CD244 or

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Analysis of NK cell-mediated cytotoxicity was performed using a standard 4-h 51chromium release assay. Target cells (K562 or primary AML cells) were plated at 5 · 103 per well. All assays were performed in triplicate. Where indicated, NK cells were negatively isolated on days 0 and 7 of culture using an NK cell isolation kit (Miltenyi Biotec) and separated on an autoMACS separator. The % specific lysis was calculated using the following formula: % lysis = [(experimental release ) spontaneous release)/(total release ) spontaneous release)] · 100.

Statistical analysis

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The comparison of NK cell receptor expression between AML patients and healthy donors was performed using an independent samples t-test. A non-parametric matched paired analysis (Wilcoxon-signed rank test) was used to examine the change in NK cell receptor expression for AML patients at presentation with CR and the change in receptor expression between days 0 and 7 of culture. Statistical analysis was performed using the Statistical Package for the Social Science (SPSS) version 14.0 (SPSS, Chicago, IL, USA) with statistical significance set at P < 0Æ05. Graphs were produced using GRAPHPAD PRISM (version 5.0).

Results Down-regulation of NK cell activation and inhibitory receptors in AML The percentage of cells expressing and the MFI of the NK activation receptors (NKp30, NKp46, CD244, DNAM-1, NKG2D, NKG2C, CD16) and the inhibitory receptors

Fig 1. NK cell receptor expression in AML patients and healthy controls. (A) The percentage expression and (B) MFI for each of the indicated NK cell activation and inhibitory receptors was analysed by multi-colour flow cytometry in the peripheral blood of AML patients at presentation and healthy controls. The mean and standard error of the mean (SEM) for each of the NK cell receptors is shown. *Represents a statistically significant difference between each of the NK cell receptors between the time of presentation of AML and healthy volunteer controls. Statistical significance was set at P < 0Æ05. (C) Paired analysis of the change in receptor expression of DNAM-1 in AML patients at presentation and complete remission (CR).


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W. Ingram et al NKG2C. Interestingly, the expression of the KIR receptors, KIR2DL1 (% P = 0Æ002, MFI P = 0Æ008), KIR2DL2/3 (MFI P = 0Æ005) and KIR3DL1 (MFI P = 0Æ029) were also significantly lower in AML patients at presentation. We next used a matched paired analysis to compare NK cell receptor expression in 8 patients who achieved CR with that at presentation. Of particular note, a significant increase in the expression of DNAM-1 (median 16% vs. 79%, P = 0Æ012), NKG2D (median 7% vs. 27%, P = 0Æ036) and CD16 (median 6% vs. 73%, P = 0Æ012) was detected at CR. A representative example of the change in receptor expression of DNAM-1 is shown in Fig 1C. We subsequently examined whether CD80/IL2-transduced cells could augment NK activity in healthy donors and patients who achieved CR following combination chemotherapy.

CD80 and IL2 transgene expression following lentivirus transduction of AML cells The cell lines U937/HL60 and primary AML cells were transduced with CD80/IL2 lentivirus (LV-CD80/IL2). Efficient transduction of the cell lines was achieved with a median CD80 transgene expression of 91% (range 82–100%) and secreted IL2 of 10Æ2 ng/106 cells/24 h (range 2Æ0–45Æ7 ng). As a result of their efficient transduction, the cell lines were used as stimulator cells for initial cultures. Transgene expression of LV-CD80/IL2 primary AML cells was more variable, with a median CD80 expression of 47% (range 17–85%) and IL2 secretion 1Æ29 ng/106 cells/24 h (range 0Æ29–47Æ62 ng). All cell lines and primary AML cells had 95% expression of HLA class I (1 AML had 72% expression). NK cells used for in-vitro culture showed no evidence of CD28 expression. Despite higher transgene expression by the cell lines, no significant difference in NK cell activation was detected compared with stimulation with primary AML cells (data not shown) and as such, the results of all allogeneic cultures are reported together. As shown in Fig 2A and B, a significant increase in the number of NK cells was observed following allogeneic stimulation with LV-CD80/IL2 AML cells as compared with day 0 (P = 0Æ007) or unmodified AML (P < 0Æ001). In 9/25 cultures a less than twofold increase in NK cell numbers was 752

observed with CD80/IL2 stimulation (Fig. 2B). The variation in the magnitude of increase in NK cell numbers showed no correlation with type of AML or cell line, CD80 transgene expression or level of secreted IL2 by transduced AML cells. Nonetheless, it is likely that in-vitro secretion of IL2 by stimulated T cells in the allogeneic cultures may contribute to the variation in NK cell numbers. The effect of stimulation on autologous NK cell activity in 13 cultures was then examined to assess responses in patients who had previously received chemotherapy. As expected, autologous AML cells provided a weaker stimulus to NK cells, therefore in contrast to allogeneic stimulation, the number of NK cells following stimulation with autologous LV-CD80/IL2 AML cells was lower than the number at day 0 (P = 0Æ019). This number of NK cells was however higher following LVCD80/IL2 stimulation than cultures with unmodified AML (P = 0Æ002) (Fig 2C and D).

LV-CD80/IL2-transduced AML cells promote NK cell activation We next examined the surface expression of a panel of NK activation (NKp30, NKp44, NKp46, CD244, DNAM-1, NKG2D, NKG2C, CD16, CD25, CD69, HLA-DR) and inhibitory (KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A, CD85j) receptors in order to analyse the impact of stimulation with LV-CD80/IL2 AML cells on allogeneic and autologous NK cell activity. The surface expression of CD107a on NK cells was examined as a marker of cytolytic activity. The percentage and MFI for each of the receptors was analysed on days 0 and 7 following allogeneic (n = 8) and autologous (n = 4) stimulation with unmodified or LV-CD80/IL2 AML cells. A matched paired analysis was used to examine the change in receptor expression compared with day 0. Receptors in which a significant change was observed following allogeneic stimulation are shown in Fig 3. In terms of percentage expression, a significant up-regulation of the activation markers NKp30, NKp44, CD244, CD25, CD69, HLA-DR and CD107a was detected following LV-CD80/IL2 stimulation compared with day 0. In addition, a significantly higher percentage expression of CD25, CD69, HLA-DR and CD107a was detected following LV-CD80/IL2 stimulation compared with unmodified AML cells. We also detected an increased MFI for NKp30, NKp44, CD25, CD69, HLA-DR and CD107a following allogeneic LV-CD80/IL2 stimulation (Fig 3). With regards to NKp44, CD69 and HLA-DR an increase in the MFI was detected in seven out of nine experiments; however, as no increase was detected in two of the experiments the detected difference was not statistically significant. Of particular note was the absence of a significant change in the percentage expression of the inhibitory receptors KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A or CD85j, with an increase in just the MFI of the inhibitory KIR receptors KIR2DL2/3 and KIR3DL1 following LV-CD80/IL2 stimulation compared with unmodified AML cells.


CD80/IL2-transduced AML Cells Stimulate NK Cell Activity

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Fig 2. Effect of in-vitro culture with CD80/IL2 lentivirus-transduced AML cells on NK cell number. The absolute cell count (ACC) of NK cells (CD3)CD56+) was examined by multi-colour flow cytometry on day 0 then at day 7 following co-culture with unmodified (UM) or LVCD80/IL2-transduced AML cells. The ACC and median value (–) following (A) allogeneic stimulation and (C) autologous stimulation are shown. A paired comparison of ACC NK cells following (B) allogeneic and (D) autologous stimulation with UM or LV-CD80/IL2 AML cell stimulation is shown.

As a result of limited cell numbers obtained from patients post chemotherapy, the effect of LV-CD80/IL2 on activation and inhibitory receptor expression was analysed in only four autologous cultures. Using a matched paired analysis, a consistent change in receptor expression was observed following LV-CD80/IL2 stimulation (Fig 4). An increase in percentage expression of the activation markers NKp30, NKp44, NKp46, NKG2D, NKG2C, CD69 and CD107a was detected following LV-CD80/IL2 compared with day 0, with a consistently higher expression of NKp30 and CD69 compared with unmodified AML cells. Furthermore, the MFI of NKp30, NKp46 and CD69 was consistently higher following culture with LV-CD80/IL2 AML cells than they were either prior to or following culture with unmodified AML cells. As was seen following allogeneic stimulation, no change in percentage expression of the inhibitory receptors was observed, with only an increase in the MFI of the inhibitory KIR receptors KIR2DL1 and KIR3DL1 following autologous LV-CD80/IL2 stimulation. These results demonstrated that LV-CD80/IL2modified AML cells stimulate allogeneic and autologous NK cell activation.

LV-CD80/IL2 stimulated NK cells exhibit enhanced cytotoxicity against K562 and primary AML cells

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The cytolytic activity of NK cells following LV-CD80/IL2 stimulation was examined against the NK sensitive cell line K562 and against primary AML cells. Unfractionated PBMCs were initially used as effector cells against K562 in order to evaluate the cytolytic activity of the whole population containing both T cell and NK cells. The median lysis of K562 on day 0 was 3Æ6% vs. 2Æ3% following allogeneic stimulation with unmodified AML and 52Æ5% with LV-CD80/ IL2 AML cells (Fig 5A). The enhanced lysis against K562 in part probably reflects the increased number of NK cells in the LV-CD80/IL2 stimulated culture. Therefore in order to directly evaluate the NK cell cytolytic activity, NK cells were isolated on days 0 and 7 and used as effector cells in the 51Cr release assay. Indeed, lysis against K562 increased from a median of 46Æ7% on day 0 to 90Æ4% following LV-CD80/IL2 stimulation (Fig 5B). Importantly, NK cell cytotoxicity against primary AML blasts was demonstrated in three separate experiments following allogeneic and one autologous co-culture (Fig 5C and D).


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Fig 3. Change in NK receptor expression following allogeneic culture with lentivirus-transduced AML cells. (A) The percentage expression and (B) MFI for each of the NK activation and inhibitory receptors was analysed on day 0 and day 7 following allogeneic co-culture with either unmodified (UM) or LV-CD80/IL2-transduced AML cells. Statistical significance in (A) is represented by *P < 0Æ05 and **P < 0Æ001. The MFI ratio in (B) represents the MFIday 7/MFIday 0. *Represents a significant change in MFI following LV-CD80/IL2 stimulation compared with day 0 and following stimulation with unmodified AML using a matched paired analysis. The mean and SEM for each of the receptors is displayed.

The median lysis of AML blasts was 11Æ8% on day 0, 8Æ7% following allogeneic stimulation with unmodified AML and 20Æ1% with LV-CD80/IL2 AML cells. Due to limited cell numbers, only an effector: target (E:T) ratio of 5:1 was tested. Of particular importance was the enhanced cytolytic activity against autologous AML as shown in Fig 5D. Using an E:T ratio of 10:1, lysis of autologous AML was 0Æ4% on day 0, 1Æ8% following stimulation with unmodified AML and 22Æ5% after 7 d of culture with autologous LV-CD80/IL2 expressing AML cells. 754

LV-CD80/IL2 dual vector promotes high NK cell activation and cytotoxicity despite production of relatively low amounts of IL2 The effect of IL2 on NK cell activation is well described, therefore in order to assess the degree of NK cell activation and cytotoxicity following LV-CD80/IL2 stimulation, healthy donor NK cells were stimulated with either LV-IL2 AML cells or rhIL2 at a dose of 100 IU/ml or 1000 IU/ml on day 1 of culture. These two doses of rhIL2 were chosen because they


CD80/IL2-transduced AML Cells Stimulate NK Cell Activity

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Fig 4. Change in NK receptor expression following autologous culture with lentivirus-transduced AML cells. (A) The percentage expression and (B) MFI for each of the NK activation and inhibitory receptors was analysed on day 0 and day 7 following autologous culture with either unmodified (UM) or LV-CD80/IL2-transduced AML cells. The MFI ratio in (B) represents the MFIday 7/MFIday 0. (A) *Represents a significant increase in % expression following LV-CD80/IL2-transduced AML cells compared with day 0. **Represents a significant increase in % expression following LVCD80/IL2 stimulation compared with day 0 or stimulation with unmodified AML cells. (B) *Represents a consistent increase in MFI Ratio following LV-CD80/IL2 stimulation compared with day 0. **Represents a change in MFI Ratio compared with day 0 or following stimulation with unmodified AML cells. The mean and SEM for each of the receptors is displayed.

represented the range of secreted IL2 from lentivirus-transduced AML cells. NK cell activation and inhibitory receptor expression was analysed as previously described. Comparison of LV-IL2- and LV-CD80/IL2-stimulated NK cells demonstrated no significant difference in the expression of NK cell receptors or lysis of K562 or primary AML cells despite the median level of secreted IL2 being fourfold higher for the AML cells expressing IL2 alone compared with AML cells expressing CD80 and IL2 (data not shown). The results therefore suggest a potential role for CD80 in NK cell activation. Furthermore,

NK cell lysis of K562 following stimulation with rhIL2 alone displayed a dose effect. Up to 1Æ5-fold higher lysis of K562 following stimulation with 1000 IU/ml rhIL2 and up to eightfold lower lysis of K562 following stimulation with 100 IU/ml rhIL2 was detected when compared with NK cell lysis after LV-CD80/IL2 AML cell culture. This data highlights the dose effect of IL2 on NK cell activity, however it also demonstrates that effective killing of target cells was achieved with a relatively lower amount of IL2 following LV-CD80/IL2 AML cell culture.


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W. Ingram et al Fig 5. NK cell cytotoxicity is enhanced following in-vitro stimulation with LV-CD80/IL2 AML cells. NK cell cytotoxicity was analysed on day 0 and day 7 following in-vitro stimulation with unmodified or LV-CD80/IL2 AML cells, using a standard 4-h 51chromium release assay. The median % specific lysis and interquartile range is shown. (A) PBMCs from day 0 or day 7 following allogeneic culture were used as effector cells against K562 at an E:T ratio of 10:1. Separated NK cells were isolated on day 0 or 7 following allogeneic culture and used as effectors against (B) K562 at an E:T ratio of 10:1 and (C) Primary AML blasts at an E:T ratio of 5:1. (D) Separated NK cells were isolated on day 0 or day 7 following autologous stimulation with either unmodified (UM) or LV-CD80/IL2 modified AML cells. The % specific lysis against autologous AML blasts using an E:T ratio of 10:1 is shown. The % specific lysis and interquartile range for (D) represents the results of triplicate wells performed in one experiment.

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Discussion NK cell effector function is governed by the expression of activation and inhibitory receptors that, when disrupted, culminate in impaired cytotoxicity. This study demonstrated 756

depressed NK cell activity at presentation of AML and, more importantly, showed that NK cell activity can be enhanced following both allogeneic and autologous stimulation with CD80/IL2 lentivirus-transduced AML cells. A significant reduction in the absolute number of NK cells was detected at the time of presentation of AML, with associated impaired expression of the activating receptors DNAM-1, NKG2D and CD16. Of particular interest is the finding of a significantly higher expression of all three activation receptors on induction of complete remission. DNAM-1 is a potent inducer of cytotoxicity which has not previously been studied in AML and further studies are required to determine its role in the cytolytic response against leukaemia. Indeed, previous reports demonstrated impaired NK cell cytolytic function in patients with AML (Costello et al, 2002; Fauriat et al, 2007). Furthermore, in keeping with the data presented in the current study, normal NK cell receptor expression may be established on achievement of CR and has been shown to be associated with enhanced cytolytic responses (Fauriat et al, 2007). The potential for NK function to be restored at a time of minimal residual disease therefore provides an opportunity to harness autologous NK cell effector responses. Indeed, conventional therapeutic strategies have limited efficacy in patients with poor risk AML with overall survival of less than 20% reported at 5 years, thereby highlighting the need for novel therapies (Goldstone et al, 2001; Grimwade et al, 2001, 1998; Slovak et al, 2000; Wheatley et al, 1999). T cells play an important role in the elimination of leukaemia, as highlighted by the use of donor leucocyte infusions (DLI) following allogeneic transplantation (Collins et al, 1997; Kolb et al, 1995). The effectiveness of DLI is however limited by the high incidence of graft-versus-host disease (GvHD) and, as such, several novel strategies are currently being explored, including the generation of leukaemia-specific or lineage-restricted cytotoxic T cells or ex-vivo activation of DLI (Porter, 2003). In addition, the role of NK cells in AML cell lysis is currently being explored. The effect of KIR incompatibility on transplant outcome has yielded inconsistent results, however the significantly reduced relapse rates with absence of GvHD reported by some groups supports


CD80/IL2-transduced AML Cells Stimulate NK Cell Activity a potential role for NK cells in elimination of leukaemia (Ruggeri et al, 2002, 2007). Here we propose a strategy in which autologous blasts transduced ex-vivo with a self-inactivating lentivirus vector encoding CD80 (B7Æ1) and IL2 may be used to vaccinate patients with poor prognosis AML (Chan et al, 2005). This strategy not only has the potential to stimulate T cell activity but may also promote NK cell activation and cytotoxicity thereby maximising the potential anti-leukaemia immune response. The effect of CD80/IL2-transduced AML cells on in-vitro T cell activation has previously been reported (Chan et al, 2005). We also demonstrated increased lytic activity by stimulated cytotoxic T cells (unpublished observations), whereas the effect on NK cell activity and the role of NK cells in the cytolytic response against AML has not been previously described. As expected, allogeneic culture provides a strong stimulus to NK cells. As a result, a significant increase in the number of NK cells was detected following stimulation with allogeneic LVCD80/IL2 AML cells. In addition, expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR together with the cytolytic receptor CD107a was significantly higher following LV-CD80/IL2 stimulation. In contrast, the weaker stimulation provided by autologous AML cells led to a fall in the number of NK cells on day 7 in cultures stimulated with both unmodified and LV-CD80/IL2 AML cells. NK cell numbers were however higher in LV-B7Æ1/IL2 stimulated cultures, supporting the notion that IL2 is required to support in-vitro maintenance of NK cells. Despite a lower NK cell number, up-regulation of the activation receptors; NKp30, NKp44, NKp46, NKG2D and CD69 as well as CD107a were detected following co-culture with LV-CD80/IL2 expressing autologous AML cells. The results therefore illustrate that NK cell activity can be enhanced even in patients who have recently received chemotherapy. Fewer changes in inhibitory receptor expression were detected following either allogeneic or autologous culture, as compared with the changes in activatory receptors. An increase in MFI of the KIRs was the only change detected following stimulation with LV-CD80/IL2 AML cells. However, each KIR molecule exists as a pair of inhibitory and activatory receptors, the function of which is determined by the length of the cytoplasmic tail. As such, altered expression of KIR receptors (as detected by the use of monoclonal antibodies CD158a, CD158b and CD158e) may not only reflect a change in the inhibitory KIR, but also the activatory molecule. An increase in inhibitory KIR expression may potentially inhibit NK cell killing of HLA class I target cells. However, as previously discussed, NK cell function is dependent on the balance of NK cell activatory and inhibitory receptors. Even though it is possible that activatory and/or inhibitory KIR expression is increased by LV-CD80/IL2 AML cell stimulation, the data suggests that the balance of receptor expression is altered in favour of enhanced NK cell activation, which is supported by the results of functional killing assays.

Indeed, cytotoxicity against the NK cell sensitive cell line K562 was higher following LV-CD80/IL2 stimulation. An increase from 46Æ7% to 90Æ4% was detected when purified NK cells were used as effectors. Furthermore, of particular importance was the finding that killing of primary AML cells was enhanced following autologous as well as allogeneic stimulation and, even at a low E:T ratio of 5:1, an increase in lysis of allogeneic AML cells from 11Æ8% to 20Æ1% was detected. Our preliminary studies of autologous stimulation achieved an E:T ratio of 10:1 with a resulting increase in lysis from 0Æ4% on day 0 to 22Æ5% following co-culture with LVCD80/IL2 expressing AML cells. The difference in E:T ratios used precludes a direct comparison of the results of allogeneic and autologous killing assays, nonetheless, the results illustrate that NK cell cytolytic activity is enhanced following LV-CD80/ IL2 stimulation and that the stimulated NK cells have the capacity to lyse primary unmodified AML cells even in the autologous setting. An increasing number of reports have described the existence of cancer stem cells, which are thought to be responsible for the maintenance of cancers, including leukaemia (Dick & Lapidot, 2005; Luo & Han, 2006). The ability of the immune system to eliminate cancer stem cells is currently unknown and may be critical to eradicating the disease. Studies are ongoing to address this question with respect to CD80/ IL2-stimulated immune responses. Immunotherapeutic approaches are likely to be most effective when disease bulk is minimal and, as such, the phase I clinical study will be focused on vaccinating patients at a time of minimal residual disease (MRD) i.e.