Introduction

The prognosis for acute myeloid leukemia (AML) has improved considerably in the past 2 decades due to advances in intensive chemotherapy and allogeneic bone marrow transplantation (BMT). However, outcome for the majority of AML patients remains bleak. The need to improve prognosis in this disease has therefore led to the search for alternative therapies. This includes harnessing the graft versus leukemia effect using reduced intensity conditioned allogeneic hematopoietic stem cell transplantation (for review, see ¹). However, to date this has been of limited effectiveness.

In contrast to solid tumors, large numbers of viable AML blasts are obtainable from patients by leukaphoresis. Therefore this provides a readily available source of cells for potential use as cancer vaccines. In addition, AML blasts are of the same lineage as professional antigen-presenting cells (APCs); hence, unlike many other tumor cells, they express high levels of MHC-II and adhesion molecules such as ICAM-1. Therefore AML presents a unique opportunity for immune-based therapies (for review see Galea-Lauri ²). However, unlike professional APCs, most AML blasts lack the costimulatory molecule B7.1 (CD80) ^3,4,5,6 and secrete immunosuppressive factors ⁷. A logical approach is therefore to modify AML blasts to express B7.1 plus a stimulatory cytokine.

There is evidence in both solid tumor models ^8,9 and leukemia ^10,11,12 that B7.1 is an effective stimulator of anti-tumor immune responses. Furthermore, evidence in nonleukemic models reported by us ^13,14 and others ^15,16 shows that this can be enhanced by the combined use of IL-2. To date there are limited reports of the B7.1 and IL-2 combination in leukemia ¹⁷ but a recent case study in a single AML patient is encouraging ¹⁸. A Th1 cytokine, such as IL-2, is a logical choice, as CTL responses, rather than antibody responses, are considered to offer the greatest promise for successful immunotherapy. The hyporesponsive state of T cell anergy is thought to aid tumor evasion of the immune response. Stimulation of anergic T cells with IL-2 results in proliferation and a complete reversal of the state ¹⁹. In addition, systemic IL-2 therapy has been reported to have beneficial effects in AML patients but problems of toxicity prevent its widespread application ²⁰.

Until recently, expression of transgenes in human AML blasts has been limited to the use of retroviral ^3,21 and adenoviral vectors ²¹, which are poorly effective due to the low proliferation of primary AML blasts and variable coxsackie adenovirus receptor expression ^22,23, respectively. In addition, the use of vectors based on herpes simplex virus and adeno-associated virus gives highly variable transduction efficiencies (our own unpublished observations). This has hampered development of immune gene therapy for AML.

HIV-1-based lentiviral vectors, in contrast to classical retroviral vectors, readily transduce mitotic as well as a number of postmitotic targets, owing to the HIV matrix protein as well as the accessory protein Vpr ^24,25. While optimized attenuation of viral genes improves the safety of the packaged lentiviral vector ²⁶, the incorporation of the HIV central polypurine tract (cppt) and termination sequence (cts) improves nuclear import in otherwise nonpermissive targets ^27,28. In addition, the presence of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) improves intracellular messenger RNA stability ²⁹. Enhanced lentiviral vector-derived transgene expression in hematopoietic cells has been observed by the incorporation of these elements individually and in combination ^30,31. The absence of any viral-encoded proteins warrants lentiviral vectors as suitable vehicles for immune gene therapy strategies. The effectiveness of lentiviral transduction on leukemic cells has been previously demonstrated by several groups ^{32,33,34,35,36}, including the transfer of B7.1/GM-CSF in combination, which resulted in autologous T cell stimulation.

In our development of whole cell vaccines for AML, we show for the first time an efficient transduction protocol to modify primary AML blasts to express B7.1 and IL-2 as a single cistron from a myeloid efficient self-inactivating lentiviral backbone. Furthermore, we show that primary AML blasts modified in this way are capable of stimulating enhanced in vitro responses in allogeneic, chimeric, and autologous settings.

Results

Improved self-inactivating (SIN) lentiviral expression vectors

The lentiviral (LV) expression vectors were derived from the self-inactivating vector backbone carrying a deletion in the HIV U3 promoter region of the 3' LTR, which results in its inactivation following reverse transcription in the target cells ³⁷. To achieve efficient transgene expression, we incorporated the HIV flap region encompassing the cppt and cts, as well as the WPRE. To promote transgene expression, we used the U3 promoter sequence of the spleen focus-forming virus (SFFV) LTR, which is highly active in myeloid lineage-committed cells ³⁸. The resulting vector, HR'SINctwSV, was used to express: (1) GFP (LV.GFP), (2) B7.1 (LV.B7.1), (3) soluble IL-2 (LV.IL-2), and (4) IL-2 plus B7.1 as a chimeric monocistronic transcript (LV.IL-2/B7.1) with the leader sequence located at the N-terminus of IL-2 and separated from the mature B7.1 by a furin endoprotease cleavage site—Arg.Gly.Arg.Arg. The fusion protein encoded by LV.IL-2/B7.1 is postsynthetically cleaved in the Golgi to generate the biologically active constituents ³⁹. See Fig. 1 for vector details.

Figure 1.

Schematic representation of SIN lentiviral vectors expressing GFP, B7.1, IL-2, or B7.1 + IL-2. Common features of the lentiviral backbone include 5' LTR, —packaging signal, RRE (Rev-responsive element), flap—HIV central polypurine tract and terminal sequence, SFFV (spleen focus-forming virus) LTR U3 region, WPRE (woodchuck hepatitis posttranscriptional regulatory element), and the U3-deleted HIV 3' LTR, which renders the vector self-inactivating. HR'SINctwSVGFP (LV.GFP) expresses the enhanced green fluorescence protein, HR'SINctwSVB71 (LV.B7.1) expresses the 1074-bp B7.1 cDNA, HR'SINctwSVIL2 (LV.IL-2) expresses the 459-bp human IL-2, and HR'SINctwSVIL2B71 (LV.IL-2/B7.1) expresses the fusion IL-2/B7.1 separated by a furin cleavage site (Arg.Gly.Arg.Arg). ER transport of chimeric IL-2/B7.1 is mediated by the IL-2 signaling peptide located at the N-terminus.

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In vitro characteristics of primary myeloid leukemia cells

We included in the study only samples that thrived in culture, as defined by at least 80% viable cells after 1 week of culture, as determined by flow cytometry based on forward and side scatter characteristics (data not shown). We examined samples for FAB subtypes and karyotypes (Fig. 2) and for the expression of the following cell surface markers: CD34, CD14, CD19, CD3, MHC-I, MHC-II, CD80, and CD86 (data not shown). We included only samples with a high level of purity, as defined by at least 70% CD34-positive cells. All AML blasts expressed high levels of MHC-I, MHC-II, and CD86 (data not shown) but undetectable levels of CD80 (Fig. 4A).

Figure 2.

Infection of U937, NB4, and K562 leukemic cell lines by SIN lentiviral vectors. FACS profiles of leukemia cells infected with LV.GFP. (A) U937, K562, and NB4 inoculated at an m.o.i. of 0.2 (gray lines, clear background) and 2 (black lines, clear background), analyzed 8 days after infection. (B) Primary AML sample from Patient 1 infected at an m.o.i. of 3 and analyzed 4 days after infection.

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Figure 4.

Allogeneic mixed lymphocyte reaction with modified primary AML blasts. (A) Eight primary AML samples were modified with lentiviral vectors (LV.GFP, LV.B7.1, LV.IL-2/B7.1, LV.IL-2) and assessed for transgene expression (mean values are shown for % positive cells as determined by FL-1 fluorescence, IL-2 as ng/10⁶ cells/24 h). —, not detectable. Modified cells were then tested for their ability to stimulate the proliferation of allogeneic T cells compared to unmodified cells. Unstimulated T cells were included as a control. Proliferation of stimulated T cells is expressed relative to this control value, as Stimulation Index (S.I.). Horizontal lines represent the mean values of eight samples. ^*Gene transduction data obtained from three AML samples. (B) Proliferation of allogeneic T cells in response to recombinant IL-2: in the absence of AML blasts (diamonds), in the presence of unmodified AML blasts (squares), and in the presence of LV.B7.1-modified AML blasts (triangles). Proliferation is expressed as S.I.

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Lentiviral vectors mediate efficient transduction of both established and primary myeloid leukemia cells

We tested the efficiency of lentiviral vectors first on leukemic cell lines to determine optimal m.o.i. We infected U937, K562, and NB4 cells with LV.GFP and analyzed them by FACS 8 days later to exclude the possibility of pseudotransduction ⁴⁰. We observed similar efficiencies in gene transduction for all three cell lines, providing between 20 and 99% modification at m.o.i. of 0.2 and 2, respectively (Fig. 2A). GFP transgene expression was remarkably high, resulting in between 3 and 4 log increases in fluorescence. When we used the same virus preparation to infect primary AML blasts, we observed an equally efficient gene transduction at an m.o.i. of 3 (Fig. 2B shows Patient 1 as a representative sample). These data demonstrate the suitability of the constructed lentivirus for efficient transduction in both established and primary myeloid leukemia cells at relatively low m.o.i. with a single round of infection.

Modification of AML blasts to express B7.1 and IL-2 alone or in combination by lentiviral vectors

We evaluated LV.B7.1, LV.IL-2, and LV.IL-2/B7.1 on eight primary AML samples and on U937 and NB4 cell lines (Fig. 3A). Lentiviral transduction had no significant effect on viability of either primary or established cells, as represented by the Viability Index. Similar to LV.GFP transduction, the established U937 and NB4 cell lines were readily transduced by both LV.B7.1 and LV.IL-2/B7.1 at an m.o.i. of 10, producing greater than 95% transduced cells as determined by the surface detection of B7.1. Transduction of primary AML blasts by LV.B7.1 and LV.IL-2/B7.1 was, however, more variable, with B7.1 modification ranging from 60 to 98% for LV.B7.1 and 41 to 98% for LV.IL-2/B7.1. This sample-to-sample variation appeared to be consistent between the two vectors and could not be overcome either by applying a higher m.o.i. (up to 40) or by the additional expression of the HIV accessory proteins (Vpr, Vpu, Vif, Nef) (Figs. 3B and 3C).

Figure 3.

Transduction of established and primary leukemic samples by lentiviral vectors expressing B7.1 (LV.B7.1), IL-2 (LV.IL-2), and IL-2/B7.1 (LV.IL-2/B7.1). (A) Eight primary AML samples (Pats. 1–8) and U937 and NB4 cell lines were analyzed for lentiviral transduction with LV.B7.1 and LV.IL-2/B7.1 at an m.o.i. of 10 and LV.IL-2 at an equivalent p24 concentration of 1 g/10⁶ cells, except for Pat. 8 (+), for whom a lower p24 concentration was used. (B) The effects of increased LV.B7.1 m.o.i. on primary AML infectivity for Pat. 6. (C) The effects of inclusion of accessory proteins (Vpr, Vpu, Vif, and Nef) in addition to Tat and Rev (using the packaging construct pCMVR8.2 instead of pCMVR8.91 in LV production) on LV.B7.1 transduction in Pat. 6. B7.1 surface detection was performed with FITC-conjugated antibody except for samples from Pats. 5, 7, and 8 (^*), which were treated with PE-conjugated antibody. PE-conjugated antibody consistently excites a higher level of fluorescence compared to FITC-conjugated antibody independent of transgene expression (data not shown). Viability index was determined by SSC/FSC in flow cytometry by comparing the fractions of live intact cells between sample populations exposed to virus supernatant or control medium.

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The levels of IL-2 transgene expression following LV.IL-2/B7.1 transduction also displayed variation between patient samples, from 1.5 to 17.4 ng/10⁶ cells/24 h, with samples 1, 2, 5, and 8 expressing IL-2 at levels markedly higher than samples 3, 4, 6, and 7 in a pattern similar to B7.1 expression.

Infection with LV.IL-2 at equivalent m.o.i. (Pats. 1 to 7) resulted in significantly higher levels of IL-2 transgene expression compared with LV.IL-2/B7.1. In U937, this was 118 ng/10⁶ cells/24 h, 10-fold higher than in LV.IL-2/B7.1-infected cells, and in NB4, the increase was 25-fold, from 9.5 to 238 ng/10⁶ cells/24 h. In primary cells, the enhancement was between 17- and 21-fold for samples 1, 2, and 5 but only 2- to 9-fold for samples 3, 6, and 7.

The infectability of AML patient samples appeared to be vector independent as all three vectors provide similar levels of transduction efficiencies within the same sample population. The overall levels of transgene expression in primary AML blasts were comparable to the levels observed in the established cell lines. We detected B7.1 and IL-2 transgene expression only following gene transduction and not as a result of in vitro culture or infection with the control vector, LV.GFP (Fig. 4A).

Allostimulatory activity of modified primary AML blasts

Using a panel of eight primary AML samples, we compared the ability of the transduced and unmodified AML blasts to stimulate allogeneic T cells (Fig. 4A). LV.IL-2/B7.1-modified AML blasts induced a significant increase in allo T cell stimulation compared to unmodified or GFP-modified cells (P = 0.002 and P = 0.0056, respectively). LV.IL-2/B7.1-modified AML blasts induced higher levels of allo T cell stimulation (mean 54) than AML blasts modified with either LV.B7.1 or LV.IL-2 alone (mean 31 and 40, respectively). Since AML blasts infected with LV.IL-2.B7.1 expressed lower levels of B7.1 and IL-2 than cells infected with a single gene, this shows that the combination provided superior T cell stimulation than when B7.1 or IL-2 was expressed alone (Fig. 4A). The enhanced T cell stimulation was partially blocked by the presence of anti-CD80 antibody by an average of 50% for LV.B7-infected cells and an average of 40% for LV.IL-2/B7.1-infected cells (data not shown).

To investigate the possibility that the allogeneic T cells were proliferating nonspecifically in response to IL-2, we carried out experiments using several doses of recombinant IL-2 (Fig. 4B). T cells cultured with IL-2 showed proliferation in the absence of AML cells; however, this was much greater when AML cells were also present. This proliferation was further increased by B7.1 modification of the AML blasts. This suggests that although some nonspecific T cell proliferation was being induced, it is unlikely that the response elicited by IL-2/B7.1 AML cells was totally nonspecific.

Stimulation of patients' peripheral blood mononuclear cells (PBMCs) by modified primary AML blasts

We then assessed the ability of AML blasts to stimulate the proliferation of the patients' own PBMCs. We tested two autologous PBMC samples, one obtained from a patient at diagnosis and one obtained from a patient in chemotherapy-induced remission (Patient 9). In addition we tested PBMCs from two patients post-allogeneic BMT, after the establishment of donor chimerism. Fig. 5 shows the levels of transgene expression and the simulation index (S.I.) values for these patients. Although there was notable variation in the S.I. values in different patients, a general trend is apparent. LV.B7.1 modification caused slight increases in stimulation (mean S.I. = 18) compared to unmodified cells (mean S.I. = 13). LV.IL-2 modification caused greater increases in stimulation (mean S.I. = 140). However, the greatest stimulation was achieved by AML blasts modified with LV.IL-2/B7.1 (mean S.I. = 227). As with the allogeneic MLR, this was despite lower levels of B7.1 and IL-2 than in AML blasts modified to express either B7.1 or IL-2 individually. Therefore, the expression of IL-2/B7.1 resulted in greater stimulation of PBMCs than could be achieved by either B7.1 or IL-2 alone. Proliferation was partially blocked by the presence of anti-CD80 antibody by 60% for LV.B7-infected cells and 25% for LV.IL-2/B7.1-infected cells. In addition, proliferation was partially blocked by the presence of anti-MHC-I but not by the presence of anti-MHC-II (Fig. 5B).

Figure 5.

Mixed lymphocyte reaction (MLR) with modified primary AML blasts and patients' PBMCs. Primary AML blasts (uninfected, LV.B7.1, LV.IL-2 and LV.IL-2/B7.1 infected) were tested for their ability to stimulate patients' own PBMCs. PBMCs alone were included and proliferation was related to this control value, expressed as S.I. B7.1 modification is expressed as % positive cells and IL-2 as ng/10⁶ cells/24 h. (A) MLR results from four different patients: Patient 7—AML blasts obtained at diagnosis and PBMCs obtained after BMT with one MHC mismatch, Patient 9—AML blasts obtained at diagnosis and autologous PBMCs obtained during chemotherapy induced remission (no BMT), Patient 5—AML blasts obtained at diagnosis and PBMCs obtained after BMT from a matched unrelated donor, Patient 8—AML blasts and nonremission PBMCs harvested at diagnosis. (B) MLR with PBMCs and AML cells from Patient 5 in the presence of blocking antibodies (anti-CD80, anti-MHC-I, and anti-MHC-II).

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Stimulation of Post-BMT PBMCs with LV.IL-2/B7.1 AML blasts promotes the expansion of activated CD8⁺ cells with increased secretion of Th1 cytokines

We stimulated PBMCs from bone marrow-transplanted patients with medium alone (unstimulated control), unmodified AML blasts, or LV.IL-2/B7.1-modified cells. At day 0 and day 13 we determined cell numbers and analyzed surface marker expression (Fig. 6A). PBMCs cultured in medium alone or stimulated with unmodified AML blasts did not show an increase in numbers. Conversely, cultures stimulated with LV.IL-2/B7.1-infected AML blasts showed an approximate fourfold expansion in numbers. In Patient 5 a clear expansion of CD8⁺ cells and an increase in CD25 and CD69 expression was detectable when the stimulation was with LV.IL-2/B7.1-modified cells. In Patient 7 there was an expansion of CD8⁺ cells in all three cultures, but activation markers were detected only on PBMCs stimulated with LV.IL-2/B7.1-modified AML blasts.

Figure 6.

Analysis of post-BMT PBMCs at day 13 after two rounds of stimulation with patients' own AML blasts. PBMCs from Patient 5 and Patient 7 were cocultured with medium alone (unstimulated), irradiated unmodified AML blasts, or irradiated LV.IL-2/B7.1-modified AML blasts. On day 0 and day 13 viable cell numbers were counted and frequencies of CD4⁺, CD8⁺, CD25⁺, and CD69⁺ cells were determined by flow cytometry, expressed as % +ve cells (A). Th1/Th2 cytokine levels in culture supernatant on day 13 were determined by cytometric bead analysis, expressed as pg cytokine/5 10⁵ cells (B).

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Analysis of Th1 and Th2 cytokines in these cultures shows that stimulation of PBMCs with LV.IL-2/B7.1 AML blasts caused increased levels of cytokine secretion, most notably the Th1 cytokines: IFN-, TNF-, and IL-2 (Fig. 6B).

The activated lymphocytes proliferate preferentially in response to unmodified AML blasts compared to normal bone marrow cells

We then determined the specificity of the preactivated PBMCs by further proliferation against two different targets: the patients' own unmodified AML blasts and bone marrow cells obtained during chemotherapy-induced remission prior to allogeneic BMT (Fig. 7).

Figure 7.

Proliferation assay showing the response of expanded PBMCs to the patients' AML blasts or remission bone marrow cells. PBMCs from Patient 5 and Patient 7 were initially primed by coculture with LV.IL-2/B7.1-infected AML blasts, unmodified AML blasts, or medium only (unstimulated), with subsequent restimulation carried out on days 7 and 14. On day 20 the specificity of the expanded cells was examined by testing their ability to proliferate in response to the patient's unmodified AML or remission bone marrow cells in a 5 day MLR. Unstimulated responder cells were included as a control for each group. Proliferation of stimulated responders was related to this control value and expressed as S.I.

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In Patient 5 proliferation was low; however, cells did show marginally higher proliferation in response to AML cells compared to remission bone marrow.

In Patient 7, all three lymphocyte populations showed low proliferation against remission BM cells. Lymphocytes initially stimulated with unmodified cells also showed a correspondingly low proliferation rate against unmodified AML blasts. However, lymphocytes previously stimulated with LV.IL-2/B7.1-infected cells showed a marked preferential proliferation against unmodified AML blasts compared to bone marrow (S.I. = 25 compared to 1.08). This suggests that a population of AML-specific responder cells had been expanded by stimulation with the IL-2/B7.1-expressing AML blasts.

Discussion

The therapeutic effects of allogeneic bone marrow transplantation and donor leukocyte infusion, together with the detection of leukemia-specific cytotoxic T lymphocytes in myeloid malignancies ^41,42, provide optimism for the development of immune gene therapy strategies against AML. However, ubiquitously expressed leukemia-associated or leukemia-specific antigens have not been identified. Therefore autologous whole cell vaccines provide an attractive strategy for the stimulation of immune responses in AML. The induction of immunological responses against AML blasts, particularly in the context of allogeneic bone marrow transplants, may enhance the graft versus leukemia effect or the efficacy of donor leukocyte infusions resulting in prolonged duration of remission. To this end we have developed a self-inactivating lentiviral vector for the efficient transduction of primary AML blasts with immune regulatory factors B7.1 and IL-2. Important vector features contributing to this efficiency are the presence of factors that enhance nuclear import ^27,28 and RNA stability ²⁹, as well as the use of a myeloid efficient promoter derived from spleen focus-forming virus long terminal repeats ³⁸. We coupled these features with our previously developed "fusagene" strategy, which utilized the ubiquitously expressed endoprotease furin to process fusion proteins in the Golgi to generate the biologically active constituents ³⁹. This enables the monocistronic expression of multiple proteins as a single precursor. Therefore, we have shown for the first time with this vector system the genetic modification of between 40 and 99% of primary AML blasts with both B7.1 and IL-2 as a result of a single round of infection at an m.o.i. of 10. The expression of the encoded proteins (GFP, B7.1, or IL-2) in these cells was at least 1 log above background. These are consistently higher than the transduction efficiencies we could previously achieve with multiple rounds of infection with retroviral ³, adenoviral, and adeno-associated virus vectors at substantially higher m.o.i. (unpublished data). The levels of lentiviral-AML transduction observed here are superior to those previously shown for CMV-driven constructs without the cppt/WPRE enhancer combination ^32,35 and comparable to CMV-driven constructs with cppt/WPRE, but at a substantially higher m.o.i. of 50 ³⁶. However, within some primary samples there were subpopulations of cells that appeared to be less susceptible to transduction (Patients 3, 4, 6, 7). This resistance was independent of the proteins encoded by the vector (Fig. 3A) and could not be overcome by the use of higher m.o.i. (up to 40, Fig. 3B), the presence of HIV accessory proteins (Fig. 3C), or the addition of deoxynucleosides to the virus stock or the infection mixture ⁴³ (data not shown). The cause of this block to infection remains unclear, but is unlikely to be associated with virus entry because of the ubiquitous cell entry pathway through phospholipids ⁴⁴ utilized by VSV-G. In addition, the use of an amphotropic envelope pseudotype instead of, or in addition to, VSV-G did not provide any improvement in the transduction rates (manuscript in preparation). Despite the ability of lentiviral vectors to transduce postmitotic cells, we still found a correlation between proliferation and infectability as demonstrated by the positive influence of SCF and IL-3 (up to 20% increase in infection, data not shown). This is in agreement with findings previously reported with noncycling T cells ⁴⁵ and hematopoietic stem cells ⁴⁶.

The levels of both IL-2 and B7.1 in blast cells infected with LV.IL-2/B7.1 were almost always lower than in cells infected with the single protein vectors (LV.IL-2 or LV.IL-2/B7.1). This may be the product of reduced rates of transcription, translation, or processing of the fusion protein and the present studies do not discriminate between these possibilities. However, we were able to detect IL-2 that remained bound to B7.1 on the cell surface (data not shown), indicating the incomplete proteolytic cleavage of the precursor IL-2/B7.1 fusion protein. Nonetheless this is consistent with our previous findings that endogenous furin expression varies between tissue type ³⁹, and despite high levels of soluble IL-2 being secreted, a proportion remain attached to the membrane-anchored B7.1. Whether the uncleaved IL-2 attached to B7.1 acts as a novel antigen is not clear at present. However, the data presented here and in our previous study ³⁹ demonstrate biological activities of B7.1 and IL-2 generated by this strategy. In addition, the levels of IL-2 secretion in our lentiviral fusagene constructs remain substantially higher and more stable than those achieved in IRES configurations (unpublished data by us and others ³⁶).

The efficient transduction of primary AML blasts allowed us to assess the effects of B7.1 and IL-2 expression on the immunogenicity of primary AML blasts. This was performed using lymphocytes obtained from: (i) healthy donors (allogeneic), (ii) patients prior to or following chemotherapy-induced remission (autologous), and (iii) patients post-allogeneic BMT following reduced intensity conditioning (chimeric). In all cases, levels of B7.1 and IL-2 were lower in AML blasts transfected with both genes than in cells transfected with a single gene. Despite this, all the responder cells tested showed the greatest proliferation against LV.IL-2/B7.1-transduced AML blasts. The levels of autologous stimulation observed here were greater than those previously reported by Stripeke et al., who first described the use of LV transduction of primary AML with B7.1/GM-CSF to generate whole cell vaccines ³⁵. Higher levels of autologous T cell stimulation, comparable to ours, have been achieved by the transduction of primary AML with IL-7 in combination with cytokine differentiation to a DC phenotype ⁴⁷.

It is worth noting that effector cells isolated from patients prior to induction of remission would have been recently exposed to a highly immunosuppressive environment due to large numbers of AML blasts ⁷. Despite this, they showed enhanced proliferation in response to LV.IL-2/B7.1-transduced cells (Patient 8).

In the autologous setting (Patients 8 and 9) the IL-2/B7.1-expressing AML blasts would have stimulated the proliferation of either nonspecific T cells or T cells directed against AML-associated/specific targets. Stimulation of nonspecific proliferation by IL-2 was investigated in separate experiments using recombinant IL-2. Donor T cells cultured with IL-2 showed proliferation in the absence of AML cells, but this was much greater when AML cells were also present. This proliferation was further increased by B7.1 modification of the AML blasts (Fig. 4B). Therefore as expected, IL-2 induces nonspecific T cell proliferation, but this cannot account for the entire proliferative response elicited by the IL-2/B7.1-expressing AML cells.

With lymphocytes from patients following allogeneic BMT patients (5 and 7), the expanded T cells may also be directed against alloantigens. Our preliminary analysis suggests greater specificity of these T cells against AML-associated antigens, rather than antigenic determinants of normal hematopoietic cells. This is indicated by the higher proliferative response of T cells stimulated by the IL-2/B7.1-expressing AML blasts in the subsequent presence of unmodified AML blasts coma compared to remission bone marrow cells (Fig. 7). Although the level of T cell proliferation in Patient 5 was too low to show this difference convincingly, Patient 7 show substantially greater stimulation in the presence of the patient's own unmodified AML blasts than the remission bone marrow cells (S.I. 25 vs 1). This is similar to the findings of Mutis and colleagues, who stimulated autologous T cells with B7.1-transfected AML cells, resulting in the expansion of T cell clones that showed preferential proliferation against AML cells compared to normal hematopoietic cells (immortalized B cells) ⁴⁸. We are now examining the cytotoxic activity of the in vitro stimulated T cells against autologous unmodified AML blasts.

The data presented demonstrate efficient transduction of 40 to 99% (mean 54%, n = 12) in primary AML blasts, resulting in high levels of IL-2 and B7.1 transgene expression. This is achieved by a single round of infection with the constructed self-inactivating lentiviral vector. The genetically modified AML blasts stimulate proliferation of both allogeneic and autologous lymphocytes, as well as chimeric lymphocytes from bone marrow-transplanted AML patients. This stimulation results in expansion, primarily of CD8⁺ cells, upregulated expression of activation markers, and increased secretion of Th1 cytokines. Therefore the LV.IL-2/B7.1-modified AML blasts are capable of stimulating the conditions required for the generation of immunological responses with a potential therapeutic effect.

Materials and methods

Cell Culture

Culture media for 293T (DMEM), U937, K562, and NB4 (RPMI 1640) were supplemented with L-glutamine, 10% FBS (Sigma), and penicillin G (50 U/ml)/streptomycin (50 mg/ml).

Primary AML blasts were obtained from peripheral blood of adult patients with high-count acute myeloid leukemia at diagnosis and prior to initiation of chemotherapy. PBMCs were obtained from AML patients prior to or following chemotherapy-induced remission and also from healthy volunteers. AML blasts and PBMCs were purified by Histopaque (Sigma) density gradient centrifugation and cryopreserved in X-VIVO 15 (BioWhittaker) with 10% DMSO and 50% human AB serum (Sigma). Cryopreserved bone marrow samples from patients in remission were obtained from the Stem Cell Laboratory (King's College Hospital, London, UK). T cells were isolated from healthy donor PBMCs with a T Cell Negative Isolation Kit (Dynal, Oslo, Norway). All primary cells were cultured in X-VIVO 15 medium. AML cultures were supplemented with rhSCF (20 ng/ml) and rhIL-3 (10 ng/ml) (R&D Systems, UK). FAB subtyping and karyotyping were carried out by the Cytogenetics Unit (KCH, London).

Informed consent was obtained from all patients prior to collection of primary cells.

Lentiviral Vector Construction

The improved self-inactivating lentiviral vector backbone HR'SINctwSV was made by inserting an XhoI/XbaI fragment containing the WPRE sequence from HR'PGKGFPWSIN-18 into HR'CMVGFPSIN (both kind gifts from Didier Trono, University of Geneva, Switzerland), followed by substitution of the CMV promoter with a BamHI/NotI fragment carrying the cppt/SFFV U3 promoter from HR'SIN-cPPT-SEW (kindly provided by Adrian Thrasher, Institute of Child Health, London, UK). The universal backbone—HR'SINctwSV—was used to express GFP, B7.1, IL-2, and IL-2/B7.1 (all constructs were verified by sequencing analysis, further construction details available on request).

Lentiviral Vector Production and Titration

Lentiviral vectors were produced from transient calcium phosphate-transfected 293T cells. Cells were cultured to 80% confluence in 150-cm² triple-layer flasks (Nalge Nunc) and transfected with 26 g pCMVR8.91, 14 g pMD.G, and 40 g vector construct. Virus harvests were concentrated by an overnight centrifugation (8600g, 4°C) followed by ultracentrifugation (183,000g, 90 min, 4°C). Virus pellets were resuspended in HBSS (with 1% FBS) and stored at -80°C. Titer was determined by serial dilutions of virus inoculates on target U937 leukemia cells and by calculating representative populations of transduced cells analyzed 96 h after infection. This protocol produces titers of 5–7 10⁸ transduction units (tu)/ml from a starting volume of 450 ml. When correlated with HIV p24 concentration (ELISA, reagents from SAIC–Frederick, Inc., MD, USA), approximately 10⁷ tu/g p24 was observed. This correlation was used to determine dosage loading of LV.IL-2, which cannot be titered otherwise. Where stated, the packaging construct pCMVR8.2 was used instead of pCMVR8.91 to express additional HIV accessory proteins: Vpr, Vpu, Vif, and Nef.

Lentiviral-Mediated Gene Transfer

Thawed AML blasts were cultured for 3 days in the presence of SCF and IL-3 prior to LV infection. Target cells were plated at 5 10⁵/ml (U937, K562, NB4) or 1 10⁶/ml (primary AML) and supplemented with 10 g/ml DEAE dextran (Amersham Pharmacia) for viral complexing ⁴⁹, which we found to be 10-fold more efficient than Polybrene (unpublished data). Aliquots of LV were thawed and infections performed overnight under standard culture conditions. The following day cells were washed twice and cultured for 72 h prior to analysis of IL-2 (ELISA) and B7.1 and GFP (FACS) transgene expression. Except where stated, an m.o.i. of 10 was used for primary AML infection. For comparative immunological assays, the m.o.i. of LV.IL-2 was lowered to adjust for comparable IL-2 secretion with LV.IL-2/B7.1.

Flow Cytometry

For analysis of GFP expression, cells were washed with HBSS, 1% FBS and fixed in 150 l 2% paraformaldehyde solution. For analysis of B7.1 transgene expression cells were stained with directly conjugated CD80–FITC antibody or, where stated, CD80–PE antibody (Becton–Dickinson, Oxford, UK). PBMCs were stained with the following conjugated antibodies: CD4–FITC, CD8–FITC, CD25–FITC, and CD69–FITC (Becton–Dickinson). All labeling was performed with matched isotype controls for each sample and analyzed using a FACSCalibur cytometer (Becton–Dickinson).

Analysis of IL-2 Production

IL-2 levels were determined with a DuoSet ELISA kit (R&D).

Mixed Lymphocyte Reaction (MLR)

Stimulators

Three days after LV infection, AML blasts were washed and resuspended in fresh X-VIVO 15 medium at 5 10⁵/ml, -irradiated at 30 Gy, and seeded in flat-bottom 96-well plates at 5 10⁴ per well. Where required, remission bone marrow cells were irradiated and seeded in the same way. To test the role of B7.1, MHC-I, and MHC-II, primary AML cells were incubated with the anti-CD80 antibody clone l307.4; anti-HLA-DR, DP, DQ clone TU39 (Becton–Dickinson); and anti-HLA-ABC clone W6/32 (Serotec, Oxford) at a concentration of 20 g/ml for 30 min prior to the addition of responder cells. In experiments addressing nonspecific IL-2-induced proliferation, various concentrations of recombinant human IL-2 (R&D Systems, UK) were added to the wells prior to the addition of responder cells.

Responders

T cells from healthy volunteers (allogeneic assays) or PBMCs from AML patients in remission (Patients 5, 7, and 9) were used. For assays on Patient 8 responder cells were obtained from peripheral blood at the time of diagnosis. AML blasts were removed from the sample using the Dynal CD34 progenitor cell selection system (Dynal Biotech). In all experiments responder cells were added at 1 10⁵ per well, giving a stimulator-to-responder ratio of 1:2. Subsets of responder cells were also cultured alone, providing a control for unstimulated responders. MLR cultures were incubated for 5 days. One microcurie of [methyl-³H]thymidine (sp act 5.0 Ci/mmol; Amersham Pharmacia) was added to each well for the last 16 h of culture. Incorporated thymidine was assessed by harvesting cells onto glass fiber filters and drying them for 1 h at 80°C, and radioactivity was quantified using a Beta counter. Mean cpm values were calculated from four wells, and the results were expressed as S.I. = (cpm AMLs + responders)/cpm responders alone.

Analysis of Post-BMT PBMCs Stimulated with Modified AML Blasts

Post-BMT PBMCs (1 10⁶) from AML patients in remission were seeded into 3 wells of a 12-well plate, in a volume of 1 ml. LV.IL-2/B7.1-modified or unmodified AML blasts from the same patients were prepared at 5 10⁵/ml and irradiated at 30 Gy. PBMC cultures received 1 ml of medium only (unstimulated control), unmodified AML, or LV.IL-2/B7.1 AML. PBMC cultures were restimulated by further addition of 5 10⁵ irradiated AML blasts (modified or unmodified) on days 7 and 14. Supernatants were removed on day 13 for cytokine analysis and the lymphocytes gently resuspended and counted to assess proliferation. PBMC were stained with antibodies for CD4, CD8, CD25, and CD69 expression. On day 20 cells were washed and tested for their ability to proliferate against unmodified autologous AML or bone marrow cells in a secondary MLR (see above).

Analysis of Cytokine Production by Stimulated PBMCs

Supernatants were removed from the cocultures described above and stored at -20°C until analysis. Levels of IFN-, TNF-, IL-10, IL-6, IL-4, and IL-2 were measured with the Cytometric Bead Array Human Th1/Th2 cytokine kit (BD Pharmingen). Supernatant from cultures containing AML blasts alone were included as a control, to subtract transgene IL-2 released by modified AML blasts.

Statistical Analysis

Significance values were calculated using GraphPad Prism version 3.02 for windows (GraphPad Software, San Diego, CA, USA) using a two-tailed test. P values of less than 0.05 were taken as significant and values less than 0.005 as highly significant.

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Acknowledgements

We thank Mary Collins for her generosity and her assistance in our lentiviral work. We are grateful to Dr. Adrian Thrasher (Institute of Child Health) and Dr. Didier Trono (University of Geneva) for providing the lentiviral vectors and packaging constructs. Our thanks also go to Michelle Kenyon for her invaluable assistance in obtaining patient samples and to Taylor Mackey, Debbie King, and Maddy Hayes for p24 assays. The bone marrow aspirates used in this study were provided by KCH Stem Cell Laboratory.