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Prepublished online as a Blood First Edition Paper on October 3, 2002; DOI 10.1182/blood-2002-08-2351.
GENE THERAPY
From the Cancer Immunotherapy and Gene Therapy Program
and Bone Marrow Transplantation Unit, Istituto Scientifico H. S. Raffaele, Milan, Italy; Program in Immunology, Fred
Hutchinson Cancer Research Center, Seattle, WA; and Molmed S.p.A.,
Milan, Italy.
We have previously shown that the infusion of donor lymphocytes
expressing the herpes simplex virus thymidine kinase
(HSV-tk) gene is an efficient tool for controlling
graft-versus-host disease (GVHD) while preserving the
graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the
administration of donor HSV-tk+ cells could have a clinical
impact in promoting immune reconstitution after T-cell-depleted stem
cell transplantation (SCT). To explore this hypothesis, we have
investigated whether in vitro polyclonal activation, retroviral
transduction, immunoselection, and expansion affect the immune
competence of donor T cells. We have observed that, after appropriate
in vitro manipulation, T cells specific for antigens relevant in the
context of SCT are preserved in terms of frequency, expression of
T-cell receptor, proliferation, cytokine secretion, and lytic activity.
A reduction in the frequency of allospecific T-cell precursors is
observed after prolonged T-cell culture, suggesting that cell
manipulation protocols involving a short culture time and high
transduction efficiency are needed. Finally, the long-term persistence
of HSV-tk+ cells was observed in a patient treated in the
GVL clinical trial, and a reversion of the phenotype of
HSV-tk+ cells from CD45RO+ to
CD45RA+ was documented more than 2 years after the
infusion. Based on all this evidence, we propose a clinical study of
preemptive infusions of donor HSV-tk+ T cells after SCT
from haploidentical donors to provide early immune reconstitution
against infection and potential immune protection against disease recurrence.
(Blood. 2003;101:1290-1298) In allogeneic stem cell transplantation (allo-SCT),
delayed donor lymphocyte infusion (DLI) plays a crucial therapeutic
role in promoting an antitumor effect.1-3 However, the
efficacy of DLI is limited by the development of graft-versus-host
disease (GVHD).2,4 We and others have previously shown
that GVHD can be selectively treated by the transfer of the
HSV-tk gene into donor lymphocytes to confer ganciclovir
(GCV) sensitivity.5,6 In these clinical trials, the
antitumor activity of transduced lymphocytes was observed in a
significant number of patients, resulting in complete and durable
clinical remission6-8 (F.C. et al, manuscript in
preparation). In addition to the above clinical context, the early
administration of donor lymphocytes genetically engineered for the
expression of a suicide phenotype could have a clinical impact in
promoting immune reconstitution after SCT from haploidentical donors
(haplo-SCT). In this context the intensive T-cell depletion required to
avoid lethal GVHD has been associated with a delayed immune recovery of
CD3+ cells to 7 to 9 months after transplantation and a
prolonged risk for infection after transplantation.9,10
The early infusion of HSV-tk-transduced donor lymphocytes could
provide a rapid reconstitution of a wide T-cell repertoire while
providing a tool for the selective control of GVHD.
We and others previously demonstrated that human lymphocytes can be
efficiently transduced by retroviral vectors after the induction of
active cell proliferation.11,12 The cell manipulation protocol proposed consists of polyclonal T-cell activation and culture
in the presence of recombinant human interleukin-2 (rhIL-2). This protocol allows the transduction of a polyclonal T-cell
population, potentially representative of all T-cell precursors
circulating in healthy donors.7,13 However, the
variability in response to activation stimuli, activation-induced cell
death (AICD), exposure to rhIL-2, and proliferation-induced T-cell
exhaustion could alter the immune potential of donor T cells. Indeed,
other investigators previously reported that some gene transfer
protocols skew the V Transduction and selection of human lymphocytes with
retroviral vectors
Flow cytometric analysis of genetically engineered T cells
Transduced cells were detected in vitro and ex vivo by staining with
the mouse antihuman LNGFR monoclonal antibody (mAb) followed by
FITC-conjugated goat antimouse antibody (DAKO, Glostrup, Denmark). Analysis of the T-cell receptor (TCR)-V Intracytoplasmic staining for cytokine production To determine the cytokine production profile, intracellular cytokines were quantified by flow cytometry as described,18 with some modifications. Pairs of RV cells and PBLs from the same donors (1 × 106/mL) were stimulated with 10 µg/mL immobilized OKT3 and 10 ng/mL phorbol myristate acetate (TPA). After 3 hours of activation, 1 ng/mL brefeldin A (Sigma Chemical, Milan, Italy) was added. After 6 hours of activation, T cells were washed in phosphate-buffered saline (PBS), stained with anti-CD4 and anti-CD8 mAbs, and fixed in 2% formaldehyde for 20 minutes at 20°C. After fixation, T cells were permeabilized in PBS supplemented with 2% fetal calf serum (FCS) and 0.5% saponin (Sigma). T cells were incubated with PE-labeled anti-hIL-4, anti-hIL-2, or anti-hIL-10 and FITC-coupled anti-interferon (IFN-) (PharMingen). After washing, cells were analyzed on FACScalibur.
Analysis of antigen-specific T-cell frequency on genetically engineered T cells and PBLs using peptide-major histocompatibility complex tetramers The nonapeptide pp65495-503, derived from the structural protein pp65 of cytomegalovirus (CMV), which binds HLA-A0201,19 was synthesized. Tetramers were made according to the protocol of Altman et al.20 In brief, human 2-microglobulin and the soluble domain of the HLA-A0201 heavy
chain linked at its COOH terminus to BirA substrate peptide and
2-microglobulin subunits were solubilized and refolded in the
presence of the peptide. Folded material was biotinylated by BirA
enzyme. HLA-A0201-peptide complexes were purified on gel filtration
and ion exchange columns. Tetrameric complexes of biotinylated
HLA-A0201 peptide were produced by mixing purified, biotinylated
heterodimer with NeutrAvidin-PE (Molecular Probes) at a molar ratio of
4:1. To compare the frequency of antigen-specific T cells on RV cells
and PBLs, 1 × 106 cells were stained with PE-conjugated
pp65-tetramer, anti-CD8-Quantum Red (1/60) and anti-CD3-FITC (1/60)
antibodies for 30 minutes at 4°C. The frequency of pp65-specific
cytotoxic T lymphocytes (CTLs) is presented as a fraction of
tetramer-positive, CD3+/CD8+ lymphocytes over
the total of CD3+/CD8+ lymphocytes.
Induction of EBV-specific cytotoxic T lymphocytes For the induction of CTLs specific to EBV, EBV IgG-seropositive healthy donors were used. Autologous EBV cells lines (EBV-LCL) were obtained by infecting PBLs with the B95-8 EBV strain for 3 hours.21 To compare RV cells with PBLs for the ability to lyse autologous EBV-LCL, CD8+ cells were obtained by positive separation using anti-CD8 magnetic-activated cell separation (MACS) microbeads (Miltenyi Biotec GmbH) according to the manufacturer's recommendations. CD8+ RV cells (1.5 × 106) and the same number of CD8+ PBLs were stimulated in vitro with 5 × 105 previously irradiated (100 Gy) autologous EBV-LCL, in Iscove modified Dulbecco medium (IMDM) supplemented with 10% human serum, glutamine, and antibiotics in the presence of 50 U/mL rhIL-2 (mixed lymphocyte reaction [MLR] medium). Effector cells were tested against autologous EBV-LCL in a standard cytotoxicity assay 10 days later. As negative controls, effectors were tested against autologous PHA-activated lymphocytes. As a control for specific cytotoxic activity, cold inhibition assay was used adding unlabeled K562 cells in 30 times excess to chromium Cr 51-labeled target cells. As described elsewhere,22 this proportion of K562 cells resulted in complete inhibition of nonspecific natural killer/lymphokine-activated killer cell (NK/LAK) activity.Induction of CMV-specific cytotoxic T lymphocytes To isolate specific CTLs against CMV, autologous fibroblasts from IgG CMV-positive healthy donors were infected with AD169 strain CMV for 4 hours. RV cells (2 × 106) and the same number of PBLs chosen for a similar CD4+/CD8+ ratio were cultured in the presence of 5 × 104 autologous CMV-infected fibroblasts and 1 × 106 autologous peripheral blood mononuclear cells (PBMCs) previously irradiated at 60 Gy in MLR medium. Cells were restimulated at day 7 using the same culture conditions. Effector cells were tested after 7 to 10 days against autologous CMV-infected fibroblasts in a cold inhibition cytotoxicity assay. Autologous mock-infected fibroblasts were used as a negative control. To determine the target of the anti-CMV immune response, autologous EBV-LCLs were infected for 8 to 12 hours with recombinant vaccinia virus (rVV) encoding the CMV matrix proteins pp65, pp150, and pp72 at an MOI of 10:1. Infected EBV-LCLs were then used as targets of a cold inhibition cytotoxicity assay. Autologous EBV-LCLs infected with an irrelevant rVV were used as a negative control.Induction of flu-specific cytotoxic T lymphocytes To isolate flu-specific cytotoxic T lymphocytes, 5.6 × 106 PHA-activated lymphocytes from HLA-A0201 donors were pulsed with 50 µM class 1 HLA-A0201-restricted M58-66 peptide derived from the flu matrix protein (Neosystem Laboratoire, Strasburg, France) in 1 mL IMDM in the presence of 2 µg/mL2-microglobulin (Sigma) and 100 U/mL rhIL-2 for 4 hours
at room temperature. Cells were then irradiated at 60 Gy, washed, and
cultured with CD8+ RV cells or CD8+ PBLs at a
responder-to-stimulator ratio of 1.5:1. Effector cells were tested 7 to
10 days later in a cold inhibition cytotoxicity assay as described. To
this purpose, M58-66-pulsed HLA-A0201 TAP-deficient T2
cells23 were labeled with 51Cr and were used
as targets. Unpulsed T2 cells were used as negative controls. Because
M58-66 flu peptide is usually recognized in HLA-A0201-positive patients by V17-TCR+ T cells,
staining with a V17 antibody was used to monitor the expansion of
V17+ cells by flow cytometry.
Cytotoxic T-lymphocyte precursors frequency against HLA-mismatched target cells To isolate cytotoxic T lymphocytes specific for allogeneic cells, 1.5 × 106 CD8+ RV cells and CD8+ PBLs from the same donor were stimulated in MLR against 1 × 106 60-Gy-irradiated, fully allogeneic PBMCs in MLR medium. Effector cells were tested against PHA-activated lymphocytes from the same donor in a standard cytotoxicity assay performed in cold inhibition 10 days later. Autologous PHA-activated lymphocytes were used as negative controls. To establish the frequency of antiallogeneic cytotoxic T precursor (CTLp), CD8+ RV cells and CD8+ PBLs from the same donor were plated in limiting dilution at 50 × 103, 40 × 103, 30 × 103, 20 × 103, 10 × 103, 5 × 103, 2.5 × 103, and 1.25 × 103 cells per well (96-well plate) with 30 × 103 fully mismatched PBMCs as stimulators and 30 × 103 autologous PBMCs as feeder cells; both types were 60 Gy irradiated in MLR medium. Twenty wells were plated for each dilution of responders. Medium was replaced on day 7, and cells were tested in a cold inhibition cytotoxicity assay on day 10. As negative controls, effectors were tested against autologous PHA-derived lymphocytes. Statistical analysis was performed with the 2 Poisson distribution method.
Human lymphocytes activated with different stimuli can be efficiently transduced by cell-free, retroviral-containing supernatant To identify the protocol of T-cell activation resulting in the highest transduction efficiency, different activation signals, such as PHA, anti-CD3 mAb, and the combination of anti-CD3 mAb and anti-CD28 mAb, were compared. Cells were transduced 48 hours after stimulation by 2 cycles of 2-hour centrifugation in the presence of cell-free retroviral supernatant. All the activation signals tested resulted in efficient and similar transduction efficiency (Figure 1B). In 3 independent experiments performed with cells from different donors, the difference in transduction efficiency observed with the 3 activation protocols was less than 5%. No differences in gene transfer efficiency were observed if transduction was delayed by 24 hours (data not shown). Such gene transfer efficiency is similar to the efficiency obtained by coculture of T cells with irradiated packaging cell lines.13 However, the use of a cell-free supernatant is more suitable to clinical use.
Retrovirally transduced cells were immunoselected for Transduction/selection procedure results in T cells with a mixed activation phenotype To analyze the effects of cell manipulation on different cell subsets, 3 pairs of PBLs and RV cells were analyzed for the expression of surface markers. As shown in Table 1, most RV cells expressed the panlymphocytic CD2 marker. Although most RV cells were represented by T lymphocytes (CD3+ cells), a variable number of cells (ranging from 3% to 19%) showed an NK phenotype (CD2+CD3 CD56+). Among
CD3+ T cells, an inversion of the
CD4+/CD8+ ratio was usually observed in RV
cells, whereas B lymphocytes (CD19+) were lost during
culture. Interestingly, the inversion of the CD4+/CD8+ ratio was not caused by the
preferential transduction of CD8+ cells but by culture
conditions because it was seen also in cultured nontransduced cells.
Different activation signals resulted in a similar
CD4+/CD8+ ratio. Polyclonality of transduced
cells was verified by immunophenotyping for TCR V repertoire. All
TCR analyses were represented in RV cells, and comparative analysis of
RV cells and autologous PBLs revealed no gross alterations of the
TCR repertoire.
The in vitro stimulation required for transduction induces polyclonal
activation of T cells. To characterize the level of cell activation in
RV cells, the expression of activation molecules such as CD25, HLA-DR,
CD69, CD95, CD45RA, CD45RO, and CD28 was investigated. More than 91%
of RV cells had an antigen-experienced phenotype
(CD45RA To obtain the in vivo induction of an effective immune response, T cells are supposed to travel to T-cell areas of secondary lymphoid organs to find antigen presented by professional antigen-presenting cells (APCs).24,25 A substantial proportion of RV cells expressed CD62L, a crucial molecule for lymphocyte migration to lymph nodes,25 suggesting that the in vitro manipulation does not interfere with homing abilities. Finally, a variable but consistent proportion of T cells maintained expression of CD28 after in vitro manipulation. Because CD28 engagement in CD28+ T cells recruits membrane microdomains containing kinases and adapters to the triggered TCR, allowing the activation threshold to be reached more rapidly and at lower doses of antigen,26 the maintained expression of this molecule should facilitate the activation of RV cells. Interestingly, the same results were observed using untransduced cells stimulated and cultured in the same conditions (data not shown), suggesting that the in vitro culture conditions and not the transduction/selection procedures were responsible for the alterations in cell phenotype. Transduction/selection procedure results mainly in TC1 and TH1 subsets To determine whether the gene transfer procedure skews toward different T-cell subsets, CD4+ and CD8+ RV cells from 3 different donors were analyzed for intracytoplasmic cytokine production. IL-2 and interferon (IFN)- were chosen as representative of helper T-cell/cytotoxic T-cell
(TH1/TC1) subsets, and IL-4 and IL-10 were used
to detect, respectively, TH2/TC2 and regulatory
T cells.18,27 All cytokines were detected, though in
different amounts. Analysis performed on RV cells obtained from 3 different donors showed a higher proportion of
CD4+/IL-2+/TH1 (41%-50%) and
CD4+/IFN-+/TH1 (37%-51%) than
CD4+/IL-4+/TH2 (12%-19%) subsets
and a higher proportion of
CD8+/IL-2+/TC1 (44%-46%) and
CD8+/IFN-+/TC1 (64%-70%) than
CD8+/IL-4+/TC2 (7%-13%) cells.
Less than 3% IL-10+ regulatory cells were detected in RV
cells (Figure 2). These results suggest a
more rapid and increased production of type 1 and type 2 cytokines than
peripheral blood mononuclear cells and a preferential skewing toward
CD4+TH1 and
CD8+TC1,28 probably because of
culture in the presence of rhIL-2.
Cytotoxic T cells specific for CMV antigens are preserved after gene transfer and selection Immune competence of RV cells was studied using antigens relevant in allogeneic stem cell transplantation. In particular CMV, a significant cause of illness and death after SCT,29 was used as a model antigen. To determine whether the gene transfer/selection procedure resulted in a reduced frequency of CTLs specific for CMV, pairs of RV cells and PBLs were stained with a PE-conjugated tetramer specific for the immunodominant HLA-A0201-restricted CMV pp65495-503 peptide. The frequency of CD8+ pp65 tetramer-binding cells in PBLs ( LNGFR) was preserved in RV cells
(LNGFR+) (Figure 3). The
high level of circulating tetramer+ cells shown in Figure 3
is suggestive of a recent CMV infection and T-cell activation. To
evaluate whether resting memory T cells specific for pp65 are preserved
after gene manipulation, cells from 2 CMV-seropositive donors with a
lower frequency of circulating tetramer+ cells were
studied. A similar frequency of CD8+ pp65 tetramer-binding
cells in PBLs and RV cells (0.59% vs 0.56% of total
CD3+/CD8+ cells in donor 2; 0.3% vs 0.5% in
donor 3) was observed. In spite of interindividual differences
highlighting the heterogeneity of T-cell responses, these results
suggest that CTLs specific for pp65 are preserved in terms of frequency
and level of TCR expression after in vitro manipulation.
Because the polyclonal T-cell activation required for gene transfer could potentially result in a refractory phase of nonresponse to additional stimuli or in AICD secondary to TCR religation,30 experiments were designed to assess the ability of RV cells to recognize and lyse cells expressing antigens relevant in the SCT setting. Immune response to viral antigens is preserved after gene transfer and selection To evaluate the reactivity of RV cells to viral antigens, EBV, CMV, and influenza virus were used. RV CD8+ cells and PBL CD8+ cells were stimulated either against autologous EBV-LCLs or autologous fibroblasts infected with CMVs, and specific lytic activity was tested in a cold cytotoxic inhibition assay. RV cells (Figure 4B) were always as efficient as PBLs (Figure 4A) in lysing viral-infected autologous targets.
To identify potential modifications in the CMV-specific T-cell repertoire induced by in vitro manipulation, immunity against single CMV-derived proteins was studied. To this purpose, autologous EBV-LCLs were infected with rVV coding for pp65, pp150, and pp72 CMV proteins. Infected cells were used in a cytotoxic assay as targets of the CMV-specific T-cell lines. Autologous EBV-LCLs infected with rVV coding for green fluorescent protein (GFP) were used as a negative control. Stimulation of PBLs and RV cells resulted in the expansion of CTLs specific for the same CMV antigen, most frequently a pp65-derived peptide for HLA-A2+ donors (data not shown) or a pp150-derived peptide for HLA-A3+ donors (Figure 4C). To better correlate proliferation of specific viral CTLs with cytolytic
activity, immune response to the M58-66 matrix flu peptide
was investigated. Because the M58-66 peptide is presented by HLA-A0201 molecules to cytotoxic effectors V
RV lymphocytes maintain an antiallogeneic immune repertoire The immune reactivity of genetically modified donor T cells against allogeneic targets was used to further characterize the immune potential of cultured cells. Repeat MLRs confirmed that RV cells have the same lytic activity as PBLs against mismatched target (Figure 6). To investigate whether prolonged in vitro manipulation results in alterations of the immune repertoire with a reduction of antiallogeneic effector cells in addition to PBLs and
RV cells transduced with the standard 14-day culture
protocolgenetically modified cells obtained by prolonging the culture
time to a total of 28 days were tested as source of effectors (RV 28).
An MLR was set by stimulating PBLs, RV cells, and RV 28 cells with
fully HLA-mismatched irradiated PBMCs. On stimulation, PBL and RV cell effectors exhibited a similar amount of specific lysis, whereas RV
28-derived effectors showed a significant reduction in lytic activity
(Figure 7A). To precisely quantify the
loss in antiallogeneic precursors induced by prolonged T-cell culture,
a more sensitive and quantitative assay, the cytotoxic T-lymphocyte
precursor frequency assay (CTLp) was used. Although the frequency of
antiallogeneic precursors was already 3- to 4-fold reduced in RV cells
(Figure 7C) compared with PBLs (Figure 7B), a more pronounced reduction in the CTLp frequency was observed in RV 28 cells (Figure 7D).
The moderate difference in antiallogeneic precursor frequency observed between RV cells and PBLs by the CTLp assay, and not by the MLR assay, probably reflects the different level of sensitivity of the 2 methods. The profound reduction in the CTLp frequency observed using RV 28, detectable already in the MLR assay, suggests that prolonged T-cell culture results in skewing of the antiallogeneic immune repertoire toward oligoclonality. Ex vivo detection of circulating CD45RA+/LNGFR+ T cells long term after infusion In a clinical study designed to take advantage of the graft-versus-leukemia (GVL) activity of HSV-tk-transduced donor T cells while controlling GVHD, we have shown that the infusion of donor lymphocytes expressing HSV-tk allows efficient control of GVHD while preserving antitumor potential7 (F.C. et al, manuscript in preparation). In this trial, GVHD did not develop in a significant number of patients, and they did not require GCV-mediated elimination of transduced cells. Some of these patients developed immunity against genetically modified cells, resulting in the elimination of circulating HSV-tk cells7,13 (C.T. et al, manuscript in preparation). However, in patients in whom an immune response against HSV-tk did not develop, long-term persistence of genetically modified cells (more than 60 months) could be documented. Circulating genetically modified T cells were detected at low levels long term after infusion by PCR for HSV-tk, and, when the number of circulating transduced donor lymphocytes reached the levels of FACS sensitivity, characterization of LNGFR+ cells was performed.7 In one patient of this series, characterization of LNGFR+ cells was possible up to 28 months after the last infusion, showing that circulating LNGFR+ T cells were usually CD45RA+ (Figure 8). This observation suggests that HSV-tk donor T cells produced under the described manipulation conditions and almost exclusively represented by memory CD45RO+ T cells (Table 1) can persist long term and can revert to a phenotype of terminally differentiated CD45RA+ effectors in vivo. This antigen-dependent differentiation and proliferation process25,32 suggests that genetically modified cells are able to persist long term and to properly react to antigens in vivo. Future studies will be conducted to estimate the frequency and the clinical relevance of this phenomenon.
The possibility of exploiting the different kinetics of GVL and GVHD and selectively treating GVHD makes HSV-tk+ donor lymphocytes a potential tool for controlling GVHD while preserving GVL.4,6 Administration of HSV-tk+ donor lymphocytes could have a clinical impact in promoting early immune reconstitution after T-depleted transplantation from a haploidentical donor.10,33 As an alternative strategy to provide immune reconstitution and to reduce the risk for GVHD, T cells specific for viral antigens, such as EBV34,35 or CMV,29 have been successfully infused in patients who have undergone transplantation. In contrast to these approaches, the HSV-tk-DLI approach aims at providing immunity against a wide range of pathogens, and potentially neoplastic cells, while controlling GVHD. Moreover, the HSV-tk-DLI approach relies on a faster and easier manipulation method than approaches based on antigen-specific T cells. Some limitations intrinsic to HSV-tk led to recent investigations of novel, less immunogenic suicide genes such as dimerized Fas36 and CD20.37 However, the HSV-tk/GCV suicide strategy is to date the only suicide switch that reached clinical trials, proving safety and efficacy in controlling GVHD. Given that the infusion of donor lymphocytes after haplo-SCT may require an efficient killing system and may not benefit from a nonimmunogenic protein because of the immune incompetence of recipients, HSV-tk+ RV cells were used to evaluate the immune function of RV cells in this study. However, some of the experiments described in this report were repeated with lymphocytes expressing an alternative suicide gene, resulting in similar findings. To completely exploit the use of RV cells, protocols of in vitro manipulation able to fully preserve T-cell potential are required. In this work, we investigated whether the polyclonal activation, retroviral transduction, immune selection, and expansion in the presence of rhIL-2 affects the immune reactivity of donor CD3+ T cells. The main concerns were AICD, which could skew the immune repertoire,38 and the 14 days of culture in the presence of rhIL-2, which could alter the T-cell repertoire, lead to T-cell exhaustion, or affect the proliferative and effector machinery of T cells.14,39 To reduce the level of AICD, different activation signals were compared. We observed that costimulation with anti-CD3 alone or with anti-CD3 plus anti-CD28 is as efficient as PHA in inducing T-cell proliferation and transduction. In contrast to the findings of Pollock et al,40 we observed no differences in the level of transgene expression after transduction following different stimulation signals. These different results could be attributed to the use of soluble instead of immobilized forms of antibodies, to the different promoters used in the 2 studies, or, less likely, to fibronectin, used by Pollock et al.40 Recent studies reported that AICD could be partially prevented by CD28 engagement through Bcl-Xl up-regulation in activated T cells.26,40 Indeed, results obtained with Annexin V showed that stimulation with anti-CD3 plus anti-CD28 mAb induce a lower level of AICD than PHA (data not shown). Furthermore, because CD28 costimulation provides T cells with signals that prevent the induction of anergy and that promote IL-2 production and clonal expansion,41,42 anti-CD3 in combination with anti-CD28 mAb is to date the ideal activation signal. Although antigen-specific cytotoxic CD8+ T cells may be the principal effectors for most antitumor and antiviral immune responses, CD4+ cells provide crucial helper functions required to sustain the CD8+ response.43 Even though the CD4/CD8 ratio is inverted in RV cells, both T cell populations are present after gene transfer. Furthermore, RV cells proved to have a broad cytokine secretion pattern with a preferential skew toward TH1/TC1, the T-cell subsets most likely responsible for GVL.44 An efficient T-cell-mediated immune response relies on the presence of a sufficient number of antigen-specific T cells and on the ability of T cells to be specifically activated by exposure to the antigen. Tetramers of the peptide-MHC complex20,45 were used to directly analyze the frequency of T cells of a defined specificity. Furthermore, antigens relevant in allo-SCT were used to evaluate the proliferative and lytic activities of RV cells. Anti-CMV and -EBV immunity were investigated because CMV and EBV cause significant illness and death after SCT46,47 and because viral reactivation can be efficiently treated by donor T cells.29,34,35 Results of this work showed that CTLs specific for viral antigens are preserved after in vitro manipulation, in terms of frequency and level of TCR expression and in terms of ability to proliferate and to kill antigen-expressing cells with the same efficiency and kinetics of PBLs. Among the antigens relevant in allo-SCT, allogeneic targets are particularly important because they are involved in GVHD,48,49 in the conversion to full donor chimerism,50 and in the prevention and treatment of disease relapse.1,2,34,51 Quantitative assays measuring the frequency of antigen-specific T-lymphocyte precursors by limiting dilution analyses showed a moderate reduction in the frequency of allospecific T cells in RV cells compared with unmodified lymphocytes. The reduction became more relevant when cells were kept in culture for longer times (RV 28). Because no additional stimulation was performed between RV cells and RV 28, AICD should not be implicated in this phenomenon. Conversely, this is probably because of the preferential growth advantage of some clones, resulting in progressive skewing toward oligoclonality.14 However, we cannot exclude that the reduced alloreactivity observed by prolonging the T-cell culture to 28 days could also occur in vivo. Indeed, in a murine model, Drobyski et al39 observed reduced alloreactivity and GVHD potential in transduced lymphocytes compared with unmodified cells. Based on this possibility and in addition to further improvements in gene transfer efficiency and cell manipulation methods, future clinical protocols will ideally involve the use of alternative gene transfer technologies that do not require cell proliferation, such as lentiviral vectors.52 Taken together, these results suggest that a cell manipulation
protocol consisting of appropriate cell activation, high transduction efficiency, and short culture results in the preservation of human T
cells specific for antigens relevant in the context of allo-SCT. However, the clinical relevance of these observations can only be
evaluated by analysis of the in vivo behavior of genetically modified
cells. Indeed, in a series of patients who received RV donor
lymphocytes to treat disease relapse after SCT,7 we
documented the long-term persistence of RV cells. In one patient we
could characterize LNGFR+ cells long after infusion and
could observe the reversion of the phenotype of infused cells from a
"memory" CD45RO phenotype to a CD45RA phenotype. This phenomenon
was already observed in HIV+ patients 10 weeks after the
infusion of genetically marked T cells from their syngeneic
twins.53,54 Antigen-experienced T cells with a CD45RA
phenotype have been described,32 and phenotypic and
functional analyses have characterized this T-cell subset as terminally
differentiated effector T cells.25 In our study, Based on these results and on our previous experience in terms of safety and efficacy of the HSV-tk/GCV strategy,5,7 we propose the use of HSV-tk genetically engineered donor lymphocytes to promote early immune reconstitution after T-cell-depleted SCT from haploidentical donors. The possibility of selectively eliminating cells expressing HSV-tk with GCV could allow the anticipation of immune reconstitution and improvement of the GVL effect by an early infusion of genetically modified cells, potentially in association with immune modulators such as IL-2.57
We thank the physicians and nurses of the Bone Marrow Transplantation Unit, Istituto Scientifico San Raffaele, for patient care, and we thank all the patients and marrow donors who participated in the clinical study. We thank Dr A. Aiuti for helpful discussion and D. Maggioni and E. Zino for excellent technical assistance.
Submitted August 13, 2002; accepted September 24, 2002.
Prepublished online as Blood First Edition Paper, October 3, 2002; DOI 10.1182/blood-2002-08-2351.
Supported by the European Commission (Biomed contract BMH4-CT97-2760, BMH4-CT97-2074, and QLK3-CT-2001-01265); the Italian Association for Cancer Research (AIRC), Milan, Italy; and the Cancer Research Institute, New York, NY.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Chiara Bonini, Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H. S. Raffaele, via Olgettina 58, 20132, Milan, Italy; e-mail: bonini.chiara{at}hsr.it.
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Abstract
Introduction
