Abstract

Background.The profound immunodeficiency associated with allogeneic hematopoietic stem cell transplantation is permissive to uncontrolled replication of latent human herpesviridae such as cytomegalovirus. Morbidity and mortality associated with viral dissemination or its treatment are significant. Although adoptive cellular therapy with virus-specific T cells offers the potential for accelerating pathogen-specific immune reconstitution, the risk of induction of graft-versus-host disease and the logistics of production of clonal T cell populations restrict application.

Methods.We investigated the ability of cytomegalovirus-specific mixed CD4+and CD8+T cell lines, generated by short-term ex vivo culture of donor lymphocytes with donor monocyte-derived dendritic cells pulsed with virus lysate, to restore antiviral immunity in 30 allogeneic transplant recipients at high risk of both uncontrolled viral replication and of graft-versus-host disease.

Results.There were no immediate toxicities and no excess of graft-versus-host disease. Massive in vivo expansions of cytomegalovirus-specific T lymphocytes occurred, temporally associating with periods of viral replication, suggesting that antigen exposure was necessary for optimal cytomegalovirus-specific immune reconstitution. The expanding populations maintained functional competence in ex vivo re-stimulation assays, promoting reconstitution of durable functional cytomegalovirus-specific immunity and effectively preventing recurrent viral infection and late cytomegalovirus disease.

Conclusions.These data confirm the ability of cellular immunotherapy to hasten reconstitution of antiviral immunity following allogeneic transplantation, indicating that significant clinical benefits may be conferred in terms of reduction of secondary viral infection episodes, potentially reducing exposure to the toxicities of antiviral drugs.

Infection with human cytomegalovirus (CMV) is a potentially lethal complication following allogeneic hematopoietic stem cell transplantation [1]. It usually results from reactivation of latent virus and is defined by detection of CMV in peripheral blood samples (usually by polymerase chain reaction). Although reactivation events are probably common during long-term CMV carriage, their detection is relatively uncommon in immunocompetent individuals. Significant suppression of host antiviral immunity, which is affected by both CD8+and CD4+T cells, allows reactivation events to become detectable. Uncontrolled viral replication and dissemination can result in the development of end-organ damage (CMV disease). Prophylactic administration of antiviral drugs effectively reduces the incidence of CMV disease following allogeneic hematopoietic stem cell transplantation (HSCT) [2, 3], but their use is associated with significant morbidity due to suppression of myelopoeisis or induction of renal toxicity. Hence, pre-emptive rather than prophylactic treatment strategies have generally become routine [4]. These approaches are associated with an increased incidence of late-onset CMV disease [5, 6].

Because the morbidity associated with CMV infection may be caused either directly by the virus or indirectly through the toxicities associated with antiviral drugs [7], reduction of drug usage is a valuable therapeutic aim. Furthermore, prior exposure of recipient and/or donor to CMV remains inextricably linked to poor overall survival. This effect seems restricted to recipients of T cell-depleted allografts and/or transplants from unrelated or human leukocyte antigen (HLA)-mismatched donors [7, 8]. These considerations have particular current relevance. The increasingly widespread application of reduced intensity conditioning in a more elderly transplant population has been coupled with an expanded use of unrelated donors. Many receive T cell-depleting serotherapy, such as alemtuzumab or ATG, to limit graft-versus-host disease (GvHD). Such patients are at a higher risk of CMV infection and may benefit the most from restoration of anti-CMV immunity, but they are also at greatest risk of GvHD subsequent to infusion of alloreactive T cells.

Attempts to restore antiviral immunity following allogeneic HSCT with cell-based immunotherapies initially focused on strategies that required prolonged culture in vitro [9, 10], and translation into routine clinical practice was limited by the logistics and costs associated with such heroic cell expansions. Subsequent approaches have focused on more rapid generation of cellular therapy products, either with more efficient short-term culture techniques [11–13], direct selection of antigen-specific T cells from the donor [14], or a combination of these strategies [15, 16]. We previously reported an early phase I clinical study of pre-emptive infusion of polyclonal CMV-specific T cell lines following detection of CMV DNAemia [12]. The majority of these patients received grafts from matched related donors. In a subsequent phase II study, we treated 30 patients with T cell lines administered early after transplantation. This cohort included a higher number of patients at risk of GvHD following adoptive cell therapy (ACT) by virtue of inclusion of more mismatched or unrelated donors (47%).

Methods

Eligibility. Patients undergoing allogeneic HSCT from a CMV-seropositive donor were eligible. The institutional Ethics Committee approved the study protocol, and procedures followed were in accordance with the ethical standards of the Helsinki Declaration. Patients and donors gave written informed consent. Patients were excluded from receiving CMV-specific cellular therapy (ACT) if they had active GvHD Grade I or higher at the planned time of infusion.

Study design. The trial was a single-arm open-label phase II design in which all patients were intended to receive ACT 28 days after transplantation. Primary endpoints were the number of CMV-related treatment episodes and incidence of GvHD. Antiviral drugs (ganciclovir or foscarnet) were administered according to institutional guidelines if CMV titers increased over 2 consecutive weeks, and treatment was continued until clearance of viral DNAemia. Subsequent infection episodes were treated if CMV titers increased over 3 consecutive weeks. Initial treatment episodes were classified as primary, whereas additional treatment episodes following viral clearance were classified as secondary. Thus, patients receiving CMV-specific immunotherapy either before or after a single episode of viral DNAemia (prophylactic and pre-emptive groups, respectively) were evaluable for an impact on the number of primary episodes. Those surviving >4 weeks after discontinuation of antiviral drugs, including those receiving ACT after commencement of antiviral drug therapy (concurrent group), were considered evaluable for secondary episodes. Acute GvHD was assessed using Glucksberg criteria [17], and chronic GvHD was graded as limited or extensive [18]. Using a 1-stage Fleming trial design and on the basis of historical institutional data on the incidence of CMV infection (secondary treatment episodes in 52% of patients requiring primary treatment), we determined that a sample size of 20 patients evaluable for secondary treatment was required to suggest that ACT was sufficiently active (secondary therapy rate of ⩽20%) to warrant further testing (a=0.05; b=0.1).

Generation of T cell lines. T cell lines were generated from a single 70-mL blood draw with use of a CMV cell lysate. Details of the methods, including the antigen and testing for CMV infectivity, have been reported elsewhere [19, 20]. Recombinant human CD40L was introduced for maturation of dendritic cells, which was validated by analysis of phenotype, and ability to induce T cell proliferation and interferon (IFN)-γ secretion (Figure 1). CD40L-matured dendritic cells increased T cell proliferation in co-cultures without increasing non-CMV-specific proliferation and enhanced induction of IFN-γ-secreting CD4+and CD8+T cells, allowing a reduction in the duration of culture from 3 to 2 weeks.

Figure 1

Optimization and characterization of CD40L-matured dendritic cell co-cultures.

Figure 1

Optimization and characterization of CD40L-matured dendritic cell co-cultures.

Release criteria. Each cell culture was screened for microbiological and fungal contamination before being released for clinical use. On the basis of prior preclinical and phase I clinical data, there were no specific requirements for the assessment of individual cultures for alloreactivity, cytotoxicity, or level of CMV-specific T cells. The target cell dose was 1×105total cultured cells/kg.

Patient monitoring. Patients were reviewed weekly until day 100–120 after transplant, then every 2–4 weeks until day 180. Surveillance monitoring for CMV was performed by quantitative polymerase chain reaction. Reconstitution of CMV-specific CD8+T cell responses was evaluated using pp65-specific Class I HLA-pentamers (Proimmune) in appropriate patients (with the HLA-A*0201-restricted NLVPMVATV epitope and/or the HLA-B*0702-restricted TPRVTGGGAM or RPHERNGFTVL epitopes). Peripheral blood mononuclear cells were stained with PE-conjugated CMV-specific pentameric complex, then washed and counter-stained with FITC-conjugated anti-CD3 mAb (Dako) and PerCP-conjugated anti-CD8 mAb (Dako) prior to flow cytometric analysis (Beckman Coulter). Negative controls included cells from CMV-seronegative individuals expressing the appropriate HLA antigen and from CMV seropositive individuals not expressing the appropriate HLA antigen.

IFN-γ staining. Peripheral blood mononuclear cells were stained with Class I HLA pentamers, then restimulated overnight with the corresponding peptide (NLV, TPR, or RPH), control antigen, or no antigen. The frequency of IFN-γ-secreting T cells was then measured using a cytokine secretion assay kit (Miltenyi Biotec GmbH). Cells were costained with CD8-FITC and CD14 PerCP-Cy5.5. Propidium iodide+cells and CD14+monocytes were excluded by negative gating.

Results

Patient and cell line characteristics. Patients were enrolled from February 2004 through May 2007. None have been previously reported. Thirty-one patient/donor pairs consented to the study. CMV-specific T cell lines were generated in all cases. One patient developed GvHD prior to ACT and was excluded from receiving cell therapy. Cultured cells consisted largely of CD3+T cells (median, 91%; range, 73%–97%), skewed toward the CD4+compartment (CD3+CD4+median, 78%; range, 46%–91%; CD3+CD8+median, 14%; range, 4%–50%). NLV-specific CD8+cells represented 0.1%–4.3% (median, 1.1%) of the post-culture CD8+T cells, TPR-specific cells represented 9.8%–18%, and RPH-specific cells represented 2.0%–7.6%. Patient characteristics are outlined in Table 1. T cell depletion was performed using alemtuzumab in 24 cases. At the time of ACT, 28 patients were receiving cyclosporine A at therapeutic levels.

Table 1

Patient Characteristics

Table 1

Patient Characteristics

CMV-specific T cell lines can be administered early after transplantation without clinically significant toxicities. CMV-specific T cells were returned according to protocol in 28 patients at day 25–34; delays occurred while possible GvHD was excluded (Table 2). Two patients received cells earlier at their physician's discretion. The majority of patients received cells either pre-emptively (n=10) or concurrent with antiviral drugs (n=10), reflecting the pattern of early infection following T cell depletion. Notably, all the seronegative patients received ACT prophylactically.

Table 2

Graft-versus-Host Disease (GvHD) and Current Status

Table 2

Graft-versus-Host Disease (GvHD) and Current Status

No infusional toxicities occurred. All patients were evaluable for the development of acute GvHD. Twenty-six had either no (n=19) or Grade I (n=7) disease (Table 2). Only 4 (13%) developed Grade II-III GvHD; 2 in those receiving T cell-depleted grafts (8%) and 2 with T cell replete grafts (33%). None of the 14 patients with matched unrelated, mismatched unrelated, or mismatched sibling donors developed Grade II or higher disease (Table 2). Twenty-eight patients were evaluable for the development of chronic GvHD. Ten developed limited disease, and 2 developed extensive disease (both in patients receiving T cell-replete grafts).

Reconstitution of CMV pp65-specific CD8+T cell immunity following adoptive cellular therapy is brisk in the presence of viral DNAemia. CD8+T cell numbers increased into the normal reference interval within 4 weeks in 28 patients and to supranormal levels in 13 (Figure 2). CD4+T cell counts increased less rapidly but were >200×106cells/L in 13 patients within 4 weeks and in an additional 4 patients by 8 weeks (Figure 2). Peak levels lagged behind peak CMV titers (Figures 2and 3), suggesting that antigenic challenge is a strong stimulus to T cell expansion. Fourteen patients expressed HLA-A*0201, allowing evaluation of reconstitution of CMV-specific immunity with class-I HLA pentamers (Figure 3). Four patients coexpressed HLA-B*0702. Marked differences were observed between those with CMV DNAemia (n=11) and those in whom it remained undetectable (n=3) (Figure 3E and 3F ). In the former, NLV-specific T cells peaked at 3.6%–36.0% (median, 14.0%) of CD3+CD8+T cells (excluding those coexpressing HLA-B*0702), compared with 0.5%–0.8% (median, 0.6%) in the latter (P=.017, by 2-tailed Mann-Whitney test). The corresponding peak absolute NLV-specific cell counts were 70.2×106−240.9×106cells/L, compared with <1×106cells/L (P=.017, by 2-tailed Mann-Whitney test). In patients coexpressing HLA-B*0702, the responses directed toward HLA-B*0702-restricted epitopes predominated in three-quarters of the cases, both in terms of maximal percentage of the CD3+CD8+T cell compartment and absolute cell counts (Figure 3E and 3F ). The peak absolute NLV-specific T cell counts in these patients were 1.8×106−109.8×106cells/L. The temporal dynamics of expansion and contraction of these different CMV-specific T cell populations varied (Figure 3D ).

Figure 2

Reconstitution of lymphocyte subsets. The temporal kinetics of reconstituting lymphocyte subsets and viral load are shown for patient 05 (A) and patient 23 (B) . Total lymphocyte (triangles) , CD8+T cell (squares) , and CD4+T cell counts (diamonds) are shown. Circles represent viral load. T cell reconstitution was typically faster in the CD8+population, compared with the CD4+population, and appeared to occur in response to antigenic challenge. Peak numbers of CD8+(C) and CD4+(D) T cells within 8 weeks of adoptive transfer rose significantly (paired t tests). Bars in panels C and D represent mean values. E and F , For comparison, the temporal kinetics of lymphocyte reconstitution and association with viral load are shown for 2 control patients undergoing reduced intensity T cell depleted transplantation from unrelated donors. Both were human cytomegalovirus (hCMV)-seropositive donor/recipient pairs, and neither developed more than grade I graft-versus-host disease.

Figure 2

Reconstitution of lymphocyte subsets. The temporal kinetics of reconstituting lymphocyte subsets and viral load are shown for patient 05 (A) and patient 23 (B) . Total lymphocyte (triangles) , CD8+T cell (squares) , and CD4+T cell counts (diamonds) are shown. Circles represent viral load. T cell reconstitution was typically faster in the CD8+population, compared with the CD4+population, and appeared to occur in response to antigenic challenge. Peak numbers of CD8+(C) and CD4+(D) T cells within 8 weeks of adoptive transfer rose significantly (paired t tests). Bars in panels C and D represent mean values. E and F , For comparison, the temporal kinetics of lymphocyte reconstitution and association with viral load are shown for 2 control patients undergoing reduced intensity T cell depleted transplantation from unrelated donors. Both were human cytomegalovirus (hCMV)-seropositive donor/recipient pairs, and neither developed more than grade I graft-versus-host disease.

Figure 3

Human cytomegalovirus (hCMV)-specific immune reconstitution. A , NLV-specific HLA-pentamer labeling following adoptive cell therapy (ACT) in patient 19. The percentage of the CD3+population labeling with pentamer is shown within the flow plot, and the percentage of the CD3+CD8+population labeling with pentamer is shown below. CMV-specific T cell reconstitution relative to viral load is shown for 3 further representative patients in panels B-D . B and C , Patient 12 and patient 25 expressed HLA-A*0201 but not HLA-B*0702. Symbols represent viral load (circles) , total lymphocyte count (triangles) , and CD3+CD8+T cell count (squares) . NLV-specific T cell counts (diamonds) are shown in absolute numbers (×109/500 mL, so that they can be discerned in the same graph). D , Patient 16 co-expressed HLA-A*0201 and HLA-B*0702. Absolute levels of cells labeling with 3 pp65-specific pentamers (NLV, diamonds; TPR, squares; RPH, triangles ) are shown. E and F , Peak levels of pentamer-labeling cells occurring during the first 3 months following infusion are shown as percentage of CD8+T cells (E) and absolute counts (F) , divided according to pentamer labeling and whether CMV replication was detected. Circles indicate patients expressing HLA-A*0201 but not HLA-B*0702, whereas diamonds indicate those expressing both alleles. Closed symbols represent those with sibling donors, and open symbols represent those with unrelated donors. G , Absolute levels of pentamer-labeling cells at 3-4 months post infusion. Symbols shown on the x -axis equate to levels of <0.1×106/L. Bars in panels E-G represent mean values.

Figure 3

Human cytomegalovirus (hCMV)-specific immune reconstitution. A , NLV-specific HLA-pentamer labeling following adoptive cell therapy (ACT) in patient 19. The percentage of the CD3+population labeling with pentamer is shown within the flow plot, and the percentage of the CD3+CD8+population labeling with pentamer is shown below. CMV-specific T cell reconstitution relative to viral load is shown for 3 further representative patients in panels B-D . B and C , Patient 12 and patient 25 expressed HLA-A*0201 but not HLA-B*0702. Symbols represent viral load (circles) , total lymphocyte count (triangles) , and CD3+CD8+T cell count (squares) . NLV-specific T cell counts (diamonds) are shown in absolute numbers (×109/500 mL, so that they can be discerned in the same graph). D , Patient 16 co-expressed HLA-A*0201 and HLA-B*0702. Absolute levels of cells labeling with 3 pp65-specific pentamers (NLV, diamonds; TPR, squares; RPH, triangles ) are shown. E and F , Peak levels of pentamer-labeling cells occurring during the first 3 months following infusion are shown as percentage of CD8+T cells (E) and absolute counts (F) , divided according to pentamer labeling and whether CMV replication was detected. Circles indicate patients expressing HLA-A*0201 but not HLA-B*0702, whereas diamonds indicate those expressing both alleles. Closed symbols represent those with sibling donors, and open symbols represent those with unrelated donors. G , Absolute levels of pentamer-labeling cells at 3-4 months post infusion. Symbols shown on the x -axis equate to levels of <0.1×106/L. Bars in panels E-G represent mean values.

The median absolute pp65-specific T cell numbers had decreased by 3 months following transfer but were maintained at levels consistent with those thought to be protective against uncontrolled viral replication [21, 22] in at least one of the measurable HLA restrictions (median, 50×106cells/L for NLV, 15×106cells/L for TPR, and 9×106cells/L for RPH) (Figure 3G ).

CMV-specific T cells maintain functional competence following expansion in vivo. The functional competence of CD8+pp65-specific T cells was measured by colabeling with Class I HLA pentamers and intracellular staining for IFN-γ after peptide stimulation in 5 patients. Between 27% and 77% of pentamer-labeling cells were capable of production of IFN-γ in an antigen-specific manner when sampled within 2 weeks after viral clearance (Figure 4A and 4B ). The median absolute number of CD8+IFN-γ+T cells measured within 2 weeks of CMV clearance was 24.7×106cells/L (range, 1.8×106−99.6×106cells/L) (Figure 4C ).

Figure 4

Functional competence of cytomegalovirus-specific T cells. Cells collected from patients following adoptive transfer were assessed for their ability to produce interferon (IFN)-γ in response to antigenic challenge. A , Pentamer-labeling cells were capable of IFN-γ production in an antigen-specific manner. The number of antigen-specific IFN-γ-secreting cells measured within 2 weeks of viral clearance was assessed in 5 patients and shown as percentage of the pentamer-labeling population (B) following subtraction of background staining (circles represent NLV-, diamonds RPH-, and triangles TPR-specific populations), with absolute levels (C) .

Figure 4

Functional competence of cytomegalovirus-specific T cells. Cells collected from patients following adoptive transfer were assessed for their ability to produce interferon (IFN)-γ in response to antigenic challenge. A , Pentamer-labeling cells were capable of IFN-γ production in an antigen-specific manner. The number of antigen-specific IFN-γ-secreting cells measured within 2 weeks of viral clearance was assessed in 5 patients and shown as percentage of the pentamer-labeling population (B) following subtraction of background staining (circles represent NLV-, diamonds RPH-, and triangles TPR-specific populations), with absolute levels (C) .

CMV-specific T cell therapy effectively prevents the requirement for recurrent treatment and the occurrence of late CMV disease. Of those receiving ACT prophylactically, 3/10 (30%) had a primary episode of CMV infection requiring antiviral therapy (1 of 6 seronegative recipients, compared with 2 of 4 seropositive recipients) (Table 3). In 2 of these cases, therapy was necessary only following institution of systemic steroids (2 mg/kg methylprednisolone). In both cases, the introduction of steroid was followed by a significant reduction in lymphocyte count. An additional patient had a single isolated episode of CMV DNAemia. All 10 patients treated pre-emptively required antiviral drugs because of increasing viral titers. The median duration of antiviral therapy in these 13 patients was 21 days (mean, 21 days), compared with 28 days (mean, 29 days) in those receiving antiviral drug at the time of ACT (P=.027, by 1-tailed Mann-Whitney test).

Table 3

Cytomegalovirus (CMV)-directed Treatment According to Adoptive Cell Therapy (ACT) Group

Table 3

Cytomegalovirus (CMV)-directed Treatment According to Adoptive Cell Therapy (ACT) Group

Three patients survived <4 weeks after cessation of initial therapy and were considered not to be evaluable for secondary treatment episodes. The 20 remaining patients survived at least 100 days after initial viral clearance. None of these patients had another episode of CMV infection requiring antiviral drug therapy. Seven had CMV DNA detectable for 1–3 periods (median, 1 period) with durations of 1–5 consecutive weeks (median, 3 weeks), but none reached the threshold for treatment. CMV titers were >2000 copies/mL in only 2 patients, for a single week in each patient. There were no episodes of CMV disease.

Discussion

CMV-specific cellular therapy has the potential to accelerate pathogen-specific immune reconstitution and reduce morbidity (and ultimately perhaps mortality) following allogeneic transplantation. A number of investigators have published early phase clinical studies of cellular immunotherapy in small numbers of patients [11–14, 23–25]. We performed a phase I-II clinical study with use of polyclonal CMV-specific T cell lines containing both CD4+and CD8+T cells generated by short-term ex vivo culture. Our data are important because they show, in a relatively large cohort, (1) that polyclonal CMV-specific T cell lines can be infused safely in groups of allogeneic transplant recipients who are at high risk of both GvHD and of CMV infection and associated morbidities and (2) that viral replication and increasing antigenic load appear to induce massive expansion of infused cells, (3) allowing infusion of very small numbers of CMV-specific T cells to enable reconstitution of durable functional CMV-specific immunity, effectively preventing recurrent viral infection and late CMV disease.

GvHD is one of the most significant potential toxicities of cellular therapy. The low predicted background incidence following alemtuzumab-based T cell depletion allows particularly stringent testing for GvHD-related toxicity. It is therefore notable that GvHD rates (2 of 24 patients in the T cell-depleted cohort experienced Grade II-IV acute GvHD) were no higher than would be predicted in the absence of ACT (5%–24% [26–28]) and, more specifically, that 0 of 14 patients with unrelated or HLA-mismatched donors developed >Grade I GvHD. These data support the conclusion that CMV-specific T cell therapy with polyclonal T cell lines can be safely administered, even to high-risk patients, at early time points following transplantation without an excessive risk of GvHD.

The CMV-specific cell lines were not marked before transfer, but clinical evidence strongly suggests that they formed the basis for the expansions observed in vivo. Although endogenous CMV-specific immune reconstitution can be brisk in patients transplanted with T cell-replete grafts and sibling donors, transplants from unrelated donors are associated with markedly impaired reconstitution within the first 100 days following transplantation [21, 22, 29]. In the current study, all 7 unrelated donor transplant recipients experiencing episodes of viral DNAemia for whom CMV epitope-specific immune reconstitution was available showed prompt immune reconstitution after ACT, reaching maximum absolute levels of 55.3×106−226.8×106cells/L. Furthermore, we have previously demonstrated that expanding populations share the same T cell receptor BV usage and CDR3 lengths as the transfused populations [12].

The absolute levels of CMV-specific T cells reached are well above those thought to be protective against CMV infection (10×106−20×106cells/L for NLV-specific T cells [21, 22]). Coupled with the demonstration that these cells maintain functional competence in terms of peptide-specific IFN-γ production, these data lend biological plausibility to the apparent protection against subsequent viral replication/dissemination events. If transfused cells are largely responsible for the increased numbers of CMV-specific T cells, these cells must be capable of expansion of many orders of magnitude in vivo. Delivery of virus-specific T cells into a lymphopenic environment at the time of rapidly increasing viral load may be permissive to this degree of expansion. We estimate that cells expanded up to 5 log over a period as short as 10 days following transfer (assuming a 5 L circulating volume and that 2% of the lymphocyte pool is located in the peripheral blood), with lymphocyte doubling times possibly as short as every 12 h. The tempo of in vivo expansion, compared with that of viral replication, is likely to be an important determinant in the efficacy during the initial post-transplantation infection episode, and it is possible that virus may have been cleared without addition of antiviral drugs in at least a proportion of cases. It is also likely that this accounts for the universal requirement for antiviral therapy in the pre-emptive group, because the time between ACT and initial sampling to assess increasing viral load was only 3 days. Reconstitution of epitope-specific CD4+T cell responses was not routinely assessed, and further comment on their contribution to restoration of anti-CMV immunity is therefore not possible [30, 31].

CMV infection rates are high following T cell depletion with alemtuzumab, approaching 85%–90% in seropositive and 50%–60% in seronegative patients with seropositive donors, allowing consideration of prophylactic strategies [32]. Although only 3 of 10 patients receiving ACT as prophylaxis required antiviral drugs, all 6 CMV-seronegative recipients were within this group reflecting their lower overall predicted risk of infection. Larger numbers of patients receiving prophylactic ACT are needed before drawing definitive conclusions regarding an impact on primary treatment episodes. The impact of CMV-specific ACT on secondary treatment episodes was more striking. None of the 20 patients considered evaluable for a secondary treatment episode required subsequent therapy, suggesting that further studies of adoptive cellular therapy for CMV are warranted (based on the 1-stage Fleming design) and helping to inform the design of a prospective randomized study comparing standard surveillance and pre-emptive antiviral drug therapy with or without ACT (CMV-IMPACT). Although current regulation favors direct selection strategies (targeting a more restricted range of viral epitopes) rather than in vitro culture, our use of a viral lysate has the potential advantage of generating a broader repertoire of response. This may be important given the heterogeneity of immune responses demonstrated among healthy donors because not all have responses directed toward any specific epitope [33], and these data provide a useful comparator for the next generation of studies. Whether pathogen-specific immunotherapy can influence overall mortality rates remains unclear, but it is notable that whereas 0 of 27 evaluable patients developed late CMV disease in our study, other groups have identified late CMV disease as a growing problem in the era of reduced intensity transplantation and pre-emptive anti-CMV therapy, reporting disease rates of 15%–20% with associated mortality rates approaching 50% [5, 6].

In summary, our study demonstrates that CMV-specific cellular therapy can be safely applied in patients at high risk of GvHD-associated complications and confirms that virus-specific expansions can occur in a profoundly lymphopenic environment in the presence of therapeutic levels of cyclosporine A. The absolute levels of CMV-specific T cells achieved and lack of secondary infection episodes or late CMV disease suggest that durable functional immunity to CMV has been restored, and the magnitude of the effect provides the rationale for prospective randomized studies addressing efficacy.

Acknowledgments

Financial support. This work was undertaken at University College London Hospitals/University College London, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding Scheme, and at the Royal Free Hospital, London. K.S.P. receives funding from Leukaemia Research (London, UK). The funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

Potential conflicts of interest. All authors: no conflicts.

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