In this issue of Blood, Barker et al demonstrate that third-party non-neonatal T cells specific for Epstein-Barr virus (EBV) can be safely used to treat EBV-associated disease after allogeneic umbilical cord blood (UCB) transplantation (UCBT).1  This is made possible by the a priori generation of cryopreserved banks of EBV-specific T cells from peripheral blood.

The clinical benefits of infusing third-party T cells is predicted from extensive nonhuman and human experiences demonstrating that the adoptive transfer of antigen-specific T cells from a donor can successfully augment an immune response protecting and treating a designated recipient against pathogens and tumors after allogeneic hematopoietic stem cell transplantation (HSCT). What is only recently becoming apparent is that one donor can be used to generate antigen-specific T cells that can be infused into multiple recipients.

The report in this issue joins a growing list of clinical trials in which previously generated cryopreserved third-party EBV-specific T cells have been adoptively transferred.2-4  Multiple infusions were administered to achieve a clinical response with thawed T cells given every week, every 2 weeks, or every 3 weeks, at intravenous doses of 106/kg/infusion to 2 × 106/kg/infusion based on recipient weight. These data describe clinical responses to opportunistic EBV infection and associated disease that were apparently long-lasting and significantly were not associated with severe adverse effects. The anonymity, small size, and functional naiveté of the UCB graft typically preclude isolation of clinical-grade T cells with desired specificity and suitable for adoptive immunotherapy. Thus, the testing of third-party EBV-specific T cells from non-neonatal donors is particularly compelling in this clinical context. In aggregate, Barker's data highlight that third-partycryopreserved T cells can be successfully infused and that anti-EBV responses occur with sufficient rapidity despite the likely elimination of the infused T cells due to immune recognition by the recipient of mismatched major histocompatibility complex (MHC) molecules.

What is particularly heartening is that all published reports to date demonstrate an absence of clinically significant graft-versus-host-disease (GVHD) after infusion of off-the-shelf EBV-specific T cells that are at best only partially MHC matched. This is somewhat surprising considering that only up to ∼ 104/kg of donor-derived lymphocytes can be infused and reinfused after haploidentical HSCT without causing severe GVHD.5  Alloreactive T cells are described as having intrinsic affinity for the surface of disparate MHC molecules as well as maintaining conventional recognition for cognate peptides presented in the context of MHC.6  As shown by Amir et al,7  populations of T cells bearing αβ T-cell receptors with either unknown specificity or defined specificity for viral antigens both demonstrate alloreactivity in vitro. The absence of GVHD after the infusions of EBV-specific T cells perhaps reflects that the tissue-culturing process (coculture of T cells with recurrent additions of autologous γ-irradiated EBV-transformed B cells) to obtain EBV-specific T cells reduces the potential for alloreactivity. The apparent lack of alloreactivity in clinical practice was recently confirmed in recipients of allogeneic HLA-matched and HLA-mismatched HSCT who received donor-derived viral-specific T cells, despite in vitro data to the contrary.8 

The future clinical impact regarding infusion of third-party EBV-specific T cells after UCBT will be interpreted regarding competing approaches and technologies. To generate T-cell effectors capable of long-lived immunosurveillance, investigators are developing methodologies to generate viral-specific T cells derived from the infused UCB allograft.9  For example, populations of UCB-derived “trivirus”-specific T cells can be generated with specificity for adenovirus, cytomegalovirus, and EBV10  by adapting an approach that has shown beneficial clinical activity upon infusing trivirus-specific T cells obtained from peripheral blood mononuclear cells (PBMC).11  Indeed, trivirus-specific PBMC-derived T cells are being evaluated in a multi-institution clinical trial as anoff-the-shelf therapy (ClinicalTrials.gov Identifier: NCT00711035). To broaden the therapeutic potential of UCB-derived T-cell therapy investigators are using gene-transfer approaches to redirect the specificity of T cells to introduce chimeric antigen receptor (CAR) for a tumor-associated antigen and expressing CAR on trivirus-specific T cells.12,13 

The field of T-cell therapy is changing so that a given T-cell product can be infused into multiple recipients. Preclinical data are being generated to avoid immune recognition by down-regulating the expression of disparate MHC14  and using T-cell precursors from MHC-mismatched donors that can be infused across transplantation barriers.15  Regarding the clinical application of off-the-shelf T cells, questions remain for infrastructure, governance, and clinical conduct. Who will prepare these banks? What procedures will govern the distribution of cryopreserved T cells within state and across state lines? What are the avenues for reimbursement? Will these cells be dispensed using blood-banking practices? What informatics is needed to match candidate recipients with banked T cells?

There are also compelling questions that remain unanswered for how best to deploy off-the-shelf T cells. For example, should third-party effectors be infused after a lymphodepleting regimen to enable lymphopenia-induced proliferation of the adoptively transferred T cells as well as reduce the potential for immune-mediated rejection? Should the dose of T cells be increased per infusion rather than administering multiple infusions on the basis of reducing the chance of immunizing the recipient against disparate MHC?

The current approach of pairing a patient with cryopreserved T cells is based upon matching the recipient with the most closely matched MHC in the bank, with preference given to the cryopreserved product having the greatest number of shared MHC loci. In addition to typing, a bank's in vitro data should be queried so that priority is given to T-cell lines that indeed recognize the desired antigen through a restricting MHC molecule that is shared by the recipient.

Off-the-shelf T-cell therapy is currently being used when other therapeutic options are exhausted. A decision tree (see figure) is offered that integrates immunotherapy with chemotherapy for the treatment of EBV-associated disease after solid organ and HSCT. Trials will be needed to compare third-party T cells with other treatment options. In this context, it is revealing to consider that the cost to generate and release clinical-grade T cells in facilities that operate in compliance with current good manufacturing practice continues to fall due to improved processing and economies of scale. Indeed, manufacturing and releasing a T-cell line with specificity for EBV costs approximately $6000 which may be used for multiple infusions.16  When one considers the cost of treating EBV-disease with rituximab (cumulatively $9000 per dose) or chemotherapy, there is considerable financial merit, in addition to scientific rationale, for infusing off-the-shelf EBV-specific T cells.

An algorithm for treating EBV-associated clinical disease after transplantation. Frontline treatment (Option #1) and second-line therapies (Option #2) are widely practiced. Option #3 is to be considered experimental, but encouraging, at this time.

An algorithm for treating EBV-associated clinical disease after transplantation. Frontline treatment (Option #1) and second-line therapies (Option #2) are widely practiced. Option #3 is to be considered experimental, but encouraging, at this time.

In summary, medical centers around the world are now stacking the shelves of their freezers with viral-specific T cells ready to be infused as third-party effectors. These off-the-self therapies are the first steps to transforming T cells as drugs that can be prepared a priori and infused on demand.

Conflict-of-interest disclosure: The author declares no competing financial interests. ■

1
Barker
 
JN
Doubrovina
 
E
Sauter
 
C
, et al. 
Successful treatment of Epstein-Barr virus (EBV)–associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes.
Blood
2010
, vol. 
116
 
23
(pg. 
5045
-
5049
)
2
Wynn
 
RF
Arkwright
 
PD
Haque
 
T
, et al. 
Treatment of Epstein-Barr-virus-associated primary CNS B cell lymphoma with allogeneic T-cell immunotherapy and stem-cell transplantation.
Lancet Oncol
2005
, vol. 
6
 
5
(pg. 
344
-
346
)
3
Haque
 
T
Wilkie
 
GM
Taylor
 
C
, et al. 
Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells.
Lancet
2002
, vol. 
360
 
9331
(pg. 
436
-
442
)
4
Haque
 
T
Wilkie
 
GM
Jones
 
MM
, et al. 
Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial.
Blood
2007
, vol. 
110
 
4
(pg. 
1123
-
1131
)
5
Lewalle
 
P
Triffet
 
A
Delforge
 
A
, et al. 
Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study.
Bone Marrow Transplant
2003
, vol. 
31
 
1
(pg. 
39
-
44
)
6
Felix
 
NJ
Allen
 
PM
Specificity of T-cell alloreactivity.
Nat Rev Immunol
2007
, vol. 
7
 
12
(pg. 
942
-
953
)
7
Amir
 
AL
D'Orsogna
 
LJ
Roelen
 
DL
, et al. 
Allo-HLA reactivity of virus-specific memory T cells is common.
Blood
2010
, vol. 
115
 
15
(pg. 
3146
-
3157
)
8
Melenhorst
 
JJ
Leen
 
AM
Bollard
 
CM
, et al. 
Allogeneic virus-specific T cells with HLA alloreactivity do not produce GVHD in human subjects.
Blood
2010
, vol. 
116
 
22
(pg. 
4700
-
4702
)
9
Park
 
KD
Marti
 
L
Kurtzberg
 
J
Szabolcs
 
P
In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes.
Blood
2006
, vol. 
108
 
5
(pg. 
1770
-
1773
)
10
Hanley
 
PJ
Cruz
 
CR
Savoldo
 
B
, et al. 
Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes.
Blood
2009
, vol. 
114
 
9
(pg. 
1958
-
1967
)
11
Leen
 
AM
Myers
 
GD
Sili
 
U
, et al. 
Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.
Nat Med
2006
, vol. 
12
 
10
(pg. 
1160
-
1166
)
12
Micklethwaite
 
KP
Savoldo
 
B
Hanley
 
PJ
, et al. 
Derivation of human T lymphocytes from cord blood and peripheral blood with antiviral and antileukemic specificity from a single culture as protection against infection and relapse after stem cell transplantation.
Blood
2010
, vol. 
115
 
13
(pg. 
2695
-
2703
)
13
Serrano
 
LM
Pfeiffer
 
T
Olivares
 
S
, et al. 
Differentiation of naive cord-blood T cells into CD19-specific cytolytic effectors for posttransplantation adoptive immunotherapy.
Blood
2006
, vol. 
107
 
7
(pg. 
2643
-
2652
)
14
Gonzalez
 
S
Castanotto
 
D
Li
 
H
, et al. 
Amplification of RNAi-targeting HLA mRNAs.
Mol Ther
2005
, vol. 
11
 
5
(pg. 
811
-
818
)
15
Zakrzewski
 
JL
Suh
 
D
Markley
 
JC
, et al. 
Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors.
Nat Biotechnol
2008
, vol. 
26
 
4
(pg. 
453
-
461
)
16
Heslop
 
HE
Slobod
 
KS
Pule
 
MA
, et al. 
Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.
Blood
2010
, vol. 
115
 
5
(pg. 
925
-
935
)