Chapter 16: Cellular Therapy in Allogeneic Hematopoietic Cell Transplantation

The efficacy of allogeneic hematopoietic cell transplantation (HCT) in hematologic malignancies can in large part be attributed to a graft-versus-tumor (GVT) effect, by which the donor immune system achieves immunologic control of the tumor. As such, it is the prototype of cellular therapy. The human leukocyte antigen (HLA) system is fundamental to transplant biology. The HLAs are highly polymorphic proteins that have a key role in antigen presentation and immunoregulation. Class I HLAs are expressed on the surfaces of all nucleated cells; class II are expressed on specialized antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells.

Peptides derived from microbes are presented on class I HLAs to CD8⁺ T cells and result in immunologic destruction of infected cells; class II HLAs are recognized by CD4⁺ T cells. T-cell activation requires costimulatory signals from the APC, specifically CD80/86 binding to CD28 or LFA-3 binding to CD2 (¹). Absence of a costimulatory signal results in T-cell anergy, which is a key mechanism of peripheral immune tolerance to self-antigen in normal immunoregulation. Early posttransplantation, there is a “cytokine storm”; release of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), is induced by tissue damage from the conditioning regimen, activating the host innate immune system (Fig. 16-1). Donor T cells interact with host APCs and recognize foreign peptides; helper T cells produce further cytokines, especially IL-2, and prime host APCs via CD40:CD40L interaction. Differentiation of naïve donor T cells into effector cells subsequently occurs, resulting in immunologic attack on host tissues and the potential development of graft-versus-host disease (GVHD) (²). Increasing numbers of HLA mismatches are associated with higher incidence of GVHD and transplant-related mortality (TRM) (³). However, even in a fully HLA matched HCT, GVHD still occurs due to donor T cells directed against minor histocompatibility antigens (MiHAs), polymorphic peptides displayed on host HLA molecules.

There are three key barriers to successful HCT: GVHD, infectious complications, and relapse due to failure of immunologic control of the underlying disease. Herein, we review these issues in greater detail with an emphasis on recent cellular therapeutic approaches to address these complications.

Pathophysiology of the Graft-Versus-Tumor Effect

The GVT effect occurs due to a predominantly T-cell-mediated immunologic attack on tumor cells. In HLA-identical transplants, GVT effect is mediated by naïve T cells; for development of effector function, these must first be primed by host APCs. This requires the following: presentation of MiHAs or tumor-specific antigens on HLA; appropriate costimulatory molecules, including CD28, OX40, CD40L, and 41BB; and an appropriate “third signal,” provided by IL-12, interferon gamma (IFN-γ) or adjuvant (⁴). Restraining influences limiting the degree of immune activation are present to protect the host from an excessive immune response and include expression of CTLA4 (which competes with CD28 for binding to CD80/86) and programmed cell death protein 1 (PD1) and its interactions with its lig and PDL1, which limit T-cell activation and expansion during normal, pathogen-directed immune responses.

Soon after transplant, there is activation and expansion of MiHA-reactive T cells, followed by a decline, similar to that seen in pathogen-directed immune reactions. This may in part relate to the development of peripheral tolerance/anergy and also to replacement of host hematopoiesis, with resulting loss of host APCs. For tumor-associated antigen presentation to continue, there must be cross presentation on donor APCs. In addition, the initial alloresponse to MiHAs results in recruitment of T cells targeting either tumor-associated antigens or nonpolymorphic genes, which are either overexpressed or aberrantly expressed by the tumor (⁴).

Some tumor-associated antigens of importance include pathogenesis related protein (PR1), an epitope shared by proteinase-3 and elastase proteins, which is expressed in normal neutrophils and overexpressed in myeloid leukemias; and PR1-specific CD8⁺ T-cell responses, which can be detected in a range of myeloid and nonmyeloid malignancies post-HCT and correlate with outcome in chronic myelogenous leukemia (CML) (⁵). CD8⁺ T-cell responses to WT1, which is frequently overexpressed in acute myelogenous leukemia (AML), can be induced with vaccination strategies in both the autologous and allogeneic settings (^6,7,8). Clinical responses from tumor vaccines have thus far been suboptimal.

Natural killer (NK) cells can also mediate antitumor effects; this is discussed in further detail in this chapter.

Tumor Escape From Immunologic Destruction

Tumors utilize numerous mechanisms to escape immunological destruction, including the following:

• Induction of regulatory T cells

• Production of inhibitory cytokines

• Downregulation of costimulatory molecules (⁹) and HLA I (¹⁰)

• Induction of coinhibitory molecules

• Induction of myeloid-derived suppressor cells in the microenvironment that inhibit immune responses through multiple mechanisms.

• Invasion of immunologically privileged sites (¹¹).

Cellular therapeutic approaches are designed with the intent to abrogate these escape mechanisms.

Cellular Therapy to Induce Graft-Versus-Tumor Effect

Donor Lymphocyte Infusions

Donor lymphocyte infusion (DLI) may induce remissions in patients with molecular or overt relapse of their malignancy and can reverse CD8⁺ T-cell exhaustion. (¹²). However, the likelihood of success varies significantly according to the underlying disease. Chronic myelogenous leukemia is most sensitive; follicular lymphoma (FL) and Hodgkin lymphoma (HL) are also highly responsive (^13,14). Responses to DLI in AML or myelodysplastic syndrome (MDS) are less frequent, and durability is often poor. Acute lymphoblastic leukemia (ALL) is the least responsive to DLI. Reported response rates are 60% to 73% in CML, 15% to 29% in AML, and 0% to 18% in ALL (^15,16).

Problems and Challenges Associated With Donor Lymphocyte Infusion

Development of Graft-Versus-Host Disease

Graft-versus-host disease occurs in 40% to 60% of patients with HCT (^15,16) and is more likely to occur in unrelated donor recipients (¹³). Lympho-depleting chemotherapy given prior to DLI enhances alloreactive T-cell proliferation, potentiating the GVT effect but increasing GVHD (¹⁷). Approaches to reduce the GVHD incidence include the following:

1. Reducing T-cell dose. A dose-response relationship exists for both GVT and GVHD effects. In CML, no increased response is seen with cell doses greater than 4.5 × 10⁸ CD3⁺ cells/kg. In AML, response rates plateau beyond 1.5 × 10⁸/kg; higher doses increase GVHD. Gradual dose escalation schedules have been successfully utilized in relapses of indolent diseases. Follicular lymphoma appears highly sensitive to DLI after T-cell-depleted HCT, responding to low-dose DLI at doses from 1 to 10 × 10⁶/kg with less than 20% incidence of clinically significant acute GVHD (aGVHD) and chronic GVHD (cGVHD) (¹⁴). A similar strategy has shown success in HL, mainly in the setting of low-volume disease detected on surveillance positron emission tomographic (PET) imaging (¹⁸). More sensitive surveillance techniques, such as deep sequencing, to detect low-volume disease may allow earlier institution of DLI and maximize efficacy; utilization of these techniques remains experimental.

2. Transduction of donor lymphocytes with a suicide gene (¹⁹).

3. Selection of T cells to target tumor-associated antigens/antigens with restricted or differential expression (analogous to the use of viral-specific T cells [VSTs]). Infusion of MiHA-specific T cells was effective at eradicating tumor in mouse models (²⁰). In addition, tumor-infiltrating lymphocytes (TILs) recognizing tumor-associated antigens have been successfully used for selected metastatic solid tumors (²¹). However, the technology to isolate TILs remains restricted to a few specialized centers and is yet to be applied in large-scale clinical studies.

Marrow Hypoplasia

If there is insufficient residual donor hematopoiesis prior to DLI, eradication of host hematopoiesis by the infused lymphocytes can result in marrow aplasia; chimerism studies should therefore be performed prior to DLI to ensure adequate donor hematopoiesis (²²).

Delayed Onset of Action

Responses to DLI may not be seen for up to 2 months (¹¹). In indolent diseases (eg, FL), this may not be problematic, but in overt relapse of aggressive diseases (eg, AML), chemotherapy may be required first to achieve disease control (²³).

Prophylactic Donor Lymphocyte Infusion in High-Risk Patients

The delayed onset of action of DLI has led to the use of preemptive DLI to prevent relapse in high-risk patients. Mixed donor/recipient chimerism within the T-cell lineage is frequently seen in T-cell-depleted transplants and is associated with higher rates of relapse in CML (²⁴), FL (¹⁴), and HL (¹⁸), likely due to development of bidirectional tolerance with resulting tumor escape from immunologic control. Donor lymphocyte infusion can induce full donor chimerism in both FL and HL, and subsequent relapse rates are low; no formal comparison to similar groups not receiving DLI has been performed.

Bi-Specific T-Cell-Engaging Antibodies

The topic of bi-specific T-cell-engaging antibodies has been discussed in detail elsewhere. Bi-specific T-cell-engaging antibodies are single-chain antibodies that engage T cells via CD3 and direct them to an antigenic target present on tumor cells, typically CD19, resulting in T-cell redistribution, activation, expansion, and perforin-mediated killing of target cells (²⁵). Blinatumomab is highly efficacious in ALL in the setting of persistent MRD (²⁵), overt relapse, or refractory disease (²⁶).

Chimeric Antigen Receptor-Modified T Cells

The ideal cellular therapy for a malignant disease should expand and persist in vivo and selectively target cancer cells. This can be achieved by modifying autologous T cells with a chimeric antigen receptor (CAR). The CAR consists of a single-chain monoclonal antibody (scFv) targeted to a tumor-associated antigen, which is thus recognized in a major histocompatibility complex (MHC)–independent fashion (unlike unmodified T-cell-mediated GVT effect); the scFv is coupled via an extracellular hinge domain and transmembrane domain to an intracellular signaling domain, typically the CD3ζ chain (Fig. 16-2) (²⁷). Autologous T cells are collected from peripheral blood (PB) via a steady-state blood draw or apheresis procedure and transduced with the CAR construct via a lentiviral or retroviral vector or using electroporation and a transposon/transposase system (²⁸). Cells are cultured and expanded ex vivo using either CD3/28 beads (²⁹) or artificial APCs (³⁰) and specific cytokines prior to infusion. The persistence and clinical activity of CAR T cells (CAR-T) in vivo can be enhanced by the addition of a costimulatory molecule to the CAR construct (³¹), usually CD28 (^31,32). An initial report in a patient with highly refractory CLL treated with an anti-CD19 CAR-T utilizing CD137 (4-1BB) as the costimulatory domain generated great excitement (³³): Infused T cells expanded more than 3 log, the patient developed a cytokine-release syndrome (CRS) and tumor lysis syndrome (TLS), and achieved complete remission (CR); long-term persistence of CAR-T and persistent normal B-cell aplasia (a predictable, on-target effect when targeting CD19) were demonstrated.

Subsequent results in CLL have been heterogeneous; updated results from the University of Pennsylvania showed that 5 of 24 patients treated achieved durable CRs, 7 had partial responses (PRs), and 12 had no response (³⁴). The variables underlying response to treatment are not well understood, but in vivo CAR-T expansion is a prerequisite and appears to be more important than the dose of infused cells (³⁰). In ALL, results have been particularly impressive, with a CR rate of 86% in children treated for relapsed/refractory disease; patients were MRD negative even when tested with highly sensitive deep-sequencing techniques (³⁴). Long-term survival rates are not yet known, particularly as in many cases the treatment has been used as a “bridge to transplant.” At least two patients with ALL have relapsed with CD19-negative disease (³⁴). Similarly impressive CR rates have been reported by the groups at the National Institutes of Health (³⁵) and Memorial Sloane Kettering Cancer Center (³⁶). All three groups reported a similar toxicity profile (see Table 16-1). It is unclear whether this treatment can replace HCT in a proportion of patients; long-term survival outcomes in patients ineligible for HCT will be important in answering this question. The CAR-T targeting CD19 have now been used in a range of B-cell malignancies, with responses seen in both aggressive and indolent lymphomas, CLL, and ALL (Table 16-1).

While this therapy shows great promise, many aspects require optimization. Due to the heterogeneity in technique for CAR-T production, it is difficult to compare across trials to determine optimal manufacturing methods. The controversies are numerous: First, while it has been shown that addition of a costimulatory domain to the CAR enhances expansion and persistence (³¹), it is unclear whether CD28 or 4-1BB, or both, is superior. Second, lympho-depleting chemotherapy may enhance CAR-T expansion and persistence (³²), but the optimal drugs and schedule are unknown. Third, the epitope targeted by the antibody fragment is likely important, in terms of both the spatial location of epitope binding and binding affinity. Fourth, the hinge and transmembrane domains are important in determining interaction with antigen and formation of the immunologic synapse, but little is known about optimizing this aspect of the CAR design. Fifth, the method of T-cell transduction (viral vs transposon) may be important in determining efficacy. Sixth, the ex vivo culture technique and duration (eg, CD3/28 beads vs artificial APCs and which supplemental cytokines to provide) may be important; for example, culture after transduction using the Sleeping Beauty system is relatively prolonged, which is problematic in kinetically active diseases such as ALL (³⁷). The dose and composition (unselected vs specific ratios of CD4/8 cells) of T-cell product infused are also variables requiring consideration. Finally, the bulk of tumor present at the time of CAR-T infusion may potentially affect the in vivo proliferation of the infused CAR-T, and the ideal time of infusion (eg, in an MRD state vs overt relapse) has not been elucidated.

Adverse Events and Optimizing Safety

All responding patients have had some degree of CRS and have developed B-cell aplasia (³⁸). Cytokine-release syndrome is characterized by fever with variable systemic symptoms, including hypotension, and high levels of inflammatory cytokines, of which IL-6 appears particularly important (³⁷). Macrophage activation syndrome (MAS) may accompany CRS. Major neurological symptoms, including seizures, have occurred. The mechanism of neurological events is unclear; it may be cytokine mediated, associated with MAS, or due to direct CAR-T infiltration. Unexpectedly, CD19 CAR-T have been found in the cerebrospinal fluid of some patients without central nervous system disease (³⁷). Cytokine-release syndrome can be managed with the anti-IL-6 antibody tocilizumab, with prompt responses in the majority of patients (³⁷). Corticosteroids, while potentially efficacious in managing CRS/MAS, are toxic to the infused cells and may limit efficacy.

Unanticipated on-target toxicity may occur; for example, a toxic death occurred in a patient treated with a CART-T directed against Human Epidermal Growth Factor Receptor 2, or ErbB2, due to unanticipated low-level pulmonary epithelial expression (³⁹). The inclusion of a suicide gene within the CAR, such as inducible caspase 9 (iCaspase9), which could be triggered in the event of severe toxicity, would provide an added safety measure.

Most human trials to date have focused on CD19 as a target in B-cell diseases, but a number of novel targets show potential in different diseases.

Adoptive Transfer of Natural Killer Cells to Enhance Antitumor Effect

In contrast to T and B lymphocytes, NK cells do not express rearranged, antigen-specific receptors; rather, NK effector function is dictated by the integration of signals received through germ-line-encoded receptors that can recognize ligands on their cellular targets. Functionally, NK cell receptors are classified as activating or inhibitory. Natural killer cell function, including cytotoxicity and cytokine release, is governed by a balance between inhibitory receptors, notably the killer immunoglobulin-like receptors (KIRs) and the heterodimeric C-type lectin receptor (NKG2A), and activating receptors, in particular the natural cytotoxicity receptors (NCRs) NKp46, NKp30, NKp44, and the membrane protein NKG2D (⁴⁰). Inhibitory receptors bind to HLA class I molecules, expressed on the surface of normal cells, resulting in signals that block NK cell triggering and inhibit killing. In the setting of malignancy or viral infection, HLA class I is often downregulated or has altered peptide expression, resulting in failure of KIR-mediated recognition by the NK cell and resultant cell killing (⁴⁰). Activating receptors, such as NKG2D, bind ligands that are induced by cellular stress (eg, viral infection and malignant transformation); binding results in NK cell activation and target lysis (⁴¹) (Fig. 16-3).

Early NK cell recovery (within 30 days) postallogeneic stem cell transplant has been associated with reduced rates of both relapse and aGVHD, with resultant improved survival (⁴²). This dual benefit makes allogeneic NK cells an attractive option for adoptive cellular therapy peritransplant. Adoptive transfer of NK cells has previously been limited by the small numbers of circulating NK cells (5%-15% of the total lymphocytes) and consequently the low numbers obtained in an apheresis procedure (⁴³).

Use of allogeneic NK cells may be more efficacious than autologous NK cells due to inhibition of autologous NK cell activity by recognition of host HLA. Adoptive transfer of ex vivo–expanded haploidentical NK cells after lympho-depleting chemotherapy is safe. High-dose, but not low-dose, chemotherapy facilitates in vivo NK cell expansion, likely due to both prevention of host T-cell-mediated rejection and reduction in competition for cytokines, particularly IL-15. Persistence for at least 4 weeks has been achieved in some patients and responses have been observed in high-risk AML without inducing GVHD (⁴⁴). The NK cell expansion ex vivo has traditionally included culture with cytokines (IL-2 or IL-15) and cell selection (CD3 depletion) (⁴⁴); the use of “feeder cells” (Epstein-Barr virus [EBV]–transformed lymphoblastic cell lines or gene-modified, irradiated K562 cells), and large-scale expansion flasks have dramatically increased NK cell yield and activation status. Clinically relevant NK cell numbers can be obtained from both cord blood (CB) and adult donors (⁴³). The CB-derived NK cells show a similar phenotype and are similarly active against leukemic targets as PB-derived NK cells (⁴⁵).

There are several potential limitations of NK cell adoptive transfer, particularly limited persistence and the potential for passenger lymphocyte-related complications. The NK cells may rapidly develop exhaustion in vivo after adoptive transfer, despite initial expansion and activity (⁴⁶). In part, this may relate to NK cells’ exquisite sensitivity to cytokines such as IL-2 and IL-15. In vivo use of IL-2 (which can expand NK cell numbers) can lead to severe toxicity and to T-regulatory (T-reg) expansion, which limits NK cell activation (⁴⁷). In contrast, IL-15/IL-15Rα complexes promoted NK cell activation and enhanced function without the detrimental effects of IL-2 (⁴⁸). Whether in vivo use of IL-15/IL-15Rα will rescue NK cells from this phenomenon is not known. Lymphocyte contamination of the infused product can be avoided by proper selection techniques. T-cell contamination should be limited to less than 1 to 5 × 10⁵/kg (⁴⁹) to minimize the risk of GVHD; this can be achieved by CD3 depletion (⁴⁴). Addition of CD56⁺ selection reduces B-cell contamination to less than 1%, minimizing passenger B-lymphocyte-mediated EBV-posttransplant lymphoproliferative disorder (PTLD) and acute hemolytic anemia (⁵⁰).

Optimizing Natural Killer Cell Efficacy

The NK cells have a range of highly polymorphic KIRs, which are divided into inhibitory and activating subtypes. The KIRs are inherited as haplotypes (KIR-A and KIR-B). The KIR-A haplotypes, found in one-third of adult Caucasians, have one activating receptor, while the KIR-B haplotypes have 2 or more. Transplantation in AML from a KIR-B haplotype donor is associated with lower relapse rates and superior survival (⁵¹). Donor KIR2DS1 (an activating KIR) and recipient HLA-C type influence relapse risk. The KIR2DS1-associated reduction in the rate of AML relapse is restricted to donors with HLA-C1/C1 or C1/C2, in whom KIR2DS1-expressing NK cells are presumed to be “educated,” and the benefit was eliminated in transplants from donors with HLA-C2/C2, where KIR2DS1-expressing NK cells are expected to be tolerized in the setting of self HLA-C2 (⁵²). Selection of adult or CB donors for ex vivo NK cell expansion based on KIR genotype may therefore enhance NK cell efficacy.

Other future strategies to enhance NK cell tumor killing include the use of immunomodulatory drugs such as lenalidomide and the use of bi-specific killer engagers (BiKEs), which consist of a single-chain Fv against CD16 and a tumor-associated antigen. A CD16x33 BiKE has been shown to have activity in refractory AML (⁵³).

PD1/PDL1 Antibodies

Interaction of PD1/PDL1 induces T-cell dysfunction in CLL (⁵⁴). Anti-PD1 antibodies are efficacious in a subset of patients with metastatic solid tumors as monotherapy and in combination with the anti-CTLA4 antibody ipilimumab (⁵⁵). They have also shown remarkable activity in relapsed/refractory HL (⁵⁶).

The potential importance of immune checkpoints has been demonstrated in AML. CD8⁺ T-cell responses directed against the MiHA Liver receptor homolog-1 (LRH-1) have resulted in remission post-DLI. Despite persistence of CD8⁺ T-memory cells specific for LRH-1, subsequent relapse with LRH-1-positive blasts occurs, unaccompanied by LRH-1-specific T-cell expansion, suggesting anergy/functional impairment. LRH-1-specific T cells from patients with relapsed AML have elevated levels of PD-1. The addition of anti-PD1 antibody to a coculture system resulted in marked LRH-1-specific T-cell expansion, IFN-γ production and cytotoxicity, suggesting a specific inhibitory effect induced by the PD-L1/PD1 interaction (⁵⁷). Blockade of the PD-L1/PD1 axis may therefore represent an immunomodulatory target for patients with persistent/relapsed AML post-alloHCT.

Cellular Therapy for Prevention of Graft-Versus-Host Disease

Grades II-IV aGVHD occur in 25% to 60% of matched related donors and 45% to 70% of MUDs (⁵⁸). Corticosteroid-based therapy for grades II-IV aGVHD is unsatisfactory, with fewer than 50% of patients showing a durable complete response (⁵⁹). Increasing severity of aGVHD is associated with incremental TRM and inferior survival. Corticosteroid-refractory GVHD has a poor prognosis. Numerous additional agents have been studied in combination with corticosteroids or as second line therapies and showed uniformly poor response rates and numerous complications, particularly viral reactivation (⁵⁹). Prevention of GVHD is therefore of paramount importance.

T-Cell Depletion

A T-cell-replete transplant usually contains 1 to 5 × 10⁷ T cells/kg recipient weight (⁵⁸). T-cell depletion is the most potent method of preventing aGVHD but is associated with increased rates of graft rejection; delayed immune reconstitution; infectious complications, including EBV-driven PTLD; and increased relapse risk (⁵⁸). T-cell depletion can be accomplished ex vivo immunologically (T-cell antibodies, positive CD34 selection) or by the use of physical separation (eg, density gradients) or in vivo by the use of anti-T-cell antibodies such as antithymocyte globulin (ATG) or alemtuzumab. In vivo T-cell depletion with ATG reduces severe aGVHD and extensive cGVHD without increasing relapse but does not reduce TRM or improve survival (⁶⁰), likely due to increased infection risk. Alemtuzumab-based GVHD prophylaxis achieves low rates of severe aGVHD and extensive cGVHD, but results in high rates of mixed chimerism and viral infections, particularly cytomegalovirus (CMV) reactivation (⁶¹).

T-Cell Depletion With Planned Postengraftment Donor Lymphocyte Infusion Utilizing a “Suicide Gene” in the Infused T Cells

Transduction of donor T cells with a “suicide gene,” which can be activated in the event of developing severe aGVHD, may enhance the safety of adoptively transferred cellular therapy products. One method involves incorporating a herpes simplex virus thymidine kinase (HSV-TK) within the cellular product. Ganciclovir, a prodrug, is then administered, activated by HSV-TK and incorporated into replicating DNA, inhibiting DNA polymerase and resulting in cell death. Patients given HSV-TK-transduced DLI from haploidentical donors who developed GVHD had prompt resolution of GVHD after ganciclovir administration (⁶²).

A more recent technique involves T-cell transduction with an inducible human caspase 9 protein, modified to remove its endogenous caspase activation and recruitment domain and conjugated to a sequence of human FK-binding protein, which binds to an otherwise-inert dimerizing agent, AP1903. Dimerization of the iCaspase9 protein by AP1903 activates the intrinsic apoptotic pathway and induces apoptosis. A pilot study using this technique in patients undergoing haplo-HCT showed robust in vivo expansion of gene-modified T cells and 90% and 99% killing of transduced cells within 30 minutes and 24 hours of AP1903 administration, respectively (^19,63). Concurrently, manifestations of aGVHD in both skin and liver rapidly resolved. Intriguingly, alloreactive T cells were preferentially eliminated by the dimerizing agent, potentially due to greater expression of the transgene in these cells. There was relative sparing of antiviral T cells, with polyclonal CD3/19⁺ transduced T cells with antiviral specificity detectable within 1 to 2 weeks postadministration of AP1903.

Adoptive Transfer of Regulatory T Cells

The level of CD4⁺CD25⁺FOXP3⁺ T-reg cells in the graft and after HCT correlates inversely with aGVHD and cGVHD (⁶⁴). Adoptive transfer of T-reg cells could therefore ameliorate GVHD.

The most efficient method for obtaining a pure T-reg population is sorting based on flow cytometry. Some effector T cells will be present in the product if CD4⁺/CD25⁺(high) cells are selected. Removal of CD127 (IL-7R)-positive cells, which are not expressed on T-reg cells, achieves a more purified product (⁶⁵). Following sorting, T-reg cells can be expanded using CD3/28-coated microbeads and IL-2 and maintain suppressive function (^66,67). Murine xenogenic GVHD models have shown that infusion of human CB-derived T-reg cells confers protection from aGVHD and improves survival (⁶⁸).

Potential concerns with T-reg cell infusions relate to impairment of immune reconstitution and GVT effect. However, by inhibiting GVHD-mediated thymic destruction (⁶⁹), T-reg cells may actually facilitate functional immune reconstitution (⁷⁰). Indeed, immune function is preserved in murine models of T-reg cell infusion (⁷¹). Data from mouse models regarding impairment of GVT effect is contradictory. Tumor regression after IL-2–diphtheria toxin–mediated T-reg cell depletion was seen in one model, suggesting that T-reg cells may adversely affect disease control (⁷²).

In contrast, simultaneous adoptive transfer of T-reg cells and unselected donor T cells in a mismatched murine transplant model showed protection from GVHD without impairing tumor control (⁷³). Timing of adoptive transfer is likely important; efficacy may be greatest if infused peritransplant to limit initial alloreactive T-cell expansion rather than if infused to treat established aGVHD (⁷⁴). An early-phase clinical trial has shown posttransplant adoptive transfer of T-reg cells to be safe, with a reduction in risk of grades II-IV aGVHD relative to historical controls and similar disease-free survival (⁷⁵). In this study, there did not appear to be an excess risk of disease relapse or infection.

Delayed recovery of cellular and humoral immunity results in morbidity and mortality from infection. Figure 16-4 shows the approximate time course of numeric recovery of immune cells post-HCT; Fig. 16-5 shows the time course of infections.

Cellular Therapy for the Prevention and Treatment of Infectious Complications Postallogeneic Stem Cell Transplantation

Viral infection is a major cause of mortality post-HCT, resulting from cellular and humoral immune deficiency; risk factors include umbilical CB transplantation, T-cell depletion, and GVHD requiring systemic immunosuppression. Viral infections of particular relevance post-HCT are CMV, EBV, the polyoma virus BK, adenovirus (ADV), and human herpesvirus 6 (HHV-6). Pharmacotherapy for these infections has limited efficacy and substantial toxicity (^76,77). Consequently, adoptive immunotherapy for the treatment and prevention of viral reactivation/infection postallograft is attractive.

General Principles of Generating Viral-Specific T Cells

Generation of VSTs is most straightforward from an immune-experienced, adult donor who will have viral-specific memory T cells specific in their PB. The VSTs from such donors can be generated in two ways (⁷⁸):

1. T-cell coculture, in the presence of specific cytokines, with an artificial APC modified to express the immunodominant antigens of the target virus and costimulatory molecules. Rapid culture techniques using overlapping peptide pools rather than live virus have shortened culture time to 7 to 14 days.

2. Rapid selection strategies without ex vivo culture. These rely on the presence of sufficient numbers of VSTs in the donor PB and hence are limited to CMV and EBV (⁷⁶).

Generation of VSTs from immunologically naïve donors (eg, CB) is more challenging. However, multivirus-specific T cells against EBV, CMV, and ADV can be generated from naïve CB cells by genetically modifying EBV lymphoblastoid cell lines transduced with an adenoviral vector expressing the CMVpp65 transgene (⁷⁹). These are highly active against virus, despite recognition of noncanonical CMV and EBV epitopes (⁷⁹), and early clinical results are encouraging (⁸⁰).

Third-party, banked VSTs from adult donors could also be used to treat viral reactivation in patients without an available adult donor or in whom rapid disease progression precludes waiting for generation of VSTs from their donor. Suitable lines (which are dependent on the recipient expressing immunodominant viral peptides on an HLA antigen shared by a donor VST line) are available in approximately 90% of patients and can be rapidly identified and made available. In an early-phase clinical trial, these VSTs demonstrated high response rates in patients with refractory CMV, ADV, and EBV-related PTLD (⁸¹). Interestingly, despite the theoretical risk of inducing GVHD due to HLA mismatch, no severe cases of de novo aGVHD were seen in initial studies, and re-treatment was successful in several cases despite initial immunologic rejection of the transferred cells (⁸¹).

Five virus-specific T cells (EBV, CMV, ADV, BK, and HHV-6) can now be produced from adult donors after culture with a peptide mix containing immunodominant antigens of the FIVE viruses (⁸²).

Treatment of Specific Infections With Viral-Specific T Cells

Epstein-Barr Virus Reactivation and Posttransplant Lymphoproliferative Disorder

Posttransplant lymphoproliferative disorder related to EBV occurs in the setting of severe transplant-related immunosuppression when the EBV-specific T-cell response is insufficient to control latent EBV infection within recipient or donor B cells. The biology and pathology of EBV-related PTLD have been reviewed previously (⁸³). Risk factors for infection predominantly relate to the degree of immunosuppression in recipients of T-cell-depleted transplants. Patients typically present with high fever and lymphadenopathy, with an elevated serum lactate dehydrogenase (⁸³).

Initial therapy for EBV reactivation is reduction in immunosuppression (⁸³). However, there are no randomized studies to guide the best therapy of established EBV-associated PTLD. In patients with frank PTLD, the largest study (⁸⁴) showed a 70% CR/CRu rate with four weekly doses of rituximab monotherapy, but there were poor responses to subsequent treatment (chemotherapy, DLI, or both) in nonresponders. Chemotherapy is associated with greater toxicity in HCT recipients and may increase infection risk.

Cellular therapy shows great promise in the management of EBV-related PTLD. Unmanipulated donor T-cell infusions can control established PTLD in approximately 70% of patients but can induce severe or fatal GVHD (⁸⁵). Therefore, when available, VSTs are preferred. The EBV-specific or polyvirus-specific cytotoxic T lymphocytes (CTLs) can be generated as described previously and have proven successful in overt PTLD in over 80% of patients, including patients with rituximab-refractory disease (⁷⁶). Figure 16-6 demonstrates a dramatic clinical response in a patient with chemorefractory PTLD to infusion of EBV-specific CTLs.

Cytomegalovirus Infection

Risk factors for CMV reactivation and disease post-HCT include receiving umbilical CB grafts, T-cell-depleted grafts, T-cell antibody therapy for GVHD prophylaxis, or high-dose steroids (⁸⁶). In addition, CMV-seropositive patients with seronegative donors are at particularly high risk due to the lack of memory T cells against CMV from the seronegative donor (⁸⁶). While ganciclovir prophylaxis reduces the risk of CMV disease, it prolongs neutropenia, increases invasive bacterial and fungal infections, and does not improve survival (⁸⁶). Close monitoring for CMV reactivation in blood, followed by preemptive therapy with ganciclovir when assays in blood become positive, reduces the incidence of CMV disease. Overall, rates of CMV disease have declined from 30% to 35% to 8% to 10% with ganciclovir prophylaxis or preemptive therapy (⁸⁶). Gastrointestinal disease, pneumonia, and retinitis are the most common manifestations; pneumonia has a high mortality despite treatment with ganciclovir or foscarnet and CMV-immunoglobulin (⁸⁶). Given the toxicity and expense associated with pharmacological interventions and their imperfect efficacy, cellular therapy as treatment or prophylaxis of CMV in high-risk patients is attractive.

Adenovirus Infection

Adenovirus infection occurs in up to 21% of transplant recipients, with manifestations of adenovirus disease in 20% to 89% of infected patients (⁸⁷). Four clinically significant syndromes are seen: pneumonitis, nephritis, hemorrhagic colitis, and hemorrhagic cystitis. Disseminated disease with multiorgan failure also occurs and has a poor outcome despite antiviral therapy; most successfully treated cases are respiratory or urinary tract infections. The ADV-specific T cells can now be generated from both CB (^79,80) and adult donors (⁸⁸), with a high clinical response rate in cidofovir-refractory cases. Rapid isolation strategies are not applicable for generation of ADV-specific CTLs due to the low numbers of circulating ADV-specific T cells in donor blood.

BK Virus Infection

BK virus reactivation occurs in 5% to 68% of hematopoietic stem cell transplantation recipients. It can cause severe hematuria, urinary obstruction, renal failure, and increased mortality. It is more frequent in patients with grades III and IV aGVHD and Umbilical cord blood transplant (UCBT) recipients (⁸⁹). Pharmacologic therapy is toxic and poorly efficacious. BK virus-specific CTLs can be generated from PB, but rapid overnight generation is not possible due to the low frequency of BK virus-specific T cells in PB; hence, culture with peptide mix or APCs is required for generation.

Human Herpes Virus 6 Infection

Infection with HHV-6 is virtually universal before age 2 years (⁹⁰). Reactivation occurs in more than 50% of allograft recipients and can result in encephalitis, delayed engraftment, and increased rate of GVHD, with increased mortality (⁹⁰). Production of HHV-6 VSTs using a peptide mix and 10-day culture with IL-4 and IL-7 is possible from adult donors with prior exposure (⁹⁰). Production of HHV-6-specific VSTs has yet to be performed from CB, and rapid isolation methods are not possible given the low frequency of VSTs in blood. Clinical studies of five virus-specific CTLs (EBV, CMV, ADV, BKV, and HHV-6) are ongoing (NCT 01570283).

Allogeneic HCT, initially performed in a twin patient with leukemia by Dr. E. Donnall Thomas in the late 1950s, was one of the first elegant demonstrations of the power of cellular therapy. Much has been learned since then, and our increased understanding of the immune system has translated into exciting new therapeutic approaches, especially relevant to transplantation, leading to continued better outcomes for patients.