Cellular Therapies in Transplantation – Regulatory T Cell Therapies and Virus Specific Therapies

Abstract

Purpose of Review

Cellular therapies have shown great promise in enhancing immune tolerance and managing opportunistic infections in transplant recipients. This review explores the latest advancements in regulatory T cell (Treg) and virus-specific T cell (VST) therapies in solid organ transplantation.

Recent Findings

Treg-based therapies, including polyclonal Tregs, donor antigen-reactive Tregs (darTregs), and chimeric antigen receptor Tregs (CAR-Tregs) are being studied to minimize conventional, systemic immunosuppression while preventing graft rejection. Clinical trials demonstrated the safety and feasibility of ex vivo-expanded Tregs in kidney and liver transplantation, supporting reduced rejection rates and lower infection risks. The clinical applicability of CAR-T cell therapies extends to autoimmune diseases. Additionally, VSTs targeting BK virus, cytomegalovirus, Epstein-Barr virus, and adenovirus offer a novel approach for refractory viral infections in transplant recipients. Advances in third-party, “off-the-shelf” and multi-VSTs allow faster availability and standardized, scalable manufacturing compared to conventional VSTs.

Summary

By reducing dependence on conventional immunosuppression, cellular therapies provide a promising approach in transplantation. To establish their role in clinical transplantation, further research is needed to optimize dosing and manufacture, improve antigen specificity, and address long-term safety concerns.

Introduction

Advancements in organ transplantation have significantly improved patients’ outcomes over the past decades. Kidney transplantation offers better survival, quality of life, and cost effectiveness to patients with end-stage kidney disease (ESKD) compared to dialysis [1, 2]. While life-long immunosuppression is necessary to prevent rejection and improve allograft survival, complications associated with prolonged immunosuppression remain a significant clinical concern. The accumulating burden of immunosuppression in transplant recipients results in an increased risk of infection and cancer. Reduction of immunosuppression may help treat infection and cancer, but it can increase the risk of graft rejection, which casts a substantial clinical conundrum. Given these challenges, there is growing clinical data for cellular therapies to enhance tolerance and treat viral infections.

Unlike conventional immunosuppressive drugs, cellular therapies offer greater specificity in modulating immune responses. Regulatory T cells (Tregs) and regulatory chimeric antigen receptors T cells (CAR-Tregs) can be utilized to promote allograft tolerance by targeting allospecific antigens, enabling minimization of conventional immunosuppression, thereby decreasing the risk of infection and cancer while preventing graft rejection. In addition, virus-specific T-cell therapies (VSTs) can target specific pathogens that arise due to chronic immunosuppression and are often refractory to conventional antiviral therapy. These therapies represent an emerging and promising approaches to promote graft tolerance and treating opportunistic infections in transplant recipients. This review aims to examine the current clinical data on the use of cellular therapies to enhance graft tolerance via Tregs and mitigate immunosuppression-related complications via VSTs, while exploring potential applications beyond transplantation.

Polyclonal Treg Therapy

T regulatory cells (Tregs) were discovered as a subset of CD4 + T cells that exhibit immune suppressive function [3]. Foxp3 is a transcriptional master regulator of Treg development, and Foxp3 deletion leads to severe autoimmune disorder both in humans and mice [4, 5]. Tregs suppress immune responses through various mechanisms including the secretion of anti-inflammatory mediators, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), and the expression of co-inhibitory molecules such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) [6]. Through these mechanisms, Tregs elicit antigen-specific suppression of effector T cells and create tolerogenic immune microenvironment. Peripheral Treg activity was shown to be reduced in various autoimmune conditions and acute allograft rejections [7]. Moreover, Tregs mediate immune suppression not only against a specific donor antigen but also against other antigens present in the same transplanted organ, a phenomenon known as linked suppression. This process helps extend tolerance beyond the initially recognized antigen, promoting graft acceptance and immune regulation [8]. Thus, Tregs have been thought to be a promising target for achieving immunological tolerance in autoimmune disease and transplantation (Fig. 1).

Fig. 1.

Polyclonal Treg, darTreg, and CAR-Treg therapies. Several approaches have been developed for regulatory T cell (Treg)-based therapies. First, blood is collected from the patient. Magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) can be used for Treg isolation. Treg markers used for isolation include CD4⁺, CD25⁺, CD127^low. Isolated Tregs are cultured with a combination of stimulatory signals, including anti-CD3/CD28 antibodies, interleukin-2 (IL-2), and rapamycin. This approach leads to (1) polyclonal Tregs. To generate donor-antigen reactive Tregs (darTregs) (2). Isolated Tregs are co-cultured with donor-specific APCs and further expanded. The last approach involves engineering Tregs expressing chimeric antigen receptors (CARs) (3) through retroviral or lentiviral transduction. CARs specific for HLA-A2 and CD19 are being studied in transplantation [9, 10]. CARs specific for drivers of autoimmunity, such as insulin β chain 10–23 peptide in autoimmune diabetes and myelin oligodendrocyte glycoprotein in multiple sclerosis, have shown promise in the management of autoimmune conditions. Abbreviations: Treg = T regulatory cell, CAR = chimeric antigen receptor, darTreg = donor-reactive antigen T regulatory cell, MACS = Magnetic-activated cell sorting, FACS = fluorescence-activated cell sorting, IL-2 = interleukin-2, MOG = myelin oligodendrocyte glycoprotein, MHC = major histocompatibility complex. (Created in BioRender)

In Vivo Treg Expansion by Low Dose IL-2: Clinical Challenges

Several clinical approaches have been used to enhance Treg function and quantities. In vivo expansion of Treg cells by administration of low dose interleukin 2 (IL-2) was one of the first strategies undertaken. Taking advantage of the expression of high-affinity IL-2 receptors on Tregs [11], low dose IL-2 administration [12] successfully expanded Treg in vivo in multiple diseases, such as hepatitis C-associated vasculitis and graft-versus host disease (GvHD) [13, 14]. While low dose IL-2 has shown some clinical efficacy in the initial trials and systemic lupus erythematosus (SLE) [14, 15], this approach was unable to promote tolerance in transplant recipients despite increasing circulatory Tregs [16–18]. It turned out that low-dose IL-2 led to unintended expansion of effector T cells and natural killer (NK) cells, which hampered the tolerogenic effect of expanded Tregs, making the IL-2-mediated approach clinically challenging due to its narrow therapeutic window.

More recently, engineered mutant IL-2 (IL-2 muteins) have been used in pre-clinical models and clinical trials. IL-2 muteins carry targeted mutation in IL-2 molecule to enhance its affinity to IL-2 receptor alpha chain (CD25) and lower its affinity to IL-2 receptor beta chain (CD122), to achieve more selective expansion of Tregs compared to effector T cells or NK cells. Success in mouse type 1 diabetes (T1D) model and mouse transplant models are encouraging [19, 20].

Ex Vivo Expanded Polyclonal Treg Clinical Trials

Ex vivo expansion of polyclonal Tregs starts with obtaining Tregs from either peripheral blood mononuclear cells (PBMC) via leukaphereses or other sources (e.g. cord blood or pediatric thymus), followed by magnetic or flow cytometry-based enrichment. Isolated Tregs are then expanded ex vivo with CD3/CD28 beads (or artificial antigen presenting cells; K562 CD64/CD86 and anti-CD3 [21]), in the presence of high concentration of IL-2, with or without rapamycin (Fig. 1) [22]. Good Manufacturing Practice (GMP) compliant ex vivo polyclonal expansion has been used in several clinical trials including Regulatory Cell Therapy in Kidney Transplantation (The ONE Study) consortium [23–25]. Despite the initial concerns on in vivo persistence after infusion, adoptively transferred polyclonal Tregs were detectable even a year after infusion, as demonstrated in a clinical trial involving type 1 diabetes patients, by tracking a deuterium (²H)-loaded Treg product [26].

Polyclonal Treg adoptive cell therapy (ACT) was first tested in a clinical setting to prevent GvHD post-hematopoietic stem cell transplantation (HSCT) [27, 28]. Autologous, ex vivo-expanded Treg cell therapies have been trialed in solid organ transplant recipients as part of tolerance induction (Table 1). In the TReg Adoptive Cell Therapy (TRACT) trial, autologous, polyclonally expanded Tregs were administered to 9 kidney transplant recipients (KTRs) after 60 days from transplantation [29]. Alemtuzumab (anti-CD52 antibody) was used for induction, and tacrolimus and mycophenolate were used as perioperative immunosuppression. To enhance Treg survival as well as lineage and functional stability, tacrolimus was later switched with sirolimus prior to Treg infusion [30, 31]. Doses up to 5 × 10⁹ cells were safe and tolerable. Treg proportion was sustained 12 months post-transplant, and no clinical allograft rejections were observed after two years. Fueyo et al. tested autologous Tregs in 9 deceased liver transplant patients [32]. Before Treg infusion, tacrolimus levels were decreased to 2–5 ng/mL and sirolimus was added to target a level of 5–8 ng/ml. None of the patients developed rejection during the follow-up period. Of note, Treg collection was possible even after transplantation and in the presence of immunosuppression. Among all trials involving autologous, polyclonal Tregs (Table 1), none experienced graft loss, increased risk of infection, or de novo cancer at one year of follow-up [29, 32–34].

Table 1.

Treg adoptive cellular therapy published trials in solid organ transplantation

Trial	Population	Follow-up	Treg product and dose	Manufacturing process	Induction	Results	Safety
Todo, 2016 [35]	10 living-donor liver transplant Patients	3–5 Years post-tx	Non-isolated, donor-specific 3.39 × 106 Tregs/kg	Co-culture with irradiated donor cells and anti-CD80/86 mAB	CP	(7/10) patients successfully underwent weaning and completed cessation of IS at 18 months	(3/10) patients developed acute cellular rejection during weaning.
Chandran (TASKp), 2017 [33]	3 kidney transplant patients	1 year post-infusion	Isolated, polyclonal autologous 3.20 × 106 Tregs/kg	Stimulated with anti-CD3, anti-CD28, IL-2, and deuterated glucose	BX	(3/3) had graft survival at 1 year (1/3) developed acute cellular rejection + DSA	(1/3) developed leukopenia
Mathew (TRACT), 2018 [29]	9 living donor renal transplant patients	2 year post-tx	Isolated, polyclonal autologous Tregs Three dosages per 3 recipients (0.5 × 109), (1 × 109), and (5 × 109)	Stimulated using anti-CD3, anti-CD28, IL-2, TGFβ, and Rapamycin	AZ	(9/9) graft survival at 2 years	(1/9) sublclinical biopsy-proven rejection at 1 year due to noncompliance (1/9) developed DSA at 1-year post-tx similar clinical and safety profiles to reference group
(ONE) Roemhild, 2020 [34]	11 living donor kidney transplant recipients	3 years post-tx	Isolated, polyclonal three dosages per group: 0.5–3 × 106 Treg/kg	Stimulated with anti-CD3, anti-CD28, IL-2, and Rapamycin	None	(11/11) Treg group threeyear allograft survival (8/11) Treg group successfully weaned off triple IS to monotherapy with TAC
Sanchez Fueyo, 2020 [32]	9 deceased liver transplant recipients	1 year post-infusion	Isolated, polyclonal 1 × 106 Tregs/Kg (n = 3) or 4.5 × 106 Tregs/Kg (n = 6)	Stimulated with anti-CD3, anti-CD28, IL-2, and Rapamycin	ThymoG (Later stopped)	(9/9) did not have rejection during follow-up period	(1/9) experienced an infusion reaction, classed as a dose-limiting toxicity
Koyoma, 2020 [36]	16 HLA mismatched living kidney transplant recipients	7-year post-tx	Non-isolated, donor-specific, 3.39 × 106 Tregs/kg	cocultured with donor cells in the presence of anti-CD80/CD86 mAb	CP + SP (A); CP + RX (B); RX + rATG (C)	(7/16) developed acute rejection during first year	No Treg-related significant adverse events
(ONE) Harden, 2021 [37]	12 living kidney transplant recipients	4 years post-tx	Isolated, autologous 1–10 × 106 Treg/kg	Stimulated with anti-CD3, anti-CD28, IL-2, and Rapamycin	None	(12/12) AR-free survival - Treg group vs. (16/19) - reference group at 48 months post-Tx (4/12) successful weaning off IS and left on TAC monotherapy	Similar clinical and safety profiles to reference group
(ARTEMIS) 2022 [38]	5 liver transplant recipients	1-year post infusion	100 × 106 isolated, autologous donor-specific Tregs	Stimulated with anti-CD3, anti-CD28, IL-2.		“2 participants reached the primary endpoint (reduction of CNI by 75%)	No adverse events
(ONE) Guinan, 2023 [39]	3 living kidney transplant recipients	6–8 years post-tx	Isolated, donor-antigen reactive autologous 0.86–1.9 × 104 Treg/kg	Stimulated with irradiated donor PBMCs and belatacept	None	(3/3) successful weaning to TAC monotherapy.	No SAE related to IS vs. 3 in the control group
(TWO) Brook, 2024 [40]	7 kidney transplant recipients	1.5 years post Tx	5–10 × 106 cells per kg isolated, autologous, cryopreserved Tregs	Stimulated with anti-CD3/anti-CD28, rapamycin, and IL-2	AZ	3/3 patientson TAC + MMF had successful weaning to TAC monotherapy from

Abbreviations: Tx = transplant; Tregs = T regulatory cells; PBMCs = peripheral blood mononuclear cells; IL-2 = interleukin-2; mAB = monoclonal antibodies; TGFβ = transforming growth factor-beta; IS = immunosuppresion; MMF = mycophenolate mofetil; TAC = tacrolimus; AZ = Alemtuzumab; CP = cyclophosphamide; RX = Rituximab; rATG = rabbit anti-thymocyte globulin; CNI = calcineurin inhibitor; SAE = serious adverse event; VST = viral specific therapy; DSA = donor specific antibodies

The recent ONE study included trials from 8 international centers that demonstrated the safety and preliminary efficacy of polyclonal Treg cellular therapy in KTRs when compared to standard of care with a median follow-up of 60 months [23]. In the Treg arm, mycophenolate mofetil (MMF) was tapered off while maintaining low-dose tacrolimus. While previous studies have used induction therapy, no induction was used in the Treg arm compared to basiliximab in the control group. The choice of using lymphocyte depleting induction regimens remains an open question. Despite the reduced immunosuppression in patients receiving Treg cellular therapy, there was no difference in biopsy-proven rejection, but were six times less likely to have viral infections.

The Transplantation Without Overimmunosuppression (TWO) study plans to evaluate the efficacy of autologous Treg ACT with alemtuzumab induction in a phase 2b randomized controlled trial [41]. Due to the COVID-19 pandemic, the TWO protocol was amended and alemtuzumab was substituted with basiliximab. The results of 7 KTRs from the TWO study before its amendments were recently published [40]. The Treg arm included 3 patients receiving alemtuzumab induction, followed by tacrolimus and MMF at week 12. MMF was stopped at 6 months 72 h before Treg infusion was given. The three patients had successful weaning of immunosuppression to tacrolimus monotherapy. Patient and transplant survival was 100% at 18 months posttransplantation. Despite challenges in production, clinical trials have shown that polyclonal Treg therapy is feasible and permits minimizing conventional immunosuppression. Additionally, Treg-based approaches may reduce viral infections while maintaining graft stability. Though, longer follow-up is needed to confirm durable tolerance.

Safety

Using ex vivo expanded polyclonal Tregs carries potential safety concerns. While cellular therapy-associated adverse events such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were not observed in clinical trials involving polyclonal Tregs in transplantation [42], Tregs’ plasticity to convert to conventional T cells poses potential safety concerns that adoptively transferred Treg could precipitate allograft rejection in transplant recipients. Exposure to chronic or acute inflammation can lead to epigenetic silencing of Foxp3 in some Treg cells, allowing the release of proinflammatory cytokines [43]. Some groups attempted to introduce Foxp3 into CD4⁺ T cells [44, 45]. In contrast, others engineered CD4⁺ cells to express synthetic Notch (synNotch) regulatory circuits to induce a local suppressive effect similar to Tregs but with better cellular stability [46]. Currently, safety measures can be taken in many products using suicide genes and surface proteins that can manage unstable Treg cells if they were to develop [47–49]. Overall, Treg-based therapy has demonstrated a favorable safety profile, with no cases of CRS, ICANS, allograft loss, or increased risk of infection at one-year follow up. Strategies such as Foxp3 gene engineering, synNotch circuits, and safety switches can help mitigate safety concerns regarding Treg plasticity.

Improving Treg Specificity

Only a fraction of ex vivo expanded polyclonal T cells are donor-specific, raising potential efficacy concerns and the possibility of off-target suppression. Improved alloantigen specificity of Tregs can enhance its efficacy and reduce the potential risk of off-target effects [50]. Antigen specificity plays a key role in transplant tolerance and autoimmune diseases. In transplantation, several studies have shown that alloantigen-specific Tregs can also suppress immune responses against other unrelated antigens that are co-presented on the same antigen presenting cells (APCs) [8, 51, 52]. This phenomenon was first demonstrated when mice that had developed tolerance to allograft from one strain were later transplanted with allograft from another strain that carries shared antigens, it led to prolonged acceptance of the new allograft through linked suppression [53]. This tolerizing potential of a single alloantigen, which then achieve non-responsiveness against other antigens present within the graft, gives us rational to target single- or oligoalloantigens as a strategy to achieve allograft tolerance.

The ability to enhance Treg antigen specificity involves several approaches. Some studies expanded donor-antigen reactive Tregs (darTregs) by co-culturing Tregs with transplant donor cells (Fig. 1). Todo et al. conducted the first trial to examine darTregs in 10 living donor liver transplant recipients [35]. T cells obtained from recipients’ peripheral blood were co-cultured with irradiated donor lymphocytes in the presence of anti-CD80 and CD86 antibodies. The goal was to start weaning tacrolimus and cyclosporine at 6 months to reach a complete withdrawal of immunosuppression at 18 months. Only 7 out of 10 patients reached the goal. Koyama et al. used a similar culturing technique to produce darTregs for KTRs [54]. However, 7 out of 16 patients developed acute rejection during the first year. In this study, post-transplant immunosuppression between three groups was similar: cyclosporine, MMF, and methylprednisolone, whereas induction regimens differed: cyclophosphamide (CP) plus splenectomy (A), CP plus rituximab (B), and rituximab plus rabbit anti-thymocyte globulin (C). Two groups that didn’t include anti-thymocyte globulin developed higher rates of rejection, which might be due to insufficient T-cell suppression during induction [54, 55].

Autologous darTregs were also used on 5 living-donor kidney transplant recipients in the ONE study. The Boston and San Francisco groups successfully weaned 5 living-donor kidney transplant recipients to tacrolimus monotherapy, after infusion of ex vivo-expanded Tregs by co-culturing with irradiated donor PBMCs [23]. Yet, optimal conditions for culturing and expansion are still to be determined. While darTreg infusion seems clinically well tolerated, it requires access to the donor cells, long ex vivo expansion duration, and the presence of HLA mismatches [56]. Despite the long processing time of darTregs, their increased potency enabled us to minimize the number of cells needed for maintaining efficacy. darTregs enriched by alloantigen stimulation were superior in preventing GvHD and graft rejection through alloantigen-specific suppression and were approximately 100-fold more potent when compared to polyclonally cytokine-expanded Tregs [57]. It is notable that these darTregs expanded ex-vivo against donor cells work primarily through direct allorecognition mechanisms. However, as time passes after transplant, indirect allorecognition will predominate and darTregs might lose effect over time. Strategies to generate darTregs with indirect allorecognition might pave the way for more durable effects of alloantigen-specific tolerance.

Chimeric Antigen Receptor (CAR)-Treg Adoptive Cellular Therapy

While darTreg therapy remains a promising approach, it is currently saddled by the need for generation of individualized cell product. A potential alternative cell therapy involves the use of CAR-T cells, which was initially developed in the field of oncology. The traditional method of CAR-T therapy involves leukapheresis and enrichment of autologous T cells from the patient, followed by genetic engineering to express CARs. First generation CAR-T therapies were developed in the 1990s has an extra-cellular antigen binding domain and intracellular CD3 signaling domain. Due to limited in vivo proliferation and persistence, the first-generation CAR-T cells demonstrated limited clinical efficacy. To overcome this challenge, Krause et al. used the intracellular costimulatory domain of CD28 to enhance survival and proliferation of T cells against tumor antigens [58]. These second generation CARs, which have an intracellular costimulatory domain, such as CD28, 4-1BB, or OX40 [59], have shown better efficacies. However, the third generation CARs, with two tandem costimulatory domains, were not approved for clinical use due to increased toxicity such as CRS and ICANS [60, 61]. Modified CAR-T cells that express key cytokines or caspase-9 suicide genes became the fourth generation CARs or T cells redirected for universal cytokine-mediated killing (TRUCKs). The use of CAR-T cell therapies revolutionized the treatment of B-cell lymphomas and multiple myeloma. To this date, six 2nd-generation CAR T cell products targeting either CD19 or B cell maturation antigen have been FDA-approved for the treatment of B-cell lymphomas and multiple myeloma [62–67].

Advancements in CAR-T cell therapy and gene editing paved the way for designing CAR constructs for Tregs. While CAR-T therapy is engineered as effector T cells to eliminate target cells by cytotoxicity and pro-inflammatory cytokines, CAR-Treg promotes regulatory immune responses in an HLA-independent manner to help prevent immune pathology. CAR-Tregs are produced by isolating Tregs (CD4 + CD25hi) by magnetic beads or flow cytometry, activating them with CD3/CD28 (sometimes in the presence of sirolimus and IL-2 to maintain higher purity of Tregs), and transducing them with a second-generation CAR. CAR-Tregs were shown to be more effective than polyclonal Tregs in various preclinical studies involving GvHD, skin allografts, and heart transplants [9, 68, 69]. It is remarkable that CAR-Treg with single antigen specificity towards HLA-A2 could provide effective tolerance in solid organ transplant models that involve multiple alloantigens. One can speculate that infectious tolerance, a mechanism where tolerance to one antigen is spread to other antigens, may play a role. CAR-Treg to one antigen (e.g. HLA-A2) could create tolerogenic immune microenvironment in allograft draining lymph nodes and allograft itself, and might promote tolerance against other alloantigens. Currently, a multicenter trial (Safety and Tolerability Study of Chimeric Antigen Receptor T-Reg Cell Therapy in Living Donor Renal Transplant Recipients (STEADFAST)) is evaluating the safety of CAR-Treg therapy targeting HLA-A2, the most frequent HLA class I allele, in 25 living-donor KTRs [70]. While CAR-Tregs are a promising frontier, there is a room to improve their stability and efficacy. For example, CAR-Tregs are not yet able to induce tolerance when animal recipients were pre-sensitized to donor antigens [71].

CAR-Tregs offer a promising approach for autoimmune diseases as well. A recent study explored CAR-Tregs, specific for insulin β chain 10–23 peptide which is present on islet cells of non-obese diabetic mice. The study demonstrated CAR-Treg’s ability to prevent autoimmune diabetes in preclinical models [72]. Another approach involves depleting autoreactive T cells using CAR-T cells. Eliminating peptide-reactive CD4 + T cells in murine model of autoimmune encephalomyelitis was possible using peptide-MHC-II CAR-T cells [73]. These CAR-T cells successfully prevented disease onset and reversed ongoing neuroinflammation. Similar approaches have shown efficacy in rheumatoid arthritis and autoimmune diabetes in preclinical models [74, 75]. These findings underscore the potential of CAR-cell therapies in treating a range of autoimmune diseases and transplant tolerance.

Despite the promising preclinical data, broad implementation of CAR-Treg therapy is limited by production costs, HLA variability, and prior sensitization. Researchers aim to modulate CAR constructs to potentiate the phenotypic stability and efficacy of CAR-Treg and Treg ACT. For example, introducing the sequence encoding IL-10 increases its ability to suppress effector T cells in vitro, though suppression was not sufficient in vivo [76]. Recently, Henschel et al. explored HLA-A2 specific CAR-Tregs that co-expressed Foxp3 [77]. This strategy led to improved stability and survival of CAR-Tregs under inflammation and IL-2-deprived conditions. Moreover, eliminating endogenous TCR expression may help control CAR-Treg specificity. Advances in synthetic antigen receptor platforms now enable precise targeting of Treg activity [78]. Technologies like universal CARs (UniCARs) allow flexible antigen selection, while synNotch receptors link antigen recognition to transcriptional outputs, allowing Tregs to become active only within target (e.g. allograft) tissues [79].

Virus-specific T Cell Therapies in Immunocompromised Hosts

Opportunistic infections are serious complications postsolid organ transplant. BK virus (BKV), cytomegalovirus (CMV), adenovirus (AdV), and Epstein Barr Virus (EBV) are common viral infections that have a clinical impact on transplant patients. While some but not all opportunistic viral infections can be treated by conventional antiviral treatment, it can be limited due to resistance, toxicity, and limited efficacy. For example, CMV viremia or CMV diseases can be treated with valganciclovir/ganciclovir or maribavir. In severe cases of BK virus infections, cidofovir, brincidofovir, and intravenous immunoglobulins are used with limited efficacy. However, valganciclovir is often associated with cytopenia, and cidofovir can cause nephrotoxicity. To overcome these limitations of antiviral agents, virus-specific T cell (VST) therapies are emerging as a treatment option for some viruses, such as BKV, AdV, EBV, and CMV (Table 2) [80–84].

Table 2.

Viral specific therapy clinical trials in transplantation

Clinical trial No	Author & Year	Phase	Patients and characteristics, n	Indication	Targeted Viruses	Source	Results	Safety (GvHD, CRS, Death)
	Haque 2007 [85]	Phase II	31 SOTRs and 2 HSCT recipients with PTLD	EBV-PTLD	EBV	Third-party 2 × 106/kg	64% ORR at 5w 52% ORR at 6 m 76% overall survival at 6 m	None
NCT01070797	Gerdemann 2013 [86]	Phase I	10 HSCT	CMV, EBV, AdV post-transplant infections	CMV+EBV+AdV	Allogeneic donor-derived 0.5-2 × 107/m2	6 m 8/10 virological response	1 (G1) skin GvHD
NCT00711035	Leen 2013 [87]	Phase I/II	58 HSCT recipients	Drug-resistant viral infections	CMV+EBV+AdV	Third-party 2 × 107/m2	ORR 95% at 6w median viral load reduction of 97%	8/50 acute GvHD
NCT02108522	Tzannou 2017 [88]	Phase II	38 HSCT recipients, 45 infections	Drug-resistant viral infections	CMV+EBV+AdV+HHV-6	Third-party 2 × 107 partially HLA-matched VSTs/m2	At 6w, 2/2 EBV achieved CR. 16/17 CMV achieved CR or PR, 5/7 AdV achieved CR or PR, 2/3 HHV-6 achieved PR, 16/16 BK achieved clinical benefit	5 (G1-2) GvHD; 1 (G3) GvHD
ACTRN12613000981729	Smith 2018 [89]	Phase I	22 SOTRs (13 Renal, 8 lung, 1 heart)	Ganciclovir-resistant CMV disease	CMV	Autologous 1-2 × 107 cells/m2	11/13 (84.6%) with virologic or clinical improvement	None
NCT01498484 and NCT00002663	Prockop 2019 [90]	Phase II	13 SOTRs and 33 HCST recipients	EBV-PTLD	EBV	Third-party	ORR in 68% of HCT and 54% of SOTRs 2 year survival 57% HSCT and 54% SOT	1 (G1) acute GvHD
NCT02532452 (Cincinnati)	Nelson 2020 [91]	Phase II	41	BK viremia and/or hemorrhagic cystitis or nephropathy after transplant	CMV, EBV, AdV, BK	Allogeneic donor-derived or third-party 5 × 107 VSTs/m2	ORR 86% in BK viremia, 100% ORR in hemorrhagic cystitis; 87% ORR in both	None
NCT02479698	Olson 2021 [92]	Phase II	59 HSCT recipients	BK virus-associated hemorrhagic cystitis	CMV, EBV, BK	third-party 1-2 × 105/kg	CR (34/49) PR (6/49) NR (9/49) at 45d	(G2) skin GvHD, (G3) GI GvHD, 9 chronic GvHD
NCT03883906	Rubinstein 2022 [93]	Phase II	23	Prophylaxis	CMV, EBV, AdV, BK	Allogeneic donor-derived 2 × 107 VST/m2	(7/23) With no viremia; reactivation (11/18); 5 treatment failure	(G2-3) 2/23 GvHD
NCT02532452 (Cincinnati)	Rubinstein 2022 [94]	Phase II	4 HSCT recipients	JCV-Progressive multifocal leukoencephalopathy	CMV, EBV, AdV, BK	Third-party 5 × 107/m2	1/4 stable disease 3/4 progressive disease	None
NCT02108522	Pfeiffer 2023 [95]	Phase II	58 HSCT adult and pediatric recipients	Unresponsive viral infection or antiviral untolerable	CMV + EBV +AdV + BKV + HHV-6 + JCV	Third-party 2 × 107/m2	ORR 95% at 6w	2 (G2 GvHD) and 1 (G3 GvHD)
NCT04605484	Chandraker 2023 [96]	Phase III	61 KTRs	BK viremia	CMV + EBV +AdV + BKV + HHV-6 + JCV	Third-party 4 × 107 cells	50% (10/20) ≥ 1 log BKV viral load reduction (biweekly dosing) 30% (6/20) ≥ 1 log BKV viral load reduction (monthly dosing)	None
NCT02532452 (Cincinnati)	Khoury 2024 [97]	Phase II	98 SOTRs (52 Kidney, 18 Liver, 16 Hear, 8 Lung, 4 small bowel)	refractory viral reactivation/infection	EBV + CMV + AdV + BKV	Third-party 50 × 106 cells/m2.	ORR of 45% for BKV, 65% for CMV, 68% for AdV, and 61% for EBV.	3 episodes of acute organ rejection
NCT04832607	Feuchtinger (TRACE) 2024 [94]	Phase III	HSCT recipients	New or refractory CMV, EBV, or AdV viral infection	CMV + EBV + AdV	Allogeneic donor-derived 1.0 × 105 T cells/ kg recipient (HLA-matched) 2.5 × 104 T cells/ kg (HLA-mismatched)	Ongoing

Abbreviations: SOTRs = solid organ transplant recipients; HSCT = hematopoietic stem cell transplantation; PTLD = post-transplant lymphoproliferative disorder; EBV = Epstein-barr virus; CMV = cytomegalovirus; AdV = Adenovirus; BKV = BK virus; HHV-6 = human-herpes virus-6; JCV = JC virus; ORR = objective response rate; CR = complete response; PR = partial response; NR = no response; HLA = human leukocyte antigen; VST = viral specific therapy; GvHD = graft versus host disease; GI = gastrointestinal

Manufacturing VST Cells

Manufacturing VSTs involves enriching and/or expanding virus-specific T cells ex vivo by stimulating T cells with virus-associated peptides loaded on antigen-presenting cells [98]. The source of T cells could be from their transplant donor (donor-derived) in cases of HSCT when available, or a healthy seropositive individual (third-party), also known as “off-the-shelf” VST. Ex vivo expansion of virus antigen-specific T cells was first described in 1999 [99]. After expansion, polyclonal virus antigen-specific CD4 + and CD8 + T cells are enriched to become VST product [100, 101]. One method of isolating VST is through TCR affinity to viral peptides loaded onto HLA multimers [102]. Alternatively, a fully GMP-compatible, interferon gamma (IFN-γ) capture method can directly isolate VST after restimulating T cells obtained from leukapheresis of seropositive donors, with virus-associated antigens for short time [103]. This allows rapid production of VST within 24–48 h, without days-long expansion of T cells (Fig. 2) [104, 105]. It is also important to ensure VST maintains polyfunctionality; they should be able to produce several types of cytokines (e.g. IFN-g, TNF-α, IL-2) cytotoxic activity and maintain proliferative capacity. Polyfunctionality is critical for potent cytotoxic activity and durability of VST.

Fig. 2.

Methods of virus-specific T cell (VST) production. (A) Ex vivo expansion of VST cells from the donor-derived peripheral blood mononuclear cells (PBMCs). First, PBMCs are obtained from the donor. Next, PBMC-derived antigen-presenting cells (APCs) are pulsed with overlapping peptide libraries (pepmixes), then cocultured with autologous T cells in a gas-permeable rapid expansion bioreactor with the addition of stimulating cytokines. Lymphocytoblastic cell lines (LCLs) made by infecting B cells with Epstein-Barr virus (EBV) may be used as APCs for the manufacturing of EBV-specific VST cells. Coculture leads to the expansion of donor memory T cells contained within the PBMCs of a presensitized donor and allows sufficient expansion for clinical use after 12 to 14 days. (B) The cytokine capture system exposes donor PBMCs to a peptide library resulting in interferon gamma production by memory T cells, which is fixed to the surface of the cells by application of a matrix. Interferon gamma-producing cells are then magnetically enriched by labeling them with a magnetic bead-conjugated anti–interferon gamma antibody and magnetically sorting cells expressing membrane-bound interferon gamma. This results in an infusible product after 12 to 16 h of production. (Adopted from Schreiber et al. Semin. Nephrol. (2024) 44:151498)

VST Safety

VST infusions are well tolerated in solid organ and bone marrow transplant recipients. There are a few cases of infusion-related events including myalgia, chills, etc. Typically, the concerns associated with using T cells to treat active infection include the overstimulation of antigen-specific T cells, potentially resulting in CRS and tissue damage. In most patients, CRS will occur within the first 72 h of infusion but can happen later [106]. Across the literature, there have been no associated deaths and very rare cases of CRS amounting to less than 2% [107, 96]. There have been rare reports of GvHD, but the symptoms are typically mild and usually limited to the skin. When organ rejection occurs, it is typically due to immunosuppression reduction and not directly due to VSTs [96]. To date, multiple groups have reported the safety of infusion of virus-specific T cells in HSCT. There have been no infusion-related side effects, and GvHD reactivation has been reported in very few instances [108, 109].

VSTs in Clinical Use

Third-party, multivirus-specific T Cell Therapies

Prolonged production periods limit the clinical application of allogenic VST in an acute setting. Some patients may be unable to undergo leukapheresis due to cytopenia. Moreover, the quality of autologous T cells to function as effector cells is limited, when they are obtained from transplant recipients on immunosuppression. As a result, generating third-party “off-the-shelf” VSTs that are partially HLA matched and cryopreserved is an important step moving forward.

There are newer strategies that involve overlapping peptide pools allowed development of broad spectrum multi-VSTs. Multi-VSTs that have activity against CMV, EBV, AdV, HHV-6, BK, and JC virus have been developed [101, 88]. Donor-derived multi-VSTs achieved a virological response in 8 out of 10 HSCT patients with EBV, CMV, or AdV infections, including those with dual infections [86]. A recent trial tested donor-derived multi-VSTs in 13 patients with sickle cell disease undergoing HSCT. Four out of 5 patients achieved remission, and 6 out of 7 patients remained virus titer free after receiving VSTs prophylactically [110].

Available and accessible third-party banks of multi-VSTs with known antiviral reactivity offer a greater advantage for immediate use [111, 112]. However, disadvantages include less degree of HLA-matching which may lead to less persistence of VST. A group from Cincinnati Children’s Hospital found no significant difference in clinical response between patients receiving third-party VSTs and donor-derived VSTs in 145 HSCT recipients [113]. Several other groups have described the use of partially HLA-matched (generally less than 4 out of 10 HLA alleles), third-party multi-VSTs in transplant recipients [87, 88, 92, 95, 97, 114]. Posoleucel is a third-party T-cell therapy specific for six viral infections: AdV, BK, CMV, EBV, HHV-6, and JC. In a study by Pfeiffer et al. [95], posoleucel demonstrated 95% overall response rate 6 weeks after infusion in 58 adult and pediatric allogeneic HSCT recipients. Three patients developed GvHD during the trial. One multicenter study showed a complete or partial response of 74% across 50 HSCT recipients with refractory EBV, CMV, or AdV infections using third-party multi-VSTs [87]. A phase II trial conducted by Cincinnati Children’s Hospital involved 98 adult and pediatric solid organ transplant recipients (SOTRs) – 47 kidney, 18 liver, 16 heart, 8 lung, 4 small bowel, and 5 multi-organ transplants. They used third-party quadrivalent VSTs with response rates of 45%, 65%, 68%, and 61% for BKV, CMV, AdV, and EBV, respectively, including 20% complete and 40% partial responses in post-transplant lymphoproliferative disorder (PTLD) [97]. All patients were receiving varying doses of immunosuppression at the time of VST infusion and only 3 (3%) of patients experienced an episode of acute rejection, which occurred in the context of immunosuppression reduction.

EBV-specific T-cell Therapy for post-transplant Lymphoproliferative Disorder (PTLD)

EBV infection in solid organ transplants present with variety of symptoms, from asymptomatic infection to PTLD. EBV is very seroprevalent (> 90%) and can be latent in B cells, enabling it to evade T-cell lytic responses through multiple mechanisms [115, 116]. The first reported use of EBV-specific cytotoxic T cells (EBV-CTLs) was in a lung transplant recipient but was ineffective for the treatment of PTLD [117]. One of the first trials on EBV-refractory PTLD involved 33 patients, 31 of whom were SOTRs, treated with allogeneic EBV-CTLs. At 6 months, 14 and 3 patients achieved complete and partial response, respectively, while 26 (79%) patients remained alive [85]. A study on third-party EBV CTLs for refractory PTLD showed an overall response rate of 46% in HSCT recipients (13/28) and 75% in SOTRs (15/20) [118]. All patients were pre-treated with chemo or targeted therapies. The low response rate in HSCT recipients was likely attributed to the poor clinical status of patients, many of whom died before completing infusions. Higher HLA-matching was associated with better response rates, reaching up to 100% response rate in 8–10 HLA matches.

A meta-analysis reviewed 11 studies with a total of 76 solid organ transplant recipients who received autologous (15 out of 76) and HLA-matched third-party (61 out of 76) EBV-specific CTLs for refractory EBV-associated PTLD, which had exhausted available therapies [119]. Among solid organ transplants, 22 were liver transplants, 27 were kidney transplants, 8 were heart transplants, and 6 were lung transplants. The most predominant malignancy was B-cell type, monomorphic PTLD. The response rate was 66% (50 out of 76 patients) from which 36 achieved complete response and 14 had partial response. Overall, EBV-specific VST was well tolerated and reasonably effective for refractory EBV-associated PTLD.

Prockop et al. investigated the application of partially HLA matched, allogeneic EBV-VST derived from third-party donors in the treatment of rituximab-refractory PTLD in 33 HSCT and 13 SOT recipients [90]. Complete remission was achieved in 68% of HSCT recipients and 54% of SOT recipients. This phase 2 trial led to the Tabelecleucel for Solid Organ or Allogeneic Hematopoietic Cell Transplant Participants with Epstein-Barr Virus-Associated Post-Transplant Lymphoproliferative Disease (EBV positive PTLD) After Failure of Rituximab or Rituximab and Chemotherapy (ALLELE) trial [120], a multicenter open-label phase 3 trial that included 43 patients with rituximab-refractory or relapsed PTLD. Fifteen of 29 SOTRs and 7 of 14 HSCT recipients had an objective response. In this study there were no GvHD, graft rejection or CRS related to the treatment [120].

In the pediatric population, a study evaluated rituximab with third-party EBV-VST in newly diagnosed PTLD. Patients who achieved a complete response after rituximab induction received additional rituximab doses, while patients with a partial response or progressive disease received EBV-VST. A separate cohort of refractory PTLD only received EBV-VST. Overall response rate after the first cycle of EBV-VST was 70% (7/10) for the newly diagnosed cohort and 20% (1/5) for the refractory cohort [121].

BK virus-specific VST

Nelson et al. [100] studied donor-derived (n = 14) or third-party (n = 27) BK VST in 38 HSCT and 3 SOTRs. Without any infusion toxicity or de novo GvHD, the overall response rate was 86% and 87% in patients treated for BK viremia and both BK viremia and hemorrhagic cystitis, respectively. In Cincinnati Children’s Hospital group [97], two out of three patients with BKV attained a partial response. Five (14%) KTRs had complete resolution of BK viremia, and 10 KTRs (28%) had at least one logarithmic reduction in their viral load. None of the 20 patients who had BK nephropathy had improvement in their creatinine levels. However, the time point of assessment of eGFR was 4 weeks after the VST infusion, which might be too early to assess the estimated glomerular filtration ratio (eGFR). Enzyme-linked immunosorbent spot (ELISPOT) data showed expansion of BKPyV-specific T cells post-VST infusion [97, 100].

In a recent phase 2 trial by Chandraker et al. [96], a multi-VST posoleucel was tested on 61 KTRs who had BK viremia. Patients were randomized 1:1:1 to receive posoleucel every week for 3 weeks and subsequently every 14 days (group 1) or every 28 (group 2) days or placebo for 12 weeks. Half the patients (10 out of 20) and 30% (6 out of 20) receiving posoleucel every 14 days and 28 days, respectively, had one logarithmic reduction in viral DNA compared to only 28% (5 out of 18) of the placebo group. A single-center retrospective study found a decrease in all 17 BK viral loads with a reduction in creatinine and proteinuria in KTRs after using third-party multi-VSTs. There were no significant adverse events besides one T-cell mediated kidney rejection, which is likely due to discontinuation of MMF, and another membranoproliferative glomerulonephritis, where the direct causality of VST infusion is unclear [91]. Overall, BK virus-specific VST therapies are well tolerated, but its efficacy for BK viremia and BK nephropathy seems modest and needs further improvement.

CMV-specific VST

While CMV infection has several antiviral drug options, treating resistant CMV infection is challenging. A phase I trial analyzed autologous CMV VST in 4 kidney, 8 lung, and 1 heart transplant recipients with ganciclovir-resistant CMV. Patients either stopped antiviral therapy or remained on reduced antivirals post-infusion. While no serious adverse events occurred, 11 out of 13 patients demonstrated reduce or resolved CMV disease and improved response to antiviral drug therapy [122]. Another retrospective study evaluated donor-derived and third-party CMV and AdV VST in 10 HSCT patients across 10 years [89]. While 40% was the overall survival, 6 out of 10 patients responded to VST. Interestingly, one patient did not undergo HSCT but rather had advanced HIV-associated CMV reactivation and was successfully treated with CMV-specific VSTs. This encourages the evaluation of VST beyond transplantation.

Conclusion

Emerging T cell therapies have promising future clinical implications in transplantation. T cell therapy for desensitization, using CAR-T cells targeting antibody-producing cells via CD19 and B cell maturation antigen (BCMA) can be one direction [123, 124]. Additionally, genetically engineering T cells (e.g. glucocorticoid receptor knock-out) to be functional in immunocompromised recipients is another emerging strategy [125, 126].

Although the potential of cell therapies in transplantation is substantial, efforts are still needed to translate these experimental treatments into clinically viable options. Further large-scale clinical trials are crucial to address existing challenges and widely adopt these groundbreaking therapies for transplant recipients.

Data Availability

No datasets were generated or analysed during the current study.