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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Am J Transplant. 2018 Apr 21;18(6):1305–1311. doi: 10.1111/ajt.14747

Engineering Therapeutic T Cells to Suppress Alloimmune Responses Using TCRs, CARs or BARs

Antoine Sicard 1,2,3, Megan K Levings 1,2, David W Scott 4
PMCID: PMC5992079  NIHMSID: NIHMS955345  PMID: 29603617

Abstract

Adoptive cell therapy with therapeutic T cells has become one of the most promising strategies to stimulate or suppress immune responses. Using viral-mediated genetic manipulation, the antigen-specificity of T cells can now be precisely redirected. Tailored specificity has not only overcome technical limitations and safety concerns, but it has also considerably broadened the spectrum of therapeutic applications. Different T cell-engineering strategies have now become available to suppress alloimmune responses. We first provide an overview of the allorecognition pathways and effector mechanisms that are responsible for alloimmune injuries in the setting of vascularized organ transplantation. We then discuss the potential to use different T cell-engineering approaches to supress alloimmune responses. Specifically, expression of allospecific T-cell receptors (TCR), single-chain chimeric antigen receptors (CARs) or antigen domains recognized by B-cell receptors (B-cell antibody receptors, BARs) in regulatory or cytotoxic T cells are considered. The ability of these strategies to control the direct or indirect pathways of allorecognition and the cellular or humoral alloimmune responses is discussed. An intimate understanding of the complex interplay that occurs between the engineered T cells and the alloimmune players is a necessary prerequisite for the design of safe and successful strategies for precise immunomodulation in transplantation.

Introduction

Suppressing the alloimmune response has been the main challenge of transplantation medicine since the discovery of allograft rejection in the mid-20th century. Prevention of rejection has relied so far on immunosuppressive drugs, but despite major advances, modern immunosuppressive regimens remain responsible for high morbidity and mortality. For example, they act on immune effectors regardless of their antigen-specificity thereby inducing global immune suppression and increasing the risk of infections and cancer. In addition, current regimens only partially block the alloimmune response and often fail to prevent the development of chronic rejection, which remains the primary cause of allograft loss (1). Ideally an allosuppressive strategy should exclusively target anti-donor immune responses while preserving other aspects of normal immunity.

One of the most promising strategies to specifically control different types of immune responses is adoptive cell therapy with T cells (T-cell adoptive cell therapy, TACT). Following encouraging results in animal models, in the late 1980’s the potential of TACT to stimulate immunity in humans was demonstrated by use of ex vivo-expanded tumor-infiltrating T cells to treat melanoma (2). Transplantation has led the way with testing immunoregulatory TACT, providing evidence that transfer of CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) prevents alloimmune responses in preclinical models, with multiple clinical trials completed or in progress with polyclonal or donor-specific ex vivo expanded Tregs (3).

The possible use of gene therapy to engineer T cells prior to transfer has considerably augmented the therapeutic potential of TACT. Specifically, retroviral- or lentiviral-mediated genetic manipulations now allow precise redirection of the antigen-specificity of therapeutic T cells and/or shaping of their function (4–6). The use of antigen-specific T cells overcomes many technical limitations and safety consideration of polyclonal T cells, and also considerably broadens the spectrum of therapeutic applications. Indeed, many different T cell engineering strategies have now become conceivable as a strategy to prevent alloimmune responses. However, designing successful allosuppressive strategies requires an intimate understanding of the allorecognition pathways and effector mechanisms that are responsible for allograft loss and relevant to clinical transplantation. It is essential to identify all of the immune players to be targeted by therapeutic T cells to choose the right engineering approach.

We first provide a brief overview of the allorecognition pathways and effector mechanisms that are relevant to clinical transplantation and must be targeted to prevent alloimmune damages. We then discuss approaches to create antigen-specific Tregs by expression of specific T cell receptors (TCR), single chain chimeric antigen receptors (CARs), or antigen domains recognized by B-cell receptors (BARs) and their potential application to suppress alloimmune responses.

Alloimmune players to be targeted by therapeutic T cells

Allorecognition is initiated by recipient T cells recognizing either donor peptides bound to self MHC molecules on recipient APCs (indirect pathway) (7) or intact donor MHC molecules on donor APCs (direct pathway) (8). Activated T-cell clones with direct alloreactivity interact with donor-specific MHC molecules expressed by graft cells and induce their apoptosis via a process termed cellular rejection. Activated T cell clones with indirect alloreactivity also cause tissue injury by promoting delayed-type hypersensitivity within the allograft and by providing an indispensable helper T cell function as the cellular and humoral alloimmune responses develop (9). The latter results from activation of naïve alloreactive B cells by donor MHC molecules in secondary lymphoid organs. Activated B cells enter the germinal center reaction and differentiate into memory B cells or long-lived plasma cells which migrate to the bone marrow or remain in secondary lymphoid organs (10) and secrete high-affinity donor-specific antibodies (DSA). The binding of DSA to mismatched MHC molecules expressed by graft endothelium leads to progressive tissue destruction, a process referred to as antibody-mediated rejection (AMR).

The relevance of direct and indirect allorecognition pathways to clinical transplantation varies since the two pathways differ in the type and number of alloreactive T-cell clones being activated and in the length of the activation phase (11). The high-precursor frequency of T cells able to interact directly with donor-MHC molecules underlies the strength of the direct alloimmune response and the highly destructive potential of acute cellular rejection (8). However, it is generally accepted that direct alloreactivity is curtailed overtime by elimination of donor APC and that indirect alloreactivity dominates late after transplantation, being the driving force behind AMR (11,12).

In terms of immunoregulatory cells, although TACT using Tregs with direct donor-specificity can prevent rejection (13,14), there is evidence that Tregs with indirect donor-specificity may be more potent. Three independent teams using different approaches to generate donor-specific Tregs concluded that indirect Tregs had a higher ability to promote long-term allograft survival than direct Tregs (15–18). The humoral alloimmune response was not monitored in these studies, but cognate interactions between indirect Tregs and B cells resulting in suppression of the humoral alloimmune response may have been key for long-term allograft protection. Observations in other experimental studies that transfer of indirect Tregs abrogates GC alloantibody responses and blocks development of allograft vasculopathy (19,20) are in line with this notion.

Although both cellular and humoral rejection can lead to rapid allograft destruction, in the clinic, cellular versus humoral rejection are associated with different prognosis. Whereas cellular rejection is a rare cause of graft loss, AMR severely shortens allograft survival and is widely recognized as the leading cause of late transplant failure (1). This difference can be explained by the availability of efficient drugs to control cellular rejection (e.g. corticosteroids or thymoglobulin) but the lack of effective treatments to reverse AMR (21,22). Finding better ways to suppress the humoral alloimmune response is one of the main unmet needs of modern transplantation, both to prolong allograft survival after AMR and to reset B-cell and plasma cell memory in patients whose humoral allosensitization precludes access to transplantation.

Gene therapy approaches to engineer therapeutic antigen-specific T cells

Antigen specificity is the basis for successful TACT (23), but methods to generate antigen-specific T-cell products through long-term in vitro stimulation-based enrichment protocols is challenging and expensive because the frequency of T cells specific for a single target is low. A possible solution is to engineer specific receptors into expanded human T cells. Not only does this ensure on-target functionality, it also decreases the chances of global immunosuppression. Moreover, giving T cells specific receptors allows transfer of fewer cells and increases efficacy. Combining in vitro expansion and gene-editing technology with maintenance of function, which is particularly important with human T regulatory cells (Tregs), will allow for the development of safe therapeutic protocols in transplantation, as well as in autoimmunity and adverse immune responses to biotherapeutics (see below).

Transfer of genes encoding the TCR

The first preclinical proof of concept demonstrating the potential of engineered T cells was pioneered in the early 1990’s by the seminal studies of Zelig Eshhar in Israel. The goal was to study T cell signalling and interactions, but it quickly became apparent that the approach using chimeric receptors, at the time called “T-bodies”, had enormous clinical potential (24). Proof of principle using TCR-engineered gene therapy was obtained by several groups in the next decade, generating tumor- or virus-reactive cytotoxic T cells through retrovirus-mediated TCR gene transfer (4). T cells targeting the melanoma antigen MART-1 or the testis antigen NY-ESO-1 demonstrated clinically objective l responses in patients with melanoma, synovial cell sarcoma or myeloma (4).

Soon thereafter, the same approach was used to redirect the specificity of Tregs toward autoimmune disease relevant antigens (Figure 1, left panel). For example, manifestations of autoimmune diseases were successfully prevented in models of inflammatory arthritis and diabetes with mouse or human Treg expressing ovalbumin- or pancreatic islet-specific TCRs (25,26). The adverse immune response to therapeutic factor VIII (FVIII), which obviates the pro-coagulant activity of FVIII in hemophilia A patients treated with the recombinant molecule, was also specifically inhibited by TCR-transduced human Tregs specific for FVIII (27). Important to the field of transplantation, Tsang et al. conferred indirect allospecificity to mouse Tregs by transferring a TCR specific for H-2Kd peptide presented by H-2Ab MHC class II molecules (16). These TCR-transduced Tregs induced long-term survival of MHC-mismatched heart grafts.

Figure 1. The TCR and Chimeric Antigen Receptor (CAR) approaches.

Figure 1

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Left panel. The TCR approach: exogenous TCR genes are transferred to Tregs. The TCRs are specific for a donor peptide in the context of a recipient MHC class II molecule to confer indirect alloreactivity to therapeutic Tregs. The limit of this approach in the setting of transplantation is the class II MHC-restriction of the TCR and the huge diversity of possible recipient MHC molecule-donor peptides combinations. Right panel. The CAR approach: an extracellular single-chain antigen–binding domain (scFv) derived from an antibody is fused to costimulatory and T-cell signaling domains. The scFv is specific for a mismatched donor HLA molecule and confers a direct allospecificity to the Treg. This approach enables the Tregs to prevent the cellular alloimmune response. The impact on the humoral alloimmune response remains to be determined.

One important limitation of the transgenic TCR approach is the risk of mispairing with the endogenous TCR that can cause unwanted reactivity, off-target effects and low efficacy. CRISPr/Cas9 technology could now be used to eliminate endogenous TCR chains (28), but an alternative strategy is to use single chain chimeric antigen receptor (CAR) technology based on antibody recognition, as described below.

Transfer of genes encoding Chimeric-Antigen Receptors (CARs)

CARs are composed of an extracellular single-chain antigen–binding domain (scFv) derived from an antibody fused to intracellular signalling domains, usually encoding components of CD3ζ as well as one or more costimulatory domains (Figure 1, right panel (6)). Antibodies recognize conformational or linear epitopes; thus, unlike TCR-engineered T cells, CARs are not MHC-restricted. Since their first description by Eshhar et al in 1993 (29), CAR-engineered T cells have become a major option in the therapeutic arsenal against hematological malignancies when engineered into cytotoxic T cells (30). In addition, CAR technology has rapidly been extended to generate redirected Tregs to suppress unwanted immune responses (5,6,31). Elinav et al. provided the first proof of concept in 2008 with the demonstration that Tregs from a transgenic mouse expressing a 2,4,6-trinitrophenol (TNP)-specific CAR protected mice from experimental colitis induced by 2,4,6-trinitrobenzene sulfonic acid (32). Retrovirally-transduced mouse CAR Tregs specific for the carcinoembryonic antigen or for myelin oligodendrocyte glycoprotein successfully prevented colitis and experimental autoimmune encephalomyelitis, respectively (33,34) and Kim et al., submitted 2018).

Hypothesizing that this approach could also be used to suppress the alloimmune response, three teams created CARs directed against the HLA-A2 molecule, one of the most commonly mismatched antigen in organ transplantation (35–37). MacDonald et al. reported that expression of an HLA-A2-specific CAR in human Tregs enabled antigen-specific recognition of HLA-A2 and activation of Tregs (35) without any significant induction of cytotoxic activity. Importantly, A2-CAR Tregs were markedly better than polyclonal Tregs at inhibiting xenogeneic GVHD mediated by HLA-A2+ T cells in vivo. Two other studies confirmed that A2-CAR Tregs were more suppressive than polyclonal Tregs in humanized mouse models with HLA-A2+ skin xenografts (36,37).

As this approach is developed for clinical transplantation, it is important to consider the possible types of alloimmune responses that could be targeted. For example, CARs targeting class I or class II MHC molecules could be used: class I CAR Tregs would be activated by both graft cells and donor APCs, whereas class II CAR Tregs would be principally activated by donor APC. In addition, since donor endothelium expresses both class I and class II molecules, both class I and II-specific CARs should be able to transmigrate into the allograft where local active or bystander suppression could occur. Indeed Boardman et al. reported that Tregs expressing an A2-CAR preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers (36). In addition, the intragraft ratio of FOXP3: CD3 cells was higher with A2-CARs than with polyclonal Tregs, suggesting that CAR expression facilitated homing and retention of the Tregs in allografts.

HLA-A2 is commonly mismatched in the Caucasian transplant population so A2-specific CARs will be applicable to many HLA-A2 negative recipients with an HLA-A2+ organ. Although not currently available, CARs with specificities for other HLA proteins can also be easily developed so that a wide variety of HLA-mismatch scenarios would be covered by a bank of pre-manufactured lentivirus. With pre-existing virus, or other pre-designed genome engineering strategies, CAR-Tregs can easily be manufactured within 2-3 weeks, making their delivery shortly after induction therapy and transplantation feasible even in the deceased donor setting. Moreover, with increasing interest in developing third-party T cell (and Treg) therapy (38), a bank of “off the shelf” Tregs which are pre-engineered with a series of CARs can also be envisioned.

An important consideration is that the effect of CAR-Tregs on the indirect pathway and the humoral alloimmune response has not yet been investigated. However, recent data from other systems sheds light on their potential to modulate antibody responses. Of particular interest in this regard is a study which assessed the impact of TCR versus CAR-Tregs on an antigen-specific humoral responses (31). Yoon et al. demonstrated that human Tregs expressing either a CAR that recognized a FVIII domain, or a TCR recognizing an HLA-DRB1-restricted FVIII epitope, prevent the anti-FVIII immune response in HLA-DRB1 transgenic hemophilic mice. Importantly, both CAR- and TCR-transduced Tregs inhibited T-cell and B-cell primary antibody responses to FVIII. However, antibody recall responses were not well controlled by these Tregs, perhaps because by that time (56 days after cell injection) the human Tregs did not survive long enough in this xenogeneic model.

Transwell experiments with TCR-engineered Tregs demonstrated that these Tregs block T-cell responses indirectly through contactless inhibition of T effector cells, but only in the presence of activated effectors that provide a source of IL-2 ((39) and Kim et al. submitted). Thus, like unmodified Tregs, optimal T-cell inhibition by engineered Tregs requires cell proximity.

Interactions of TCR-Treg with antibody-secreting plasma cells are uncertain, but theoretically possible since isotype-switched plasma cells have been shown to present sufficient antigens in vivo to interact with cognate T effector cells and shape their function (40). Concerning donor-specific CAR-Tregs, they may not interact directly with recipient plasma cells that do not express donor antigens, possibly limiting their therapeutic potential in previously sensitized recipients. To overcome this possible limitation, an alternate strategy designed to engineer T cells to directly interact with antigen-specific B cells and plasma cells expressing immunoglobulin receptors has recently been developed, as described below.

Transfer of genes encoding B cell antibody chimeric receptors (BAR) into cytotoxic and regulatory T cells

The B-cell antibody chimeric receptor (BAR) strategy was first described in 2016. Aiming to suppress pathogenic autoimmune cells while sparing protective immunity, Ellebrecht et al created a novel CAR construct, which they termed “chimeric auto-antibody receptor” or CAAR (41). In that construct, the scFv of the CAR (which we refer to as BAR for B-cell antibody receptor) was replaced by the antigen itself (Figure 2), with the intracellular portion encoding T-cell signalling domains as in conventional CARs. The authors showed in the antibody-mediated autoimmune disease pemphigus vulgaris (PV) model that cytotoxic T cells expressing a BAR encoding the PV-autoantigen, desmoglein3, exhibited specific cytotoxicity against hybridoma cells expressing a PV-specific BCR in vitro. These BAR T cells homed to the spleen and bone marrow, expanded, persisted, and specifically eliminated autoantigen-specific hybridomas in vivo without off-target toxicity, although their effect on primary B cells was not tested. In parallel, Parvathaneni et al. engineered BARs containing the immunodominant domains of FVIII into murine or human CD8 T cells and confirmed their direct cytotoxicity on anti-FVIII hybridomas in vitro (42). Importantly, these cytotoxic BARs specifically suppressed LPS-stimulated B cells production of anti-FVIII in vitro and the FVIII-induced primary response in vivo.

Figure 2. The B-cell Antibody Receptor (BAR) approach.

Figure 2

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B-cell antibody receptor (BAR) Tregs are comprised of an antigen or its domains expressed on the cell surface and fused with intracellular costimulatory and T-cell signaling domains. BAR have been designed by the Scott lab to be expressed either in cytotoxic T cells or in Tres as potential therapeutics to ameliorate antibody-mediated autoimmune diseases and/or immune responses to biotherapeutics.

Would BAR-expressing Tregs be able to directly suppress B-cell responses by directly interacting with specific B cells? Because studies in IPEX patients suggested that Tregs suppress pathogenic B cells (43), we hypothesized that BAR engineered Tregs could directly suppress FVIII-specific B cells. Thus, Tregs were engineered by our team to express a BAR comprising either the immunodominant domains of FVIII (termed FVIII-A2 [not to be confused with HLA-A2] or FVIII-C2). We found that FVIII-A2 and FVIII-C2 BAR Tregs suppressed FVIII primary antibody response in FVIII-immunized hemophilic mice (submitted and (44)). Recall antibody-responses to FVIII in vitro were also inhibited by BAR-Tregs while T-cell responses remained intact, suggesting a specific tolerization of the FVIII-specific B cell compartment. These results encourage the potential use of BAR cytotoxic cells to prevent anti-FVIII “inhibitor” production in patients in the future, and could possibly be applied to block AMR in a transplant setting.

Discussion and Future Perspectives

The possible use combined cell and gene therapy has paved the way for several promising T cell-engineering strategies to prevent alloimmune responses. Depending on the strategy, different alloimmune players may be targeted. The CAR-Treg approach holds promise for inhibiting the direct pathway of allorecognition and cellular rejection. The effect of CAR-Treg on the indirect pathway and the humoral alloimmune response remains to be investigated.

In contrast, the ability of the TCR approach to confer indirect allospecificity to Tregs has already been used successfully to promote allograft tolerance in mice (16). A consideration, however, is that it will be difficult to translate the TCR approach to clinical transplantation because of the class II MHC-restriction of the TCR and the large diversity of possible recipient MHC molecule-donor peptides combinations.

The promising results of the BAR approach in the context of autoimmunity and hemophilia suggest that BARs may also be used in transplantation. For example, HLA-encoding BAR Tregs might be able to prevent the alloimmune humoral response and antibody-mediated rejection (AMR) in non-sensitized recipients through direct tolerogenic interactions with allospecific-B cells. It would also be conceivable to use HLA-encoding BAR-transduced cytotoxic T cells to target alloantigen-specific memory B cells or plasma cells. This method could reverse an established AMR process in transplant patients, stop new donor-specific antibody generation, as well as re-set humoral alloimmune memory in highly sensitized patients.

An important question is what effect circulating antibodies would have on BAR-expressing cells since soluble-protein-mediated BAR stimulation could cause tonic and/or suboptimal signalling and result in an anergic or exhausted phenotype, and/or lead to Fc-mediated cell clearance (5). Alternatively, antibody crosslinking of the BAR potentially may stimulate expansion of these Tregs. In addition, the possible “rejection” of the HLA-encoding BAR Tregs, which carry donor HLA molecules on their surface, may also be a limitation of this approach, especially in previously sensitized patients, and has to be considered.

In conclusion, it has become clear that cell therapy with engineered T cells represents a powerful approach to control alloimmune responses. An important consideration is that a critical aspect of this therapy will be assurance that there is no risk of de-differentiation of CAR-Tregs into CAR-expressing effector T cells which would have the ability to kill allograft tissue. Manufacturing strategies should focus on engineering homogeneous populations of thymic Tregs which are less prone to lineage instability (45). The engineered receptor could also encode a cell-fate control system, for example by including an epitope that is recognized by a clinically-approved antibody which could be delivered for acute cell clearance, or by designing regulatable receptors which are only active in the presence of a drug (46). Moreover, in addition to cell manufacturing challenges, there remain many outstanding questions about the mechanisms by which these therapies will work. Designing successful and safe strategies will require an accurate understanding of their impact on the allorecognition pathways and effector mechanisms responsible for alloimmune injuries.

Acknowledgments

AS is supported by the European Commission H2020 program (Marie Skłodowska-Curie actions) and by the Canadian Institutes of Health Research. MKL’s own work in this area is supported by grants from the Canadian Institutes of Health Research (FDN-154304), Canadian National Transplant Research Program (TFU 127880) and TxCell. MKL receive salary awards from the BC Children’s Hospital Research Institute. DWS has grant support from the NIH (HL126727).

Abbreviations

AMR

antibody-mediated rejection

BAR

B-cell antibody receptor

CAR

chimeric antigen receptor

DSA

donor-specific antibody

TACT

T-cell adoptive cell therapy

TCR

T-cell receptor

Treg

regulatory T cell

Footnotes

Dedication

This review is dedicated to the memory to Professor Johannes (Jan) van Rood, Leiden University Medical Center, whose pioneering work on HLA was critical in advancing transplant science

Author contributions

AS wrote the primary draft and created the figures; MKL and DWS wrote and edited the final version of the manuscript.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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