T-cell receptor (TCR) transgenic regulatory T cells (Tregs) may represent a promising personalized treatment for T-cell-mediated autoimmune diseases such as type 1 diabetes. A curative therapy that targets the underlying immunological cause of disease to restore antigen-specific immunological tolerance represents an essential objective for the preservation of β-cell mass and function in the treatment of type 1 diabetes (1). Non-antigen-specific therapies involving hematopoietic stem cell transplantation combined with T-cell depletion, via high-dose anti-thymocyte globulin (ATG) or fludarabine, plus immunomodulation with cyclosporine and granulocyte-colony stimulating factor (G-CSF) have been shown to preserve β-cell function (2, 3), but the risks associated with these aggressive protocols preclude common clinical use. Comparatively, non-specific polyclonal immunotherapies, including immunoregulatory or depleting agents [e.g., alefacept (human LFA-3/IgG1-Fc fusion protein), teplizumab or otelixizumab (anti-CD3), and rituximab (anti-CD20)], have been better tolerated and offered some temporary efficacy but not long-term induction of tolerance (4–10). Until recently, most antigen-specific tolerance induction efforts have involved mucosal or peripheral administration of autoantigen(s), but thus far, such attempts have yielded limited efficacy in only a subset of patients, again with no indication for long-term tolerance induction (11, 12). Indeed, a safe treatment that controls persistent immune memory and induces long-term tolerance is needed.

Islet cell antigen-reactive Tregs, isolated from BDC2.5 TCR transgenic mice, could be expanded in vitro, and following adoptive transfer, migrate to the pancreatic draining lymph node/nodes (13). These Treg prevent and reverse autoimmune diabetes in non-obese diabetic (NOD) mice (14). In contrast, Tregs isolated and expanded from GAD286 TCR transgenic mice could suppress responder T cells (Tresp) in vitro but did not proliferate in vivo after transfer into recipient animals (14). Moreover, expression of cognate autoantigen is required for efficient trafficking of Tregs to the target organ and suppression of diabetes in NOD mice (15). These preclinical data support the notions that autoantigen-specific Tregs may offer an important therapy for type 1 diabetes, but also that intrinsic factors such as TCR specificity and/or avidity may play an important role in determining the capacity for immunomodulation and efficacy. The need for continued autoantigen expression by the host may render insulin-reactive TCRs less effective in patients with long-standing type 1 diabetes and support a need to investigate additional, potentially bystander, TCRs specific for additional/alternative autoantigen targets such as glutamic acid decarboxylase (GAD). Moreover, antigen localization, density, and persistence in β-cells along with risk of effector cell reprogramming support the use of alternative TCRs (16).

Genetically modified T cells with TCRs specific for tumor or viral antigens have become a valuable tool for the treatment of certain cancers or infections in humans (17–19). We previously demonstrated successful HLA class I-restricted TCR gene transfer in human Tregs using a high-affinity model receptor specific for the melanoma antigen tyrosinase presented by HLA-A*02:01 (20). We also generated a murine form of these tyrosinase-specific Tregs, and when transferred in vivo, the cells were capable of suppressing anti-tumor immunity in murine tumor models (20). This prompted us to ask whether candidate TCRs specific for type 1 diabetes-related autoantigens could be used to generate regulatory TCR avatars for human therapy.

Two TCR clones (R164 and 4.13) specific for the same β-cell peptide (GAD_555–567) presented by HLA-DR4, but with different binding affinities, have been identified from the peripheral blood of subjects with or at-risk for T1D (21–23). Indeed, we recently identified T cells expressing the TCR β-chain complementarity determining region (CDR3β) of the GAD 4.13 clone from tissues of seven organ donors with type 1 diabetes, including the pancreatic islets of one type 1 diabetes subject. Interestingly, for one donor with long-standing disease, the TCR CDR3β was highly enriched in the pLN (>25% of all productive sequences), representing the most prevalent clone in both the Treg and conventional CD4⁺ T-cell (Tconv) populations (24). Interestingly, 4.13 TCR transgenic HLA-DR4 mice were reported to contain a mixture of Th1 and Tr1 cells capable of producing IL-10 (21). Conversely, R164 TCR transgenic HLA-DR4 mice exhibited greater thymic negative selection, and the T cells that escaped the thymus were skewed toward a Th1 phenotype (21). These observations support the notion that TCR avidity may impart important functional distinctions.

In a recent report by Ali et al., human CD4⁺ T cells were engineered to express the R164 TCR clone, and importantly, when administered to NSG-Ab⁰ DRB*04:01 mice, these R164 cells established long-term engraftment and islet infiltration, up to 12 weeks, without graft versus host disease (GvHD) (25). The creation of these autoreactive T-cell avatars presents the exciting possibility of autologous Treg therapy for type 1 diabetes with the benefit of antigen specificity to potentially enhance Treg trafficking to the target organ and associated draining lymph nodes. These antigen-specific Tregs would likely represent a significant improvement upon autologous polyclonal Treg therapy, which has already been shown to be safe for use in human subjects (26, 27). Indeed, antigen-specific Tregs offer the potential for long-term tolerance to the target antigen and possibly, to other key β-cell epitopes via bystander suppression and infectious tolerance (14, 28). To expand on these efforts, we generated primary human Tregs expressing the two GAD_555–567-reactive TCR clones (R164 and 4.13), and investigated the pre-transfer conditions needed to optimize suppressive activity for potential use in adoptive cell therapy.

For tolerogenic adoptive cell therapy, autologous polyclonal Tregs can be expanded from peripheral blood which provides an attractive Treg source given the abundant cell numbers, allowing for repeat dosing if needed, and the safety associated with autologous cell therapy (26, 27). Concerns remain, however, regarding the lack of antigen specificity by administrating polyclonal Tregs. Indeed, Tregs have a highly diverse repertoire (24), which indicates the precursor frequency of autoreactive Tregs will likely be quite low in peripheral blood, especially considering that Tregs do not enrich to the extent that is observed for Tconv during conversion to effector T cells and expansion. Hence, we expect polyclonal Treg therapy to confer potentially limited efficacy and trafficking to the pancreas or PLN to induce immunological tolerance for β-cell antigens. To address this, we utilized LV TD to generate primary human T-cell avatars expressing two GAD_555–567-reactive TCRs (R164 and 4.13) originally identified from the peripheral blood of subjects with or at risk for type 1 diabetes (21–23). These clones, which differ by only 10 amino acids in TRAV and TRBV genes and only three amino acid charge differences in the CDR3 region (Table 1), exhibit different binding affinities for their cognate antigen peptide (21).

Regulatory T cell avatars maintained FOXP3 positivity, indicating that LV TD did not impair Treg stability. Functionally, 4.13 Treg avatars effectively suppressed antigen-specific 4.13 CD4⁺ Tresp. Beyond this, 4.13 Treg avatars exhibited a moderate ability to suppress MART-1 CD8⁺ Tresp in a bystander suppressive mechanism that required Treg activation. Interestingly, when the high-avidity R164 or lower avidity 4.13 Treg avatars were cultured with R164 CD4⁺ Tresp in the presence of GAD_555–567 peptide presented by CD3-depleted HLA-DRB1*04:01/HLA-A*02:01 APCs, R164 TCR were significantly more suppressive. This suggests that Treg TCR avidity affects suppressive ability and importantly, that the optimal avidity of TCR or CAR signals may be required for effective Treg avatar cellular therapy. Importantly, however, there is the potential for heterologous TCR chain pairing with the endogenous receptors, and further experiments are needed to empirically determine this for each receptor. Recent developments in gene editing technologies could be used to correct for this potential caveat. Specifically, knockout of endogenous TCR α- and β-chains via CRISPR/Cas9, silencing of endogenous TCR via shRNA with expression of a codon optimized de novo TCR, or the domain-swap approach described by Bethune et al. (41) could be applied.

Although preproinsulin (PPI) and alternative forms of this antigen (e.g., hybrid insulin peptides, alternative mRNA transcripts) (42, 43) are considered key type 1 diabetes autoantigens, we anticipate continued expression of cognate antigen will be imperative for Treg survival and trafficking to the target organ (15, 44). Hence, we focused our efforts on the development of Tregs against GAD65, which exhibits a high autoantigen density in T1D (45) and is the target of persistent autoimmunity, as evidenced by maintenance of autoantibodies (46). We anticipate that adoptive cell therapy with GAD-specific Tregs will lead to bystander suppression and infectious tolerance (47) with the hope for inducing long-term antigen-specific tolerance to GAD as well as other β-cell antigens. A recent report by Hull et al. described the generation and in vitro characterization of peripheral blood-derived human Tregs expressing TCRs specific for insulinoma-associated protein-2 (IA-2) and insulin (48). Although the authors did not conduct functional comparisons of TCR avidity, in vivo investigations of these and the GAD-specific clones generated herein are certainly warranted to determine the optimal clone(s) for tolerogenic cell therapy as we move forward toward clinical testing.

Chimeric antigen receptor (CAR) Treg therapy should also be considered given the promising outcomes observed from CAR effector T cells in cancer immunotherapy (49, 50). CAR Treg therapy could be particularly advantageous given that CAR T cells are not constrained by HLA restriction, hence, offering the opportunity for off-the-shelf clinical utility. However, the need for surface expression of the target antigen on islets or β-cells represents a clear limitation compared with TCR gene transfer, which allows for recognition of intracellular antigens in the context of class II HLA. An additional approach to potentially address this challenge could involve the use of a CD8-restricted TCR that functions independently of the CD8 co-receptor. In fact, this type of activity has been demonstrated previously with a high-affinity melanoma antigen tyrosinase-reactive TCR expressed by CD4⁺ T cells (20). Yet an additional approach could involve the identification of a CAR capable of recognizing an islet epitope in the context of HLA-A2, given the observation that beta cells hyperexpress class I HLA in settings of type 1 diabetes (51).

RNA TCR or CAR gene transfer has been demonstrated as one potential approach to confer T-cell antigen-specificity (52), and could be further explored in the context of tolerogenic adoptive Treg therapy for type 1 diabetes. Specifically, mRNA encoding the TCR or a CAR of choice can be introduced to T cells via electroporation, thereby eliminating the need for LV TD and associated safety requirements. This approach would, of course, be transient with transgene expression lasting only a few days (53), but might be accomplished with multiple autologous dosings. Temporary transgene expression presents lower risk of off-target effects such as bystander suppression of anti-tumor or anti-infection immunity. However, lentivirus transduced Treg avatars likely offer greater potential for long-term efficacy in clearing islet infiltration/inflammation and leading to persistent engraftment.

Importantly, adoptive cell therapy with polyclonal autologous peripheral blood Tregs has been demonstrated to be safe in Phase I clinical trials (26, 27). While we anticipate a similar safety profile with TCR transgenic Treg therapy, tolerogenic cell therapies always carry with them possible associations with increased risk of infection or cancer due to bystander suppression and infectious tolerance mechanisms. Thus, there is a need to perform Phase I safety studies and simultaneously, investigate co-transfection of suicide genes for inducible apoptosis of TCR transgenic Tregs—a biological “off-switch” (54).

We recently demonstrated that cryopreserved umbilical cord blood Tregs (cryoCB Tregs) could be isolated and expanded efficiently while retaining their naïve phenotype as well as suppressive capacity (55). Beyond the possibility for polyclonal autologous cryoCB Treg therapy, these cells offer the potential to generate antigen-specific Treg avatars from precursors with an optimal naïve phenotype and without the need for a large-volume peripheral blood draw and leukapheresis, which is generally contraindicated in pediatric patients. This is a goal currently being pursued by our lab and others. Additional optimization, such as further genetic manipulation of TCR transgenic Tregs, could be implemented to correct intrinsic single-nucleotide polymorphisms (SNPs) with putative implications for Treg function and known associations with type 1 diabetes as identified by genome-wide association studies (GWAS) (56). Beyond this, there is potential for delivery of tissue repair factors directly to the pancreas via production by antigen-specific Tregs or via conjugation to the Treg surface using poly lactic-co-glycolic acid (PLGA) nanoparticles (57–59). For these approaches to be successful, we expect that Treg survival in vivo and trafficking to the target organ will depend largely upon TCR specificity. Hence, we anticipate the functional effects of TCR avidity on human Treg phenotype and function, as demonstrated herein, will be extremely important as we refine adoptive cell therapies to reverse autoimmunity in type 1 diabetes.

Healthy control blood donors provided written informed consent prior to inclusion in the study in accordance with the Declaration of Helsinki and according to Institutional Review Board-approved protocols at the University of Florida (Protocol no. IRB201500059) and the University of Colorado Denver (Protocol no. COMIRB92-292).

As the guarantor of this work, Todd Brusko assumes responsibility for ethical completion of the study, integrity of the data, and accuracy of the data analysis reported herein.

W-IY researched and analyzed the data and wrote the manuscript. HS and BN researched the data and reviewed/edited the manuscript. AP and FM contributed to discussion and wrote the manuscript. AM and CM contributed to discussion and reviewed/edited the manuscript. JB conceived of the study and reviewed/edited the manuscript. TB conceived of the study, researched the data, and wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.