Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes

Abstract

The ability to alter antigen specificity by T-cell receptor (TCR) or chimeric antigen receptor (CAR) gene transfer has facilitated personalized cellular immune therapies in cancer. Inversely, this approach can be harnessed in autoimmune settings to attenuate inflammation by redirecting the specificity of regulatory T cells (Tregs). Herein, we demonstrate efficient protocols for lentiviral gene transfer of TCRs that recognize type 1 diabetes-related autoantigens with the goal of tissue-targeted induction of antigen-specific tolerance to halt β-cell destruction. We generated human Tregs expressing a high-affinity GAD555–567-reactive TCR (clone R164), as well as the lower affinity clone 4.13 specific for the same peptide. We demonstrated that de novo Treg avatars potently suppress antigen-specific and bystander responder T-cell (Tresp) proliferation in vitro in a process that requires Treg activation (P < 0.001 versus unactivated Tregs). When Tresp were also glutamic acid decarboxylase (GAD)-reactive, the high-affinity R164 Tregs exhibited increased suppression (P < 0.01) with lower Tresp-division index (P < 0.01) than the lower affinity 4.13 Tregs. These data demonstrate the feasibility of rapid expansion of antigen-specific Tregs for applications in attenuating β-cell autoimmunity and emphasize further opportunities for engineering cellular specificities, affinities, and phenotypes to tailor Treg activity in adoptive cell therapies for the treatment of type 1 diabetes.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood

“…These results coupled with our data support the notion that CB Tregs represent a population with increased phenotypic homogeneity alongside increased receptor diversity compared to APB Treg, thus serving as an ideal candidate for ACT in autoimmune diseases. Additionally, the potential to generate TCR redirected or chimeric antigen receptor (CAR) Treg has become a topic of great interest in the immunotherapy space (138)(139)(140). Our data suggest that the phenotypic stability and homogeneity of CB Tregs relative to APB might ameliorate concerns over lineage stability with TCR or CAR-directed Treg therapies.…”

Treg Enhancing Therapies to Treat Autoimmune Diseases

“…Such technologies have the capacity to sequence and identify the TCRs alongside the whole transcriptome and TCR–peptide–MHC dextramer binding data of tens of thousands of single cells simultaneously [ 93 , 94 ]. Numerous TCRs have been used to generate human TCR-Tregs to target various autoimmune diseases, such as T1D, MS and acquired factor VIII deficiency [ 95 , 96 , 97 ].…”

“…The advantageous effect of Tregs transduced with an auto-antigen specific, high-affinity TCR is their increased potency in suppressive function compared to Tregs transduced with a non-specific TCR or polyclonal Tregs. These more potent Tregs are better equipped to restore dominant immune tolerance, leading to complete remission of the targeted autoimmune disease [ 95 ].…”

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

“…To ascertain antigen specificity of TCRs identified by single cell sequencing, T cells are engineered to express TCRs of interest by retroviral or lentiviral gene transduction systems (47)(48)(49) and are used to test reactivity to peptide-MHC complexes. Simultaneous examinations of multiple TCRs in a single well saves both time and reagents, which is beneficial when numerous TCRs and antigens are examined.…”