Eras of designer Tregs: Harnessing synthetic biology for immune suppression.

Since their discovery, CD4+ CD25hi FOXP3hi regulatory T cells (Tregs) have been firmly established as a critical cell type for regulating immune homeostasis through a plethora of mechanisms. Due to their immunoregulatory power, delivery of polyclonal Tregs has been explored as a therapy to dampen inflammation in the settings of transplantation and autoimmunity. Evidence shows that Treg therapy is safe and well-tolerated, but efficacy remains undefined and could be limited by poor persistence in vivo and lack of antigen specificity. With the advent of new genetic engineering tools, it is now possible to create bespoke "designer" Tregs that not only overcome possible limitations of polyclonal Tregs but also introduce new features. Here, we review the development of designer Tregs through the perspective of three 'eras': (1) the era of FOXP3 engineering, in which breakthroughs in the biological understanding of this transcription factor enabled the conversion of conventional T cells to Tregs; (2) the antigen-specificity era, in which transgenic T-cell receptors and chimeric antigen receptors were introduced to create more potent and directed Treg therapies; and (3) the current era, which is harnessing advanced genome-editing techniques to introduce and refine existing and new engineering approaches. The year 2022 marked the entry of "designer" Tregs into the clinic, with exciting potential for application and efficacy in a wide variety of immune-mediated diseases.

Multiplex Imaging for Cell Phenotyping of Early Human Atherosclerosis.

BACKGROUND: Previous studies using animal models and cultured cells suggest that vascular smooth muscle cells (SMCs) and inflammatory cytokines are important players in atherogenesis. Validating these findings in human disease is critical to designing therapeutics that target these components. Multiplex imaging is a powerful tool for characterizing cell phenotypes and microenvironments using biobanked human tissue sections. However, this technology has not been applied to human atherosclerotic lesions and needs to first be customized and validated. METHODS AND RESULTS: For validation, we created an 8-plex imaging panel to distinguish foam cells from SMC and leukocyte origins on tissue sections of early human atherosclerotic lesions (n=9). The spatial distribution and characteristics of these foam cells were further analyzed to test the association between SMC phenotypes and inflammation. Consistent with previous reports using human lesions, multiplex imaging showed that foam cells of SMC origin outnumbered those of leukocyte origin and were enriched in the deep intima, where the lipids accumulate in early atherogenesis. This new technology also found that apoptosis or the expression of pro-inflammatory cytokines were not more associated with foam cells than with nonfoam cells in early human lesions. More CD68+ SMCs were present among SMCs that highly expressed interleukin-1ß. Highly inflamed SMCs showed a trend of increased apoptosis, whereas leukocytes expressing similar levels of cytokines were enriched in regions of extracellular matrix remodeling. CONCLUSIONS: The multiplex imaging method can be applied to biobanked human tissue sections to enable proof-of-concept studies and validate theories based on animal models and cultured cells.

Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Sex biased human thymic architecture guides T cell development through spatially defined niches.

Within the thymus, regulation of the cellular cross-talk directing T cell development is dependent on spatial interactions within specialized niches. To create a holistic, spatially defined map of tissue niches guiding postnatal T cell development we employed the multidimensional imaging platform CO-detection by indEXing (CODEX), as well as CITE-seq and ATAC-seq. We generated age-matched 4-5-month-old postnatal thymus datasets for male and female donors, and identify significant sex differences in both T cell and thymus biology. We demonstrate a crucial role for JAG ligands in directing thymic-like dendritic cell development, reveal important functions of a novel population of ECM- fibroblasts, and characterize the medullary niches surrounding Hassall's corpuscles. Together, these data represent a unique age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and T cell development arise, and provide an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

Manufacturing next-generation regulatory T-cell therapies.

Regulatory T-cell (Treg) therapy has shown promise in treating autoimmune diseases, transplant rejection, or graft-versus-host disease in early clinical trials. These trials have demonstrated that cell therapy using polyclonal Tregs is feasible and safe, however, the field has been limited by the lack of polyclonal cell specificity and consequent large cell numbers required, and the difficulty in generating autologous products for some patients. Thus, the field is moving toward 'next generation' Treg cell therapies that include genetic modification strategies to engineer specificity and/or modify function, as well as methods to generate Tregs in vitro. In this review, we describe how genetic modification of Tregs using viral transduction or gene editing may be incorporated into Treg manufacturing protocols. We also describe how Tregs may be generated via FOXP3 gene editing or overexpression, or by differentiation from pluripotent stem cells. The application of these various types of engineered Tregs is discussed.

Optogenetic Control of Heterologous Metabolism in E. coli.

Multiobjective optimization of microbial chassis for the production of xenobiotic compounds requires the implementation of metabolic control strategies that permit dynamic distribution of cellular resources between biomass and product formation. We addressed this need in a previous study by engineering the T7 RNA polymerase to be thermally responsive. The modified polymerase is activated only after the temperature of the host cell falls below 18 °C, and Escherichia coli cells that employ the protein to transcribe the heterologous lycopene biosynthetic pathway exhibit impressive improvements in productivity. We have expanded our toolbox of metabolic switches in the current study by engineering a version of the T7 RNA polymerase that drives the transition between biomass and product formation upon stimulation with red light. The engineered polymerase is expressed as two distinct polypeptide chains. Each chain comprises one of two photoactive components from Arabidopsis thaliana, phytochrome B (PhyB) and phytochrome-integrating factor 3 (PIF3), as well as the N- or C-terminus domains of both, the vacuolar ATPase subunit (VMA) intein of Saccharomyces cerevisiae and the polymerase. Red light drives photodimerization of PhyB and PIF3, which then brings together the N- and C-terminus domains of the VMA intein. Trans-splicing of the intein follows suit and produces an active form of the polymerase that subsequently transcribes any sequence that is under the control of a T7 promoter. The photodimerization also involves a third element, the cyanobacterial chromophore phycocyanobilin (PCB), which too is expressed heterologously by E. coli. We deployed this version of the T7 RNA polymerase to control the production of lycopene in E. coli and observed tight control of pathway expression. We tested a variety of expression configurations to identify one that imposes the lowest metabolic burden on the strain, and we subsequently optimized key parameters such as the source, moment, and duration of photostimulation. We also identified targets for future refinement of the circuit. In summary, our work is a significant advance for the field and greatly expands on previous work by other groups that have used optogenetic circuits to control heterologous metabolism in prokaryotic hosts.