Nishith R. Reddy

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

To engineer cells that can specifically target the central nervous system (CNS), we identified extracellular CNS-specific antigens, including components of the CNS extracellular matrix and surface molecules expressed on neurons or glial cells. Synthetic Notch receptors engineered to detect these antigens were used to program T cells to induce the expression of diverse payloads only in the brain. CNS-targeted T cells that induced chimeric antigen receptor expression efficiently cleared primary and secondary brain tumors without harming cross-reactive cells outside of the brain. Conversely, CNS-targeted cells that locally delivered the immunosuppressive cytokine interleukin-10 ameliorated symptoms in a mouse model of neuroinflammation. Tissue-sensing cells represent a strategy for addressing diverse disorders in an anatomically targeted manner.

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

T cells with synthetic Notch (synNotch) receptors driving antigen-triggered production of anti-inflammatory payloads. Screening a diverse library of suppression programs, we observed the strongest suppression of cytotoxic T cell attack by the production of both anti-inflammatory factors (interleukin-10, transforming growth factor-β1, programmed death ligand 1) and sinks for proinflammatory cytokines (interleukin-2 receptor subunit CD25). Engineered cells with bespoke regulatory programs protected tissues from immune attack without systemic suppression. Synthetic suppressor T cells protected transplanted beta cell organoids from cytotoxic T cells. They also protected specific tissues from unwanted chimeric antigen receptor (CAR) T cell cross-reaction. Synthetic suppressor T cells are a customizable platform to potentially treat autoimmune diseases, organ rejection, and CAR T cell toxicities with spatial precision.