Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.

NK cell exhaustion (NCE) due to sustained proliferation results in impaired NK cell function with loss of cytokine production and lytic activity. Using murine models of chronic NK cell stimulation, we have identified a phenotypic signature of NCE, characterized by upregulation of the terminal differentiation marker KLRG1 and by downregulation of eomesodermin and the activating receptor NKG2D. Chronic stimulation of mice lacking NKG2D resulted in minimal NCE compared with control mice, thus identifying NKG2D as a crucial mediator of NCE. NKG2D internalization and downregulation on NK cells have been previously observed in the presence of tumor cells with high expression of NKG2D ligands (NKG2DL) due to the activation of the DNA damage repair pathways. Interestingly, our study revealed that during NK cell activation, there is an increase of MULT1, an NKG2DL, that correlates with an induction of DNA damage. Treatment with the ATM DNA damage repair pathway inhibitor KU55933 (KU) during activation reduced NCE by improving expression of activation markers and genes involved in cell survival, through sustaining NKG2D expression and preserving cell functionality. Importantly, NK cells expanded ex vivo in the presence of KU displayed increased antitumor efficacy in both NKG2D-dependent and -independent mouse models. Collectively, these data demonstrate that NCE is caused by DNA damage and is regulated, at least in part, by NKG2D. Further, the prevention of NCE is a promising strategy to improve NK cell–based immunotherapy.

Key Points: TNF-α produced during aGVHD is a strong and selective activator of CD4+CD25+FoxP3+ Tregs. In vitro TNF-α priming enhances CD4+CD25+FoxP3+ Treg proliferation and their ability to protect from GVHD.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

The potential of dendritic cell (DC) vaccination against cancer is not fully achieved. Little is known about the precise nature of the anti-cancer immune response triggered by different natural DC subsets and their relevance in preventing postsurgical tumor recurrence. Here, we use mouse splenic conventional DC1s (cDC1s) or cDC2s pulsed with tumor cell lysates to generate DC vaccines. cDC1-based vaccination induces a stronger effector and memory CD4⁺ and CD8⁺ anti-tumor T cell response, leading to a better control of tumors treated either therapeutically or prophylactically. Using an experimental model of tumor relapse, we show that adjuvant or neoadjuvant cDC1 vaccination improves anti-tumor immune memory, particularly by increasing the infiltrates of CD4⁺ tissue resident memory (Trm) and CD8⁺ memory T cells. This translates into complete prevention of tumor relapses. Moreover, elevated abundance of cDC1s positively correlates with CD4⁺ Trm presence, and both associate with enhanced survival in human breast cancer and melanoma. Our findings suggest that cDC1-based vaccination excels at immune memory induction and prevention of cancer recurrence.