Single-mitosis dissection of acute and chronic DNA mutagenesis and repair.

How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.

Rgs16 promotes antitumor CD8+ T cell exhaustion.

T cells become functionally exhausted in tumors, limiting T cell-based immunotherapies. Although several transcription factors regulating the exhausted T (Tex) cell differentiation are known, comparatively little is known about the regulators of Tex cell survival. Here, we reported that the regulator of G protein signaling 16 (Rgs-16) suppressed Tex cell survival in tumors. By performing lineage tracing using reporter mice in which mCherry marked Rgs16-expressing cells, we identified that Rgs16+CD8+ tumor-infiltrating lymphocytes (TILs) were terminally differentiated, expressed low levels of T cell factor 1 (Tcf1), and underwent apoptosis as early as 6 days after the onset of Rgs16 expression. Rgs16 deficiency inhibited CD8+ T cell apoptosis and promoted antitumor effector functions of CD8+ T cells. Furthermore, Rgs16 deficiency synergized with programmed cell death protein 1 (PD-1) blockade to enhance antitumor CD8+ T cell responses. Proteomics revealed that Rgs16 interacted with the scaffold protein IQGAP1, suppressed the recruitment of Ras and B-Raf, and inhibited Erk1 activation. Rgs16 deficiency enhanced antitumor CD8+ TIL survival in an Erk1-dependent manner. Loss of function of Erk1 decreased antitumor functions of Rgs16-deficient CD8+ T cells. RGS16 mRNA expression levels in CD8+ TILs of patients with melanoma negatively correlated with genes associated with T cell stemness, such as SELL, TCF7, and IL7R, and predicted low responses to PD-1 blockade. This study uncovers Rgs16 as an inhibitor of Tex cell survival in tumors and has implications for improving T cell-based immunotherapies.

Skeletal muscle antagonizes antiviral CD8+ T cell exhaustion.

CD8+ T cells become functionally impaired or "exhausted" in chronic infections, accompanied by unwanted body weight reduction and muscle mass loss. Whether muscle regulates T cell exhaustion remains incompletely understood. We report that mouse skeletal muscle increased interleukin (IL)-15 production during LCMV clone 13 chronic infection. Muscle-specific ablation of Il15 enhanced the CD8+ T cell exhaustion phenotype. Muscle-derived IL-15 was required to maintain a population of CD8+CD103+ muscle-infiltrating lymphocytes (MILs). MILs resided in a less inflamed microenvironment, expressed more T cell factor 1 (Tcf1), and had higher proliferative potential than splenic T cells. MILs differentiated into functional effector T cells after reentering lymphoid tissues. Increasing muscle mass via muscle-specific inhibition of TGFß signaling enhanced IL-15 production and antiviral CD8+ T cell responses. We conclude that skeletal muscle antagonizes T cell exhaustion by protecting T cell proliferative potential from inflammation and replenishing the effector T cell progeny pool in lymphoid organs.

T Cell Factor 1 Suppresses CD103+ Lung Tissue-Resident Memory T Cell Development.

T cell factor 1 (Tcf1) promotes the central memory CD8+ T (TCM) cell differentiation and stemness in lymphoid tissues after systemic infections. It remains unclear whether Tcf1 regulates the CD103high tissue-resident memory CD8+ T (TRM) cell formation in non-lymphoid tissues after mucosal infections. We find that Tcf1 is progressively decreased during lung TRM cell formation. Abrogation of transforming growth factor ß (TGF-ß) signaling is associated with a loss of CD103+ and reciprocal gain of Tcf1+ cells among TRM precursors in vivo. T-cell-specific ablation of Tcf7 enhances CD103 protein expression in TRM cells and precursors and increases TRM cell numbers after primary and secondary infections. Tcf1 directly binds to the Itgae (encoding CD103) locus and partly inhibits TGF-ß-induced CD103 expression. Our study suggests that memory T cell tissue residency and homeostatic proliferation are reciprocally regulated by Tcf1. Tcf1 may play either immunosupportive or immunosuppressive roles in CD8+ T cells, depending on systemic or mucosal infections.