Highlights d Longitudinal scRNA-seq of tumor-specific TCF-1 + CD8 + T cells in KP lung adenocarcinoma d Identified a proliferative Slamf6 + TCF-1 + T cell subset and a non-cycling SlamF6 À subset d The lymph node contains a recruitable reservoir of functional TCF-1 + CD8 + T cells d Flt3L+CD40 boosts cDC1, increases TCF-1 + CD8 + T cell frequencies, decreases tumor burden

Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the "immunopeptidome". Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the Kras LSL-G12D/+ ; p53 fl/fl (KP) mouse model (KP; K b Strep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the K b Strep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

23Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes 24 cellular homeostasis through the degradation of cytosolic components, also known as cargo. 25During autophagy, cargo is sequestered into double-membrane vesicles called 26 autophagosomes, which are predominantly transported in the retrograde direction to the 27 perinuclear region to fuse with lysosomes, thus ensuring cargo degradation [1]. The 28 mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family 29 of proteins associate with autophagosome membranes [2] and play key roles in autophagy, 30 such as the movement of autophagosomes. This is achieved via the interaction of ATG8 with 31 adaptor proteins, including FYCO1, a protein involved in the anterograde transport of 32 autophagosomes toward the cell periphery [1,[3][4][5]. We previously reported that phosphorylation 33 of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4 is required for autophagy 34 through unknown mechanisms [6]. Here, we show that LC3B-T50 phosphorylation decreases 35 the interaction between LC3B and FYCO1, which in turn regulates the starvation-induced 36 perinuclear positioning of autophagosomes. Moreover, non-phosphorylatable LC3B-T50A 37 aberrantly switches the predominant retrograde movement of autophagosomes to anterograde 38 movement towards the cell periphery in multiple cell types, including in mouse primary 39 hippocampal neurons. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis 40 in the regulation of the directional transport of autophagosomes via the post-translational 41 regulation of LC3B. Given that autophagy is impaired in many human conditions, including 42 neurodegenerative diseases, our findings may highlight new principles of vesicle transport 43 regulation critical for disease etiology.44 3 RESULTS 46 LC3B-T50 Phosphorylation Regulates the Interaction Between LC3B and FYCO1 47We recently reported that phosphorylation of LC3B-T50 by STK4 is required for autophagy [6]. 48Specifically, depletion of Stk4 or expression of a non-phosphorylatable LC3B-T50A mutant 49 causes accumulation of autophagosomes throughout the cell and clustering of lysosomes 50 around the nucleus, leading to a block in autophagy in mouse embryonic fibroblasts (MEFs) and 51 C2C12 mouse myoblasts [6]. We hypothesized that phosphorylation of LC3B might modulate its 52 interaction with binding partners such as autophagy adaptor proteins. To test this, we transiently 53 expressed HA-tagged wild-type (WT) LC3B or the phospho-mutant forms LC3B-T50A (T50A, 54 phospho-deficient) and LC3B-T50E (T50E, phospho-mimetic) in human 293T cells, affinity 55 purified the HA-tagged LC3B proteins, and identified the associated proteins by mass 56 spectrometry (Figure 1A). Of the LC3B interactors identified in this analysis, FYCO1, a known 57 autophagy adaptor protein [3,4], showed the most LC3B-T50 phosphorylation-sensitive binding; 58 specifically, FYCO1 binding was ~2-fold higher to the T50A mutant and ~5-fold ...

Highlights d STK4-mediated LC3B phosphorylation lowers LC3B binding to the transport protein FYCO1 d LC3B phosphorylation inhibition decreases retrograde transport of autophagosomes d Block of LC3B phosphorylation also compromises autophagosome-lysosome association d The STK4-LC3B-FYCO1 axis is a nutrient-sensitive, autophagy-regulatory pathway