Jax P. Dunning

Abstract Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance 1,2 . The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity 3–6 . However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8 + T cell function by enhancing JAK–STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.

Abstract Description Memory T cells display heterogeneous migratory patterns. Some recirculate through the blood (TCIRC) while others reside within solid tissues (TRM). Classical models of memory T cell surveillance define TRM and TCIRC as distinct cellular lineages and presume that TRM are permanent residents of their host tissue, persisting there until death. However, recent evidence indicates that divisions between these subsets are not absolute, and some CD8+ TRM egress from their host tissues and join the TCIRC pool (termed ex-TRM). To precisely track the development and maintenance of ex-TRM, we are leveraging a mouse model in which tamoxifen administration permanently fluorescently labels cells expressing CD103, a marker of TRM in barrier tissues. When labeling is induced at a memory post-infection time point, labeled cells gradually accumulate in the blood, lymph nodes, spleen, and liver but decline or remain stable in barrier tissues. This suggests that as tissue-localized TRM naturally wane in the memory phase, they seed a portion of the TCIRC compartment with ex-TRM. These ex-TRM lose expression of some tissue residency markers, including CD103, but retain others, implying partial dedifferentiation from their residency programming. Measuring ex-TRM quality and quantity may provide a strategy to assess regional tissue immunity via blood samples. Ongoing studies are further characterizing ex-TRM ontogeny and differentiation and their functional relevance during recall responses. Funding Sources Supported by NIH 2R37AI084913 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Description For over six decades, LNs have been considered sites of lymphocyte recirculation, with central memory T cells (TCM) assuming the role of primary surveillants. We and others have identified CD8+ memory T cells that establish residency within the LNs (LN TRM), expanding our view of LN immunosurveillance. However, the contribution of LN TRM to host immunity is largely unknown. Here we utilized numerous murine infection models, quantitative immunofluorescent microscopy, and parabiosis surgery to investigate LN TRM abundance, longevity, and function. We demonstrated that mechanical dissociation underestimates the density of LN TRM, and enzymatic digestion revealed that LN TRM account for over 50% of memory T cells in some LNs. Parabiosis surgery revealed that TRM occupy distinct niches in LNs including B cell follicles and the subcapsular sinus. Functionally, compared to TCM, LN TRM more robustly react to recurrent antigen via proinflammatory cytokine production. Further, LN TRM orchestrate intranodal recall responses by bolstering TCM activation, innate cell recruitment, and myeloid cell activation. Despite residency at homeostasis, LN TRM constitutively express tissue-specific homing molecules perhaps positioning them for rapid deployment. Following adoptive transfer and recall, compared to TCM, LN TRM preferentially home to upstream tissues. Thus, in the event of pathogen reemergence, LN TRM make specialized contributions to LN immunosurveillance and regional recall responses. Funding Sources Supported by NIH/NIAID F31 AI176750 Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Antigen-experienced lymphocytes can be equilibrating (continuously migratory between blood and peripheral tissues) or resident (stably surveilling within tissues). Because migration is difficult to measure outside of mice, it is common to extrapolate phenotypic proxies of residence derived from mouse studies to other species. We wished to more rigorously assess the differentiation state and function of equilibrating vs. resident memory CD8+ T cells (TRM) in nonhuman primates (NHP). To this end, we delivered a heterologous prime-boost-boost (HPBB) vaccine to both mice and Indian rhesus macaques that resulted in preternaturally abundant memory CD8 T cells that were distributed in over 30 anatomical sites. We then conducted a constellation of assays to infer migration properties of primate T cells: through single cell genomic analyses of paired mouse vaccinees that underwent parabiotic migration tests, through staged intravascular staining in NHP, and through comparisons of systemic and local routes of immunization. These data informed a cross-species signature that correlated with T cell migration properties. Functional assays were performed in NHP by reactivating memory CD8 T cells in situ and ex vivo, revealing that Trm are uniquely poised to communicate reactivation events to neighboring immune and stromal cells. Funding Sources Bill and Melinda Gates Foundation (OPP1116224, D.M.) NIH grants AI090732 and 5R01AI084913-14 (D.M.) U19AI096187, UM1AI124436 and UM1AI169662 (E.H and R.R.A) NCRR/NIH base grant P51 OD011132 to Emory National Primate Research Center. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Description Antigen experienced T cell subsets are heterogenous, and can be described based on their phenotype, function, and migration properties. Whether antigen stimulation regulates the migration, survival, differentiation, or developmental plasticity of T cell subsets remains unclear. Therefore, we developed a new mouse model that allows dissection between antigen stimulation dynamics and the trajectories of T cell differentiation and maintenance. Specifically, we engineered a novel tamoxifen-inducible TCR-knockout transgenic mouse using a rAAV6 vector encoding for the floxed P14 TCR gene, which allows for the in vivo excision of TCR on a specific fraction of tumor-, acute virus-, or chronic virus-specific T cells. After acute infection with LCMV Armstrong, elimination of the TCR in the effector phase affected the T cell phenotype, promoting differentiation of memory precursor effector cells. Established memory T cells survived without a TCR with no discernable change in phenotype, showing that steady-state memory programs are remarkably TCR-independent, including among resident memory T cells. TCR deletion during chronic infection (30 days after LCMV Clone 13) or cancer caused most cells to die, affecting both the population structure and phenotype. Based on these results, we propose that memory and exhausted T cells can be delineated based on their antigen dependence for survival. Funding Sources JK is supported through the Walter Benjamin-Fellowship of the German Research Foundation (DFG) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)