233 Human PD-L2 triggers a unique T cell inhibitory program through PD-1 engagement distinct from that of PD-L1

BackgroundPD-1/PD-L1 blockade is responsible for the majority of the success of cancer immunotherapy.1 However, only 14% of patients eligible to receive checkpoint blockade achieve objected clinical responses.2 3 The reason for the failure of PD-L1 blockade may be attributed to the recently appreciated widespread expression of PD-L2 across human cancers and its immunosuppresive stromal cells.4 PD-L2 expression was shown to be as or more predictive of response to PD-1 blockade than PD-L14. PD-L2 traditionally was dismissed as functionally redundant to PD-L1 varying only in pattern of expression. We hypothesize that PD-L2 engages PD-1 to generate a distinct inhibitory signal from that of PD-L1, and antibody mediated blockade and depletion of PD-L2+ cells may promote anti-tumor immunity that is superior to PD-L1 blockade alone.MethodsCell based bioluminescent assay demonstrated the nature of regulation mediated by human PD-L2 through the PD-1 co-receptor. RNA-sequencing identified key differences in the signaling pathways generated in Jurkat T cells by PD-1 binding to PD-L1 or PD-L2. Multidimensional flow cytometry determined the differential effects of PD-L1 and PD-L2 on human T cell proliferation and effector function. Western blot elucidated the temporal kinetics of inhibition mediated by PD-L1 and PD-L2. Survival studies in murine syngeneic lymphoma model evaluated the efficacy of antibody mediated blockade and depletion of PD-L2+ cells.ResultsWe validated that human PD-L2, unlike murine PD-L2, generates a purely co-inhibitory signal in human T cells, albeit with a reduced inhibitory potential relative to PD-L1. We discovered significant differences in downstream T cell signaling pathways generated by PD-L1 versus PD-L2 through PD-1 engagement. Human PD-L1 and PD-L2 differentially modulated T cell effector function and proliferation with PD-L2 preferentially arresting T cells in S-phase of cell cycle. PD-L1 and PD-L2 also differed in the temporal kinetics of dephosphorylation of the membrane proximal proteins in the TCR-CD3 signaling complex. We observed that combination blockade of PD-L1 and PD-L2 improves on blockade of PD-L1 alone resulting in increased production of IL-2 and IFNγ in primary human mixed lymphocyte reactions. Our data in a syngeneic murine model of EL4 showed that effector-function capable PD-L2 blocking antibodies are therapeutically superior to PD-L1 or PD-L2 blockade alone.ConclusionsWe are the first to report on T cell immunoregulatory functions of PD-L2 which are distinct from those of PD-L1, and demonstrate that the more tumor-selective expression pattern of PD-L2 relative to PD-L1 provides a therapeutic advantage to effector-function capable PD-L2 antibodies.AcknowledgementsAS was supported by the CPRIT Research Training Grant(RP170067)ReferencesRibas A, Wolchok JD (2018). Cancer immunotherapy using checkpoint blockade. Science 359:1350–1355.Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A, McClanahan TK, Tomassini JE, Loboda A, Kaufman D (2018). Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science 362.Haslam A, Prasad V (2019). Estimation of the percentage of US patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs. JAMA Netw Open 2:e192535.Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, Lunceford J, Cheng J, Chow LQM, Seiwert TY, Handa M, Tomassini JE, McClanahan T (2017). PD-L2 expression in human tumors: relevance to anti-PD-1 therapy in cancer. Clin Cancer Res 23:3158–3167.

Task context determines whether common or separate inhibitory signals underlie the control of eye-hand movements

Whereas inhibitory control of single effector movements has been widely studied, the control of coordinated eye-hand movements has received less attention. Nevertheless, previous studies have contradictorily suggested that either a common or separate signal/s is/are responsible for inhibition of coordinated eye-hand movements. In continuation of our previous study, we varied behavioral contexts and used a stochastic accumulation-to-threshold model, which predicts a scaling of the mean reaction time distribution with its variance, to study the inhibitory control of eye-hand movements. Participants performed eye-hand movements in different task conditions, and in each condition they had to redirect movements in a fraction of trials. Task contexts where the behavior could be best explained by a common initiation signal had similar error responses for eye and hand, despite having different mean reaction times, indicating a common inhibitory signal. In contrast, behavior that could be best explained by separate initiation signals had dissimilar error responses for eye and hand indicating separate inhibitory signals. These behavioral responses were further validated using electromyography and computational models having either a common or separate inhibitory control signal/s. Interestingly, in a particular context, whereas in majority trials a common initiation and inhibitory signal could explain the behavior, in a subset of trials separate initiation and inhibitory signals predicted the behavior better. This highlights the flexibility that exists in the brain and in effect reconciles the heterogeneous results reported by previous studies. NEW & NOTEWORTHY Prior studies have contradictorily suggested either a single or separate inhibitory signal/s underlying inhibition of coordinated eye-hand movements. With the use of different tasks, we observed that when eye-hand movements were initiated by a common signal, they were controlled by a common inhibitory signal. However, when the two effectors were initiated by separate signals, they were controlled by separate inhibitory signals. This highlights the flexible control of eye-hand movements and reconciles the heterogeneous results previously reported in the literature.

Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120

ABSTRACT Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning. IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis

During cytokinesis, a signal from the central spindle that forms between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.

A CD200R-CD28 fusion protein appropriates an inhibitory signal to enhance T-cell function and therapy of murine leukemia

Key Points IFPs can convert signals from inhibitory ligands into activating signals. Costimulation was most effectively achieved by engineering the IFP to promote the ability to localize in the immunological synapse.