Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire. CTLA-4 imposes major boundaries on CD4+ T cell phenotypes, whereas PD-1 subtly limits CD8+ T cell phenotypes. By computationally reconstructing T cell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in T cell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of T cell differentiation and suggest that checkpoint blockade might work in part by altering the limits of T cell phenotypes.

Post-immunotherapy CTLA-4 Ig treatment improves antitumor efficacy.

Immune checkpoint therapies (ICT) improve overall survival of patients with cancer but may cause immune-related adverse events (irAEs) such as myocarditis. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4 Ig), an inhibitor of T cell costimulation through CD28, reverses irAEs in animal models. However, concerns exist about potentially compromising antitumor response of ICT. In mouse tumor models, we administered CTLA-4 Ig 1) concomitantly with ICT or 2) after ICT completion. Concomitant treatment reduced antitumor efficacy, while post-ICT administration improved efficacy without affecting frequency and function of CD8 T cells. The improved response was independent of the ICT used, whether CTLA-4 or PD-1 blockade. The frequency of Tregs was significantly decreased with CTLA-4 Ig. The resulting increased CD8/Treg ratio potentially underlies the enhanced efficacy of ICT followed by CTLA-4 Ig. This paradoxical mechanism shows that a CTLA-4 Ig regimen shown to reduce irAE severity does not compromise antitumor efficacy.

Combination anti-CTLA-4 plus anti-PD-1 checkpoint blockade utilizes cellular mechanisms partially distinct from monotherapies.

Immune checkpoint blockade therapy targets T cell-negative costimulatory molecules such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1). Combination anti-CTLA-4 and anti-PD-1 blockade therapy has enhanced efficacy, but it remains unclear through what mechanisms such effects are mediated. A critical question is whether combination therapy targets and modulates the same T cell populations as monotherapies. Using a mass cytometry-based systems approach, we comprehensively profiled the response of T cell populations to monotherapy and combination anti-CTLA-4 plus anti-PD-1 therapy in syngeneic murine tumors and clinical samples. Most effects of monotherapies were additive in the context of combination therapy; however, multiple combination therapy-specific effects were observed. Highly phenotypically exhausted cluster of differentiation 8 (CD8) T cells expand in frequency following anti-PD-1 monotherapy but not combination therapy, while activated terminally differentiated effector CD8 T cells expand only following combination therapy. Combination therapy also led to further increased frequency of T helper type 1 (Th1)-like CD4 effector T cells even though anti-PD-1 monotherapy is not sufficient to do so. Mass cytometry analyses of peripheral blood from melanoma patients treated with immune checkpoint blockade therapies similarly revealed mostly additive effects on the frequencies of T cell subsets along with unique modulation of terminally differentiated effector CD8 T cells by combination ipilimumab plus nivolumab therapy. Together, these findings indicate that dual blockade of CTLA-4 and PD-1 therapy is sufficient to induce unique cellular responses compared with either monotherapy.

Optogenetic probing of functional brain circuitry.

Recently developed optogenetic technologies offer the promise of high-speed mapping of brain circuitry. Genetically targeted light-gated channels and pumps, such as channelrhodopsins and halorhodopsin, allow optical control of neuronal activity with high spatial and temporal resolution. Optogenetic probes of neuronal activity, such as Clomeleon and Mermaid, allow light to be used to monitor the activity of a genetically defined population of neurons. Combining these two complementary sets of optogenetic probes will make it possible to perform all-optical circuit mapping. Owing to the improved efficiency and higher speed of data acquisition, this hybrid approach should enable high-throughput mapping of brain circuitry.

Checkpoint Blockade + Chemotherapy: the Right Combination for AML?

An emerging strategy to enhance the efficacy of immune checkpoint blockade in relapsed/refractory cancers is increasing immunogenic cell death via combination with cytotoxic therapies. Understanding the effects of cytotoxic and immunotherapeutic agents on immune cell populations will enable improved mechanism-based design of combination therapies to maximum efficacy and minimum toxicity.See related article by Zeidner et al., p. 616.

A Genetic Mouse Model Recapitulates Immune Checkpoint Inhibitor-Associated Myocarditis and Supports a Mechanism-Based Therapeutic Intervention.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis. SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4-Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521.

Distribution of RGS9-2 in neurons of the mouse striatum.

Regulators of G protein signaling (RGS) proteins negatively modulate G protein-coupled receptor (GPCR) signaling activity by accelerating G protein hydrolysis of GTP, hastening pathway shutoff. A wealth of data from cell culture experiments using exogenously expressed proteins indicates that RGS9 and other RGS proteins have the potential to down-regulate a significant number of pathways. We have used an array of biochemical and tissue staining techniques to examine the subcellular localization and membrane binding characteristics of endogenous RGS9-2 and known binding partners in rodent striatum and tissue homogenates. A small fraction of RGS9-2 is present in the soluble cytoplasmic fraction, whereas the majority is present primarily associated with the plasma membrane and structures insoluble in non-ionic detergents that efficiently extract the vast majority of its binding partners, R7BP and G(beta5). It is specifically excluded from the cell nucleus in mouse striatal tissue. In cultured striatal neurons, RGS9-2 is found at extrasynaptic sites primarily along the dendritic shaft near the spine neck. Heterogeneity in RGS9-2 detergent solubility along with its unique subcellular localization suggests that its mechanism of membrane anchoring and localization is complex and likely involves additional proteins beside R7BP. An important nuclear function for RGS9-2 seems unlikely.

Analyzing outcomes of neoadjuvant and adjuvant treatment for borderline-resectable pancreatic adenocarcinoma in the perioperative period at an academic institution.

BACKGROUND: Only 15-20% of pancreatic ductal adenocarcinoma (PDAC) patients are upfront surgical candidates at presentation, and for this cohort of patients, the 5-year survival is a mere 20% despite adjuvant therapy. Previous data indicate that in clinical practice most of these cases are "borderline-resectable," and there is currently no mature data on perioperative treatment. METHODS: We performed a retrospective electronic chart review of patients with "borderline-resectable"PDAC treated at an academic comprehensive cancer center, dividing them into groups based on surgery alone, surgery plus neoadjuvant, adjuvant, or neoadjuvant plus adjuvant perioperative treatment groups. The objectives were to determine the median overall survival (mOS), progression-free survival (PFS) and disease-free survival (DFS). Statistical analysis was performed to assess the association of demographic, tumor traits, and interventions with OS, PFS and DFS. RESULTS: Only surgery followed by adjuvant therapy showed an increase in mOS [hazard ratio (HR) 0.22; 95% CI, 0.09-0.51; P<0.001), after adjustment for radiation (yes vs. no), resection margins (R0 vs. R1 or R2), and tumor location (head vs. body or tail). Patients who received adjuvant therapy after surgery had 2.1 times greater odds to be alive at 24 months after diagnosis than those who had surgery alone (P=0.015). PFS and DFS were not statistically significantly different among treatment groups after adjustment. Those whose disease was located in the head of the pancreas had a significantly improved OS (HR =0.27; 95% CI, 0.11-0.64; P=0.003), PFS (HR =0.40; 95% CI, 0.17-0.94; P=0.035), and DFS (HR =0.30; 95% CI, 0.13-0.67; P=0.004). Negative margins led to a significant improvement in PFS (HR =0.30; 95% CI, 0.16-0.57; P<0.001) and DFS (HR =0.30; 95% CI, 0.16-0.57; P<0.001). Those who received radiation had a non-significantly improved OS, PFS, and DFS (P>0.05). CONCLUSIONS: Our study corroborated that patients treated with adjuvant therapy after surgical resection had an mOS benefit as reported on prior phase III clinical trials. Patients with "borderline-resectable" pancreatic cancer are encouraged to participate in a clinical trial or clinically be treated with adjuvant therapy until more mature results from the ongoing perioperative prospective study are available.

STAT3 Inhibits CD103+ cDC1 Vaccine Efficacy in Murine Breast Cancer.

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103+ cDC1s in mice) are necessary to mount CD8+ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103+ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10; thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103+ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3∆/∆) CD103+ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3∆/∆ CD103+ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103+ cDC1s. In addition, vaccination with Stat3∆/∆ CD103+ cDC1s elicited increased amounts of tumor antigen-specific CD8+ T cells and IFN-γ+ CD4+ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103+ cDC1s controlled tumor growth to a similar degree as Stat3∆/∆ CD103+ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103+ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103+ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs.

STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.

QTL analysis of self-selected macronutrient diet intake: fat, carbohydrate, and total kilocalories.

The present study investigated the inheritance of dietary fat, carbohydrate, and kilocalorie intake traits in an F(2) population derived from an intercross between C57BL/6J (fat-preferring) and CAST/EiJ (carbohydrate-preferring) mice. Mice were phenotyped for self-selected food intake in a paradigm which provided for 10 days a choice between two macronutrient diets containing 78/22% of energy as a composite of either fat/protein or carbohydrate/protein. Quantitative trait locus (QTL) analysis identified six significant loci for macronutrient intake: three for fat intake on chromosomes (Chrs) 8 (Mnif1), 18 (Mnif2), and X (Mnif3), and three for carbohydrate intake on Chrs 17 (Mnic1), 6 (Mnic2), and X (Mnic3). An absence of interactions among these QTL suggests the existence of separate mechanisms controlling the intake of fat and carbohydrate. Two significant QTL for cumulative kilocalorie intake, adjusted for baseline body weight, were found on Chrs 17 (Kcal1) and 18 (Kcal2). Without body weight adjustment, another significant kcal locus appeared on distal Chr 2 (Kcal3). These macronutrient and kilocalorie QTL, with the exception of loci on Chrs 8 and X, encompassed chromosomal regions influencing body weight gain and adiposity in this F2 population. These results provide new insight into the genetic basis of naturally occurring variation in nutrient intake phenotypes.

Integration of multiscale dendritic spine structure and function data into systems biology models.

Comprising 10(11) neurons with 10(14) synaptic connections the human brain is the ultimate systems biology puzzle. An increasing body of evidence highlights the observation that changes in brain function, both normal and pathological, consistently correlate with dynamic changes in neuronal anatomy. Anatomical changes occur on a full range of scales from the trafficking of individual proteins, to alterations in synaptic morphology both individually and on a systems level, to reductions in long distance connectivity and brain volume. The major sites of contact for synapsing neurons are dendritic spines, which provide an excellent metric for the number and strength of signaling connections between elements of functional neuronal circuits. A comprehensive model of anatomical changes and their functional consequences would be a holy grail for the field of systems neuroscience but its realization appears far on the horizon. Various imaging technologies have advanced to allow for multi-scale visualization of brain plasticity and pathology, but computational analysis of the big data sets involved forms the bottleneck toward the creation of multiscale models of brain structure and function. While a full accounting of techniques and progress toward a comprehensive model of brain anatomy and function is beyond the scope of this or any other single paper, this review serves to highlight the opportunities for analysis of neuronal spine anatomy and function provided by new imaging technologies and the high-throughput application of older technologies while surveying the strengths and weaknesses of currently available computational analytical tools and room for future improvement.