Characterization of murine lymphocyte activation and exhaustion markers by a 14-color flow cytometry panel.

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8 T cells, helper and regulatory CD4 T cells and natural killer cells in murine whole blood, lymph nodes and tumor.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

Unlabelled: Intratumoral delivery of plasmid IL12 via electroporation (IT-tavo-EP) induces localized expression of IL12 leading to regression of treated and distant tumors with durable responses and minimal toxicity. A key driver in amplifying this local therapy into a systemic response is the magnitude and composition of immune infiltrate in the treated tumor. While intratumoral IL12 typically increases the density of CD3+ tumor-infiltrating lymphocytes (TIL), this infiltrate is composed of a broad range of T-cell subsets, including activated tumor-specific T cells, less functional bystander T cells, as well as suppressive T regulatory cells.

Enhancing clinical and immunological effects of anti-PD-1 with belapectin, a galectin-3 inhibitor.

Background: PD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC).

Methods: We performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404).

Galectin-3 inhibition with belapectin combined with anti-OX40 therapy reprograms the tumor microenvironment to favor anti-tumor immunity.

Treatment with an agonist anti-OX40 antibody (aOX40) boosts anti-tumor immunity by providing costimulation and driving effector T cell responses. However, tumor-induced immune suppression contributes significantly to poor response rates to aOX40 therapy, thus combining aOX40 with other agents that relieve tumor-mediated immune suppression may significantly improve outcomes. Once such target is galectin-3 (Gal-3), which drives tumor-induced immunosuppression by increasing macrophage infiltration and M2 polarization, restricting TCR signaling, and inducing T cell apoptosis.

Enhancing the Generation of Eomes CD8 T Cells Augments the Efficacy of OX40- and CTLA-4-Targeted Immunotherapy.

CTLA-4 blockade in combination with an agonist OX40-specific monoclonal antibody synergizes to augment antitumor immunity through enhanced T-cell effector function, leading to increased survival in preclinical cancer models. We have shown previously that anti-OX40/anti-CTLA-4 combination therapy synergistically enhances the expression of Eomesodermin (Eomes) in CD8 T cells. Eomes is a critical transcription factor for the differentiation and memory function of CD8 T cells.

Arginase Therapy Combines Effectively with Immune Checkpoint Blockade or Agonist Anti-OX40 Immunotherapy to Control Tumor Growth.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors.

Intracellular Galectin-3 Is Essential for OX40-Mediated Memory CD8 T Cell Development.

CD8 T cells are critical mediators of adaptive immunity, and enhancing their function can promote robust responses against invading pathogens and neoplastic cells. In addition to TCR stimulation, the provision of costimulation through ligation of TNFR family members, such as OX40 (CD134), provides essential signals driving T cell differentiation, survival, and memory in part through enhanced IL-2/IL-2R signaling. Interestingly, TCR stimulation in the presence of IL-2 upregulates intracellular expression of the β-galactoside binding protein, Galectin-3 (Gal-3).

NKTR-214 immunotherapy synergizes with radiotherapy to stimulate systemic CD8 T cell responses capable of curing multi-focal cancer.

Background: High-dose radiotherapy (RT) is known to be immunogenic, but is rarely capable of driving clinically relevant abscopal antitumor immunity as monotherapy. RT is known to increase antigen presentation, type I/II interferon responses, and immune cell trafficking to irradiated tumors. Bempegaldesleukin (NKTR-214) is a CD122-preferential interleukin 2 (IL-2) pathway agonist that has been shown to increase tumor-infiltrating lymphocytes, T cell clonality, and increase PD-1 expression.

A bacterial immunomodulatory protein with lipocalin-like domains facilitates host-bacteria mutualism in larval zebrafish.

Stable mutualism between a host and its resident bacteria requires a moderated immune response to control bacterial population size without eliciting excessive inflammation that could harm both partners. Little is known about the specific molecular mechanisms utilized by bacterial mutualists to temper their hosts' responses and protect themselves from aggressive immune attack. Using a gnotobiotic larval zebrafish model, we identified an secreted immunomodulatory protein, AimA.

The role of Galectin-3 in modulating tumor growth and immunosuppression within the tumor microenvironment.

The efficacy of cancer immunotherapy is limited, in part, by the multitude of immunosuppressive mechanisms present within the tumor microenvironment (TME). Galectin-3 (Gal-3) is a lectin that contributes to TME immunosuppression and regulates diverse functions including cellular homeostasis and cancer biology. Increased Gal-3 expression during cancer progression augments tumor growth, invasiveness, metastatic potential, and immune suppression, which highlights the potential use of Gal-3 as a therapeutic target capable of modulating anti-tumor immunity.

The enteric nervous system promotes intestinal health by constraining microbiota composition.

Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition.

Individual Members of the Microbiota Disproportionately Modulate Host Innate Immune Responses.

Predicting host health status based on microbial community structure is a major goal of microbiome research. An implicit assumption of microbiome profiling for diagnostic purposes is that the proportional representation of different taxa determine host phenotypes. To test this assumption, we colonized gnotobiotic zebrafish with zebrafish-derived bacterial isolates and measured bacterial abundance and host neutrophil responses.

Spatial and temporal features of the growth of a bacterial species colonizing the zebrafish gut.

Unlabelled: The vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of an Aeromonas species, representing an abundant genus in the zebrafish gut.

A twist in the tail.

Lipopolysaccharide molecules released by the bacteria Vibrio fischeri when it rotates its flagella prompts its host, the Hawaiian bobtail squid, to prepare for its arrival.

The degree of Helicobacter pylori-triggered inflammation is manipulated by preinfection host microbiota.

Helicobacter pylori infects over 3 billion people worldwide and is the primary risk factor for gastric cancer. Most individuals infected with H. pylori develop only asymptomatic gastritis; however, some develop ulcers or gastric adenocarcinoma.

Helicobacter pylori requires TlpD-driven chemotaxis to proliferate in the antrum.

Different disease outcomes of Helicobacter pylori infection correlate with distinct inflammation patterns. These different inflammatory distributions may be initiated by differences in bacterial localization. One H.

Bacterial chemotaxis modulates host cell apoptosis to establish a T-helper cell, type 17 (Th17)-dominant immune response in Helicobacter pylori infection.

The host inflammatory response to chronic bacterial infections often dictates the disease outcome. In the case of the gastric pathogen Helicobacter pylori, host inflammatory responses result in outcomes that range from moderate and asymptomatic to more severe with concomitant ulcer or cancers. It was found recently that H.

A cytoskeletal-based perimeter fence selectively corrals a sub-population of cell surface Kv2.1 channels.

The Kv2.1 delayed-rectifier channel trafficks to 1-3 microm(2) clusters on the surface of neurons and transfected HEK cells. Single quantum dot (Qdot) tracking and FRAP approaches were used to quantify the diffusion of GFP-labeled Kv2.

Kv2.1 potassium channels are retained within dynamic cell surface microdomains that are defined by a perimeter fence.

Ion channel localization to specific cell surface regions is essential for proper neuronal function. The Kv2.1 K+ channel forms large clusters on the plasma membrane of hippocampal neurons and transfected human embryonic kidney (HEK) cells.