IMMU-15. INVESTIGATING CD8 T CELL EXHAUSTION STATES GLIOBLASTOMA

BACKGROUND Glioblastoma (GBM) is the most common primary brain cancer in adults and remains universally lethal. Median survival remains a bleak 15-17 months from time of diagnosis, and current immunotherapeutic efficacy continues to be hindered by the robust immunosuppression present in the GBM microenvironment. T cells, critical for tumor clearance, are particularly affected, and many take on a functionally exhausted phenotype within the tumor. Importantly, two exhaustion states, progenitor and terminal, have been identified in models of chronic infection and cancer. This distinction is particularly relevant, as progenitor exhausted T cells can respond favorably to immune checkpoint blockade, while terminally exhausted T cells are resistant. To date, the dynamics and characteristics of these exhausted populations in GBM remain unclear. RESULTS In an orthotopic murine model of GBM, progenitor and terminal exhausted CD8 T cells were identified by flow cytometry as PD1+SLAMF6+ and PD1+TIM3+, respectively. Using a time-course approach, we detected progenitor exhaustion by day 8 in the tumor, but not in draining lymph nodes. Additionally, we show that the frequency of progenitor exhaustion is highest during early tumor progression, while terminal exhaustion is the most abundant in more advanced tumors ( >14 days). Functional differences between subsets were evaluated via intracellular staining of IFNγ, TNFα, granzyme B, and Ki67. Terminally exhausted T cells displayed higher cytotoxic molecule expression than progenitor exhausted T cells, similar to what has been documented in melanoma models. CONCLUSIONS Our findings identify T cell exhaustion subsets within GBM that require further investigation and may be relevant to overcome current barriers to immunotherapeutic efficacy.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn’s disease

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.

Responses to chemical cross-talk between the Mycobacterium ulcerans toxin, mycolactone, and Staphylococcus aureus

Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa, but without typical pathology associated with those pathogens’ colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co-colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not affect S. aureus growth. RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and significant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecific interactions that may play a role in regulation of responses such as effects between M. ulcerans, mycolactone, and S. aureus virulence that will be useful for treatment and prevention.

Trastuzumab-emtansine induced pleural and pericardial effusions

Introduction Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate which combine trastuzumab (T), a monoclonal antibody targeting the human epidermal growth factor receptor-2 (HER2), and a cytotoxic molecule derived from maytansine (DM1). Case report We report the first case of T-DM1-associated pleural and pericardial effusions three weeks after the second course of T-DM1 in a patient with breast cancer. Drug-induced pleural and pericardial effusions was implicated in the absence of other etiologies. The Naranjo Scale indicated a probable drug-induced adverse reaction. Management & outcome: The patient fully recovered after thoracentesis and discontinuation of T-DM1. The patient has reported no side effect after the sixth course of trastuzumab. Discussion To our knowledge, this is the first case in the literature of bilateral pleural and pericardial effusions in a patient treated with T-DM1. The successful initiation of treatment with trastuzumab following withdrawal of T-DM1 suggests that emtansine played a role in the development of bilateral pleural and pericardial effusions. We hypothesize that the patient’s condition was a result of a local inflammatory reaction to emtansine by direct toxicity.

CD38 identifies pre-activated CD8+ T cells which can be reinvigorated by anti-PD-1 blockade in human lung cancer

Background CD38 has been observed expressing in activated T cells, while the features and functions of CD38+ T cells in human NSCLC are still unclear. Methods Here we uncovered the correlation between CD38 expression and survival and immune infiltration levels in tumor of NSCLC. Then, we collected samples from 51 NSCLC patients to study the biological feature and response to anti-PD-1 of tumor-infiltrating CD38+ CD8+ T cells in vitro. Results We found CD38 expression correlated with the survival and immune infiltration levels of NSCLC. It is interesting that CD38+ CD8+ T cells enriched in the tumors expressed higher level of cytotoxic molecule, cytokines and PD-1 than CD38− CD8+ T cells. Moreover, PD-1+ subset in tumor-infiltrating CD38+ CD8+ T cells expressed higher level of activated markers than PD-1+ CD38− CD8+ T cells. Next, we found tumor-infiltrating CD38+ CD8+ T cells expressed higher level of CD103, IFN-γ, TNF-α and perforin than CD38− CD8+ T cells when were reactivated in vitro. Finally, we observed that CD38+ CD8+ T cells isolated from tumors could be reinvigorated by anti-PD-1 in vitro. Conclusions Our findings demonstrate that CD38 expression defines a subset of CD8+ T cells enriched in tumors of NSCLC which have paradoxical phenotypes and response to anti-PD-1. Our results suggest a pre-priming of these cells is may exist in tumor and consequentially facilitate it acquiring both anti-tumor potency and exhausted phenotype which can be reinvigorated by PD-1 blockade.

Cystathionine-β-synthase: Molecular Regulation and Pharmacological Inhibition

Cystathionine-β-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used “CBS inhibitors” (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models.

Development of a Split Esterase 1 for Protein-Protein 2 Interaction Dependent Small Molecule Activation

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.