Cattle T Cell Phenotyping by an 8-Colour, 10-Parameter Panel

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve, TAV, TCM, TEM, TTE and γδ-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.

Nicotinamide Inhibits T Cell Exhaustion and Increases Differentiation of CD8 Effector T Cells

One of the limitations of immunotherapy is the development of a state referred to as T cell exhaustion (TEx) whereby T cells express inhibitory receptors (IRs) and lose production of effectors involved in killing of their targets. In the present studies we have used the repeated stimulation model with anti CD3 and anti CD28 to understand the factors involved in TEx development and treatments that may reduce changes of TEx. The results show that addition of nicotinamide (NAM) involved in energy supply to cells prevented the development of inhibitory receptors (IRs). This was particularly evident for the IRs CD39, TIM3, and to a lesser extent LAG3 and PD1 expression. NAM also prevented the inhibition of IL-2 and TNFα expression in TEx and induced differentiation of CD4+ and CD8 T cells to effector memory and terminal effector T cells. The present results showed that effects of NAM were linked to regulation of reactive oxygen species (ROS) consistent with previous studies implicating ROS in upregulation of TOX transcription factors that induce TEx. These effects of NAM in reducing changes of TEx and in increasing the differentiation of T cells to effector states appears to have important implications for the use of NAM supplements in immunotherapy against cancers and viral infections and require further exploration in vivo.

Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection

First recognized more than 30 years ago, glycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target and mechanism of glycine cytoprotection, however, remain entirely elusive. We hypothesized that glycine targets ninjurin-1 (NINJ1), a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and apoptotic cell death. This common terminal effector is thought to cluster within the plasma membrane to cause cell rupture. Here, we first demonstrate that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages stimulated to undergo lytic cell death. Glycine treatment in NINJ1 knockout cells provides no additional protective effect. Next, we show that glycine treatment prevents NINJ1 clustering within the plasma membrane thereby preserving its integrity. By identifying NINJ1 as a glycine target, our data help resolve the long-standing mechanism of glycine cytoprotection. This new understanding will inform the development of cell and tissue preservation strategies for pathologic conditions associated with lytic cell death pathways.

Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis

The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.

IMMU-01. CHARACTERIZATION OF TUMOR-INFILTRATING CD8+ T CELLS IN BRAIN METASTASES

BACKGROUND Use of immune checkpoint blockade (ICB) therapy has prolonged overall survival in patients with metastatic cancer. One potential strategy to improve the effectiveness of ICB is to target additional inhibitory receptors on exhausted CD8+ T cells, which may promote rescue of CD8+ T cells that do not respond to PD-1 pathway blockade alone. This therapeutic strategy requires knowledge of the inhibitory molecules expressed on tumor-specific CD8+ T cells. Toward achieving this goal, we characterized the phenotype of CD8+ T cells infiltrating brain metastases. METHODS We performed flow cytometry on 45 brain metastases samples, single cell RNA sequencing with T cell receptor (TCR) sequencing on 5 samples, and spatially-resolved transcriptomics on 8 samples. RESULTS Analysis of our scRNA-seq data revealed 4 populations of PD-1+ CD8+ T cells infiltrating brain metastases. One of these populations (cluster A) has a terminal effector exhausted phenotype suggesting that this population contains tumor-specific CD8+ T cells. Two of the other populations (clusters B and C) have a transcriptional profile that suggests they may contain stem-like CD8+ T cells. TCR sequencing shows that cells in cluster A do not express the same TCRs as cells in clusters B and C, suggesting that stem-like cells in clusters B and C are not the progenitors of the terminal effector cells in cluster A. Bystander cells expressing TCRs specific for viral antigens are found predominantly in clusters B and C, further supporting the hypothesis that cluster A contains tumor-specific cells. Spatial transcriptomics reveals that cluster A cells are infiltrating the tumor parenchyma while cluster B and C cells are predominantly in peri-tumoral inflammatory tissue. CONCLUSIONS Brain metastases are infiltrated by a population of terminally-differentiated effector CD8+ T cells which express co-inhibitory molecules that may be potential therapeutic targets to improve control of metastatic disease in the brain.

Fungal gasdermin-like proteins are controlled by proteolytic cleavage

Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death-inducing toxins in the context of conspecific non-self discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction, controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina. We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a ~5 kDa C-terminal fragment during the cell death reaction in presence of a subtilisin-like serine protease, termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in a heterologous host (Saccharomyces cerevisiae) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analysing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2-like proteins carry either subtilisin-like or caspase-related proteases, which in some cases correspond to the N-terminal effector domain of NOD-like receptor proteins (NLRs). This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways.

The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

Necroptosis is an inflammatory form of lytic programmed cell death that is thought to have evolved to defend against pathogens. Genetic deletion of the terminal effector protein—MLKL—shows no overt phenotype in the C57BL/6 mouse strain under conventional laboratory housing conditions. Small molecules that inhibit necroptosis by targeting the kinase activity of RIPK1, one of the main upstream conduits to MLKL activation, have shown promise in several murine models of non-infectious disease and in phase II human clinical trials. This has triggered in excess of one billion dollars (USD) in investment into the emerging class of necroptosis blocking drugs, and the potential utility of targeting the terminal effector is being closely scrutinised. Here we review murine models of disease, both genetic deletion and mutation, that investigate the role of MLKL. We summarize a series of examples from several broad disease categories including ischemia reperfusion injury, sterile inflammation, pathogen infection and hematological stress. Elucidating MLKL’s contribution to mouse models of disease is an important first step to identify human indications that stand to benefit most from MLKL-targeted drug therapies.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector–specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule–mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.