Apoptosis and Clearance of Apoptotic Cells.

The human body generates 10-100 billion cells every day, and the same number of cells die to maintain homeostasis in our body. Cells infected by bacteria or viruses also die. The cell death that occurs under physiological conditions mainly proceeds by apoptosis, which is a noninflammatory, or silent, process, while pathogen infection induces necroptosis or pyroptosis, which activates the immune system and causes inflammation. Dead cells generated by apoptosis are quickly engulfed by macrophages for degradation. Caspases are a large family of cysteine proteases that act in cascades. A cascade that leads to caspase 3 activation mediates apoptosis and is responsible for killing cells, recruiting macrophages, and presenting an "eat me" signal(s). When apoptotic cells are not efficiently engulfed by macrophages, they undergo secondary necrosis and release intracellular materials that represent a damage-associated molecular pattern, which may lead to a systemic lupus-like autoimmune disease.

Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

An exhausted-like microglial population accumulates in aged and APOE4 genotype Alzheimer's brains.

The dominant risk factors for late-onset Alzheimer's disease (AD) are advanced age and the APOE4 genetic variant. To examine how these factors alter neuroimmune function, we generated an integrative, longitudinal single-cell atlas of brain immune cells in AD model mice bearing the three common human APOE alleles. Transcriptomic and chromatin accessibility analyses identified a reactive microglial population defined by the concomitant expression of inflammatory signals and cell-intrinsic stress markers whose frequency increased with age and APOE4 burden. An analogous population was detectable in the brains of human AD patients, including in the cortical tissue, using multiplexed spatial transcriptomics. This population, which we designate as terminally inflammatory microglia (TIM), exhibited defects in amyloid-ß clearance and altered cell-cell communication during aducanumab treatment. TIM may represent an exhausted-like state for inflammatory microglia in the AD milieu that contributes to AD risk and pathology in APOE4 carriers and the elderly, thus presenting a potential therapeutic target for AD.

Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.

Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.

The inhibitory receptor TIM-3 limits activation of the cGAS-STING pathway in intra-tumoral dendritic cells by suppressing extracellular DNA uptake.

Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.

Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1-CD8+ Tumor-Infiltrating T Cells.

An improved understanding of the anti-tumor CD8+ T cell response after checkpoint blockade would enable more informed and effective therapeutic strategies. Here we examined the dynamics of the effector response of CD8+ tumor-infiltrating lymphocytes (TILs) after checkpoint blockade therapy. Bulk and single-cell RNA profiles of CD8+ TILs after combined Tim-3+PD-1 blockade in preclinical models revealed significant changes in the transcriptional profile of PD-1- TILs. These cells could be divided into subsets bearing characterstics of naive-, effector-, and memory-precursor-like cells. Effector- and memory-precursor-like TILs contained tumor-antigen-specific cells, exhibited proliferative and effector capacity, and expanded in response to different checkpoint blockade therapies across different tumor models. The memory-precursor-like subset shared features with CD8+ T cells associated with response to checkpoint blockade in patients and was compromised in the absence of Tcf7. Expression of Tcf7/Tcf1 was requisite for the efficacy of diverse immunotherapies, highlighting the importance of this transcriptional regulator in the development of effective CD8+ T cell responses upon immunotherapy.

Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.

Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

Next generation of immune checkpoint molecules in maternal-fetal immunity.

Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.

Intermembrane space-localized TbTim15 is an essential subunit of the single mitochondrial inner membrane protein translocase of trypanosomes.

All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.

Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity.

TIM22 pathway cargos are essential for sustaining mitochondrial homeostasis as an excess of these proteins leads to proteostatic stress and cell death. Yme1 is an inner membrane metalloprotease that regulates protein quality control with chaperone-like and proteolytic activities. Although the mitochondrial translocase and protease machinery are critical for organelle health, their functional association remains unexplored. The present study unravels a novel genetic connection between the TIM22 complex and YME1 machinery in Saccharomyces cerevisiae that is required for maintaining mitochondrial health. Our genetic analyses indicate that impairment in the TIM22 complex rescues the respiratory growth defects of cells without Yme1. Furthermore, Yme1 is essential for the stability of the TIM22 complex and regulates the proteostasis of TIM22 pathway substrates. Moreover, impairment in the TIM22 complex suppressed the mitochondrial structural and functional defects of Yme1-devoid cells. In summary, excessive levels of TIM22 pathway substrates could be one of the reasons for respiratory growth defects of cells lacking Yme1, and compromising the TIM22 complex can compensate for the imbalance in mitochondrial proteostasis caused by the loss of Yme1.

Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria.

Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis.

BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

Regulatory B cells: TIM-1, transplant tolerance, and rejection.

Regulatory B cells (Bregs) ameliorate autoimmune disease and prevent allograft rejection. Conversely, they hinder effective clearance of pathogens and malignancies. Breg activity is mainly attributed to IL-10 expression, but also utilizes additional regulatory mechanisms such as TGF-ß, FasL, IL-35, and TIGIT. Although Bregs are present in various subsets defined by phenotypic markers (including canonical B cell subsets), our understanding of Bregs has been limited by the lack of a broadly inclusive and specific phenotypic or transcriptional marker. TIM-1, a broad marker for Bregs first identified in transplant models, plays a major role in Breg maintenance and induction. Here, we expand on the role of TIM-1+  Bregs in immune tolerance and propose TIM-1 as a unifying marker for Bregs that utilize various inhibitory mechanisms in addition to IL-10. Further, this review provides an in-depth assessment of our understanding of Bregs in transplantation as elucidated in murine models and clinical studies. These studies highlight the major contribution of Bregs in preventing allograft rejection, and their ability to serve as highly predictive biomarkers for clinical transplant outcomes.

The physiological role of TRP channels in sleep and circadian rhythm.

TRP channels, are non-specific cationic channels that are involved in multiple physiological processes that include salivation, cellular secretions, memory extinction and consolidation, temperature, pain, store-operated calcium entry, thermosensation and functionality of the nervous system. Here we choose to look at the evidence that decisively shows how TRP channels modulate human neuron plasticity as it relates to the molecular neurobiology of sleep/circadian rhythm. There are numerous model organisms of sleep and circadian rhythm that are the results of the absence or genetic manipulation of the non-specific cationic TRP channels. Drosophila and mice that have had their TRP channels genetically ablated or manipulated show strong evidence of changes in sleep duration, sleep activity, circadian rhythm and response to temperature, noxious odours and pattern of activity during both sleep and wakefulness along with cardiovascular and respiratory function during sleep. Indeed the role of TRP channels in regulating sleep and circadian rhythm is very interesting considering the parallel roles of TRP channels in thermoregulation and thermal response with concomitant responses in growth and degradation of neurites, peripheral nerves and neuronal brain networks. TRP channels provide evidence of an ability to create, regulate and modify our sleep and circadian rhythm in a wide array of physiological and pathophysiological conditions. In the current review, we summarize previous results and novel recent advances in the understanding of calcium ion entry via TRP channels in different sleep and circadian rhythm conditions. We discuss the role of TRP channels in sleep and circadian disorders.

NOX4-TIM23 interaction regulates NOX4 mitochondrial import and metabolic reprogramming.

Pulmonary fibrosis is a progressive lung disease characterized by macrophage activation. Asbestos-induced expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (NOX4) in lung macrophages mediates fibrotic progression by the generation of mitochondrial reactive oxygen species (ROS), modulating mitochondrial biogenesis, and promoting apoptosis resistance; however, the mechanism(s) by which NOX4 localizes to mitochondria during fibrosis is not known. Here, we show that NOX4 localized to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 and NOX4 interaction was found in lung macrophages from human subjects with asbestosis, while it was absent in mice harboring a conditional deletion of NOX4 in lung macrophages. This interaction was localized to the proximal transmembrane region of NOX4. Mechanistically, TIM23 augmented NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. These observations highlight the important role of the mitochondrial translocase TIM23 interaction with NOX4. Moreover, this interaction is required for mitochondrial redox signaling and metabolic reprogramming in lung macrophages.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.