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Proteasome inhibitors bortezomib, carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM), but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites, $\beta$5i, $\beta$1i, and $\beta$2i, which are different from their constitutive $\beta$5c, $\beta$1c, and $\beta$2c counterparts. Bortezomib and carfilzomib block $\beta$5c and $\beta$5i sites. We report here that pharmacologically relevant concentrations of $\beta$5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition, increasing immunoproteasome expression by interferon-$\gamma$ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102, an inhibitor of $\beta$2 sites, dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus, immunoproteasome inhibitors, currently in clinical trials for the treatment of autoimmune diseases, should also be considered for the treatment of MM.

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse, we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding, at the molecular level, how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here, we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally, we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization, as well as T cell migration in vitro. Altogether, our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility.

Hematopoietic stem/progenitor cells (HSPC) are characterized by their unique capacities of self-renewal and multi-differentiation potential. This second property makes them able to adapt their differentiation profile depending on the local environment they reach. Taking advantage of an animal model of peritonitis, induced by injection of the TLR-2 ligand, zymosan, we sought to study the relationship between bone marrow-derived hematopoietic stem/progenitor cells (BM-HSPCs) and innate lymphoid cells (ILCs) regarding their emergence and differentiation at the site of inflammation. Our results demonstrate that the strength of the inflammatory signals affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections trigger the appearance of mature ILCs in the peritoneal cavity where the inflammation occurs. Herein, we show that only in low-dose injected mice, the recovered ILCs are dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose, the stronger inflammatory environment seems to be able to induce the emergence of ILCs independently of BM-derived HSPCs. We suggest that a relationship between HSPCs and ILCs seems to be affected by the strength of the inflammatory stimuli opening new perspectives in the manipulation of these early hematopoietic cells.

Cellular senescence, which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2, can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing, including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however, a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis, hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition, and degeneration of cortical and hippocampal neurons, thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively, these results show that senescent cells have a role in the initiation and progression of tau-mediated disease, and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies.

Neurologic melioidosis occurs in both human and animals; however, the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however, during melioidosis, lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines, chemokines and cell adhesion molecules; the activation of astrocytes, microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans LPS-induced brains. Accordingly, different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice, B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular, these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption.

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.

The T cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the major histocompatibility complex (MHC). To convert this binding event into downstream signaling, the TCR complex contains immunoreceptor tyrosine-based activation motifs (ITAMs) that act as docking sites for the cytoplasmic tyrosine kinase ZAP-70. Unique among antigen receptors, the TCR complex uses 10 ITAMs to transduce peptide-MHC binding to the cell interior. Using synthetic, drug-inducible receptor-ligand pairs, it was found that greater ITAM multiplicity primarily enhanced the efficiency with which ligand binding was converted into an intracellular signal. This manifested as an increase in the fraction of cells that became activated in response to antigen, and a more synchronous initiation of TCR-proximal signaling, rather than direct amplification of the intracellular signals. Exploiting these findings, the potency and selectivity of chimeric antigen receptors targeted against cancer were substantially enhanced by modulating the number of encoded ITAMs.

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article, we show that EPCR-positive UM171-treated cells, as opposed to EPCR-negative cells, exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers, such as CD38, EPCR expression is maintained when cells are introduced in culture, irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity, its expression is required for the repopulating activity of human HSCs. Altogether, our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs.

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly, the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid, sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine, and a sensitive single cell assay to monitor System L amino acid transport.

Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3$\zeta$ or 4-1BB/CD3$\zeta$ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3$\zeta$ and 4-1BB/CD3$\zeta$ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3$\zeta$ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell-like phenotype and function. In contrast, 4-1BB/CD3$\zeta$ CAR T cells preferentially expressed T cell memory-associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3$\zeta$ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.

The complete resolution of inflammation requires the uptake of apoptotic polymorphonuclear cells (PMN) by local macrophages (efferocytosis) and the consequent reprogramming of the engulfing phagocytes to reparative and pro-resolving phenotypes. The tyrosine kinase receptors TYRO3, AXL, and MERTK (collectively named TAM) are fundamental mediators in regulating inflammatory responses and efferocytosis. Protein S (PROS1) is a ligand for all TAM receptors that mediates various aspects of their activity. However, the involvement of PROS1 in the resolution of inflammation is incompletely understood. Here, we report the upregulation of Pros1 in macrophages during the resolution of inflammation. Selective knockout of Pros1 in the myeloid lineage significantly downregulated macrophage pro-resolving properties. Hence, Pros1-deficient macrophages engulfed fewer apoptotic PMN remnants in vivo, and exogenous PROS1 rescued impaired efferocytosis ex vivo. Moreover, Pros1-deficient peritoneal macrophages secreted higher levels of the pro-inflammatory mediators TNF$\alpha$ and CCL3, while they secreted lower levels of the reparative/anti-inflammatory IL-10 following exposure to lipopolysaccharide in comparison to their WT counterparts. Moreover, Pros1-deficient macrophages expressed less of the anti-inflammatory/pro-resolving enzymes arginase-1 and 12/15-lipoxygenase and produced less of the specialized pro-resolving mediator resolvin D1. Altogether, our results suggest that macrophage-derived PROS1 is an important effector molecule in regulating the efferocytosis, maturation, and reprogramming of resolution phase macrophages, and imply that PROS1 could provide a new therapeutic target for inflammatory and fibrotic disorders.