Academic literature on the topic 'Differentiation checkpoint'

ABSTRACT Using a subtractive cDNA library hybridization approach, we found that receptor interacting protein 2 (RIP2), a tumor necrosis factor receptor 1 (TNFR-1)-associated factor, is a novel early-acting gene that decreases markedly in expression during myogenic differentiation. RIP2 consists of three domains: an amino-terminal kinase domain, an intermediate domain, and a carboxy-terminal caspase activation and recruitment domain (CARD). In some cell types, RIP2 has been shown to be a potent inducer of apoptosis and an activator of NF-κB. To analyze the function of RIP2 during differentiation, we transduced C2C12 myoblasts with retroviral vectors to constitutively produce RIP2 at high levels. When cultured in growth medium, these cells did not show an enhanced rate of proliferation compared to controls. When switched to differentiation medium, however, they continued to proliferate, whereas control cells withdrew from the cell cycle, showed increased expression of differentiation markers such as myogenin, and began to differentiate into multinucleated myotubes. The complete RIP2 protein appeared to be necessary to inhibit myogenic differentiation, since two different deletion mutants lacking either the amino-terminal kinase domain or the carboxy-terminal CARD had no effect. A mutant deficient in kinase activity, however, had effects similar to wild-type RIP2, indicating that phosphorylation was not essential to the function of RIP2. Furthermore, RIP proteins appeared to be important during myogenic differentiation in vivo, as we detected a marked decrease in expression of the RIP2 homolog RIP in several muscle tissues of the dystrophic mdx mouse, a model for continuous muscle degeneration and regeneration. We conclude that RIP proteins can act independently of TNFR-1 stimulation by ligand to modulate downstream signaling pathways, such as activation of NF-κB. These results implicate RIP2 in a previously unrecognized role: a checkpoint for myogenic proliferation and differentiation.

Differentiation therapy is directed to the self-renewing cancer stem cells, as well as their progeny transit amplifying cells, to force them to mature to terminal differentiation. Differentiation therapy is effective in treatment of neuroblastomas and myeloid leukemias. Checkpoint inhibition therapy removes blocks to cancer reactive T-killer cells and allows them to react to malignant cells and limit the growth of cancer. The percentage of patients with a given cancer that responds to either therapy is less than hoped for, and the duration of response is variable. Multiplying the response rate (percentage of patients responding to therapy) by the duration of response may be used to derive a survival score for patients treated with differentiation therapy or checkpoint inhibition. By this criterion, differentiation therapy gives better survival scores than checkpoint inhibition. Yet, checkpoint inhibition is considered a great success, mostly because it may be applied to many different types of cancer, and differentiation therapy is considered relatively ineffective because it is limited to a few specific cancers. On the other hand, the cost of checkpoint inhibition treatment is 10–20 times more per patient than that of differentiation therapy. Hopefully, future combined treatments and advances in both approaches will increase the effectiveness of these cancer treatments.

Abstract Myelofibrosis (MF) is a progressive, myeloid malignancy characterized by deposition of collagen and reticulin fibers in the bone marrow (BM). Previous studies have shown that monocytosis is associated with poor prognosis in MF, highlighting a potential pathogenic role for monocytes in MF. Although many studies have addressed the role of cell-intrinsic and soluble extracellular factors in MF development, it is currently unknown if mechanical properties of fibrotic BM contribute to aberrant differentiation of myeloid cells and of monocytes in particular. We first defined the stiffness and viscoelastic properties of healthy and fibrotic BM. Stiffness is defined as the resistance of a matrix to deformation, while viscoelasticity is the rate of dissipation of an applied stress over time. Independent of stiffness, an applied stress relaxes rapidly in a more viscous, liquid-like matrix, whereas in a more elastic, solid-like material, stress relaxes slowly. We next generated a cohort of fibrotic and non-fibrotic mice by transplanting retrovirally transduced JAK2V617F or empty vector (EV) control hematopoietic stem and progenitor cells (HSPCs) into lethally irradiated recipients. Femurs from these mice were harvested seven months post-transplant, as well as from age- and sex-matched healthy primary mice. Nanoindentation was performed to measure BM stiffness and viscoelasticity. Fibrotic BM showed higher stiffness, as well as trending higher elastic, solid-like properties, compared to BM of control mice. We then aimed to study the effect of matrix stiffness and viscoelasticity on monocytes. Human BM-derived monocytes were encapsulated in stiff, viscous or stiff, elastic hydrogels and cultured in the presence of GM-CSF, IL-4, and PGE2 for 3 days, followed by nanoString and flow cytometry analyses. Cells in elastic gels upregulated gene sets associated with co-stimulatory molecules and cytokine receptor signaling, MHC class II antigen presentation, and regulation of extracellular matrix (ECM), compared to cells in viscous gels of the same stiffness. The fraction of dendritic cells (DCs) was significantly upregulated, as indicated by double-positive CD11c+CD1c+ (40.9% viscous vs 69.5% elastic of CD11b+HLA-DR+ cells) and CD80+ cells (20.9% viscous vs 62.7% elastic of CD11b+HLA-DR+ cells), and surface expression of HLA-DR (gMFI 2587 viscous vs 6334 elastic). Consistent with these findings, the fraction of pro-fibrotic SLAMF7+ cells (4.2% viscous vs 17.3% elastic) were also significantly higher in elastic gels. Together, these data suggest that stiff, elastic ECM drives pro-inflammatory polarization and differentiation of monocytes into antigen-presenting cells. Next, we examined the role of the cytoskeleton on human monocyte differentiation. Cortical F-actin was significantly upregulated in cells in stiff, elastic gels compared to viscous gels. Cells were exposed to a highly selective small molecular inhibitor of the γ-isoform of PI3K. Treatment with the PI3Ky inhibitor significantly reduced F-actin staining of cells in elastic gels, upregulated immature monocyte markers, reduced surface expression of HLA-DR, and downregulated the cytokines IL6, IL8, CCL4, which have previously been associated with disease progression in myelofibrosis. In line with the above human ex vivo data, BM isolated from fibrotic mice (described above) showed skewing towards Ly6G-Ly6C+ monocytes (a population enriched for inflammatory monocytes) within the CD11b myeloid compartment compared to control transplanted mice or to non-fibrotic mice that were transplanted with endogenously expressing Jak2V617F cells. Additionally, the percentage of conventional DCs (cDCs) was increased in fibrotic Jak2V617F mice compared to control mice. Importantly, 16 day in vivo treatment with the PI3Ky inhibitor significantly reduced the fraction of Ly6G-Ly6C+ monocytes within the CD11b compartment as well as the fraction of cDCs, compared to vehicle-treated Jak2V617F mice. In summary, fibrotic BM is stiffer and more elastic than normal BM. Our studies show that a stiff, elastic BM environment drives monocytes towards a more pro-inflammatory state which can in part be suppressed by PI3K-γ inhibition. Our results have relevance for human MF by demonstrating that a fibrotic BM niche is not just a consequence of chronic inflammation but is also inflammation-promoting. KHV and AEM contributed equally to this work. Disclosures Pozdnyakova: Scopio Labs: Consultancy. Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy. Wucherpfennig: Novartis: Research Funding; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees. Mooney: Novartis: Patents & Royalties: Licensed IP, Research Funding; Sirenex: Patents & Royalties: Licensed IP; Samyang Corp: Membership on an entity's Board of Directors or advisory committees; IVIVA: Membership on an entity's Board of Directors or advisory committees; Attivare: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Revela: Membership on an entity's Board of Directors or advisory committees; Amend Surgical: Patents & Royalties: Licensed IP; Lyell: Current equity holder in publicly-traded company, Patents & Royalties.

The alternative or noncanonical nuclear factor kappa B (NF-κB) pathway regulates the osteoclast (OC) response to receptor activator of nuclear factor kappa B ligand (RANKL) and thus bone metabolism. Although several lines of evidence support the emerging concept that nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12 (NLRP12) impedes alternative NF-κB activation in innate immune cells, a functional role for NLRP12 outside an inflammatory disease model has yet to be reported. Our study demonstrates that NLRP12 has a protective role in bone via suppression of alternative NF-κB–induced osteoclastogenesis and is down-modulated in response to osteoclastogenic stimuli. Here, we show that retroviral overexpression of NLRP12 suppressed RelB nuclear translocation and OC formation. Conversely, genetic ablation of NLRP12 promoted NIK stabilization, RelB nuclear translocation, and increased osteoclastogenesis in vitro. Using radiation chimeras, we demonstrated these in vitro observations dovetail with our in vivo findings that NLRP12 deficiency leads to enhanced OC numbers accompanied by a significant decline in bone mass under physiological conditions. Consistent with the basal bone phenotype, we also observed an enhanced osteolytic response following RANKL injection over the calvaria of NLRP12-deficient chimeric mice compared with wild-type control mice. Thus, modulation of NLRP12 levels controls alternative NF-κB signaling in OC precursors, altering bone homeostasis and osteolytic responses.

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.

V-domain Ig Suppressor of T cell Activation (VISTA) is a negative immune checkpoint that is expressed on multiple immune cell subsets and has been characterized in T cells, macrophages, and myeloid-derived suppressor cells. As the only immune checkpoint expressed on naïve T cells, VISTA contributes to the maintenance of T cell quiescence and tolerance. VISTA also regulates multiple myeloid cell activities such as chemotaxis, differentiation, and migration. In the context of cancer, antagonistic monoclonal antibody targeting of VISTA has been shown to aid anti-tumor immunity. Furthermore, combination therapies that include other immune checkpoints such as PD-1 or CTLA-4 with VISTA blockade may enhance therapeutic efficacy in a variety of cancers. Combination therapy may help overcome adaptive resistance to individual checkpoint therapies, thereby improving patient outcomes and survival. Here, we summarize the role of VISTA in myeloid cells and T cells within the tumor microenvironment. We discuss the proposed counter-receptors for VISTA, VISTA antibodies currently in development, and the potential for combination therapies with checkpoint inhibitors such as PD-1 and CTLA-4.

AUF1 promotes rapid decay of mRNAs containing 3′ untranslated region (3′UTR) AU-rich elements (AREs). AUF1 depletion in mice accelerates muscle loss and causes limb girdle muscular dystrophy. Here, we demonstrate that the selective, targeted degradation by AUF1 of key muscle stem cell fate-determining checkpoint mRNAs regulates each stage of muscle development and regeneration by reprogramming each myogenic stage. Skeletal muscle stem (satellite) cell explants show that Auf1 transcription is activated with satellite cell activation by stem cell regulatory factor CTCF. AUF1 then targets checkpoint ARE-mRNAs for degradation, progressively reprogramming the transcriptome through each stage of myogenesis. Transition steps in myogenesis, from stem cell proliferation to differentiation to muscle fiber development, are each controlled by fate-determining checkpoint mRNAs, which, surprisingly, were found to be controlled in their expression by AUF1-targeted mRNA decay. Checkpoint mRNAs targeted by AUF1 include Twist1, decay of which promotes myoblast development; CyclinD1, decay of which blocks myoblast proliferation and initiates differentiation; and RGS5, decay of which activates Sonic Hedgehog (SHH) pathway-mediated differentiation of mature myotubes. AUF1 therefore orchestrates muscle stem cell proliferation, self-renewal, myoblast differentiation, and ultimately formation of muscle fibers through targeted, staged mRNA decay.

Abstract E2f-2 is a member of the E2F superfamily of transcription factors that plays multiple roles in regulating cell cycle progression, cell cycle exit and checkpoint control [1]. While over-expression studies have ascribed a role for E2f-2 as a transcriptional activator of cell cycle genes, our loss of function studies performed in vivo have revealed a novel role for E2f-2 in repressing cellular proliferation and mediating differentiation. Our expression analyses demonstrate that E2f-2 is up-regulated in hematopoietic tissues, and is most strongly expressed in differentiating erythroblasts. Furthermore, E2f-2 is the major pRb-associated E2F in hematopoietic tissues, suggesting E2f-2 can function as a co-repressor of cell cycle genes during end-stage differentiation. Compound Rb−/−;E2f2−/− mice were generated to understand how deregulated E2f-2 activity contributes to the erythroid defects in Rb null mice. Loss of E2f-2 restored end-stage erythroid differentiation and enucleation to Rb null erythroblasts and extended survival of Rb null mice. Loss of E2f-2 was sufficient to bypass the mitotic arrest observed in Rb null erythroblasts [2] and was associated with reduced expression of known E2f target genes involved in M phase checkpoint control, such as Aurora B and Survivin. In addition, we observed a significant increase in caspase-3 expression in E2f-2 null erythroblasts and demonstrate, for the first time, direct regulation of caspase-3 expression by E2f-2. Given known roles for caspases in promoting erythroid differentiation and enucleation, we have over-expressed caspase-3 in Rb null erythroblasts and are currently analyzing its effects on cell cycle and maturation. Thus, our work identifies caspase-3 as a key downstream target of E2f-2 during end-stage erythroid differentiation whose expression may alleviate enucleation and mitotic defects in Rb null erythroblasts. Future work aims to understand which E2f-2 target genes ensure mitotic checkpoint control, and the molecular mechanism by which caspase-3 coordinates cell cycle control with erythroid differentiation.

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

Le cycle cellulaire est l'ensemble des étapes qui conduisent une cellule mère à se diviser en deux cellules filles. La protéine Checkpoint kinase 1 (Chk1) est importante pour sa progression. Nous avons d'une part cherché à savoir si Chk1 intervenait lors des mécanismes de production des plaquettes, car ces cellules permettant la coagulation du sang sont issues d'un cycle cellulaire particulier. Par ailleurs, nous avons étudié le rôle de Chk1 dans la Leucémie Aiguë Myéloïde (LAM), cancer des cellules sanguines. Les patients atteints de LAM sont traités par une chimiothérapie visant à endommager l'ADN afin d'entrainer la mort des cellules cancéreuses. Chk1 est garante du contrôle de la réparation des dommages de l'ADN, ce qui contrecarre l'effet de la chimiothérapie. Elle pourrait donc favoriser l'apparition de résistance. Son rôle dans les LAM étant peu connu, l'objectif de ce projet est donc de vérifier si Chk1 favorise la résistance des cellules leucémiques aux chimiothérapies
The cell cycle is a series of events that takes place in a mother cell, leading to its division into two daughter cells. The protein Checkpoint kinase 1 (Chk1) is mandatory for its coordinated progression. In this PhD projet, we wondered on the one hand whether Chk1 could be involved in the platelets production process, because these componants of blood that enables coagulation are produced due to a particular cell cycle dedicated to this end. On the other hand, we studied the role of Chk1 in Acute Myeloid Leukemia (LAM) physiopathology. LAM is a cancer of blood cells, in which patients are treated with drugs that create DNA damages, causing the death of tumoral cells. The role of Chk1 in the drug response in LAM is not well studied, but, as it enables DNA repair, it may render theses medicines less efficient, leading to relapses to therapies. So the goal of this project is to check wether Chk1 favors the resistance of some LAM cells to chemotherapeutic treatments

La protéine Checkpoint kinase 1 (CHK1) est un acteur clé du cycle cellulaire qui agit comme un agent double, à la fois dans un contexte cellulaire normal mais également en condition de stress tel que l'apparition de dommages à l'ADN. Ma thèse a consisté à évaluer les fonctions et la régulation de cette protéine dans ces deux contextes, en m'intéressant à l'hématopoïèse normale et leucémique. Plusieurs études mettent déjà en avant un rôle de CHK1 dans l'hématopoïèse normale, mais une meilleure compréhension de l'importance de CHK1 dans la différenciation, notamment dans la voie mégacaryocytaire, est nécessaire. De plus, CHK1 se révèle importante dans les Leucémies Aiguës Myéloïdes (LAM), notamment dans la résistance à la chimiothérapie. Pour permettre une meilleure compréhension de la biologie des LAM et déterminer comment cibler CHK1, il est aujourd'hui nécessaire de définir comment cette kinase est régulée dans cette pathologie. Ces deux axes, ont constitué mon travail de thèse, dont les objectifs ont été de mieux définir les fonctions de la kinase CHK1 dans ces deux contextes cellulaires. Ainsi, ce travail met en lumière le rôle de CHK1 dans la différenciation mégacaryocytaire normale, possiblement en régulant l'activité du facteur de transcription NF-E2. De plus, dans le contexte des Leucémies Aiguës Myéloïdes, ce travail implique la déubiquitinase USP7 comme régulateur majeur du niveau protéique de CHK1 dans les LAM, et comme nouvelle cible thérapeutique d'intérêt dans cette pathologie
The protein Checkpoint kinase 1 (CHK1) acts as a double agent. Indeed, CHK1 is a key player in the cell cycle, both in a normal cellular context and stress conditions such as DNA damage. The goal of my PhD project was to evaluate the functions and regulation of this protein in these two contexts, working on normal and leukemic hematopoiesis. Several studies have already described a role of CHK1 in normal hematopoiesis, but the mechanisms behind the importance of CHK1 in differentiation, particularly in megakaryopoiesis, remain to be elucidated. In addition, CHK1 has proven to be important in Acute Myeloid Leukemia (AML), particularly in the context of resistance to chemotherapy. To better understand the biology of AML, and identify ways to target CHK1, it is necessary to decipher how this kinase is regulated in this context. These two axes have been the backbone of my thesis work, which aims to better define the CHK1 kinase role in these two cellular contexts. It highlights a role for CHK1 in normal megakaryopoiesis, possibly by regulating the activity of the NF-E2 transcription factor. In addition, in the context of Acute Myeloid Leukemia, this work identifies the USP7 deubiquitynase as a major regulator of CHK1 protein levels in AML, and as a new interesting therapeutic target in this pathology

Mes études doctorales ont permis de démontrer que les souris déficientes en VIP présentent une microcéphalie ayant principalement une origine maternelle qui affecte secondairement le développement de la substance blanche. Cette production placentaire par les lymphocytes T pourrait être affectée dans des pathologies du système immunitaire. De plus, nos données indiquent qu’une déficience en VIP prédispose à l'apparition de troubles sensoriels, en particulier de la nociception. Il est donc possible que les déficits précoces de développement du cerveau murin et l'apparition de l'hypersensibilité cutanée mécanique et thermique froide soient deux facettes d'une même pathologie. Des mesures d'activité de décharge spontanée des neurones dans le thalamus sensoriel chez des mâles adultes anesthésiés ont montré que les neurones des animaux KO sont hyper-excités, ce qui suggère un traitement aberrant des informations, notamment nociceptives, ou que l'activité inhibitrice des interneurones des réseaux locaux est réduite
The studies carried out during my PhD demonstrate that VIP-deficient mice suffer from microcephaly and as well as white matter deficits mainly due to the absence of maternal VIP during embryogenesis, Placental secretion of VIP is dependent on T lymphocytes and could be altered in pathologies of the immune system. Moreover, our data links VIP deficiency to sensory alterations, specifically, the nociceptive system. Thus, it is possible that early developmental defects and hypersensitivity to mechanical and cold stimuli are two manifestations of the same pathology. This hypothesis was reinforced following analysis of spontaneous firing patterns of neurons in the sensory thalamus of anesthetized adult males. Neurons from VIP-KO mice are hyperactive, which suggests aberrant local processing of nociceptive input or that the inhibitory inputs from local interneuron networks is reduced

Carcinomatose péritonéale est un terme utilisé pour désigner la dissémination métastatique généralisée du cancer dans la cavité péritonéale. Il se caractérise par l’accumulation de liquide appelé « ascite » et est considéré comme étant au stade terminal du cancer, car il est difficile à traiter. L'ascite accumulée dans la PC comprend des cellules tumorales, cytokines et cellules immunitaires. Les cellules cancéreuses expriment des protéines spécifiques qui les aident à supprimer les cellules immunitaires et à survivre, appelées points de contrôle immunitaires. Des points de contrôle immunitaires sont présents pour réguler le système immunitaire et sont cruciaux contre la tolérance de soi. PD-1 / PD-L1 et CTLA-4 sont des voies de contrôle immunitaire bien établies adaptées au cancer pour échapper à l'immunité. Récemment, HLA-G a été reconnu comme un point de contrôle et il a été constaté que la survie globale était diminuée dans plusieurs types de cancers solides.Au cours de ma thèse, nous avons évalué l'expression de HLA-G dans la carcinomatose ovarienne. Nous avons constaté que les cellules cancéreuses dans l'ascite de presque tous les patients atteints de carcinomatose ovarienne exprimaient HLA-G. De plus, des taux croissants de sHLA-G1 et de HLA-G5 ont été trouvés dans les ascites. Cette présence de sHLA-G s'est révélée être corrélée positivement avec les Tregs et en corrélation négative avec les cellules T cytotoxiques (CD8) et les cellules NK. De plus, nous avons constaté que les ascites peuvent induire l’expression de HLA-G dans des «Hospicells» via des cytokines inflammatoires. Parmi les cytokines inflammatoires, le TGF-β et IL-1β ont une importance capitale dans l’induction de HLA-G. En outre, nous avons constaté que IL-1β implique la voie NF-κB. Dans une cohorte distincte de carcinomatose péritonéale, composée de patients atteints de PC d'origine différente, nous avons constaté que le groupe de cellules cancéreuses dans l'ascite avait une expression génique hétérogène de PD-L1, CTLA-4 et HLA- G. En outre, nous avons constaté que tous les patients présentaient des taux solubles de HLA-G et PD-L1 dans leur ascite. Cependant, seulement 5 patients présentaient des taux de CTLA-4 solubles dans leur ascite. De plus, nous avons trouvé une très forte corrélation positive entre le niveau de gène de PD-L1 et de CTLA-4, alors qu'aucune corrélation n'a été trouvée pour HLA-G avec PD-11 et CTLA-4 suggérant que HLA-G agit indépendamment des deux points de contrôle immunitaires. En outre, nous avons évalué l'expression de ces points de contrôle immunitaires par des nodules de cancer présents sur la membrane péritonéale. Nous avons trouvé une faible expression de HLA-G et PD-L1, mais la moitié des échantillons étaient fortement positifs pour sHLA-G. Nous avons également constaté que le sHLA-G pouvait être absorbé par l'ascite par la couche mésothéliale. Cette sHLA-G absorbée peut fournir un environnement immunosuppresseur pour la fixation des grappes de cellules cancéreuses à la membrane péritonéale. In vitro, nous avons constaté que l'ascite peut exercer une action immunosuppressive et retarder la lyse des cellules cancéreuses par les cellules immunitaires.De plus, nous avons constaté que la différenciation des cellules cancéreuses se traduit par une augmentation des propriétés immunosuppressives par une expression accrue de HLA-G ou PD-L1. En outre, l'expression de HLA-G et PD-L1 dépend de la phase du cycle cellulaire. Les cellules cancéreuses, si elles sont bloquées dans les cellules mitotiques, expriment des niveaux élevés de HLA-G et de PD-L1, tandis qu'une expression plus faible a été observée en phase G1. Par conséquent, nous suggérons d’éviter l’utilisation d’inhibiteurs de la mitose car ils pourraient augmenter la suppression immunitaire du cancer. De plus, le Ki-67 étant directement lié à l'index mitotique, nous suggérons de développer une échelle de Ki-67 pour évaluer le profil d'immunosuppresseur des patients cancéreux
Peritoneal carcinomatosis (PC) is a term used for widespread metastatic dissemination of cancer to the peritoneal cavity. It is characterized by the accumulation of fluid called “ascites” and is considered a terminal stage of cancer, as it is hard to treat. The overall survival rate for untreated patients is six-months. However, owing to modern techniques like HIPEC, the survival rate can be increased up to five years. The ascites accumulated in PC, consists of tumor cells, cytokines and immune cells. Cancer cells express specific proteins to suppress immune cells activity and their attack, known as immune checkpoints. PD-1/PD-L1 and CTLA-4 are well established immune checkpoint pathways adapted by cancer in evading immunity. Recently, HLA-G has been recognized as an immune checkpoint and has been found to decrease overall survival in several types of solid cancers. We evaluated the expression of HLA-G in ascites from ovarian carcinomatosis. We found that HLA-G is expressed by cancer cells in ascites from all of the patients(n=16) with ovarian carcinomatosis. Moreover, increased levels of sHLA-G1 and HLA-G5 were found in ascites. This presence of sHLA-G isoforms was found to be positively correlated with Tregs and negatively correlated with cytotoxic T-cells (CD8) and NK-cells suggesting the role of HLA-G in immune suppression. Further, we found that ascites can induce the expression of HLA-G in “Hospicells” via inflammatory cytokines. Among the inflammatory cytokines, TGF-β and IL-1β are of crucial importance in HLA-G induction with IL-1β being more potent compared to TGF-β. Further, we found that IL-1β induces HLA-G expression through NF-κB pathway.In a separate cohort of peritoneal carcinomatosis(n=27), consisting of patients with cancer from a different origin, we found that cancer cell cluster in ascites (n=23) had a heterogeneous gene expression of PD-L1, CTLA-4 and HLA-G. Further, we found that all of the patients presented soluble levels of HLA-G in their ascites. However, one patient was negative for soluble PD-L1 and only 5 patients presented soluble CTLA-4 levels in their ascites. This heterogeneity explains why some of the patients respond to immune therapy while others don’t. This also suggests the need for prescreening patients before immune therapy. Moreover, we found a very strong positive correlation (rs=0.793) between gene level of PD-L1 and CTLA-4, while no correlation was found for HLA-G with PD-L1 and CTLA-4 suggesting that HLA-G acts independently of both the immune checkpoints. Also, we evaluated the expression of these immune checkpoints by cells in peritoneal tissue (n=20). We found low expression of HLA-G and PD-L1, but the majority of the samples were found strongly positive for sHLA-G presence. This sHLA-G can provide an immune-suppressive environment for the attachment of the cancer cell clusters to the peritoneal membrane to form cancer nodule. Additionally, we developed an in-vitro cytotoxicity assay to show that the ascites can provide the immune-suppressive action by interfering with immune cell interaction and delaying the lysis of cancer cells by the immune cells.In parallel, we found that the differentiation of the cancer cells results in increased expression of immune checkpoints like HLA-G or PD-L1. This may render these cells more immune resistant and can protect against immune attack. However, in-vivo mice model is needed to study the oncogenic potential of these differentiated cells. Further, we report that the expression of HLA-G and PD-L1 is dependent on the cell cycle phase. The cancer cells, if blocked in mitotic phase express high levels of HLA-G and PD-L1, while lowest expression was observed in G1-phase. Therefore, we suggest avoiding the use of mitotic inhibitors as it may increase the immune suppression of cancer. Moreover, as Ki-67 is directly related to the mitotic index, we suggest developing a Ki-67 scale to evaluate the immune-suppressive profile of cancer patients

This chapter discusses how checkpoints were the most obvious signs of the militarization of southern Thailand. In general terms, the installation of road blocks from the beginning of the conflict has marked Patani as a different country within Thailand. More specifically, in day-to-day encounters between soldiers and those crossing, racialized ideas have emerged as a key marker to differentiate peaceful from dangerous subjects. The chapter outlines how the racialization of the southern population has historically extended itself to include certain ideas of dress and language, and it details how these differentiations are drawn in contemporary checkpoint interactions. It also shows the gendering of ideas of the Malay khaek, which entails attributing Orientalized and often sexualized beauty to young local girls.

It is well established that allergic asthma is T cell-driven disease where CD4+ T cells of Th2 phenotype play a critical role in disease initiation and maintenance. There are several critical steps in the induction of Th2 type immune response to the allergen. The first critical step is the antigen processing and presentation of allergen-derived peptides in the context of specific major histocompatibility Class II (MHCII) molecules by antigen-presenting cells (APC). Recognition of this complex by T cell receptor (TCR) and interaction of costimulatory ligands with corresponding receptors represents the second step in T cell activation. As the third part of optimal T cell differentiation, proliferation, and expansion, several cytokines, integrins, and chemokines get involved in the fine-tuning of DC-T cell interaction and activation. Multiple recent evidences point to the selected members of B7 and semaphorin families as important checkpoints providing a fine-tuning regulation of immune response. In this book chapter, we discuss the properties of costimulatory molecules and address their roles in allergic asthma.

Metaplastic breast carcinoma (MpBC) is a rare and morphologically diverse group of tumors in which a variable proportion or the entire tumor is composed of nonglandular epithelium or mesenchymal cells. It is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. It is composed of ductal, squamous, and/or chondroid, and spindle elements, with squamous cell carcinoma being the most frequent histological subtype. MpBC represents 0.2%–5% of all breast cancers and it is very aggressive. This type of breast cancer is typically triplenegative and is therefore not targetable with hormone therapy or anti-HER2 therapies, leaving only chemotherapeutics for management. MpBCs are known for their aggressive course and poor response to chemotherapy. PDL1/PD1 expression is a predictor of the effectiveness of immune checkpoint therapy in breast cancer. Finally, there are currently no standardized treatment guidelines specifically for MpBC2. A 42-year-old female patient, lactating, who had her only pregnancy at age 40, visited a Mastology Clinic on July 16, 2019, complaining of huge left breast pain. She did not know about her family background, as she was adopted. On physical examination, she had lactating breasts and two palpable lumps of hard consistency, contiguous, and mobile in the upper outer quadrant of the left breast, measuring 3 and 2.5 cm. Mammography described dense breasts, with no other changes and breast ultrasound revealed two solid nodules, measuring 2.7 and 0.6 cm, and a simple cyst measuring 3.4 cm, all of which were contiguous in the upper outer quadrant of the left breast — BIRADS 4. A fine-needle aspiration puncture was performed in the simple cyst, with a histopathological result of poorly differentiated malignant neoplasm with pleomorphic focus, and a core-needle biopsy, with histopathological result of breast tissue infiltrated by pleomorphic malignant neoplasm. The immunohistochemical analysis showed positive for pan cytokeratin AE1/AE3 and negative for CD45, S100, myogenin, and myodio; bringing the conclusion of poorly differentiated carcinoma, suggestive of MpBC. She received neoadjuvant chemotherapy, with doxorubicin + cyclophosphamide, but had rapid local tumor progression. A new ultrasound revealed a heterogeneous and partially delimited mass, measuring 8.8×6.1 cm — BIRADS 6. The patient underwent a left total mastectomy and axillary lymph node dissection on September 23, 2019 — without breast reconstruction, and confirmed invasive metaplastic carcinoma with chondroid-type mesenchymal differentiation, measuring 7 cm, histological grade III, nuclear grade III, associated with solid and cribriform ductal carcinoma in situ, with comedonecrosis — grade III; free surgical margins, but with axillary lymph node metastasis (8/20). The immunohistochemical analysis of the surgical specimen revealed a triple-negative carcinoma: estrogen and progesterone receptors negative, and HER2 negative. The patient had a good postoperative recovery and received radiotherapy (50 Gy). Thereafter, she received adjuvant chemotherapy with capecitabine, within which she evolved with axillary, supraclavicular, and pulmonary lymph node metastasis. The PDL1 marker showed a negative result; therefore, palliative paclitaxel and bevacizumab were prescribed. The patient rapidly evolved with worsening of the lung lesions and was hospitalized on March 9, 2020, with serious dyspnea, progressing to death on March 19, 2020.