Cytogenetics and genomics in CML and other myeloproliferative neoplasms.

Chronic myeloid leukemia is defined by the presence of the Philadelphia translocation t (9; 22) resulting in the BCR::ABL1 fusion. The other myeloproliferative neoplasms (MPN) subtypes also carry typical chromosomal abnormalities, which however are not pathognomonic for a specific entity of MPN. According to the WHO classification the distinction between these entities is still based on the integration of cytological, histopathological and molecular findings. Progression of CML into accelerated and blastic phase is usually driven by additional chromosome abnormalities and ABL1 kinase mutations. In the other MPN subtypes the additional mutations besides driver gene mutations in JAK2, MPL and CALR have a decisive impact on the propensity for progression. In addition, the sequence in which the driver mutations and risk conveying additional mutations have been acquired appears to play an important role. Here, we review cytogenetic and molecular changes in CML and MPN that should be evaluated during diagnosis and disease monitoring.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Clonal evolution process from essential thrombocythemia to acute myeloid leukemia in the original patient from whom the CALR-mutated Marimo cell line was established.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

Paradoxical cell targeting of calreticulin-empowered, protein-only nanoparticles.

Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4+ cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand.

Novel tetrasubstituted 5-Arylamino pyrazoles able to interfere with angiogenesis and Ca2+ mobilization.

In the last years, 5-pyrazolyl ureas and 5-aminopyrazoles have been investigated for their antiangiogenetic properties and their potential interaction with the ubiquitous Ca2+ binding protein Calreticulin. Based on the structure of the active compounds I and GeGe-3, novel 5-arylamino pyrazoles 2 and 3 were synthesized through a stepwise procedure. In MTT assays, all the new derivatives proved to be non-cytotoxic against eight different tumor cell lines, normal fibroblasts, and endothelial cells. Furthermore, selected derivatives showed relevant antiangiogenetic properties, resulting more effective than reference molecules I and GeGe-3 in inhibiting HUVEC endothelial tube formation. 5-Arylamino pyrazoles 2a and 2d were identified as the most interesting compounds and significantly prevented tube formation of tumor secretome-stimulated HUVEC. Furthermore, the two compounds inhibited HUVEC migration in wound healing assay and altered cell invasion capability. Additionally, 2a and 2d strongly affected Ca2+ mobilization and cytoskeletal organization of HUVEC cells, being as active as the reference compound GeGe-3. Differently from previous studies, molecular docking simulations suggested a poor affinity of 2a towards Calreticulin, one of the interacting partners of the lead compound GeGe-3. Collectively, this new amino-pyrazole library further extends the structure-activity relationships of the previously prepared derivatives and confirmed the biological attractiveness of this chemical scaffold as antiangiogenetic agents.

Calreticulin-Enigmatic Discovery.

Calreticulin (CRT) is an intrinsically disordered multifunctional protein that plays essential roles intra-and extra-cellularly. The Michalak laboratory has proposed that CRT was initially identified in 1974 by the MacLennan laboratory as the high-affinity Ca2+-binding protein (HACBP) of the sarcoplasmic reticulin (SR). This widely accepted belief has been ingrained in the scientific literature but has never been rigorously tested. In our report, we have undertaken a comprehensive reexamination of this assumption by meticulously examining the majority of published studies that present a proteomic analysis of the SR. These analyses have utilized proteomic analysis of purified SR preparations or purified components of the SR, namely the longitudinal tubules and junctional terminal cisternae. These studies have consistently failed to detect the HACBP or CRT in skeletal muscle SR. We propose that the existence of the HACBP has failed the test of reproducibility and should be retired to the annals of antiquity. Therefore, the scientific dogma that the HACBP and CRT are identical proteins is a non sequitur.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

NGS panel enhance precise diagnosis of myeloid neoplasms under WHO-HAEM5 and International Consensus Classification: An observational study.

This study aimed to assess hematological diseases next-generation sequencing (NGS) panel enhances the diagnosis and classification of myeloid neoplasms (MN) using the 5th edition of the WHO Classification of Hematolymphoid Tumors (WHO-HAEM5) and the International Consensus Classification (ICC) of Myeloid Tumors. A cohort of 112 patients diagnosed with MN according to the revised fourth edition of the WHO classification (WHO-HAEM4R) underwent testing with a 141-gene NGS panel for hematological diseases. Ancillary studies were also conducted, including bone marrow cytomorphology and routine cytogenetics. The cases were then reclassified according to WHO-HAEM5 and ICC to assess the practical impact of these 2 classifications. The mutation detection rates were 93% for acute myeloid leukemia (AML), 89% for myelodysplastic syndrome (MDS), 94% for myeloproliferative neoplasm (MPN), and 100% for myelodysplasia/myeloproliferative neoplasm (MDS/MPN) (WHO-HAEM4R). NGS provided subclassified information for 26 and 29 patients with WHO-HAEM5 and ICC, respectively. In MPN, NGS confirmed diagnoses in 16 cases by detecting JAK2, MPL, or CALR mutations, whereas 13 "triple-negative" MPN cases revealed at least 1 mutation. NGS panel testing for hematological diseases improves the diagnosis and classification of MN. When diagnosed with ICC, NGS produces more classification subtype information than WHO-HAEM5.

Proteasome inhibitors FHND6091 enhance the ability of NK cells to kill tumor cells through multiple mechanisms.

The immune system has a strong connection to tumors. When a tumor cell is recognized as an abnormal cell by the immune system, the immune system may initiate an immune response to kill the tumor cell. In this study, RNA sequencing was performed on multiple myeloma (MM) cells treated with the proteasome inhibitor FHND6091. The transcriptional changes induced by FHND6091 in RPMI8226 cells aligned notably with immune response activation and results indicated upregulation of cGAS-STING pathway-related genes in the FHND6091-treated group. In vivo and in vitro experiments had demonstrated that FHND6091 stimulated the immunoreaction of MM cells via activation of the cyclic guanosine monophosphate-adenosine synthase/stimulator of interferon genes (cGAS-STING) pathway. This activation resulted in the generation of type-I interferons and the mobilization of natural killer (NK) cells. Notably, FHND6091 upregulated the levels of calreticulin and the protein ligands UL16-binding protein 2/5/6, MHC class I chain-related A (MICA), and MICB on the surface of MM cells. Subsequently, upon engaging with the surface activation receptors of NK cells, these ligands triggered NK cell activation, leading to the subsequent elimination of tumor cells. Thus, our findings elucidated the mechanism whereby FHND6091 exerted its immunotherapeutic activity as a STING agonist, enhancing the killing ability of NK cells against tumor cells.

Clinical impact of mutated JAK2 allele burden reduction in polycythemia vera and essential thrombocythemia.

The variant allele frequency (VAF) of driver mutations (JAK2, CALR) in myeloproliferative neoplasms is associated with features of advanced disease and complications. Ruxolitinib and interferon were reported to variably reduce the mutant VAF, but the long-term impact of molecular responses (MR) remains debated. We prospectively measured changes in JAK2 and CALR VAF in 77 patients with polycythemia vera and essential thrombocythemia, treated with ruxolitinib for a median of 8 years, and assessed correlation with complete clinical and hematological response (CCHR) and outcomes. At last observation time, JAK2 VAF reduced overall from a median of 68% (range, 20%-99%) to 3.5% (0%-98%). A profound and durable MR (DMR; defined as a VAF stably ≤2%), including complete MR in 8%, was achieved in 20% of the patients, a partial MR (PMR; VAF reduction >50% of the baseline level) in 25%, and 56% had no molecular response (NMR). A CCHR was reached by 69% overall, independently of any degree of MR achieved; conversely, a DMR correlated with longer duration of CCHR and, most importantly, with reduced rate of progression to myelofibrosis and with longer myelofibrosis-free, event-free and progression-free survival. Achievement of PMR also had some favorable impact on outcomes, compared to NMR. A baseline JAK2 VAF <50%, and a VAF reduction of ≥35% after 2 years of treatment, predicted for the achievement of DMR and reduced progression to myelofibrosis. Overall, these findings support the clinical value of achieving profound, durable MR and its consideration as surrogate endpoint in future clinical trials.

The characteristics of CALR mutations in myeloproliferative neoplasms: a clinical experience from a tertiary care center in Qatar and a literature review.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are hematological disorders characterized by abnormal production of myeloid cells due to genetic mutations. Since 2013, researchers have identified somatic mutations in the Calreticulin (CALR) gene, primarily insertions or deletions, in two Philadelphia chromosome-negative MPNs; essential thrombocytosis (ET) and primary myelofibrosis (PMF), and occasionally in chronic myelomonocytic leukemia (CMML). This study aims to identify the various types of CALR mutations and their impact on CALR-positive MPN patients' clinical manifestations and outcomes. METHODS: A single-center retrospective study was conducted. The data was collected from pre-existing records. The study was carried out on Philadelphia-negative MPN patients who were being followed up on at the NCCCR (National Center for Cancer Care and Research) to assess the clinical manifestation and outcome of disease treatment. All patients included, were followed in our center between January 1, 2008, and November 20, 2021. RESULTS: A total of 50 patients with CALR-positive MPN were reviewed with a median follow-up of three years (1-11). This cohort included 31 (62%) patients with ET, 10 (20%) patients with PMF, and 9 (18%) patients with prefibrotic myelofibrosis (pre-MF). The study involved 38 (76%) male and 12 (24%) female patients. There were 16 (32%) patients diagnosed before the age of 40, 24 (48%) patients diagnosed between the ages of 40 and 60; and 10 (20%) patients diagnosed after the age of 60. Molecular analysis showed 24 (48%) patients with CALR type 1, 21 (42%) patients with CALR type 2, and 5 (10%) patients with none Type 1, none Type 2 CALR mutations. Two patients have double mutations; 1(2%) with none Type 1, none Type 2 CALR and JAK2 mutations, and 1(2%) with CALR type 1 and MPL mutations. The thrombotic events were 3 (6%) venous thromboembolisms, 3 (6%) abdominal veins thromboses, 2 (4%) strokes, and 4 (8%) ischemic cardiac events. Only 4 (8%) patients progressed to Myelofibrosis and were carrying CALR 1 mutations, and 1 (2%) patient progressed to AML with CALR 2 mutation. CONCLUSION: The data shows a significant rise in CALR-positive MPN diagnoses in younger people, emphasizing the need for a better assessment tool to improve disease management and reduce complications.

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

Immunophenotype of myeloid granulocytes in Chinese patients with BCR::ABL1-negative myeloproliferative neoplasms.

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

Clinically relevant GABARAP deficiency abrogates bortezomib-induced immunogenic cell death in multiple myeloma.

Recently, it was revealed that the high-risk, poor-prognosis downregulation of GABA type A receptor-associated protein (GABARAP) causes a defect in both autophagy and surface exposure of calreticulin (CALR) in multiple myeloma (MM) cells responding to bortezomib. Hence, GABARAP-defective MM cells fail to undergo immunogenic cell death.

Calreticulin regulates the expression of MMP14 and ADAR1 through EIF2AK2 signaling to promote the proliferation and progression of malignant melanoma cells.

It has been demonstrated that calreticulin (CALR) is expressed abnormally in various tumors and is involved in the occurrence and development of tumors. In this study, CALR and EIF2AK2 expression was measured in the clinical specimens of 39 patients with melanoma. Then, we constructed knockdown and overexpression cell models of CALR and EIF2AK2 and used wound healing and Transwell assays to observe cell migration and invasion. Apoptosis, EDU, and ROS assays were used to measure cell apoptosis and proliferation, as well as ROS levels. The effect of CALR on endoplasmic reticulum stress was detected using endoplasmic reticulum fluorescent probes. Western blotting was used to detect protein levels of CALR, EIF2AK2, ADAR1, and MMP14. The results indicated that CALR and EIF2AK2 expression levels were significantly higher in human melanoma tissues than in adjacent non-tumor tissue. In addition, we found a correlation between CALR and the expression of EIF2AK2 and MMP14, and the experimental results indicated that overexpression of CALR significantly upregulated the expression of EIF2AK2, MMP14, and ADAR1, while knockdown of CALR inhibited their expression. Notably, the knockdown of EIF2AK2 in the CALR overexpression group blocked the upregulation of MMP14 and ADAR1 expression by CALR, and the knockdown of both CALR and EIF2AK2 significantly inhibited MMP14 and ADAR1 expression. In conclusion, CALR and EIF2AK2 play a promoting role in melanoma progression, and knockdown of CALR and EIF2AK2 may be an effective anti-tumor target, and its mechanism may be through MMP14, ADAR1 signaling.

Risk factors for ischemic stroke in patients with Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms.

BACKGROUND: Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-negative MPNs) are linked with various complications, notably ischemic stroke. The study aims to identify risk factors for ischemic stroke in Ph-negative MPNs patients. METHODS: Patients were categorized into two groups based on whether they had experienced ischemic stroke. Subsequently, an analysis of demographics, biochemical makers, and genetic mutations (JAK2V617F and CALR mutations), was conducted to identify potential associations with an elevated risk of ischemic stroke in individuals with Ph-negative MPNs. RESULTS: A total of 185 patients diagnosed with Ph-negative MPNs participated in the study, including 82 with essential thrombocythemia (ET), 78 with polycythemia vera (PV), and 25 with primary myelofibrosis (PMF). Among these, 57 patients (30.8 %) had a history of ischemic stroke. Independent risk factors associated with ischemic stroke in Ph-negative MPNs patients included hypertension (OR = 5.076) and smoking (OR = 5.426). Among ET patients, smoking (OR = 4.114) and an elevated percentage of neutrophils (OR = 1.080) were both positively correlated with ischemic stroke incidence. For PV patients, hypertension (OR = 4.647), smoking (OR = 6.065), and an increased percentage of lymphocytes (OR = 1.039) were independently associated with ischemic stroke. Regardless of the presence of the JAK2V617F mutation, hypertension was the sole positively and independently associated risk factor for ischemic stroke. The odds ratios for patients with the JAK2V617F mutation was 3.103, while for those without the mutation, it was 11.25. CONCLUSIONS: Hypertension was a more substantial factor associated with an increased incidence of ischemic stroke in Ph-negative MPNs patients.

Calreticulin regulates hepatic stellate cell activation through modulating TGF-beta-induced Smad signaling.

Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) as a wound healing process. Activated hepatic stellate cells (HpSCs) are the major producer of the ECM and play a central role in liver fibrogenesis. It has been widely accepted that elimination of activated HpSCs or reversion to a quiescent state can be a feasible strategy for resolving the disease, further highlighting the urgent need for novel therapeutic targets. Calreticulin (CRT) is a molecular chaperone that normally resides in the endoplasmic reticulum (ER), important in protein folding and trafficking through the secretory pathway. CRT also plays a critical role in calcium (Ca2+) homeostasis, with its Ca2+ storage capacity. In the current study, we aimed to demonstrate its function in directing HpSC activation. In a mouse liver injury model, CRT was up-regulated in HpSCs. In cellular experiments, we further showed that this activation was through modulating the canonical TGF-ß signaling. As down-regulation of CRT in HpSCs elevated intracellular Ca2+ levels through a form of Ca2+ influx, named store-operated Ca2+ entry (SOCE), we examined whether moderating SOCE affected TGF-ß signaling. Interestingly, blocking SOCE had little effect on TGF-ß-induced gene expression. In contrast, inhibition of ER Ca2+ release using the inositol trisphosphate receptor inhibitor 2-APB increased TGF-ß signaling. Treatment with 2-APB did not alter SOCE but decreased intracellular Ca2+ at the basal level. Indeed, adjusting Ca2+ concentrations by EGTA or BAPTA-AM chelation further enhanced TGF-ß-induced signaling. Our results suggest a crucial role of CRT in the liver fibrogenic process through modulating Ca2+ concentrations and TGF-ß signaling in HpSCs, which may provide new information and help advance the current discoveries for liver fibrosis.

Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Glioblastoma Phagocytic Cell Death: Balancing the Opportunities for Therapeutic Manipulation.

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).