Tomasz M Grzywa

Interleukin-6 signal transducer (IL6ST) encodes the GP130 protein which transduces the proinflammatory signaling of the IL6 cytokine family through Janus kinase signal transducers and activators of transcription pathway (JAK/STAT) activation. Biallelic loss-of-function IL6ST variants cause autosomal recessive hyper-IgE syndrome or a variant of the Stuve–Wiedemann syndrome. Somatic gain-of-function IL6ST mutations, in particular, small monoallelic in-frame deletions of which the most prevalent is the IL6ST Ser187_Tyr190del, are an established cause of inflammatory hepatocellular tumors, but so far, no disease caused by such mutations present constitutively has been described. Herein, we report a pediatric proband with a novel syndrome of neonatal onset immunodeficiency with autoinflammation and dysmorphy associated with the IL6ST Tyr186_Tyr190del variant present constitutively …

Psoriasis is one of the most common inflammatory skin diseases with a chronic, relapsing-remitting course. The last decades of intense research uncovered a pathological network of interactions between immune cells and other types of cells in the pathogenesis of psoriasis. Emerging evidence indicates that dendritic cells, TH17 cells, and keratinocytes constitute a pathogenic triad in psoriasis. Dendritic cells produce TNF-α and IL-23 to promote T cell differentiation toward TH17 cells that produce key psoriatic cytokines IL-17, IFN-γ, and IL-22. Their activity results in skin inflammation and activation and hyperproliferation of keratinocytes. In addition, other cells and signaling pathways are implicated in the pathogenesis of psoriasis, including TH9 cells, TH22 cells, CD8+ cytotoxic cells, neutrophils, γδ T cells, and cytokines and chemokines secreted by them. New insights from high-throughput analysis of lesional skin …

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR FMC63 as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

ObjectiveThis study aimed to assess the utility of circulating miR-125b-5p, miR-199a-3p, miR-451a, and miR-3613-5p as biomarkers of endometriosis.Study designPatients with stage III or IV of endometriosis according to the revised American Society of Reproductive Medicine (rASRM) staging classification, as well as control women, were recruited. We created a prospective study conducted on a group of 48 patients (n = 25 controls, n = 24 endometriosis) who had laparoscopic surgery. Blood samples were taken and plasma miRNA levels were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and assessed with AUC and ROC curves.ResultsMiR-451a and miR-3613-5p were significantly decreased in the plasma of endometriosis patients. miR-451a had a receiver-operating characteristic (ROC) area under the curve 0.8283 and miR-3613-5p had a ROC area under the curve 0.7617. The …

Immunotherapy revolutionized cancer treatment in the last decade. Natural killer (NK) cells are one of the key host immunity components against malignant cells. Thus, they are currently extensively investigated in the field of immunotherapy of cancer. Different approaches have been developed to improve the antitumor activity of NK cells. Nonetheless, tumor microenvironment remains an obstacle to effective NK cell-based therapies. Here, we demonstrated that a cancer-conditioned medium suppresses the anti-tumor activity of NK cells. Further, we found that ammonia, a by-product of cancer cell metabolism, accumulates in the cancer-conditioned medium and tumor microenvironment. We identified that ammonia impairs the cytotoxicity of NK cells as well as the effectiveness of antibody-based and chimeric antigen receptor (CAR)-NK-based therapies in vitro. Inhibited activity of NK cells was caused by decreased levels of perforin. This effect was dependent on the lysosomotropic features of ammonia and its ability to increase pH in acidic compartments. In consequence, upon contact with ammonia the mature form of perforin was decreased in NK cells leading to their dysfunction. Our findings demonstrate that in addition to its previously described role of promoting tumor growth as a nitrogen source for tumor biomass ammonia could promote tumor escape as an NK cells immune checkpoint.

ResultsIn Vk* MYC-bearing mice we observed an accumulation of CECs in the blood and spleen in time, correlating with the development of anemia. At the late stage of the disease MM-associated CECs showed less differentiated phenotype and increased expression of arginase-1 (ARG1) and ARG2. MM-associated CECs inhibited T cells activation and proliferation in an ARG and reactive oxygen species-dependent manner. Interestingly, MM development was delayed in ARG2 KO vs WT mice. In the BM aspirates of MM patients we observed increased percentage of CECs expressing high levels of both ARG1 and ARG2 comparing with healthy controls. Moreover, MM impaired control of L. monocytogenes infection as we observed an elevated bacterial burden in the livers and spleens of Vk* MYC-bearing vs control mice. Infected MM-bearing mice had fewer NK cells and activated CD4+ and CD8+ T cells in the …

Endometrial cancer (EC) is one of the most common types of cancer of the female reproductive system. EC is classified into two types (EC1 and EC2). MiRNAs are single-stranded RNA molecules that regulate gene expression posttranscriptionally. They have aberrant expression profiles in cancer, including EC. This study aimed to assess the level of expression of a panel of 16 miRNAs in both types of EC and healthy endometrium (HE). A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). RNA was isolated from collected material and the expression of selected miRNAs was determined using the real-time qPCR. We found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and …

MiRNAs are short non-coding RNAs that regulate gene expression post-transcriptionally contributing to the development of different diseases including cancer. The miR-200 family consists of five members, miR-200a, miR-200b, miR-200c, miR-141, and miR-429. Their expression is dysregulated in cancer tissue and their level is altered in the body fluids of cancer patients. Moreover, the levels of miR-200 family members correlate with clinical parameters such as cancer patients’ survival which makes them potentially useful as diagnostic and prognostic biomarkers. MiRNAs can act as either oncomiRs or tumor suppressor miRNAs depending on the target genes and their role in the regulation of key oncogenic signaling pathways. In most types of cancer, the miR-200 family acts as tumor suppressor miRNA and regulates all features of cancer. In this review, we summarized the expression pattern of the miR-200 family in different types of cancer and their potential utility as biomarkers. Moreover, we comprehensively described the role of miR-200 family members in the regulation of all hallmarks of cancer proposed by Hanahan and Weinberg with the focus on the epithelial-mesenchymal transition, invasiveness, and metastasis of tumor cells.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 (Tmcc2) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2−/− pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2−/− nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71+TER119+ erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of …

Melanoma is a life-threatening disease due to the early onset of metastasis and frequent resistance to the applied treatment. For now, no single histological, immunohistochemical or serological biomarker was able to provide a precise predictive value for the aggressive behavior in melanoma patients. Thus, the search for quantifying methods allowing a simultaneous diagnosis and prognosis of melanoma patients is highly desirable. By investigating specific molecular interactions with some biosensor-based techniques, one can determine novel prognostic factors for this tumor. In our previous study, we have shown the possibility of a qualitative in vitro distinguishing the commercially available melanoma cells at different progression stages based on the measurements of the lectin Concanavalin A interacting with surface glycans present on cells.Here, we present the results of the quantitative diagnostic and …

Background: For decades, CD71+ erythroid cells (CECs) were considered to be mere progenitors and precursors of oxygen-transporting erythrocytes. However, increasingly more observations indicate that they are also crucial regulators of the immune response in various stages of life. CECs use various mechanisms to regulate immune response in different niches, including arginase-2, reactive oxygen species, or checkpoint molecules. However, factors regulating immunomodulatory properties of CECs remain largely unknown, making it difficult to develop effective therapies exploiting immunomodulatory functions of these cells. A common condition under which CECs potently suppress the immune response in fetal life, pregnancy, or advanced cancer is hypoxia. Thus, we hypothesized that hypoxia regulates the immunoregulatory functions of CECs.Methods: Murine CECs were isolated from the spleens of anemic …

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more …

Simple Summary Tumor immune evasion is one of the hallmarks of tumor progression that enables tumor growth despite the activity of the host immune system. It is mediated by various types of cells. Recently, immature red blood cells called erythroid progenitor cells (EPCs) were identified as regulators of the immune response in cancer. EPCs expand in cancer as a result of dysregulated erythropoiesis and potently suppress the immune response. Thus, targeting dysregulated EPC differentiation appears to be a promising therapeutic strategy. Abstract Cancer cells harness normal cells to facilitate tumor growth and metastasis. Within this complex network of interactions, the establishment and maintenance of immune evasion mechanisms are crucial for cancer progression. The escape from the immune surveillance results from multiple independent mechanisms. Recent studies revealed that besides well-described myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) or regulatory T-cells (Tregs), erythroid progenitor cells (EPCs) play an important role in the regulation of immune response and tumor progression. EPCs are immature erythroid cells that differentiate into oxygen-transporting red blood cells. They expand in the extramedullary sites, including the spleen, as well as infiltrate tumors. EPCs in cancer produce reactive oxygen species (ROS), transforming growth factor β (TGF-β), interleukin-10 (IL-10) and express programmed death-ligand 1 (PD-L1) and potently suppress T-cells. Thus, EPCs regulate antitumor, antiviral, and antimicrobial immunity, leading to immune …

Simple Summary Endometrial cancer (EC) is one of the most frequent cancers with increasing annual death rates. Therefore, it is of great clinical importance to understand the mechanisms of endometrial cancer invasiveness and metastasis. MiRNAs are small single-stranded RNAs that regulate gene expression. They were discovered to play a role in all steps of cancer development. This study aimed at conducting a systematic review of the role of miRNAs in endometrial cancer invasiveness and metastasis. The collected data demonstrate that miRNAs regulate EC invasiveness and metastasis by different targets. MiRNAs seem to be potential candidates for diagnostic and prognostic biomarkers, as well as possible therapeutic targets. Abstract Endometrial cancer (EC) is the most common genital cancer in women with increasing death rates. MiRNAs are short non-coding RNAs that regulate gene expression on the post-transcriptional levels. Multiple studies demonstrated a fundamental role of miRNAs in the regulation of carcinogenesis. This systematic review is a comprehensive overview of the role of miRNAs in the regulation of cancer cell invasiveness and metastasis in EC. The literature was searched for studies investigating the role of miRNAs in the regulation of invasiveness and metastasis in EC. We explored PubMed, Embase, and Scopus using the following keywords: miRNA, metastasis, invasiveness, endometrial cancer. Data were collected from 163 articles that described the expression and role of 106 miRNAs in the regulation of EC invasiveness and metastasis out of which 63 were tumor suppressor …

Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition …

Complex regulation of the immune response is necessary to support effective defense of an organism against hostile invaders and to maintain tolerance to harmless microorganisms and autoantigens. Recent studies revealed previously unappreciated roles of CD71+ erythroid cells (CECs) in regulation of the immune response. CECs physiologically reside in the bone marrow where erythropoiesis takes place. Under stress conditions, CECs are enriched in some organs outside of the bone marrow as a result of extramedullary erythropoiesis. However, the role of CECs goes well beyond the production of erythrocytes. In neonates, increased numbers of CECs contribute to their vulnerability to infectious diseases. On the other side, neonatal CECs suppress activation of immune cells in response to abrupt colonization with commensal microorganisms after delivery. CECs are also enriched in the peripheral blood of …

Melanoma tumors are the most heterogeneous of all tumor types. Tumor heterogeneity results in difficulties in diagnosis and is a frequent cause of failure in treatment. Novel techniques enable accurate examination of the tumor cells, considering their heterogeneity. The study aimed to determine the somatic variations among high and low proliferating compartments of melanoma tumors. In this study, 12 archival formalin-fixed paraffin-embedded samples of previously untreated primary cutaneous melanoma were stained with Ki-67 antibody. High and low proliferating compartments from four melanoma tumors were dissected using laser-capture microdissection. DNA was isolated and analyzed quantitatively and qualitatively. Libraries for amplicon-based next-generation sequencing (NGS) were prepared using NEBNext Direct Cancer HotSpot Panel. NGS detected 206 variants in 42 genes in melanoma samples. Most of them were located within exons (135, 66%) and were predominantly non-synonymous single nucleotide variants (99, 73.3%). The analysis showed significant differences in mutational profiles between high and low proliferation compartments of melanoma tumors. Moreover, a significantly higher percentage of variants were detected only in high proliferation compartments (39%) compared to low proliferation regions (16%, p < 0.05). Our results suggest a significant functional role of genetic heterogeneity in melanoma.

Simple Summary The present paper describes a simple, non-enzymatic and effective method of melanocyte or metastatic melanoma cell isolation from skin or lymph node explants, respectively. The method is based on selective harvesting of melanocytes or melanoma cells emigrating from the explants. Thus, isolated cells display specific phenotypical and functional features of melanocytes/melanoma cells such as tyrosinase and Melan-A expression and melanin production. Furthermore, melanocyte or melanoma cell cultures are not contaminated by keratinocytes and/or fibroblasts. The method appears to be a useful tool for studies on the biology of melanocytes and malignant melanoma. Abstract The development of an effective method of melanocyte isolation and culture is necessary for basic and clinical studies concerning skin diseases, including skin pigmentation disorders and melanoma. In this paper, we describe a novel, non-enzymatic and effective method of skin melanocyte and metastatic melanoma cell isolation and culture (along with the spontaneous spheroid creation) from skin or lymph node explants. The method is based on the selective harvesting of melanocytes and melanoma cells emigrating from the cultured explants. Thereby, isolated cells retain their natural phenotypical features, such as expression of tyrosinase and Melan-A as well as melanin production and are not contaminated by keratinocytes and fibroblasts. Such melanocyte and melanoma cell cultures may be very useful for medical and cosmetology studies, including studies of antitumor therapies.

MicroRNAs are involved in various pathologies including cancer. The aim of the study was to assess the level of expression of miR-96-5p, -134-5p, -181b-5p, -200b-3p in FFPE samples of prostate cancer, adjacent cancer-free tissue, and benign prostatic hyperplasia. Samples of 23 FFPE prostate cancer and 22 benign prostatic hyperplasias were dissected and HE stained. Compartments of tumor tissue and adjacent healthy glandular tissue were isolated from each sample using Laser Capture Microdissection. Total RNA was isolated from dissected tissues. Expression of miR-96-5p, miR-134-5p, 181b-5p, and miR-200b-3p was determined by real-time RT-qPCR method. The expression of miR-200b-3p was significantly higher in cancerous prostate: both in adenocarcinomatous glands and in the adjacent, apparently unaffected glands compared to BPH samples. The expression of miR-181b-5p was lower …