Last year marked the 100th anniversary of the birth of two giants of world immunology-Milan Hašek and Jaroslav Šterzl. The following text is one of the articles in a special issue of Folia Microbiologica commemorating the "golden age of Czechoslovak immunology" associated with these two figures.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Low-molecular weight (MW) amphiphilic copolymers have been recently introduced as a powerful tool for the detergent-free isolation of cell membrane proteins. Herein, a screening approach is used to identify a new copolymer type for this application. Via a two-step ATRP/acidolysis procedure, a 3 × 3 matrix of well-defined poly[(butyl methacrylate)-co-(methacrylic acid)] copolymers (denoted BMAA) differing in their MW and ratio of hydrophobic (BMA) and hydrophilic (MAA) units is prepared. Subsequently, using the biologically relevant model (T-cell line Jurkat), two compositions of BMAA copolymers are identified that solubilize cell membranes to an extent comparable to the industry standard, styrene-maleic acid copolymer (SMA), while avoiding the potentially problematic phenyl groups. Surprisingly, while only the lowest-MW variant of the BMA/MAA 2:1 composition is effective, all the copolymers of the BMA/MAA 1:1 composition are found to solubilize the model membranes, including the high-MW variant (MW of 14 000). Importantly, the density gradient ultracentrifugation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting experiments reveal that the BMA/MAA 1:1 copolymers disintegrate the Jurkat membranes differently than SMA, as demonstrated by the different distribution patterns of two tested membrane protein markers. This makes the BMAA copolymers a useful tool for studies on membrane microdomains differing in their composition and resistance to membrane-disintegrating polymers.

In connection with the drastically deteriorating pandemic situation in the Czech Republic, Jan Culik talks again to Professor Vaclav Hořejsi, a leading Czech immunologist, about what to do next. The interview was broadcast by the Czech cable TV station Regionalnitelevize.cz from Friday 8th January 2021.

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both in vitro and in vivo. Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

Complete tumour devascularisation (CTD) is a surgical technique which entails the complete disruption by ligation or cutting of afferent and efferent tumour vasculature which remains in situ. In some animal models, CTD induces immune responses that lead to regression of distant metastases and protective immunity.

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (> 250 nm) than those containing non-raft proteins (< 20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L-o phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

Since publication of this article, the authors identified an error in the acknowledgments section. The original sentence in acknowledgments reads as follows:

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

C/EBPalpha and its target genes are known as important regulators of myeloid differentiation and maintenance of hematopoietic stem cells. Here, we identified a novel C/EBPalpha target gene, EVI2B, and demonstrated its abundant expression in the majority of hematopoietic populations, with remarkably high levels in myeloid lineage cells. We demonstrated that Evi2b depletion alters myeloid lineage development in vitro, both in murine and human models. Further, we observed that Evi2b-depleted hematopoietic stem and progenitor cells (HSPC) demonstrate impaired colony forming ability as well as decreased capacity to reconstitute the hematopoietic system of lethally irradiated mice. In addition, we demonstrated increased apoptosis and decreased proliferation in progenitor cells with reduced levels of EVI2B, which explains the affected functionality of these cells. To deepen our understanding of the mechanistic role of EVI2B in hematopoiesis, we aimed to identify Evi2b interacting proteins. Using immunoprecipitation followed my mass spectrometry analysis we identified a list of potential EVI2B binding partners. In within those, CD97, a G protein-coupled receptor involved in adhesion and migration, was present. The interaction between EVI2B and CD97 was verified by western blot analysis in murine and human cells. Both EVI2B and CD97 localize in the cellular membrane, and we hypothesize that EVI2B interacts with CD97 and possibly regulates its function. In addition, alterations in Evi2b-depleted HSPCs might be caused by impaired performance of the EVI2B /CD97 complex leading to changes in cell adhesion and/or migration. Altogether, our work provides an insight into the role of EVI2B in myeloid differentiation and functionality of hematopoietic progenitors, and suggests its involvement in cellular adhesion and/or migration.

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

BACKGROUND Immune system is principally capable to recognize and eliminate tumor cells, using several mechanisms (phagocytes, antibodies, NK-cells, cytotoxic Tlymphocytes). These immune weapons are usually not sufficiently efficient as tumor cells are mostly evaluated by the immune system as too similar to normal cells and the tumor microenvironment is very immunosuppressive. CONCLUSION Recent marked progress in elucidation of mechanisms underlying the relationships between the immune system and tumor cells made it possible to develop a number of very promising immunotherapeutic approaches, including monoclonal antibodies recognizing tumor antigens, antibodies blocking T cell inhibitory receptors, bispecific antibody constructs, in vitro expansion and stimulation of tumor specific Tlymphocytes, chimeric antigenic receptors expressed in T cells or dendritic cellbased vaccines. Immunotherapy of neoplastic diseases is apparently becoming reality.