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CAR T cell therapies face challenges in combating solid tumors due to their single-target approach, which becomes ineffective if the targeted antigen is absent or lost. Universal CAR T cells (UniCAR Ts) provide a promising solution by utilizing molecular tags (linkers), such as biotin conjugated to monoclonal antibodies, enabling them to target a variety of tumor antigens. Recently, we showed that conventional CAR T cells could penetrate the extracellular matrix (ECM) of ADCC-resistant tumors, which forms a barrier to therapeutic antibodies. This finding led us to investigate whether UniCAR T cells, targeted by soluble antibody-derived linkers, could similarly tackle ADCC-resistant tumors where ECM restricts antibody penetration. We engineered UniCAR T cells by incorporating a biotin-binding monomeric streptavidin 2 (mSA2) domain for targeting HER2 via biotinylated trastuzumab (BT). The activation and cytotoxicity of UniCAR T cells in the presence or absence of BT were evaluated in conventional immunoassays. A 3D spheroid coculture was set up to test the capability of UniCAR Ts to access ECM-masked HER2 + cells. For in vivo analysis, we utilized a HER2 + xenograft model in which intravenously administered UniCAR T cells were supplemented with intraperitoneal BT treatments. In vitro , BT-guided UniCAR T cells showed effective activation and distinct anti-tumor response. Upon target recognition, IFNγ secretion correlated with BT concentration. In the presence of BT, UniCAR T cells effectively penetrated HER2 + spheroids and induced cell death in their core regions. In vivo , upon intravenous administration of UniCAR Ts, circulating BT linkers immediately engaged the mSA2 domain and directed effector cells to the HER2 + tumors. However, these co-treated mice died early, possibly due to the lung infiltration of UniCAR T cells that could recognize both native biotin and HER2. Our results suggest that UniCAR T cells guided with soluble linkers present a viable alternative to conventional CAR T cells, especially for patients resistant to antibody therapy and those with solid tumors exhibiting high antigenic variability. Critical to their success, however, is the choice of an appropriate binding domain for the CAR and the corresponding soluble linker, ensuring both efficacy and safety in therapeutic applications.

In engineered T cells the CAR is co-expressed along with the physiological TCR/CD3 complex, both utilizing the same downstream signaling machinery for T cell activation. It is unresolved whether CAR-mediated T cell activation depends on the presence of the TCR and whether CAR and TCR mutually cross-activate upon engaging their respective antigen. Here we demonstrate that the CD3ζ CAR level was independent of the TCR associated CD3ζ and could not replace CD3ζ to rescue the TCR complex in CD3ζ KO T cells. Upon activation, the CAR did not induce phosphorylation of TCR associated CD3ζ and, vice versa, TCR activation did not induce CAR CD3ζ phosphorylation. Consequently, CAR and TCR did not cross-signal to trigger T cell effector functions. On the membrane level, TCR and CAR formed separate synapses upon antigen engagement as revealed by total internal reflection fluorescence (TIRF) and fast AiryScan microscopy. Upon engaging their respective antigen, however, CAR and TCR could co-operate in triggering effector functions through combinatorial signaling allowing logic “AND” gating in target recognition. Data also imply that tonic TCR signaling can support CAR-mediated T cell activation emphasizing the potential relevance of the endogenous TCR for maintaining T cell capacities in the long-term.

Background The COVID-19 pandemic led to widespread public health interventions that significantly affected the transmission of various pathogens, including enteroviruses (EVs). EVs exhibit considerable genetic diversity and can cause clinical manifestations ranging from mild illnesses to severe diseases. Our present study aimed to evaluate the diversity of circulating EV types in Hungary and assess the impact of lockdown measures on EV prevalence based on testing clinical samples obtained from symptomatic patients. Methods As part of the routine enterovirus diagnosis, we conducted quantitative reverse transcription polymerase chain reaction (RT-qPCR) on clinical samples obtained from patients presenting with symptoms corresponding to EV infection. Positive samples were then subjected to virus isolation in cell culture and next-generation sequencing (NGS). Phylogenetic analysis was performed to place the newly generated sequences within the global diversity of EV strains for comparison. Results During this period, an overall number of 125 patients tested positive for EVs, mostly children under the age of 15 years. The most common symptoms were fever, hand-foot-mouth disease, encephalitis, and meningitis. The temporal distribution of EV-positive cases showed strong seasonality, with peaks in the summer and autumn months. The lowest number of confirmed cases occurred during the lockdown years, attributed to limited sample collection and reduced personal contacts. However, following the easing of restrictions, the number of cases significantly increased, with the highest incidence observed in 2022. The distribution of EV genotypes shifted notably after easing the lockdowns. While only coxsackievirus (CV)A6 was detected during 2021, a broader range of genotypes emerged afterwards, including CVA10, CVA16, echovirus E9, and E11. Conclusions Next-generation sequencing analysis revealed notable genotypic diversity, providing valuable insights into the evolution of EVs in Hungary and across Europe. These findings underscore the importance of continued surveillance of enterovirus infections, particularly in the context of pandemic recovery, as the shifting EV genotype landscape may impact disease severity and spread, highlighting the need for adaptive public health responses.

Chimeric antigen receptor (CAR)-modified T cells brought a paradigm shift in the treatment of chemotherapy-resistant lymphomas. Conversely, clinical experience with CAR T cells targeting solid tumors has been disheartening, indicating the necessity of their molecular-level optimization. While incorporating CD28 or 41BB costimulatory domains into CARs in addition to the CD3z signaling domain improved the long-term efficacy of T cell products, their influence on early tumor engagement has yet to be elucidated. We studied the antigen-independent self-association and membrane diffusion kinetics of first- (.z), second- (CD28.z, 41BB.z), and third- (CD28.41BB.z) generation HER2-specific CARs in the resting T cell membrane using super-resolution AiryScan microscopy and fluorescence correlation spectroscopy, in correlation with RoseTTAFold-based structure prediction and assessment of oligomerization in native Western blot. While .z and CD28.z CARs formed large, high-density submicron clusters of dimers, 41BB-containing CARs formed higher oligomers that assembled into smaller but more numerous membrane clusters. The first-, second-, and third-generation CARs showed progressively increasing lateral diffusion as the distance of their CD3z domain from the membrane plane increased. Confocal microscopy analysis of immunological synapses showed that both small clusters of highly mobile CD28.41BB.z and large clusters of less mobile .z CAR induced more efficient CD3ζ and pLck phosphorylation than CD28.z or 41BB.z CARs of intermediate mobility. However, electric cell-substrate impedance sensing revealed that the CD28.41BB.z CAR performs worst in sequential short-term elimination of adherent tumor cells, while the .z CAR is superior to all others. We conclude that the molecular structure, membrane organization, and mobility of CARs are critical design parameters that can predict the development of an effective immune synapse. Therefore, they need to be taken into account alongside the long-term biological effects of costimulatory domains to achieve an optimal therapeutic effect.

Eredményeinket összefoglalva megállapíthatjuk, hogy míg a HER2.z és a HER2.CD28.z CAR dimerek nagy méretű és denzitású szubmikronos doméneket alkottak, a 41BB-t tartalmazó CAR oligomerek kisméretű, de nagyobb számú membrán klaszterré álltak össze. Ezzel párhuzamosan az I., II. és III. generációba tartozó CAR-ok a CD3z domén membrán síkjától mért távolságával arányosan gyorsuló laterális diffúziót mutattak. Immunfluoreszcenciával vizsgálva az első tumorsejttel való találkozáskor mind a HER2.CD28.41BB.z rendkívül mobilis oligomerjei és azok kisméretű klaszterei, mind a HER2.z CAR-ok előre összeszerelődött nagyméretű klaszterei hatékonyabban indukáltak CD3z foszforilációt és gyűjtöttek pLck-t az immunológiai szinapszisba, mint a közepesen mobilis HER2.CD28.z és HER2.41BB.z CAR-ok. Ugyanakkor tumorsejtkultúrák ECIS-szel mért impedanciaváltozása alapján hosszabb, egy napos távon a CD28.41BB.z CAR eredményezte a legkisebb ölést és a HER2.z CAR T sejtek bizonyultak a leghatékonyabbnak. Mindezek alapján a molekuláris szerkezet, a membránbeli szerveződés és a mobilitás olyan fontos paraméterei a CAR-ok tervezésének, amelyek következtetni engednek az immunszinapszis kialakulásának és a célsejt elpusztításának hatékonyságára.Egy másik lehetséges beavatkozási pont a szolid tumorokat célzó CAR T terápia optimalizálásában a kostimulációs domének közvetítette aktivációs jelátvitel és a natív T sejt funkciók összefonódásának vizsgálata. Kutatásunk során demonstráltuk, hogy a CAR T készítmények előállításának metodikája befolyásolja az elkészült heterogén sejttermékek fenotipikus összetételét, és igazoltuk ezen jellemzők jelentős hatását a CAR T sejtek működésére. Eredményeink szerint a jobban differenciált, effektor memória domináns CAR T sejttermékek erősebb in vitro citotoxicitással rendelkeznek és kevésbé merülnek ki hosszan tartó antigénstimuláció alatt, mint azok a sejtcsoportok, amelyek differenciációját az előállítási szakaszban korlátoztuk. Megfigyeltük továbbá, hogy a 41BB kostimuláció hatására, valamint a transzdukciót megelőző T sejt expanzió során alkalmazott RetroNectin stimuláció mellékhatásaként a CD8+ citotoxikus CAR T sejtek aránya jelentős növekedést mutatott. Preklinikai modellünkben demonstráltuk, hogy az effektor memória irányú differenciáció, és a kiegyensúlyozott CD4+ /CD8+ arány kölcsönzi a legerőteljesebb expanziós és citolitikus hatékonyságot a HER2+ szolid tumorokat célzó CAR T sejteknek. Ez a felismerés azt a világos üzenetet közvetíti a klinikumnak, hogy CAR T sejtkészítmények előállításnak optimalizálása és az ideális fenotípusprofil meghatározása a kezelt tumor típusának és antigénprofiljának függvényében elengedhetetlen feltétele a sikeres klinikai kipróbálásnak.