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The differentiation of naive CD8+ T cells into effector cells is important for establishing immunity. However, the effect of heterogeneous naive CD8+ T cell populations is not fully understood. Here, we demonstrate that steady-state naive CD8+ T cells are composed of functionally heterogeneous subpopulations that differ in their ability to differentiate into type 17 cytotoxic effector cells (Tc17) in a context of murine inflammatory disease models, such as inflammatory bowel disease and graft-versus-host disease. The differential ability of Tc17 differentiation is not related to T-cell receptor (TCR) diversity and antigen specificity but is inversely correlated with self-reactivity acquired during development. Mechanistically, this phenomenon is linked to differential levels of intrinsic TCR sensitivity and basal Suppressor of Mothers Against Decapentaplegic 3 (SMAD3) expression, generating a wide spectrum of Tc17 differentiation potential within naive CD8+ T cell populations. These findings suggest that developmental self-reactivity can determine the fate of naive CD8+ T cells to generate functionally distinct effector populations and achieve immense diversity and complexity in antigen-specific T-cell immune responses.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

Programmed death-ligand 1 (PD-L1) is a promising target for cancer immunotherapy due to its ability to inhibit T cell activation; however, its expression on various noncancer cells may cause on-target off-tumor toxicity when designing PD-L1-targeting Chimeric Antigen Receptor (CAR) T cell therapies. Combining rational design and directed evolution of the human fibronectin-derived monobody scaffold, PDbody was engineered to bind to PD-L1 with a preference for a slightly lower pH, which is typical in the tumor microenvironment. PDbody was further utilized as a CAR to target the PD-L1-expressing triple negative MDA-MB-231 breast cancer cell line. To mitigate on-target off-tumor toxicity associated with targeting PD-L1, a Cluster of Differentiation 19 (CD19)-recognizing SynNotch IF THEN gate was integrated into the system. This CD19-SynNotch PDbody-CAR system was then expressed in primary human T cells to target CD19-expressing MDA-MB-231 cancer cells. These CD19-SynNotch PDbody-CAR T cells demonstrated both specificity and efficacy in vitro, accurately eradicating cancer targets in cytotoxicity assays. Moreover, in an in vivo bilateral murine tumor model, they exhibited the capability to effectively restrain tumor growth. Overall, CD19-SynNotch PDbody-CAR T cells represent a distinct development over previously published designs due to their increased efficacy, proliferative capability, and mitigation of off-tumor toxicity for solid tumor treatment.

Cellular signaling, crucial for biological processes like immune response and homeostasis, relies on specificity and fidelity in signal transduction to accurately respond to stimuli amidst biological noise. Kinetic proofreading (KPR) is a key mechanism enhancing signaling specificity through time-delayed steps, although its effectiveness is debated due to intrinsic noise potentially reducing signal fidelity. In this study, we reformulate the theory of kinetic proofreading (KPR) by convolving multiple intermediate states into a single state and then define an overall processing time required to traverse these states. This simplification allows us to succinctly describe kinetic proofreading in terms of a single waiting time parameter, facilitating a more direct evaluation and comparison of KPR performance across different biological contexts such as DNA replication and T cell receptor (TCR) signaling. We find that loss of fidelity for longer proofreading steps relies on the specific strategy of information extraction and show that in the first-passage time (FPT) discrimination strategy, longer proofreading steps can exponentially improve the accuracy of KPR at the cost of speed. Thus, KPR can still be an effective discrimination mechanism in the high noise regime. However, in a product concentration-based discrimination strategy, longer proofreading steps do not necessarily lead to an increase in performance. However, by introducing activation thresholds on product concentrations, can we decompose the product-based strategy into a series of FPT-based strategies to better resolve the subtleties of KPR-mediated product discrimination. Our findings underscore the importance of understanding KPR in the context of how information is extracted and processed in the cell.

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation.

Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti -tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen -containing liposomes conjugated with CD169- or DC -SIGN -specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169 + and DC -SIGN + APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC -SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR b-chain (TCR-b) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-b and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-b clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternaletal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL, Burroughs Wellcome Fund, National Institutes of Health, Ulla og Mogens Folmer Andersens Fond, Depto. de Estadística y Ciencia de los Datos, Fac. de Estudios Estadísticos, TRUE, pub

Background: We assessed the laboratory diagnosis and treatment of invasive fungal disease (IFD) in Italy to detect limitations and potential for improvement. Methods: The survey was available online at www.clinicalsurveys.net/uc/IFI management capacity/, and collected variables such as (a) institution profile, (b) perceptions of IFD in the respective institution, (c) microscopy, (d) culture and fungal identification, (e) serology, (f) antigen detection, (g) molecular tests, (h) susceptibility testing and (i) therapeutic drug monitoring (TDM). Results: The laboratory capacity study received responses from 49 Italian centres, with an equitable geographical distribution of locations. The majority of respondents (n = 36, 73%) assessed the occurrence of IFD as moderate-high, with Aspergillus spp. being the pathogen of highest concern, followed by Candida spp. and Mucorales. Although 46 (94%) of the institutions had access to microscopy, less than half of them performed direct microscopy on clinical specimens always when IFD was suspected. Cultures were available in all assessed laboratories, while molecular testing and serology were available in 41 (83%), each. Antigen detection tests and antifungal drugs were also generally accessible (> 90%) among the participating institutions. Nevertheless, access to TDM was limited (n = 31, 63%), with a significant association established between therapeutic drug monitoring availability and higher gross domestic product per capita. Conclusions: Apart from TDM, Italy is adequately prepared for the diagnosis and treatment of IFD, with no significant disparities depending on gross domestic product. Future efforts may need to focus on enhancing the availability and application of direct microscopic methods, as well as TDM, to promote optimal treatment and better patient outcomes.

Prostate cancer is a disease of significant public health importance and is the most common in the male population starting from the age of 50, with its peak occurring after the age of 65. Its incidence has increased in recent years due to improvements in diagnostic techniques, increased life expectancy, and other less-known factors such as environmental exposure, lifestyle, and genetics. The most common type of prostate cancer is adenocarcinoma, which typically localizes in the peripheral zone of the prostate, exhibits slow growth, and often remains asymptomatic in the early stages of the disease. There are various treatment options available for addressing the different stages and levels of aggressiveness of this neoplasm. Achieving early detection and applying appropriate treatment are essential steps to enhance survival rates in prostate cancer patients., El cáncer de próstata es una enfermedad de gran relevancia en la salud pública. Es el más común en la población masculina a partir de los 50 años, y su pico máximo es después de los 65 años. Su incidencia ha aumentado en los últimos años debido a la mejora en las técnicas diagnósticas, el aumento en la esperanza de vida y otros factores no tan conocidos como la exposición ambiental, el estilo de vida y la genética. El tipo más común de cáncer de próstata es el adenocarcinoma, el cual generalmente se localiza en la zona periférica de la próstata, cuyo crecimiento es lento y se presenta de manera asintomática en estadios tempranos de la enfermedad. Existen diversas opciones de tratamiento para el abordaje según las diferentes etapas y grados de agresividad de esta neoplasia. Lograr una detección temprana y aplicar un tratamiento adecuado son pasos esenciales para mejorar las tasas de supervivencia en pacientes con cáncer de próstata.

The T cell receptor (TCR) complex is a naturally occurring antigen sensor that detects, amplifies and coordinates cellular immune responses to epitopes derived from cell surface and intracellular proteins. Thus, TCRs enable the targeting of proteins selectively expressed by cancer cells, including neoantigens, cancer germline antigens and viral oncoproteins. As such, TCRs have provided the basis for an emerging class of oncology therapeutics. Herein, we review the current cancer treatment landscape using TCRs and TCR-like molecules. This includes adoptive cell transfer of T cells expressing endogenous or engineered TCRs, TCR bispecific engagers and antibodies specific for human leukocyte antigen (HLA)-bound peptides (TCR mimics). We discuss the unique complexities associated with the clinical development of these therapeutics, such as HLA restriction, TCR retrieval, potency assessment and the potential for cross-reactivity. In addition, we highlight emerging clinical data that establish the antitumour potential of TCR-based therapies, including tumour-infiltrating lymphocytes, for the treatment of diverse human malignancies. Finally, we explore the future of TCR therapeutics, including emerging genome editing methods to safely enhance potency and strategies to streamline patient identification.

The coordinated (dis)engagement of the membrane-bound T cell receptor (TCR)-CD3-CD4 complex from the peptide-major histocompatibility complex (pMHC) is fundamental to TCR signal transduction and T cell effector function. As such, an atomic-scale understanding would not only enhance our basic understanding of the adaptive immune response but would also accelerate the rational design of TCRs for immunotherapy. In this study, we explore the impact of the CD4 coreceptor on the TCR-pMHC (dis)engagement by constructing a molecular-level biomimetic model of the CD3-TCR-pMHC and CD4-CD3-TCR-pMHC complexes within a lipid bilayer. After allowing the system complexes to equilibrate (engage), we use steered molecular dynamics to dissociate (disengage) the pMHC. We find that 1) the CD4 confines the pMHC closer to the T cell by 1.8 nm at equilibrium; 2) CD4 confinement shifts the TCR along the MHC binding groove engaging a different set of amino acids and enhancing the TCR-pMHC bond lifetime; 3) the CD4 translocates under load increasing the interaction strength between the CD4-pMHC, CD4-TCR, and CD4-CD3; and 4) upon dissociation, the CD3-TCR complex undergoes structural oscillation and increased energetic fluctuation between the CD3-TCR and CD3-lipids. These atomic-level simulations provide mechanistic insight on how the CD4 coreceptor impacts TCR-pMHC (dis)engagement. More specifically, our results provide further support (enhanced bond lifetime) for a force-dependent kinetic proofreading model and identify an alternate set of amino acids in the TCR that dominate the TCR-pMHC interaction and could thus impact the design of TCRs for immunotherapy.

Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.

Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.

Remarkable complete response rates have been shown with tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell therapy targeting CD19, in patients up to age 26 years with refractory/relapsed B-cell acute lymphoblastic leukemia; it is US Food and Drug Administration approved for this indication. Currently, patients receive a single dose of tisagenlecleucel across a wide dose range of 0.2 to 5.0 × 106 and 0.1 to 2.5 × 108 CAR T cells per kg for patients ≤50 and >50 kg, respectively. The effect of cell dose on survival and remission is not yet well established. Our primary goal was to determine if CAR T-cell dose affects overall survival (OS), event-free survival (EFS), or relapse-free-survival (RFS) in tisagenlecleucel recipients. Retrospective data were collected from Pediatric Real World CAR Consortium member institutions and included 185 patients infused with commercial tisagenlecleucel. The median dose of viable transduced CAR T cells was 1.7 × 106 CAR T cells per kg. To assess the impact of cell dose, we divided responders into dose quartiles: 0.134 to 1.300 × 106 (n = 48 [27%]), 1.301 to 1.700 × 106 (n = 46 [26%]), 1.701 to 2.400 × 106 (n = 43 [24%]), and 2.401 to 5.100 × 106 (n = 43 [24%]). OS, EFS, and RFS were improved in patients who received higher doses of tisagenlecleucel (P = .031, .0079, and .0045, respectively). Higher doses of tisagenlecleucel were not associated with increased toxicity. Because the current tisagenlecleucel package insert dose range remains broad, this work has implications in regard to targeting higher cell doses, within the approved dose range, to optimize patients' potential for long-standing remission.

The development of CD4+ T cells and CD8+ T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8+ or CD4+ T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during a first wave of TCR signaling, followed by a second TCR signaling wave that coincides with CD8+ T cell lineage specification. CITE-seq and pharmaceutical inhibition experiments implicated a TCR-calcineurin-NFAT-GATA3 axis in driving the CD4+ T cell fate. Our data provide a resource for understanding cell fate decisions and implicate a sequential selection process in guiding lineage choice.

CD4+ T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8+ T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8+ T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4+ T cells is less well understood. We have characterized the murine CD4+ T cell response against a validated NeoAg (CLTCH129>Q) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTCH129>Q-specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4+ T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8+ T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4+ T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (TSCM)-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with TSCM-like CD4+ T cells results in lower PD-1 expression by CD8+ T cells in the tumor microenvironment and an increased frequency of PD-1+CD8+ T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4+ T cells in mediating antitumor immunity via providing help to CD8+ T cells and highlight their therapeutic potential in ACT.

Coccidioidomycosis is a fungal disease associated with soil exposure that frequently goes undiagnosed due at least in part to its nonspecific presentation and the lack of clinical suspicion by health care providers. Currently available diagnostics for coccidioidomycosis offer qualitative results that can suffer from low specificity, while semiquantitative assays are labor-intensive and complex and can require multiple days to complete. Furthermore, significant confusion exists regarding the optimal diagnostic algorithms and appropriate usage of available diagnostic tests. This review aims to inform clinical laboratorians and treating clinicians about the current diagnostic landscape, appropriate diagnostic strategies, and future diagnostic directions for coccidioidomycosis, which is expected to become more prevalent due to increased migration into areas of endemicity and climate changes.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

IntroductionMuscle-invasive bladder cancer (MIBC) is a heterogeneous disease with several taxonomic molecular subtypes showing different genetic, clinical, and epidemiological profiles. It has been suggested that MIBC-subtypes follow different tumorigenesis pathways playing decisive roles at different stages of tumor development, resulting in distinct tumor microenvironment containing both innate and adaptive immune cells (T and B lymphocytes). We aim to characterize the MIBC tumor microenvironment by analyzing the tumor-infiltrating B and T cell repertoire according to the taxonomic molecular subtypes.MethodsRNAseq data from 396 MIBC samples included in TCGA were considered. The subtype information was collected from the international consensus taxonomic classification describing six subtypes: Basal/Squamous-like (Ba/Sq), Luminal papillary (LumP), Luminal non-Specify (LumNS), Luminal unstable (LumU), Stroma-rich, and Neuroendocrine-like (NE-like). Using MiXCR, we mapped the RNA read sequences to their respective B-cell receptor (BCR) and T-cell receptor (TCR) clonotypes. To evaluate the BCR and TCR differences among subtypes, we compared diversity measures (richness and diversity) using a Wilcoxon test and we performed a network analysis to characterize the clonal expansion. For the survival analysis stratified by subtypes, Cox regression models adjusted for age, region, and pathological stage were performed.ResultsOverall, we found different patterns of tumor-infiltrating immune repertoire among the different MIBC subtypes. Stroma-rich and Ba/Sq tumors showed the highest BCR and TCR infiltration while LumP showed the lowest. In addition, we observed that the Ba/Sq and Stroma-rich tumors were more clonally expanded than the Luminal subtypes. Moreover, higher TCR richness and diversity were significantly associated with better survival in the Stroma-rich and Ba/Sq subtypes.DiscussionThis study provides evidence that MIBC subtypes present differences in the tumor mic

Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells.

Chimeric antigen receptor-associated hemophagocytic lymphohistiocytosis (HLH)-like toxicities (LTs) involving hyperferritinemia, multiorgan dysfunction, coagulopathy, and/or hemophagocytosis are described as occurring in a subset of patients with cytokine release syndrome (CRS). Case series report poor outcomes for those with B-cell acute lymphoblastic leukemia (B-ALL) who develop HLH-LTs, although larger outcomes analyses of children and young adults (CAYAs) with B-ALL who develop these toxicities after the administration of commercially available tisagenlecleucel are not described. Using a multi-institutional database of 185 CAYAs with B-ALL, we conducted a retrospective cohort study including groups that developed HLH-LTs, high-grade (HG) CRS without HLH-LTs, or no to low-grade (NLG) CRS without HLH-LTs. Primary objectives included characterizing the incidence, outcomes, and preinfusion factors associated with HLH-LTs. Among 185 CAYAs infused with tisagenlecleucel, 26 (14.1%) met the criteria for HLH-LTs. One-year overall survival and relapse-free survival were 25.7% and 4.7%, respectively, in those with HLH-LTs compared with 80.1% and 57.6%, respectively, in those without. In multivariable analysis for death, meeting criteria for HLH-LTs carried a hazard ratio of 4.61 (95% confidence interval, 2.41-8.83), controlling for disease burden, age, and sex. Patients who developed HLH-LTs had higher pretisagenlecleucel disease burden, ferritin, and C-reactive protein levels and lower platelet and absolute neutrophil counts than patients with HG- or NLG-CRS without HLH-LTs. Overall, CAYAs with B-ALL who developed HLH-LTs after tisagenlecleucel experienced high rates of relapse and nonrelapse mortality, indicating the urgent need for further investigations into prevention and optimal management of patients who develop HLH-LTs after tisagenlecleucel.

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.

BackgroundThe tumor antigen NY-ESO-1 has been shown to be an effective target for transgenic adoptive cell therapy (ACT) for the treatment of sarcoma and melanoma. However, despite frequent early clinical responses, many patients ultimately develop progressive disease. Understanding the mechanisms underlying treatment resistance is crucial to improve future ACT protocols. Here, we describe a novel mechanism of treatment resistance in sarcoma involving loss of expression of NY-ESO-1 in response to transgenic ACT with dendritic cell (DC) vaccination and programmed cell death protein-1 (PD-1) blockade.MethodsA HLA-A*02:01-positive patient with an NY-ESO-1-positive undifferentiated pleomorphic sarcoma was treated with autologous NY-ESO-1-specific T-cell receptor (TCR) transgenic lymphocytes, NY-ESO-1 peptide-pulsed DC vaccination, and nivolumab-mediated PD-1 blockade.ResultsPeripheral blood reconstitution with NY-ESO-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. There was initial tumor regression, and immunophenotyping of the peripheral transgenic T cells showed a predominantly effector memory phenotype over time. Tracking of transgenic T cells to the tumor sites was demonstrated in on-treatment biopsy via both TCR sequencing-based and RNA sequencing-based immune reconstitution, and nivolumab binding to PD-1 on transgenic T cells was confirmed at the tumor site. At the time of disease progression, the promoter region of NY-ESO-1 was found to be extensively methylated, and tumor NY-ESO-1 expression was completely lost as measured by RNA sequencing and immunohistochemistry.ConclusionsACT of NY-ESO-1 transgenic T cells given with DC vaccination and anti-PD-1 therapy resulted in transient antitumor activity. NY-ESO-1 expression was lost in the post-treatment sample in the setting of extensive methylation of the NY-ESO-1 promoter region.Biological/clinical insightAntigen loss represents a novel mechanism of immune escape in sarcoma and

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

The primary utility of the VWF antigen (VWF:Ag) test is to diagnose von Willebrand disease (VWD), which is a bleeding disorder caused by due to a reduction or qualitative alteration of VWF, which may be hereditary in most cases, or acquired when secondary to other pathologies. On the other hand, the VWF:RCo test measures the ability of VWF to agglutinate platelets exogenous normals fixed in formaldehyde, in the presence of the cofactor ristocetin antibiotic. The amount of ristocetin-induced agglutination is directly related to the VWF concentration, and it is determined as a percentage of activity through of an aggregometer. Finally, the von Willebrand factor multimers test is based on the technique of electrophoresis and immunofixation in agarose gel, which allows to evaluate the distribution and presence of VWF multimers in plasma according to their molecular weight. In the gel, the multimers are separated and immunoprecipitated with a specific anti-VWF antiserum. The use of a peroxidase-labeled antibody and a specific substrate makes it possible to visualize the different bands on the gel., La utilidad principal de la prueba antígeno del FVW (FVW:Ag) es diagnosticar la enfermedad de von Willebrand (EVW), la cual es un trastorno hemorrágico causado por una reducción o alteración cualitativa del FVW, que puede ser hereditario en la mayoría de los casos, o adquirido cuando es secundario a otras patologías. Por su parte, la prueba FVW:RCo mide la capacidad del FVW de aglutinar plaquetas exógenas normales fijadas en formaldehído, ante la presencia del cofactor antibiótico ristocetina. La cantidad de aglutinación inducida por la ristocetina está directamente relacionada con la concentración del FVW, y se determina como porcentaje de actividad a través de un agregómetro. Finalmente, la prueba de multímeros de factor von Willebrand se basa en la técnica de electroforesis e inmunofijación en gel de agarosa, que permite evaluar la distribución y presencia de los multímeros del FVW en el plasma de acuerdo a su peso molecular. En el gel, los multímeros son separados e inmunoprecipitados con un antisuero específico anti-FVW. La utilización de un anticuerpo marcado con peroxidasa y un substrato específico, hace que se puedan visualizar las diferentes bandas en el gel.

von Willebrand disease (VWD) is the most common hereditary bleeding disorder, and is characterized by a decreased ability of the von Willebrand factor (VWF) to bind to platelets and extracellular matrix collagen during primary hemostasis, due to quantitative or qualitative defects. VWD is classified into three main phenotypes: 1 and 3, which are quantitative disorders, and 2 (2A, 2B, 2M and 2N) that reflects qualitative disorders. Several steps are necessary for its diagnosis: 1) evaluation of the patient's personal and family bleeding history, 2) initial screening tests for bleeding disorders, 3) tests for the detection of VWD, 4) tests for the classification of VWD, and 5) molecular analysis. Both the subclassification of VWD and its diagnosis continue to represent important challenges, which we aimed to describe in this review, so that health professionals have a guide to assist them when they have patients with a bleeding disorder that requires exclusion of VWD, and implementation of an appropriate treatment., La enfermedad de von Willebrand (EVW) es el trastorno hemorrágico hereditario más común, y se caracteriza por presentar disminución de la capacidad del factor von Willebrand (FVW) de unirse a las plaquetas y al colágeno de la matriz extracelular durante la hemostasia primaria, debido a defectos cuantitativos o cualitativos. La EVW se clasifica en tres fenotipos principales: el 1 y el 3 que son trastornos cuantitativos, y el 2 que se subclasifica en 2A, 2B, 2M y 2N, y refleja los trastornos cualitativos. Para su diagnóstico son necesarios varios pasos: 1) la evaluación del historial de sangrado personal y familiar del paciente, 2) detección inicial de trastornos hemorrágicos, 3) pruebas para la detección de la EVW, 4) pruebas para la tipificación de la EVW, y 5) el análisis molecular. Tanto la subclasificación de la EVW como su diagnóstico continúan planteando desafíos importantes, motivo por el cual se realiza esta revisión, de manera que los profesionales de la salud tengan una guía que los oriente al momento de tener pacientes con algún trastorno hemorrágico que amerite descartar una EVW e implementar un tratamiento adecuado.

Activation of the immune system is a needed for designing new antigen/drug delivery systems to develop new therapeutics and for developing animal disease models to study the disease pathogenesis. A weak antigen alone is insufficient to activate the immune system. Sometimes, assistance in the form of polymers is needed to control the release of antigens under in vivo conditions or in the form of an adjuvant to activate the immune system efficiently. Many kinds of polymers from different functional groups are suitable as microbial antigens for inducing therapeutic immune responses against infectious diseases at the preclinical level. The choice of the functionality of polymer varies as per the application type. Polymers from the acid and ester groups are the most common types investigated for protein-based antigens. However, electrostatic interaction-displaying polymers like cationic polymers are the most common type for nucleic acid-based antigens. Metal coordination chemistry is commonly used in polymers designed for cancer immunotherapeutic applications to suppress inflammation and induce a protective immune response. Amide chemistry is widely deployed in polymers used to develop antigen-specific disease models like the experimental autoimmune arthritis murine model. © 2023 by the authors.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Every infection consists of a battle between the invading pathogen and the resisting host. To be successful, a pathogen must escape the many defenses of the host immune system until it can replicate and spread to another host. A pathogen must prevent one of three stages of immune function: detection, activation, or effector function. Examples of disease-specific immune evasion and the mechanisms used by pathogens to prevail over their hosts' immune systems are discussed. Also considered is what these host-pathogen interactions reveal about the normal function of the immune system and basic cell biological processes, such as protein maturation and degradation.

In this course, evolutionary pathways that have led to the development of innate and adaptive immunity are analyzed, the conserved and unique features of the immune response from bacteria to higher vertebrates is traced, and factors, such as adaptive changes in pathogens that have shaped the evolution of immune system are identified.This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting.

Every infection consists of a battle between the invading pathogen and the resisting host. To be successful, a pathogen must escape the many defenses of the host immune system until it can replicate and spread to another host. A pathogen must prevent one of three stages of immune function: detection, activation, or effector function. Examples of disease-specific immune evasion and the mechanisms used by pathogens to prevail over their hosts' immune systems are discussed. Also considered is what these host-pathogen interactions reveal about the normal function of the immune system and basic cell biological processes, such as protein maturation and degradation.

In this course, evolutionary pathways that have led to the development of innate and adaptive immunity are analyzed, the conserved and unique features of the immune response from bacteria to higher vertebrates is traced, and factors, such as adaptive changes in pathogens that have shaped the evolution of immune system are identified.This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting.

Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization of immunosequencing, also known as high-throughput sequencing of the T-cell receptor (HTS-TCR), enables identification and quantification of the precise genetic signature of dominant T-cell clones. Although immunosequencing is more sensitive than commonly used methods such as polymerase chain reaction (PCR) paired with capillary electrophoresis or flow cytometry, it remains underutilized for CTCL management. Nonetheless, incorporation of HTS-TCR in clinical practice offers distinct advantages compared to other molecular analyses that may improve diagnostic evaluation, prognostication, and disease monitoring in CTCL. The objective of this comprehensive review is to provide a thorough explanation of the application of immunosequencing in the context of CTCL. We describe the significance of T-cell clonality and the methods used to detect it, including a detailed comparison between PCR paired with capillary electrophoresis and HTS-TCR. The utilization of immunosequencing in the blood and skin of CTCL patients is discussed in depth, specifically outlining how HTS-TCR can assist in diagnosing CTCL, predicting outcomes, and tracking disease progression. Finally, we address the potential applications of immunosequencing in clinical management and research as well as the novel challenges it presents.

Autoimmune diseases affect many people worldwide. Current treatment modalities focus on the reduction of disease symptoms using anti-inflammatory drugs which can lead to side effects due to systemic immune suppression. Restoration of immune tolerance by down-regulating auto-reactive cells in an antigen-specific manner is currently the “holy grail” for the treatment of autoimmune diseases. A promising strategy is the use of nanoparticles that can deliver antigens to antigen-presenting cells which in turn can enhance antigen-specific regulatory T cells. In this review, we highlight some promising cell targets (e.g. liver sinusoidal endothelial cells and splenic marginal zone macrophages) for exploiting natural immune tolerance processes, and several strategies by which antigen-carrying nanoparticles can target these cells. We also discuss how nanoparticles carrying immunomodulators may be able to activate tolerance in other antigen-presenting cell types. Finally, we discuss some important aspects that must be taken into account when translating data from animal studies to patients.

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

UNAIDS ma’lumotlariga ko’ra dunyoda 36,7 million odam va kuniga 5000 dan ortiq kasallanish soni qayd etilgan. OITS ni davolab bo’lmaganligi sababli asosiy choralar uni profilaktikasiga qaratilgan edi. Lekin hozirda tibbiyot va biotexnologiyada rivojlanib katta yutuqlarga erishilmoqda. Tuzatib bo’lmaydigan deb hisoblangan kasalliklarga yangi davolash usullari va preparatlar ishlab chiqarilmoqda

UNAIDS ma’lumotlariga ko’ra dunyoda 36,7 million odam va kuniga 5000 dan ortiq kasallanish soni qayd etilgan. OITS ni davolab bo’lmaganligi sababli asosiy choralar uni profilaktikasiga qaratilgan edi. Lekin hozirda tibbiyot va biotexnologiyada rivojlanib katta yutuqlarga erishilmoqda. Tuzatib bo’lmaydigan deb hisoblangan kasalliklarga yangi davolash usullari va preparatlar ishlab chiqarilmoqda

Mixed Connective Tissue Disease is an autoimmune disease characterized by Raynaud’s phenomenon and arthritis among other symptoms. It is primarily caused by antibodies that target the U1-RNP 70K peptide. The treatment proposed in this paper uses competitive inhibition to prevent the binding of the anti-U1-RNP 70K antibodies with the U1-RNP 70K peptide. A method for testing the designed treatment in silico is proposed using AutoDock Vina docking software.

Infants with B-cell acute lymphoblastic leukemia (B-ALL) have poor outcomes because of chemotherapy resistance leading to high relapse rates. Tisagenlecleucel, a CD19-directed chimeric antigen receptor T-cell (CART) therapy, is US Food and Drug Administration approved for relapsed or refractory B-ALL in patients ≤25 years; however, the safety and efficacy of this therapy in young patients is largely unknown because children <3 years of age were excluded from licensing studies. We retrospectively evaluated data from the Pediatric Real-World CAR Consortium to examine outcomes of patients with infant B-ALL who received tisagenlecleucel between 2017 and 2020 (n = 14). Sixty-four percent of patients (n = 9) achieved minimal residual disease-negative remission after CART and 50% of patients remain in remission at last follow-up. All patients with high disease burden at time of CART infusion (>M1 marrow) were refractory to this therapy (n = 5). Overall, tisagenlecleucel was tolerable in this population, with only 3 patients experiencing ≥grade 3 cytokine release syndrome. No neurotoxicity was reported. This is the largest report of tisagenlecleucel use in infant B-ALL and shows that this therapy is safe and can be effective in this population. Incorporating this novel immunotherapy into the treatment of infant B-ALL offers a promising therapy for a highly aggressive leukemia.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

This special edition summarizes major advances in our understanding of signaling by T lymphocytes. T cell interactions with antigen-presenting cells (APCs) and other immune cells are characterized by changes in T cell adhesion and major rearrangements of the actin cytoskeleton. This issue describes some of the mediators of these changes both within the T cells and on the T cell surface. The five articles focus on "inside-out integrin signaling" in T cells, components of the immunological synapse between lymphocyte and APCs, an unexpected role for T cell receptor (TCR) signaling from endosomes, transfer of membrane constituents from APCs to T cells via trogocytosis, immune deficiencies in these T cell signaling pathways, and the role of thymocyte-expressed molecule involved in selection (THEMIS) in thymocyte development and peripheral T cell function.

Syftet med projektet var att utveckla en multiplexanalys för att detektera antigen från SARS-CoV-2, influensa och respiratoriskt syncytial virus genom att använda en masspektrometrisk metod som involverar antigenspecifika bindare. Bindarna klonades, renades och biotinylerades innan de användes i en analys utvecklad genom en målinriktad metod som involverade antigenerna. En slutsats som drog var att det var möjligt för Avi-märkta bindare att specifikt binda antigen-härledda peptidmål i multiplexanalysen., The purpose of project was to develop a multiplex assay capable of detecting antigens from SARSCoV-2, influenza and respiratory syncytial virus by utilizing a mass spectrometric approach involving antigen-specific binders. Binders were cloned, purified and biotinylated before being employed in an assay developed by though a targeted method involving the antigens. It was concluded to be possible for Avi-tagged binders to specifically bind antigen-derived peptide targets in the multiplex assay.

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to <4.9 log copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.

This special edition summarizes major advances in our understanding of signaling by T lymphocytes. T cell interactions with antigen-presenting cells (APCs) and other immune cells are characterized by changes in T cell adhesion and major rearrangements of the actin cytoskeleton. This issue describes some of the mediators of these changes both within the T cells and on the T cell surface. The five articles focus on "inside-out integrin signaling" in T cells, components of the immunological synapse between lymphocyte and APCs, an unexpected role for T cell receptor (TCR) signaling from endosomes, transfer of membrane constituents from APCs to T cells via trogocytosis, immune deficiencies in these T cell signaling pathways, and the role of thymocyte-expressed molecule involved in selection (THEMIS) in thymocyte development and peripheral T cell function.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Chimeric antigen receptor (CAR) T cells have transformed the therapeutic options for relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia. Data for CAR therapy in extramedullary (EM) involvement are limited. Retrospective data were abstracted from the Pediatric Real World CAR Consortium (PRWCC) of 184 infused patients from 15 US institutions. Response (complete response) rate, overall survival (OS), relapse-free survival (RFS), and duration of B-cell aplasia (BCA) in patients referred for tisagenlecleucel with EM disease (both central nervous system (CNS)3 and non-CNS EM) were compared with bone marrow (BM) only. Patients with CNS disease were further stratified for comparison. Outcomes are reported on 55 patients with EM disease before CAR therapy (CNS3, n = 40; non-CNS EM, n = 15). The median age at infusion in the CNS cohort was 10 years (range, <1-25 years), and in the non-CNS EM cohort it was 13 years (range, 2-26 years). In patients with CNS disease, 88% (35 of 40) achieved a complete response vs only 66% (10 of 15) with non-CNS EM disease. Patients with CNS disease (both with and without BM involvement) had 24-month OS outcomes comparable to those of non-CNS EM or BM only (P = .41). There was no difference in 12-month RFS between CNS, non-CNS EM, or BM-only patients (P = .92). No increased toxicity was seen with CNS or non-CNS EM disease (P = .3). Active CNS disease at time of infusion did not affect outcomes. Isolated CNS disease trended toward improved OS compared with combined CNS and BM (P = .12). R/R EM disease can be effectively treated with tisagenlecleucel; toxicity, relapse, and survival rates are comparable to those of patients with BM-only disease. Outcomes for isolated CNS relapse are encouraging.

T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen-binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tiseher and Weiner, 2019). Here, we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. Leveraging the ability of our system to rapidly disengage ligand binding, we also measure slower reset rates for downstream signaling events. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at slower resetting downstream signaling complexes that could help balance antigen sensitivity and discrimination.