T Cell Receptor Research Articles

Membraneless organelles in health and disease: exploring the molecular basis, physiological roles and pathological implications

AbstractOnce considered unconventional cellular structures, membraneless organelles (MLOs), cellular substructures involved in biological processes or pathways under physiological conditions, have emerged as central players in cellular dynamics and function. MLOs can be formed through liquid-liquid phase separation (LLPS), resulting in the creation of condensates. From neurodegenerative disorders, cardiovascular diseases, aging, and metabolism to cancer, the influence of MLOs on human health and disease extends widely. This review discusses the underlying mechanisms of LLPS, the biophysical properties that drive MLO formation, and their implications for cellular function. We highlight recent advances in understanding how the physicochemical environment, molecular interactions, and post-translational modifications regulate LLPS and MLO dynamics. This review offers an overview of the discovery and current understanding of MLOs and biomolecular condensate in physiological conditions and diseases. This article aims to deliver the latest insights on MLOs and LLPS by analyzing current research, highlighting their critical role in cellular organization. The discussion also covers the role of membrane-associated condensates in cell signaling, including those involving T-cell receptors, stress granules linked to lysosomes, and biomolecular condensates within the Golgi apparatus. Additionally, the potential of targeting LLPS in clinical settings is explored, highlighting promising avenues for future research and therapeutic interventions.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Staphylococcal superantigens evoke temporary and reversible T cell anergy, but fail to block the development of a bacterium specific cellular immune response.

Superantigens (sAgs) are bacterial virulence factors that induce a state of immune hyperactivation by forming a bridge between certain subsets of T cell receptor (TCR) β chains on T lymphocytes, and class II major histocompatibility complex (MHC-II) molecules; this cross-linking leads to indiscriminate T cell activation, cytokine storm and toxic shock. Here we show that sAg exposure drives the preferential expansion of naive and central memory T cell subsets, but not effector or resident memory T cells, which instead, hyper release pro-inflammatory cytokines. A targeted therapeutic approach to minimise cytokine release by effector memory T cells attenuated sAg-induced cytokine release. Irrespective of antigen experience, sAg activation does not render mature T cells permanently dysfunctional, and full restoration of effector function is observed following a transient and reversible anergy. Moreover, we show that in the face of sAg induced immune hyperactivation, an intact bacterium-specific CD4+ T cell response can be mounted.

Proteomic profile of the antibody diversity in circulating extracellular vesicles of lung adenocarcinoma.

Immunoglobulin diversity encompasses B-cell receptor, T-cell receptor, and antibody diversity. Existing studies have focused more on the role of B-cell and T-cell receptor diversity in tumor immunity, while the role of antibody diversity is less understood. This study examined and compared the blood extracellular vesicles (EVs) of lung cancer patients and healthy individuals using proteomics and bioinformatics analyses. The results revealed that among the 270 identified proteins, those involved in defense mechanisms were the most abundant. Most of these were antibody subtypes, accounting for 50.00%. Similarly, of the 40 identified EVs differentially expressed proteins (DEPs), 29 were involved in defense mechanisms (72.50%), with a higher proportion being antibody subtypes (82.76%). Furthermore, 24 DEP antibody subtypes were implicated in 18 immune reaction-related signaling pathways. These findings suggest that human serum EVs contain a significant number of antibody subtypes, and the antibody subtypes from lung cancer serum EVs differ from those of healthy controls (HCs). The variations in antibody diversity may be closely associated with lung cancer tumor immunity.

Cell Division Cycle 42 Improves Renal Functions, Fibrosis, Th1/Th17 Infiltration and Inflammation to Some Degree in Diabetic Nephropathy.

Our two previous studies observed that cell division cycle 42 (CDC42) was lower and correlated with improved renal function and inflammation in diabetic nephropathy (DN) patients, and CDC42 inhibited renal tubular epithelial cell fibrosis and inflammation under high glucose condition. Sequentially, this current study aimed to investigate the effect of CDC42 on improving renal function, fibrosis, and inflammation in DN mice, and its interaction with T cell receptor (TCR) related pathways. Mice were treated by streptozotocin to construct early-stage DN model, then transfected with CDC42 overexpression adenovirus, followed by simultaneous treatment of LY294002 (PI3K/AKT inhibitor) and CI-1040 (ERK inhibitor), respectively. CDC42 reduced blood glucose, creatinine, and 24h urine protein in DN mice, but only showed a tendency to decrease blood urea nitrogen without statistical significance. Hematoxylin&eosin staining revealed that CDC42 descended the glomerular volume, basement membrane thickness, and inflammatory cell infiltration in kidney. Meanwhile, CDC42 lowered fibronectin, TGF-β1, and Collagen I expressions in kidney, but not decreased α-SMA significantly. Besides, CDC42 decreased T-helper (Th) 1 and Th17 cells in kidney, and reduced serum IFN-γ, IL-1β, IL-17A, and TNF-α but not IL-6. Regarding TCR-related pathways, CDC42 activated AKT and ERK pathways but not JNK pathway. However, the treatment of LY294002 and CI-1040 had limited effect on attenuating CDC42's functions on renal function and fibrotic markers. CDC42 improves renal functions, fibrosis, Th1/Th17 infiltration and inflammation to some degree in DN mice, these functions may be independent to AKT and ERK pathways.

A Modular Genetic Approach to Newborn Screening from Spinal Muscular Atrophy to Sickle Cell Disease—Results from Six Years of Genetic Newborn Screening

Background/Objectives: Genetic newborn screening (NBS) has already entered the phase of common practice in many countries. In Germany, spinal muscular atrophy (SMA), severe combined immunodeficiency (SCID) and sickle cell disease (SCD) are currently a mandatory part of NBS. Here, we describe the experience of six years of genetic NBS including the prevalence of those three diseases in Germany. Methods: Samples and nucleic acids were extracted from dried blood spot cards, commonly used for NBS. A qPCR assay was used to detect disease-causing variants for SMA and SCD, and the detection of T-cell receptor excision circles (TRECs) was performed for SCID screening. Results: The results of the NBS of over 1 million newborns for SMA, approximately 770,000 for SCID and over 410,000 for SCD are discussed in detail. In these newborns, we have identified 121 cases of SMA, 15 cases of SCID and syndrome-based immunodeficiencies and 77 cases of SCD or β-thalassemia. Conclusions: The flexibility of multiplex qPCR is assessed as an effective tool for incorporating different molecular genetic markers for screening. The processing of dried blood spot (DBS) filter cards for molecular genetic assays and the assays are described in detail; turn-around times and cost estimations are included to give an insight into the processes and discuss further options for optimization. The identified cases are in the range expected for the total number of screened newborns, but present a more exact view on the actual prevalences for Germany.

Abstract A075: Circulating T-cell receptor repertoire analysis improves cancer early detection

Abstract Introduction: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current ctDNA-based approaches detect only a minority of early-stage cancers. For liquid biopsy to realize its full potential for cancer early detection, sensitivity for earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy by exploiting tumor recognition by T cells through sequencing of the circulating T-cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 471 lung cancer patients (85% stage I) and 600 subjects without cancer enriched for older individuals with a history of smoking. We performed TCR beta chain sequencing to yield a median of 101,899 TCR clonotypes per sample (median 81,602 cancer / 112,966 controls) and built a TCR sequence similarity graph to cluster clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using an SVM model into a cancer score. Cancer RFU levels were also correlated to imputed subject HLA types for 32 HLA Class I and 38 Class II alleles. The model was evaluated by 10-fold cross-validation and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes and to 17 lung cancer related protein biomarkers in 86 subjects. Results: We identified 372 cancer-associated TCR RFUs at FDR≤0.1, including 198 enriched in cancer samples (fold-change range 1.03 to 3.13) and 174 enriched in controls (fold- change 0.96 to 0.30). Levels of 265/372 (71%) RFUs were correlated to presence of an HLA allele at FDR ≤ 0.1. Of the total 2,770 significant HLA-RFU associations, 479 (17%) involved an HLA Class I allele, while 2,291 (83%) involved an HLA Class II allele, highlighting a potential role for CD4 helper or regulatory T cell antigen recognition in the cancer RFUs found. The RFU cancer score achieved a cross-validated test ROC AUC of 0.71 for cancer status prediction (0.71 Stage I / 0.70 Stage II-IV) with 49% of Stage I cancers detected at a specificity of 80%. Importantly, the cancer score was not dependent on sample source site, technical or biological variation in TCR repertoire depth, or lung cancer histology subtype. The TCR RFU score also distinguished lung cancer patients from control subjects with other conditions such as benign lung nodules and COPD. We observed an up to ∼20%-point gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins in a potential multi-analyte cancer screening test. By contrast, TCR analysis did not provide additional sensitivity for stage II-IV disease. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. A similar study for the detection of early colorectal cancer and advanced adenomas is under way. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jasper Braun, María Antonia Fortuño, María-del-Mar Ocón, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M Montuenga, Roman Yelensky. Circulating T-cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A075.

Characterization of the T-cell receptor repertoire associated with lymph node metastasis in colorectal cancer

PurposeColorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with lymph node (LN) metastasis playing a pivotal role in disease progression. This study aimed to explore the T-cell receptor (TCR) repertoire among CRC patients, distinguishing those with LN metastasis from those without, in order to uncover potential biomarkers for predicting metastasis.MethodsWe analyzed the TCR repertoire in CRC patients with and without LN metastasis. A classification model utilizing random forest analysis was developed to assess the predictive potential of the TCR repertoire.ResultsThe findings demonstrated a significant increase in the number of V-J combinations and immune CDR3 sequences in patients with LN metastasis compared to the control group. The classification model achieved high accuracy in differentiating patients with LN metastasis, with AUC values ranging from 0.514 to 0.794. Specific V-J combinations and CDR3 sequences were identified as significant predictors of the model’s predictive accuracy.ConclusionThese results suggest that the TCR repertoire is altered in CRC patients exhibiting LN metastasis, potentially influencing disease progression. This study highlights the importance of TCR repertoire analysis as a non-invasive biomarker for predicting LN metastasis in CRC patients.

In utero human cytomegalovirus infection expands NK-like FcγRIII+ CD8+ T cells that mediate Fc antibody functions.

Human cytomegalovirus (HCMV) profoundly impacts host T and natural killer (NK) cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells following HCMV exposure in utero. Most FcγRIII+ CD8+ T cells express the canonical αβ T cell receptor (TCR) but a proportion express non-canonical γδ TCR. FcγRIII+ CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+ CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+ CD8+ T cells mediate antibody-dependent IFNγ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+ CD8+ T cell Fc effector functions were further enhanced by interleukin-15 (IL-15), as has been observed in neonatal NK cells. Our study reveals that FcγRIII+ CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

IMMU-12. TRANSLATING COMBINED PD1 AND LAG3 INHIBITION FROM PRECLINICAL MODELS TO PATIENTS WITH REFRACTORY, DNA REPLICATION REPAIR DEFICIENT (RRD) GLIOBLASTOMA: AN IRRDC STUDY

Abstract BACKGROUND AND AIMS RRD-glioblastoma harbour high tumor mutation burden (TMB) and respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority progress, highlighting the need for combinatorial therapies for sustained immune-surveillance. METHODS We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combined ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. RESULTS Human RRD-glioblastoma (n=80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2LoxP/LoxP-POLES459F/+) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1-/-/NestinCre+/Trp53LoxP/LoxP and therapy-induced hypermutant ENU/Trp53-/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming, in-vivo, that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Seven children with refractory RRD-glioblastoma who had progressed after anti-PD1 treatment were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition for similar patients. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. CONCLUSIONS LAG3 is an effective target in refractory RRD-glioblastoma. Combined inhibition with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses. The combination will now be tested in biomarker-driven clinical trials in RRD-glioblastoma and other immune-inflamed solid tumors.

RBIO-06. STEREOTACTIC RADIOSURGERY ENHANCES T-CELL RECEPTOR REPERTOIRES AND INDUCES IMMUNOGENICITY IN BRAIN METASTASIS

Abstract BACKGROUND Brain metastasis is a frequent and deadly complication of systemic malignancy. The incidence rate is increasing and the prognosis remains poor, in contrast to recent advances in the management of systemic malignancy. Our previous work revealed an immunosuppressive microenvironment in brain metastasis and correlation between T-cell status and patient outcomes, shared across the primary tumor histologies. Therefore, we hypothesize that induction of immunogenicity has potential to improve brain metastasis patient outcome. METHODS Pre-operative stereotactic radiosurgery (SRS) (n = 44) and post-operative SRS (n = 47) brain metastasis specimens were collected from an ongoing clinical trial (MDACC protocol 2018-0552) designed to determine optimal timing of SRS for the treatment of brain metastasis. The patient materials were extracted DNA and RNA, and subjected to T-cell receptor (TCR) repertoire sequencing and mRNA-seq to interrogate radiation-induced immunogenicity. RESULTS In a preliminary analysis of the first 21 specimens, Gene Set Enrichment Analysis revealed that SRS significantly enhanced multiple immune-related signaling pathways including interferon response, along with apoptotic pathways, in brain metastasis. We also found that TCR signaling and PD-1 signaling was upregulated by radiotherapy. TCR repertoire analysis further identified increased density and diversity of TCRs induced by SRS treatment. Profiling of the remaining sample set is currently ongoing. CONCLUSIONS In light of our previous finding regarding the correlation between immune status and outcomes of patients with brain metastases, reversing immunosuppression in brain metastasis could be a key factor on patient prognosis and treatment. Our TCR repertoire and mRNA sequencing analyses with pre-operative and post-operative SRS brain metastasis cohorts support a role for radiation-induced immunogenicity, pointing to a prospective therapeutic approach for brain metastasis management.

TMIC-22. MYELOID CELLS INDUCE IMMUNOSUPPRESSION AND HYPORESPONSIVENESS OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA

Abstract Chimeric antigen receptor T (CART)-cell therapy has shown little success in glioblastoma and other solid tumors, unlike in hematologic malignancies. The immunosuppressive milieu and intratumoral heterogeneity are some obstacles limiting the effectiveness of CART-cell therapy in glioblastoma. Our goal in this study is to investigate how CART-cell transcriptional adaptations and cellular interactions in the glioblastoma microenvironment drive T-cell hyporesponsiveness. Using a human neocortical slice model, we performed physically interacting cell sequencing (PIC-seq) to investigate the transcriptional diversity of NKG2D CAR-T cells and control T-cells in the glioblastoma ecosystem. Through the integration of spatially resolved T-cell receptors sequencing and PIC-seq, we investigated the cellular and receptor-ligand interactions in CART-treated glioblastoma cells. Gene regulatory networks were reconstructed to identify key transcriptional regulators of CD8 CART-cell dysfunction. We used in-silico perturbation to understand transcriptional drivers that could potentially enhance the cytotoxic response of CAR-Ts. Primary patient-derived glioma cells were inoculated into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CARTs or control T-cells. We observed that after 2 days of anti-tumor T-cell response in the CART group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CARTs (tomato+) and tumor cells (GFP+) as well as physically connected cells (+/+). We identified a transcriptional shift towards T-cell exhaustion in CART cells compared to T-cells lacking the NKG2D construct. This transcriptional phenotype was driven by myeloid-CART and myeloid-tumor interaction. We identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T-cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CART-cell function. Myeloid-tumor and myeloid-T-cell interactions promote an immunosuppressive microenvironment and hyporesponsive T-cells in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CART-cell therapies with sustained cytotoxic effect against glioblastoma.

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

Rapid parallel reconstruction and specificity screening of hundreds of T cell receptors.

The ability to screen the reactivity of T cell receptors (TCRs) is essential to understanding how antigen-specific T cells drive productive or dysfunctional immune responses during infections, cancer and autoimmune diseases. Methods to profile large numbers of TCRs are critical for characterizing immune responses sustained by diverse T cell clones. Here we provide a medium-throughput approach to reconstruct dozens to hundreds of TCRs in parallel, which can be simultaneously screened against primary human tissues and broad curated panels of antigenic targets. Using Gibson assembly and miniaturized lentiviral transduction, individual TCRs are rapidly cloned and expressed in T cells; before screening, TCR cell lines undergo combinatorial labeling with dilutions of three fluorescent dyes, which allows retrieval of the identity of individual T cell effectors when they are organized and tested in pools using flow cytometry. Upon incubation with target cells, we measure the upregulation of CD137 on T cells as a readout of TCR activation. This approach is scalable and simultaneously captures the reactivity of pooled TCR cell lines, whose activation can be deconvoluted in real time, thus providing a path for screening the reactivity of dozens of TCRs against broad panels of synthetic antigens or against cellular targets, such as human tumor cells. We applied this pipeline to systematically deconvolute the antitumoral and antiviral reactivity and antigenic specificity of TCRs from human tumor-infiltrating lymphocytes. This protocol takes ~2 months, from experimental design to data analysis, and requires standard expertise in cloning, cell culture and flow cytometry.

TCR-CD3 signal strength regulates plastic coexpression of IL-4 and IFN-γ in Tfh-like cells

The development of T follicular helper (Tfh) cells is an ongoing process resulting in the formation of various Tfh subsets. Despite advancements, the precise impact of T cell receptor (TCR) stimulation on this process remains incompletely understood. This study explores how TCR-CD3 signaling strength influences naive CD4+ T cell differentiation into Tfh-like cells and the concurrent expression of interleukin-21 (IL-21), interleukin-4 (IL-4), and interferon-gamma (IFN-γ). Strong TCR-CD3 stimulation induces proliferation and increased IL-21 expression in Tfh-like cells, which exhibit a characteristic phenotype expressing CXCR5 and PD1. The coexpression of IL-4 and IFN-γ in IL-21-producing Tfh-like cells is controlled by the strength TCR-CD3 stimulation; low stimulation favors IL-4, while strong stimulation enhances IFN-γ secretion. Exogenous addition of the effector cytokines IL-21 and IL-4 further modulate cytokine coexpression. These findings highlight the intricate regulatory mechanisms governing cytokine production and plasticity in Tfh-like cells, providing insights into B cell response modulation. In vivo, antigen availability may regulate Tfh cell plasticity, impacting subsequent B cell differentiation, emphasizing the need for further exploration through animal models or antigen-specific Tfh cell analyses in human lymph node biopsies

Unlocking Intracellular Oncology Targets: The Unique Role of Antibody-Based T-Cell Receptor Mimic (TCRm) Therapeutics in T-Cell Engagers (TCEs) and Antibody-Drug Conjugates (ADCs)

Effectively targeting intracellular tumor-associated proteins presents a formidable challenge in oncology, as they are traditionally considered inaccessible to conventional antibody-based therapies and CAR-T cell therapies. However, recent advancements in antibody engineering have revolutionized this field, offering promising new strategies to combat cancer. This review focuses on the innovative use of T-cell receptor mimic (TCRm) antibodies within the therapeutic frameworks of T-cell engagers (TCE) and antibody-drug conjugates (ADCs). TCRm antibodies, designed to recognize peptide-MHC complexes rather than cell surface proteins, integrate the capacity of T-cells to reach intracellular targets with the unique strengths of antibodies. When incorporated into T-cell engaging therapeutics, TCRms redirect T cells to cancer cells, facilitating direct cytotoxicity. In ADCs, TCRm antibodies deliver cytotoxic agents with highly specific targeting to cancer cells, sparing healthy tissues. Together, these antibody-based strategies represent a significant leap forward in oncology, opening new avenues for the treatment of cancers previously deemed untreatable, with other potential applications in autoimmune diseases. This review discusses the mechanisms, clinical advancements, and future prospects of these cutting-edge therapies, highlighting their potential to transform the landscape of cancer treatment.

Unravelling T-cell dynamics and immune responses in initial and recurrent uveitis.

This study aimed to identify novel serological targets and investigate immune responses in patients with non-infectious uveitis, focusing on differences between initial onset and recurrent episodes. Differential gene expression analysis, immunocyte typing and T-cell receptor (TCR) gene analysis were conducted on RNA-sequenced peripheral blood samples from healthy individuals (n = 6) and non-infectious uveitis patients (n = 12), divided into 6 patients each at initial onset and recurrent stages. Peripheral blood T-cell types were analysed using flow cytometry. Bioinformatics methods included tools for RNA sequencing data processing, CIBERSORT for immune cell type prediction and specialized software for TCR repertoire analysis. Findings indicated that individuals with recurrent uveitis demonstrated a stronger adaptive immune response and a more pronounced immune imbalance compared to those with initial onset. Memory T cells were predominant in recurrent episodes, suggesting their potential role as biomarkers for disease progression. Significant differences in TCR diversity and V(D)J gene usage were observed between the various uveitis groups and healthy controls. Importantly, 38 uveitis-specific TCR sequences showed substantial expansion in the uveitis patients compared to controls. An elevated expansion of these specific TCR sequences was associated with an increased risk of uveitis development. The study highlights the critical role of adaptive immune responses and specific immune cell types in the pathogenesis of recurrent uveitis. Identification of the uveitis-specific TCR repertoire set could provide deeper insights into the disease and facilitate the development of targeted therapies for uveitis patients.

In situ cell-surface conformation of the TCR-CD3 signaling complex.

The extracellular molecular organization of the individual CD3 subunits around the αβ T cell receptor (TCR) is critical for initiating T cell signaling. In this study, we incorporate photo-crosslinkers at specific sites within the TCRα, TCRβ, CD3δ, and CD3γ subunits. Through crosslinking and docking, we identify a CD3ε'-CD3γ-CD3ε-CD3δ arrangement situated around the αβTCR in situ within the cell surface environment. We demonstrate the importance of cholesterol in maintaining the stability of the complex and that the 'in situ' complex structure mirrors the structure from 'detergent-purified' complexes. In addition, mutations aimed at stabilizing extracellular TCR-CD3 interfaces lead to poor signaling, suggesting that subunit fluidity is indispensable for signaling. Finally, employing photo-crosslinking and CD3 tetramer assays, we show that the TCR-CD3 complex undergoes minimal subunit movements or reorientations upon interaction with activating antibodies and pMHC tetramers. This suggests an absence of 'inactive-active' conformational states in the TCR constant regions and the extracellular CD3 subunits, unlike the transmembrane regions of the complex. This study contributes a nuanced understanding of TCR signaling, which may inform the development of therapeutics for immune-related disorders.

Spontaneous high clonal expansion of Wilms’ tumor gene 1-specific cytotoxic T-lymphocytes in patients with Wilms’ tumor gene 1-expressing solid tumor

Wilms’ tumor protein 1 (WT1)-targeted immunotherapy has been used in patients with leukemia and solid tumors. However, the spontaneous WT1-specific immune response before WT1 peptide vaccination in patients with WT1-expressing tumors (PTs) remains unclear. Therefore, we investigated whether WT1-specific cytotoxic CD8+ T-lymphocytes (CTLs) are clonally expanded in the peripheral blood outside of tumor sites. Clonal expansion of WT1126 peptide (a.a.126–134)-specific CTLs (WT1126-CTLs) was compared between seven PTs and five healthy volunteers (HVs), and their T-cell receptors (TCRs) were analyzed at the single-cell level. Overall, 433 and 351 TCR β-chains of WT1126-CTLs were detected from PTs and HVs, respectively, and complementarity-determining region 3 was sequenced for clonality analysis. The frequencies of WT1126-CTLs were higher in human leukocyte antigen (HLA)-A*02:01+ PTs than in HLA-A*02:01+ HVs, although the difference was not statistically significant. WT1126-CTLs of differentiated types, including memory and effector, were higher in PTs than in HVs; whereas, those of the naïve type were higher in HVs than in PTs. WT1126-CTL clonality was significantly higher in PTs than in HVs. Furthermore, the frequency of effector WT1126-CTLs positively correlated with WT1126-CTL clonality in PTs; whereas, the frequency of naïve phenotype WT1126-CTLs tended to be negatively correlated with clonality. In conclusion, these results suggest that the WT1 protein in tumor cells is highly immunogenic, thereby stimulating endogenous naïve-type WT1126-CTLs and enabling them to clonally expand and differentiate into effector-type WT1126-CTLs.