In pancreatic cancer patients, chemotherapy reshapes the gene expression profile and antigen receptor repertoire of T lymphocytes and enhances their effector response to tumor-associated antigens.

Introduction: Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 13%. Less than 20% of patients have a resectable tumor at diagnosis due to the lack of distinctive symptoms and reliable biomarkers. PDA is resistant to chemotherapy (CT) and understanding how to gain an anti-tumor effector response following stimulation is, therefore, critical for setting up an effective immunotherapy. Methods: Proliferation, and cytokine release and TCRB repertoire of from PDA patient peripheral T lymphocytes, before and after CT, were analyzed in vitro in response to four tumor-associated antigens (TAA), namely ENO1, FUBP1, GAPDH and K2C8. Transcriptional state of PDA patient PBMC was investigated using RNA-Seq before and after CT. Results: CT increased the number of TAA recognized by T lymphocytes, which positively correlated with patient survival, and high IFN-γ production TAA-induced responses were significantly increased after CT. We found that some ENO1-stimulated T cell clonotypes from CT-treated patients were expanded or de-novo induced, and that some clonotypes were reduced or even disappeared after CT. Patients that showed a higher number of effector responses to TAA (high IFN-γ/IL-10 ratio) after CT expressed increased fatty acid-related transcriptional signature. Conversely, patients that showed a higher number of regulatory responses to TAA (low IFN-γ/IL-10 ratio) after CT significantly expressed an increased IRAK1/IL1R axis-related transcriptional signature. Conclusion: These data suggest that the expression of fatty acid or IRAK1/IL1Rrelated genes predicts T lymphocyte effector or regulatory responses to TAA in patients that undergo CT. These findings are a springboard to set up precision immunotherapies in PDA based on the TAA vaccination in combination with CT.

BertTCR: a Bert-based deep learning framework for predicting cancer-related immune status based on T cell receptor repertoire.

The T cell receptor (TCR) repertoire is pivotal to the human immune system, and understanding its nuances can significantly enhance our ability to forecast cancer-related immune responses. However, existing methods often overlook the intra- and inter-sequence interactions of T cell receptors (TCRs), limiting the development of sequence-based cancer-related immune status predictions. To address this challenge, we propose BertTCR, an innovative deep learning framework designed to predict cancer-related immune status using TCRs. BertTCR combines a pre-trained protein large language model with deep learning architectures, enabling it to extract deeper contextual information from TCRs. Compared to three state-of-the-art sequence-based methods, BertTCR improves the AUC on an external validation set for thyroid cancer detection by 21 percentage points. Additionally, this model was trained on over 2000 publicly available TCR libraries covering 17 types of cancer and healthy samples, and it has been validated on multiple public external datasets for its ability to distinguish cancer patients from healthy individuals. Furthermore, BertTCR can accurately classify various cancer types and healthy individuals. Overall, BertTCR is the advancing method for cancer-related immune status forecasting based on TCRs, offering promising potential for a wide range of immune status prediction tasks.

Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαß+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

TCR clustering by contrastive learning on antigen specificity.

Effective clustering of T-cell receptor (TCR) sequences could be used to predict their antigen-specificities. TCRs with highly dissimilar sequences can bind to the same antigen, thus making their clustering into a common antigen group a central challenge. Here, we develop TouCAN, a method that relies on contrastive learning and pretrained protein language models to perform TCR sequence clustering and antigen-specificity predictions. Following training, TouCAN demonstrates the ability to cluster highly dissimilar TCRs into common antigen groups. Additionally, TouCAN demonstrates TCR clustering performance and antigen-specificity predictions comparable to other leading methods in the field.

Advances in manufacturing chimeric antigen receptor immune cell therapies.

Biomedical research has witnessed significant strides in manufacturing chimeric antigen receptor T cell (CAR-T) therapies, marking a transformative era in cellular immunotherapy. Nevertheless, existing manufacturing methods for autologous cell therapies still pose several challenges related to cost, immune cell source, safety risks, and scalability. These challenges have motivated recent efforts to optimize process development and manufacturing for cell therapies using automated closed-system bioreactors and models created using artificial intelligence. Simultaneously, non-viral gene transfer methods like mRNA, CRISPR genome editing, and transposons are being applied to engineer T cells and other immune cells like macrophages and natural killer cells. Alternative sources of primary immune cells and stem cells are being developed to generate universal, allogeneic therapies, signaling a shift away from the current autologous paradigm. These multifaceted innovations in manufacturing underscore a collective effort to propel this therapeutic approach toward broader clinical adoption and improved patient outcomes in the evolving landscape of cancer treatment. Here, we review current CAR immune cell manufacturing strategies and highlight recent advancements in cell therapy scale-up, automation, process development, and engineering.

The amalgam of naive CD4+ T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response.

Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.

Parsing digital or analog TCR performance through piconewton forces.

αß T cell receptors (TCRs) principally recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP366-374/Db and PA224-233/Db, respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker up-regulation in vitro. Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies.

Chemical complementarity of tumor resident, T-cell receptor CDR3s and renalase-1 correlates with increased melanoma survival.

Overexpression of the secretory protein renalase-1 negatively impacts the survival of melanoma and pancreatic cancer patients, while inhibition of renalase-1 signaling drives tumor rejection by promoting T-cell activation. Thus, we investigated the chemical complementarity between melanoma-resident, T-cell receptor (TCR) complementarity-determining region 3 (CDR3) amino acid sequences (AAs) and the renalase-1 protein. Increasing complementarity of TCR CDR3s to renalase-1 AAs, as assessed by a chemical complementarity scoring algorithm, was associated with improved overall survival (OS) in melanoma patients. The expression levels of several immune signature genes were significantly, positively correlated with increasing TCR CDR3-renalase-1 complementarity scores. Additionally, the survival association observed with high complementarity of TCR CDR3s to renalase-1 AAs was more robust in cases with low renalase-1 gene expression levels. Mapping of TCR CDR3-renalase-1 in silico interaction sites identified major epitope candidates including RP220, the signaling module of the renalase-1 protein, consistent with the fact that a monoclonal antibody to RP220 is a potent inhibitor of melanoma growth. These findings indicate that renalase-1 is a potential antigen for TCR recognition in melanoma and could be considered as a target for immunotherapy.

Lipidomic scanning of self-lipids identifies headless antigens for natural killer T cells.

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

Inhibition of PCSK9 enhances the anti-hepatocellular carcinoma effects of TCR-T cells and anti-PD-1 immunotherapy.

T cells play important roles in antitumor immunity. However, given that the hepatocellular carcinoma (HCC) tumor microenvironment confers resistance to T cell-based immunotherapies, novel strategies to boost T cell-mediated antitumor efficacy are urgently needed for the treatment of HCC. Here, we show that high proprotein convertase subtilisin/kexin type9 (PCSK9) expression was negatively associated with HCC patient's overall survival and markers of CD8+ T cells. Pharmacological inhibition of PCSK9 enhanced tumor-specific killing and downregulated PD-1 expression of AFP-specific TCR-T. Inhibition of PCSK9 significantly enhances the anti-HCC efficacy of TCR-T cells and anti-PD-1 immunotherapy in vivo. Moreover, PCSK9 inhibitor suppressed HCC growth dependent on CD8+ T cells. Mechanically, pharmacological inhibition of PCSK9 promoted low-density lipoprotein receptor (LDLR)-mediated activation of mTORC1 signaling in CD8+ T cells. LDLR deficiency was shown to impair cellular mTORC1 signaling and the anti-HCC function of CD8 T cells. On the basis of our findings in this study, we propose a potential metabolic intervention strategy that could be used to enhance the antitumor effects of immunotherapy for HCC.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Regulation of MAIT cells through host-derived antigens.

Mucosal-associated invariant T (MAIT) cells are a major subset of innate-like T cells that function at the interface between innate and acquired immunity. MAIT cells recognize vitamin B2-related metabolites produced by microbes, through semi-invariant T cell receptor (TCR) and contribute to protective immunity. These foreign-derived antigens are presented by a monomorphic antigen presenting molecule, MHC class I-related molecule 1 (MR1). MR1 contains a malleable ligand-binding pocket, allowing for the recognition of compounds with various structures. However, interactions between MR1 and self-derived antigens are not fully understood. Recently, bile acid metabolites were identified as host-derived ligands for MAIT cells. In this review, we will highlight recent findings regarding the recognition of self-antigens by MAIT cells.

A non-invasive nanobody probe for high precision mapping of Lck spatial distribution.

The tyrosine kinase Lck is mandatory for initiating signaling responses downstream the antigenic T cell receptor (TCR). Numerous studies have shown that a prerequisite for efficient and well-balanced Lck regulation and function is its finely orchestrated spatial distribution pattern, especially at the plane of the plasma membrane. There is a wealth of knowledge on Lck localization sites, preference for specialized lipid microenvironments and colocalization partners. However, several questions concerning the spatial organization of its differentially phosphorylated conformers and the dynamics of their juxtaposition in relation to ligated and non-ligated TCRs remain elusive. In this brief report we introduce a non-invasive nanobody-based approach for mapping Lck subcellular allocation with high precision. Our initial data using this methodology, provide insight into the topology of Lck in resting T cells and its confined localization in a strictly delimited environment within the plane of the plasma membrane.

Preclinical studies show that Co-STARs combine the advantages of chimeric antigen and T cell receptors for the treatment of tumors with low antigen densities.

Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.

GTE: a graph learning framework for prediction of T-cell receptors and epitopes binding specificity.

The interaction between T-cell receptors (TCRs) and peptides (epitopes) presented by major histocompatibility complex molecules (MHC) is fundamental to the immune response. Accurate prediction of TCR-epitope interactions is crucial for advancing the understanding of various diseases and their prevention and treatment. Existing methods primarily rely on sequence-based approaches, overlooking the inherent topology structure of TCR-epitope interaction networks. In this study, we present $GTE$, a novel heterogeneous Graph neural network model based on inductive learning to capture the topological structure between TCRs and Epitopes. Furthermore, we address the challenge of constructing negative samples within the graph by proposing a dynamic edge update strategy, enhancing model learning with the nonbinding TCR-epitope pairs. Additionally, to overcome data imbalance, we adapt the Deep AUC Maximization strategy to the graph domain. Extensive experiments are conducted on four public datasets to demonstrate the superiority of exploring underlying topological structures in predicting TCR-epitope interactions, illustrating the benefits of delving into complex molecular networks. The implementation code and data are available at https://github.com/uta-smile/GTE.

The molecular basis underlying T cell specificity towards citrullinated epitopes presented by HLA-DR4.

CD4+ T cells recognising citrullinated self-epitopes presented by HLA-DRB1 bearing the shared susceptibility epitope (SE) are implicated in rheumatoid arthritis (RA). However, the underlying T cell receptor (TCR) determinants of epitope specificity towards distinct citrullinated peptide antigens, including vimentin-64cit59-71 and α-enolase-15cit10-22 remain unclear. Using HLA-DR4-tetramers, we examine the T cell repertoire in HLA-DR4 transgenic mice and observe biased TRAV6 TCR gene usage across these two citrullinated epitopes which matches with TCR bias previously observed towards the fibrinogen ß-74cit69-81 epitope. Moreover, shared TRAV26-1 gene usage is evident in four α-enolase-15cit10-22 reactive T cells in three human samples. Crystal structures of mouse TRAV6+ and human TRAV26-1+ TCR-HLA-DR4 complexes presenting vimentin-64cit59-71 and α-enolase-15cit10-22, respectively, show three-way interactions between the TCR, SE, citrulline, and the basis for the biased selection of TRAV genes. Position 2 of the citrullinated epitope is a key determinant underpinning TCR specificity. Accordingly, we provide a molecular basis of TCR specificity towards citrullinated epitopes.

Harnessing the tumor microenvironment to boost adoptive T cell therapy with engineered lymphocytes for solid tumors.

Adoptive cell therapy (ACT) using Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) engineered T cells represents an innovative therapeutic approach for the treatment of hematological malignancies, yet its application for solid tumors is still suboptimal. The tumor microenvironment (TME) places several challenges to overcome for a satisfactory therapeutic effect, such as physical barriers (fibrotic capsule and stroma), and inhibitory signals impeding T cell function. Some of these obstacles can be faced by combining ACT with other anti-tumor approaches, such as chemo/radiotherapy and checkpoint inhibitors. On the other hand, cutting edge technological tools offer the opportunity to overcome and, in some cases, take advantage of TME intrinsic characteristics to boost ACT efficacy. These include: the exploitation of chemokine gradients and integrin expression for preferential T-cell homing and extravasation; metabolic changes that have direct or indirect effects on TCR-T and CAR-T cells by increasing antigen presentation and reshaping T cell phenotype; introduction of additional synthetic receptors on TCR-T and CAR-T cells with the aim of increasing T cells survival and fitness.

T cell receptor repertoire deciphers anti-tuberculosis immunity.

T cell induced cellular immunity is considered to be extremely important for the control of tuberculosis (TB). T cell receptor (TCR), the key component responsible for the specificity and clustering of T cells, holds the potential to advance our understanding of T cell immunity against TB infection. This review systematically expounded the study progressions made in the field of TB-relevant TCRs based on single cell sequencing together with GLIPH2 technology and initiated a comparison of the T cell distribution between peripheral blood and infected organs. We divided clonal expanded T cell clones into recirculation subsets and local subsets to summarize their distinctions in clonal abundance, TCR sequences and antigenic specificity. Notably, local expansion appears to drive the primary variances in T cell subsets between these two contexts, indicating the necessity for further exploration into the functions and specificity of local subsets.

SARS-CoV-2 spike does not interact with the T cell receptor or directly activate T cells.

SARS-CoV-2infection can induce multisystem inflammatory syndrome in children, which resembles superantigen-induced toxic shock syndrome. Recent work has suggested that the SARS-CoV-2 spike (S) protein could act as a superantigen by binding T cell receptors (TCRs) and inducing broad antigen-independent T cell responses. Structure-based computational modeling identified potential TCR-binding sites near the S receptor-binding domain, in addition to a site with homology to known neurotoxins. We experimentally examined the mechanism underpinning this theory-the direct interaction between the TCR and S protein. Surface plasmon resonance of recombinantly expressed S protein and TCR revealed no detectable binding. Orthogonally, we pseudotyped lentiviruses with SARS-CoV-2 S in both wild-type and prefusion-stabilized forms, demonstrated their functionality in a cell line assay, and observed no transduction, activation, or stimulation of proliferation of CD8+ T cells. We conclude that it is unlikely that the SARS-CoV-2 spike protein engages nonspecifically with TCRs or has superantigenic character.

[Clinicopathological features of primary mucosal CD30-positive T-cell lymphoproliferative disorders].

Objective: To investigate the clinicopathological features and differential diagnosis of primary mucosal CD30-positive T-cell lymphoproliferative disorders (pmCD30+TLPD). Methods: Eight cases of pmCD30+TLPD diagnosed from 2013 to 2023 at the Department of Pathology, Beijing Friendship Hospital Affiliated to Capital Medical University and Beijing Ludaopei Hospital were retrospectively collected. The immunophenotype, EBV infection status and T-cell receptor (TCR) clonability of tumor cells were examined. The clinicopathological features were analyzed and related literatures were reviewed. Results: There were 5 females and 3 males, aged 28 to 73 years, without B symptoms, lack of trauma and autoimmune diseases. Seven cases occurred in oral mucosa and one in anal canal mucosa. Submucosal nodules with ulcerations were presented in all cases except one, which only submucosal nodule. Morphologically, there was different distribution of allotypic lymphocytes in inflammatory background. Four cases showed "kidney-shaped", "embryonic" and "horseshoe-shaped" cells, and one case resembled Hodgkin and Reed/Sternberg (HRS) cells. Allotypic lymphocytes expressed CD3 (7/8), CD4+/CD8-(7/8) and CD4-/CD8-(1/8). CD30 was uniformly strongly positive while ALK and CD56 were negative. In situ hybridization of EBER was negative in five cases (5/5). Clonal TCR gene rearrangement was positive in two cases. Four patients did not receive radiotherapy or chemotherapy. All the seven patients survived without disease except one died due to concurrent leukopenia. Conclusions: pmCD30+TLPD had a broad morphological spectrum and could be easily confused with primary cutaneous CD30+TLPD and systemic ALK-negative anaplastic large cell lymphoma involving mucosa, which may lead to misdiagnosis. Although the majority of the cases had a favorable prognosis, a few cases relapsed or progressed to lymphoma.