Cell Receptor Research Articles

EXTH-21. WINNING THE TUG OF WAR BETWEEN VIRUS AND TUMOR IMMUNODOMINANCE IN GLIOMAS TREATED WITH ONCOLYTIC ADENOVIRUSES USING TOLEROGENIC STRATEGIES

Abstract Phase 1 and 2 clinical trials of our oncolytic adenovirus Delta-24-RGD for patients with recurrent malignant gliomas have demonstrated its safety and efficacy, and showed that 20% of the patients experienced long-term tumor remissions often associated with inflammatory responses and tumor infiltration of lymphocytes. Based on our preliminary data, we expected that the most frequent tumor-infiltrating CD8+ T cell clones will target adenoviral hexon, with a single virus-specific clone representing up to 5% of the total population. Competition between T cells for resources may limit the expansion of subdominant clones recognizing tumor antigens. We hypothesized that mitigating virus-specific immunity allows the expansion of tumor-specific T cells and results in better therapeutic efficacy. In mice, intravenously injected nanoparticles encapsulating adenoviral antigens preferentially accumulated in the liver (P<0.0001), where self-reactive immune cells are deleted through peripheral tolerance. RNA-sequencing of antigen-specific T cells isolated after nanoparticle injection revealed enriched expression of gene sets involved in immune tolerance through clonal deletion and T cell anergy, including increased expression of immune checkpoint molecules (P<0.001). In glioma-bearing mice treated with virotherapy in combination with nanoparticle administration, tumor infiltrating lymphocytes exhibited decreased IFNγ secretion against viral antigens and increased secretion against tumor antigens, suggesting a redirection of the immunity from anti-viral to anti-tumoral (P<0.05). Importantly, virotherapy with nanoparticles increased the overall survival of tumor-bearing mice compared to virus treatment alone (P<0.001). To further characterize the roles of virus- and tumor-specific immune cells during virotherapy we are analyzing the T cell receptor repertoire of surgical specimens from nine glioma patients treated with Delta-24-RGD in a phase 1 clinical trial. In summary, our data provide the basis for a future clinical trial using tolerogenic strategies to redirect immunodominance of the viral to the glioma antigens, and represent a novel strategy to enhance anti-tumor immunity during virotherapy of brain tumors.

Structure-guided design and evaluation of CRM197-scaffolded vaccine targeting GnRH for animal immunocastration

Immunocastration is a humane alternative to surgical castration for controlling population and estrous behaviors in animals. Gonadotropin-releasing hormone (GnRH), the pivotal initiating hormone of the hormonal cascade in mammals, is the optimal target for immunocastration vaccine development. Cognate antigen-mediated cross-linking of B cell receptors (BCRs) is a strong activation signal for B cells and is facilitated by repetitive surface organizations of antigens. In this study, we describe the structure-guided design of highly immunogenic chimeric proteins with variant numbers of GnRH peptide insertion by epitope grafting. Linear B-cell epitopes of cross-reacting material 197 (CRM197) were replaced with multiple copies of GnRH peptide, and the inserts were displayed on the surface of the designs while maintaining the overall folding of CRM197. Among the seven designs, TCG13, which carries 13 copies of GnRH peptide, was the most immunogenic, and its immunocastration efficacy was evaluated in male mice. Vaccination with the BFA03-adjuvanted TCG13 induced potent humoral immunity and reduced the serum testosterone concentration in mice. It could significantly decrease sperm quality and severely impair gonadal function and fertility. These results demonstrate that CRM197 holds great value as a scaffold for epitope presentation in peptide-based vaccine development and supports TCG13 as a promising vaccine candidate for animal immunocastration.Key points• Provide a feasible way to design chimeric immunogen targeting GnRH by epitope grafting.• CRM197 can accommodate the insertion of multiple copies of heterologous epitope peptides.• Administration with the most immunogenic design led to effective immunocastration in male mice.

Antiviral efficacy of heparan sulfate and enoxaparin sodium against SARS‐CoV‐2

AbstractAs the world transitions from the acute phase of the COVID‐19 pandemic caused by SARS‐CoV‐2, the scientific community continues to explore various therapeutic avenues to control its spread and mitigate its ongoing effects. Among the promising candidates are heparan sulfate (HS) and enoxaparin (EX), which have emerged as potential virus inhibitors. HS, a type of glycosaminoglycan, plays a prominent role in the attachment of the virus to host cells. At the same time, EX, a low‐molecular‐weight heparin, is being investigated for its ability to disrupt the interaction between the spike protein of SARS‐CoV‐2 and the ACE2 receptor in human cells. Understanding the mechanisms through which these substances operate could lay the foundation for new strategies in the ongoing management of COVID‐19. This study aimed to examine the details of SARS‐CoV‐2's entry mechanisms and the role of HS in this process. Furthermore, it examines EX's mechanism of action, highlighting how it potentially inhibits SARS‐CoV‐2. The interactions between HS and the virus, alongside in‐vitro and in‐silico inhibition studies with HS and EX, are critically analyzed to assess their antiviral efficacy. Additionally, the antiviral activity of sulfated polysaccharides and the potential therapeutic applications of these findings are discussed.

Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstractα‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2. The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.

Pep2TCR: Accurate prediction of CD4 T cell receptor binding specificity through transfer learning and ensemble approach

Pep2TCR: Accurate prediction of CD4 T cell receptor binding specificity through transfer learning and ensemble approach

B Cells With Complementary B Cell Receptors Can Kill Each Other

ABSTRACTB cells differentiate from hematopoietic stem cells in the bone marrow (BM) and migrate as transitional cells to the spleen where final maturation takes place. Due to the enormous diversity in variable (V) regions of B cell receptors for antigen (BCR), B cells with complementary BCRs are likely to be generated. These could encounter each other in the BM or in secondary lymphoid organs. The outcome of such an event is unknown. To study this issue, we used two strains of gene‐modified mice whose B cells display complementary BCRs. B cells of one strain express an idiotype+ (Id+) BCR while B cells of the other strain display an anti‐idiotypic (αId) BCR. In vitro, B cells with complementary BCRs killed each other in a mechanism that required physical binding between BCR V‐regions. In contrast, killing was unilateral in vivo: αId B cells with a follicular (FO) B cell phenotype were expanded, while Id+ B cells with a marginal zone (MZ) phenotype became deleted. The results show that B cells with complementary BCRs can recognize and regulate each other in vivo. This mechanism should be taken into account in theories for idiotypic regulation of the immune system.

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...

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

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

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

The role of polyreactive memory B cells in systemic lupus erythematosus

Abstract In systemic lupus erythematosus (SLE), the production of autoantibodies is a crucial characteristic, and B cells play a significant role in its pathogenesis. B cells are the immune cells most associated with the genetic predispositions of SLE, and recent clinical studies showing that anti-CD19 CAR-T cell therapy induces drug-free remission have underscored the importance of B cells in SLE. Meanwhile, various B cell subsets exist across different stages of differentiation, from naive B cells to plasma-cells, and identifying the important subpopulations within SLE remains a critical future challenge. Years of B cell repertoire analyses have revealed the importance of polyreactive B cell receptors (BCRs) and autoantibodies that react to a various self-antigens and microbial antigens. Particularly, memory B cells with polyreactive BCRs, which play a crucial role in biological defense during the fetal stage, are characteristically differentiated in SLE. Type I interferon-mediated expression of CXCL13 and IL21 in CD4+ T cells is associated with the development of polyreactive memory B cells. The expansion of the polyreactive B cell repertoire, vital for defending against infections such as viruses, may exert an intrinsic function in SLE.

Mineralocorticoid Receptor in Endothelial Cells Contributes to VEGF Receptor Inhibitor-Induced Vascular and Kidney Damage

Abstract BACKGROUND Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer patient survival by inhibiting tumor angiogenesis. However, VEGFRis induce treatment-limiting hypertension which has been associated with impaired vascular endothelial cell (EC) function and kidney damage. The mineralocorticoid receptor (MR) regulates blood pressure via effects on the vasculature and the kidney. Thus, we interrogated the role of the MR in EC dysfunction, renal impairment, and hypertension in a mouse model of VEGFRi-induced hypertension using sorafenib. METHODS EC dysfunction in mesenteric arterioles was assessed by immunoblotting for phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. Renal damage was measured by assessing glomerular endotheliosis histologically. Blood pressure (BP) was measured using implanted radiotelemetry. RESULTS Six days of sorafenib treatment significantly impaired mesenteric resistance vessel EC function, induced renal damage, and increased BP. Pharmacologic MR blockade with spironolactone prevented the sorafenib-induced decline in eNOS phosphorylation and the renal glomerular endotheliosis, without affecting systolic or diastolic BP. Mice with the MR knocked out specifically in ECs (EC-MR-KO) were protected from sorafenib-induced EC dysfunction and glomerular endotheliosis, whereas smooth muscle cell-specific MR (SMC-MR) knockout mice were not. Neither EC-MR knockout nor SMC-MR knockout affected the degree to which sorafenib increased systolic or diastolic BP. CONCLUSIONS These results reveal that the MR, specifically in EC but not in SMCs, is necessary for VEGFRi-induced renal and vascular injury. While ineffective at lowering SBP, these data suggest potential therapeutic benefits of MR antagonists, like spironolactone, to protect the vasculature and the kidneys from VEGFRi-induced injury.

Dysfunction of exhausted T cells is enforced by MCT11-mediated lactate metabolism

AbstractCD8+ T cells are critical mediators of antitumor immunity but differentiate into a dysfunctional state, known as T cell exhaustion, after persistent T cell receptor stimulation in the tumor microenvironment (TME). Exhausted T (Tex) cells are characterized by upregulation of coinhibitory molecules and reduced polyfunctionality. T cells in the TME experience an immunosuppressive metabolic environment via reduced levels of nutrients and oxygen and a buildup of lactic acid. Here we show that terminally Tex cells uniquely upregulate Slc16a11, which encodes monocarboxylate transporter 11 (MCT11). Conditional deletion of MCT11 in T cells reduced lactic acid uptake by Tex cells and improved their effector function. Targeting MCT11 with an antibody reduced lactate uptake specifically in Tex cells, which, when used therapeutically in tumor-bearing mice, resulted in reduced tumor growth. These data support a model in which Tex cells upregulate MCT11, rendering them sensitive to lactic acid present at high levels in the TME.

Broadening alloselectivity of T cell receptors by structure guided engineering

Specificity of a T cell receptor (TCR) is determined by the combination of its interactions to the peptide and human leukocyte antigen (HLA). TCR-based therapeutic molecules have to date targeted a single peptide in the context of a single HLA allele. Some peptides are presented on multiple HLA alleles, and by engineering TCRs for specific recognition of more than one allele, there is potential to expand the targetable patient population. Here, as a proof of concept, we studied two TCRs, S2 and S8, binding to the PRAME peptide antigen (ELFSYLIEK) presented by HLA alleles HLA-A*03:01 and HLA-A*11:01. By structure-guided affinity maturation targeting a specific residue on the HLA surface, we show that the affinity of the TCR can be modulated for different alleles. Using a combination of affinity maturation and functional T cell assay, we demonstrate that an engineered TCR can target the same peptide on two different HLA alleles with similar affinity and potency. This work highlights the importance of engineering alloselectivity for designing TCR based therapeutics suitable for differing global populations.

Immune responses in checkpoint myocarditis across heart, blood and tumour.

Immune checkpoint inhibitors are widely used anticancer therapies1 that can cause morbid and potentially fatal immune-related adverse events such as immune-related myocarditis (irMyocarditis)2-5. The pathogenesis of irMyocarditis and its relationship to antitumour immunity remain poorly understood. Here we sought to define immune responses in heart, tumour and blood in patients with irMyocarditis by leveraging single-cell RNA sequencing coupled with T cell receptor (TCR) sequencing, microscopy and proteomics analyses of samples from 28 patients with irMyocarditis and 41 unaffected individuals. Analyses of 84,576 cardiac cells by single-cell RNA sequencing combined with multiplexed microscopy demonstrated increased frequencies and co-localization of cytotoxic T cells, conventional dendritic cells and inflammatory fibroblasts in irMyocarditis heart tissue. Analyses of 366,066 blood cells revealed decreased frequencies of plasmacytoid dendritic cells, conventional dendritic cells and B lineage cells but an increased frequency of other mononuclear phagocytes in irMyocarditis. Fifty-two heart-expanded TCR clones from eight patients did not recognize the putative cardiac autoantigens α-myosin, troponin I or troponin T. Additionally, TCRs enriched in heart tissue were largely nonoverlapping with those enriched in paired tumour tissue. The presence of heart-expanded TCRs in a cycling blood CD8 T cell population was associated with fatal irMyocarditis case status. Collectively, these findings highlight crucial biology driving irMyocarditis and identify putative biomarkers.

Systemic hormone therapy after breast and gynecological cancers: an Italian expert group consensus opinion.

The specific Italian Group of Study of the Menopause formulated a consensus opinion on the use of estrogen therapy (ET) or combined estro-progestin hormone therapy (HT) after breast and gynecological cancers. This consensus is based on the risk of recurrence of the specific cancer during ET/HT, the presence of steroid receptors in cancer cells, the use of adjuvant hormone therapies and data on the use of ET/HT after cancer. The following positions were reached. ET/HT can be used after vulvar cancers and melanoma, but with great caution after the rare adenocarcinomas. ET/HT can be used after cervical cancer, but ET should be used with caution after adenocarcinomas. ET/HT can be used after International Federation of Obstetrics and Gynecology (FIGO) stage I-II estrogen-dependent endometrial cancers, except in Black women, and can probably be used after estrogen-independent endometrial cancers. ET/HT cannot be administered or should be used with great caution after most uterine sarcomas. ET/HT can probably be used after ovarian neoplasms except for granulosa cell tumors, and with great caution after low-grade serous ovarian carcinoma and serous borderline ovarian tumors. ET/HT can be used with great caution in women after estrogen receptor (ER)/progesterone receptor (PR)-positive breast cancer and is probably allowed after ER/PR-negative breast cancer.

Food for thought: Nutrient metabolism controlling early T cell development.

T cells develop in the thymus by expressing a diverse repertoire of either αβ- or γδ-T cell receptors (TCR). While many studies have elucidated how TCR signaling and gene expression control T cell ontogeny, the role of nutrient metabolism is just emerging. Here, we discuss how metabolic reprogramming and nutrient availability impact the fate of developing thymic T cells. We focus on how the PI3K/mTOR signaling mediates various extracellular inputs and how this signaling pathway controls metabolic rewiring during highly proliferative and anabolic developmental stages. We highlight the role of the hexosamine biosynthetic pathway that generates metabolites that are utilized for N- and O-linked glycosylation of proteins and how it impacts TCR expression during T cell ontogeny. We consider the dichotomy in metabolic needs during αβ- versus γδ-T cell lineage commitment as well as how metabolism is also coupled to molecular signaling that controls cell fate.

Effect of NK cell receptor genetic variation on allogeneic stem cell transplantation outcome and in vitro NK cell cytotoxicity

Natural killer (NK) cells recognize and may kill malignant cells via their cell surface receptors. Killer cell immunoglobulin-like receptor (KIR) genotypes of donors have been reported to adjust the risk of relapse after allogeneic stem cell transplantation (HSCT), particularly in patients with acute myeloid leukemia. To test whether non-KIR NK cell receptors have a similar effect, we screened 1,638 genetic polymorphisms in 21 non-KIR NK cell receptor genes for their associations with relapse and graft-versus-host disease (GVHD) after HSCT in 1,491 HSCT donors (from Finland, the UK, Spain, and Poland), divided into a discovery and replication cohort. Eleven polymorphisms regulating or located in CD226, CD244, FCGR3A, KLRD1, NCR3, and PVRIG were associated with the risks for relapse and GVHD. These associations could not be confirmed in the replication cohort. Blood donor NK cells carrying alleles showing genetic protection for relapse had a higher in vitro NK cell killing activity than non-carriers whereas those with alleles genetically protective for GVHD had lower cytotoxicity, potentially indicating functional effects. Taken together, these results show no robust effects of genetic variation in the tested non-KIR NK cell receptors on the outcome of HSCT.

A novel immunomodulating peptide with potential to complement oligodeoxynucleotide-mediated adjuvanticity in vaccination strategies

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

Mitochondrial Iron Metabolism as a Potential Key Mediator of PD-L1 Thermal Regulation

Glioblastoma (GBM) is the most common primary brain malignancy in the U.S. with a 5-year overall survival < 5% despite an aggressive standard of care. Laser interstitial thermal therapy (LITT) is a surgical approach to treating GBM that has gained traction, providing a safe option for reducing intracranial tumor burden. LITT is believed to potentially modulate GBM immune responses; however, the biochemical mechanisms underlying the modulation of immune checkpoints in GBM cells have been poorly characterized. The present study aimed to preliminarily evaluate the effects of thermal therapy and radiation on PD-L1 modulation in vitro, as a function of IDH mutational status. U87 cells and their IDH-mutant counterpart (U87R132H), which was generated using a crispr-cas9 knock-in approach, were utilized for this preliminary evaluation. Cell heating was achieved by harvesting with trypsin centrifugation where the cell pellets were treated on a heat block for the associated time and temperature. Following thermal therapy, cells were resuspended and irradiated using a 37-Cesium irradiator at 0.6 Gy min−1. Immediately following treatment, cells were either plated as single cells to allow colonies to form, and stained with Coomassie blue to be counted approximately 10–14 days later or harvested for Western blot analysis. Cell lysates were analyzed for PD-L1 expression with respect to various iron metabolic parameters (mortalin (HSPA9), transferrin receptor, and ferritin heavy chain) using a Western blotting approach. In both U87 and U87R132H cell lines, thermal therapy showed a temperature-dependent cell-killing effect, but U87R132H cells appeared more sensitive to thermal treatment when treated at 43 °C for 10 min. Moreover, thermal therapy had minimal effects on cell responses to 2 Gy irradiation. Treatment with thermal therapy downregulated PD-L1 expression in U87R132H cells, which was associated with increased expression of the mitochondrial iron metabolic enzyme, HSPA9. Thermal therapy reversed the radiation-induced overexpression of PD-L1, transferrin receptor, and ferritin heavy chain in U87R132H cells. No effects were observed in wild-type U87 cells. Moreover, Ga(NO3)3 depleted mitochondrial iron content which, in turn, significantly enhanced the sensitivity of U87R132H cells to thermal therapy and 2 Gy irradiation and caused a significant increase in PD-L1 expression. These results suggest that thermal therapy alone can modulate the immune checkpoint PD-L1. This effect was more pronounced when thermal therapy was combined with radiation. Mechanistically, mitochondrial iron trafficking through HSPA9 may coordinate the regulation of PD-L1 in the context of thermal therapy and ionizing radiation, which can be targeted with gallium-based therapy. These novel, preliminary findings warrant further mechanistic investigations in pre-clinical models of LITT.

Identifying T-cell clubs by embracing the local harmony between TCR and gene expressions.

T cell receptors (TCR) and gene expression provide two complementary and essential aspects in T cell understanding, yet their diversity presents challenges in integrative analysis. We introduce TCRclub, a novel method integrating single-cell RNA sequencing data and single-cell TCR sequencing data using local harmony to identify functionally similar T cell groups, termed 'clubs'. We applied TCRclub to 298,106 T cells across seven datasets encompassing various diseases. First, TCRclub outperforms the state-of-the-art methods in clustering T cells on a dataset with over 400 verified peptide-major histocompatibility complex categories. Second, TCRclub reveals a transition from activated to exhausted T cells in cholangiocarcinoma patients. Third, TCRclub discovered the pathways that could intervene in response to anti-PD-1 therapy for patients with basal cell carcinoma by analyzing the pre-treatment and post-treatment samples. Furthermore, TCRclub unveiled different T-cell responses and gene patterns at different severity levels in patients with COVID-19. Hence, TCRclub aids in developing more effective immunotherapeutic strategies for cancer and infectious diseases.