Modeling and predicting the overlap of B- and T-cell receptor repertoires in healthy and SARS-CoV-2 infected individuals

Adaptive immunity's success relies on the extraordinary diversity of protein receptors on B and T cell membranes. Despite this diversity, the existence of public receptors shared by many individuals gives hope for developing population wide vaccines and therapeutics. Yet many of these public receptors are shared by chance. We present a statistical approach, defined in terms of a probabilistic V(D)J recombination model enhanced by a selection factor, that describes repertoire diversity and predicts with high accuracy the spectrum of repertoire overlap in healthy individuals. The model underestimates sharing between repertoires of individuals infected with SARS-CoV-2, suggesting strong antigen-driven convergent selection. We exploit this discrepancy to identify COVID-associated receptors, which we validate against datasets of receptors with known viral specificity. We study their properties in terms of sequence features and network organization, and use them to design an accurate diagnosis tool for predicting SARS-CoV-2 status from repertoire data.

Systems immune profiling of variant-specific vaccination against SARS-CoV-2

Lipid-nanoparticle(LNP)-mRNA vaccines offer protection against COVID-19. However, multiple variant lineages caused widespread breakthrough infections. There is no report on variant-specific vaccines to date. Here, we generated LNP-mRNAs specifically encoding wildtype, B.1.351 and B.1.617 SARS-CoV-2 spikes, and systematically studied their immune responses in animal models. All three LNP-mRNAs induced potent antibody responses in mice. However, WT-LNP-mRNA vaccination showed reduced neutralization against B.1.351 and B.1.617; and B.1.617-specific vaccination showed differential neutralization. All three vaccine candidates elicited antigen-specific CD8 and CD4 T cell responses. Single cell transcriptomics of B.1.351-LNP-mRNA and B.1.617-LNP-mRNA vaccinated animals revealed a systematic landscape of immune cell populations and global gene expression. Variant-specific vaccination induced a systemic increase in reactive CD8 T cell population, with a strong signature of transcriptional and translational machineries in lymphocytes. BCR-seq and TCR-seq unveiled repertoire diversity and clonal expansions in vaccinated animals. These data provide direct systems immune profiling of variant-specific LNP-mRNA vaccination in vivo.

Immunosenescence and Autoimmunity: Exploiting the T-Cell Receptor Repertoire to Investigate the Impact of Aging on Multiple Sclerosis

T-cell receptor (TCR) repertoire diversity is a determining factor for the immune system capability in fighting infections and preventing autoimmunity. During life, the TCR repertoire diversity progressively declines as a physiological aging progress. The investigation of TCR repertoire dynamics over life represents a powerful tool unraveling the impact of immunosenescence in health and disease. Multiple Sclerosis (MS) is a demyelinating, inflammatory, T-cell mediated autoimmune disease of the Central Nervous System in which age is crucial: it is the most widespread neurological disease among young adults and, furthermore, patients age may impact on MS progression and treatments outcome. Crossing knowledge on the TCR repertoire dynamics over MS patients’ life is fundamental to investigate disease mechanisms, and the advent of high- throughput sequencing (HTS) has significantly increased our knowledge on the topic. Here we report an overview of current literature about the impact of immunosenescence and age-related TCR dynamics variation in autoimmunity, including MS.

T Cell Repertoire Abnormality in Immunodeficiency Patients with DNA Repair and Methylation Defects

Both DNA damage response and methylation play a crucial role in antigen receptor recombination by creating a diverse repertoire in developing lymphocytes, but how their defects relate to T cell repertoire and phenotypic heterogeneity of immunodeficiency remains obscure. We studied the TCR repertoire in patients with the mutation in different genes (ATM, DNMT3B, ZBTB24, RAG1, DCLRE1C, and JAK3) and uncovered distinct characteristics of repertoire diversity. We propose that early aberrancies in thymus T cell development predispose to the heterogeneous phenotypes of the immunodeficiency spectrum. Shorter CDR3 lengths in ATM-deficient patients, resulting from a decreased number of nucleotide insertions during VDJ recombination in the pre-selected TCR repertoire, as well as the increment of CDR3 tyrosine residues, lead to the enrichment of pathology-associated TCRs, which may contribute to the phenotypes of ATM deficiency. Furthermore, patients with DNMT3B and ZBTB24 mutations who exhibit discrepant phenotypes present longer CDR3 lengths and reduced number of known pathology-associated TCRs.

740 Radiotherapy-activated NBTXR3 nanoparticles Increase CD8+ T cell infiltration and diversity in tumors, and modulate the immunopeptidome of cancer cells

BackgroundWhen exposed to radiotherapy (RT), NBTXR3 nanoparticles increase radiation dose deposition from within the cancer cells. NBTXR3 is intended for a single intratumor injection. Results from a phase II/III clinical trial in patients with locally advanced Soft Tissue Sarcoma demonstrated significant superiority and clinical benefits of NBTXR3 activated by RT compared to RT alone, and was well tolerated. NBTXR3 is currently being evaluated in several other tumors including head and neck, liver, and pancreatic cancer as a single agent or in combination with anti-PD1. Moreover, preclinical studies have demonstrated that NBTXR3 can produce a significant abscopal effect, whereas RT alone cannot. Here, we explored the impact of NBTXR3 activated by RT on CD8+ infiltrates and TcR repertoire diversity change, and the effect on the immunopeptidome of cancer cells.MethodsCT26 (murine colorectal cancer cells) were subcutaneously injected in BALB/c mice in one flank. Then, tumors were intratumorally injected with NBTXR3 (or vehicle) and irradiated 24 hours later with 4Gy per fraction for 3 consecutive days. Tumors were collected 3 days after the last RT fraction and immune cell infiltrates were measured using immunohistochemistry (IHC) and digital pathology. For TcR repertoire sequencing, the same workflow was followed, except cells were injected in both flanks. Only right tumors received treatment, while left tumors remained untreated. For immunopeptidome analysis, in vitro cells were irradiated by 4Gy. After one day, cells were collected for isolation and sequencing of MHC I-loaded peptides.ResultsIHC analyses showed a significant increase of CD8+ T cell infiltrates in tumors of mice treated with NBTXR3+RT, while RT alone had no significant effect. In addition, NBTXR3+RT treatment was able to increase TcR repertoire diversity, both in treated and untreated tumors, compared to RT alone. Finally, analysis of immunopeptidome showed that NBTXR3+RT changed the profile of MHC-I-loaded peptides.ConclusionsOur in vivo data indicate that NBTXR3+RT can modulate the microenvironment of treated tumors, leading to enhanced CD8+ T cell infiltration as well as modification of the TcR repertoire, both in treated and distant untreated tumors. These NBTXR3+RT-induced responses may be related to changes in the immunopeptidome of cancer cells triggered by this treatment.

Impact of HLA type, age and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals

The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, factors such as thymic selection and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRβ variable region of 426 healthy individuals, we find that, on average, fewer than 1% of TCRβ clones are shared between individuals, consistent with largely private TCRβ repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRβ clones, with each additional shared HLA allele contributing to an increase in ~0.01% of the total shared TCRβ clones, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRβ clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRβ clone sharing, indicating that the pattern of private TCRβ clonal expansion is a general feature of the T-cell response to other infectious antigens as well. However, increased age also correlates with increased sharing among the lowest frequency clones, consistent with decreased repertoire diversity in older individuals. Together, all of these factors contribute to shaping the TCRβ repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.

Butyrophilin-like 2 regulates site-specific adaptations of intestinal γδ intraepithelial lymphocytes

Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vγ TCR+ IELs, however, the mechanisms that control γδ IEL maintenance within discrete intestinal segments are unclear. Here, we show that Btnl2 suppressed homeostatic proliferation of γδ IELs preferentially in the ileum. High throughput transcriptomic characterization of site-specific Btnl2-KO γδ IELs reveals that Btnl2 regulated the antimicrobial response module of ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ repertoire diversity of ileal γδ IELs. During DSS-induced colitis, Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ IEL frequencies and TCR specificities in response to site-specific homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.