Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy

Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response

A Phase I/II first-line study of R-CHOP plus B-cell receptor/NF-κB-double-targeting to molecularly assess therapy response

The ImbruVeRCHOP trial is an investigator-initiated, multicenter, single-arm, open label Phase I/II study for patients 61-80 years of age with newly diagnosed CD20+ diffuse large B-cell lymphoma and a higher risk profile (International Prognostic Index ≥2). Patients receive standard chemotherapy (CHOP) plus immunotherapy (Rituximab), a biological agent (the proteasome inhibitor Bortezomib) and a signaling inhibitor (the Bruton's Tyrosine Kinase-targeting therapeutic Ibrutinib). Using an all-comers approach, but subjecting patients to another lymphoma biopsy acutely under first-cycle immune-chemo drug exposure, ImbruVeRCHOP seeks to identify an unbiased molecular responder signature that marks diffuse large B-cell lymphoma patients at risk and likely to benefit from this regimen as a double, proximal and distal B-cell receptor/NF-κB-co-targeting extension of the current R-CHOP standard of care. EudraCT-Number: 2015-003429-32; ClinicalTrials.gov identifier: NCT03129828

A Novel Statistical Method for Decontaminating T-Cell Receptor Sequencing Data

The T-cell receptor (TCR) repertoire is highly diverse among the population and plays an essential role in initiating multiple immune processes. TCR sequencing (TCR-seq) has been developed to profile the T cell repertoire. Similar to other high-throughput experiments, contamination can happen during several steps of TCR-seq, including sample collection, preparation and sequencing. Such contamination creates artifacts in the data, leading to inaccurate or even biased results. Most existing methods assume \u27clean\u27 TCR-seq data as the starting point with no ability to handle data contamination. Here, we develop a novel statistical model to systematically detect and remove contamination in TCR-seq data. We summarize the observed contamination into two sources, pairwise and cross-cohort. For both sources, we provide visualizations and summary statistics to help users assess the severity of the contamination. Incorporating prior information from 14 existing TCR-seq datasets with minimum contamination, we develop a straightforward Bayesian model to statistically identify contaminated samples. We further provide strategies for removing the impacted sequences to allow for downstream analysis, thus avoiding any need to repeat experiments. Our proposed model shows robustness in contamination detection compared with a few off-the-shelf detection methods in simulation studies. We illustrate the use of our proposed method on two TCR-seq datasets generated locally

Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70

Various subsets of invariant natural killer T (iNKT) cells with different cytokine productions develop in the mouse thymus, but the factors driving their differentiation remain unclear. Here we show that hypomorphic alleles of Zap70 or chemical inhibition of Zap70 catalysis leads to an increase of IFN-gamma-producing iNKT cells (NKT1 cells), suggesting that NKT1 cells may require a lower TCR signal threshold. Zap70 mutant mice develop IL-17-dependent arthritis. In a mouse experimental arthritis model, NKT17 cells are increased as the disease progresses, while NKT1 numbers negatively correlates with disease severity, with this protective effect of NKT1 linked to their IFN-gamma expression. NKT1 cells are also present in the synovial fluid of arthritis patients. Our data therefore suggest that TCR signal strength during thymic differentiation may influence not only IFN-gamma production, but also the protective function of iNKT cells in arthritis

A pan-immunotherapy signature to predict intratumoral CD8+ T cell expansions.

Effective cancer immunotherapy relies on the clonal proliferation and expansion of CD8+ T cells in the tumor. However, our insights into clonal expansions are limited, owing to an inability to track the same clones in tumors over time. Here, we develop a multi-site tumor mouse model system to track hundreds of expanding and contracting CD8+ T cell clones over multiple timepoints in tumors of the same individual. Through coupling of clonal expansion dynamics and single-cell RNA/TCR-seq data, we identify a transcriptomic signature in PD-1+Ly108+ precursor exhausted cells that strongly predicts rates of intratumoral clone expansion. The signature correlates with expansion in mice, both with and without immunotherapies, and in patients undergoing PD-1 blockade therapy. Expression of the signature during treatment corresponds with positive clinical outcomes. Downregulation of the signature precedes clone contraction-a phase in which clones contract but maintain revivable precursor exhausted cells in the tumor. LAG-3 blockade re-activates the expansion signature, re-expanding pre-existing clones, including previously contracted clones. These findings reveal how the study of clonal expansion dynamics provide a powerful 'pan-immunotherapy' signature for monitoring immunotherapies with implications for their future development

A pan-immunotherapy signature to predict intratumoral CD8⁺ T cell expansions

Effective cancer immunotherapy relies on the clonal proliferation and expansion of CD8+ T cells in the tumor. However, our insights into clonal expansions are limited, owing to an inability to track the same clones in tumors over time. Here, we develop a multi-site tumor mouse model system to track hundreds of expanding and contracting CD8+ T cell clones over multiple timepoints in tumors of the same individual. Through coupling of clonal expansion dynamics and single-cell RNA/TCR-seq data, we identify a transcriptomic signature in PD-1+Ly108+ precursor exhausted cells that strongly predicts rates of intratumoral clone expansion. The signature correlates with expansion in mice, both with and without immunotherapies, and in patients undergoing PD-1 blockade therapy. Expression of the signature during treatment corresponds with positive clinical outcomes. Downregulation of the signature precedes clone contraction-a phase in which clones contract but maintain revivable precursor exhausted cells in the tumor. LAG-3 blockade re-activates the expansion signature, re-expanding pre-existing clones, including previously contracted clones. These findings reveal how the study of clonal expansion dynamics provide a powerful 'pan-immunotherapy' signature for monitoring immunotherapies with implications for their future development. </p

Features of the intratumoral T cell receptor repertoire associated with antigen exposure in cancer patients

The clinical success of immunotherapies demonstrates the importance of the immune system in tumour control, but the response rates remain low and many biological mechanisms underlying how these therapies work are still uncharacterised. In particular, the specificity of the anti-tumour immune response pre-existing in treatment-naive patients or induced by treatment remains poorly described. In this thesis, I explore how T cell receptor (TCR) sequencing data in multi-omics contexts can be utilised to identify features associated with antigen exposure in cancer patients. In treatment-naive non-small cell lung cancer (NSCLC) patients, multi-region TCR sequencing revealed a pattern of heterogeneity in the TCR repertoire resembling the heterogeneity observed in the mutational profile of these tumours and a range of clonotype frequency values associated with tumour specificity. A novel method was built in order to identify distinct TCR populations that spatially follow the pattern of the well-established clonal/subclonal mutational dichotomy. The impact of immune checkpoint blockade therapy on the TCR repertoire distribution was assessed in advanced renal cell carcinoma in the context of anti- PD1 treatment. TCRs with frequency distribution characteristics similar to what was observed in NSCLC were maintained upon treatment and associated with clinical response. In addition, RNA-sequencing analysis identified a gene expression profile consistent with specific activation of T cells through TCR signalling. Finally, the same methodology was applied to bone marrow samples harvested from B cell acute lymphoblastic leukaemia (B-ALL) patients. A statistical framework was developed in order to efficiently distinguish leukaemic re-arrangements from the non- leukaemic TCR repertoire of B-ALL patients. Subsequently, longitudinal analysis revealed TCR distributions that suggested the presence of cytotoxic T cells which was further characterised in matched single-cell RNA sequencing data

Randomized trial of neoadjuvant vaccination with tumor-cell lysate induces T cell response in low-grade gliomas

BACKGROUND. Long-term prognosis of WHO grade II low-grade gliomas (LGGs) is poor, with a high risk of recurrence and malignant transformation into high-grade gliomas. Given the relatively intact immune system of patients with LGGs and the slow tumor growth rate, vaccines are an attractive treatment strategy. METHODS. We conducted a pilot study to evaluate the safety and immunological effects of vaccination with GBM6-AD, lysate of an allogeneic glioblastoma stem cell line, with poly-ICLC in patients with LGGs. Patients were randomized to receive the vaccines before surgery (arm 1) or not (arm 2) and all patients received adjuvant vaccines. Coprimary outcomes were to evaluate safety and immune response in the tumor. RESULTS. A total of 17 eligible patients were enrolled — 9 in arm 1 and 8 in arm 2. This regimen was well tolerated with no regimen-limiting toxicity. Neoadjuvant vaccination induced upregulation of type-1 cytokines and chemokines and increased activated CD8+ T cells in peripheral blood. Single-cell RNA/T cell receptor sequencing detected CD8+ T cell clones that expanded with effector phenotype and migrated into the tumor microenvironment (TME) in response to neoadjuvant vaccination. Mass cytometric analyses detected increased tissue resident–like CD8+ T cells with effector memory phenotype in the TME after the neoadjuvant vaccination. CONCLUSION. The regimen induced effector CD8+ T cell response in peripheral blood and enabled vaccine-reactive CD8+ T cells to migrate into the TME. Further refinements of the regimen may have to be integrated into future strategies