Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Hierarchical Clustering Can Identify B Cell Clones with High Confidence in Ig Repertoire Sequencing Data.

Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data. Although several hierarchical clustering-based methods have been proposed, they vary in distance and linkage methods and have not yet been rigorously compared. In this study, we use a combination of human experimental and simulated data to characterize the performance of hierarchical clustering-based methods for partitioning sequences into clones. We find that single linkage clustering has high performance, with specificity, sensitivity, and positive predictive value all >99%, whereas other linkages result in a significant loss of sensitivity. Surprisingly, distance metrics that incorporate the biases of somatic hypermutation do not outperform simple Hamming distance. Although errors were more likely in sequences with short junctions, using the entire dataset to choose a single distance threshold for clustering is near optimal. Our results suggest that hierarchical clustering using single linkage with Hamming distance identifies clones with high confidence and provides a fully automated method for clonal grouping. The performance estimates we develop provide important context to interpret clonal analysis of repertoire sequencing data and allow for rigorous testing of other clonal grouping algorithms.

VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements.

BACKGROUND: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. RESULTS: To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. CONCLUSIONS: The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/ . We welcome participation from the community in developing the file format standard, as well as code contributions.

Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data.

UNLABELLED: Advances in high-throughput sequencing technologies now allow for large-scale characterization of B cell immunoglobulin (Ig) repertoires. The high germline and somatic diversity of the Ig repertoire presents challenges for biologically meaningful analysis, which requires specialized computational methods. We have developed a suite of utilities, Change-O, which provides tools for advanced analyses of large-scale Ig repertoire sequencing data. Change-O includes tools for determining the complete set of Ig variable region gene segment alleles carried by an individual (including novel alleles), partitioning of Ig sequences into clonal populations, creating lineage trees, inferring somatic hypermutation targeting models, measuring repertoire diversity, quantifying selection pressure, and calculating sequence chemical properties. All Change-O tools utilize a common data format, which enables the seamless integration of multiple analyses into a single workflow. AVAILABILITY AND IMPLEMENTATION: Change-O is freely available for non-commercial use and may be downloaded from http://clip.med.yale.edu/changeo. CONTACT: steven.kleinstein@yale.edu.

Designing meaningful continuous representations of T cell receptor sequences with deep generative models.

T Cell Receptor (TCR) antigen binding underlies a key mechanism of the adaptive immune response yet the vast diversity of TCRs and the complexity of protein interactions limits our ability to build useful low dimensional representations of TCRs. To address the current limitations in TCR analysis we develop a capacity-controlled disentangling variational autoencoder trained using a dataset of approximately 100 million TCR sequences, that we name TCR-VALID. We design TCR-VALID such that the model representations are low-dimensional, continuous, disentangled, and sufficiently informative to provide high-quality TCR sequence de novo generation. We thoroughly quantify these properties of the representations, providing a framework for future protein representation learning in low dimensions. The continuity of TCR-VALID representations allows fast and accurate TCR clustering and is benchmarked against other state-of-the-art TCR clustering tools and pre-trained language models.

LTßR Agonism Promotes Anti-Tumor Immune Responses via Modulation of the Tumor Microenvironment.

The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune-checkpoint blockade (ICB) in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses may facilitate development of improved treatment strategies. Lymphotoxin beta receptor (LTßR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and ICB treatment to augment tumor-associated HEV formation. Here, we demonstrated that LTßR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTßR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTßR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTßR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner, and the LTßR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTßR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTßR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments.

Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

BACKGROUND: Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated cytotoxicity independent of T-cell/major histocompatibility complex interaction. Phase I results in patients with R/R B-NHL demonstrated that odronextamab monotherapy could achieve deep and durable responses with a generally manageable safety profile (ELM-1; NCT02290951). As part of a biomarker analysis of the same study, we investigated potential biomarkers and mechanisms of resistance to odronextamab. METHODS: Patients with R/R B-NHL enrolled in ELM-1 received one time per week doses of intravenous odronextamab for 4×21 day cycles, then doses every 2 weeks thereafter. Patient tumor biopsies were obtained at baseline, on-treatment, and at progression. Immune cell markers were analyzed by immunohistochemistry, flow cytometry, single-cell RNA sequencing, and whole genome sequencing. RESULTS: Baseline tumor biopsies showed that almost all patients had high proportions of B cells that expressed the CD20 target antigen, whereas expression of other B-cell surface antigens (CD19, CD22, CD79b) was more variable. Responses to odronextamab in patients with diffuse large B-cell lymphoma were not related to the relative level of baseline CD20 expression, cell of origin, or high-risk molecular subtype. A potential link was observed between greater tumor programmed cell death-ligand 1 expression and increased likelihood of response to odronextamab. Similarly, a trend was observed between clinical response and increased levels of CD8 T cells and regulatory T cells at baseline. We also identified an on-treatment pharmacodynamic shift in intratumoral immune cell subsets. Finally, loss of CD20 expression through inactivating gene mutations was identified as a potential mechanism of resistance in patients who were treated with odronextamab until progression, as highlighted in two detailed patient cases reported here. CONCLUSIONS: This biomarker analysis expands on clinical findings of odronextamab in patients with R/R B-NHL, providing verification of the suitability of CD20 as a therapeutic target, as well as evidence for potential mechanisms of action and resistance.

Rapid TCR:Epitope Ranker (RAPTER): a primary human T cell reactivity screening assay pairing epitope and TCR at single cell resolution.

Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

Neoadjuvant cemiplimab for resectable hepatocellular carcinoma: a single-arm, open-label, phase 2 trial.

BACKGROUND: Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma. METHODS: For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8+ T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing. FINDINGS: Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks. INTERPRETATION: This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma. FUNDING: Regeneron Pharmaceuticals.

A framework for highly multiplexed dextramer mapping and prediction of T cell receptor sequences to antigen specificity.

T cell receptor (TCR) antigen-specific recognition is essential for the adaptive immune system. However, building a TCR-antigen interaction map has been challenging due to the staggering diversity of TCRs and antigens. Accordingly, highly multiplexed dextramer-TCR binding assays have been recently developed, but the utility of the ensuing large datasets is limited by the lack of robust computational methods for normalization and interpretation. Here, we present a computational framework comprising a novel method, ICON (Integrative COntext-specific Normalization), for identifying reliable TCR-pMHC (peptide-major histocompatibility complex) interactions and a neural network-based classifier TCRAI that outperforms other state-of-the-art methods for TCR-antigen specificity prediction. We further demonstrated that by combining ICON and TCRAI, we are able to discover novel subgroups of TCRs that bind to a given pMHC via different mechanisms. Our framework facilitates the identification and understanding of TCR-antigen-specific interactions for basic immunological research and clinical immune monitoring.

Absence of central tolerance in Aire-deficient mice synergizes with immune-checkpoint inhibition to enhance antitumor responses.

The endogenous anti-tumor responses are limited in part by the absence of tumor-reactive T cells, an inevitable consequence of thymic central tolerance mechanisms ensuring prevention of autoimmunity. Here we show that tumor rejection induced by immune checkpoint blockade is significantly enhanced in Aire-deficient mice, the epitome of central tolerance breakdown. The observed synergy in tumor rejection extended to different tumor models, was accompanied by increased numbers of activated T cells expressing high levels of Gzma, Gzmb, Perforin, Cxcr3, and increased intratumoural levels of Cxcl9 and Cxcl10 compared to wild-type mice. Consistent with Aire's central role in T cell repertoire selection, single cell TCR sequencing unveiled expansion of several clones with high tumor reactivity. The data suggest that breakdown in central tolerance synergizes with immune checkpoint blockade in enhancing anti-tumor immunity and may serve as a model to unmask novel anti-tumor therapies including anti-tumor TCRs, normally purged during central tolerance.

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response.

CD4+ follicular regulatory T (Tfr) cells suppress B cell responses through modulation of follicular helper T (Tfh) cells and germinal center (GC) development. We found that Tfr cells can also promote the GC response through provision of interleukin-10 (IL-10) after acute infection with lymphocytic choriomeningitis virus (LCMV). Sensing of IL-10 by B cells was necessary for optimal development of the GC response. GC B cells formed in the absence of Treg cell-derived IL-10 displayed an altered dark zone state and decreased expression of the transcription factor Forkhead box protein 1 (FOXO1). IL-10 promoted nuclear translocation of FOXO1 in activated B cells. These data indicate that Tfr cells play a multifaceted role in the fine-tuning of the GC response and identify IL-10 as an important mediator by which Tfr cells support the GC reaction.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.