Advances in cancer immunotherapy, particularly immune checkpoint inhibitors, have dramatically improved the prognosis for patients with metastatic melanoma and other previously incurable cancers. ...However, patients with pancreatic ductal adenocarcinoma (PDAC) generally do not respond to these therapies. PDAC is exceptionally difficult to treat because of its often late stage at diagnosis, modest mutation burden, and notoriously complex and immunosuppressive tumor microenvironment. Simultaneously interrogating features of cancer, immune, and other cellular components of the PDAC tumor microenvironment is therefore crucial for identifying biomarkers of immunotherapeutic resistance and response. Notably, single-cell and multiomics technologies, along with the analytical tools for interpreting corresponding data, are facilitating discoveries of the systems-level cellular and molecular interactions contributing to the overall resistance of PDAC to immunotherapy. Thus, in this review, we will explore how multiomics and single-cell analyses provide the unprecedented opportunity to identify biomarkers of resistance and response to successfully sensitize PDAC to immunotherapy.
Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell–mediated ...autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43–mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR–mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8
T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.
Post-translational modifications (PTMs) on proteins can be targeted by antibodies associated with autoimmunity. Despite a growing appreciation for their intrinsic role in disease, there is a lack of ...highly multiplexed serological assays to characterize the fine specificities of PTM-directed autoantibodies.
In this study, we used the programmable phage display technology, Phage ImmunoPrecipitation Sequencing (PhIP-Seq), to profile rheumatoid arthritis (RA) associated anti-citrullinated protein antibody (ACPA) reactivities.
Using both unmodified and peptidylarginine deiminase (PAD)-modified phage display libraries consisting of ~250,000 overlapping 90 amino acid peptide tiles spanning the human proteome, PTM PhIP-Seq robustly identified antibodies to citrulline-dependent epitopes.
PTM PhIP-Seq was used to quantify key differences among RA patients, including PAD isoform specific ACPA profiles, and thus represents a powerful tool for proteome-scale antibody-binding analyses.
This research is based upon work supported in part by the Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA). The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of ODNI, IARPA, or the US Government. The US Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation therein. This study was made possible by a National Institute of General Medical Sciences (NIGMS) grant R01 GM136724 (HBL). MFK was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) grant T32AR048522. ED was supported by the Rheumatology Research Foundation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Myositis, or idiopathic inflammatory myopathy (IIM), is a group disorders of unknown etiology characterized by the inflammation of skeletal muscle. The role of T cells and their antigenic targets in ...IIM initiation and progression is poorly understood. T cell receptor (TCR) repertoire sequencing is a powerful approach for characterizing complex T cell responses. However, current TCR sequencing methodologies are complex, expensive, or both, greatly limiting the scale of feasible studies.
Here we present Framework Region 3 AmplifiKation sequencing (“FR3AK-seq”), a simplified multiplex PCR-based approach for the ultra-efficient and quantitative analysis of TCR complementarity determining region 3 (CDR3) repertoires. By using minimal primer sets targeting a conserved region immediately upstream of CDR3, undistorted amplicons are analyzed via short read, single-end sequencing. We also introduce the novel algorithm Inferring Sequences via Efficiency Projection and Primer Incorporation (“ISEPPI”) for linking CDR3s to their associated variable genes.
We find that FR3AK-seq is sensitive and quantitative, performing comparably to two different industry standards. FR3AK-seq and ISEPPI were used to efficiently and inexpensively characterize the T cell infiltrates of surgical muscle biopsies obtained from 145 patients with IIM and controls. A cluster of closely related TCRs was identified in samples from patients with sporadic inclusion body myositis (IBM).
The ease and minimal cost of FR3AK-seq removes critical barriers to routine, large-scale TCR CDR3 repertoire analyses, thereby democratizing the quantitative assessment of human TCR repertoires in disease-relevant target tissues. Importantly, discovery of closely related TCRs in muscle from patients with IBM provides evidence for a shared antigen-driven T cell response in this disease of unknown pathogenesis.
This work was supported by NIH grant U24AI118633 and a Prostate Cancer Foundation Young Investigator Award.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Assays linking cellular phenotypes with T cell or B cell antigen receptor sequences are crucial for characterizing adaptive immune responses. Existing methodologies are limited by low sample ...throughput and high cost. Here, we present INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS), an approach that combines molecular indexing of receptor repertoires within intact cells and fluorescence-activated cell sorting (FACS). We demonstrate that INCERTS enables efficient processing of millions of cells from pooled human peripheral blood mononuclear cell (PBMC) samples while retaining robust association between T cell receptor (TCR) sequences and cellular phenotypes. We used INCERTS to discover antigen-specific TCRs from patients with cancer immunized with a novel mutant KRAS peptide vaccine. After ex vivo stimulation, 28 uniquely barcoded samples were pooled prior to FACS into peptide-reactive and non-reactive CD4+ and CD8+ populations. Combining complementary patient-matched single-cell RNA sequencing (scRNA-seq) data enabled retrieval of full-length, paired TCR alpha and beta chain sequences for future validation of therapeutic utility.
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•INCERTS uses intracellular barcoding of cDNA prior to pooled cell sorting•Upstream sample pooling efficiently links immune repertoires with cell phenotypes•INCERTS readily scales to millions of cells from a large number of samples
Existing methodologies that integrate T or B cell phenotypes with their antigen receptor sequences are limited by low sample throughput and high cost. Such approaches include single-cell RNA sequencing (scRNA-seq) and flow cytometric sorting of cells into subpopulations for bulk repertoire sequencing, both of which are difficult to scale across many samples and millions of cells. Therefore, we developed INtraCEllular Reverse Transcription with Sorting and sequencing (INCERTS). INCERTS successfully links cellular phenotype with antigen receptor sequence via barcoding during reverse transcription within intact cells. Subsequent cell sorting, sequencing, and demultiplexing enables high-throughput characterization of phenotype-coupled antigen receptor sequences across millions of cells.
Jayaraman et al. develop INCERTS, a scalable and efficient method for linking cell phenotypes to immune receptor sequences. INCERTS uses intracellular cDNA barcoding, which enables pooling of samples prior to cell sorting, thereby linking cell phenotypes with immune receptor repertoire sequences.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Recent advances have enabled the integration of T cell receptor (TCR) sequencing into spatial transcriptomics. Current approaches yield critical information about in situ T cell responses ...directly from relevant tissues, yet are largely limited to alpha/beta TCRs, leaving the clonal dynamics of B cells and gamma/delta T cells underexplored. We therefore modified our previously reported ultra-efficient immune receptor repertoire sequencing (IRR-seq) method, Framework Region 3 AmplifiKation Sequencing (FR3AK-seq), for compatibility with the 10X Genomics Visium spatial transcriptomics platform. Here we report simultaneous acquisition of alpha/beta TCR, gamma/delta TCR, and immunoglobulin (B cell receptor, BCR) heavy/light chain sequences from a single multiplexed FR3AK-seq reaction using Visium transcriptomics-generated cDNA libraries. More specifically, we used the Visium spatial transcriptomics protocol to generate cDNA from serial sections of a lymph node with a metastatic lesion obtained from a patient with pancreatic ductal adenocarcinoma previously treated with immunotherapy. Notably, we detected sequences from all IRR chains as expected from the abundant immune infiltrates within these sections. Furthermore, we found that multiplexed FR3AK-seq yields technically reproducible repertoires, with the TCR beta chain repertoires also comparable to those obtained using a previously published method. We anticipate that this simple extension of the Visium technology will accelerate the simultaneous and comprehensive examination of in situ IRR dynamics beyond alpha/beta T cells.
Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy necessitates optimization and maintenance of activated effector T cells (Teff). We prospectively collected and applied multi-omic ...analyses to paired pre- and post-treatment PDAC specimens collected in a platform neoadjuvant study of granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDAC vaccine (GVAX) vaccine ± nivolumab (anti-programmed cell death protein 1 PD-1) to uncover sensitivity and resistance mechanisms. We show that GVAX-induced tertiary lymphoid aggregates become immune-regulatory sites in response to GVAX + nivolumab. Higher densities of tumor-associated neutrophils (TANs) following GVAX + nivolumab portend poorer overall survival (OS). Increased T cells expressing CD137 associated with cytotoxic Teff signatures and correlated with increased OS. Bulk and single-cell RNA sequencing found that nivolumab alters CD4+ T cell chemotaxis signaling in association with CD11b+ neutrophil degranulation, and CD8+ T cell expression of CD137 was required for optimal T cell activation. These findings provide insights into PD-1-regulated immune pathways in PDAC that should inform more effective therapeutic combinations that include TAN regulators and T cell activators.
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•Prospectively collected PDAC specimens from a neoadjuvant platform clinical trial•Identified sensitivity and resistance mechanisms to anti-PD-1 therapy in PDAC•Informed studies of additional immune-modulating agents in the ongoing platform trial•Generated hypotheses of reprogramed TME signals for combination immunotherapy strategies
Li et al. perform multi-omic analyses on pre- and post-treatment specimens from a pancreatic cancer neoadjuvant platform trial, and identify sensitivity and resistance mechanisms associated with anti-PD-1 combination therapy. Results associate tumor-associated neutrophils with poor outcomes but CD137+CD8+ T cells with better outcomes, suggesting treatment strategies for future interventions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Objective
Sjögren's syndrome (SS) patients may be affected by the neuromyelitis optica spectrum disorder (NMOSD), a severe demyelinating syndrome associated with anti–aquaporin 4 antibodies ...(anti‐AQP‐4 antibodies). The relationship between SS and NMOSD has been a sustained focus of investigation. Among SS patients, anti‐AQP‐4 antibodies have been detected exclusively in those with NMOSD. It has therefore been speculated that NMOSD is not a neurologic complication of SS. However, such studies evaluated small numbers of SS patients, often mixed with other inflammatory disorders.
Methods
We compared frequencies of anti‐AQP‐4 and SS‐associated antibodies in 109 SS patients, including 11 with NMOSD, 8 with non‐NMOSD demyelinating syndromes, and 90 without demyelinating syndromes.
Results
When assessed using a fluorescence‐activated cell sorting (FACS) assay, anti‐AQP‐4 antibodies were seen exclusively in those SS patients with NMOSD (72.7%), but not in SS patients without NMOSD (P < 0.01). In contrast, anti–Ro 52, anti–Ro 60, and other autoantibodies were not more prevalent in SS patients with NMOSD versus those without. Anti‐AQP‐4 antibodies were detected more frequently among NMOSD patients by FACS assay than with a commercial immunohistochemical assay (72.7% versus 54.5%), despite assessment after a more prolonged period of immunosuppressive therapy (median 38 months versus 5 months; P < 0.01).
Conclusion
The syndrome‐specificity of anti‐AQP‐4 antibodies, along with an otherwise similar antibody profile in SS NMOSD patients, indicates that NMOSD is not a direct central nervous system manifestation of SS. Anti‐AQP‐4 antibodies can persist and be refractory to prolonged immunosuppressive therapy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract Background: In pancreatic ductal adenocarcinoma (PDAC), rare long-term survivors correlate with high intratumoral tertiary lymphoid structure (TLS) density. This finding prompted our ...investigation of clinically viable strategies to induce TLS in patients with immune-excluded tumors. We previously reported the induction of intratumoral TLS following administration of a neoadjuvant GM-CSF-secreting allogeneic vaccine (GVAX) to PDAC patients (NCT00727441). However, no clinical benefit was observed, likely owing to immune tolerance mechanisms governing the PDAC tumor microenvironment (TME). We previously observed upregulation of both the PD-1 and 4-1BB pathways with GVAX, and thus in a subsequent neoadjuvant trial combined GVAX with PD-1 blockade and 4-1BB agonism (NCT02451982) which was associated with pathologic responses. We hypothesized this combination strategy induced TLS of higher maturity and anti-tumor activity compared to GVAX alone. Methods: To explore how this therapeutic strategy affected TLS morphology and intercellular crosstalk, we leveraged the Visium spatial transcriptomics platform and a 35-marker customized TLS panel for imaging mass cytometry. We generated cellular and molecular maps of the TME after neoadjuvant treatment in 26 PDAC patients (GVAX n=19, GVAX+aPD1 n=2, GVAX+aPD1+a41BB n=5). To compare TLS maturation in parallel with secondary lymphoid organ-mediated tumor immunity, we also profiled tumor-adjacent lymph nodes. We applied unsupervised learning with non-negative matrix factorization (NMF) and trained AI-enabled image classification models to characterize cellular states within tissue structures of interest. Results: We identified spatial gene expression NMF patterns in PDAC TLS spanning across distinct morphologies and neoadjuvant treatment arms. Intratumoral TLS after GVAX were found to propagate activated B cells expressing immunoglobulins that infiltrated into malignant cellular niches. TLS NMF patterns were also associated with autoimmune disease signatures, such as diabetes, in a subset of patients. We scored TLS using tumor-draining lymph nodes as a reference and found increased maturation of TLS after PD-1 blockade, while addition of 4-1BB agonism significantly boosted the cytotoxic NK/T cell compartment compared to GVAX alone. Conclusions: We present machine learning approaches for spatial multi-omics analysis to characterize the TLS-enriched TME. We mined genome-wide spatial TLS gene expression patterns elucidating the spatial dynamics of humoral immunity of rare immunotherapy pathologic responders in PDAC. Altogether our findings shed light on the plasticity of TLS in neoadjuvant immunotherapy and suggest future immunotherapy approaches should target both humoral and cytotoxic NK/T cell compartments to augment responses in solid tumors. Citation Format: Dimitrios N. Sidiropoulos, Sarah M. Shin, Alexander Girgis, Daniel H. Shu, Janelle Montagne, Atul Deshpande, Jeanette A. Johnson, Lucie Dequiedt, Victoria Jacobs, Aleksandra Ogurtsova, Guanglan Mo, Xuan Yuan, Genevieve Stein-O’Brien, Mark Yarchoan, Qingfeng Zhu, Ashley Kiemen, Elizabeth M. Jaffee, Lei Zheng, Won Jin Ho, Robert Anders, Elana J. Fertig, Luciane T. Kagohara. Machine learning integrating spatial omics uncovers humoral immunity patterns in intratumoral tertiary lymphoid structures in pancreatic cancer pathologic responders abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1159.
Abstract
The evolution of pancreatic ductal adenocarcinoma (PDAC) from tumor initiation to metastases requires years to decades, providing a window of opportunity for the prevention of premalignant ...progression. One of the first genetic mutations to occur early in development is in KRAS, an oncogene that is expressed by over 90% of PDACs. KRAS mutations (mKRAS) are attractive targets as they are recurrent hot–spot drivers that are expressed by, and drive the growth of all PDAC cells. Preclinical data provide compelling evidence that targeting mKRAS proteins using a Listeria-based vaccination approach in KPC mouse model results in prevention of progression from precursor lesions to PDAC. Based on these data, we hypothesize that targeting mKRAS in combination with bypassing early immune–suppressive signals will slow or even halt the progression of precursor lesions to PDA in subjects who are at high–risk. We have thus developed a clinical–grade long peptide vaccine targeting the six most common mKRAS proteins (G12V, G12R, G12A, G12C, G12D, and G13D) in PDAC patients. To establish initial safety and immunogenicity, our KRAS vaccine has been tested in 11 patients thus far in a Phase 1 clinical trial (NCT04117087) in combination with checkpoint blockade in patients who have undergone surgery and peri–operative chemotherapy and remained disease–free on imaging. Longitudinal immune data demonstrates an induction of activated and poly-functional mKRAS-specific CD4+ and CD8+ T cells, as well as effector and central memory T cells post–vaccination. In parallel, we performed paired single-cell RNA and TCR sequencing in PBMCs from a patient to obtain greater resolution of the vaccine–induced cytotoxic anti–tumor T cells. We captured 66,869 immune cells across three timepoints for analysis. Notably, we similarly observed an expansion of central memory CD4+ and CD8+ T cells post–vaccination. Using a novel computational algorithm (developed in collaboration with Dr. Elana Fertig’s lab) that takes into account physiochemical similarity of known mKRAS TCRs, we used our single–cell dataset to identify mKRAS-reactive TCRs` in the periphery that were functionally validated in vitro using CRISPR-Cas12a-based genome editing of human T cells. Overall, these studies suggest the induction of de novo, high quality mutant KRAS–specific T cells in the peripheral blood post–vaccination. Based on these immune data, we are enrolling onto a second clinical trial testing our mKRAS peptide vaccine in patients who are at high risk for developing PDAC (NCT05013216). These high-risk groups include those with strong family histories of PDAC or known pathogenic germline variants with a lifetime risk of up to 10% of developing PDA. Our preliminary data in our first three vaccinated patients shows an induction of mKRAS–specific T cell responses in the peripheral blood. Further studies characterizing the T cell response will lay the foundation for peripheral T cell–based biomarkers that could be used to predict response in a prevention setting.
Citation Format: Neeha Zaidi, Amanda Huff, Emily Marcisak-Davis, Daniel Haldar, Thatcher Heumann, Max Konig, Brian Mog, Janelle Montagne, Gabriella Longway, Lalitya Andaloori, Melissa Lyman, Ludmila Danilova, Julie Nauroth, Luciane Kagohara, Elana Fertig, Nilo Azad, Elizabeth Jaffee. Intercepting pancreatic cancer development with mutant KRAS-targeted immunotherapy abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA013.
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