Spatial multi-omics reveal intratumoral humoral immunity niches associated with tertiary lymphoid structures in pancreatic cancer immunotherapy pathologic responders.

Pancreatic adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. We generated a spatial multi-omics atlas encompassing 26 PDAC tumors from patients treated with combination immunotherapies. Using machine learning-enabled H&E image classification models and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within TLS niches spanning across distinct morphologies and immunotherapies. Unsupervised learning generated a TLS-specific spatial gene expression signature that significantly associates with improved survival in PDAC patients. These analyses demonstrate TLS-associated intratumoral B cell maturation in pathological responders, confirmed with spatial proteomics and BCR profiling. Our study also identifies spatial features of pathologic immune responses, revealing TLS maturation colocalizing with IgG/IgA distribution and extracellular matrix remodeling. HIGHLIGHTS: Integrated multi-modal spatial profiling of human PDAC tumors from neoadjuvant immunotherapy clinical trials reveal diverse spatial niches enriched in TLS.TLS maturity is influenced by tumor location and the cellular neighborhoods in which TLS immune cells are recruited.Unsupervised machine learning of genome-wide signatures on spatial transcriptomics data characterizes the TLS-enriched TME and associates TLS transcriptomes with survival outcomes in PDAC.Interactions of spatially variable gene expression patterns showed TLS maturation is coupled with immunoglobulin distribution and ECM remodeling in pathologic responders.Intratumoral plasma cell and immunoglobin gene expression spatial dynamics demonstrate trafficking of TLS-driven humoral immunity in the PDAC TME. Significance: We report a spatial multi-omics atlas of PDAC tumors from a series of immunotherapy neoadjuvant clinical trials. Intratumorally, pathologic responders exhibit mature TLS that propagate plasma cells into malignant niches. Our findings offer insights on the role of TLS-associated humoral immunity and stromal remodeling during immunotherapy treatment.

Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis.

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and hypothesized to drive the loss of immune tolerance to self-antigens in autoimmunity. Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines. Here, we investigate the hypothesis that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage. We show that citrullination alters processing and presentation of autoantigens, resulting in the generation of a unique citrullination-dependent repertoire composed primarily of native sequences. This repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than controls. The generation of this unique repertoire is achieved through altered protease cleavage and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA.

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3 Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8 In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.