Charlotte Reiche

A fundamental challenge for cancer vaccines is to generate long-lived functional T cells that are specific for tumour antigens. Here we find that mRNA–lipoplex vaccines against somatic mutation-derived neoantigens may solve this challenge in pancreatic ductal adenocarcinoma (PDAC), a lethal cancer with few mutations. At an extended 3.2-year median follow-up from a phase 1 trial of surgery, atezolizumab (PD-L1 inhibitory antibody), autogene cevumeran1 (individualized neoantigen vaccine with backbone-optimized uridine mRNA–lipoplex nanoparticles) and modified (m) FOLFIRINOX (chemotherapy) in patients with PDAC, we find that responders with vaccine-induced T cells (n = 8) have prolonged recurrence-free survival (RFS; median not reached) compared with non-responders without vaccine-induced T cells (n = 8; median RFS 13.4 months; P = 0.007). In responders, autogene cevumeran induces CD8+ T cell clones with an average estimated lifespan of 7.7 years (range 1.5 to roughly 100 years), with approximately 20% of clones having latent multi-decade lifespans that may outlive hosts. Eighty-six percent of clones per patient persist at substantial frequencies approximately 3 years post-vaccination, including clones with high avidity to PDAC neoepitopes. Using PhenoTrack, a novel computational strategy to trace single T cell phenotypes, we uncover that vaccine-induced clones are undetectable in pre-vaccination tissues, and assume a cytotoxic, tissue-resident memory-like T cell state up to three years post-vaccination with preserved neoantigen-specific effector function. Two responders recurred and evidenced fewer vaccine-induced T cells. Furthermore, recurrent PDACs were pruned of vaccine-targeted cancer clones. Thus, in PDAC, autogene cevumeran induces de novo CD8+ T cells with multiyear longevity, substantial magnitude and durable effector functions that may delay PDAC recurrence. Adjuvant mRNA–lipoplex neoantigen vaccines may thus solve a pivotal obstacle for cancer vaccination. In a phase 1 trial, patients with pancreatic ductal adenocarcinoma who were treated with surgery and bespoke neoantigen mRNA vaccines combined with anti-PD-L1 and chemotherapy exhibited marked long-lived persistence of neoantigen-specific CD8+ T cell clones, which correlated with prolonged recurrence-free survival at a 3.2-year follow-up.

Abstract Pancreatic ductal adenocarcinoma (PDAC) responds poorly to checkpoint blockade, such as anti-CTLA-4 and anti-PD-1. Galectin-9, a β-galactoside-binding lectin, promotes immune suppression through T-cell inhibition, and programming of tolerogenic macrophages. Of all cancers tested, PDAC showed the highest expression of LGALS9 (galectin-9) mRNA. We analyzed formalin-fixed and paraffin-embedded specimens from 83 patients with PDAC stained for galectin-9. Using flow cytometry, we determined galectin-9 expression on immune cells from tumor and matched blood samples from 12 patients with resectable PDAC. Furthermore, we analyzed galectin-9 serum levels by enzyme-linked immunosorbent assay using serum samples from 70 patients with PDAC, from 36 individuals with benign pancreatic disease, and from 28 healthy controls. Galectin-9 was highly expressed in human PDAC compared with normal pancreas and present on both tumor and immune cells. Tumor-infiltrating immune cells, especially CD3 + T cells, showed upregulation of galectin-9 compared with immune cells from matched blood. Blood γδ T cells from PDAC patients had higher galectin-9 expression than γδ T cells from healthy individuals. Galectin-9 polarized macrophages toward a protumoral M2 phenotype leading to suppressed T-cell cytokine secretion. Furthermore, serum concentration of galectin-9 was able to discriminate PDAC from benign pancreatic disease and healthy individuals, and was prognostic for stage IV patients. Galectin-9 is a new biomarker for the detection of PDAC.

Tertiary lymphoid structures (TLSs) are de novo ectopic lymphoid aggregates that regulate immunity in chronically inflamed tissues, including tumours. Although TLSs form due to inflammation-triggered activation of the lymphotoxin (LT)–LTβ receptor (LTβR) pathway1, the inflammatory signals and cells that induce TLSs remain incompletely identified. Here we show that interleukin-33 (IL-33), the alarmin released by inflamed tissues2, induces TLSs. In mice, Il33 deficiency severely attenuates inflammation- and LTβR-activation-induced TLSs in models of colitis and pancreatic ductal adenocarcinoma (PDAC). In PDAC, the alarmin domain of IL-33 activates group 2 innate lymphoid cells (ILC2s) expressing LT that engage putative LTβR+ myeloid organizer cells to initiate tertiary lymphoneogenesis. Notably, lymphoneogenic ILC2s migrate to PDACs from the gut, can be mobilized to PDACs in different tissues and are modulated by gut microbiota. Furthermore, we detect putative lymphoneogenic ILC2s and IL-33-expressing cells within TLSs in human PDAC that correlate with improved prognosis. To harness this lymphoneogenic pathway for immunotherapy, we engineer a recombinant human IL-33 protein that expands intratumoural lymphoneogenic ILC2s and TLSs and demonstrates enhanced anti-tumour activity in PDAC mice. In summary, we identify the molecules and cells of a druggable pathway that induces inflammation-triggered TLSs. More broadly, we reveal a lymphoneogenic function for alarmins and ILC2s. IL-33 induces tertiary lymphoid structures.

PURPOSE: Immunotherapy has led to a fundamental shift in the treatment of several cancers. However, its efficacy in pancreatic ductal adenocarcinoma (PDAC) is limited. Understanding the expression of inhibitory immune checkpoint receptors (ICR) by intratumoral T cells may help to unravel their involvement in insufficient T-cell-mediated antitumor immunity. EXPERIMENTAL DESIGN: Using multicolor flow cytometry, we analyzed circulating and intratumoral T cells from blood (n = 144) and matched tumor samples (n = 107) of patients with PDAC. We determined the expression of programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) by CD8+ T-cells, conventional CD4+ T-cells (Tconv) and regulatory T cells (Treg) and their association with T-cell differentiation, tumor reactivity, and cytokine expression. A comprehensive follow-up was used to determine their prognostic value. RESULTS: Intratumoral T cells were characterized by increased PD-1 and TIGIT expression. Both markers delineated distinct T-cell subpopulations. PD-1+TIGIT- T cells highly expressed proinflammatory cytokines and markers of tumor reactivity (CD39, CD103), whereas TIGIT expression was linked to antiinflammatory and exhausted phenotypes. In addition, the enhanced presence of intratumoral PD-1+TIGIT- Tconv was associated with improved clinical outcomes, while high ICR expression on blood T cells was a significant hazard for overall survival (OS). CONCLUSIONS: Our results uncover the association between ICR expression and T-cell functionality. PD-1 and TIGIT characterized intratumoral T cells with highly divergent phenotypes linked to clinical outcomes, further underscoring the relevance of TIGIT for immunotherapeutic approaches in PDAC. The prognostic value of ICR expression in patient blood may be a valuable tool for patient stratification.