Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (T _reg cells) in αPD-L1 therapy–resistant solid tumor–bearing mice. T _reg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating T _reg cells in human patients with skin cancer, and in patients with non–small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1–induced PD-1 ⁺ T _reg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of T _reg cells, resulting in therapy resistance, suggesting that T _reg cell targeting is an important adjunct strategy to enhance therapeutic efficacy.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere–encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Background: Patients with locally advanced pancreatic cancer (LAPC) are treated with chemotherapy. In selected cases, stereotactic body radiotherapy (SBRT) can be added to the regimen. We hypothesized that adding an adjuvant containing a heat-killed mycobacterium (IMM-101) to SBRT may lead to beneficial immuno-modulatory effects, thereby improving survival. This study aims to investigate the safety of adding IMM-101 to SBRT and to investigate the immuno-modulatory effects of the combination treatment in the peripheral blood of LAPC patients. Methods: LAPC patients were treated with SBRT (40 Gy) and six intradermal vaccinations of one milligram IMM-101. The primary endpoint was an observed toxicity rate of grade 4 or higher. Targeted gene-expression profiling and multicolor flow cytometry were performed for longitudinal immune-monitoring of the peripheral blood. Results: Twenty patients received study treatment. No treatment-related adverse events of grade 4 or higher occurred. SBRT/IMM-101 treatment induced a transient decrease in different lymphocyte subsets and an increase in CD14+CD16−CD11b+HLA−DR^low myeloid-derived suppressor cells. Importantly, treatment significantly increased activated ICOS+, HLA-DR+ and Ki67+PD1+ T and NK cell frequencies. This was not accompanied by increased levels of most inhibitory markers, such as TIM-3 and LAG-3. Conclusions: Combination therapy with SBRT and a heat-killed mycobacterium vaccine was safe and had an immune-stimulatory effect.

Systemic anti-tumour immunity results from T cell priming in tumour-draining lymph nodes (TDLNs) ^1–4 . Although the suppression of T cells in tumours is well characterized ^5–8 , whether this occurs in TDLNs—and if so, through which mechanisms—remains poorly understood. Here, using imaging mass cytometry of TDLNs from patients with melanoma, we identify a spatial neighbourhood in the TDLN paracortex that is linked to the development of distant metastases. Targeted spatial transcriptomics of cells inside this neighbourhood revealed activated CD8 ⁺ T cells engaging with myeloid cells that expressed high levels of the immunosuppressive secretory phospholipase PLA2G2D. PLA2G2D ⁺ myeloid cells were substantially more abundant in TDLNs than they were in primary tumours or metastases. Genetic loss-of-function or antibody-mediated inhibition of PLA2G2D reduced tumour growth markedly, and single-cell transcriptomics in melanoma-bearing mice revealed that expression of Pla2g2d is confined to lymph-node macrophages. Mechanistically, PLA2G2D directly suppressed the early proliferation of T cells in vitro, and inhibiting PLA2G2D resulted in an expansion of tumour-specific T cells in TDLNs, leading to an increase in these T cells in the circulation and subsequently in tumours. Notably, PLA2G2D and PD-1 act as non-redundant immune checkpoints, with combination treatment showing additive or synergistic efficacy in humanized mice treated with human-specific antibodies. Collectively, our in-depth spatial profiling identifies PLA2G2D as a TDLN-centred targetable immune checkpoint for cancer immunotherapy.

Cytotoxic T lymphocytes screen cells for signs of infection and transformation by recognizing peptides displayed on MHC class I molecules. Next to canonical ATG-initiated open reading frames (ORF), noncanonical translation can result in synthesis of nonconventional or “cryptic” polypeptides. These can originate from translation initiation at noncanonical start codons, a process previously associated with inflammation and oncogenic transformation. Cryptic translation products are efficiently presented on MHC class I molecules and therefore increasingly recognized as potential targets for cancer immunotherapy. In this study, we studied the impact of localization of a CTG-initiated ORF relative to a canonical ATG start codon on cryptic expression after innate immune stimulation. We generated immortalized C57BL/6J mouse–derived bone marrow progenitor cells (HoxB8) expressing tandem minigene constructs, which encoded a CTG-driven chicken ovalbumin–derived SIINFEKL (S8L) epitope (CTG-S8L; H-2Kb restriced) either up- or downstream of a canonical ATG-initiated UTY-derived peptide WI9. The treatment of HoxB8-derived macrophages with Toll-like receptor agonists enhanced position-independent CTG-S8L translation, without affecting ATG-driven expression. Downstream CTG-S8L translation was driven by leaky scanning or ribosome re-initiation rather than read-through translation. Mouse AE17 mesothelioma and B16F10 melanoma cells expressing cryptic S8L either up- or downstream of a canonical ORF were efficiently killed by H-2Kb/S8L-restriced OT-I T cells in vitro, even though their antigen expression levels were extremely low. Mice implanted with tumors expressing cryptic S8L showed delayed tumor progression in vivo. In summary, our study contributes to the characterization of noncanonical start codon–driven cryptic antigen translation and highlights its potential for cancer immunotherapy.

Supplementary Table 2 from CTG-Initiated Cryptic Peptide Translation Up- and Downstream of a Canonical ATG Start Codon Is Enhanced by TLR Stimulation and Induces Tumor Regression in Mice