The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

ABSTRACTInhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-γ induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-γ production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-γ in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

Validation of a Traditional Italian-Style Salami Manufacturing Process for Control of Salmonella and Listeria monocytogenes†

Italian-style salami batter (formulated with pork shoulder) was inoculated with ca. 7.0 log CFU/g of either Salmonella or Listeria monocytogenes. Salami links (55-mm cellulose casings) were fermented at 30°C for 24, 40, or 72 h and then dried to target moisture/protein ratios (MPRs) of 1.9:1 or 1.4:1. Links were sampled after fermentation (24, 40, and 72 h) and after combined fermentation-drying treatments (MPRs of 1.9:1 and 1.4:1 for all fermentation periods), and microbiological and proximate analyses were performed at each sampling. Pathogen populations were enumerated by direct plating on selective agar and by an injured-cell recovery method. When enumerated by the injured-cell recovery method, Salmonella populations were reduced by 1.2 to 2.1 log CFU/g after fermentation alone (24 to 72 h) and by 2.4 to 3.4 log CFU/g when fermentation was followed by drying. Drying to an MPR of 1.4:1 was no more effective than drying to an MPR of 1.9:1 (P > 0.05). When enumerated directly on selective media, Salmonella populations were reduced from 1.6 to 2.4 log CFU/g and from 3.6 to 4.5 log CFU/g for fermentation alone and fermentation followed by drying, respectively. L. monocytogenes populations were reduced by <1.0 log CFU/g following all fermentation and combined fermentation-drying treatments, regardless of the enumeration method. These results suggest that the Italian-style salami manufacturing process evaluated does not adequately reduce high pathogen loads. Processors may thus need to consider supplemental measures, such as raw material specifications and a final heating step, to enhance the lethality of the overall manufacturing process.

Cytolysis of Eosinophils in Nasal Secretions

It is still unknown how eosinophils degranulate in nasal mucus. Currently, cytolysis is being reevaluated as the mode of degranulation of eosinophils in allergic nasal mucosa. To examine whether eosinophils migrating to the nasal mucus degranulate by cytolysis, we sampled nasal mucus from 9 patients with nasal allergy and observed it under electron and light microscopes. Both intact and necrotic eosinophils were observed in the nasal mucus. Although the total eosinophil count in the nasal mucus was not correlated with the frequency of sneezes, there was a significant correlation (p = .0025) between the rate of eosinophil lysis and the frequency of sneezes. Whereas extracellular release of eosinophil peroxidase was not detected from the eosinophils with intact cell membranes, large quantities of eosinophil peroxidase were found outside the eosinophils with injured cell membranes. We concluded that eosinophils migrating to the nasal mucus degranulate mainly by cytolysis, and that granular proteins released from the necrotic eosinophils into the nasal mucus are one of the important factors causing hypersensitivity in the nasal mucosa.

Modification du pH vacuolaire des cellules épidermiques foliaires de Solanum dulcamara soumises à l'action d'un acarien cécidogène

Leaf epidermis cells of Solanum dulcamara cultured for several hours show, after cell wall perforation by Eriophyes cladophthirus, an increase of the vacuolar pH (≥8) both in susceptible and resistant plants. This reaction is more rapid on susceptible plants and the injured cell remains alive. In contrast, on resistant plants, the reaction starts later and the injured cell collapses and dies.

Changes in leaves of susceptible and resistant Solanum dulcamara infested by the gall mite Eriophyes cladophthirus (Acarina, Eriophyoidea)

Eriophyes cladophthirus perforates the wall of epidermis cells during feeding on young susceptible leaves and provokes the formation of cone-shaped feeding punctures. Callose is detected near the puncture after 20 min and the injured cells are transformed into nutritive cells. Nutritive cells are induced near the feeding sites on susceptible plants, whereas only necrotic cells appear near feeding sites on resistant plants.Mites feeding on resistant plants rapidly initiate a hypersensitive response detectable after 10 min on injured epidermal cells which then leads to severe necrosis of surrounding tissues after 1 h. No typical feeding punctures or callose deposits appear on injured cell walls. Polyphenolic compounds are detected in the necrotic region after 4 h.