Adapting the sequential infection mouse model to improve the translation of preclinical cancer research and immunotherapy 2257903

Abstract Introduction Specific Pathogen Free (SPF) mice have been critical in understanding cancer-immune interactions and immunotherapies. However, they often fail to recapitulate the complex immune environment of human tumors. A main reason is their immature immune system, which lacks microbiota adaptations, infections, and vaccinations. The use of “dirty” mice through co-housing has started to address this issue, but practical issues preclude their widespread use. To overcome this, we utilized a sequential infection (SI) model to establish a more complex immunity in SPF mice for cancer research. Methods Starting from 4 weeks old, SPF mice were sequentially exposed to MHV68, MCMV, influenza A (H1N1), and Citrobacter rodentium at 2-week intervals. SI mice were subjected to experiments at 18-22 weeks old. Results SI mice exhibited a more experienced immune system enriched in effector memory and tissue-resident memory T cells, changes in bone marrow progenitors, and elevated circulating antibody, compared to mock-infected (mi)SPF mice. When compared to miSPF mice, SI mice treated with an anti-CD28 super-agonist reacted similarly to adult humans, including limited Treg expansion and elevated serum inflammatory cytokines. In tumor models, SI mice exhibited accelerated growth of orthotopic breast cancer and melanoma models. Further, SI mice showed decreased responsiveness to anti-PD1 and anti-CTLA4 blockade, better recapitulating the response rates observed in humans. Compared to miSPF mice, tumors in SI mice were infiltrated with less tumor-specific CD8 T cells and increased memory “bystander” CD8 T cells, with additional alterations in the myeloid cells. Conclusion These observations suggest that prior pathogen encounter significantly influences the tumor and response to immunotherapy, which better resembles adult humans. We propose that SI mice can enhance the translatability of preclinical cancer studies and reveal novel cancer-mediated immune regulation. Funding Source NIH R21, 2025-2027 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)