Probes for CD4

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

The gastrointestinal microbiota begins to be acquired at birth and continually matures through early adolescence. Despite the relevance for gut health, few studies have evaluated the impact of pathobiont colonization of neonates on the severity of colitis later in life. LF82 is an adherent invasive E. coli strain associated with ileal Crohn's disease. The aim of this study was to evaluate the severity of dextran sodium sulfate (DSS)-induced colitis in mice following E. coli LF82 colonization. Gnotobiotic mice harboring the altered Schaedler flora (ASF) were used as the model. While E. coli LF82 is neither adherent nor invasive, it was been demonstrated that adult ASF mice colonized with E. coli LF82 develop more severe DSS-induced colitis compared to control ASF mice treated with DSS. Therefore, we hypothesized that E. coli LF82 colonization of neonatal ASF mice would reduce the severity of DSS-induced inflammation compared to adult ASF mice colonized with E. coli LF82. To test this hypothesis, adult ASF mice were colonized with E. coli LF82 and bred to produce offspring (LF82N) that were vertically colonized with LF82. LF82N and adult-colonized (LF82A) mice were given 2.0% DSS in drinking water for seven days to trigger colitis. More severe inflammatory lesions were observed in the LF82N + DSS mice when compared to LF82A + DSS mice, and were characterized as transmural in most of the LF82N + DSS mice. Colitis was accompanied by secretion of proinflammatory cytokines (IFNγ, IL-17) and specific mRNA transcripts within the colonic mucosa. Using 16S rRNA gene amplicon sequencing, LF82 colonization did not induce significant changes in the ASF community; however, minimal changes in spatial redistribution by fluorescent in situ hybridization were observed. These results suggest that the age at which mice were colonized with E. coli LF82 pathobiont differentially impacted severity of subsequent colitic events.