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Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. Celiac disease is associated with either HLA-DQ2 or HLA-DQ8 restricted responses to specific antigenic epitopes of gliadin, and may be treated by induction of antigen-specific tolerance. We investigated whether oral administration of LL-delivered DQ8-specific gliadin epitope induces antigen-specific tolerance. L. lactis was engineered to secrete a deamidated DQ8 gliadin epitope (LL-eDQ8d) and the induction of antigen-specific tolerance was studied in NOD AB° DQ8 transgenic mice. Tolerance was assessed by delayed-type hypersensitivity reaction, cytokine measurements, eDQ8d-specific proliferation and regulatory T cell analysis. Oral administration of LL-eDQ8d induced suppression of local and systemic DQ8 restricted T-cell responses in NOD AB° DQ8 transgenic mice. Treatment resulted in an antigen-specific decrease of the proliferative capacity of inguinal lymph node cells and lamina propria cells. Production of IL-10 and TGF-β and a significant induction of Foxp3+ regulatory T-cells were associated with the eDQ8d-specific suppression induced by LL-eDQ8d. These data provide support for the development of effective therapeutic approaches for gluten-sensitive disorders using orally administered antigen-secreting LL. Such treatments may be effective even in the setting of established hypersensitivity.

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-β1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-β1 and TGF-β2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-β may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.

GATA-4, -5, and -6 zinc finger and hepatocyte nuclear factor-1␣ (HNF-1␣) homeodomain transcription factors are expressed in the intestinal epithelium and synergistically activate the promoter of intestinal genes. Here, we demonstrate that GATA-5 and HNF-1␣ physically associate both in vivo and in vitro and that this interaction is necessary for cooperative activation of the lactasephlorizin hydrolase promoter. Furthermore, physical association is mediated by the C-terminal zinc finger of GATA factors and the homeodomain of HNF-1␣. Deletion of HNF-1␣ activation domains or interruption of HNF-1-binding sites in the lactase-phlorizin hydrolase promoter resulted in a complete loss of cooperativity, whereas deletion of GATA-5 activation domains or interruption of GATA-binding sites resulted in a reduction, but not an elimination, of cooperativity. We hypothesize that GATA/HNF-1␣ cooperativity is mediated by HNF-1␣ through its activation domains, which are oriented for high levels of activation through binding to DNA and physical association with GATA factors. These data suggest a paradigm whereby intestine-specific gene expression is regulated by unique interactions among tissuerestricted transcription factors coexpressed in the intestine. Parallel mechanisms in other tissues as well as in Drosophila suggest that zinc finger/homeodomain interactions are an efficient pathway of cooperative activation of gene transcription that has been conserved throughout evolution.The intestinal epithelium is a dynamic structure that undergoes a highly regulated process of cell division, migration, cell fate determination, and differentiation (1-3). During intestinal development, interactions between visceral endoderm and mesoderm at E8 1 in mice result in the formation of a primitive foregut that rapidly undergoes cytodifferentiation, so that by E19, an epithelial monolayer overlies nascent villi. During the first 2 weeks of postnatal life, a proliferating compartment develops into the crypts of Lieberkü hn. Stem cells located near the base of crypts rapidly divide and give rise to four terminally differentiated cell types, which migrate both basally and apically. Cells migrating to the base of crypts become Paneth's cells, whereas those migrating up the crypt toward villi become absorptive enterocytes, goblet cells, and enteroendocrine cells. At the crypt-villus junction, the proliferative phase ends, and cells acquire a differentiated phenotype characterized by the synthesis of functionally relevant proteins. The cells continue to migrate up the villi, enter an apoptotic cycle, and are shed into the intestinal lumen ϳ3 days after their initial appearance on villi. The molecular mechanisms underlying the dynamic processes of intestine-specific gene expression and cellular differentiation during development are poorly understood.Absorptive enterocytes compose ϳ95% of epithelial cells on villi and are the cells responsible for the terminal digestion and absorption of nutrients. Lactase-phlorizin hydrolase (LPH), the enzyme critical ...

3511 Background: The combination of PD-1 and CTLA4 blockade has changed the treatment landscape for several cancer types. Although this treatment is highly effective in metastatic mismatch-repair deficient (dMMR) colorectal cancers, metastatic MMR-proficient (pMMR) tumors do not respond. The NICHE study was the first neoadjuvant immunotherapy study in colon cancer (CC) to show impressive responses in 100% of dMMR ( n= 20) and 27% of pMMR ( n= 15) CC. In contrast, pathologic response to neoadjuvant chemotherapy using standard of care folfox is approximately 5% in dMMR tumors. Here we present the final efficacy data for the original NICHE study cohorts. Methods: Patients with non-metastatic, resectable dMMR or pMMR CC were treated with a single dose of ipilimumab 1mg/kg and two doses of nivolumab 3mg/kg and underwent surgery within 6 weeks. In addition, patients with pMMR tumors were randomized to receive celecoxib. The primary endpoints were safety and feasibility, and secondary endpoints included pathologic response rate and disease-free survival in 30 patients with dMMR and 30 with pMMR tumors. Pathologic response was defined as 50% or less viable tumor rest (VTR), and major pathologic response (MPR) as <10% VTR. Results: Thirty patients with pMMR and 32 with dMMR tumors were evaluable for the efficacy analyses. In the pMMR cohort, pathologic response was observed in 9/30 (30%, 95% CI 14-46%) patients, consisting of 7 MPR (including 3 pathologic complete responses {pCR}) and 2 partial responses. Four out of 9 pMMR responders had received celecoxib. Five patients received adjuvant chemotherapy. At a median follow-up of 25 months (IQR 12-35 months), 3 patients (all non-responders) in the pMMR group had disease recurrence. In the 32 patients with dMMR tumors, pathologic response was observed in 100% of patients, with 31/32 MPR (97%, 95% CI 91-100%) and one partial response. Pathologic complete response was observed in 22/32 (69%, 95% CI 53-85%) patients. None of the patients in the dMMR cohort had disease recurrence. Surgery was delayed in one patient with a pMMR tumor due to myositis. Grade 3 immune-related adverse events were observed in 12% of patients, consistent with our previous report on the primary endpoint. There were no grade 4 immune-related AEs nor unexpected surgical complications. Conclusions: These data confirm our previously published results of the NICHE study, with responses to neoadjuvant nivolumab plus ipilimumab in 30% of pMMR and 100% of dMMR CC in the completed original cohorts. Validation of the dMMR responses in a large group of dMMR patients is ongoing and has the potential to change current practice. Clinical trial information: NCT03026140.

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BackgroundRoux-en-Y gastric bypass (RYGB) causes several alterations in gastrointestinal function. We hypothesized that levels of three commonly used fecal tests change after RYGB.MethodsFecal levels of calprotectin, elastase, and alpha-1-antitrypsin were determined in 122 patients without signs of gastrointestinal disease 1 to 2 years after RYGB. Medians, distribution of values, and the percentage of patients with levels above or below reference values were determined.ResultsMedian fecal calprotectin level was 163.5 (<30–1587) μg/g; in 87 % of patients, it was above the reference value (<50 μg/g). Median fecal elastase level was 444 (<15–647) μg/g; 13 % was below the reference value (>200 μg/g). Median fecal alpha-1-antitrypsin level was 0.51 (<0.20–2.20) mg/g, comparable to the reference values.ConclusionsFecal calprotectin levels are significantly higher than the reference value in most patients after RYGB. Fecal elastase is significantly lower. This might indicate that the validity of fecal calprotectin testing is impaired after RYGB and the specificity for fecal elastase is decreased. Clinical awareness of altered fecal markers after RYGB is essential to prevent unnecessary diagnostic tests, such as colonoscopy. Fecal alpha-1-antitrypsin is not influenced by RYGB.