title: Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells.
abstract: The introduction of β-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-β partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Takiishi
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Dana Paulina
authors.initials: DP
authors.lastname: Cook
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Hannelie
authors.initials: H
authors.lastname: Korf
authors.affiliation: Diabetes Unit, Department of Internal Medicine, Endocrine and Metabolic Sciences and Biochemistry, University of Siena and Fondazione Umberto Di Mario ONLUS, Toscana Life Science Park, Siena, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Sebastiani
authors.affiliation: Diabetes Unit, Department of Internal Medicine, Endocrine and Metabolic Sciences and Biochemistry, University of Siena and Fondazione Umberto Di Mario ONLUS, Toscana Life Science Park, Siena, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Mancarella
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: João Paulo Monteiro Carvalho Mori
authors.initials: JP
authors.lastname: Cunha
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL.
authors.forename: Clive
authors.initials: C
authors.lastname: Wasserfall
authors.affiliation: Immunology and Immunotherapy Program, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Casares
authors.affiliation: Immunology and Immunotherapy Program, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors.forename: Juan José
authors.initials: JJ
authors.lastname: Lasarte
authors.affiliation: Intrexon ActoBiotics NV, Zwijnaarde, Ghent, Belgium.
authors.forename: Lothar
authors.initials: L
authors.lastname: Steidler
authors.affiliation: Intrexon ActoBiotics NV, Zwijnaarde, Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Rottiers
authors.affiliation: Diabetes Unit, Department of Internal Medicine, Endocrine and Metabolic Sciences and Biochemistry, University of Siena and Fondazione Umberto Di Mario ONLUS, Toscana Life Science Park, Siena, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Dotta
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium conny.gysemans@kuleuven.be.
authors.forename: Conny
authors.initials: C
authors.lastname: Gysemans
authors.affiliation: Laboratory of Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Chantal
authors.initials: C
authors.lastname: Mathieu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001786:Blood Glucose / Q000187:drug effects* / Q000378:metabolism; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D060908:CTLA-4 Antigen / Q000187:drug effects / Q000276:immunology; D003920:Diabetes Mellitus / Q000378:metabolism*; D004195:Disease Models, Animal; D051858:Forkhead Transcription Factors / Q000276:immunology; D005951:Glucose Tolerance Test; D007108:Immune Tolerance / Q000187:drug effects* / Q000276:immunology; D016753:Interleukin-10 / Q000378:metabolism*; D007778:Lactobacillus / Q000378:metabolism*; D008213:Lymphocyte Activation; D051379:Mice; D016688:Mice, Inbred NOD; D010179:Pancreas / Q000187:drug effects / Q000473:pathology; D011384:Proinsulin / Q000378:metabolism*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000276:immunology
chemical_list: D000906:Antibodies; D057134:Antibodies, Neutralizing; D001786:Blood Glucose; D017252:CD3 Complex; D060908:CTLA-4 Antigen; C556707:Ctla4 protein, mouse; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D016212:Transforming Growth Factor beta; D016753:Interleukin-10; D011384:Proinsulin