Treatment of T1D via optimized expansion of antigen-specific Tregs induced by IL-2/anti-IL-2 monoclonal antibody complexes and peptide/MHC tetramers.

Type 1 diabetes can be overcome by regulatory T cells (Treg) in NOD mice yet an efficient method to generate and maintain antigen-specific Treg is difficult to come by. Here, we devised a combination therapy of peptide/MHC tetramers and IL-2/anti-IL-2 monoclonal antibody complexes to generate antigen-specific Treg and maintain them over extended time periods. We first optimized treatment protocols conceived to obtain an improved islet-specific Treg/effector T cell ratio that led to the in vivo expansion and activation of these Treg as well as to an improved suppressor function. Optimized protocols were applied to treatment for testing diabetes prevention in NOD mice as well as in an accelerated T cell transfer model of T1D. The combined treatment led to robust protection against diabetes, and in the NOD model, to a close to complete prevention of insulitis. Treatment was accompanied with increased secretion of IL-10, detectable in total splenocytes and in Foxp3− CD4 T cells. Our data suggest that a dual protection mechanism takes place by the collaboration of Foxp3+ and Foxp3− regulatory cells. We conclude that antigen-specific Treg are an important target to improve current clinical interventions against this disease

Transcriptional re-programming of insulin B-chain epitope-specific T-follicular helper cells into anti-diabetogenic T-regulatory type-1 cells

Systemic delivery of nanoparticles (NPs) coated with mono-specific autoimmune disease-relevant peptide-major histocompatibility complex class II (pMHCII) molecules can resolve organ inflammation in various disease models in a disease-specific manner without impairing normal immunity. These compounds invariably trigger the formation and systemic expansion of cognate pMHCII-specific T-regulatory type 1 (TR1) cells. By focusing on type 1 diabetes (T1D)-relevant pMHCII-NP types that display an epitope from the insulin B-chain bound to the same MHCII molecule (IAg7) on three different registers, we show that pMHCII-NP-induced TR1 cells invariably co-exist with cognate T-Follicular Helper (TFH)-like cells of quasi-identical clonotypic composition and are oligoclonal, yet transcriptionally homogeneous. Furthermore, these three different TR1 specificities have similar diabetes reversal properties in vivo despite being uniquely reactive against the peptide MHCII-binding register displayed on the NPs. Thus, pMHCII-NP treatment using nanomedicines displaying different epitope specificities results in the simultaneous differentiation of multiple antigen-specific TFH-like cell clones into TR1-like cells that inherit the fine antigenic specificity of their precursors while acquiring a defined transcriptional immunoregulatory program

Estudio de la respuesta autoreactiva de linfocitos B anti-periferina en la diabetes tipo I

[spa] La diabetes tipo 1 (DT1) es una enfermedad de origen multifactorial, caracterizada por la destrucción selectiva de células ß productoras de insulina localizadas en los islotes pancreáticos. Estudios realizados en el modelo de ratón NOD, han revelado que la respuesta autoinmune en esta enfermedad está dirigida principalmente por poblaciones autoreactivas de linfocitos T CD4 y CD8 infiltrantes de islote. Durante muchos años, el papel de los linfocitos B en la DT1 ha sido relegado a un segundo plano, sin embargo, a día de hoy se sabe que estas células son importantes en el desarrollo del proceso, y que posiblemente su papel en la presentación de autoantígenos sea clave en las primeras etapas de la respuesta autoinmune. Recientemente, se ha descrito la presencia de linfocitos B autoreactivos infiltrantes de islote frente a una proteína con patrón de expresión neuroendocrino, identificada como periferina. Estos datos, junto con trabajos recientes donde se describe la presencia de un ataque inicial en páncreas frente a elementos del sistema nervioso, ponen de manifiesto la importancia del análisis de esta respuesta. El estudio presentado en esta memoria describe y caracteriza por primera vez, la región antigénica presente en la periferina reconocida por linfocitos B autoreactivos en ratones NOD. La respuesta de estos linfocitos B, esta dirigida frente a la región L465-S495, un epítopo lineal de 17 aminoácidos de longitud localizado en la región C terminal de las isoformas 61 y 58. Por otra parte, mediante el desarrollo de una molécula de detección de linfocitos B específica, hemos podido llevar a cabo un estudio del desarrollo y fenotipo de esta población de linfocitos en ratones NOD. Los resultados obtenidos, muestran la presencia de una respuesta temprana de linfocitos B anti-periferina detectable a partir de las 4 semanas, con un perfil heterogéneo de linfocitos B1 y B2. De forma importante, hemos comprobado como el peritoneo es una región clave en la dinámica de esta población autoreactiva. En este compartimento, la frecuencia de linfocitos B anti-periferina es superior a la detectada en otros órganos, y una parte de la población presenta signos de cambio de isotipo. Por otra parte, experimentos llevados a cabo en ratones diabéticos NOD, han revelado un aumento en el número y frecuencia de estas células en esta región, apuntando a que el compartimento peritoneal podría ser clave en el desarrollo y mantenimiento de la respuesta autoreactiva anti-periferina durante el transcurso de la DT1.[eng] Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. In this thesis, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target antigen of islet-infiltrating B cells. PRPH autoreactive B cells only recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cell during disease progression. Before extended insulitis established, anti-PRPH B cells preferentially accumulated in the peritoneum. As disease unfolded, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not pre-diabetic mice. Isotype switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes establishes. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis

Treatment of T1D via optimized expansion of antigen-specific Tregs induced by IL-2/anti-IL-2 monoclonal antibody complexes and peptide/MHC tetramers.

Type 1 diabetes can be overcome by regulatory T cells (Treg) in NOD mice yet an efficient method to generate and maintain antigen-specific Treg is difficult to come by. Here, we devised a combination therapy of peptide/MHC tetramers and IL-2/anti-IL-2 monoclonal antibody complexes to generate antigen-specific Treg and maintain them over extended time periods. We first optimized treatment protocols conceived to obtain an improved islet-specific Treg/effector T cell ratio that led to the in vivo expansion and activation of these Treg as well as to an improved suppressor function. Optimized protocols were applied to treatment for testing diabetes prevention in NOD mice as well as in an accelerated T cell transfer model of T1D. The combined treatment led to robust protection against diabetes, and in the NOD model, to a close to complete prevention of insulitis. Treatment was accompanied with increased secretion of IL-10, detectable in total splenocytes and in Foxp3− CD4 T cells. Our data suggest that a dual protection mechanism takes place by the collaboration of Foxp3+ and Foxp3− regulatory cells. We conclude that antigen-specific Treg are an important target to improve current clinical interventions against this disease

In Vivo Detection of Peripherin-Specific Autoreactive B Cells during Type 1 Diabetes Pathogenesis.

Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. In this study, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target Ag of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cells during disease progression. Before extended insulitis was established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not prediabetic mice. Isotype-switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes is established. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis. J Immunol 2014 Apr 1; 192(7):3080-90

A Monoclonal Antibody Against the Extracellular Domain of Mouse and Human Epithelial V-like Antigen 1 Reveals a Restricted Expression Pattern Among CD4- CD8- Thymocytes

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44lowCD25+) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging.Fil: Garabatos, Nahir. Institut d'Investigacions Biomèdiques August Pi i Sunyer; EspañaFil: Blanco, Jesus. Institut d'Investigacions Biomèdiques August Pi i Sunyer; EspañaFil: Fandos, Cesar. Institut d'Investigacions Biomèdiques August Pi i Sunyer; EspañaFil: Lopez, Elena. Institut d'Investigacions Biomèdiques August Pi i Sunyer; EspañaFil: Santamaria, Pere. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. University of Calgary; CanadáFil: Ruiz, Andrea. Institut d'Investigacions Biomèdiques August Pi i Sunyer; EspañaFil: Perez Vidakovics, Maria Laura Anabella. Institut d'Investigacions Biomèdiques August Pi i Sunyer; España. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benveniste, Patricia. University of Toronto; CanadáFil: Galkin, Oleksandr. University of Toronto; CanadáFil: Zuñiga Pflucker, Juan Carlos. University of Toronto; CanadáFil: Serra, Pau. Institut d'Investigacions Biomèdiques August Pi i Sunyer; Españ