Parasite Proximity Drives the Expansion of Regulatory T Cells in Peyer's Patches following Intestinal Helminth Infection

Helminth infections are typically chronic in nature; however, the exact molecular mechanisms by which these parasites promote or thwart host immunity remain unclear. Worm expulsion requires the differentiation of CD4(+) T cells into Th2 cells, while regulatory T cells (Tregs) act to dampen the extent of the Th2 response. Priming of T cells requires drainage or capture of antigens within lymphoid tissues, and in the case of intestinal helminths, such sites include the mucosa-associated Peyer's patches (PPs) and the draining mesenteric lymph nodes (MLN). To gain insight into when and where the activation of the adaptive T cell response takes place following intestinal helminth infection, we analyzed Th2 and Treg responses in the PPs and MLN following infection with the murine intestinal helminth Heligmosomoides polygyrus bakeri. Protective Th2 responses were observed to be largely restricted to the MLN, while a greater expansion of Tregs occurred within the PPs. Interestingly, those PPs that formed a contact with the parasite showed the greatest degree of Treg expansion and no evidence of type 2 cytokine production, indicating that the parasite may secrete products that act in a local manner to selectively promote Treg expansion. This view was supported by the finding that H. polygyrus bakeri larvae could promote Treg proliferation in vitro. Taken together, these data indicate that different degrees of Treg expansion and type 2 cytokine production occur within the PPs and MLN following infection with the intestinal helminth H. polygyrus bakeri and indicate that these organs exhibit differential responses following infection with intestinal helminths.

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

The pancreas plays a central role in metabolism. The exocrine pancreas, composed of ductal and acinar cells, secretes and delivers digestive enzymes into the duodenum where they contribute to food digestion. The endocrine pancreas, constituted by the islets of Langerhans, secretes hormones in the blood to control glucose levels. In particular, insulin is the hormone produced by β-cells that instructs the peripheral tissues to uptake circulating glucose. Diabetes mellitus is a heterogeneous disease characterized by deregulated glucose homeostasis due to an impaired insulin function. The advancements in clinical practice have made of diabetes a chronic, instead of a lethal, disease; yet no definitive cure is available. Beta-cell transplantation offers promising results, especially for type 1 diabetes where β-cells are selectively eliminated by an autoimmune attack. Unfortunately its application suffers from the scarcity of transplantable pancreas/islets from cadaveric donors. Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) would constitute unlimited sources of transplantable β-cells, but the available differentiation protocols are not yet optimal, especially with regards to the numbers and the functionality of the generated β-cells. It is anticipated that a stepwise protocol would succeed in generating mature β-cells in vitro if it is capable of fully mimicking the embryonic development of these cells. Many aspects of pancreatic organogenesis have been elucidated and could serve as a guide for driving the commitment to β-cells. Unfortunately, pancreatic progenitors in vitro do not behave as in the intricate context of an embryo. This severely limits both the understanding of pancreatic development at the single-cell level and our ability in manipulating the process. The aim of our work was to develop in vitro methodologies to sustain pancreatic progenitor culture and expansion and to establish conditions to manipulate their progeny. To this aim, we combined an informed approach based on developmental biology and an empirical one. We first observed that pancreatic epithelial explants from embryonic day 10.5 mice could be cultured in presence of Matrigel™ and exogenous FGFs even in the absence of the mesenchyme that normally surrounds the epithelium. Notably, the removal of mesenchyme did not affect the endocrine commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the expansion of dissociated embryonic pancreatic progenitors in a three-dimensional Matrigel™-based environment. Under these conditions, progenitors proliferated and self-organized to generate pancreatic organoids containing both progenitors and differentiated cells, mostly exocrine, after 7 days of culture. When grafted into recipient pancreatic explants, the cultured progenitors contributed to the three epithelial pancreatic lineages (ductal, endocrine, acinar), integrating seamlessly into the endogenous cellular network. Thus, cultured progenitors retained their potential to differentiate into endocrine cells, but expressed it only in the appropriate niche. Subsequent removal of single components from the culture system led to the identification of some essential requirements for the maintenance/expansion of dissociated pancreatic progenitors, such as a strong FGF signaling and the Rho-associated kinase (ROCK) inhibitor Y-27632. In addition to this, we observed that only small clusters of pancreatic progenitors, but not single cells, were able to generate pancreatic organoids, suggesting a pivotal role for intercellular signaling in progenitor maintenance and expansion. By manipulating the components of the medium, we could affect fate commitment. In particular, the removal of FGF1 increased the yield of endocrine cells generated in vitro. In our search for a full control over the in vitro niche, we explored chemically defined matrices to replace Matrigel™. We showed that pancreatic progenitors could be cultured on laminin-functionalized hydrogels, although less efficiently than in Matrigel™. Moreover, differentiation into exocrine and endocrine cells occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and two-dimensional culture systems, we uncovered the importance of a tridimensional architecture for pancreatic progenitor maintenance. We showed that pancreatic progenitors lost their identity as they flattened onto 2D surfaces, whereas 3D culture systems maintained the epithelial character of pancreatic progenitors and allowed the acquisition of apical polarization. To conclude, our work provides the first detailed characterization of in vitro culture systems for expansion and manipulation of dissociated embryonic pancreatic progenitors. Further implementation will hopefully allow the establishment of a fully-defined in vitro niche for developing pancreas with potential fundamental implications for the expansion of ES-derived pancreatic progenitors and their differentiation into functional β-cells.

EPFL2011

Redundant Notch1 and Notch2 Signaling Is Necessary for IFNγ Secretion by T Helper 1 Cells During Infection with Leishmania major

Floriane Auderset, Ute Koch, Freddy Radtke

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

2012

Improving Immunogenicity of Subunit Vaccines Using Nanocarrier Systems

Armando Stano

New generation vaccines, which are subunit vaccines, have considerably improved safety profiles, but are often poorly immunogenic, especially when administered through mucosal routes. Also, the diversity of the mechanisms of action of infectious pathogens requires the tuning of vaccine formulations towards humoral or cell mediated immunity, systemic or mucosal immunity, Th1 or Th2 immune responses. To modulate the immune response of subunit vaccines, the new field of immunobioengineering is developing a broad spectrum of nanocarrier systems. In this work, we describe the potential of two different fully synthetic nanocarrier systems to increase and modulate the immune responses of subunit vaccines. We show that upon intranasal administration, 50 nm solid-core NPs penetrated the nasal mucosae, transited via M cells, and were taken up by antigen- presenting cells in the nasopharynx-associated lymphoid tissue (NALT). Ovalbumin-conjugated NPs induced antigen-specific cytotoxic T lymphocytic responses in lung and spleen tissues, as well as humoral responses in mucosal airways. Co-conjugation of the TLR5 ligand flagellin to the NPs further enhanced the humoral responses in the airway as well as in the distant vaginal and rectal mucosal compartments, and induced cellular immune responses with a Th1 bias, in contrast to free flagellin. Furthermore, by increasing the size of the NPs from 30 to 200 nm, the conjugated protein antigen was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with larger ovalbumin-conjugated NPs increased the magnitude of CD4 T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4 T cells as compared to 30 nm NPs, resulting in a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4 T cells for protection, such Mycobacterium tuberculosis. In the last part of this thesis, the role of the nanocarrier polymersomes in the induction of adaptive immunity is characterized. Mice subcutaneously immunized with antigen-loaded into polymersomes and CpG displayed enhanced induction of antigen-specific CD4 T cells in spleen, lymph nodes and lungs, as compared to mice immunized not only with free antigen and CpG but also to the solid-core NP formulations. In contrast, unlike NPs, polymersomes did not enhance clonal expansion of antigen-specific CD8 T cells, nor cytokine production by effector CD8 T cells. Interestingly, a formulation with both polymersomes and NPs elicited T cell immunity characteristic of the two nanocarriers at the same time. In conclusion, this thesis proposes solid-core NPs and polymersomes as effective nanocarrier systems for the fine-tuning of the immune response towards a protein antigen. These results have significant implications for the next generation of rationally designed subunit vaccines.