The aryl hydrocarbon receptor (AhR) ligand VAF347 selectively acts on monocytes and naïve CD4(+) Th cells to promote the development of IL-22-secreting Th cells.

The low molecular weight compound VAF347, and its pro-drug version VAG539, interact with the transcription factor aryl hydrocarbon receptor (AhR) on monocytes to mediate its anti-inflammatory activity in vitro and in vivo. AhR is a crucial factor for IL-22 production, which regulates skin and gut homeostasis. Here we investigated whether VAF347 might control the differentiation of naïve T cells into IL-22-secreting cells and/or regulate IL-22 production by memory T cells. Human monocytes exposed to VAF347 differentiated into dendritic cells capable of instructing a naïve CD4(+) T cell differentiation program that promoted IL-22 secretion and concomitantly inhibited IL-17 production. Whilst AhR ligation by VAF347 on naïve CD4(+) T cells favored the development of single IL-22-secreting cells (Th22), it suppressed the generation of T cells secreting either IL-22 and IFN-γ or IL-17 and IFN-γ. In contrast, memory T cells were refractory to AhR regulation since VAF347, AhR antagonist or AhR gene silencing did not modulate the production of any of these cytokines. Interfering with AhR functions using VAF347 may provide an efficient way to intervene with autoimmune disease since it would enhance the host protective function mediated by IL-22 while preventing the development of Th cells secreting pro-inflammatory cytokines.

Flt3 ligand-receptor interaction is important for maintenance of early thymic progenitor numbers in steady-state thymopoiesis.

T-cell production throughout life depends on efficient colonization and intrathymic expansion of BM-derived hematopoietic precursors. After irradiation-induced thymic damage, thymic recovery is facilitated by Flt3 ligand (FL), expressed by perivascular fibroblasts surrounding the thymic entry site of Flt3 receptor-positive progenitor cells. Whether intrathymic FL-Flt3 interactions play a role in steady-state replenishment of T cells remains unknown. Here, using competitive BM transplantation studies and fetal thymic organ cultures we demonstrated the continued numerical advantage of Flt3+ intrathymic T-cell precursors. Sub-kidney capsule thymic transplantation experiments, in which WT and FL-/- thymic lobes were grafted into FL-/- recipients, revealed that FL expression by the thymic microenvironment plays a role in steady-state thymopoiesis. The deficiency of the most immature thymic T-cell precursors correlated to upregulation of FL by thymic MTS15+ fibroblasts, suggesting that the number of Flt3+ progenitor cells may regulate the thymic expression of this cytokine. Together, these results show that FL expression by thymic stromal fibroblasts interacting with Flt3+ T-cell progenitors is important for the physiological maintenance of early T-cell development.

Intrathymic expression of Flt3 ligand enhances thymic recovery after irradiation.

Hematopoietic stem cell transplantation (HSCT) requires conditioning treatments such as irradiation, which leads to a severely delayed recovery of T cell immunity and constitutes a major complication of this therapy. Currently, our understanding of the mechanisms regulating thymic recovery is limited. It is known that a subpopulation of bone marrow (BM)-derived thymic immigrant cells and the earliest intrathymic progenitors express the FMS-like tyrosine kinase 3 (Flt3) receptor; however, the functional significance of this expression in the thymus is not known. We used the BM transplant model to investigate the importance of Flt3 ligand (FL) for the regeneration of the T cell compartment. We show that FL is expressed in the adult mouse thymus on the surface of perivascular fibroblasts. These cells surround the proposed thymic entry site of Flt3 receptor-positive T cell progenitors. After irradiation, perivascular FL expression is up-regulated and results in an enhanced recovery of thymic cellularity. Thymic grafting experiments confirm an intrathymic requirement for FL. Collectively, these results show that thymic stromal cell-mediated FL-Flt3 receptor interactions are important in the reconstitution of thymopoiesis early after lethal irradiation and HSCT, and provide a functional relevance to the expression of the Flt3 receptor on intrathymic T cell progenitors.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor-negative cells that facilitate bone marrow repopulation with hematopoietic stem cells.

Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8(+) cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8(+) cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8alpha(+)CD3epsilon(+) T-cell receptor- negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34(+) cells. In vitro, cFCs cocultured with CD34(+) cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8(+)CD3(+)TCR(-) cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

Three-dimensional perfusion culture of human bone marrow cells and generation of osteoinductive grafts.

Three-dimensional (3D) culture systems are critical to investigate cell physiology and to engineer tissue grafts. In this study, we describe a simple yet innovative bioreactor-based approach to seed, expand, and differentiate bone marrow stromal cells (BMSCs) directly in a 3D environment, bypassing the conventional process of monolayer (two-dimensional [2D]) expansion. The system, based on the perfusion of bone marrow-nucleated cells through porous 3D scaffolds, supported the formation of stromal-like tissues, where BMSCs could be cocultured with hematopoietic progenitor cells in proportions dependent on the specific medium supplements. The resulting engineered constructs, when implanted ectopically in nude mice, generated bone tissue more reproducibly, uniformly, and extensively than scaffolds loaded with 2D-expanded BMSCs. The developed system may thus be used as a 3D in vitro model of bone marrow to study interactions between BMSCs and hematopoietic cells as well as to streamline manufacture of osteoinductive grafts in the context of regenerative medicine.

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity.

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.

Characterization of a mouse model of allergy to a major occupational latex glove allergen Hev b 5.

Allergen-specific immunotherapy is a clinically proven effective treatment for many allergic diseases, including asthma; however, it is not currently available for latex allergy because of the high risk of anaphylaxis. There is, therefore, a crucial need for an animal model of latex allergy in which to develop effective immunotherapy. Previous mouse models of latex allergy either did not characterize the allergic pulmonary immune response or used crude latex extracts, making it difficult to quantify the contribution of individual proteins and limiting their usefulness for developing specific immunotherapy. We immunized mice with recombinant Hev b 5, a defined major latex allergen, or latex glove protein extract, representing the range of occupationally encountered processed latex allergens. The immune response was compared with that seen in ovalbumin-immunized mice. Immunization with Hev b 5 or glove extract elicits hallmarks of allergic pulmonary Th2-type immune responses, comparable to those for ovalbumin, including (1) serum antigen-specific IgE, (2) an eosinophilic inflammatory infiltrate in the lung, (3) increased interleukin-5 in lung bronchoalveolar lavage fluid, and (4) mucus hypersecretion by epithelial cells in the lung airways. This mouse model will aid the development of potentially curative treatments for latex-sensitized individuals, including those with occupational asthma.