Loss of Zbtb32 in NOD mice does not significantly alter T cell responses.

Background: We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2 + dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2 + DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods: To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results: Although NOD.Zbtb32 -/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32 -/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions: The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype.

Low CD25 on autoreactive Tregs impairs tolerance via low dose IL-2 and antigen delivery.

Dendritic cell (DC)-mediated T cell tolerance deficiencies contribute to the pathogenesis of autoimmune diseases such as type 1 diabetes. Delivering self-antigen to dendritic-cell inhibitory receptor-2 (DCIR2)+ DCs can delay but not completely block diabetes development in NOD mice. These DCIR2-targeting antibodies induce tolerance via deletion and anergy, but do not increase islet-specific Tregs. Because low-dose IL-2 (LD-IL-2) administration can preferentially expand Tregs, we tested whether delivering islet-antigen to tolerogenic DCIR2+ DCs along with LD-IL-2 would boost islet-specific Tregs and further block autoimmunity. But, surprisingly, adding LD-IL-2 did not increase efficacy of DC-targeted antigen to inhibit diabetes. Here we show the effects of LD-IL-2, with or without antigen delivery to DCIR2+ DCs, on both polyclonal and autoreactive Treg and conventional T cells (Tconv). As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required both antigen and IL-2 stimulation for optimal expansion. Also, islet-specific Tregs had lower CD25 expression and IL-2 sensitivity, while islet-specific Tconv had higher CD25 expression, compared to polyclonal populations. LD-IL-2 increased activation and expansion of Tconv, and was more pronounced for autoreactive cells after treatment with IL-2 + islet-antigen. Therefore, LD-IL-2 therapy, especially when combined with antigen stimulation, may not optimally activate and expand antigen-specific Tregs in chronic autoimmune settings.

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia.

BACKGROUND: Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. Epidemiologic studies associate the risk of vulvodynia with a history of atopic disease. We used an established model of hapten-driven contact hypersensitivity to investigate the underlying mechanisms of allergy-provoked prolonged sensitivity to pressure. METHODS: We sensitized female ND4 Swiss mice to the hapten oxazolone on their flanks, and subsequently challenged them four days later with oxazolone or vehicle for ten consecutive days on the labia. We evaluated labiar sensitivity to touch, local mast cell accumulation, and hyperinnervation after ten challenges. RESULTS: Oxazolone-challenged mice developed significant tactile sensitivity that persisted for over three weeks after labiar allergen exposures ceased. Allergic sites were characterized by mast cell accumulation, sensory hyper-innervation and infiltration of regulatory CD4+CD25+FoxP3+ T cells as well as localized early increases in transcripts encoding Nerve Growth Factor and nerve-mast cell synapse marker Cell Adhesion Molecule 1. Local depletion of mast cells by intra-labiar administration of secretagogue compound 48/80 led to a reduction in both nerve density and tactile sensitivity. CONCLUSIONS: Mast cells regulate allergy-provoked persistent sensitivity to touch. Mast cell-targeted therapeutic strategies may provide novel means to manage and limit chronic pain conditions associated with atopic disease.

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.