Continuous Subcutaneous Insulin Infusion (CSII) is superior to conventional insulin therapy as it improves glycemic control thus reducing the probability of diabetic complications. Notwithstanding CSII's benefits, insulin dependent diabetic patients rarely achieve optimal glucose control. Moreover, CSII is only FDA approved for 3 days and often fails prematurely for reasons that have not been fully elucidated. We hypothesize that phenolic compounds, such as m‐cresol and phenol, which are present in all commercial insulin formulations are responsible for the tissue reaction occurring at the insulin infusion site. This hypothesis was examined with in vitro cell cultures and a mouse air‐pouch model to determine cellular and tissue reactions following infusions with saline, phenolic compounds, (i.e., commercial diluent), and insulin. We demonstrated that diluent and insulin were cytotoxic to cells in culture at sub‐clinical concentrations (e.g., >1:10 of commercial insulin). Air pouch studies demonstrated that infusion of either diluted insulin or diluent itself induced three to five‐fold level of recruited leukocytes as compared to saline. At both 3‐ and 7‐days post infusion, these were predominantly neutrophils and macrophages. We conclude that phenolic compounds in commercial insulin preparations are cell and tissue toxic, which contributes to the failure of effective insulin infusion therapy.

Summary CD8+ regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune‐privileged site. Although these CD8+ regulatory T cells perform an antigen‐specific regulatory function for an immune response to self and non‐self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8+ regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP‐BSA) amplified AC‐induced splenic CD8+ regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8+ regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)‐deficient CD8+ regulatory T‐cell function was rescued by inclusion of exogenous interferon‐γ (IFN‐γ), demonstrating that the expression of FasL by CD8+ regulatory T cells was dispensable, but IFN‐γ was not. Ultimately, we demonstrated that the generation of these CD8+ regulatory T cells occurred independently of IFN‐γ, but their suppressor function required IFN‐γ receptor stimulation.

Summary The introduction of antigen into the anterior chamber of an eye induces the antigen‐specific suppression of cell‐mediated immunity and the antigen‐induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell‐mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti‐F4/80 and/or anti‐CD11c, anti‐CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP‐BSA) into an anterior chamber and Percoll‐enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP‐BSA‐immunized or naive recipients. The recipients were challenged to induce delayed‐type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)‐transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti‐F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP– mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP‐BSA transferred the suppression of DTH when injected intravenously into TNP‐BSA‐immunized recipients, activated immunoregulatory thymocytes and activated antigen‐specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.

The introduction of antigen into the anterior chamber (AC) of the eye induces the production of antigen-specific splenic CD8+ regulatory T cells (AC-SPL cells) that suppress a delayed-type hypersensitivity (DTH) reaction in immunized mice. Because the generation of these regulatory T cells is also induced by exposure to transforming growth factor (TGF)-β and antigen or F4/80+ cells exposed to TGF-β and antigen in vitro, we investigated (i) whether these cells are produced in dominant negative receptor for transforming growth factor β receptor type II (dnTGFβRII) or Cbl-b−/− mice whose T cells are resistant to TGF-β, (ii) whether DTH is suppressed by wild type (WT) CD8+ AC-SPL cells in Cbl-b−/− and dnTGFβRII mice and (iii) the effect of antibodies to TGF-β on the suppression of DTH by CD8+ AC-SPL cells. DnTGFβRII immunized and Cbl-b−/− mice produced splenic CD8+ regulatory cells after the intracameral injection of antigen and immunization. The suppression of a DTH reaction by CD8+ AC-SPL cells in WT mice was blocked by the local inclusion of antibodies to TGF-β when WT splenic CD8+ AC-SPL cells were injected into the DTH reaction site. Moreover, the DTH reaction in immunized dnTGFβRII and Cbl-b−/− mice was not suppressed by the transfer of WT CD8+ AC-SPL cells to the site challenged with antigen. In aggregate, these observations suggest that T cell sensitivity to TGF-β is not an obligate requirement for the in vivo induction of CD8+ AC-SPL T cells but the suppression of an in vivo DTH reaction by CD8+ AC-SPL cells is dependent on TGF-β.

The injection of antigen into the anterior chamber (AC, intracameral injection)2 of a murine eye induces the generation of splenic CD8+ regulatory T cells (AC-SPL cells) that effect the antigen-specific suppression of a Delayed-Type Hypersensitivity (DTH) reaction. Here we show (i) for the first time that the local antigen-specific suppression of DTH-induced swelling in immunized mice by either an intracameral injection of antigen or by the direct injection of CD8+ AC-SPL cells into an antigen-challenged site is associated with an absence of infiltrated mononuclear cells, (ii) the local antigen-specific suppression of the DTH reaction by CD8+ AC-SPL cells requires compatibility between the Qa-1 but not H2 antigen haplotype of the immunized recipient and the injected AC-SPL regulatory T cells, (iii) The suppression of the DTH reaction by CD8+ AC-SPL cells requires the expression of Qa-1 but not H2 antigens and is not due to bystander suppression.