Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3–glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1β, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1α (MIP-1α), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte–CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan–initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.

Natural CD4+CD25+ regulatory T (CD4+CD25+ T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4+CD25+ T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4+CD25+ T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFβ1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4+CD25+ T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4+CD25+CD62Lhi T cells, mediated by TGFβ, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFβ1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4+CD25+CD62Lhi T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4+CD25+ T reg cells in NOD mice express TGFβ1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D.

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by ∼95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcγRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 μg) in NOD mice, FcγRIII and FcγRIV compensated for inadequate FcγRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcγR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigenspecific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65؉pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4 ؉ T-cells secreting IL-4, IL-10, or interferon-␥ were detected in mice treated with pGAD65؉pIL4؉pIL10 and pGAD65؉pIL10. However, the frequency of FoxP3-expressing CD4؉ CD25 ؉ CD62L hi T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65؉pIL4؉pIL10. These immunoregulatory CD4 ؉ CD25 ؉ T-cells (CD4 ؉ CD25 ؉ Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-␤ dependent. Furthermore, duration of islet graft protection in pGAD65؉pIL4؉pIL10-vaccinated diabetic recipients correlated with the persistence of CD4 ؉ CD25 ؉ Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4 ؉ CD25 ؉ Treg influence antigen-induced suppression of ongoing ␤-cell autoimmunity in diabetic recipients.

We have previously reported that multiple injections of soluble MHC class I tetramers assembled with wild-type HY peptide induces unresponsiveness to male skin grafts in naive female C57BL/6 (B6) mice. Induction of unresponsiveness is dependent on a population of unresponsive allospecific CD8 lo T cells. Reduced expression of CD8 acts to limit a T cell response to HY peptide by limiting the avidity window of effective signal transduction. We and others have demonstrated that CD8 lo T cells are an alternative stable phenotype of CD8ab + T cells in vitro and in vivo after antigen stimulation. We show here that CD8 lo T cells can suppress naive CD8 + T cell responses to HY antigen in vitro and male skin graft rejection in vivo after adoptive transfer into female recipients. These novel regulatory T cells express surface TGF-b1 and secrete T cytotoxic 2 cytokines after antigen-specific stimulation. Anti-TGF-b antibody and latency-associated peptide inhibit the suppressive effects in vitro. We also show that HY-specific memory CD8 + T cells overcome regulation by CD8 lo T cells. These data define a novel peripheral regulatory CD8 + T cell population that arises after repeated antigen encounter in vivo. These cells have implications in the maintenance of tolerance and memory.See accompanying commentary http://dx.doi.org/10.1002/eji200535797 IntroductionDuring the past few years, mechanisms of regulation of T cell activity have attracted much attention. There has been a resurgence of interest in regulatory T cells in autoimmunity, and more recently, harnessing their potential as a means to prevent graft rejection [1], albeit largely unsuccessful to date. While it is clear that CD4 + CD25 + T cells play a critical role in the control of T cell responses [2,3], studies of CD8 + T cell-mediated regulation have been sparse to date, and have revealed different forms of regulation utilizing both deletional and non-deletional mechanisms [4]. For instance, active immunosuppression by transfusion of donor-specific lymphocytes into a murine TCR transgenic mice induced regulatory TCR + CD4 -CD8 -T cells (double negative T cells, DNTC) population [5]. These T cells induced apoptosis in activated donor-specific CD8 + T cells via a Fas-dependent pathway, but not T cells activated to a third-party alloantigen. It appears that these regulatory T cells require antigen-specific recognition. Indeed, DNTC were found to acquire MHC-allopeptide complexes from APC that were presented on their surface. As a result these DNTC functioned as "killer APC" in conjunction with up-regulated FasL induced by activation [5,6]. In contrast to deletional regulation, a distinct population of CD8 + CD28 -T suppressor (Ts) cells were found to be generated in vitro after multiple rounds of stimulation of human peripheral blood mononuclear cells with allogeneic donor APC or self-APC pulsed with allogeneic peptide [7]. Interestingly, such cells suppressed CD4 + T cells responses and induced regulatory CD4 + CD25 + T cells. Immunoregulation was dependent on th...

Recurrent autoimmune destruction of the insulin-producing β cells is a key factor limiting successful islet graft transplantation in type I diabetic patients. In this study, we investigated the feasibility of using an Ag-specific plasmid DNA (pDNA)-based strategy to protect pro-islets that had developed from a neonatal pancreas implanted under the kidney capsule of nonobese diabetic (NOD) mice. NOD recipient mice immunized with pDNA encoding a glutamic acid decarboxylase 65 (GAD65)-IgFc fusion protein (JwGAD65), IL-4 (JwIL4), and IL-10 (pIL10) exhibited an increased number of intact pro-islets expressing high levels of insulin 15 wk posttransplant, relative to NOD recipient mice immunized with pDNA encoding a hen egg lysozyme (HEL)-IgFc fusion protein (JwHEL)+JwIL4 and pIL10 or left untreated. Notably, the majority of grafted pro-islets detected in JwGAD65+JwIL4- plus pIL10-treated recipients was free of insulitis. In addition, administration of JwGAD65+JwIL4+pIL10 provided optimal protection for engrafted islets compared with recipient NOD mice treated with JwGAD65+JwIL4 or JwGAD65+pIL10, despite effective protection of endogenous islets mediated by the respective pDNA treatments. Efficient protection of pro-islet grafts correlated with a marked reduction in GAD65-specific IFN-γ reactivity and an increase in IL-10-secreting T cells. These results demonstrate that pDNA vaccination can be an effective strategy to mediate long-term protection of pro-islet grafts in an Ag-specific manner and that conditions are more stringent to suppress autoimmune destruction of grafted vs endogenous islets.

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