It is highly desirable that immature dendritic cells (DC) used for tolerance induction maintain steady immature state with predominant interleukin (IL)-10 production. In this study, we attempted to develop DC with durable immaturity and other tolerogenic features by using dexamethasone (Dex). We found DC derived from human monocytes in the presence of 10 À7 M Dex were negative for CD1a. Compared with control transduced DC (Ctrl-DC), Dex-DC expressed lower CD40, CD80 and CD86 but equivalent human leucocyte antigen-DR. Both immature Dex-and Ctrl-DC did not express CD83. Nevertheless, upon stimulation of lipopolysaccharide (LPS) or CD40 ligand, the expression of CD40, CD80, CD83 and CD86 was upregulated on Ctrl-DC but not on Dex-DC. The immaturity of Dex-DC was durable following Dex removal. Interestingly, Dex-DC maintained production of large amount of IL-10 and little IL-12 five days after Dex removed. Further study indicated that high-level IL-10 production by Dex-DC was associated with high-level phosphorylation of extracellular signal-regulated kinase (ERK) as blockade of this enzyme markedly attenuated IL-10 production. Furthermore, Dex-DC sustained the capability of high phosphorylation of ERK and IL-10 production 5 days after Dex removal. In addition, Dex-DC had significantly lower activity in stimulating T-cell proliferation. Neutralization of IL-10, to some extent, promoted DC maturation activated by LPS, as well as T-cell stimulatory activity of Dex-DC. The above findings suggest that IL-10-producing Dex-DC with durable immaturity are potentially useful for induction of immune tolerance.

In vivo induction of ␤-cell apoptosis has been demonstrated to be effective in preventing type 1 diabetes in NOD mice. Based on the notion that steady-state cell apoptosis is associated with self-tolerance and the need for developing a more practical approach using apoptotic ␤-cells to prevent type 1 diabetes, the current study was designed to investigate apoptotic ␤-cells

Increasing evidence suggests that type 1 interferon (IFN-α/β) is associated with pathogenesis of Th1-mediated type 1 diabetes (T1D). A major source of IFNα/β is plasmacytoid dendritic cells (pDCs). In this study, we analyzed peripheral blood pDC numbers and functions in at-risk, new onset and established T1D patients and controls. We found that subjects at risk for T1D, new onset and established T1D subjects possessed significantly increased pDCs but similar number of myeloid DCs (mDCs) when compared with controls. pDC numbers were not affected by age in T1D subjects but declined with increasing age in control subjects. It was demonstrated that IFN-α production by PBMCs stimulated with influenza viruses was significantly higher in T1D subjects than in controls, and IFN-α production was correlated with pDC numbers in PBMCs. Of interest, only T1D-associated Coxsackie virus serotype B4 but not B3 induced majority of T1D PBMCs to produce IFN-α which was confirmed to be secreted by pDCs. Finally, in vitro studies demonstrated IFN-α produced by pDCs augmented Th1 responses, with significantly greater IFN-γ-producing CD4+ T cells from T1D subjects. These findings indicate that increased pDCs and their IFN-α/β production may be associated with this Th1-mediated autoimmune disease, especially under certain viral infections linked to T1D pathogenesis.

Recent advances allow accurate quantification of peripheral blood (PB) myeloid and plasmacytoid dendritic cell (DC) populations (mDC and pDC, respectively), although the response to renal transplantation (RT) remains unknown. Using flow cytometry, PBDC levels were quantified in patients with end stage renal disease (ESRD) undergoing RT. PBDC levels were significantly reduced in ESRD patients pre-RT compared to healthy controls, with further reduction noted immediately following a hemodialysis session. RT resulted in a dramatic decrease in both subsets, with a greater reduction of pDC levels. Both subset levels were significantly lower than in control patients undergoing abdominal surgery without RT. Subgroup analysis revealed significantly greater mDC reduction in RT recipients receiving anti-lymphocyte therapy, with preferential binding of antibody preparation to this subset. Samples from later time points revealed a gradual return of PBDC levels back to pre-transplant values concurrent with overall reduction of immunosuppression (IS). Finally, PBDC levels were significantly reduced in patients with BK virus nephropathy compared to recipients with stable graft function, despite lower overall IS. Our findings suggest that PBDC levels reflect the degree of IS in renal allograft recipients. Furthermore, PBDC monitoring may represent a novel strategy to predict important outcomes such as acute rejection, long-term graft loss and infectious complications.

Our results identify PBDC deficiency as a previously unrecognized risk factor for BKV reactivation after renal transplantation. Pretransplant PBDC monitoring may prove to be a useful clinical tool in the assessment of patient vulnerability to BKVN posttransplant, which may allow more focused screening.

Type 1 diabetes (T1D) is a T cell-mediated disease. Various DC populations play important roles in initiating and directing T cell responses and thus may be critical for T1D pathogenesis. We thus examined peripheral blood DC1 and DC2 populations by flow cytometry in healthy controls, subjects at risk for T1D, new-onset patients, and established T1D patients. We found a significant increase in the number of DCs (including DC1 and DC2) in at-risk subjects and those with new-onset T1D versus healthy controls and established T1D patients (ANOVA; p < 0.0001). Analysis of DC1 and DC2 subsets in these same groups demonstrated a significant decrease in the ratio of DC1 and DC2 in subjects at risk and new-onset and established T1D patients in contrast with healthy controls (p < 0.0001). Both subsets of peripheral blood DCs from T1D patients expressed significantly higher levels of HLA-DR than healthy controls. Peripheral blood mononuclear cells from T1D patients secreted significantly higher amounts of IFN-alpha than controls, and IFN-alpha production correlated inversely with the DC1/DC2 ratio. This study demonstrates a marked increase in peripheral blood DC numbers that occurs during a time of active autoimmunity in at-risk subjects and patients with new-onset T1D, but is lost in established diabetes. However, the abnormal distribution of peripheral blood DC populations appears to be a persistent phenotype in all stages of T1D.

We previously demonstrated that adoptive transfer of NOD pancreatic lymph node (PLN) DC protected recipients from diabetes. Our recent studies showed that the tolerogenic DC population presented islet antigens and were mature myeloid DC that did not produce IL-12, suggestive of exhausted or fully mature DC. Extensive characterization of the DC population in vivo in NOD and control mice demonstrated a specific deficiency of PLN tolerogenic DC in older mice. These findings suggest autoimmunity might arise in NOD mice secondary to deficient maturation of myeloid DC to a tolerogenic state. To address this issue, we characterized maturation and function at development of bone marrow-derived myeloid DC from NOD and several control strains. We found that NOD DC were highly resistant to several maturation stimuli and maintained an immature phenotype (average % immature DC: 75% in NOD versus 15% in B6, p< 0.01). A survey of congenic NOD mice with various NOD diabetes susceptibility loci demonstrated that the IDD10/17/18 region on chromosome 3 controlled approximately 50% of the NOD DC maturation defect. The defect also affected NOD DC that underwent phenotypic maturation. These cells appeared to arrest in a "maturing" phase as they produced 5- to 7-fold more IL-12 than control strains and significantly less IL-10. The cytokine defect was completely corrected in NOD IDD10/17/18 mice. In addition, the IDD10/17/18 locus limited DC accumulation in islets and significantly increased tolerogenic DC in the PLN. Together, the above findings suggest that polygenic regulation of DC maturation defects in NOD mice promotes islet inflammation while limiting the generation of tolerogenic DC.

Dendritic cell (DC) immunotherapy has been effective for prevention of type 1 diabetes (T1D) in NOD mice but fails to protect if initiated after active autoimmunity. As autoreactivity expands inter- and intramolecularly during disease progression, we investigated whether DCs unpulsed or pulsed with β cell antigenic dominant determinants (DD), subdominant determinants (SD), and ignored determinants (ID) could prevent T1D in mice with advanced insulitis. We found that diabetes was significantly delayed by DC therapy. Of interest, DCs pulsed with SD or ID appeared to provide better protection. T lymphocytes from DC-treated mice acquired spontaneous proliferating capability during in vitro culture, which could be largely eliminated by IL-2 neutralizing antibodies. This trend maintained even 29 weeks after discontinuing DC therapy and appeared antigen-independent. Furthermore, CD4+Foxp3+ T regulatory cells (Tregs) from DC-treated mice proliferated more actively in vitro compared to the controls, and Tregs from DC-treated mice showed significantly enhanced immunosuppressive activities in contrast to those from the controls. Our study demonstrates that DC therapy leads to long-lasting immunomodulatory effects in an antigen-dependent and antigen-independent manner and provides evidence for peptide-based intervention during a clinically relevant window to guide DC-based immunotherapy for autoimmune diabetes.