Ruihua Peng

Dexamethasone Induces IL-10-Producing Monocyte- Derived Dendritic Cells with Durable Immaturity

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 10−7 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.

Transfusion of Apoptotic β-Cells Induces Immune Tolerance to β-Cell Antigens and Prevents Type 1 Diabetes in NOD Mice

In vivo induction of β-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 β-cells to prevent type 1 diabetes, the current study was designed to investigate apoptotic β-cells induced ex vivo in preventing type 1 diabetes. The NIT-1 cell line serves as a source of β-cells. Apoptotic NIT-1 cells were prepared by ultraviolet B (UVB) irradiation. Three weekly transfusions of UVB-irradiated NIT-1 cells (1 × 105/mouse) or PBS were used to determine whether transfusions of UVB-irradiated NIT-1 cells induce immune tolerance to β-cell antigens in vivo and prevent type 1 diabetes. The suppression of anti–β-cell antibodies, polarization of T-helper (Th) cells, and induction of regulatory T-cells by UVB-irradiated NIT-1 cell treatment were investigated. The transfusions of apoptotic NIT-1 cells suppress anti–β-cell antibody development and induce Th2 responses and interleukin-10–producing regulatory type 1 cells. Importantly, this treatment significantly delays and prevents the onset of diabetes when 10-week-old NOD mice are treated. Adoptive transfer of splenocytes from UVB-irradiated NIT-1 cell–treated mice prevents diabetes caused by simultaneously injected diabetogenic splenocytes in NOD-Rag−/− mice. Moreover, the proliferation of adoptively transferred carboxyfluorescein diacetate succinimidyl ester–labeled β-cell antigen–specific T-cell receptor–transgenic T-cells in UVB-irradiated NIT-1–cell treated mice is markedly suppressed. The transfusion of apoptotic β-cells effectively protects against type 1 diabetes in NOD mice by inducing immune tolerance to β-cell antigens. This approach has great potential for immune intervention for human type 1 diabetes.

The effects of renal transplantation on peripheral blood dendritic cells

Abstract:  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.

Dendritic cell deficiency associated with development of BK viremia and nephropathy in renal transplant recipients

Background. BK virus nephropathy (BKVN) is a significant cause of renal allograft loss. Although overall intensity of immunosuppression is the greatest risk factor, recipient immune factors likely also play a role in the pathogenesis. Dendritic cells (DC) are potent antigen-presenting cells important for the induction of anti-viral cytotoxic T-cell responses. In a previous univariate analysis, we demonstrated a peripheral blood DC (PBDC) deficiency in patients with biopsy-proven BKVN, raising the possibility that reduction in DC predisposed to BK reactivation. Methods. In this study, we refined our previous analysis by comparing random posttransplant PBDC levels between an expanded group of patients with BKVN and controls without viremia using a multivariate analysis that accounted for factors known to influence PBDC levels. Next, we compared pretransplant PBDC levels between patients stratified by the presence or absence of posttransplant viremia. Finally, we assessed the predictive value of pretransplant PBDC levels for the development of posttransplant viremia. Results. Analyses revealed a PBDC level deficiency not only posttransplant in patients with BKVN but also pretransplant in patients who subsequently developed posttransplant BK viremia. Furthermore, we identified a pretransplant PBDC level that is a reasonable predictor for the development of posttransplant viremia. Conclusions. 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.

Increased IFN-α–Producing Plasmacytoid Dendritic Cells (pDCs) in Human Th1-Mediated Type 1 Diabetes: pDCs Augment Th1 Responses through IFN-α Production

Increasing evidence suggests that type 1 IFN (IFN-αβ) is associated with pathogenesis of Th1-mediated type 1 diabetes (T1D). A major source of IFN-αβ 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 and new-onset and established T1D subjects possessed significantly increased pDCs but similar number of myeloid DCs 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 Coxsackievirus 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-αβ production may be associated with this Th1-mediated autoimmune disease, especially under certain viral infections linked to T1D pathogenesis.

Rabbit Polyclonal Mouse Antithymocyte Globulin Administration Alters Dendritic Cell Profile and Function in NOD Mice to Suppress Diabetogenic Responses

Mouse antithymocyte globulin (mATG) prevents, as well as reverses, type 1 diabetes in NOD mice, through mechanisms involving modulation of the immunoregulatory activities of T lymphocytes. Dendritic cells (DC) play a pivotal role in the generation of T cell responses, including those relevant to the autoreactive T cells enabling type 1 diabetes. As Abs against DC are likely generated during production of mATG, we examined the impact of this preparation on the phenotype and function of DC to elucidate novel mechanisms underlying its beneficial activities. In vivo, mATG treatment transiently induced the trafficking of mature CD8− predominant DC into the pancreatic lymph node of NOD mice. Splenic DC from mATG-treated mice also exhibited a more mature phenotype characterized by reduced CD8 expression and increased IL-10 production. The resultant DC possessed a potent capacity to induce Th2 responses when cultured ex vivo with diabetogenic CD4+ T cells obtained from BDC2.5 TCR transgenic mice. Cotransfer of these Th2-deviated CD4+ T cells with splenic cells from newly diabetic NOD mice into NOD.RAG−/− mice significantly delayed the onset of diabetes. These studies suggest the alteration of DC profile and function by mATG may skew the Th1/Th2 balance in vivo and through such actions, represent an additional novel mechanism by which this agent provides its beneficial activities.

Infusion of UVB-treated splenic stromal cells induces suppression of β cell antigen-specific T cell responses in NOD mice

Our previous study has demonstrated that transfusion of UVB-irradiation-induced apoptotic beta cells effectively prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the limitation of beta cell source would preclude the clinical application of this approach. Therefore, in the present study, we have attempted to establish a more practical approach by utilizing apoptotic non-beta cells to prevent T1D. We find that apoptotic splenic stromal cells significantly suppress beta cell antigen-reactive T cell proliferation in vitro and in vivo. Moreover, beta cell antigen-specific T cells primed by beta cell antigens in the presence of apoptotic stromal cells have markedly reduced responsiveness to the re-stimulation of the same beta cell antigen. We also find that beta cell antigen-specific IL-10-producing CD4+ T cells are induced in the presence of apoptotic splenic stromal cells. As expected, transfusion of apoptotic stromal cells effectively protected NOD mice from developing T1D. Furthermore, the proliferation of adoptively transferred beta cell antigen-specific TCR-transgenic T cells in pancreatic draining lymph nodes is markedly suppressed in UVB-stroma-treated mice, indicating that UVB-stroma treatment induces immune tolerance to multiple beta cell antigens. This study provides an effective and convenient approach for managing T1D by utilizing apoptotic non-beta cells.

Pre-transplant myeloid dendritic cell deficiency associated with cytomegalovirus infection and death after kidney transplantation

Dendritic cells (DCs) are potent antigen‐presenting cells critical for immunity. We previously demonstrated a significant association between pre‐transplant blood myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) deficiency and post‐transplant BK viremia in renal transplant recipients. In the current post‐hoc analysis, we studied the association of these same pre‐transplant DC levels with other post‐transplant outcomes.

Apoptotic Non–β Cells Suppress β Cell Antigen–Reactive T Cells and Induce β Cell Antigen–Specific Regulatory T Cells

Steady‐state cell apoptosis plays an important role in maintenance of self‐tolerance. Based on this notion, the use of apoptotic cells to restore self‐tolerance to β cell antigens is a rational approach to type 1 diabetes (T1D) prevention. Our previous study demonstrated that transfusion of apoptotic β cells induced immune tolerance to β cell antigens in NOD mice. However, concerned about the limited β cell source for future clinical applications, we attempted in the present study to develop a more practical approach for T1D prevention using apoptotic non–β cells. We found that UVB‐irradiation‐induced apoptotic NOD splenic stromal cells significantly suppressed β cell antigen‐specific T cell proliferation in vitro and in vivo. Furthermore, TCR‐transgenic CD4+ T cells primed by the antigens to which they were specific in the presence of UVB‐irradiated stromal cells were rendered unresponsive to the antigen restimulation, a result that was partially attributed to the induced IL‐10‐producing regulatory T cells. Of more interest, transfusion of UVB‐irradiated stromal cells appeared to induce β cell antigen–responding IL‐10‐producing regulatory T cells in vivo. Most importantly, transfusion of UVB‐irradiated stromal cells effectively prevented T1D in NOD mice, which is consistent with these findings. This study suggests that it is possible to use apoptotic non–β cells such as peripheral blood mononuclear cells to induce β cell antigen–specific tolerance, thereby preventing T1D in humans.

Immature Dendritic Cell Therapy Confers Durable Immune Modulation in an Antigen-Dependent and Antigen-Independent Manner in Nonobese Diabetic Mice

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.