Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+Foxp3− T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFβ. TGFβ-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.

This unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen‐presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique, bone marrow cells are cultured in the presence of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) to yield 5–10 × 10⁶ cells, 60% of which express DC surface markers (e.g., B‐7‐2/CD86). Additional techniques for isolating DCs from mouse spleens or other mouse tissues, as well as from human tissues, are also discussed. Curr. Protoc. Immunol. 86:3.7.1‐3.7.19. © 2009 by John Wiley & Sons, Inc.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Dendritic cells (DC) are potent inducers of immunity to foreign Ags, but also contribute to self-tolerance by induction of regulatory T cells or deletion/anergy of self-reactive T cells. In this study, we have studied the capacity of DC to activate naturally occurring CD4⁺CD25⁺ regulatory T cells as well as the ability of CD4⁺CD25⁺ T cells to suppress the DC-mediated activation of CD4⁺CD25⁻ T cells. Mature bone marrow-derived dendritic cells, but not splenic DC, were able to induce the proliferation of CD4⁺CD25⁺ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4⁺CD25⁺ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25⁺ effector cells. Because bone marrow-derived dendritic cells induce proliferation of both CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells in vitro, it was impossible to assay the suppressive function of the CD4⁺CD25⁺ T cells using [³H]TdR uptake or CFSE dilution. We therefore measured IL-2 production in cocultures of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells using the IL-2 secretion assay. Surprisingly, CD4⁺CD25⁺ T cells markedly suppressed IL-2 secretion by the CD4⁺CD25⁻ T cells without inhibiting their proliferation. Collectively, these results suggest that Ag presentation by DC can induce the expansion of CD4⁺CD25⁺ T cells while simultaneously activating their ability to suppress cytokine secretion by effector T cells.

The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.10⁵ cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated.In vitrostudies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2·6×10⁵to 1:1×10⁶). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.