Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells

An important pathway for immune tolerance is provided by thymic-derived CD25+ CD4+ T cells that suppress other CD25- autoimmune disease-inducing T cells. The antigen-presenting cell (APC) requirements for the control of CD25+ CD4+ suppressor T cells remain to be identified, hampering their study in experimental and clinical situations. CD25+ CD4+ T cells are classically anergic, unable to proliferate in response to mitogenic antibodies to the T cell receptor complex. We now find that CD25+ CD4+ T cells can proliferate in the absence of added cytokines in culture and in vivo when stimulated by antigen-loaded dendritic cells (DCs), especially mature DCs. With high doses of DCs in culture, CD25+ CD4+ and CD25- CD4+ populations initially proliferate to a comparable extent. With current methods, one third of the antigen-reactive T cell receptor transgenic T cells enter into cycle for an average of three divisions in 3 d. The expansion of CD25+ CD4+ T cells stops by day 5, in the absence or presence of exogenous interleukin (IL)-2, whereas CD25- CD4+ T cells continue to grow. CD25 + CD4+ T cell growth requires DC-T cell contact and is partially dependent upon the production of small amounts of IL-2 by the T cells and B7 costimulation by the DCs. After antigen-specific expansion, the CD25 + CD4+ T cells retain their known surface features and actively suppress CD25- CD4+ T cell proliferation to splenic APCs. DCs also can expand CD25+ CD4+ T cells in the absence of specific antigen but in the presence of exogenous IL-2. In vivo, both steady state and mature antigen-processing DCs induce proliferation of adoptively transferred CD25+ CD4+ T cells. The capacity to expand CD25+ CD4+ T cells provides DCs with an additional mechanism to regulate autoimmunity and other immune responses

SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1+ cells representing ∼5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla

CD80+CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells

Foxp3+CD25+CD4+ regulatory T cells (Treg) mediate immunological self-tolerance and suppress immune responses. A subset of dendritic cells (DCs) in the intestine is specialized to induce Treg in a TGF-β- and retinoic acid-dependent manner to allow for oral tolerance. In this study we compare two major DC subsets from mouse spleen. We find that CD8+ DEC-205/CD205+ DCs, but not the major fraction of CD8- DC inhibitory receptor-2 (DCIR2)+ DCs, induce functional Foxp3+ Treg from Foxp3- precursors in the presence of low doses of Ag but without added TGF-β. CD8 +CD205+ DCs preferentially express TGF-β, and the induction of Treg by these DCs in vitro is blocked by neutralizing Ab to TGF-β. In contrast, CD8+DCIR2+ DCs better induce Foxp3+ Treg when exogenous TGF-β is supplied. In vivo, CD8 +CD205+ DCs likewise preferentially induce Treg from adoptively transferred, Ag-specific DO11.10 RAG-/- Foxp3 -CD4+ T cells, whereas the CD8-DCIR2 + DCs better stimulate natural Foxp3+ Treg. These results indicate that a subset of DCs in spleen, a systemic lymphoid organ, is specialized to differentiate peripheral Foxp3+ Treg, in part through the endogenous formation of TGF-β. Targeting of Ag to these DCs might be useful for inducing Ag-specific Foxp3+ Treg for treatment of autoimmune diseases, transplant rejection, and allergy

CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes

In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet β cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 105 polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens

TLR2-Dependent Induction of IL-10 and Foxp3+CD25+CD4+ Regulatory T Cells Prevents Effective Anti-Tumor Immunity Induced by Pam2 Lipopeptides In Vivo

16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2) lipopeptides act as toll-like receptor (TLR)2/6 ligands and activate natural killer (NK) cells and dendritic cells (DCs) to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3+CD4+ regulatory T (T reg) cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine

CD25+ CD4+ T Cells, Expanded with Dendritic Cells Presenting a Single Autoantigenic Peptide, Suppress Autoimmune Diabetes

In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet β cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 105 polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens

Direct Expansion of Functional CD25+ CD4+ Regulatory T Cells by Antigen-processing Dendritic Cells

An important pathway for immune tolerance is provided by thymic-derived CD25+ CD4+ T cells that suppress other CD25− autoimmune disease–inducing T cells. The antigen-presenting cell (APC) requirements for the control of CD25+ CD4+ suppressor T cells remain to be identified, hampering their study in experimental and clinical situations. CD25+ CD4+ T cells are classically anergic, unable to proliferate in response to mitogenic antibodies to the T cell receptor complex. We now find that CD25+ CD4+ T cells can proliferate in the absence of added cytokines in culture and in vivo when stimulated by antigen-loaded dendritic cells (DCs), especially mature DCs. With high doses of DCs in culture, CD25+ CD4+ and CD25− CD4+ populations initially proliferate to a comparable extent. With current methods, one third of the antigen-reactive T cell receptor transgenic T cells enter into cycle for an average of three divisions in 3 d. The expansion of CD25+ CD4+ T cells stops by day 5, in the absence or presence of exogenous interleukin (IL)-2, whereas CD25− CD4+ T cells continue to grow. CD25+ CD4+ T cell growth requires DC–T cell contact and is partially dependent upon the production of small amounts of IL-2 by the T cells and B7 costimulation by the DCs. After antigen-specific expansion, the CD25+ CD4+ T cells retain their known surface features and actively suppress CD25− CD4+ T cell proliferation to splenic APCs. DCs also can expand CD25+ CD4+ T cells in the absence of specific antigen but in the presence of exogenous IL-2. In vivo, both steady state and mature antigen-processing DCs induce proliferation of adoptively transferred CD25+ CD4+ T cells. The capacity to expand CD25+ CD4+ T cells provides DCs with an additional mechanism to regulate autoimmunity and other immune responses

The Peptide Sequence of Diacyl Lipopeptides Determines Dendritic Cell TLR2-Mediated NK Activation

Natural killer (NK) cells are lymphocyte effectors that are activated to control certain microbial infections and tumors. Many NK-activating and regulating receptors are involved in regulating NK cell function. In addition, activation of naïve NK cells is fundamentally triggered by cytokines or myeloid dendritic cells (mDC) in various modes. In this study, we synthesized 16 S-[2,3-bis(palmitoyl)propyl]cysteine (Pam2Cys) lipopeptides with sequences designed from lipoproteins of Staphylococcus aureus, and assessed their functional properties using mouse (C57BL/6) bone marrow-derived DC (BMDC) and NK cells. NK cell activation was evaluated by three criteria: IFN-γ production, up-regulation of NK activation markers and cytokines, and NK target (B16D8 cell) cytotoxicity. The diacylated lipopeptides acted as TLR2 ligands, inducing up-regulation of CD25/CD69/CD86, IL-6, and IL-12p40, which represent maturation of BMDC. Strikingly, the Pam2Cys lipopeptides induced mouse NK cell activation based on these criteria. Cell-cell contact by Pam2Cys peptide-stimulated BMDC and NK cells rather than soluble mediators released by stimulated BMDC induced activation of NK cells. For most lipopeptides, the BMDC TLR2/MyD88 pathway was responsible for driving NK activation, while some slightly induced direct activation of NK cells via the TLR2/MyD88 pathway in NK cells. The potential for NK activation was critically regulated by the peptide primary sequence. Hydrophobic or proline-containing sequences proximal to the N-terminal lipid moiety interfered with the ability of lipopeptides to induce BMDC-mediated NK activation. This mode of NK activation is distinctly different from that induced by polyI:C, which is closely associated with type I IFN-inducing pathways of BMDC. These results imply that the MyD88 pathway of BMDC governs an alternative NK-activating pathway in which the peptide sequence of TLR2-agonistic lipopeptides critically affects the potential for NK activation