C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Establishment of animal model of antigen-specific T lymphocyte recruitment into nasal mucosa

DO11.10 transgenic mice, expressing an ovalbumin (OVA)-specific alphabeta T-cell receptor (TCR), have been used as a model of various immune diseases associated with T lymphocytes. Some studies of immunoresponse in lung have involved adoptive transfer of DO11.10 mice. As of yet, however, there have been no studies of the adoptive transfer model in the upper airway. The purpose of this study was to establish an animal model to clarify the recruitment mechanism and the roles of Th2 cells in allergic rhinitis. In accordance with the adoptive transfer system, we generated Th0, Th1 and Th2 cells from DO11.10 mice and transferred them into wild type BALB/c mice. Following nasal OVA challenge to DO11.10 mice or to the BALB/c mice into which antigen-specific Th2 cells had been transferred, the number of local antigen-specific TCR-positive cells accompanying the local eosinophilia had significantly increased. However, nasal OVA challenge to BALB/c mice into which antigen-specific Th0 or Th1 cells were transferred failed to increase the number of local OVA-specific TCR positive cells. These observations suggest that an antigen-specific homing mechanism of Th2 cells may exist in nasal mucosa. Analysis of this model will assist in the development of new therapeutic strategy, which targets Th2 cells in allergic rhinitis.     

Killing of naive T cells by CD95L-transfected dendritic cells (DC): in vivo study using killer DC-DC hybrids and CD4(+) T cells from DO11.10 mice

Dendritic cells (DC) play the dual task of initiating cellular immunity against potentially harmful foreign antigens (Ag), while maintaining immunological tolerance to self-Ag and environmental Ag. As an approach to induce Ag-specific suppression, we and others introduced CD95 ligand (L) cDNA into DC. The resulting "killer" DC delivered apoptotic signals, instead of activation signals, to primed CD4(+) T cells in vitro and induced Ag-specific immunosuppression in vivo. To study the impact of killer DC on naive T cells, the fate of Ag-reactive T cells and the extent of their depletion after killer DC treatment, we performed in vitro and in vivo reconstitution experiments using: (a) killer DC-DC hybrids created between CD95L-transduced XS106 DC clone (A/J origin) and splenic DC from BALB/c mice, (b) CD4(+) T cells isolated from DO11.10 transgenic mice (BALB/c background), and (c) OVA(323-339) peptide as relevant Ag. Ovalbumin (OVA)-pulsed killer DC-DC hybrids inhibited DO11.10 T cell activation triggered by conventional DC, instead of inducing their activation. Rapid apoptosis of T cells was observed after co-culture with OVA-pulsed killer DC-DC hybrids, but not with non-pulsed killer DC-DC hybrids or OVA-pulsed control DC-DC hybrids. For in vivo reconstitution, (BALB/cxA/J)F1 mice received subcutaneous administration of killer DC-DC hybrids, followed by intravenous inoculation of DO11.10 T cells. Killer DC-DC hybrids migrated preferentially to draining lymph nodes albeit with relatively low efficiency (0.5-1% recovery) and they induced significant, but incomplete (30-40%) killing of DO11.10 T cells in this location. These results document the abilities of CD95L-transduced DC to trigger apoptosis of naive T cells in an Ag-specific manner, to overrule T cell activation signals delivered by conventional DC, and to reduce local frequencies of Ag-reactive T cells in vivo. Our data also uncover two major limitations (relatively low homing efficiency and incomplete elimination of Ag-reactive T cells) that remain to be overcome for clinical application of CD95L-transduced DC strategy.     

Evidence for Th2 cell-mediated suppression of antibody responses in transgenic,beef insulin-tolerant mice

Clonal deletion, anergy and suppression have all been considered mechanisms of immunological tolerance. Although adoptive transfer of immunosuppression has been shown to occur in the periphery, particularly for transplantation tolerance, it has proven difficult to characterize this phenomenon further, due to the lack of suppressor T cell clones. To characterize tolerance towards a physiological soluble antigen, we constructed beef insulin (BI) transgenic (Tg) BALB/c (H-2^d) mice, in which the BI transgene is expressed in pancreatic β cells. These Tg mice were tolerant to BI immunization at the level of both humoral and cell-mediated immune responses. Adoptive transfer of splenocytes from Tg mice into normal syngeneic BALB/c mice demonstrated that the reduction in antibody production is regulated by transferred T cells. The cytokine profile of T cell clones obtained after selection in vitro demonstrated dominant Th1 clones from normal non-Tg mice and dominant Th2 clones from Tg mice. Some Th2 clones (CD4⁺) from Tg mice produced significant suppression of antibody production after adoptive transfer into normal syngeneic BALB/c mice. These data confirm the existence of Th2 regulatory T cells in vivo in a model of peripheral tolerance to a physiological soluble antigen as a potential mechanism for self tolerance.     

Ag and IL‐2 immune complexes efficiently expand Ag‐specific Treg cells that migrate in response to chemokines and reduce localized immune responses

An intravenous administration of a high‐dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL‐2‐anti‐IL‐2 Ab immune complexes (IL‐2 ICs) efficiently expands OVA‐specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA‐specific TCR Tg‐expressing DO11.10 mice. Here, we demonstrate that the expanded OVA‐specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL‐2 ICs enhanced OVA‐specific Treg‐cell migration and inhibited OVA‐induced delayed‐type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA‐specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL‐2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA‐specific T cells. Thus, the treatment with Ag and IL‐2 ICs can efficiently expand Ag‐specific Treg cells with the capacity to migrate and reduce localized immune responses.     

NKT cells play critical roles in the induction of oral tolerance by inducing regulatory T cells producing IL-10 and transforming growth factor beta, and by clonally deleting antigen-specific T cells

Oral tolerance is the systemic unresponsiveness induced by orally administered proteins. To explore the roles of natural killer T (NKT) cells in oral tolerance, we induced oral tolerance to ovalbumin (OVA) in NKT cell-deficient mice. In CD1d-/- mice, the induction of tolerance to orally administered high- or low-dose OVA was impaired. Dendritic cells (DCs) in the Peyer's patches (PPs) of CD1d-/- mice fed OVA showed high expression of major histocompatibility complex (MHC) class II and B7 molecules, whereas DCs of control mice fed OVA expressed low levels of these molecules. The adoptive transfer of NKT cells restored oral tolerance and induction of tolerogenic DCs in the PPs and spleens of CD1d-/- mice. Moreover, interleukin (IL)-10 and transforming growth factor (TGF)-beta1 production in vitro were reduced in cells from the spleen and PPs of CD1d-/- mice compared with those of control mice fed OVA. The numbers of OVA-specific CD4+ KJ1-26+ T cells were significantly reduced in the PPs and spleens of DO11.10 mice fed OVA. In contrast, OVA-specific CD4+ KJ1-26+ T cells were not deleted in the PPs or spleens of DO11.10 CD1d-/- mice. In conclusion, NKT cells were found to play an indispensable role in oral tolerance by inducing regulatory T cells, and clonally deleting antigen-specific CD4+ T cells.     

Regulatory T cells require renal antigen recognition through the TCR to protect against injury in nephritis

Regulatory T cells (Treg) are important for maintaining immune homeostasis. Adoptive transfer of Tregs is protective in renal disease models in both immunocompetent and immunodeficient mice. However the involvement of TCR recognition of renal antigens remains to be clarified. To address this question, we made use of Tregs from the DO11.10 mouse (a TCR transgenic (Tg) mouse), that recognise the non-murine antigen Ovalbumin (OVA) and therefore are not activated by renal antigens. DO11.10 Tregs were assessed functionally in vitro and demonstrated equivalent suppression to WT BALB/c Tregs. Adriamycin Nephropathy (AN) was induced in mice which had been transfused with CD4⁺CD25⁺Tregs isolated from DO11.10 or BALB/c mice. To eliminate the memory/activation state as a cause of differences in activity, the protective capacity of DO11.10 Tregs pre-activated with OVA in vivo was assessed. Transfer of WT BALB/c Tregs significantly attenuated the development of AN with less glomerulosclerosis, tubular atrophy and macrophage infiltration as compared to AN mice without Treg transfer. However, mice receiving either naïve or pre-activated DO11.10 Tregs were not protected from AN. The lack of protection by DO11.10 Tregs was not due to failure to traffic to the affected kidney. These results suggest that antigen recognition in the kidney is important for Treg protection against injury.     

Evidence for a repertoire of functional untolerized CD4+ T cells specific for melanoma-associated antigens

Active vaccination against melanoma requires tolerance break as melanoma-associated antigens (MAA) used in vaccine formula are mostly self-antigens. While tolerance to MAA in the CD8(+) T cell compartment is well characterized, it is still not the case for the CD4(+) T cell compartment. Here, we analysed CD4(+) T cell tolerance to such antigens in mice genetically engineered to express ovalbumin (OVA) in melanocytes (Tyr-OVA mice). When we crossed Tyr-OVA mice with DO11.10 and OT-II mice transgenic for an OVA-specific TCR restricted by MHC class II, we observed different tolerization levels. Central tolerance was complete for high avidity DO11.10 CD4(+) T cells, but absent for low avidity OT-II CD4(+) T cells. OT-II CD4(+) T cells also ignored OVA in the periphery of Tyr-OVA mice, albeit being potently reactive to vaccination. OVA challenge in single transgenic Tyr-OVA mice confirmed the existence of OVA-reactive CD4(+) T cells with the induction of efficient T helper cells for antibody production and anti-tumour T cell response. In total, our study demonstrates the existence of low avidity MAA-specific CD4(+) T cells escaping by ignorance central and peripheral tolerance, but valuable in the context of vaccination against melanoma.     

Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed‐type hypersensitivity (DTH) responses? To what extent can s.l.‐induced tolerance be explained by the generation of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (T_reg)? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40‐μg doses of OVA/CTB conjugate suppressed T‐cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag‐specific T_reg in CLN, MLN and spleen and also transforming growth factor‐β (TGF‐β) levels in serum. The CLN and splenic cells from OVA/CTB‐treated BALB/c mice efficiently suppressed OVA‐specific T‐cell receptor (TCR) transgenic (DO11.10) CD25^?CD4⁺ effector T‐cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA‐specific DTH responses in vivo and T‐cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA‐specific (KJ1‐26⁺) CD4⁺ T cells from OVA/CTB‐treated mice. Thus, s.l. administration of CTB‐conjugated Ag can efficiently induce peripheral T‐cell tolerance associated with strong increases in serum TGF‐β levels and in Ag‐specific Foxp3⁺CD25⁺CD4⁺ T_reg cells.     

In vitro‐induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites

Recently, IL‐17 produced by Th17 cells was described as pro‐inflammatory cytokine with an eminent role in autoimmune diseases, e.g. rheumatoid arthritis. A lack of IL‐17 leads to amelioration of collagen‐induced arthritis. IL‐17 induction in naïve CD4⁺ T cells depends on IL‐6 and TGF‐β and is enhanced by IL‐23. The in vivo inflammatory potential of in vitro‐primed Th17 cells however, remains unclear. Here, we show that, although IL‐17 neutralisation results in amelioration of murine OVA‐induced arthritis, in vitro‐primed Th17 cells cannot exacerbate arthritic symptoms after adoptive transfer. Furthermore, Th17 cells cannot induce an inflammatory delayed type hypersensitivity reaction because they fail to migrate into inflamed sites, possibly due to the lack of CXCR3 expression. Also, re‐isolated Th17 cells acquired IFN‐γ expression, indicating instability of the Th17 phenotype. Taken together, the data show that IL‐6, TGF‐β and IL‐23 might not provide sufficient signals to induce “fully qualified” Th17 cells.     

Memory/effector T cells in TCR transgenic mice develop via recognition of enteric antigens by a second, endogenous TCR.

The majority of clonotypic CD4(+) T cells in the intestinal lamina propria of DO11.10 TCR transgenic mice have an activated/memory phenotype and produce effector cytokines despite the absence of prior exposure to ovalbumin (OVA), the transgene-specific antigen. A small number of splenic T cells have a similar phenotype. Clonotypic T cells from Peyer's patch are intermediate in both phenotype and effector cytokine production. Flow cytometric analysis of cells isolated from thymectomized, OVA-naive DO11.10 mice treated with continuous administration of BrdU indicated that a significant fraction of clonotype-positive T cells in the lamina propria and Peyer's patch were in the cell cycle, with significantly fewer cycling cells in the spleen. Most of the cycling cells from each anatomic site expressed low levels of CD45RB. Effector cytokine expression was enriched in the CD45RB(low) populations. These memory/effector cell populations were eliminated in DO11.10/SCID and DO11.10/RAG-2(-/-) mice, suggesting that recognition of non-OVA antigens through a second, non-clonotypic TCR was driving differentiation of memory/effector cells in naive BALB/c DO11.10 mice. Clonotypic CD4(+) T cells isolated from DO11.10, but not from DO11.10/SCID or DO11.10/RAG-2(-/-) mice, were stimulated to enter the cell cycle by antigen-presenting cells pulsed with an intestinal bacterial antigen extract. These data provide direct evidence that enteric bacterial antigens can activate transgenic T cells through a second, non-clonotypic TCR, and support the notion that the development and turnover of memory/effector cells in vivo is driven by the intestinal flora.     

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.     

Tumour necrosis factor-alpha but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation

Using murine spleen-derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T-cell (Th) differentiation were analysed. Immature DC (iDC) with high- or low-dose OVA(323-339) predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor-alpha (TNF/DC) induced a significantly higher Th2 response in the presence of low-dose OVA(323-339) than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high-dose OVA(323-339), LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low-dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high-dose antigen to Th1 polarization were not related to the amount of IL-12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low-dose antigen are potent inducers of Th2 differentiation.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

Enhanced antigen-specific primary CD4+ and CD8+ responses by codelivery of ovalbumin and toll-like receptor ligand monophosphoryl lipid A in poly(D,L-lactic-co-glycolic acid) nanoparticles

The purpose of this research was to investigate the use of biodegradable poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA-NP) as a vaccine delivery system to codeliver antigen, ovalbumin (OVA) along with monophosphoryl lipid A (MPLA) as adjuvant for induction of potent CD4(+) and CD8(+) T cell responses. The primary CD4(+) T responses to OVA/MPLA NP were investigated using OVA-specific T cells from DO11.10 transgenic mice. Following adoptive transfer of these cells, mice were immunized s.c. by NP formulations. For assessing the CD8(+) responses, bone marrow derived dendritic cells (DCs) were pulsed with different OVA formulations, then, cocultured with CD8(+) T cells from OT-1 mice. T cell proliferation/activation and IFN-gamma secretion profile have been examined. Particulate delivery of OVA and MPLA to the DCs lead to markedly increase in in vitro CD8(+) T cell T cell proliferative responses (stimulation index >3000) and >13-folds increase in in vivo clonal expanded CD4(+) T cells. The expanded T cells were capable of cytokine secretion and expressed an activation and memory surface phenotype (CD62L(lo), CD11a(hi), and CD44(hi)). Codelivery of antigen and MPLA in PLGA-NP offers an effective method for induction of potent antigen specific CD4(+) and CD8(+) T cell responses.     

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

UVB-irradiated dendritic cells are impaired in their APC function and tolerize primed Th1 cells but not naive CD4+ T cells

We have shown that low-dose UVB radiation converts Langerhans cells (LC) from immunogenic to tolerogenic APC. Therefore, we questioned whether low-dose UVB irradiation of bone marrow-derived dendritic cells (DC) alters their APC function, thereby inducing tolerance in T cells. To address this issue, cocultures of DC; and naive, allogeneic T cells; na?ve, OVA-specific TCR-transgenic T cells from DO11.10 mice; or primed, antigen-specific T cells using the Th1 clone AE7 were analyzed. First, we found low-dose UVB-irradiated DC (UVB-DC) to dose-dependently (50-200 J/m2) inhibit T-cell proliferation of naive and primed T cells. In addition, supernatants harvested from cocultures of UVB-DC and naive T cells showed markedly reduced levels of IL-2 and IFN-gamma and to a lesser degree of IL-4 and IL-10, suggesting a preferential down-regulation of Th1 responses by UVB-DC. FACS analysis of UVB-DC revealed no changes in surface expression of MHC, costimulatory, and adhesion molecules. To test tolerance induction, allo- or antigen-specific T cells isolated from cocultures with unirradiated DC and UVB-DC were restimulated with unirradiated DC or IL-2. It is interesting that UVB-DC induced antigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DC induced a partial inhibition of allogeneic T-cell proliferation but no tolerance with similar unresponsiveness to restimulation with IL-2 and unirradiated DC irrespective of their haplotype. Similar observations were made when naive, TCR-transgenic T cells from DO11.10 mice were used. In conclusion, UVB-DC are impaired in their APC function and tolerize the primed antigen-specific Th1 clone AE7 but not naive allo- or OVA-specific T cells.