IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

Detection of antigen-specific T cells in experimental immune-mediated blepharoconjunctivitis in DO11.10 T cell receptor transgenic mice

Antigen (Ag)-specific T cells are thought to play a key role in pathogenesis of chronic allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). In order to investigate the trafficking of Ag-specific T cells in experimental immune-mediated blepharoconjunctivitis (EC), we established a novel AC model in DO11.10 T cell receptor (TcR) transgenic (Tg) mice. DO11.10 TcR-Tg mice were challenged with eye drops of whole OVA protein, OVA peptide 1-15, 321-335, or 323-339. Their eyes were histologically examined. Conventional proliferation assay was performed against each Ag. Phenotypes of infiltrating cells and kinetics of Ag-specific T cells were investigated by immunohistochemistry. Adoptive transfer of CD4(+) Ag-specific T cells from DO11.10 TcR-Tg to WT mice was performed. The distribution of KJ1-26(+) cells was investigated in recipient mice. The challenge of OVA peptide 323-339 induced infiltration of inflammatory cells in conjunctivae in a dose dependent manner, accompanied by the proliferative responses of splenocytes. Immunohistochemical analysis revealed Agspecific/ non-Ag-specific T cells, macrophages, and eosinophils in conjunctivae. Infiltration of Ag-specific T cells increased 24 hr later. Transfer of CD4(+) cells from DO11.10 TcR-Tg to WT mice induced EC depending on the number of transferred cells. Ag-specific T cells were detected in the conjunctivae and spleens of recipient mice, though its numbers were significantly smaller compared to DO11.10 TcR-Tg mice. The challenge of OVA peptide 323-339 induced EC in DO11.10 TcR-Tg mice without prior sensitization. The response was mediated by CD4(+) Ag-specific T cells. The trafficking of Ag-specific T cells in EC was clearly visualized.     

Lung CD25 CD4 regulatory T cells suppress type 2 immune responses but not bronchial hyperreactivity

To study the effects of chronic Ag deposition in the airway mucosa on CD4(+) T cell priming and subsequent airway disease, transgenic mice were generated that expressed OVA under the control of the surfactant protein C promoter. CD4 T cells from these mice were tolerant to OVA but this was overcome among spleen CD4 T cells by crossing to OVA-specific DO11.10 TCR-transgenic mice. Lungs from the double-transgenic mice developed lymphocytic infiltrates and modest mucus cell hyperplasia. Infiltrating cells were unaffected by the absence of either Rag-1 or Stat6, although the latter deficiency led to the disappearance of mucus. In the lung of double-transgenic mice, a large number of Ag-specific CD4 T cells expressed CD25 and functioned as regulatory T cells. The CD25(+) CD4 T cells suppressed proliferation of CD25(-) CD4 T cells in vitro and inhibited type 2 immune responses induced by aerosolized Ags in vivo. Despite their ability to suppress allergic type 2 immunity in the airways, however, CD25(+) CD4 regulatory T cells had no effect on the development of bronchial hyperreactivity.     

The role of OX40 ligand interactions in the development of the Th2 response to the gastrointestinal nematode parasite Heligmosomoides polygyrus

In these studies, we examined the effects of OX40 ligand (OX40L) deficiency on the development of Th2 cells during the Th2 immune response to the intestinal nematode parasite Heligmosomoides polygyrus. Elevations in IL-4 production and total and Ag-specific serum IgE levels were partially inhibited during both the primary and memory immune responses to H. polygyrus in OX40L(-/-) mice. The host-protective memory response was compromised in OX40L(-/-) mice, as decreased worm expulsion and increased egg production were observed compared with H. polygyrus-inoculated OX40L(+/+) mice. To further examine the nature of the IL-4 defect during priming, adoptively transferred DO11.10 T cells were analyzed in the context of the H. polygyrus response. Although Ag-specific T cell IL-4 production was reduced in the OX40L(-/-) mice following immunization with OVA peptide plus H. polygyrus, Ag-specific T cell expansion, cell cycle progression, CXCR5 expression, and migration were comparable between OX40L(+/+) and OX40L(-/-) mice inoculated with OVA and H. polygyrus. These studies suggest an important role for OX40/OX40L interactions in specifically promoting IL-4 production, as well as associated IgE elevations, in Th2 responses to H. polygyrus. However, OX40L interactions were not required for serum IgG1 elevations, increases in germinal center formation, and Ag-specific Th2 cell expansion and migration to the B cell zone.     

Early events in peripheral regulatory T cell induction via the nasal mucosa

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation.

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323-339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-gamma. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.     

GIF inhibits Th effector generation by acting on antigen-presenting B cells

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50--95% reduction of IL-4 and IFN-gamma secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA(323--339) peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.     

T regulatory cells 1 inhibit a Th2-specific response in vivo

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

Generation of CD4(+)CD25(+) regulatory T cells from autoreactive T cells simultaneously with their negative selection in the thymus and from nonautoreactive T cells by endogenous TCR expression

Normal T cell repertoire contains regulatory T cells that control autoimmune responses in the periphery. One recent study demonstrated that CD4(+)CD25(+) T cells were generated from autoreactive T cells without negative selection. However, it is unclear whether, in general, positive selection and negative selection of autoreactive T cells are mutually exclusive processes in the thymus. To investigate the ontogeny of CD4(+)CD25(+) regulatory T cells, neo-autoantigen-bearing transgenic mice expressing chicken egg OVA systemically in the nuclei (Ld-nOVA) were crossed with transgenic mice expressing an OVA-specific TCR (DO11.10). Ld-nOVA x DO11.10 mice had increased numbers of CD4(+)CD25(+) regulatory T cells in the thymus and the periphery despite clonal deletion. In Ld-nOVA x DO11.10 mice, T cells expressing endogenous TCR alpha beta chains were CD4(+)CD25(-) T cells, whereas T cells expressing autoreactive TCR were selected as CD4(+)CD25(+) T cells, which were exclusively dominant in recombination-activating gene 2-deficient Ld-nOVA x DO11.10 mice. In contrast, in DO11.10 mice, CD4(+)CD25(+) T cells expressed endogenous TCR alpha beta chains, which disappeared in recombination-activating gene 2-deficient DO11.10 mice. These results indicate that part of autoreactive T cells that have a high affinity TCR enough to cause clonal deletion could be positively selected as CD4(+)CD25(+) T cells in the thymus. Furthermore, it is suggested that endogenous TCR gene rearrangement might critically contribute to the generation of CD4(+)CD25(+) T cells from nonautoreactive T cell repertoire, at least under the limited conditions such as TCR-transgenic models, as well as the generation of CD4(+)CD25(-) T cells from autoreactive T cell repertoire.     

Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo

Flagellin is secreted by many enteric bacteria and, upon reaching the basolateral membrane of the intestinal epithelium, activates Toll-like receptor 5-mediated innate immune signaling pathways. We hypothesized that any flagellin that gets beyond the epithelium might also regulate cells of the adaptive immune system. Here we demonstrate that the clonal expansion of naive DO11.10 CD4 T cells in response to OVA peptide (323-339) was enhanced 3- to 10-fold in the presence of purified bacterial flagellin in vivo. OVA-specific CD4 T cells were also shown to have undergone more cell division in vivo if flagellin was coinjected with OVA. Flagellin administration increased the expression of B7-1 on splenic dendritic cells, and coinjection of CTLA4-Ig, which is known to block B7 function in vivo, completely ablated the adjuvant effect on CD4 T cells. Therefore, a conserved bacterial protein produced by many intestinal microbes can modulate CD4 T cell activation in vivo. Such an adjuvant effect for flagellin has important implications for vaccine development and the generation of CD4 T cell responses to enteric bacteria.     

Dual TCR expression biases lung inflammation in DO11.10 transgenic mice and promotes neutrophilia via microbiota-induced Th17 differentiation

A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4(+) cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2(-/-) mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2(-/-) DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.     

Dopamine D1-like receptor antagonist attenuates Th17-mediated immune response and ovalbumin antigen-induced neutrophilic airway inflammation

Allergic airway inflammation is generally considered a Th2-type immune response. Recent studies, however, demonstrated that Th17-type immune responses also play important roles in this process, especially in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We previously reported that dendritic cells release dopamine to naive CD4(+) T cells in Ag-specific cell-cell interaction, in turn inducing Th17 differentiation through dopamine D1-like receptor (D1-like-R). D1-like-R antagonist attenuates Th17-mediated diseases such as experimental autoimmune encephalomyelitis and autoimmune diabetes. However, the effect of antagonizing D1-like-R on Th17-mediated airway inflammation has yet to be studied. In this study, we examined whether D1-like-R antagonist suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice and then elucidated the mechanism of action. DO11.10 mice were nebulized with OVA or PBS, and some mice received D1-like-R antagonist orally before OVA nebulization. D1-like-R antagonist significantly suppressed OVA-induced neutrophilic airway inflammation in DO11.10 mice. It also inhibited the production of IL-17 and infiltration of Th17 cells in the lung. Further, D1-like-R antagonist suppressed the production of IL-23 by lung CD11c(+) APCs. In contrast, D1-like-R antagonist did not increase Foxp3(+) regulatory T cells in the lung. D1-like-R antagonist neither suppressed nonspecific LPS-induced neutrophilic airway inflammation nor OVA-induced eosinophilic airway inflammation. These results indicate that D1-like-R antagonist could suppress Th17-mediated neutrophilic airway inflammation, raising the possibility that antagonizing D1-like-R serves as a promising new strategy for treating neutrophil-dominant severe asthma.     

Role of B7 in T cell tolerance

The induction of effective immune responses requires costimulation by B7 molecules, and Ag recognition without B7 is thought to result in no response or tolerance. We compared T cell responses in vivo to the same Ag presented either by mature dendritic cells (DCs) or as self, in the presence or absence of B7. We show that Ag presentation by mature B7-1/2-deficient DCs fails to elicit an effector T cell response but does not induce tolerance. In contrast, using a newly developed adoptive transfer system, we show that naive OVA-specific DO11 CD4+ T cells become anergic upon encounter with a soluble form of OVA, in the presence or absence of B7. However, tolerance in DO11 cells transferred into soluble OVA transgenic recipients can be broken by immunization with Ag-pulsed DCs only in B7-deficient mice and not in wild-type mice, suggesting a role of B7 in maintaining tolerance in the presence of strong immunogenic signals. Comparing two double-transgenic models--expressing either a soluble or a tissue Ag--we further show that B7 is not only essential for the active induction of regulatory T cells in the thymus, but also for their maintenance in the periphery. Thus, the obligatory role of B7 molecules paradoxically is to promote effective T cell priming and contain effector responses when self-Ags are presented as foreign.     

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.     

CD18 is required for intestinal T cell responses at multiple immune checkpoints

The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4(+) T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18(-/-) DO11.10 CD4(+) T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, alpha(4)beta(7) and CCR9. Interestingly, the defect in trafficking of CD18(-/-) DO11.10 CD4(+) T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.     

Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.