CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28^−/− mice, Th2-mediated airway inflammation was not induced in CD28^−/− mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS^−/− mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.     

T-helper-1 and T-helper-2 immune responses in mice infected with the intestinal fluke Neodiplostomum seoulense: their possible roles in worm expulsion and host fatality

Neodiplostomum seoulense is highly pathogenic and lethal to experimental mice; most worms are expelled within 2 mo of acquisition. In this study, T-helper (Th) cell immune responses were studied in N. seoulense-infected BALB/c mice. Spleen and mesenteric lymph node (MLN) cells of infected mice proliferated in response to parasite antigens; CD4+ T cells proliferated more than CD8+ T cells. Antigen-induced interferon (IFN)-gamma (a Th1 cytokine) secretion began to increase at day 7 postinfection (PI) in spleen and MLN cells, and this was maintained at day 28 PI, whereas interleukin (IL)-4 (a Th2 cytokine) secretion was somewhat lower. Similar results were observed for mRNA signals of IFN-gamma and IL-4. Antigen-specific serum total immunoglobulin (Ig)G, IgG1, IgM, and IgA levels (Th2-induced) were elevated from days 7 to 14 to day 28 PI, and IgG2a (Th1-induced) was elevated at days 21 to 28 PI. Interestingly, the numbers of macrophages (Th1- or Th2-induced), which were found to kill N. seoulense worms in vitro, increased remarkably during days 14-28 PI in spleens and small intestines of infected mice. This study shows that mixed Th1 and Th2 responses occur during the course of N. seoulense infection in BALB/c mice. Heavy infiltrations of macrophages in the small intestine may participate in host damage and worm expulsion.     

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-gamma. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28(-/-) mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-gamma suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28(-/-) mice with T. gondii resulted in the increased expression of ICOS by activated CD4(+) and CD8(+) T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-gamma by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-gamma production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28(-/-) mice to T. gondi associated with reduced serum levels of IFN-gamma, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.     

The characterization of intraepithelial lymphocytes, lamina propria leukocytes, and isolated lymphoid follicles in the large intestine of mice infected with the intestinal nematode parasite Trichuris muris

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effector mechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohistochemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) from resistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria after infection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, the number of infiltrating CD4+, CD8+, B220+, and F4/80+ IEL and LPL was generally highest around the time of worm expulsion from the gut, at which point the inflammation was dominated by CD4+ IEL and F4/80+ LPL. In contrast, in susceptible mice, which mount a Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At this point, CD8+ IEL and F4/80+ LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsion of worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time, we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4+ T cells and having a central zone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. muris infection.     

Cutting edge: inducible costimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis.

The CD28 family member inducible costimulator protein (ICOS) has an important role in T cell differentiation and Ig class switching. To investigate the role of ICOS in vivo, ICOS-/- mice were infected s.c. with Leishmania mexicana. While wild-type mice developed large, cutaneous lesions, the growth of lesions and tissue histopathology was significantly delayed in ICOS-/- mice. ICOS-/- mice exhibited marked decreases in both Th1 and Th2 cytokine production and profound defects in L. mexicana-specific Ig isotype class switching to IgG1 and IgG2a and reduced total IgE levels. Our findings indicate that ICOS is a key regulator of both Th1 and Th2 responses and has a role in controlling cutaneous L. mexicana infection.     

ICOS deficiency is associated with a severe reduction of CXCR5+CD4 germinal center Th cells

ICOS is expressed on activated T cells and particularly on CXCR5+ follicular Th cells in germinal centers (GC). Its deletion leads to a profound deficiency in memory B cell formation and switched Ab response in humans. Here, we show that in ICOS-deficient patients the generation of GCs is severely disturbed, and the numbers of circulating CXCR5+CD45RO+ memory CD4 T cells are significantly reduced, indicating an essential role of ICOS in the differentiation of CXCR5+CD4 T cells. The GC-specific CD57+CXCR5+ subpopulation is virtually absent. In ICOS-/- mice, the decrease of circulating CXCR5+CD4 T cells reflects the reduction of CXCR5+ follicular Th cells in lymph nodes and spleen. Therefore, in concurrence with the absence of CXCR5+ T cells in the blood of CD40L-deficient patients, these data support the hypothesis that circulating CD57+CXCR5+ T cells are GC derived and thus may serve as a surrogate marker for the presence of functional GCs in humans.     

CD2 facilitates differentiation of CD4 Th cells without affecting Th1/Th2 polarization.

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2(a) mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [(3)H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.     

Goblet cell-derived resistin-like molecule beta augments CD4+ T cell production of IFN-gamma and infection-induced intestinal inflammation

The secreted goblet cell-derived protein resistin-like molecule beta (RELMbeta) has been implicated in divergent functions, including a direct effector function against parasitic helminths and a pathogenic function in promoting inflammation in models of colitis and ileitis. However, whether RELMbeta influences CD4(+) T cell responses in the intestine is unknown. Using a natural model of intestinal inflammation induced by chronic infection with gastrointestinal helminth Trichuris muris, we identify dual functions for RELMbeta in augmenting CD4(+) Th1 cell responses and promoting infection-induced intestinal inflammation. Following exposure to low-dose Trichuris, wild-type C57BL/6 mice exhibit persistent infection associated with robust IFN-gamma production and intestinal inflammation. In contrast, infected RELMbeta(-/-) mice exhibited a significantly reduced expression of parasite-specific CD4(+) T cell-derived IFN-gamma and TNF-alpha and failed to develop Trichuris-induced intestinal inflammation. In in vitro T cell differentiation assays, recombinant RELMbeta activated macrophages to express MHC class II and secrete IL-12/23p40 and enhanced their ability to mediate Ag-specific IFN-gamma expression in CD4(+) T cells. Taken together, these data suggest that goblet cell-macrophage cross-talk, mediated in part by RELMbeta, can promote adaptive CD4(+) T cell responses and chronic inflammation following intestinal helminth infection.     

Protective effector memory CD4 T cells depend on ICOS for survival

Memory CD4 T cells play a vital role in protection against re-infection by pathogens as diverse as helminthes or influenza viruses. Inducible costimulator (ICOS) is highly expressed on memory CD4 T cells and has been shown to augment proliferation and survival of activated CD4 T cells. However, the role of ICOS costimulation on the development and maintenance of memory CD4 T cells remains controversial. Herein, we describe a significant defect in the number of effector memory (EM) phenotype cells in ICOS(-/-) and ICOSL(-/-) mice that becomes progressively more dramatic as the mice age. This decrease was not due to a defect in the homeostatic proliferation of EM phenotype CD4 T cells in ICOS(-/-) or ICOSL(-/-) mice. To determine whether ICOS regulated the development or survival of EM CD4 T cells, we utilized an adoptive transfer model. We found no defect in development of EM CD4 T cells, but long-term survival of ICOS(-/-) EM CD4 T cells was significantly compromised compared to wild-type cells. The defect in survival was specific to EM cells as the central memory (CM) ICOS(-/-) CD4 T cells persisted as well as wild type cells. To determine the physiological consequences of a specific defect in EM CD4 T cells, wild-type and ICOS(-/-) mice were infected with influenza virus. ICOS(-/-) mice developed significantly fewer influenza-specific EM CD4 T cells and were more susceptible to re-infection than wild-type mice. Collectively, our findings demonstrate a role for ICOS costimulation in the maintenance of EM but not CM CD4 T cells.     

Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.     

CD4+CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells,Leishmania major infection,and colitis in mice

Regulatory T cells play a major role in modulating the immune response. However, most information on these cells centers on autoimmunity, and there is also considerable controversy on the functional characteristics of these cells. Here we provide direct in vitro and in vivo evidence that CD4+CD25+ regulatory T cells inhibit the differentiation and functions of both Th1 and Th2 cells. Importantly, CD4+CD25+ T cells suppressed the disease development of Leishmania major infection in SCID mice reconstituted with naive CD4+CD25- T cells. Furthermore, CD4+CD25+ T cells inhibited the development of colitis induced by both Th1 and Th2 cells in SCID mice. Our results therefore document that CD4+CD25+ regulatory T cells suppress both Th1 and Th2 cells and that these regulatory T cells have a profound therapeutic potential against diseases induced by both Th1 and Th2 cells in vivo.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

Characterization of tumor-infiltrating CD4+ T cells as Th1 cells based on lymphokine secretion and functional properties

Recent studies have subdivided the Th cells into mutually exclusive Th1 subset producing IL-2 and IFN-gamma and Th2 cells producing IL-4 and IL-5. The relative role played by these two subsets in the antitumor immunity is not clear. We earlier demonstrated that treatment of C57BL/6 mice bearing a syngeneic Ia- T cell lymphoma, LSA, with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) resulted in 90 to 100% survival of the mice. Furthermore, host's T cell responses were critical for successful BCNU-mediated cures. In our study we observed that immediately after BCNU treatment, there was a dramatic increase in the percentage of CD4+ T cells at the site of tumor growth in the peritoneal cavity. The percentage of CD4+ T cells increased from approximately 3 to 4% found in normal or tumor-bearing mice to approximately 41% in BCNU-treated tumor-bearing mice. The percentage of CD8+ T cells also increased although to a lesser degree. Also, these alterations were primarily restricted to the site of tumor-growth inasmuch as they were not seen in the thymus and were less pronounced in the spleen. The tumor-infiltrating CD4+ T cells obtained after BCNU-treatment, when further characterized, were found to secrete only IL-2 and IFN-gamma but not IL-4, after tumor-specific stimulation. Furthermore, the supernatants from LSA-activated CD4+ T cell cultures failed to provide help to the B cells but were able to activate the macrophages to inhibit the tumor cell proliferation. The CD4+ T cells when adoptively transferred could also protect the nude mice from LSA tumor challenge and induced tumor-specific delayed-type hypersensitivity reaction. Together our data suggest that in the LSA tumor model, the tumor-infiltrating CD4+ T cells have the properties of Th1 cells and these cells can mediate tumor-rejection independent of the CD8+ T cells by activating the macrophages.     

Functional and quantitative analysis of splenic T cell immune responses following oral toxoplasma gondii infection in mice

Functional and quantitative analysis of splenic T cell immune responses following oral Toxoplasma gondii infection in mice. Experimental Parasitology 91, 212-221. Immunity to Toxoplasma gondii is mediated primarily by the host T cell response. Although there is considerable information regarding host immunity following intraperitoneal infection with tachyzoites, little information is available regarding naturally acquired infection following peroral infection with bradyzoites. In this study, a sequential quantitative analysis of the cell-mediated immune response was performed at the single cell level. To assess the kinetics of this response and parasitic loads, inbred mice were orally infected with the 76K strain bradyzoites of T. gondii. Within 24 h of infection, follicular hyperplasia followed by infiltration with histiocytes, macrophages, and apoptotic bodies was observed in the spleens of infected mice. T. gondii were detected from day 1, and counts increased gradually during the experimental period. Splenocyte DNA synthesis to antigen and mitogen was severely suppressed at days 7 and 10. The percentages of NK1.1(+) or delta gamma T cells were increased from day 1, whereas CD4(+) and CD8alpha+ T cells were signficantly increased after day 7 postinfection. CD25 expression and intracellular IFN-gamma production increased in NK1.1(+) cells on day 1 and by all other T cell subsets after day 4. Intracellular IL-4 did not increase until day 7, and IL-10 production was increased in all T cell subsets after day 4. Together, these findings indicate that oral infection with T. gondii stimulates a strong cellular immune response that appears to polarize toward an early Th1 response. However, within 7 days, a strong immune Th2 regulatory response as well as high parasitic loads can be observed, with a reduction in lymphoproliferation to mitogen stimulation, increased production of IL-4 and IL-10, and evidence of T cell apoptosis in the splenic immune compartment.     

Antigen-specific Th1 but not Th2 cells provide protection from lethal Trypanosoma cruzi infection in mice

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.     

Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8+ CTL

Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8+ T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8+ T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8+ CTL in the absence of CD4+ Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8+ CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8+ T cells isolated from IFN-gamma-/- and IFN-gamma receptor-/- mice. However, BMDC1 produced higher levels of IFN-beta than other BMDC subsets, and anti-IFN-beta mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-beta, but not IFN-gamma, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8+ T cells into CTL.     

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.     

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.