Cutting edge: the NK cell receptor 2B4 augments antigen-specific T cell cytotoxicity through CD48 ligation on neighboring T cells

2B4 is expressed on all NK and a subset of memory/effector CD8(+) T cells. 2B4 binds to CD48 and activates NK cytotoxicity, but its function on CD8(+) T cells is not clear. Furthermore, two isoforms of 2B4 (2B4S and 2B4L) exist in mice but the role of individual isoforms is not known. To address these questions, we generated primary T cell cultures from L(d)-specific 2C/Rag2(-/-) TCR transgenic mice and transduced them with 2B4S or 2B4L. 2B4S- or 2B4L-transduced T cells showed greater cytotoxicity over control cells against CD48(+) and CD48(-) targets, suggesting that ligation of 2B4 by CD48 on target cells was not necessary for 2B4 function. Rather, 2B4/CD48 interaction on adjacent T cells appeared to be critical for cytotoxicity. Therefore, 2B4 functions as a costimulator of CD8(+) T cells in MHC-restricted cytotoxicity. We conclude that 2B4/CD48 interactions among T cells themselves can augment CTL lysis of their specific targets.     

Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells

Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Villous B cells of the small intestine are specialized for invariant NK T cell dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM(+) IgD(+) cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinase(xid), Galphai2(-/-)), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jalpha18(-/-) or CD1d(-/-) mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.     

CD4 T cell-mediated rejection of cardiac allografts in B cell-deficient mice

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.muMT(-/-) recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.muMT(-/-) CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.muMT(-/-) spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.muMT(-/-) mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.muMT(-/-) CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.muMT(-/-) mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-gamma production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.     

B cells and CD4-CD8- T cells are key regulators of the severity of reactivation histoplasmosis

The fungus, Histoplasma capsulatum, produces a persistent infection. Reactivation histoplasmosis is largely a result of impaired immunity, but the perturbations associated with escape of the fungus from host defenses remain ill-defined. We analyzed a murine model of reactivation to elucidate the host defects that permit reactivation. C57BL/6 mice were infected intranasally and, 42 days later, they were depleted of CD4(+) and CD8(+) cells. Elimination of these cells, but not either alone, produced a persistent infection over several weeks. Neutralization of IFN-gamma, TNF-alpha, or both did not induce reactivation. Endogenous IL-10 exacerbated reactivation. Depletion of T cells in B cell(-/-) mice induced a markedly higher burden in organs when compared with wild type. However, the infection remained persistent. Elimination of CD4(+) cells alone or neutralization of cytokines increased the fungal load. The persistent infection was not dependent on gammadelta T cells or NK cells. Elimination of Thy-1.2(+) cells in mice given mAb to CD4 and CD8 transformed reactivation into a progressive, lethal infection in B cell(-/-) and wild-type mice, but the tempo of progression was accelerated in the former. The data reveal the complex control by the host to prevent reactivation of this fungus.     

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B(-/-)) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B(-/-) C57BL mice infected with the more persisting virus, virus-specific CD8(+) T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B(-/-) BALB/c mice, virus-specific CD8(+) T cells are completely deleted, indicating that host genotype influences the severity of the T cell defect. In B(-/-) C57BL mice infected with the less persisting virus, CD8(+) T cell dysfunction was not as pronounced, although it was clearly present. Most importantly, the appearance of dysfunctional CD8(+) T cells clearly precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B(-/-) mice to produce neutralizing Abs. In contrast with CD8(+) T cells, which initially respond almost as in wild-type mice, the priming of virus-specific CD4(+) T cells was markedly impaired in B(-/-) mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects are likely to contribute to the chronic course of viral infection in B(-/-) mice.     

Mechanism of induction of NK activation by 2B4 (CD244) via its cognate ligand

We have previously shown that coincubation of purified B cells with IL-2-propagated NK cells can result in the induction of IL-13 mRNA and that the induction requires the presence of CD48 on B cells and 2B4 on NK cells. Because both of these molecules are expressed on NK cells, it is surprising that very little IL-13 mRNA can be detected in the absence of B cells. We have now found that incubation of NK cells on plates containing immobilized anti-CD48 Abs results in the clustering of CD48 and colocalization with 2B4 on the same cell. This colocalization, together with the requirement for SAP, the signal transducer for 2B4, is necessary for the induction of IL-13 mRNA expression. Activation of NK cell via CD48 on another cell may require a similar ability to alter the configuration of 2B4 to activate downstream signaling. By the use of double CD2/2B4 knockout mice, we have also shown that the induction of NK cell activation by anti-CD48 or by B cells is not due to the release of inhibitory effects of 2B4.     

TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.     

The murine NK receptor 2B4 (CD244) exhibits inhibitory function independent of signaling lymphocytic activation molecule-associated protein expression

2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-gamma production. In this report, we used CD48+/- variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.     

2B4/CD48-mediated regulation of lymphocyte activation and function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.     

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.