PERSPECTIVE
The B7/CD28/CTLA4 T-Cell Activation Pathway
Implications for Inflammatory Lung Disease

Jonathan M. Green

Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri

The molecular events that modulate the T-cell response to antigen are key determinants in the outcome of pulmonary immune responses. Over the past several years, it has become evident that the B7:CD28 costimulation pathway is a critical regulator of T-cell responses both in vitro and in vivo. Given its importance in T-cell activation and its potential as a target for immune-based therapies, this perspective will review the current data on the regulation of T-cell activation by the B7 costimulatory pathway.

In 1970 Bretscher and Cohn put forth the two-signal model of lymphocyte activation to explain self/nonself discrimination (1). This model proposes that T-cell activation requires two independent signals. The first is transduced through the T-cell receptor (TCR) after engagement by antigen; and the second, costimulatory signal is delivered by ligation of a distinct receptor present on the surface of the T cell. This model predicts that engagement of the TCR in the absence of costimulation will fail to activate the T cell. A substantial body of evidence has accumulated in support of this hypothesis. Early studies demonstrated that antigen presented by chemically fixed antigen-presenting cells (APCs) resulted in a failure of T-cell activation and rendered them unresponsive to further antigenic stimulation, a process termed induction of anergy. This was shown to be the result of the inability of the fixed APCs to engage costimulatory molecules, in particular CD28 (2). The anergic T cell fails to produce the autocrine growth factor interleukin-2 (IL-2) upon stimulation, and addition of exogenous IL-2 can reverse the anergic state (3).

The molecular basis for costimulation remained elusive until the cloning of CD28 by Arrufo and Seed in 1987 (4). First termed Tp44, crosslinking of CD28 was initially shown to augment the proliferative response of T cells in 1984 (5). CD28 enables T cells to proliferate in the presence of the immunosuppressant cyclosporine A (6). Cytokine expression is markedly enhanced after CD28 costimulation through both transcriptional activation and posttranscriptional stabilization of messenger RNA (mRNA) (7). In addition to these effects, CD28 also regulates cell survival by induction of the anti-apoptotic protein Bcl-XL and activation of the protein kinase Akt (8).

CD28 has two ligands, B7-1 (CD80) and B7-2 (CD86). B7-1 was identified as an adhesion receptor on B cells that interacted specifically with CD28 (9, 10). Subsequent work led to the identification of a second member of the B7 family, B7-2, which also bound CD28 (11). The greater complexity of the system became evident with the identification of an additional counter-receptor on T cells, cytotoxic T-lymphocyte antigen 4 (CTLA4), which could bind both B7-1 and B7-2 (14, 15). In contrast to CD28, CTLA4 is expressed only on activated T cells and is a negative regulator of T-cell function (16). CTLA4-deficient mice manifest massive lymphoproliferative disease, which is lethal by 3 wk of age (17, 18).

Recently, new members of the B7/CD28 family have been identified. The search for new tumor necrosis factor (TNF)-inducible, nuclear factor B (NFB)-dependent genes led to the cloning of B7h (19). This protein shares sequence homology with both B7-1 and B7-2 but does not bind to either CD28 or CTLA4. Instead, it binds to a newly identified CD28 homolog, inducible costimulator (ICOS), which is expressed on a subset of activated T cells (20). In addition, Dong and colleagues report the cloning of another B7 homolog, B7-H1, which may be the human ortholog of B7h (21). The role of these new B7/CD28 family members in the immune response remains to be explored.

The importance of CD28 in in vivo immune responses was highlighted by early studies examining transplant rejection. Blockade of B7 with CTLA4Ig, a soluble inhibitor of B7-1 and B7-2, led to prolonged graft survival after both heterotopic cardiac transplantation in rats and islet-cell xenografts in mice (22, 23). Intriguing results have also been found in the manipulation of B7:CD28:CTLA4 interactions in tumor immunity. Transfection of B7 into murine melanoma cells led to effective antitumor responses in vivo (24). Prevention of negative signaling through CTLA4 resulted in regression of primary tumors, as well as augmented secondary responses upon tumor rechallenge in mice (25).

In addition to its role in the initial activation of naive T cells, recent work has examined whether CD28 influences the subsequent differentiation of CD4⁺ T cells. Blockade of B7-1 in experimental autoimmune encephelomyelitis led to a reduction in disease severity and promotion of T helper (Th)2-cell development (26). In contrast, blockade of B7-2 resulted in an increase in disease severity. CD28-mediated events were protective in the development of hyperglycemia in nonobese diabetic mice, perhaps by promoting the development of Th2 cells (27). Similarly, in models of parasitic disease, CD28 has been shown to promote Th2-cell development (28). However, some conflicting results have been noted in the comparison of models that use CTLA4Ig with mice deficient in CD28 expression. For example, Corry and associates found that a single injection of CTLA4Ig could render normally susceptible BALB/c mice resistant to Leishmania major infection (29). However, examination of CD28-deficient mice in the C57Bl/6 and BALB/c background demonstrated normal Th1 and Th2 responses to L. major infection (30). Thus, the precise role that CD28 plays in Th-cell differentiation remains to be defined.

In this issue of the Red Journal, Mark and coworkers examine the development of airway inflammation in B7-deficient mice (31). They demonstrate that B7-1 and B7-2 have overlapping and complementary roles in directing the T-cell response to inhaled antigen challenge. Blockade of B7 was first shown in 1996 to completely abrogate airway inflammation and hyperresponsiveness in a murine model of allergic airway inflammation (32). Importantly, treatment with the inhibitor was effective even if the mice had been previously sensitized to the antigen in the absence of CTLA4Ig. Thus, the B7 blockade exerted effects on the effector phase of the response. These results have been subsequently confirmed by several other investigators (33). Similar to the results found in the use of B7 inhibitors in wild-type mice, CD28-deficient mice do not exhibit airway inflammation after inhaled antigen challenge ([35]; J. S. Burr and J. M. Green, unpublished results).

CTLA4 also exerts an important regulatory influence on in vivo immune responses. CTLA4 is engaged by both B7-1 and B7-2, but in contrast to CD28, CTLA4 ligation results in a decrease in proliferation and IL-2 production (36). Thus, the failure of CD28-deficient mice to respond to stimulation might be due to unopposed negative signaling through CTLA4. In vitro studies in the CD28-deficient mice were unable to detect such an effect; however, recent in vivo studies of cardiac transplantation suggest otherwise (37). B7 blockade in CD28-deficient mice that had received a heterotopic, allogeneic cardiac transplant resulted in accelerated graft rejection (38). These data suggest that the prolonged graft survival in untreated CD28-deficient mice may be the result of the inhibition of T-cell function by CTLA4.

Despite the impressive array of biologic effects mediated by CD28 and CTLA4, the precise mechanism by which these receptors function remains unclear. The topologic model of T-cell activation proposes that costimulatory molecules serve primarily to stabilize the formation of the T cell/APC contact, a structure termed the immunologic synapse (39). Alternatively, these receptors may independently initiate signal transduction pathways that are integrated with TCR-derived signals either at the level of gene transcription or at a prenuclear level.

Several observations argue against a purely topologic model. First, CD28 ligation is effective even when not colocalized with the TCR (40). Second, CD28 imparts properties to the T cell that cannot be mimicked by TCR engagement alone, such as cyclosporin resistance, enhanced cell survival, and stabilization of cytokine mRNA by induction of specific RNA-binding proteins (41).

Work by two independent groups have proposed alternative mechanisms for CD28-mediated costimulation. Ligation of CD28 promotes reorganization of the actin cytoskeleton and the directed transport of T-cell surface receptors to the TCR contact cap (42). Similarly, ligation of CD28 causes the formation of specialized membrane microdomains termed rafts (43). These structures are complexes of proteins in cholesterol-rich regions of the plasma membrane that assemble key signaling components. In this manner, CD28 might facilitate TCR signaling while initiating novel signal transduction pathways.

Two regions of the cytoplasmic tail of CD28 have been identified as important in mediating function. The first is a YMNM motif that can directly bind and activate phosphatidyl inositol 3-kinase (PI3K). However, the absolute requirement for this has been controversial, with some studies demonstrating an absolute dependence on this motif, whereas others show no such requirement (44, 45). In addition, this same region may mediate binding to the adapter protein Grb-2 and may also mediate activation of the Tec family kinase Itk (46). Studies have also identified a C terminal, proline-rich region as required for regulation of proliferation and IL-2 secretion by CD28. Deletion of this region was found to abrogate the regulation of IL-2 gene transcription in Jurkat cells (47). Recently, we have demonstrated that specific proline residues at positions 187 and 190 are absolutely required for costimulation on primary T cells by retroviral reconstitution of primary T lymphocytes from CD28-deficient mice (48). These residues appear to mediate the binding and direct activation of the Src family kinase Lck. Activation of Lck could function to amplify TCR-derived signals and integrate CD28 at the level of phosphorylation of the zeta chain of CD3, as well as lead to direct activation of the Ras/mitogen-activated protein kinase pathway.

The mechanism by which CTLA4 modulates T-cell responsiveness also remains unclear. The cytoplasmic tail of CTLA4 contains a tyrosine-based motif that can bind PI3K (49). In addition, CTLA4 can bind to the protein tyrosine phosphatase SHP-2 (50, 51). Recruitment of SHP-2 to the TCR/CD3 complex could result in the dephosphorylation of components of the signaling complex and could terminate TCR-mediated cellular activation. However, transfection of a mutant CTLA4 construct, lacking all but seven amino acids of the cytoplasmic tail, retained the ability to suppress IL-2 production in T-cell clones, raising the possibility of additional mechanisms by which CTLA4 functions (52). Alternatively, CTLA4 may inhibit T-cell activation by restricting the availability of B7 to interact with CD28. CTLA4 has approximately a 20-fold higher affinity for B7 than CD28, and the unchecked proliferation of CTLA4-deficient T cells is dependent upon CD28 engagement of B7 (53).

Although the precise biochemical mechanism through which both CD28 and CTLA4 function remains controversial, their importance in the regulation of immune responses is clear. Interaction of the TCR with the Ag/major histocompatibility complex remains the critical initiating event in T-cell activation; however, the balance of CD28:CTLA4-derived signals profoundly alter the outcome of that event. Thus, both of these pathways have tremendous potential for therapeutic manipulation in a wide range of conditions, from transplant rejection to autoimmunity to asthma.

View larger version (59K): [in this window] [in a new window]   Figure 1.   T-cell activation is modulated by B7-CD28- CTLA4 interactions. The B7 family members B7-1 and B7-2 both interact with CD28 and CTLA4 to either positively or negatively regulate the T-cell response to antigen. The balance of these signals determine whether TCR engagement results in T-cell activation and development of effector function, or unresponsiveness and programmed cell death.

	Footnotes

Abbreviations: antigen-presenting cells, APC; cytotoxic T-lymphocyte antigen, CTLA; interleukin 2, IL-2; messenger RNA, mRNA; T helper, Th.

(Received in original form January 3, 2000).

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