B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes.

CD28/B7 costimulation has been implicated in the induction and progression of autoimmune diseases. Experimentally induced models of autoimmunity have been shown to be prevented or reduced in intensity in mice rendered deficient for CD28 costimulation. In sharp contrast, spontaneous diabetes is exacerbated in both B7-1/B7-2-deficient and CD28-deficient NOD mice. These mice present a profound decrease of the immunoregulatory CD4+CD25+ T cells, which control diabetes in prediabetic NOD mice. These cells are absent from both CD28KO and B7-1/B7-2KO mice, and the transfer of this regulatory T cell subset from control NOD animals into CD28-deficient animals can delay/prevent diabetes. The results suggest that the CD28/ B7 costimulatory pathway is essential for the development and homeostasis of regulatory T cells that control spontaneous autoimmune diseases.

Treatment with intact anti-B7-1 mAb during disease remission enhances epitope spreading and exacerbates relapses in R-EAE.

PLP139-151-induced experimental autoimmune encephalomyelitis in the SJL mouse is a Th1-mediated inflammatory demyelinating disease characterized by a relapsing-remitting clinical course (R-EAE). Clinical relapses are mediated by T cells specific for a non-cross reactive secondary PLP epitope (PLP178-191) induced by epitope spreading. We have previously shown that B7-1 expression is upregulated in SJL mice undergoing R-EAE and in vivo treatment during remission with F(ab) fragments of anti-B7-1 mAb, blocked epitope spreading and disease progression. In contrast, the present study shows that treatment with intact anti-B7-1 mAb exacerbated clinical disease relapses and enhanced CNS demyelination. Anti-B7-1-treated mice showed enhanced in vivo delayed-type hypersensitivity (DTH) to the relapse-associated PLP178-191 epitope and responses to the immunodominant MBP84-104 epitope which are absent in the controls. Thus, ligation of B7-1 by intact mAbs has effects opposite to those of anti-B7-1 F(ab) fragments suggesting that the mAb is directly signaling through B7-1 expressed on T cells and/or APCs.

CD28/B7 regulation of autoimmune diabetes.

The initiation and progression of autoimmune diseases, such as insulin-dependent diabetes mellitus (IDDM), are complex processes that depend on autoantigen exposure, genetic susceptibility, and secondary events that promote autoaggression. T-cell costimulation, largely mediated by CD28/B7 interactions, is a major regulatory pathway in the activation and differentiation of T-cells that cause IDDM in murine models. In this article, we summarize our results in two models of IDDM: the nonobese diabetic (NOD) mouse and diabetes induced with multiple low doses of streptozotocin (MDSDM). In both of these models, blockade of CD28/B7 costimulation regulates the development of disease. The effects of blockade vary with the intensity of cognate signal delivered to the T-cells, the timing of the costimulatory signal, and perhaps even the CD28 ligand expressed on antigen-presenting cells (APCs). Our results suggest that targeting CD28/B7 signals is a feasible approach for treatment and prevention of recurrence of autoimmune diabetes. However, the dynamic nature of these interactions highlights the importance of a clear understanding of their role in regulation of the disease.

CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes.

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.

CD28/B7 system of T cell costimulation.

T cells play a central role in the initiation and regulation of the immune response to antigen. Both the engagement of the TCR with MHC/Ag and a second signal are needed for the complete activation of the T cell. The CD28/B7 receptor/ligand system is one of the dominant costimulatory pathways. Interruption of this signaling pathway with CD28 antagonists not only results in the suppression of the immune response, but in some cases induces antigen-specific tolerance. However, the CD28/B7 system is increasingly complex due to the identification of multiple receptors and ligands with positive and negative signaling activities. This review summarizes the state of CD28/B7 immunobiology both in vitro and in vivo; summarizes the many experiments that have led to our current understanding of the participants in this complex receptor/ligand system; and illustrates the current models for CD28/B7-mediated T cell and B cell regulation. It is our hope and expectation that this review will provoke additional research that will unravel this important, yet complex, signaling pathway.

Blockade of CD28/B7-1 interaction prevents epitope spreading and clinical relapses of murine EAE.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) induced with the immunodominant epitope from proteolipid protein, PLP139-151, is characterized by the development of recurrent relapses with recruitment of T cells reactive to additional myelin peptides, including PLP178-191 (epitope spreading). In this study, we have determined that the CD28/B7 costimulatory pathway is involved in this process. We found preferential up-regulation of B7-1 during the course of R-EAE and a selective increase in its functional costimulatory activity, relative to B7-2. Anti B7-1 F(ab) fragment therapy, but not anti B7-2 MAb therapy, blocked clinical relapses, ameliorated CNS pathology, and blocked epitope spreading. These results suggest that the maintenance of autoimmune reactivity in EAE depends on CD28/B7-1-dependent costimulation of newly recruited T cells responsible for epitope spreading. These studies have important implications for the role of epitope spreading in disease progression and the clinical application of costimulatory antagonists in autoimmune diseases.

Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies.

Antigen-specific T cell activation depends initially on the interaction of the T cell receptor (TCR) with peptide/MHC. In addition, a costimulatory signal, mediated by distinct cell surface accessory molecules, is required for complete T cell activation leading to lymphokine production and proliferation. CD28 has been implicated as the major receptor on T cells responsible for delivering the costimulatory signal. Although two distinct ligands for CD28, B7-1 and B7-2, have been identified on antigen-presenting cells (APC), the co-stimulatory role of each molecule during a physiological immune response remains unresolved. In the present study, the relative roles of B7-1 and B7-2 interactions were evaluated in an allogeneic pancreatic islet transplant setting. In isolation, anti-B7-2 mAbs and, to a much lesser degree, anti-B7-1 mAbs suppressed T cell proliferative responses to allogeneic islets or splenic APC in vitro. Maximal inhibition of the allogeneic response was observed using a combination of the anti-B7-1 and anti-B7-2 mAbs. Administration of anti-B7-2 but not anti-B7-1 mAbs prolonged C3H allograft survival in B6 recipients, with a combination of both mAbs significantly prolonging rejection beyond either mAb alone. The immunosuppressive effects of the in vivo mAb treatment were not manifested in in vitro analyses as T cells isolated from suppressed mice responded normally to allogeneic stimuli in terms of both proliferation and lymphokine production. However, combined mAb therapy in vivo selectively delayed CD4+ T lymphocyte infiltration into the graft. These data suggest that both B7-1 and B7-2 costimulatory molecules are active in vivo, although B7-2 plays a clearly dominant role in this allograft model. The mechanism of immune suppression in vivo remains unresolved but may occur at sites distinct from the allograft.

Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse.

Insulin-dependent diabetes mellitus (IDDM) is thought to be an immunologically mediated disease resulting in the complete destruction of the insulin-producing islets of Langerhans. It has become increasingly clear that autoreactive T cells play a major role in the development and progression of this disease. In this study, we examined the role of the CD28/B7 costimulation pathway in the development and progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse model. Female NOD mice treated at the onset of insulitis (2-4 wk of age) with CTLA4Ig immunoglobulin (Ig) (a soluble CD28 antagonist) or a monoclonal antibody (mAb) specific for B7-2 (a CD28 ligand) did not develop diabetes. However, neither of these treatments altered the disease process when administered late, at > 10 wk of age. Histological examination of islets from the various treatment groups showed that while CTLA4Ig and anti-B7-2 mAb treatment blocked the development of diabetes, these reagents had little effect on the development or severity of insulitis. Together these results suggest that blockade of costimulatory signals by CTLA4Ig or anti-B7-2 acts early in disease development, after insulitis but before the onset of frank diabetes. NOD mice were also treated with mAbs to another CD28 ligand, B7-1. In contrast to the previous results, the anti-B7-1 treatment significantly accelerated the development of disease in female mice and, most interestingly, induced diabetes in normally resistant male mice. A combination of anti-B7-1 and anti-B7-2 mAbs also resulted in an accelerated onset of diabetes, similar to that observed with anti-B7-1 mAb treatment alone, suggesting that anti-B7-1 mAb's effect was dominant. Furthermore, treatment with anti-B7-1 mAbs resulted in a more rapid and severe infiltrate. Finally, T cells isolated from the pancreas of these anti-B7-1-treated animals exhibited a more activated phenotype than T cells isolated from any of the other treatment groups. These studies demonstrate that costimulatory signals play an important role in the autoimmune process, and that different members of the B7 family have distinct regulatory functions during the development of autoimmune diabetes.

Immunomodulation of transplant rejection using monoclonal antibodies and soluble receptors.

The main objective of our studies has been to optimize the effects of monoclonal antibodies (MAbs) and other immunosuppressive reagents to enhance organ graft survival. One such agent is OKT3, a MAb that is directed against the CD3 component of the human T-cell receptor (TCR) complex. Treatment of a rejection episode with OKT3 results in a rapid and efficient clearing of circulating T cells and reversal of most rejection episodes. Its wider use in transplantation and in the treatment of immune-mediated disease is limited by adverse reactions that follow the initial dose, the production of neutralizing Abs, and the transient nature of the immunosuppression. We have engineered CDR-grafted "humanized" anti-CD3 MAbs that lack Fc-receptor binding activity through mutagenesis of amino acids in the Fc portion of the MAb. This results in an immunosuppressive anti-CD3 MAb that is less antigenic and one that does not induce the first-dose side effects. In addition, we have pursued a goal of developing a therapy that will induce donor-specific tolerance while maintaining overall recipient immune competency. Because antigen-specific T-cell activation depends not only on TCR-ligand interaction, but also on additional costimulatory signals mediated by accessory molecules such as CD28, blocking the binding of CD28 on T cells to its ligand B7, during TCR engagement, might modulate transplantation responses. Using a soluble fusion protein of human CTLA4, CTLA4-Ig, that binds B7 with high affinity, inhibition of human pancreatic islet rejection that occurs, at least in part, by affecting T-cell recognition of human B7+ antigen-presenting cells has been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential up-regulation of the B7-1 and B7-2 costimulatory molecules after Ig receptor engagement by antigen.

Ag-pulsed B cells are potent APCs, in part, because of the ability of the Ig receptor to mediate rapid and specific Ag uptake. However, it is also known that full T cell activation requires signals delivered by costimulatory molecules, which naive B cells seem to lack. This study examines the effect Ig receptor engagement has on the expression and function of a new CD28 counter-receptor, B7-2. Unlike B7-1 (B7), B7-2 was rapidly induced on the cell surface of B cells after engagement of the Ig receptor by either anti-Ig mAbs or hen egg lysozyme (HEL) on normal and HEL-specific B cell receptor transgenic B cells, respectively. Furthermore, B7-2 expression was up-regulated on tolerant B cells isolated from HEL/anti-HEL double transgenic mice after Ag stimulation, although at lower levels than on nontolerant transgenic B cells. No significant cell surface levels of B7-1(B7) were observed under these conditions. Finally, the B7-2 molecules induced by Ig cross-linking costimulated T cell proliferation in a CD28-dependent manner, independent of B7-1(B7) expression. Thus, the effectiveness of Ag-specific B cells as APCs depends on both their enhanced Ag uptake, mediated by the B cell receptor, and immediate up-regulation of a potent costimulatory molecule, B7-2.

CTLA-4 can function as a negative regulator of T cell activation.

CD28 and CTLA-4 are related glycoproteins found on T cells. Ligation of CD28 following antigen receptor engagement provides a costimulatory signal required for T cell activation. Anti-CTLA-4 antibodies were generated to examine the role of the CTLA-4 receptor on murine T cells. Expression of CTLA-4 as a homodimer is up-regulated 2-3 days following T cell activation. Anti-CTLA-4 antibodies and Fab fragments augmented T cell proliferation in an allogeneic MLR. However, when optimal costimulation and Fc cross-linking were present, anti-CTLA-4 Mabs inhibited T cell proliferation. Together, these results suggest that the MAb may obstruct the interaction of CTLA-4 with its natural ligand and block a negative signal, or directly signal T cells to down-regulate immune function.

IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2.

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.

Expression and functional significance of an additional ligand for CTLA-4.

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.

T cell co-stimulation and in vivo tolerance.

Previous studies have shown that effective T-cell activation requires the engagement of the T-cell receptor complex with MHC-peptide, in parallel with co-stimulation via cell surface adhesion molecules. Blocking these co-stimulatory interactions, in particular the signal transduction via the CD28 molecule, inhibits T-cell activation in vitro and induces T-cell clones into a state of unresponsiveness, termed T-cell anergy. Recent studies have examined the therapeutic effects of treating mice with CD28-B7 antagonists and highlighted the complexity of the CD28 co-stimulatory pathway, as illustrated by the finding that multiple cross-binding ligands for the CD28 and B7 molecules exist that may differentially regulate immune responses.

Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg.

Antigen-specific T cell activation depends on T cell receptor-ligand interaction and costimulatory signals generated when accessory molecules bind to their ligands, such as CD28 to the B7 (also called BB1) molecule. A soluble fusion protein of human CTLA-4 (a protein homologous to CD28) and the immunoglobulin (lg) G1 Fc region (CTLA4lg) binds to human and murine B7 with high avidity and blocks T cell activation in vitro. CTLA4lg therapy blocked human pancreatic islet rejection in mice by directly affecting T cell recognition of B7+ antigen-presenting cells. In addition, CTLA4lg induced long-term, donor-specific tolerance, which may have applications to human organ transplantation.