To examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4(-/-) mice resemble CD28(-/-) mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4(-/-) mice. The proliferation of CD28/CTLA4(-/-) T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4(-/-) T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4(-/-) cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

The requirement for CTLA-4 during the induction of peripheral T cell tolerance in vivo was investigated using naive TCR transgenic T cells lacking CTLA-4. CTLA-4(-/-) T cells are resistant to tolerance induction, as demonstrated by their proliferative responses, IL-2 production, and progression into the cell cycle. Following exposure to a tolerogenic stimulus in vivo and restimulation in vitro, wild-type T cells are blocked at the late G1 to S restriction point of the cell cycle. In contrast, CTLA-4(-/-) T cells enter into the S phase of the cell cycle, as shown by downregulation of p27(kip1), elevated cdk2 kinase activity, and Rb hyperphosphorylation. Thus, CTLA-4 has an essential role in determining the outcome of T cell encounter with a tolerogenic stimulus.

The inducible co-stimulatory molecule (ICOS) is a CD28 homologue implicated in regulating T-cell differentiation. Because co-stimulatory signals are critical for regulating T-cell activation, an understanding of co-stimulatory signals may enable the design of rational therapies for immune-mediated diseases. According to the two-signal model for T-cell activation, T cells require an antigen-specific signal and a second, co-stimulatory, signal for optimal T-cell activation. The co-stimulatory signal promotes T-cell proliferation, lymphokine secretion and effector function. The B7-CD28 pathway provides essential signals for T-cell activation, but does not account for all co-stimulation. We have generated mice lacking ICOS (ICOS-/- ) to determine the essential functions of ICOS. Here we report that ICOS-/- mice exhibit profound deficits in immunoglobulin isotype class switching, accompanied by impaired germinal centre formation. Class switching was restored in ICOS-/- mice by CD40 stimulation, showing that ICOS promotes T-cell/B-cell collaboration through the CD40/CD40L pathway.

Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.

CDC45 is required for the initiation of DNA replication in Saccharomyces cerevisiae and functions as a DNA polymerase alpha loading factor in Xenopus, but its role in mammalian DNA replication is unknown. To investigate the genetic and physiological functions of CDC45, we used a gene targeting strategy to generate mice lacking a functional CDC45 gene. Homozygous mutant mice lacking a functional CDC45 gene underwent uterine implantation and induced uterine decidualization but did not develop substantially thereafter. Detailed analysis of CDC45 null embryos cultured in vitro revealed impaired proliferation of the inner cell mass. These findings make CDC45 the only putative replication factor experimentally proven to be essential for mammalian development. The CDC45 gene localizes to human chromosome 22q11.2 in the DiGeorge syndrome critical region (DGCR). Almost 90% of individuals with congenital cardiac and craniofacial defects have a monoallelic deletion in the DGCR that includes CDC45. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities, so that it is unlikely that hemizygosity of CDC45 alone is responsible for the cardiac and craniofacial defects in the congenital syndromes.

SH2D1A, which encodes signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), is altered in patients with X-linked lymphoproliferative disease (XLP), a primary immunodeficiency. SAP-deficient mice infected with lymphocytic choriomeningitis virus had greatly increased numbers of CD8+ and CD4+ interferon-gamma-producing spleen and liver cells compared to wild-type mice. The immune responses of SAP-deficient mice to infection with Leishmania major together with in vitro studies showed that activated SAP-deficient T cells had an impaired ability to differentiate into T helper 2 cells. The aberrant immune responses in SAP-deficient mice show that SAP controls several distinct key T cell signal transduction pathways, which explains in part the complexity of the XLP phenotypes.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4(+) and CD8(+) T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.