Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 and plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells and impair monocyte migration, but the origin of soluble sema4D, particularly at sites of vascular injury, has been unclear. Here we show that platelets express sema4D and both of its receptors and provide evidence that these molecules promote thrombus formation. We also show that the surface expression of sema4D and CD72 increases during platelet activation, followed by the gradual shedding of the sema4D extracellular domain. Shedding is blocked by metalloprotease inhibitors and abolished in mouse platelets that lack the metalloprotease ADAM17 (TACE). Mice that lack sema4D exhibit delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro. In resting platelets, as in B lymphocytes, CD72 is associated with the protein tyrosine phosphatase SHP-1. Platelet activation causes dissociation of the complex, as does the addition of soluble sema4D. These findings suggest a dual role for sema4D in vascular responses to injury. As thrombus formation begins, platelet-associated sema4D can bind to its receptors on nearby platelets, promoting thrombus formation. As thrombus formation continues, sema4D is shed from the platelet surface and becomes available to interact with receptors on endothelial cells and monocytes, as well as continuing to interact with platelets.signaling ͉ thrombosis ͉ metalloprotease ͉ CD72 ͉ plexin-B1 P latelet activation typically begins with the exposure of collagen within a damaged vessel wall or the local generation of thrombin, but the establishment of a stable thrombus requires the recruitment of additional platelets and the development of stable contacts between platelets (1). Platelet activation also results in the release from platelets of molecules that can affect nearby cells, including endothelial cells and leukocytes as well as other platelets. In a continuing search for molecules that might contribute to contact-dependent events during thrombus formation, we screened human platelets for members of the semaphorin family. Although sempahorins are best known as regulators of neurite outgrowth and vascular development, individual family members have been shown to participate in a variety of events. Class IV semaphorin [semaphorin 4D (sema4D; CD100)] is a type I integral membrane protein first reported on T cells where it supports B cell development by binding to CD72 (2-4). However, sema4D receptors are not limited to B cells. Prior work has shown that a soluble sema4D extracellular domain fragment can activate endothelial cells by its other known receptor, plexin-B1. This causes endothelial migration, actin rearrangement, and the formation of tube-like structures in vitro, responses that are relevant for wound healing and angiogenesis (5-11). Soluble sema4D has also been shown to inhibit monocyte (12) and dendritic cell (13) migration. ...

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-␥-producing cells. As interferon-␥ is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2 a ) cardiac allografts placed into wild-type BALB/c (H-2 d ) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17 ؊/؊ recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17 ؊/؊ and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17 ؊/؊ recipients. In contrast, IL-17 ؊/؊ mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donorreactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17 ؊/؊ recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.

Antibody-mediated lymphoablation is commonly used in solid organ and hematopoietic cell transplantation. However, these strategies fail to efficiently control pathogenic memory T cells and significantly improve long term transplant outcomes. Understanding the mechanisms of T cell reconstitution is critical for enhancing the efficacy of antibody-mediated depletion in sensitized recipients. Using a murine analog of anti-thymocyte globulin (mATG) in a mouse model of cardiac transplantation, we previously showed that peritransplant lymphocyte depletion induces rapid memory T cell proliferation and only modestly prolongs allograft survival. We now report that T cell repertoire following depletion is dominated by memory CD4 T cells. Additional depletion of these residual CD4 T cells severely impairs the recovery of memory CD8 T cells after mATG treatment. The CD4 T cell help during CD8 T cell recovery depends on the presence of B cells expressing CD40 and intact CD40/CD154 interactions. The requirement for CD4 T cell help is not limited to the use of mATG in heart allograft recipients, and is observed in non-transplanted mice and after CD8 T cell depletion with mAb instead of mATG. Most importantly, limiting helper signals increases the efficacy of mATG in controlling memory T cell expansion and significantly extends heart allograft survival in sensitized recipients. Our findings uncover the novel role for helper memory CD4 T cells during homeostatic CD8 T cell proliferation and open new avenues for optimizing lymphoablative therapies in allosensitized patients.

Antibody-mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of pre-existing donor-reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that anti-donor immune responses following treatment with rabbit anti-mouse thymocyte globulin (mATG) were dominated by T cells derived from the pre-existing memory compartment. Administration of mATG one week prior to transplantation (pre-TP) was more efficient in targeting pre-existing donor-reactive memory T cells, inhibiting overall anti-donor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri-TP). The failure of peri-TP mATG to control anti-donor memory responses was due to faster recovery of pre-existing memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.