Yaki Eidelstein

Deletion of cognate CD8 T cells by immature dendritic cells: a novel role for perforin, granzyme A, TREM-1, and TLR7

Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4(+) and CD8(+) T cells after encountering cognate or noncognate imDCs. Whereas CD4(+) T cells were deleted via an MHC-independent mechanism through the NO system, CD8(+) T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforin-expressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.

Induction of tolerance to bone marrow allografts by donor-derived host nonreactive ex vivo-induced central memory CD8 T cells

Enabling engraftment of allogeneic T cell-depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti-third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44(+)CD62L(-) effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44(+)CD62L(+) central memory cell (Tcm) phenotype by anti-third-party CD8(+) cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell-mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti-third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

Murine anti–third-party central-memory CD8+ T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Deletion of Alloreactive T Cells by Veto Cytotoxic T Lymphocytes Is Mediated Through Extracellular Signal-Regulated Kinase Phosphorylation

Background. Anti third-party cytotoxic T lymphocytes (CTLs) were shown to exhibit marked veto activity, thereby inducing transplantation tolerance across major histocompatibility antigens. Elimination of effector cells requires co-expression of CD8 and FasL on the veto cells and is mediated through CD8-major histocompatibility complex (MHC) class I interaction and Fas-Fas ligand signaling. Methods. To further interrogate the signaling events induced in the effector cells on their interaction with veto cell populations, effector cells from 2C transgenic mice were preincubated with different signaling inhibitors and were subject to fluorescence-activated cell sorting and western blot analysis. Results. Screening with inhibitors revealed specific inhibition only with the map kinase (MEK)/extracellular signal regulated kinase (ERK) inhibitor, U0126. Accordingly, fluorescence-activated cell sorting and western blot analysis showed that ERK phosphorylation is induced in the effector cells within 1 hr of incubation with the veto cells. ERK phosphorylation had no effect on the Fas expression level, nor was it reduced when using effector cells from Fas KO mice. Examination of ERK phosphorylation in high and low MHC-I expressing effectors revealed marked differences, suggesting that the interaction between CD8 on the veto CTL, and MHC-I on the effector cells is likely responsible for ERK phosphorylation. Furthermore, XIAP in 2C cells is specifically reduced on binding to the cognate veto cells during the mixed lymphocyte reaction but before the appearance of Annexin V reactivity. Conclusions. These results suggest that the interaction between CD8 on veto CTL and the MHC class I &agr;3 domain on the effector cell, leads to phosphorylation of MEK/ERK in the latter cell, associated with a significant reduction of XIAP levels which, in turn, enables potent triggering of Fas-FasL mediated apoptosis on cognate binding of the veto CTLs.

A new approach for eradication of residual lymphoma cells by host nonreactive anti-third-party central memory CD8 T cells.

Generation of T cells endowed with graft-versus-leukemia (GVL) and depleted of graft-versus-host (GVH) activity represents a highly desirable goal in bone marrow transplantation (BMT). Here, we demonstrate that donor anti-third-party CD8 T cells with central memory phenotype (Tcm) exhibit marked GVL reactivity through a unique T-cell receptor-independent mechanism. Thus, in a residual disease mouse model, Tcm therapy following autologous BMT led to significant survival prolongation, with 30% to 40% of the treated mice displaying long-term tumor-free survival. A more impressive finding was that infusion of donor Tcm in an allogeneic model rapidly eliminated residual lymphoma cells and led to long-term survival of 100% in the absence of GVH disease. Collectively, the strong GVL reactivity of anti-third-party Tcm, coupled with their demonstrated enhancement of bone marrow allografting, suggests that the use of Tcm therapy in conjunction with allogeneic T-cell-depleted BMT could be of particular benefit in patients with B-cell malignancies who cannot tolerate intensive myeloablative conditioning.