Improved murine hematopoietic cell transplantation with IDO

This proposal outlines our continuing effort for the generation of allo-antigen specifically tolerized T cells, particularly in the context of hematopoietic stem cell transplantation (HSCT). The capability to ex vivo generate T cell populations, which are specifically tolerant against donor allo-antigens, may provide recipients of HSCT with immunological broadly competent T-cell immunity without aggravating the risk of graft-versus host disease (GVHD) as long as endogenous T cell reconstitution is insufficient. Such an approach may significantly contribute to overcome many of the immunological problems affecting patients in the immediate and long term post-HSCT period. In our previous project supported by the "Fonds zur Foerderung der wissenschaftlichen Forschung" (FWF; Austrian Science Foundation), no P 16764-B-13, we began to explore the tolerogenic potential of antigen presenting cells (APCs) that express high levels of the tryptophan metabolizing enzyme indoleamine 2,3 dioxygenase (IDO). By its tryptophan metabolizing activity IDO will result in local and systemic depletion of tryptophan and accumulation of tryptophan metabolites, both features affecting the immune function of activated T cells. As will be presented in "Preliminary Results", in our previous project we clearly show that in fact human dendritic cells (DCs) can be induced to express high levels of IDO and efficiently metabolize tryptophan (i.e. become IDO competent). When allogeneic T cells are exposed to these IDO competent DCs in vitro they (i) have an impaired capability to proliferate, and (ii) have an increased susceptibility to activation-induced apoptosis and (iii) acquire a regulatory phenotype. These encouraging results prompt us to now propose to translate this approach to an in vivo murine transplantation model. In this project we will begin to test the hypothesis whether donor T cells after an ex vivo exposure to recipient IDO competent DCs can be safely transferred to recipients of HSCT, which means that they effectively transfer T cell immunity while not inducing GVHD. A successful completion of this study will then set the basis to move forward to adopt this approach for clinical trials in humans and thus to contribute to the efforts to make HSCT in humans a safe and broadly applicable procedure.

Hematopoietic stem cell transplantation (HSCT) constitutes a potentially curative treatment modality for a variety of diseases, including leukemia, congenital immunodeficiencies and bone marrow failures. The goal of HSCT is to stably establish donor-type hematopoiesis, which includes donor-derived immune cells, in the recipient. In the context of HSCT, pathological allo-immune reactions mostly occur in the graft-versus-host direction, in which donor-type immune cells initiate immune reactions against recipient type organs (graft-versus-host, GvH). The time after HSCT is characterized by prolonged and profound immune dysfunction until the functional integrity of the immune system is reconstituted and often aggravated by pharmacological immunosuppression to prevent overt allo-immune reactions. Thus, after HSCT, the patient is at high risk for developing severe infections and/ or GvH disease. Donor T cells tolerant towards recipient allo-antigens but retaining immune activity against pathogenic microorganisms are critical for immune reconstitution. For T cells, the CD28:CD80/86 co-stimulatory pathway is essential to mount a full response to antigen stimulation and its blockade has been described to result in antigen- specific T cell unresponsiveness. CTLA-4Ig has been pharmacologically engineered to block this pathway and to induce transplantation tolerance. In addition, CTLA-4Ig was reported to affect dendritic cell (DC) function, i.e. to interfere with the presentation of antigens, via the induction of the tryptophan metabolizing enzyme indoleamine 2, 3 dioxygenase (IDO). IDO`s augmented activity has been associated with impaired T cell function. However, this mechanism has raised considerable debate in the scientific community. On this background, we explored the cellular mechanism of CTLA-4Ig mediated immunoregulation in a murine allogeneic setting as a means to induce tolerance in HSCT. Using C57BL/6 splenic or bone marrow derived DCs as stimulators of allogeneic Balb/c T cells, we found that CTLA4-Ig potently dampened allogeneic T cell responses when present during T cell/DC co-culture. This dampening effect, however, was neither related to the activation of the IDO pathway nor to the induction of a DC regulatory phenotype, but directly affected the DC/T cell crosstalk. CTLA-4Ig preferentially targeted the T helper (CD4+ ) response. Moreover, CD4 + T cells recovered from DC/T cell co-cultures performed in the presence of CTLA-4Ig displayed CD25 + CD62L+ FoxP3+ expression, compatible with a regulatory phenotype. Remarkably, this regulatory phenotype was preserved upon restimulation even in the absence of CTLA-4Ig. The stable generation of T regulatory cells is classified as being of key importance in the generation of transplantation tolerance. In conclusion, CTLA4-Ig inhibits allo-stimulated T cells in an IDO independent fashion. In addition to down- regulating the T cell proliferative response CTLA-4Ig propagates a CD4+ T cell regulatory phenotype. Expanding regulatory T cells as induced by CTLA-4Ig may possess therapeutic potential for successful adoptive T cell therapy to overcome immune dysfunction after HSCT.