Within this theme there is a longstanding research effort in murine models for haematopoietic stem cell transplantation, tolerance and immune reconstitution. Furthermore, human lymphoid organogenesis has become an important topic of research when Cupedo joined the department of Hematology. Hematopoietic stem cell transplantation (SCT) is an important therapeutic modality for many malignant hematological disorders, and it’s application is still increasing due to broader indications, eligibility of older patients, and wider use of alternative stem cell sources. Transplant-related morbidity and mortality of allogeneic SCT is still significant due to acute and chronic graft-versus-host disease (GVHD) and opportunistic infections (mainly reactivations of endogenous herpes viruses). Our research focusses on:

• The development of interventions, including cytokine intervention therapy as well innovative cellular therapies, to improve immune recovery and tolerance after transplantation.
• To elucidate cellular and molecular mechanisms that regulate Treg homeostasis
• The development of umbilical cord blood transplantation in adult patients, including new approaches to facilitate engraftment.

• Several clinical studies in the field of allogeneic stem cell transplantation and the treatment of patients with acute leukaemia were performed and reported (see literature), with emphasis on the identification of patients at high risk for complications and TRM as well as those optimally profiting from transplantation.
• Development of new therapeutic approaches to improve immune recovery after SCT. In a murine transplantation model across syngeneic or allogeneic MHC-matched and MHC-mismatched barriers (T-cell and B-cell deficient RAG-1-/- mice receive RAG-1+/+ bone marrow), thymic function was assessed by a real- time quantitative PCR assay of T-cell receptor rearrangement excision circles (TRECs). We found that the frequency of TRECs depends on age, genetic background, and input of bone marrow derived lymphocyte precursor cells. Furthermore, the administration of Interleukin-7 (IL-7), Flt3-ligand, and SCF posttransplant resulted in enhanced recovery of T-cells by improved thymopoiesis as well improved generation of thymic precursors at the bone marrow level.
• We found that keratinocyte growth factor (KGF) induces an increase in murine peripheral Treg numbers via two independent mechanisms. First, by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg. Subsequently, we showed that KGF improves engraftment of allogeneic bone marrow through a Treg dependent mechanism.
• Identification and characterization of fetal and adult human lymphoid tissue inducer cells.

Stem cell research is envisaged to offer a great potential for the development of therapeutic modalities for inherited as well as acquired diseases within as well as outside the hemopoietic system. Considering that translation of the basics into clinical therapy requires a large, multidisciplinary research effort and extensive preclinical evaluation, we concentrate on:

• Improvement of thymopoiesis by cellular and cytokine therapy, including the combination of IL-7 and stem cell factor (SCF) and Flt-3 ligand. Cellular therapeutic approaches will be focussed on the identification of epithelial precursor cells that may differentiate into cortical and medullary epithelium, able to support thymopoiesis. Functional restoration will be assessed by protection against murine viral CMV infection.
• We will continue to characterize cellular and molecular mechanisms underlying peripheral expansion of Treg. We will study the role of dendritic cells in Treg homeostasis and we aim to identify molecules involved in peripheral expansion of Treg. We will evaluate whether modulators of Treg homeostasis reduce graft rejection and Graft-versus-Host disease without negatively affecting Graft-versus-Leukemia and infectious immunity after SCT in murine models.
• In order to develop umbilical cord blood as an important alternative source of stem cells for adult patients, we will concentrate on new approaches to expand UCB derived stem cells, including the use of Wnt proteins in combination with tyrosine kinase signaling cytokines.
• Understanding the signals that drive specification of lymphoid tissue inducer cells into functionally distinct subclasses will be the basis for controlled modulation of these cells to steer immune responses. Our work is focused on understanding the differences between fetal and adult lymphoid tissue inducer cells and the signals that mediate these difference.
• The stromal microenvironment is essential for the proper formation of lymph nodes during embryogenesis but also for the formation of organized lymphoid infiltrations during chronic inflammatory diseases. We are in the process of identifying the key stromal subsets in developing human lymph nodes, which will serve as a basis for research into similar cell types during disease.

1. Cornelissen JJ. Molecular monitoring of EB-positive lymphoma. Blood 2004;104: 9.
2. Wils EJ, Braakman E, Verjans GM, Rombouts EJ, Broers AE, Niesters HG, Wagemaker G, Staal FJ, Lowenberg B, Spits H, Cornelissen JJ. Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation. J Immunol 2007;178:3551-7.
3. Broers AEC, Bruinsma M, Posthumus-van Sluijs SJ, Wils EJ, Spits H, Löwenberg B, Braakman E, Cornelissen JJ. IL-7 mediated protection against minor antigen-mismatched allograft rejection is associated with enhanced recovery of regulatory T cells. Haematologica 2007; 92: 1099-1106 
4. Cornelissen JJ, van Putten WL, Verdonck LF, Theobald M, Jacky E, Daenen SM, van Marwijk Kooy M, Wijermans P, Schouten H, Huijgens PC, van der Lelie H, Fey M, Ferrant A, Maertens J, Gratwohl A, Löwenberg B. Results of a HOVON/SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom?  Blood 2007;109:3658-66.
5. Cornelissen JJ, van der Holt B, Verhoef GE, van ’t Veer MB, van Oers MH, Schouten HC, Ossenkoppele G, Sonneveld P, Maertens J, van Marwijk Kooy M, Schaafsma MR, Wijermans PW, Biesma DH, Wittebol S, Voogt PJ, Baars JW, Zachee P, Verdonck LF, Lowenberg B, Dekker AW. Dutch-Belgian HOVON Cooperative Group.
   Myeloablative allogeneic versus autologous stem cell transplantation in adult patients with acute lymphoblastic leukemia in first remission: a prospective sibling donor versus no-donor comparison. Blood 2009;113:1375-82.
6. Bruinsma M, van Soest PL, Leenen PJM, Löwenberg B, Cornelissen JJ and Braakman E. Keratinocyte growth factor induces expansion of murine peripheral CD4⁺Foxp3⁺ regulatory T cells and increases their thymic output. J Immunol. 2007; 179: 7424-7430.
7. Bruinsma M, van Soest PL, Leenen PJM, Löwenberg B, Cornelissen JJ and Braakman E. Keratinocyte growth factor improves allogeneic bone marrow engraftment through a CD4⁺Foxp3⁺ regulatory T cell-dependent mechanism. J Immunol. 2009; 182: 7364-7369.
8. Vivier E, Spits H, Cupedo T. Interleukin-22-producing innate lymphocytes: new players in mucosal immunity and tissue repair. Nature Reviews Immunol. 2009; 9(4):229-34
9. Cupedo T, Crellin NK, Papazian N, Rombouts EJ, Weijer K, Grogan JL, Fibbe WE, Cornelissen JJ, Spits H. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells. Nature Immunol. 2009, 10(1):66-74.
10. Scheeren FA, Nagasawa M, Weijer K, Cupedo T, Kirberg J, Legrand N, Spits H. T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells. J. Exp. Med. 2008, 1;205(9):2033-42.