Georg F. Beilhack

Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.

Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (T(EM)) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.

TNF-alpha has been linked to the development of type 1 diabetes (T1D). We previously reported that neonatal treatment of nonobese diabetic (NOD) mice with TNF-alpha accelerated the onset of T1D, whereas TNF-alpha blockade in the same time period resulted in a complete absence of diabetes. The mechanisms by which TNF-alpha modulates development of T1D in NOD mice remain unclear. Here we tested the effects of TNF-alpha on the maturation of dendritic cells (DCs) in the NOD mouse. We found that neonatal treatment with TNF-alpha caused an increase in expression of maturation markers on CD11c(+)CD11b(+) DC subpopulations, whereas treatment with anti-TNF-alpha resulted in a decrease in expression of maturation markers in the CD11c(+)CD11b(+) subset. Moreover, neonatal treatment with TNF-alpha resulted in skewed development of a CD8alpha(+)CD11b(-)CD11c(+) DC subset such that TNF-alpha decreases the CD8alpha(+)CD11c(+) DC subset, increases the CD11c(+)CD11b(+) subset, and causes an increase in the expression of CD40 and CD54 on mature DCs capable of inducing immunity. Anti-TNF-alpha-treated mice had an increase in the CD8alpha(+)CD11c(+) DCs. Notably, adoptively transferred naïve CD4(+) T cells from BDC2.5 T cell receptor transgenic mice proliferated in the pancreatic lymph nodes in TNF-alpha-treated NOD mice but not in anti-TNF-alpha-treated mice. Finally, we show that anti-TNF-alpha-treated mice showed immunological tolerance to islet cell proteins. We conclude that TNF-alpha plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and, thus, the activation of islet-specific pancreatic lymph node T cells.

In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.