Applied Transplantation Immunology
Recently, we could identify the human bone marrow as a potential target of acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Acute leukemic patients and here in particular those displaying systemic acute GvHD and/or those receiving a full-intensity conditioning regimen, showed increased bone marrow T-cell infiltration associated with the loss of osteoblasts and delayed onset of B-cell neogenesis (Figure 1).
Currently, we are interested in 1) unravelling the potential mechanism of human bone marrow GvHD, 2) identifying mechanisms responsible for long-term memory B-cell deficiency post-transplant and 3) the possibility to use “long-living” memory B cells residing in the bone marrow for adoptive cell transfer in immunodeficient patients.
In the field of primary immunodeficiency, we are currently investigating potential pathogenic pathways in patients with primary selective IgM deficiency. We apply a combined approach of NGS whole exome sequencing (in collaboration with the Institute for Medical Genetics, Charité) and analysis of related immune cell function.
A) Numbers of peripheral blood total CD19+ B cells and transitional B cells, which first emigrate the bone marrow, was determined by flow cytometry at day (D)14, D28, D60 and D180 after allo-HSCT in acute leukemic patients. Shown is the mean percentage±SEM for patients, who either had an early (n=19, black line) or late (n=17, grey line) onset of B-cell reconstitution (Mann-Whitney U, **p≤0.001). (B) The number of marrow-infiltrating CD3+ T cells (brown staining left graphs) and numbers of osteoblasts along the bony trabeculae (HE staining, indicated by arrow right graphs) were determined on histological sections of bone marrow trephines obtained at D20-30 post-transplant. Representative examples of an early (upper graphs) and late (lower graphs) recovering patient are depicted.
Using bioluminescence-reporter mice we are able to easily study the in vivo migration, tumor-infiltration and activation of adoptively transferred T cells. Our aim is to optimize the efficiency of tumor rejection by adoptive T cell therapy (ATT) and to minimize the off-target organ toxicity presenting as GvHD. We have generated a transgenic reporter mouse expressing an NFAT-inducible Click-beetle luciferase (CB/NFAT) in order to be able to visualize T-cell activation in vivo (Figure 2A/B).
(A) Sorted T cells from a CB/NFAT mouse were stimulated with anti-CD3/CD28 antibodies or PMA/Ionomycin for 4h. Indicated numbers of activated T cells were injected subcutaneously into albino B6 mice. NFAT-induced Click beetle luciferase activity was assessed using an in vivo imaging system (IVIS) (top). Additionally, the luciferase signal activity (photons/s) were quantified (bottom). (B) Female CB/NFAT T cells were transferred in male or female albino B6 recipients and T cell activation-induced luciferase activity was analyzed via BLI. (C) RLuc+ MataHari T cells were transferred in HY+ tumor-bearing Rag knock out mice. CD8+ T cell migration was followed based on Renilla luciferase activity and BLI (top). Graph shows the quantification of the Renilla luciferase signal activity (mean ± SEM) in 200.09 and MB49 tumors (bottom).
As a test of our CB/NFAT reporter system in a mouse model of GvHD, we transplanted lethally irradiated male B6 mice with female B6 bone marrow and splenic CB/NFAT T cells and analyzed the transferred transgenic donor T cells via bioluminescence imaging (BLI). Two weeks after transplantation, NFAT activation could be seen predominantly in spleen and gut of male recipients most likely triggered by alloreactivity against the minor antigen HY. Thus, we are able to track adoptively transferred T cells and their spatio-temporal activation pattern mediating GvHD. Furthermore, Renilla luciferase (RLuc) transgenic mice were crossbred with H-Y specific TCR transgenic Marilyn and MataHari mice in order to obtain H-Y specific TCR, RLuc double transgenic CD4+ and CD8+ T cells respectively, used for adoptive transfer into female Rag recipients bearing H-Y antigen expressing tumors like 200.09 or MB49. Transferred RLuc+ MataHari T cells preferentially accumulated within HY antigen positive tumors (Figure 2C). Currently, we are generating a mouse triple transgenic for CB/NFAT, RLuc and the HY-TCR, in order to simultaneously visualize T cell migration and activation in an HY minor mismatched model.
Our clinically relevant transplantation model will enable us to evaluate and improve novel strategies to separate graft-versus-tumor from GvHD effects in the context of ATT.