Attenuation of graft-versus-host-disease in NOD scid IL-2Rγ(-/-) (NSG) mice by ex vivo modulation of human CD4(+) T cells.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-Host-Disease (GvHD) induced by human immune cells. We used a model of acute GvHD by transfer of human peripheral blood mononuclear cells (PBMCs) into NSG mice. The severity of GvHD was reflected by weight loss and was associated with engraftment of human cells and the expansion of leukocytes, particularly granulocytes and monocytes. Pre-treatment of PBMCs with the anti-human CD4 antibody MAX.16H5 IgG1 or IgG4 attenuated GvHD. The transplantation of 2 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 10(7) PBMCs pre-incubated with MAX.16H5 IgG1 or IgG4, GvHD development was reduced and survival was increased. Immune reconstitution was measured by flow cytometry and confirmed for human leukocytes (CD45), CD3(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) T helper cells. Human B cells (CD19) and monocytes (CD14) could not be detected. Histopathological analysis (TUNEL assay) of the gut of recipient animals showed significantly less apoptotic crypt cells in animals receiving a MAX.16H5 IgG1 pre-incubated graft. These findings indicate that pre-incubation of an allogeneic graft with an anti-human CD4 antibody may decrease the frequency and severity of GvHD after hematopoietic stem cell transplantation (HSCT) and the need of conventional immunosuppressive drugs. Moreover, this approach most probably provides a safer HSCT that must be confirmed in appropriate clinical trials in the future. © 2016 International Society for Advancement of Cytometry.

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Prevention of graft-versus-host-disease with preserved graft-versus-leukemia-effect by ex vivo and in vivo modulation of CD4(+) T-cells.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

Allogeneic non-adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment.

BACKGROUND: Non adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4(+), murine CD4(-/-), HLA-DR3(+) mice. METHODOLOGY/PRINCIPAL FINDINGS: Bone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45(+) showed depletion of CD4(+), CD8(+), CD19(+), and CD117(+) cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4(+) donor chimerism of 5.8+/-2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death. CONCLUSIONS/SIGNIFICANCE: NA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.

Shift in Th1 (IL-2 and IFN-gamma) and Th2 (IL-10 and IL-4) cytokine mRNA balance within two new histological main-types of rheumatoid-arthritis (RA).

Cytokines are the main mediators of inflammation in rheumatoid arthritis (RA). Thus, Th2 cytokines--such as IL-4 and IL-10--have protective properties to this disease. In opposite, the Th1 cytokines--such as IL-2 and IFN-gamma--are supporting proinflammatory microenvironment in joints from patients with RA. The imbalance of Th1/Th2 cytokine steady state may play an important role in the pathogenesis of rheumatoid arthritis. The evaluation of this imbalance leads up to the possibility of pathohistological discrimination in this disease. In this context, we investigated Th1- (IFN-gamma, IL-2) and Th2 (IL-10, IL-4)-cell-derived cytokine mRNA expression in two novel pathohistological main-types of RA synovial membrane (SM). These main-types are characterized by different tissue-infiltrating inflammatory cells and different extent of SM destruction. Our findings showed that expression of IL-10 mRNA was an outcome of histological main-type I (p<0.001), whereas expression of IFN-gamma and IL-2 were mainly associated with pathohistological main-type II (p<0.005, p<0.05). Surprisingly, IL4 was not differential expressed and could be associated with another special T cell subset in this disease. These results suggest that Th1/Th2 balance is biased to Th2 cytokines within main-type I and Th1 cytokines in main-type II.

From transcriptome to proteome: differentially expressed proteins identified in synovial tissue of patients suffering from rheumatoid arthritis and osteoarthritis by an initial screen with a panel of 791 antibodies.

Global scale molecular profiling of diseased tissues is an important first step to unravel candidate target molecules that are involved in the pathogenesis of a disease. We have performed a comparative molecular characterization at the transcriptome (microarray with 12 526 gene specificities) and proteome level (multi-Western blot PowerBlot with 791 antibodies) of synovial tissue from rheumatoid arthritis (RA) compared to osteoarthritis (OA) patients. From the panel of 791 antibodies, 260 (33%) detected their corresponding protein. Out of 58 unambiguous changes at the protein level only 16 coincided at the transcript level (28%). Stat1, p47phox and manganese superoxide dismutase were shown to be reproducibly overexpressed in RA versus OA synovial tissue by Western blots with a panel of 8 RA versus 8 OA samples. Cathepsin D was among the most prominent proteins scored to be underexpressed in RA by the PowerBlot whereas no differences of the respective transcript were observed. The lower abundance of cathepsin D protein in RA compared to OA tissue was also reproduced in other patient samples. Immunohistochemistry assigned the Stat1 protein in RA synovial tissue mainly to macrophages and T lymphocytes and the p47phox protein in particular to macrophages. In conclusion, our approach provided us with new candidate molecules for further analysis of rheumatic diseases and stressed the importance of studies at the protein level.

High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis.

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys-X-Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys-X-Cys ligand (CXCL)9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.