Hisaki Fujii

Anti-CD13 Abs in children with extensive chronic GVHD and their relation to soluble CD13 after allogeneic blood and marrow transplantation from a Children's Oncology Groups Study, ASCT0031

Our group previously demonstrated a strong association between elevated plasma soluble CD13 enzyme activity and newly diagnosed extensive chronic GVHD (cGVHD) in children. As cytotoxic anti-CD13 Abs have been documented after blood and marrow transplant (BMT) in association with CMV infection and cGVHD, we hypothesized that soluble CD13 contributes to cGVHD pathogenesis by induction of CD13 reactive Abs and that anti-CD13 Abs could be additional biomarkers for newly diagnosed pediatric extensive cGVHD. Using prospectively collected plasma samples from pediatric allogeneic BMT (allo-BMT) subjects with cGVHD and controls without cGVHD enrolled in a large multi-institution Childrens Oncology Group cGVHD therapeutic trial, we evaluated whether soluble CD13 correlates with induction of anti-CD13 Abs. We found that CD13 reactive Abs are present in a proportion of patients after allo-BMT, but did not seem to correlate with the presence of soluble CD13. Anti-CD13 Abs also did not meet our criteria as a diagnostic biomarker for cGVHD. These data do not confirm that induction of CD13 reactive Abs is a mechanism for cGVHD in children nor are part of the pathogenesis of cGVHD associated with elevated soluble CD13. The exact role of CD13 in cGVHD remains to be determined.

Humanized Chronic Graft-versus-Host Disease in NOD-SCID il2rγ-/- (NSG) Mice with G-CSF-Mobilized Peripheral Blood Mononuclear Cells following Cyclophosphamide and Total Body Irradiation

Chronic graft-versus-host disease (cGvHD) is the major source of late phase morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Humanized acute GvHD (aGvHD) in vivo models using NOD-SCID il2rγ-/- (NSG) mice are well described and are important tools for investigating pathogenicity of human cells in vivo. However, there have been only few reported humanized cGvHD mouse models. We evaluated if prolonged inflammation driven by low dose G-CSF-mobilized human PBMCs (G-hPBMCs) would lead to cGvHD following cyclophosphamide (CTX) administration and total body irradiation (TBI) in NSG mice. Engraftment was assessed in peripheral blood (PB) and in specific target organs by either flow cytometry or immunohistochemistry (IHC). Tissue samples were harvested 56 days post transplantation and were evaluated by a pathologist. Some mice were kept for up to 84 days to evaluate the degree of fibrosis. Mice that received CTX at 20mg/kg did not show aGvHD with stable expansion of human CD45+ CD3+ T-cells in PB (mean; 5.8 to 23.2%). The pathology and fibrosis scores in the lung and the liver were significantly increased with aggregation of T-cells and hCD68+ macrophages. There was a correlation between liver pathology score and the percentage of hCD68+ cells, suggesting the role of macrophage in fibrogenesis in NSG mice. In order to study long-term survival, 6/9 mice who survived more than 56 days showed increased fibrosis in the lung and liver at the endpoint, which suggests the infiltrating hCD68+ macrophages may be pathogenic. It was shown that the combination of CTX and TBI with a low number of G-hPBMCs (1x106) leads to chronic lung and liver inflammation driven by a high infiltration of human macrophage and mature human T cells from the graft, resulting in fibrosis of lung and liver in NSG mice. In conclusion this model may serve as an important pre-clinical model to further current understanding of the roles of human macrophages in cGvHD.

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell–mediated TNFα signaling

Blocking posttransplantation donor T cell–mediated inflammation enhances stem cell survival and accelerates blood cell reconstitution rates. TNFα tampers with stem cell success Most stem cell transplantation procedures are performed with unrelated donor/recipient pairs. One source of stem cells is umbilical cord blood, but the number of cells derived from this source can be limiting. Wang et al. examined factors that affect proliferation, engraftment, and differentiation of human umbilical cord stem cells in a preclinical model. They found that donor T cell production of TNFα was harmful to stem cell health. In the future, inhibiting TNFα after stem cell transplantation could lead to improved patient outcomes. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell–derived inflammatory signals. Donor T cell–derived tumor necrosis factor–α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell–derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell–derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

Natural Killer Cells: A Potential Therapy for Paediatric Brain Tumours

Natural killer cells are innate immune cells that are naturally involved in tumour immunosurveillance. These cells have direct cytotoxic activity and also secrete proinflammatory cytokines upon activation. The activation of natural killer cells’ cytolytic activity is dependent on a balance between inhibitory signals and activating signals provided by the host cells. Natural killer cells do not exert their activity on healthy cells, due to interactions of inhibitory receptors with self major histocompatibility class I. Inhibition may be overcome when the cell binds tumour-associated antigens on the surface of tumour cells, resulting in the polarized release of cytolytic granules toward the target cell. Natural killer cells demonstrate particular promise for cellular therapy, as they can identify and eliminate tumours that have altered major histocompatibility class I expression. Central nervous system tumours are the second most common paediatric cancer and are associated with a high mortality rate. Many of these high-grade paediatric brain tumours have very dismal prognoses, despite standard treatment regimens, and patients would substantially benefit from a novel type of therapy. Natural killer cells have been shown to be effective against many human malignancies; however, less is known about their efficacy against brain tumours. Treatment of medulloblastoma and adult glioblastoma with natural killer cells has been investigated previously and yielded promising results. Natural killer cells represent a small fraction of peripheral blood, which poses a limitation for their widespread use in adoptive cell therapy. Current research is focussed on establishing an optimal protocol for the ex vivo expansion and activation of natural killer cells. In the future, paediatric brain tumours such as glioblastoma multiforme, ependymoma and atypical teratoid/rhabdoid tumours should be examined for their susceptibility to natural killer cells.