Experimental Modeling of Acute- and Chronic-GvHD By Xenotransplanting Human Donor PBMCs or Cord Blood CD34⁺ Cells (HSC) into NSG Mice

Graft vs. host diseases (GvHD) is a life-threatening complication accounting for 15-30% of deaths following allergenic hematopoietic stem cell transplantation (allo-HSCT) for treatment of malignant diseases. There are two types of GvHD: acute (aGvHD) and chronic (cGvHD), which have different parthenogenesis and phenotype. aGvHD typically involves skin, gastrointestinal and hepatic inflammation, and occurs within 100 days of transplantation; and cGvHD involves multiple organs and occurs beyond 100 days. The former is largely due to the rapid activation of donor T cells (T_h1, CD8⁺ biased), causing tissue damage (via cell killing) and often leading to mortality; in contrast, the latter (T_h2, CD4⁺ biased) typically display autoimmune-like syndrome, involving both T- and B-cell, as well as auto antibody production and systemic fibrosis. The current understanding of the parthenogenesis, as well as developing treatment strategy, is largely based on experimental animal models, many aspects of which remain to be investigated. At present, the most commonly used GvHD models are allo-transplantation between mice, the result of which many may not all adequately translate into clinic for the donor species difference. Xeno-transplantation of human donor into mice could potential represents a better alternative GvHD model in some respects.

Methods. Human peripheral blood mononuclear cells (PBMC) derived from the consent normal donor were transplanted into NSG (other NSG-like strains) and B2M-NSG (MHC class I, b-2 micro-globulin, deficiency) mice for modeling aGvHD, while cord blood derived HSCs (hCD34⁺) (Jackson Lab, huNSG) were transplanted for modeling cGvHD. Following transplantation, the disease progress will be monitored twice weekly, including clinical observations (e.g. animal postures, activity, fur texture, and skin integrity), body weight changes, gross pathology and histo-pathology upon termination, along with human-immunological phenotype of peripheral blood, spleen, lung, and liver by flow (e.g. lymphocyte populations), multiplex, complete blood count, histo-pathology and immunohistochemistry at different time-points.

Results. NSG mice engrafted with human PBMC, or purified T-cells, from normal donors rapidly developed typical symptoms of aGvHD, onset 2~3 weeks post transplantation, including severe body loss, reduced activity, hunched posture, loss of fur in combination with severe ruffling and overall poor grooming. Mortality is observed typically from 4^th or 5^th weeks post transplantation. In correlation, we have observed significant engraftment of human CD45⁺ human leukocytes (0.5~1.5% on Day 6, 10~30% on Day 16, 30~50% on Day 26 post transplantation, with variations depending on donors, route of transplantation, IP vs. IV, etc), including dominant CD3⁺ human T-cells of single positive of either CD4⁺ or CD8⁺ T-cells. This correlated kinetics between clinical symptoms and degree of engraftment of human leukocytes, particularly T-lymphocytes, suggests xenografting human T-cells are responsible for the observed aGvHD. Further comprehensive analysis on the pathology and immunophenotypes of relevant organs are ongoing. In parallel to NSG, we assessed aGvHD in the absence of mouse MHC-I using the PBMC engrafted B2M mice. Additionally, NSG-mice were engrafted with CD34⁺ cells derived from cord blood with cGvHD high- risk and low-risk HLA haplotypes. We observed cGvHD development between 18 weeks to 39 weeks post-engraftment, in contrast to the short duration seen aGvHD; symptoms include severe weight loss, impaired movement, severe ruffling, facial/full body alopecia, and scaly skin. Interestingly, in correlation of this, human engraftment of CD45⁺, particularly T-cells including CD4⁺, CD8⁺ and their CD30⁺ subsets, are kinetically increased in blood and spleen with similar timeline, suggesting their roles in the observed cGvHD.

Conclusions. The xenograft murine model using adult human PBMC and cord blood derived CD34⁺ HSCs could be alternative experimental systems to model human aGvHD and cGvHD for investigating disease mechanisms and evaluating treatment strategy.