Graft Versus Host Disease in CD34+ Human Hematopoietic Stem Cell Treated NSG Mice

4 CONCLUSIONS• At the completion of study, a subset of treated mice demonstrated lesions consistent with GVHD. Histologic changes supportive of GVHD were

observed in skin and liver in this study.• In a previous report of GVHD in NSG mice, skin was mainly affected in animals humanized with human bone marrow, fetal liver, or thymus3. NSG

mice humanized with CD34+ CD3+ depleted stem cells developed GVHD involving skin, liver, lung, spleen, intestine, and gingiva3.• There can be variability in the presentation of GVHD and the organs involved, and none of the histologic changes are pathognomonic.• It is important to be aware of this potential in studies utilizing HCT in immunodeficient mouse strains, and it may serve as a model for GVHD in

human recipients of allogeneic HCT.

2 OBJECTIVE and METHODSObjective: The objective of this study was to evaluate the safety of intravenously (IV) administered proprietary-treated or untreated CD34+ human stem cells over 170 days in NSG mice.

1 BACKGROUNDAllogeneic hematopoietic stem cell transplantation (HCT) is used to treat hematologic malignancies, genetic diseases, and hematologic disorders.Graft versus host disease (GVHD) is the most common long term complication of HCT and is potentially fatal. Skin, liver, gastrointestinal tract, andlungs are most commonly affected. Even when the patient receives a HLA identical graft, 40% will still develop systemic acute GVHD1.

The main events in GVHD pathogenesis are activation of antigen presenting cells, donor T cell activation, proliferation and migration of donor Tcells, and target tissue damage1. Donor T cells are stimulated by foreign proteins on the recipient tissues, such as minor histocompatibilityantigens1. In most HLA-identical HCTs, both CD4+ and CD8+ T cell subsets respond to minor histocompatibility antigens and cause GVHD1.Clinical signs can include a maculopapular rash, vomiting, diarrhea, mucosal ulcers, jaundice and sicca syndrome1.

There is no histologic finding pathognomonic for GVHD 2. Histologic lesions supportive of the diagnosis of GVHD in the skin include basal cellapoptosis and vacuolation in the epidermis and hair follicle, interface dermatitis with lichenoid inflammation, lymphocytic satellitosis, andlymphocyte exocytosis1,2. More chronic changes can include dermal collagen deposition and panniculitis2. Hepatic lobular or portal inflammation,damaged bile ductules, and cholestasis can be seen in the liver1,2. More chronic changes can include ductopenia and portal fibrosis1,2.Gastrointestinal changes can include apoptosis in the crypts, gland destruction, ulceration, and fibrosis, lungs can develop bronchiolitis obliterans,often leading to fibrosis2.

Immunodeficient mice with humanized immune systems are a valuable tool to study the immunologic basis and treatment of numerous diseases.In a safety evaluation of proprietary-treated CD34+ human stem cells, NOD SCID gamma (NSG) mice(NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) developed lesions consistent with GVHD following pre-treatment with busulfan. NSG mice lack mature B and Tcells, NK cells, and complement, and have defective dendritic cells and macrophages. Busulfan pre-treatment potentiates the depletion of thesecell lines, as well as other cells such as neutrophils.

3 RESULTSAt terminal euthanasia, several mice from both stem cell treated groups developed histologic lesionssupportive of GVHD including minimal to moderate portal inflammation, and minimal to mild mononuclearinterface dermatitis with disruption of the basal cell layer of hair follicles, and vacuolation and single cellnecrosis of the basal cells of the epidermis. Histiocytic infiltrates were present in the bone marrow, spleen,and mesenteric lymph nodes of stem cell-treated animals. Minimal to moderate perivascular mononuclearinfiltrates, histiocytic infiltrates, and increased alveolar macrophages were also observed in the lungs.

Engraftment of the stem cells and establishment of a humanized immune system were supported byincreased splenic weights, increased splenic white pulp and haematopoiesis, and increased cellularity ofthe thymus and mesenteric lymph nodes in treated animals compared to controls.

Erica L. Twitchell, Shawna Jackman, Elizabeth Rainbolt, and Danielle L. Brown

Experimental DesignGroup No. Test

MaterialPretreatment

Dose Level (cells/animal)

Dose Volume(mL)

Dose Concentration (mg/mL)

No. of Animals

1 Vehiclea No treatment

0 0 0 6

2 Untreated HSCs

Busulfanb 1X106 0.2 5X106 20

3 Treated HSCs

Busulfanb 1X106 0.2 5X106 20

a. Vehicle was 50% Plasma-LyteA and 50% CryoStorTM CS10b. Busulfan solution was administered via intraperitoneal administration once a day for

2 days at 20 mg/kg prior to stem cell injection (IV administration tail vein).

Summary of Microscopic FindingsTerminal Euthanasia (Day 170)

Group 1 2 3Treatment Vehicle HSC1 HSC2

No. Animals per Group 6 20 12Site, Injection, Tail Vein (No. Examined) 6 20 12Inflammation, mononuclear cell, perifollicular 0 4 2Liver (No. Examined) 6 20 12Infiltration, histiocytic periportal 0 7 5Lung (No. Examined) 6 20 12Macrophage aggregation, alveolar 2 18 12Infiltration, histiocytic 0 3 6Infiltration, mononuclear cell, perivascular 0 8 5Bone Marrow, Sternum (No. Examined) 6 20 12Infiltration, histiocytic 0 6 3Spleen (No. Examined) 6 20 11Infiltration, histiocytic 0 5 2Increased hematopoiesis 2 20 10

REFERENCES1. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet.. 2009;373(9674):1550-61.2. Shulman HM, Kleiner D, Lee SJ, Morton T, Pavletic SZ, Farmer E, et al. Histopathologic diagnosis of chronic graft-versus-host disease: National

Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. Pathology Working Group Report. Biol Blood Marrow Transplant. 2006;12(1):31-47.

3. Sonntag K, Eckert F, Welker C, Muller H, Muller F, Zips D, et al. Chronic graft-versus-host-disease in CD34(+)-humanized NSG mice is associated with human susceptibility HLA haplotypes for autoimmune disease. J Autoimmun. 2015;62:55-66.

Skin (tail) from a HSC treatedmouse. Histiocytes andlymphocytes infiltrate the outerroot sheath of hair follicles and toa lesser extent, the epidermis.

Skin (tail) from a HSC treatedmouse. Lymphocytes infiltrate theouter root sheath of a hair follicleand epidermis, and basal cells arevacuolated.

Liver from a HSC treated mouse. Histiocytes and lymphocytes infiltrate a portal triad.