van Rijn RS, Simonetti ER, Hagenbeek A, Hogenes MCH, de Weger RA, Canninga-van Dijk MR, Weijer K, Spits H, Storm G, van Bloois L, Rijkers G, Martens AC, Ebeling SB. 
A New Xenograft Model for Graft-Versus-Host Disease by Intravenous Transfer of Human Peripheral Blood Mononuclear Cells in RAG2-/- ɣc-/- Double-Mutant Mice.
Blood. 2003 Oct 1;102(7):2522–31.
[toggle_content title=”Abstract”] The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- γc-/- mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- γc-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
2-2.5 mg/ml 69. 4 mg/ml 1 EPC/Chol/EPG 80/12/8 MLV PBS

1 Phospholipid concentration; cholesterol not assayed.

[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Adjunct Dosing? Adjunct Dosing Route
RAG2 -/- γc -/- mice, 8-34 w 200 µl intravenous systemic no NA
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. We commend the authors for assaying both clodronate and lipid concentrations in their preparation, however they selected an atypical formulation of clodronate liposomes for which they cite nor acknowledge any depletion studies performed with this particular formulation. Confirmed depletion of any population of macrophages at any time point was not reported in this paper. Depletion was assumed.
  2. The study cited for macrophage depletion was one in which rat spleen and liver macrophage depletion and repopulation was compared to those previously reported for mice. The earliest time point assessed was 2 days, while this van Rijn, et. al. study assumed depletion after one day at which time the huPBMC were injected.
  3. The clodronate-to-lipid wt ratio was significantly smaller 2.5/69 than that of other commonly used formulations (5/23 range), although the volume dosed was the same as that used for more common formulations. These animals received about half the usual clodronate dose and 3X more lipid than the usual dose.
[/toggle_content] [toggle_content title=”Results”]
  1. The goal of the authors was to develop a model of GVHD post-intravenous, rather than intraperitoneal, injection of huPBMC to more accurately mimic human GVHD caused by the intravenous introduction of donor PBMC. The engraftment rate leading to the development of GVHD was enhanced in the clodronate-liposome treated groups vs PBS-liposome treated groups suggesting that some level depletion of some population of phagocytes was critical to the development of this model. However, the authors failed to establish the extent, time of maximal depletion and repopulation rate of any phagocyte population. We believe that characterizing the phagocyte depletion parameters is absolutely necessary in establishing and optimizing this model. Additionally, it seems to us that determining which phagocyte populations are or are not involved in enhancing the engraftment rate is an important piece of information in characterizing GVHD.
  2. The authors speculate that the clodronate liposomes deplete the phagocytes in the injected huPBMC, however this is extremely unlikely, if not impossible, since the huPBMC were injected 1 day post-injection of the clodronate liposomes. These clodronate liposomes would not remain in the circulation for more than a few hours.