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. 2017 May;14(5):443-450.
doi: 10.1038/cmi.2015.92. Epub 2015 Oct 26.

Antithymocyte globulin treatment at the time of transplantation impairs donor hematopoietic stem cell engraftment

Feng Jin  1   2 , Jin He  1 , Chunhui Jin  1   3 , Wei Fan  1   2 , Yanhong Shan  1 , Zhefeng Zhang  1 , Liguang Sun  1   2 , Zheng Hu  1   2 , Yong-Guang Yang  1   2   3
Affiliations

Antithymocyte globulin treatment at the time of transplantation impairs donor hematopoietic stem cell engraftment

Feng Jin et al. Cell Mol Immunol. 2017 May.

Abstract

Antithymocyte globulin (ATG) is often included in the conditioning regimen to prevent graft vs. host disease in allogeneic hematopoietic stem cell (HSC) transplantation. However, because ATG contains antibodies targeting a wide range of antigens on human cells, its potential off-target effects remain a concern. Here, we explored this question in humanized mice that permit the analysis of human cell depletion in tissues. We showed that ATG binds to almost all lineages of human hematopoietic cells including HSCs, and accordingly it is capable of depleting almost all human hematopoietic cells. Interestingly, the efficacy of ATG was highly variable depending on the tissue of residence, with human cells in bone marrow significantly less susceptible than those in the blood and spleen. Recovery of multilineage human lymphohematopoietic reconstitution in humanized mice that received ATG 3 weeks after HSC transplantation indicates that ATG had a minimal effect on human HSCs that have settled in bone marrow niches. However, efficient human HSC depletion and engraftment failure were seen in mice receiving ATG at the time of transplantation. Our data indicate that the efficacy of ATG is tissue-dependent, and suggest a potential risk of impairing donor hematopoietic engraftment when ATG is used in preparative conditioning regimens.

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Figures

Figure 1
Figure 1
Human cell depletion by ATG in established humanized mice. Humanized mice were constructed by transplantation of human fetal thymus and CD34+ cells, and 13 weeks later received 3 injections i.v. of ATG-G (3 mg/kg−1 per injection at day 0 and day 2, and 30 mg/kg−1 at day 4) or PBS. (a) Levels (%) of human CD45+, CD3+ and CD19+ cells in peripheral blood measured 3 weeks prior to the first injection and 7 days after last injection of PBS or ATG. (b) Percentages of human CD45+, CD3+ and CD19+ cells in the spleen at day 7-post-last injection of PBS or ATG. (c) Percentages of live (PI-negative) human thymocytes in the human thymic grafts at day 7 post-last injection of PBS or ATG. (d–e) Percentages of human CD45+, CD3+ and CD19+ cells (d) and of human CD34+ cells (e) in bone marrow at day 7 post-last injection of PBS or ATG. Data presented are mean ± SEMs (n = 3–4 per group). *P < 0.05; **P < 0.01; ***P < 0.001. Similar results were obtained from an independent experiment presented in Figure S2.
Figure 2
Figure 2
Direct binding of ATG to human lymphohematopoietic cells. Shown are representative FACS profiles illustrating the binding of ATG-G to human CD3+ and CD19+ PBMCs, bone marrow cells (BMC), and purified CD34+ fetal liver cells (FLC). Binding of ATG was detected by fluorochrome-conjugated goat anti-rabbit antibody.
Figure 3
Figure 3
ATG administration at the time of human HSC transplantation induces engraftment failure. NSG mice received transplantation of human fetal thymus and CD34+ cells, followed one day later by injection i.v. of PBS or ATG-G (10 mg/kg−1). (a) Percentages of human CD45+, CD3+ and CD19+ cells in PBMCs at the indicated times after human thymus/CD34+ cell transplantation (n = 4). (b) Representative FACS profiles showing human CD45+, CD3+ and CD19+ cell chimerism in PBMCs, spleen and bone marrow at week 19.
Figure 4
Figure 4
ATG administration depletes donor HSCs prior to their settling in the bone marrow. NSG mice received transplantation of human fetal thymus and CD34+ cells, followed one day later by injection i.v. of ATG-F (10 mg/kg−1; ATG d1), or 21 days later by injection i.v. of ATG-F (30 mg/kg−1; ATG d21) or PBS. (a) Percentages of human CD45+, CD3+, and CD19+ cells in PBMCs at the indicated times. (b) Percentages of human CD45+, CD3+, and CD19+ cells in PBMCs, spleen and bone marrow (BM) at week 17. Representative staining profiles are presented in Figure S4. (c) Percentages of human CD34+ cells in bone marrow and representative staining profiles from ATG d21 and PBS control groups at week 17. N = 4 per group; N.S. indicates not significant.
Figure 5
Figure 5
Comparison of human cell depletion in peripheral blood and bone marrow in ATG-treated mice. ATG-F (10 mg/kg−1; i.v.) was administrated to NOD/SCID mice 3 days prior to (ATG d-3), or 1 (ATG d1) or 21 (ATG d21) days after human HSC transplantation. Mice in the control groups received PBS or polyclonal rabbit IgG antibodies (10 mg/kg−1) 1 day after human HSC transplantation. (a) Percentages of human CD45+ cells in PBMCs at week 3 (measured right before ATG injection in the ATG d21 group) and week 5. (b) Percentages of CD34+ human cells in bone marrow at week 5. Each group contained 3–4 mice; group means and representative FACS profiles are shown in the top and bottom panels, respectively.
Figure 6
Figure 6
NSG mice were treated with PBS (control; top panels) or ATG-F (30 mg/kg; bottom panels) 3 weeks after human CD34+ cell transplantation. Bone marrow cells were prepared one day later and analyzed for ATG binding to human cells by FACS using fluorochrome-conjugated goat anti-rabbit antibody. (a) ATG binding to human CD45+ cells. (b) ATG binding to CD34+ cells. Shown are representative FACS profiles and the number in each figure indicates the percentage of cells in the gate.
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