Disruption of Iron Regulation after Radiation and Donor Cell Infusion Ekapun Karoopongse, A. Mario Marcondes, Cecilia Yeung, Zaneta Holman, Kris V. Kowdley, Jean S. Campbell, H. Joachim Deeg  Biology of Blood and Marrow Transplantation  Volume 22, Issue 7, Pages 1173-1181 (July 2016) DOI: 10.1016/j.bbmt.2016.03.031 Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Expression of iron regulatory genes in mouse liver after allogeneic T cell infusion. Early up-regulation of IL-6 was accompanied by increased expression of Hamp and down-regulation of Fpn1 with little change in Stat3 expression, suggesting the possibility of a direct effect of T cells on Hamp. Bmp6 and Smad4 declined early (as Hamp expression rose) before returning toward or above baseline. Gene expression in hepatocytes, as assessed by mRNA levels and determined by RT-PCR. Shown is the fold change compared with expression in unmodified controls determined at 1 hour to 14 days after i.v. injection of 1 × 106 allogeneic T cells (P→F1) (mean ± SEM of 3 experiments). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Serum iron, transferrin levels, and expression of Hamp and Fpn1 after infusion of 1 × 106 CD8+ T cells from allogeneic (P→F1) or syngeneic donors. Serum iron levels increased after allogeneic and syngeneic cells, whereas transferrin levels fluctuated. However, by 7 to 14 days after T cell injection, no further changes in Hamp and Fpn1 expression were noted, whereas levels increased in recipients of allogeneic cells. This late increase in expression of both Hamp and Fpn1 was likely related to late up-regulation of Stat3 and Smad4 (see Figure 1); the mechanism of the late Fpn1 response is not clear. No up-regulation was noted after Fas ligand–deficient cells. (A) Iron levels (μg/dL) and (B) transferrin levels (mg/dL) at 1 hour to 14 days after cell injection. Shown are individual data points and the mean values. (C and D) Expression of Hamp and Fpn1 at days 7 and 14. In addition to wild-type allogeneic cells (in black) and syngeneic cells (in red), cells from Fas ligand–deficient gld mice (in yellow) were injected. Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Gene expression in mouse liver after infusion of allogeneic (P→F1) CD8+ T cells. Changes in gene expression levels were T cell dose dependent. Expression levels in hepatocytes, as assessed by mRNA levels, were determined by RT-PCR. Shown is the fold increase over expression in unmodified controls, determined at 7 and 14 days after i.v. injection of 1 × 106 or 10 × 106 allogeneic T cells (P→F1; mean ± SEM of 3 experiments). The effect, in particular on Hamp expression, was more pronounced at the higher T cell dose. Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Gene expression in mouse liver after injection of 1 × 108 T lymphocytes from Fas ligand–deficient gld donor mice. In comparison with the effect of wild-type cells (see Figure 1), there was only minimal up-regulation of IL-6, without increased expression of Hamp, and Fpn1 and Smad4 levels progressively declined, underscoring the involvement of Fas-dependent signals in the response pattern shown in Figure 1. Gene expression in hepatocyte, as assessed by mRNA levels, was determined by RT-PCR. Shown are the fold changes relative to expression in unmodified controls (mean ± SEM of 3 experiments). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Gene expression in mouse liver after TBI. Expression patterns differed significantly from those after T cell infusion. In particular, there was no early increase in IL-6 and a delayed response of Hamp. Changes in expression were more pronounced after higher doses of TBI. (A) Time course from 1 hour to 7 days after exposure to 200 cGy. (B) Comparison of the effects of 200 and 400 cGy TBI on selected genes. Gene expression in hepatocytes, as assessed by mRNA levels, was determined by RT-PCR. Shown is the fold change over expression in unmodified controls determined at 1 (1D) to 7 days (7D) (mean ± SEM of 4 experiments). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Gene expression in mouse liver after TBI and infusion of T lymphocytes with or without the addition of marrow-derived cells. Although there was an early increase in expression levels of Hamp, Fpn1, IL-6, and Stat3 followed by a decline when no marrow cells were given, there was only a minimal early increase of IL-6 in parallel with decreased expression of Hamp and Fpn1, whereas Stat3 showed little change when marrow cells were co-infused. (A) After TBI (200 cGy) followed by the infusion of 1 × 106 allogeneic (P→F1) CD8+ T lymphocytes. (B) After TBI (200 cGy) followed by the infusion of 1 × 10 6 allogeneic CD8+ lymphocytes plus bone marrow cells (see Methods). Gene expression in primary hepatocytes, as assessed by mRNA levels, was determined by RT-PCR. Shown is the fold change compared with expression in unmodified controls (mean ± SEM of 3 experiments). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 Changes in body weight of mice. No significant weight changes were observed over time after infusion of syngeneic CD8+ T cells (in fact, mice gained weight) or after 200 cGy TBI or TBI followed by transplantation of CD8+ allogeneic T lymphocytes. Only “full transplantation” of T lymphocytes plus marrow cells resulted in significant weight loss, indicative of GVHD (mean ± SEM of observations in 5 mice per group). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 8 Hepatic and duodenal histology. (A) Duodenal mucosa and (B) liver on day 7 after 200 cGy of TBI and allogeneic (B6→CC) T lymphocyte and marrow cell infusion. There were lymphocyte aggregates in the mucosa (arrow) and the liver, and rare apoptotic cells (arrowhead), as observed with GVHD, were present (original magnification, 20×). Biology of Blood and Marrow Transplantation 2016 22, 1173-1181DOI: (10.1016/j.bbmt.2016.03.031) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions