Transplantation of IL-2–transduced murine bone marrow is associated with dose- dependent toxicity  Thomas Kühr, Oliver Bechter, Stephan Dirnhofer, Stephan Geley, Anne Gächter, Georg Pall, Manfred Url, Hermann Dietrich, Peter Oberaigner, Günter Klima, Wolfgang Eisterer, Wolfgang Hilbe, Peter Lukas, Josef Thaler  Experimental Hematology  Volume 28, Issue 8, Pages 895-906 (August 2000) DOI: 10.1016/S0301-472X(00)00487-2

Figure 1 JmIL-2 retroviral vector: LTR = long terminal repeat that contains the retrovirus promotor and enhancer derived from myeloproliferative sarcoma virus; IL-2 = the coding region of the IL-2 gene; Tkneo = the coding region for the Tkneo gene Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 2 (A) Southern hybridization of cells from recipient BM (M) and/or spleen (S) obtained on days 12 and 21 after transplantation of IL-2–transduced BM. Southern hybridization was performed using a DIG-labeled IL-2 cDNA probe. The 1.7kb band in BM and spleen cells is detectable at both intervals after transplantation. (B) Analysis of total cellular RNA obtained from murine PB, BM, and spleen cells on day 12 after transplantation of IL-2–transduced and nontransduced BM: panel a indicates the total cellular RNA (5–10 μg) purified as described in the Material and methods section, subjected to electrophoresis, transferred to nitrocellulose, and hybridized to a randomly primed DIG-labeled IL-2 probe before being imaged by autoradiography; panel b indicates the ethidium bromide staining of the gel. (C) RT-PCR analysis of total cellular RNA that proved to be negative for rec. IL-2 by Northern blotting: transcription of recombinant IL-2 in BM and spleen cells obtained on day 21 post-BMT was confirmed by RT-PCR using oligonucleotide primers that bridge a sequence between the recombinant IL-2 and the neoR gene. The following samples were used as controls: S, BM = total cellular RNA of normal spleen and BM cells that was subjected to DNAse treatment while RT was omitted; − = total RNA of normal unmanipulated BM; S/IL-2 = RNA from spleen cells obtained on day 12, which proved to be positive for rec. IL-2; + = RNA obtained from JmIL-2–transduced packaging cells Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 3 Relative proportion of progenitor cells during the early post-transplant period. The impact of IL-2 on hematopoietic progenitor cells was analysed using a CFU assay performed on BM cells isolated from recipients transplanted with 50% of IL-2–transduced (BM50) or nontransduced marrow. Colony growth of CFU-GM (white bars), BFU-E (black bars), and CFU-GEMM (gray bars) was not adversely influenced by IL-2 Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 4 Comparison of cytotoxic potential generated by IL-2–transduced BM cells against C1498 cells in vitro. Following infusion of IL-2–transduced BM cells, spleen cells from BM50 recipients and nontransduced controls (BMc) were analyzed at various intervals. Values are means ± SE from six mice/interval per group from two different experiments. Data are shown as percentage of specific lysis at an effector to target ratio of 50:1 Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 5 (A) HE-stained section of liver 21 days post-transplant. Dense portal and periportal inflammatory infiltrates and frequent individual hepatocyte necrosis can be found in recipients transplanted with 100% of IL-2–transduced marrow cells. (B) HE-stained section of liver 21 days post-transplant of a nontransduced control animal Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 6 HE-stained sections of spleens 21 days post-transplant of IL-2–transduced (A) and nontransduced (B) recipients. Note the features of hemophagocytosis: large macrophages contain phagocytosed red blood cells. There is also an increase in lymphocytes, eosinophils, and polymorphonuclear leukocytes (A), while nontransduced controls were devoid of pathological features (B) Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 7 Immunohistochemical demonstration of ICAM-1 (A) and VCAM-1 (B) expression in the liver. Expression of both adhesion molecules was markedly increased in BM100 recipients early after transplantation of IL-2–transduced marrow. (C) Lack of ICAM-1 expression in the liver of a nontransduced control animal. Since sections were also negative for VCAM-1 expression, only one photograph is depicted representatively Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)

Figure 7 Immunohistochemical demonstration of ICAM-1 (A) and VCAM-1 (B) expression in the liver. Expression of both adhesion molecules was markedly increased in BM100 recipients early after transplantation of IL-2–transduced marrow. (C) Lack of ICAM-1 expression in the liver of a nontransduced control animal. Since sections were also negative for VCAM-1 expression, only one photograph is depicted representatively Experimental Hematology 2000 28, 895-906DOI: (10.1016/S0301-472X(00)00487-2)