CD4+ T Cell −Depleted Lymphocyte Infusion Impairs Neither the Recovery of Recipient Thymus nor the Development of Transplanted Thymus This information is current as of September 26, 2021. Ming Shi, Ming Li, Yunze Cui, Lin Liu, Yasushi Adachi and Susumu Ikehara J Immunol 2013; 190:2976-2983; Prepublished online 4 February 2013; doi: 10.4049/jimmunol.1201605 Downloaded from http://www.jimmunol.org/content/190/6/2976 Supplementary http://www.jimmunol.org/content/suppl/2013/02/04/jimmunol.120160 http://www.jimmunol.org/ Material 5.DC1 References This article cites 36 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/190/6/2976.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! 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The Journal of Immunology CD4+ T Cell–Depleted Lymphocyte Infusion Impairs Neither the Recovery of Recipient Thymus nor the Development of Transplanted Thymus Ming Shi,* Ming Li,* Yunze Cui,*,† Lin Liu,* Yasushi Adachi,*,‡ and Susumu Ikehara* Thymus transplantation, in conjunction with bone marrow transplantation (BMT), has been attracting attention for the treatment of various diseases. Recently, donor lymphocyte infusion (DLI) has been used as a helpful tool for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, the effects of DLI on transplanted and recipient thymuses have not been explored. We therefore performed DLI in the intrabone marrow–BMT + thymus transplantation setting. We have found that DLI leads to derangements in both recipient thymuses and transplanted thymuses; by 2 wk after BMT, we saw a decrease in total cell number, a lower percentage of CD4+CD8+ cells, and the obliteration of the thymic corticomedullary junction. Four weeks later, the Downloaded from thymic impairment became more serious. However, when we depleted the CD4+ T cells (CD42-DLI), the recipient thymic recovery and transplanted thymic development were significantly restored by the treatment. In addition, there were much greater levels of TNF-a and Fas ligand, and a lower percentage of regulatory T cells in the DLI group than in the CD42-DLI group. These findings indicate that inflammation induced by DLI, especially by CD4+ T cells, plays a crucial role in the thymic impairment. The Journal of Immunology, 2013, 190: 2976–2983. http://www.jimmunol.org/ llogeneic bone marrow transplantation (allo-BMT) is chronic GvHD (13). Thymus transplantation (TT), an attractive a potentially curative therapy for certain diseases of the method for improving T cell functions, has been applied clinically A hematopoietic system, immunodeficiencies, autoimmune for patients with DiGeorge syndrome or HIV infection, which diseases, solid malignant tumors, and so on (1–6). We have developed elicits the hypoplasia of the thymus (14). However, in mice, al- a new and powerful bone marrow transplantation (BMT) method: though T cell functions were restored or enhanced by TT, no con- intrabone marrow–BMT (IBM-BMT) (7), in which donor bone comitant GvHD was observed after TT in conjunction with allo- marrow cells (BMCs) are directly injected into the recipient’s bone BMT (15). Therefore, TT can be used to treat autoimmune diseases marrow cavity. Therefore, a much greater number of donor hemato- in chimeric-resistant MRL/lpr mice and type 2 diabetes mellitus, poietic stem cells and mesenchymal stromal cells (including mesen- and to suppress tumor growth (16–18). by guest on September 26, 2021 chymal stem cells) can be inoculated into the recipient bone marrow Donor lymphocyte infusion (DLI) is often used after allo-BMT by IBM-BMT than by conventional i.v. BMT. This results in the rapid to prevent disease relapse in the setting of T cell–depleted BMT or reconstitution of donor hematopoietic cells and permits a reduction nonmyeloablative conditioning regimens. It is also a combined in the doses of irradiation used as a conditioning regimen (8–10). method to convert from mixed chimerism to full donor chimerism The thymus is an organ for inducing T cells and maintaining (19, 20). However, DLI-induced GvHD is always associated with homeostasis. However, thymic functions are impaired by the con- an increase in therapy-related morbidity because of its uncon- ditioning regimen and the acute graft-versus-host disease (GvHD) trollable and fatal characteristics (21). It has been reported that that occurs after allo-BMT, resulting in deficient cell immunity (11, many factors are involved in the damage to the recipient thymus 12). In addition, there is a strong association between posttrans- after DLI (22, 23), whereas the effects of DLI on the transplanted plant autoimmune disease and the thymic dysfunction caused by thymus have hitherto remained unexplored. In this study, we investigate the influence of DLI on both re- *Department of Stem Cell Disorders, Kansai Medical University, 570-8506 Osaka, cipient and transplanted thymuses in the IBM-BMT + TT setting. Japan; †JIMRO Co., Ltd., 370-0021 Gunma, Japan; and ‡Division of Clinical Pathol- Because we have found that TT using newborn thymus is most ogy, Toyooka Hospital, 668-8501 Hyogo, Japan effective in tumor suppression (18), we used newborn thymus in this Received for publication June 13, 2012. Accepted for publication January 3, 2013. study. We show in this article that CD4+ T cell–depleted lympho- 2 This work was supported by the Otsuka Pharmaceutical Company, Ltd., and by grants cyte infusion (CD4 -DLI) impairs neither the recovery of recipient from the Research on Allergic Disease and Immunology Committee from the Health thymus nor the development of transplanted thymus. and Labour Sciences Research of the Ministry of Health, Labour and Welfare. Address correspondence and reprint requests to Prof. Susumu Ikehara, Department of Stem Cell Disorders, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi Materials and Methods City, Osaka 570-8506, Japan. E-mail address: [email protected] Mice The online version of this article contains supplemental material. C57BL/6 (B6), enhanced GFP (eGFP) transgenic (tg) B6, and BALB/c mice Abbreviations used in this article: allo-BMT, allogeneic bone marrow transplantation; were purchased from Shimizu Laboratory Supplies (Shizuoka, Japan). B6, C57BL/6; BMC, bone marrow cell; BMT, bone marrow transplantation; DLI, Eight- to 12-wk-old male mice were used for BMT and DLI. For TT, donor lymphocyte infusion; eGFP, enhanced GFP; FasL, Fas ligand; GvHD, graft- 1 d after birth, B6 mice were sacrificed to obtain newborn thymuses. All the versus-host disease; IBM-BMT, intrabone marrow–BMT; tg, transgenic; Treg, regu- latory T cell; TT, thymus transplantation; WSP, whole spleen. mice were maintained in a specific pathogen-free room. This article is distributed under The American Association of Immunologists, Inc., Experimental protocol Reuse Terms and Conditions for Author Choice articles. As shown in Fig. 1, BALB/c mice were lethally irradiated with 7 Gy using Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 the Gammacell 40 Exactor (MDS Nordion, Kanata, ON, Canada) with two www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201605 The Journal of Immunology 2977 [137Cs] sources, and the next day, these mice received IBM-BMT from B6 some mice were injected i.p. with 1 mg anti–TNF-a Ab and 2 mg anti-FasL mice (group I). Some mice additionally received TT from B6 mice (group Ab (WSP + Abs group) on days 0, 4, 8, and 12 as described previously (26). II). On the same day, some mice also received whole spleen (WSP-), CD42-, or CD82-DLI from B6 mice: WSP-DLI (group III), CD42-DLI Statistical analysis (group IV), and CD82-DLI (group V). The treated mice were sacrificed The results are represented as means 6 SD. The Student t test was used to 5 d, 2 wk, or 4 wk after the treatments. determine any statistical significance. A p value ,0.05 was considered to Reagents and flow cytometric analysis be a significant difference. The Abs used in this study were as follows: purified rat anti-mouse CD4 and CD8 Ab (eBioscience, San Diego, CA); FITC-conjugated anti-mouse Results CD4 and H-2Kb Ab; PE-conjugated anti-mouse H-2Kd, CD4, CD8, and In the IBM+TT setting, WSP-DLI induces serious GvHD but B220 Ab; and PerCP-Cy5.5–conjugated anti-mouse CD45 Ab (BD Phar- CD42 -DLI does not mingen, San Diego, CA). For the thymus and peripheral blood analysis, leukocytes were first gated by CD45+ cells, which were estimated as nu- We carried out IBM-BMT, TT, and DLI from B6 to BALB/c mice. clear cells. To analyze the percentage of regulatory T cells (Tregs) in the The protocol is described in detail in Fig. 1 and in Materials and spleen, we performed intracytoplasmic FoxP3 staining using an eBio- Methods. science FITC–anti-mouse/rat FoxP3 staining set in accordance with the manufacturer’s instructions. Samples were analyzed using an FACSCalibur The TT group (group II) showed similar results to group I (Fig. flow cytometer (BD Biosciences). 2), although TT provides an abundance of thymocytes (immature T cells), whereas DLI contains mature T cells and attacks the IBM-BMT and TT target organs after priming.