Alloreactivity but Failure to Reject Human Islet Transplants by Humanized Balb/c/Rag2 −/− gc −/− Mice
2009; Wiley; Volume: 71; Issue: 2 Linguagem: Inglês
10.1111/j.1365-3083.2009.02356.x
ISSN1365-3083
AutoresS. Jacobson, Frank Heuts, Julius Juaréz, Monica Hultcrantz, Olle Korsgren, Mattias Svensson, Martı́n E. Rottenberg, Malin Flodström‐Tullberg,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoScandinavian Journal of ImmunologyVolume 71, Issue 2 p. 83-90 Free Access Alloreactivity but Failure to Reject Human Islet Transplants by Humanized Balb/c/Rag2−/−gc−/− Mice S. Jacobson, S. Jacobson Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorF. Heuts, F. Heuts Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Stockholm, SwedenSearch for more papers by this authorJ. Juarez, J. Juarez Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorM. Hultcrantz, M. Hultcrantz Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorO. Korsgren, O. Korsgren Division of Clinical Immunology, Uppsala University, Uppsala, SwedenSearch for more papers by this authorM. Svensson, M. Svensson Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorM. Rottenberg, M. Rottenberg Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Stockholm, SwedenSearch for more papers by this authorM. Flodström-Tullberg, M. Flodström-Tullberg Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this author S. Jacobson, S. Jacobson Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorF. Heuts, F. Heuts Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Stockholm, SwedenSearch for more papers by this authorJ. Juarez, J. Juarez Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorM. Hultcrantz, M. Hultcrantz Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorO. Korsgren, O. Korsgren Division of Clinical Immunology, Uppsala University, Uppsala, SwedenSearch for more papers by this authorM. Svensson, M. Svensson Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this authorM. Rottenberg, M. Rottenberg Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Stockholm, SwedenSearch for more papers by this authorM. Flodström-Tullberg, M. Flodström-Tullberg Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, SwedenSearch for more papers by this author First published: 15 January 2010 https://doi.org/10.1111/j.1365-3083.2009.02356.xCitations: 12 M. Flodström-Tullberg, Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge F59, S-141 86, Stockholm, Sweden. E-mail: malin.flodstrom-tullberg@ki.se Editorial note: Conceived and designed the experiments: S.J., M.S., M.R., M.F-T. Performed the experiments: S.J., F.H., J.J., M.H., M.S., M.F-T. Analyzed the data: S.J., F.H., J.J., M.H., M.S., M.F-T. Wrote the article: S.J., M.S., M.F-T. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract A human islet transplant can cure patients with type 1 diabetes. A drawback of islet transplantation is the life-long immunosuppressive medication, often associated with severe side effects. Moreover, in spite of the immunosuppressive therapy, islets are lost in the majority of transplanted patients over time. An improved small animal model for studies on human islet allograft rejection mechanisms and the development of new measures for its prevention is clearly warranted. Here, we evaluated the potential of Balb/cRag2−/−γc−/− mice carrying a human-like immune system (so-called humanized mice) as a tool for studies on the rejection of transplanted human islets. Human T cells from Balb/cRag2−/−γc−/− mice, which as neonates had been transplanted with CD34+ human cord blood haematopoietic stem cells (HIS mice), proliferated in response to allogeneic human dendritic cells, but failed to reject a human islet allograft transplanted to the renal subcapsular space as assessed by immunohistochemistry and analysis of human serum C-peptide levels. Histological analysis revealed that few if any T cells had migrated to the grafted tissue. These observations question the usefulness of the HIS mouse model for studies on human islet allograft rejection mechanisms and highlight the need for further improvements. Introduction Type 1 diabetes (T1D) is a chronic metabolic disease resulting from a selective loss of the insulin-producing pancreatic beta cells. At present, there are no preventative treatments for the disease. Pancreatic islet transplantation is a method to restore pancreatic endocrine function in T1D patients. However, current immunosuppressive therapies fail to prevent graft rejection over time, and are associated with undesired side effects [1-3]. Given these problems and the shortage of human islet donors, the majority of T1D patients cannot be offered a transplant today. Hence, new methods preserving the transplanted tissue, including effective immunosuppressive therapies without side effects, are clearly warranted. Increased knowledge on the mechanisms involved in islet rejection would facilitate the development of tailored prevention therapies. Until now, the mouse has been the animal of choice for the majority of studies on islet rejection and its prevention. Nonetheless, despite enormous efforts by many researchers, encouraging findings in this model have seldom led to advancements in clinical islet transplantation. A reason for this may be the considerable differences between the human and murine immune systems [4, 5]. Recent advancements in the area of so-called humanized mouse models have led to expectations that it might become possible to study the detailed interactions between cells of the human immune system and transplanted human allogeneic pancreatic islets in vivo [6]. The term humanized mouse model is commonly used for immunodeficient mice that harbour components of the human immune system as a result of the transfer of human stem cells or human peripheral blood mononuclear cells (PBMC). Already in 1988, it was demonstrated that mice deficient in mature T and B cells (severe combined immunodeficient, SCID, mice) can be engrafted with human haematopoietic cells [7, 8]. A major progress in recent years is the development of SCID or Rag−/− mice lacking a functional IL-2 receptor common gamma chain (IL-2rγc), which because of their lack of natural killer (NK) cells are particularly permissive hosts for the engraftment of human haematopoietic stem cells. For example, Manz et al. reported that the transfer of human CD34+ stem cells to neonatal Balb/cRag2−/−γc−/− mice generated animals (here denoted HIS mice) that developed the major components of the mature human immune system de novo. Spleens and lymph nodes from such mice contained human CD4 and CD8 single positive T cells with a broad Vβ repertoire, as well as CD19+ IgM+ B cells. Human IgM was present in serum and several subsets of functional human dendritic cells (DC), including the plasmacytoid DC subset, were found in bone marrow, spleen, lymph nodes, thymus and liver of these mice [9, 10]. Functional assessments have shown that HIS mice are permissive to infections by the human pathogens Epstein–Barr virus (EBV) [10] and human immunodeficiency virus type 1 (HIV-1) [11-13]. Moreover, the HIS mice mount antibody responses against these viruses and different vaccination treatments, yet not to levels completely comparable to humans. The human T cells that develop in HIS mice proliferate when stimulated with human allogeneic DC, but not host mouse DC in mixed lymphocyte reactions (MLR). Finally, T cells in HIS mice infected with EBV respond to EBV-transformed B cells in vitro [10]. Efforts are required to identify which humanized mouse model(s) is the most suited for studies on allograft rejection mechanisms. Rejection of allogeneic human islet grafts has been shown in diabetic NOD/SCID [14-16] and NOD/SCIDγc−/−[17] mice (also denoted the Hu-PBL model) transplanted with human islets and subsequently injected with allogeneic PBMC. However, this is a more simplified humanized mouse model lacking thymic generation of naïve T cells and that produces low levels of B cells. Further, the Hu-PBL model develops xenoreactivity leading to graft-versus-host disease, thus limiting long-term studies [18]. Considering the promise of the HIS model for studies on the functions of the human immune system and the lack of reports on allogeneic graft rejection in this model, we set out to carefully test whether such mice reject a transplanted human islet allograft. Materials and methods Ethics. The human pancreatic islet material used in this study represents the unavoidable excess of islets generated within the Nordic Network for Clinical Islet Transplantation. Only organ donors who explicitly had agreed to donate for scientific purposes were included. Informed written consent to donate organs for medical and research purposes was obtained from donors, or relatives of donors, by the National Board of Health and Welfare (Socialstyrelsen), Sweden. Permission to obtain pancreatic islet tissue from the Nordic Network for Clinical Islet Transplantation was reviewed and approved by the local ethics committee (Regionala etikprövningsnämnden, Stockholm) in Stockholm, Sweden. The human peripheral blood and cord blood used were obtained from donors who had given their informed verbal consent to donate blood for scientific purposes. Written consent was not required as no information regarding the donors was provided (i.e. the data were analyzed anonymously). The collection of blood and experiments using the blood reported here were approved by the local ethics committee (Regionala etikprövningsnämnden, Stockholm) in Stockholm, Sweden. Animals. Balb/cRag2−/−γc−/− mice were bred and maintained at Karolinska Institutet, Stockholm, Sweden, under specific pathogen-free conditions. All animal experiments were approved by the local ethics committee (Stockholms Norra Försöksdjursetiska Nämnd) and conducted in accordance with institutional guidelines for animal care and use. Human CD34+ cells. Human cord blood was obtained from three healthy full-term newborns at the Karolinska University Hospital Huddinge, Stockholm, Sweden, with parental informed consent and CD34+ cells were enriched (70–98% purity) by MACS cell separation system using anti-CD34 microbeads according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). CD34+ cells were counted and purity was evaluated by flow cytometry. Cells were frozen in 90% foetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO) at -80 °C until transplantation. Generation of humanized mice. For the generation of HIS mice, pregnant female Balb/cRag2−/−γc−/− mice were injected subcutaneously with 0.5 mg Busulfan (Sigma, Stockholm, Sweden) dissolved in 100 μl phosphate buffered saline (PBS) and 20% DMSO 2–7 days prior to delivery [13]. Newborn Balb/cRag2−/−γc−/− mice were conditioned with sublethal whole body irradiation (550 cGy) and subsequently transplanted intrahepatically with 1 × 105 CD34+ cord blood cells in 25 μl PBS using a Hamilton syringe and 30GA needle. Human islets. Human islets were purified from four human cadaver donors at the Uppsala University Hospital, as a part of The Nordic Network for Clinical Islet Transplantation, as previously described [19]. Upon isolation, the islets were cultured in CMRL-1066 supplemented with 10 mm nicotinamide, 10 mm HEPES buffer, 0.25 μg/ml fungizone, 50 μg/ml gentamycin, 2 mm l-glutamine, 10 μg/ml ciprofloxacin and 10% heat-inactivated human serum. The islets had a purity of 46 ± 28% (mean ± SD; range 15–85%), as determined by dithizone staining, and were further purified by hand picking. The quality of the islets was evaluated by insulin release in response to higher glucose concentrations. A dynamic perfusion system was used as previously described [20], and the islets responded with a stimulation index of 14.7 ± 5.6 (range 9.2–20.5). After arrival at KI, the islets were transferred to RPMI-1640 with the same supplements as mentioned earlier but with FCS instead of human serum and without nicotinamide and incubated 8–10 days at 37 °C and 5% CO2 prior to transplantation. Islet transplantation. Prior to transplantation, recipient control and humanized Balb/cRag2−/−γc−/− mice were anesthetized using isofluran inhalation (Baxter, Kista, Sweden). A total of 300 or 500 human islets were packed into a 22GAVenflon™ (BD, Helsingborg, Sweden) and placed under the left kidney capsule (because of the scarcity of human islet material, the majority of animals received 300 islets instead of 500). DC generation and isolation. Human monocyte-derived DC were generated from PBMC collected from healthy donors. The collection and use of human PBMC was approved by the local ethical board (Regionala etikprövningsnämnden, Stockholm). Monocytes were enriched from PBMC by negative selection using RosetteSep Human Monocyte Enrichment (StemCell Technologies, Vancouver, BC, Canada). Purified monocytes were cultured in complete Dulbecco's modified Eagle medium (DMEM, 50 μm 2-mercaptoethanol, 100 U/ml penicillin, 100 g/ml Streptomycin, 10 mm Hepes, non-essential aminoacids, all from Invitrogen, 1 mm sodium pyruvate, 2 mm l-glutamine, both from Sigma–Aldrich, Poole, UK) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Sigma–Aldrich), 50 ng/ml GM-CSF (R&D Systems, Minneapolis, MN) and 12.5 ng/ml IL-4 (R&D Systems). At day 3 of culture, fresh medium supplemented with the growth factors was added. After a total of 6 days of culture, 80% CD1a+ immature DC were obtained with low to undetectable levels ( 90%) of cells in the negative fraction expressed CD4. Primary MLR were set up in round bottom 96-well plates using 1 × 105 responder cells (CD4+ T cells from HIS mice) per well and 3 × 102, 1 × 103, 3 × 103 or 104 of irradiated stimulator cells (human DC, Balb/c and C57BL/6 DC), as indicated. Alternatively, the CD4+ T cells were cultured with Concanavalin A (ConA) (2.5 μg/ml). The MLR were incubated for a total of 96 h in humidified, 5% CO2 incubators at 37 °C. The culture medium was RPMI 1640 (Invitrogen) with sodium pyruvate and L-glutamine and supplemented with 50 μm 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS (all from Invitrogen). Eight hours before termination, 0.5 μCi [3H]thymidine was added per well. Cells were harvested and the degree of [3H] thymidine incorporation in proliferating T cells was measured. Data are reported as mean counts per minute (cpm) ± SD. Immunohistochemistry. Formalin-fixed organs were embedded in paraffin and cut into 4-μm-thick sections. Before paraffin embedding, the graft bearing kidneys were cut in half at the site of the graft. For insulin staining, sections were stained with a guinea pig anti-insulin primary antibody (DakoCytomation, Denmark) followed by a biotinylated secondary antibody (goat anti-guinea pig IgG) in conjunction with Standard Vectastain ABC kit and Peroxidase Substrate kit (all purchased from Vector laboratories, Burlingame, CA, USA). For detection of immune cell markers, sections from spleens and grafted kidneys were stained with mouse anti-human CD3 (Novocastra Laboratories, Newcastle, UK) or mouse anti-human CD20 (DakoCytomation, Denmark) primary antibodies. Prior to CD3 staining, antigen retrieval was performed by heating the sections in 10 mm citric acid buffer (pH 6.0). To detect primary antibodies, a biotinylated rabbit anti-mouse IgG secondary antibody (DakoCytomation, Glostrup, Denmark) in conjunction with Elite Vectastain ABC kit and Peroxidase Substrate kit (both purchased from Vector laboratories, Burlingame, CA) were used. CD20 staining was performed using the Bond MaX automated staining system with Bond Polymer Refine Detection (Leica Microsystems, Kista, Sweden) according to manufacturer's instructions. Slides were counterstained in Mayer's Haematoxylin. Flow cytometry. After organ collection, single cell suspensions of spleens and thymi were prepared. Prior to staining, unspecific binding of the antibodies to Fc-receptors were blocked by incubation with antibodies to mouse CD16/CD32 and human immunoglobulins (Gammagard®, Baxter, Kista, Sweden). Lymphocytes were subsequently stained with antibodies against human CD45 (PB) (DakoCytomation, Denmark), CD3 (FITC) (BD Pharmingen, Sweden) and CD19 (APC-Cy7) (BD Pharmingen, Sweden). The samples were then analyzed using a Cyan FACS Instrument (BD Pharmingen) and FlowJo software (Tree Star, Ashland, OR, USA). Human C-peptide assay. Serum samples were collected from recipient mice and stored at −20 °C until analysis. The levels of human C-peptide were quantified using Mercodia Ultrasensitive C-peptide ELISA kit (Mercodia, Uppsala, Sweden) according to the manufacturer's instructions. Statistics. Statistical analyses were performed using graphpad prism version 5 software. Differences in human serum C-peptide levels were analyzed by a two-way anova. P-Values of <0.05 were considered significant. Results Establishment of the HIS mouse model and evaluation of T cell alloreactivity HIS mice were produced by transplanting human cord blood CD34+ cells intrahepatically into sublethally irradiated newborn Balb/cRag2−/−γc−/− mice [9, 10]. Approximately 6–12 weeks post-transplantation, human CD45+ leucocytes in peripheral blood were analyzed by flow cytometry. The mice showed a reconstitution level of 14.9 ± 14.2% CD45+ cells within the lymphocyte gate (mean ± SD.; range, 0.13–51.7%; n = 20). Primary MLR demonstrated that human CD4+ T cells isolated from HIS mice proliferated vigorously in response to allogeneic human DC, while the response to host (Balb/c) derived DC was low as previously reported [10]. A small degree of xenoreactivity was observed as the human CD4+ T cells proliferated to some extent in response to C57BL/6 DC (Fig. 1A and B, and data not shown). The proliferative capacity of the human CD4+ T cells was also determined by ConA stimulation (Fig. 1B). Figure 1Open in figure viewerPowerPoint Human CD4+ T cells from HIS mice proliferate in response to human allogeneic HLA and xenogeneic (C57BL/6) but not to syngeneic (Balb/c) MHC. (A) CD4+ T cells were isolated from thymi of HIS mice (n = 3) and tested for their capacity to proliferate in response to human DC, and DC from Balb/c and C57BL/6 mice in a standard mixed lymphocyte reaction (MLR). (B) CD4+ T cell proliferation in response to ConA stimulation (2.5 μg/ml). Evaluation of human islet graft rejection in the HIS mouse model To determine whether the HIS mouse model can serve as a new in vivo tool to study rejection of human islet allografts, a total of ten mice were selected for transplantation (reconstitution level 22.0 ± 16.6%; range 1.8–51.7% CD45+ cells in blood). Non-reconstituted Balb/cRag2−/−γc−/− mice were used as controls. A total of 300 (HIS mice, n = 9; control mice, n = 6) or 500 (HIS mice, n = 1; control mice, n = 1) human islets were transplanted under the left kidney capsule approximately 8–26 weeks post-CD34+ cell transplantation. The presence of CD45+ human leucocytes in spleens and thymi, as well as graft function and survival, were evaluated on days 14 (n = 4) or 35 (n = 6) post-transplantation. A mean of 38.8 ± 29.7% (mean ± SD.; range 0.8–77.3%; n = 10) human CD45+ cells was detected in the spleens of the transplanted HIS mice. Human CD3+ cells were found in the human CD45+ population of HIS mice (mean ± SD.: 6.4 ± 3.6%; range 1.1–12.1%; n = 8; Fig. S1). Human CD19+ cells were also found (mean ± SD.: 58.3 ± 41.3; range 0.3–96.1%, n = 10; Fig. S1). CD3+ and CD19+ cells accumulated in follicle-like structures in the spleens of HIS mice (Fig. S1). Of the gated lymphocytes in the thymi, a mean of 94.9 ± 7.0% (mean ± SD.; range 77.2–99.7%; n = 10) human CD45+ cells were detected in a total of eight analyzed mice of which 47.7 ± 23.7% (mean ± SD.; range 23.1–84.8%; n = 8) were human CD3+ cells. To evaluate graft rejection, sections of grafted kidneys cut at the site of graft implantation were stained for insulin. The islet grafts present in HIS mice were comparable to those in the non-reconstituted control Balb/cRag2−/−γc−/− mice (Fig. 2). To further investigate the function of the transplanted islets, serum levels of human C-peptide were measured, a standard method for monitoring islet graft viability after transplantation [22]. C-peptide is generated when proinsulin is cleaved into insulin and C-peptide, and is released together with insulin. Human C-peptide was found both in transplanted HIS and control mice, indicating that insulin was released by the transplanted beta cells (Table 1). On day 14 post-transplantation, C-peptide levels (presented as mean ± SD) in HIS mice were 717 ± 427 pmol/l (n = 7 mice) and in control mice 378 ± 178 pmol/l (n = 5 mice) (P = n.s). On day 35 post-transplantation, 832 ± 429 pmol/l was measured in HIS mice (n = 6 mice) and 223 ± 211 (n = 4 mice) in controls (P < 0.05). Serum samples drawn from HIS mice (n = 4) prior to islet transplantations were negative for human C-peptide (data not shown). Figure 2Open in figure viewerPowerPoint No islet graft rejection or infiltrating human CD3+ T cells in transplanted HIS mice. HIS mice and non-reconstituted Balb/cRag2−/−γc−/− were transplanted with 300 (HIS mice, n = 9; control mice, n = 6) or 500 (HIS mice, n = 1; control mice, n = 1) human islets under the left kidney capsule. Graft survival and the presence of graft infiltrating CD3+ T cells were evaluated on days 14 or 35 post-transplantation by immunohistochemistry. Representative stainings are presented in A-H. (A-D) Insulin staining in grafts from HIS (A, C) and non-reconstituted control (B, D) mice harvested on days 14 (A, B) or 35 (C, D) post-transplantation. Original magnification, 10×. (E–H) Human CD3 staining in grafts from HIS (E, G) and non-reconstituted control (F, H) mice harvested on days 14 (E, F) or 35 (G, H) post-transplantation. Original magnification, 25×. Arrows indicate CD3+ cells. Table 1. Overview of HIS and control mice transplanted with human islets. Human CD45+ cells in blood (%) Human CD45+ cells in spleen (%) Human islet donor Number of transplanted islets Day of sacrifice Human serum C-peptide level (pmol/L) Day 14 Day 35 Ctrl n.d. 0 A 500 14 175 – HIS 3.4 14.9 A 500 14 208 – Ctrl n.d. 0 B 300 14 277 – HIS 1.8 0.8 B 300 14 428 – Ctrl n.d. 0 B 300 35 n.d. 32 Ctrl n.d. 0 B 300 35 n.d. 76 HIS 12.9 11 B 300 35 n.d. 365* HIS 3.6 3.4 B 300 35 n.d. 537* Ctrl n.d. 0 C 300 14 528 – HIS 29.8 38.7 C 300 14 1066 – HIS 36.7 59.2 C 300 14 1218 – Ctrl n.d. 0 C 300 35 311 293 HIS 32.0 77.3 C 300 35 675 781* HIS 51.7 74.2 C 300 35 277 599* Ctrl n.d. 0 D 300 35 599 489 HIS 28.2 65.9 D 300 35 n.d. 1424* HIS 20.0 47.7 D 300 35 1145 1287* n.d., not determined. *P < 0.05 versus control, two-way anova. Finally, to determine whether human T cells infiltrated the grafts, sections from grafted kidneys were stained for human CD3 (n = 10). No or very few (1–2 per section) CD3+ T cells were detected in close proximity to or within the islet grafts (Fig. 2). Similarly, staining for CD20+ failed to reveal infiltrating B cells in the analyzed grafts (n = 4, data not shown). Collectively, these results indicate that human T cells generated in the HIS mouse model are unable to infiltrate and reject human islet grafts. Discussion The limitations of available experimental models for clinical human islet transplantation underscore the need for advancements. Humanized mice are promising candidates for studies on islet allograft rejection mechanisms, and it is therefore of importance to evaluate the suitability of presently available models. In this study, we evaluated the ability of the HIS mouse model to reject a transplanted human islet allograft. The HIS mouse model is produced by transplantation of human cord blood CD34+ cells to neonatal Balb/cRag2−/−γc−/− mice. After human haematopoietic immune cell development was established, the mice received a human islet graft under their kidney capsules and graft survival was monitored over time. As shown by immunohistochemistry and the presence of human C-peptide in serum, the grafts still produced and released insulin on day 35 post–transplantation, suggesting that graft rejection had not occurred. On day 35 post-transplantation, C-peptide levels were significantly higher in transplanted HIS mice compared to control mice. This was a counterintuitive finding suggesting that islets transplanted to humanized mice produce more insulin than islets transplanted to control animals. Future studies are required to elucidate whether the infusion of haematopoietic stem cells and/or the presence of a human immune system supports better islet engraftment/function or whether there is another explanation to this unexpected observation. A more functional study of the islet graft would include diabetic mice; however, such experiments were not conducted because of the limited numbers of produced HIS mice and the altering effect of chemically induced diabetes on the engrafted human immune system [18]. At present, it is unclear why the HIS model is suboptimal for the studies on islet allograft rejection. Our detailed analyses allowed the comparison between mice with a high and low degree of human immune cell reconstitution (Table 1). A failure to reject was found even in mice with a high reconstitution level indicating that rejection was not dependent on this variable. We confirmed that T cells from HIS mice demonstrate reactivity to human allogeneic cells ex vivo, and that their response to mouse DC is low (Fig. 1, [9, 10]). These observations demonstrate that T cells educated in the HIS model can recognise human HLA. Still, the lack of islet allograft rejection (present study) and other observations of poor T cell activities (reviewed in [23, 24]) now question the quality of the T cells developed in this mouse model. Although human DC may contribute to negative selection, the absence of human epithelial cells in the thymus of the HIS mouse model [9, 10] is likely to result in T cells being only educated in a mouse MHC context. This may preclude optimal positive selection of T cells and thereby hamper their functions. An absence of homeostatic T cell expansion, as well as high peripheral T cell turnover rates and lack of long-term T cell maintenance have been reported in Balb/cRag2−/−γc−/− mice [23, 24] and may further undermine T cell function. Interestingly, islet xenograft rejection has been reported in a new humanized mouse model where human T cells are selected on autologous foetal thymic tissue transplanted prior to human haematopoietic CD34+ cells (entitled the bone marrow–liver–thymus (BLT) model) [25, 26]. It is possible that the BLT model rejects allogeneic islet grafts; however, the labour intensity, scarcity of hu
Referência(s)