Ann Thorac Surg 2001;72:735-739
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Importance of thymus to maintain operational tolerance to fully allogeneic cardiac grafts
Masanori Niimi, MD, PhDa,
Nozomu Shirasugi, MDa,
Yosifumi Ikeda, MDa,
Shigenao Kan, MDa,
Hiroshi Takami, MDa,
Kimikazu Hamano, MD, PhDb
a First Department of Surgery, Teikyo University, Tokyo, Japan
b First Department of Surgery, Yamaguchi University, Yamaguchi, Japan
Accepted for publication May 16, 2001.
Address reprint requests to Dr Niimi, First Department of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan
e-mail: mniimi{at}med.teikyo-u.ac.jp
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Abstract
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Background. The effectiveness of donor-specific blood transfusion (DST) before transplantation has been established. Anti-CD4 monoclonal antibody (anti-CD4) augments the ability of DST to induce indefinite prolongation. Therefore, we investigated the importance of thymus to maintain the unresponsiveness to alloantigens.
Methods. CBA mice were pretreated with 0.25 mL of DST or two doses of anti-CD4 before transplantation of a C57BL/10 heart. Some mice were thymectomized.
Results. Naive CBA mice rejected C57BL/10 grafts acutely with a median survival time of 7 days. When mice were pretreated with anti-CD4 plus DST 4 weeks before transplantation, all grafts survived indefinitely (>100 days), whereas mice treated with DST alone or anti-CD4 alone rejected acutely (median survival time, 7 and 12 days, respectively). To investigate the importance of thymus, mice pretreated with anti-CD4 plus DST 4 weeks before transplantation were thymectomized or underwent a sham operation 1 day before grafting. Mice with the sham operation accepted grafts indefinitely, whereas thymectomized mice rejected the majority of the grafts (median survival time, 20 days).
Conclusions. The thymus is important in maintaining the operational tolerance induced by anti-CD4 plus DST 4 weeks before grafting.
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Introduction
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Donor-specific blood transfusions (DST) before transplantation have been shown to induce unresponsiveness in experimental models [1] and have a beneficial effect in clinical transplantation [2]. Although some experimental studies have shown that pretreatment with alloantigen can induce long-term graft survival [3], in the majority of situations the unresponsive state induced is incomplete, resulting in prolonged but not indefinite allograft survival [4]. It has been previously shown that anti-CD4 monoclonal antibody (anti-CD4) can augment the ability of DST to induce unresponsiveness resulting in the indefinite survival of fully allogeneic cardiac grafts [5].
The thymus plays a major role in the development of tolerance to self-antigens during the development of the immune system. During maturation, thymocytes become restricted to self-major histocompatibility complex (positive selection) [6] and the process of negative selection ensures that thymocytes expressing T-cell receptors with too high affinity for self-major histocompatibility complex + self-peptides do not develop into harmful T cells capable of inducing autoimmune diseases [7]. Therefore, it has been concluded that the thymus is an extremely potent organ for inducing tolerance during T-cell development.
In this study, we investigated the importance of the thymus to induce or maintain operational tolerance in our model.
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Material and methods
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Animals
CBA (H2k), C57BL/10 (H2b), and Balb/c (H2d) mice were purchased from Sankyo Ltd (Tokyo, Japan) and bred in conventional facilities of the Biomedical Services Unit, Teikyo Hospital, Tokyo, and used between 8 and 12 weeks old in accordance with the Animals Procedures of Teikyo University.
Pretreatment of recipients
Recipient mice were pretreated with 0.25 mL of DST or two doses of 50 µg of anti-CD4 (YTA 3.1; the hybridoma was kindly provided by Professor H. Waldmann, Oxford, UK) [8] either 1, 2, or 4 weeks before transplantation of C57BL/10 hearts. Some mice were thymectomized. Timing and combination of the pretreatment in all experiments was shown on Table 1.
Thymectomy
A vertical incision was made on the neck, and the muscles overlying the trachea were divided. The thymus was exposed by a median sternotomy to the second anterior rib. The left lobe of the thymus was gently retracted using forceps and the connective tissue dissected. This procedure was repeated until the left lobe was removed. The same technique was repeated to remove the right lobe.
Heart transplantation
Under anesthesia, fully vascularized heterotopic hearts were grafted into the abdomen using microsurgical techniques [9]. Graft function was followed up by palpation of the grafted hearts at least three times a week. Rejection was confirmed by electrocardiogram [10] and direct visualization of the graft. Some mice accepted cardiac grafts for more than 100 days and were transplanted with second heart from C57BL/10 or Balb/c mice into the neck [11].
Statistical analysis
Allograft survival between two groups was compared by Mann-Whitney U test using StatView SE + Graphic (Abacus Concepts Inc, Cary, NC).
Preparation of splenocytes
Single cell suspensions from spleen were depleted of erythrocytes by hypotonic lysis with water for 5 seconds.
Flow cytometry
Twenty-five microliters of viable cells (107 cells/mL) were incubated for 30 minutes on ice with primary anti-CD4 (YTS177; hybridoma was kindly provided by Professor H. Waldmann, Oxford, UK) in phosphate buffered saline supplemented with 2% fetal calf serum and 0.02% sodium azide. Then, cells were washed twice before being incubated for a further 30 minutes on ice with a FITC-labeled goat anti-rat immunoglobulin monoclonal antibody (F-6258, Sigma-Aldrich, Tokyo, Japan) blocked with 10% mouse serum. After two more washing, the fluorescence intensities of 10,000 cells were determined using a FACSort (Becton Dickinson, San Jose, CA) flow cytometer.
Adoptive transfer of cells from tolerant mice into naive mice
Naive CBA mice were pretreated with 5 x 107 splenocytes from naive or CBA mice with long-term surviving C57BL/10 grafts induced by DST plus anti-CD4. A C57BL/10 heart was transplanted 1 day later.
In vivo detection of DST cells
Carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Leiden, The Netherlands) was used to trace cells after DST administration. Splenocytes were incubated with 1 µmol/L CFSE for 10 minutes in a 37°C water bath with regular agitation and the reaction was stopped by placing the tubes on ice. The cells were washed three times for in vivo use. Splenocytes (1 x 107) were injected intravenously and the thymus and the spleen were harvested. The fluorescent intensities of thymocytes and splenocytes were determined using a FACSort flow cytometer (Becton Dickinson).
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Results
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We confirmed the effect of DST in inducing prolongation of cardiac grafts. CBA mice were pretreated with 0.25 mL of DST 1, 2, or 4 weeks before transplantation of a C57BL/10 heart. A 4-week interval between pretreatment and transplantation did not result in prolonged graft survival (median survival time [MST], 7 days) (Table 1, group 4) compared to naive control (MST, 7 days) (Table 1, group 1). Although significant graft survival was seen both in 1- and 2-week intervals (p < 0.001 and p < 0.05), the majority of grafts were rejected within 30 days (MST, 19 and 8 days, respectively) (Table 1, groups 2 and 3).
In an attempt to induce indefinite prolongation of the cardiac grafts, anti-CD4 was combined with DST. Because the anti-CD4, YTA3.1, was a depleting antibody, we examined depletion rate of CD4+ cells in the spleen. After two doses of intravenous injection of anti-CD4, CD4+ cells were immediately depleted 10% compared to the number of CD4+ cells before treatment. CD4+ cells recovered 4 days after anti-CD4 treatment and increased to 80% until 28 days after treatment (Fig 1).
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Fig 1. Depletion and recovery of CD4+ cells in the spleen after injection of anti-CD4. CBA mice were treated with depleting anti-CD4 monoclonal antibody (YTA3.1) (50 µg/dose) on two consecutive days. CD4+ cells in the spleen were counted by fluorescence activated cell sorter. Percentage of CD4+ cells compared to the original number was presented by mean ± standard deviation.
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Mice treated with anti-CD4 alone on day -1 and day 0, and day -8 and day -7 accepted their grafts indefinitely (Table 1, groups 5 and 6), whereas mice treated with anti-CD4 alone on day -15 and -14 rejected some grafts (MST, 66 days) (Table 1, group 7). When the interval between anti-CD4 treatment and grafting was 28 days, all cardiac grafts were rejected acutely (MST, 12 days; Table 1, group 8).
Pretreatment with either DST alone or anti-CD4 alone 4 weeks before grafting allowed the mice to reject the grafts (MST, 7 and 12 days, respectively; Table 1, groups 4 and 8). However, when DST and anti-CD4 treatment were combined 4 weeks before transplantation, all grafts were accepted indefinitely (Table 1, group 9). Second hearts were transplanted into the neck of the recipients that had accepted the first grafts for more than 100 days. All of the donor-specific (C57BL/10), but not third party (Balb/c), cardiac grafts were accepted (Fig 2). Moreover, when 5 x 107 splenocytes from the mice bearing allogeneic cardiac grafts indefinitely were injected into naive CBA mice and 1 day later C57BL/10 hearts were transplanted into the naive mice, all grafts were accepted indefinitely (Fig 3).
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Fig 2. Operational tolerance was induced after pretreatment with donor-specific blood transfusion (DST) plus anti-CD4. CBA mice were pretreated with anti-CD4 (50 µg/dose) in combination with 0.25 mL of donor-specific blood transfusion 4 weeks before transplantation of a C57BL/10 cardiac graft. The recipients accepted the graft indefinitely in their abdomen. Second C57BL/10 or Balb/c cardiac grafts were transplanted into the neck of the recipients 100 days after the first transplantation. *p < 0.001 between the two groups. (MST = median survival time.)
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Fig 3. Regulatory cells were generated 100 days after transplantation in the mice pretreated with donor-specific blood transfusion (DST) plus anti-CD4. CBA mice were pretreated with anti-CD4 (50 µg/dose) in combination with 0.25 mL of donor-specific blood transfusion 4 weeks before transplantation of a C57BL/10 cardiac graft. The recipients accepted their graft indefinitely. Splenocytes (5 x 107) from the recipient or naive mice were adoptively transferred into the naive CBA mice. C57BL/10 cardiac grafts were transplanted into the second recipient 1 day after the adoptive transfer. *p less than 0.001 between the two groups. (MST = median survival time.)
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Before examining the importance of the thymus to maintain the operational tolerance against the graft induced by DST plus anti-CD4, we investigated the effect of thymectomy in combination with anti-CD4 to induce graft prolongation. Mice were pretreated with anti-CD4 (50 µg/dose) 28 and 29 days before transplantation, and thymectomized either 1, 14, or 28 days before grafting. Mice thymectomized 28 days before grafting (ie, on the day of anti-CD4 injection) did not reject grafts (Table 1, group 11) and mice thymectomized 14 days before grafting (ie, 14 days after anti-CD4 injection) rejected some grafts (Table 1, group 12). When thymectomy was performed 1 day before grafting (ie, 27 days after anti-CD4 injection), all mice rejected their grafts acutely (MST, 9 days) (Table 1, group 13). Mice thymectomized without anti-CD4 treatment were also able to reject their grafts acutely (MST, 10 days) (Table 1, group 10).
To investigate the importance of the thymus to maintain the unresponsiveness induced by DST in combination with anti-CD4 treatment, mice were pretreated with DST in combination with anti-CD4 treatment 4 weeks before transplantation and underwent a sham operation or thymectomy 1 day before transplantation of a C57BL/10 heart. All of the sham-operation group accepted their grafts indefinitely (Table 1, group 15), whereas the majority of thymectomized mice rejected their grafts (MST, 20 days) (Table 1, group 14).
Finally, we investigated the fate of cells after intravenous injection. Splenocytes were stained with CFSE and injected into the naive mice. Then, the thymus and the spleen were harvested at different time points. The CFSE+ cells were examined by fluorescence activated cell sorter (FACS). In the spleen, CFSE+ cells were detected for 3 days after injection. However, no CFSE+ cells were detected in the thymus at any time after injection (Fig 4).
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Fig 4. C57BL/10 splenocytes (1 x 107) were stained with carboxyfluorescein succinimidyl ester (CFSE) before injection and injected intravenously into CBA mice. Spleens and thymus were sampled and fluorescence-positive cells were counted by fluorescence activated cell sorter. Data were presented as the mean of three mouse samples ± standard deviation and are representative of four independent experiments.
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Comment
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Donor-specific blood transfusion alone induced modest survival of fully allogeneic cardiac grafts when delivered 1 or 2 weeks before transplantation (Table 1, groups 2 and 3). The DST plus anti-CD4 induced an indefinite survival of fully allogeneic cardiac grafts in our model (Table 1, group 9). Moreover, the mice bearing C57BL/10 cardiac grafts in their abdomen accepted donor specific (C57BL/10) but not third party (Balb/c) second cardiac grafts (Fig 2), demonstrating that operational tolerance was induced after DST plus anti-CD4 treatment.
When the thymus in the mice pretreated with anti-CD4 plus DST was harvested 1 day before grafting, the majority of the cardiac grafts were rejected, suggesting that the thymus played a pivotal role in maintaining the operational tolerance. Two central explanations for the observed results are central deletion of alloreactive T cells in the thymus, for which micro/macro chimerism may play a role, and the role of recent thymic emigrants as regulatory cells.
In models of mixed allogeneic chimerism donor-derived major histocompatibility complex class II+ cells have been found in the thymus of tolerant mice (mice that accepted their grafts indefinitely) and have been shown to be responsible for the ongoing deletion of alloreactive T cells, which is required for tolerance to be maintained [12]. Recent data indicate that the thymus may play a role in the induction and maintenance of tolerance to alloantigens present in the periphery in certain transplantation models [13]. Studies performed in the 1960s indicated that unresponsiveness could be induced by intrathymic delivery of alloantigen [14, 15]. In 1990, Posselt and colleagues [16] showed that injection of allogeneic islet of Langerhans into the adult rat thymus with antilymphocyte serum resulted in operational tolerance to donor alloantigens.
However, in our model, we could not detect donor cells in the thymus (Fig 4). Moreover, even in the spleen, transfused cells disappeared 3 days after injection (Fig 4). Therefore, we speculate that donor alloantigen may be destroyed and presented on the recipient major histocompatibility complex class II molecules (ie, via the indirect pathway) [17].
The second possibility is that recent thymic emigrants from the thymus can become regulatory cells [18, 19]. In our model, regulatory cells were generated because adoptive transfer of cells from tolerant mice into naive mice induced indefinite survival of cardiac grafts in the naive mice (Fig 3). However, the relationship between recent thymic emigrants and regulatory cells is still unclear; cell surface markers of recent thymic emigrants and regulatory cells have not been discovered in mice [18].
Although 0.25 mL of DST alone or anti-CD4 alone were ineffective at inducing graft prolongation when delivered 4 weeks before transplantation (Table 1, groups 4 and 8), DST in combination with anti-CD4 induced indefinite graft prolongation in all recipients (Table 1, group 9). Bushel and colleagues [20, 21] have suggested that the mechanism involved in this prolongation is not depletion of CD4 T cell but coating of CD4 molecules by antibody, as mice pretreated with a single dose of anti-CD4 and then DST on the following day induced graft prolongation, whereas pretreatment with a single dose of anti-CD4 and DST at the same time was much less effective, suggesting that coating of CD4 molecules by antibody with anti-CD4 before DST injection is important for graft prolongation.
T cells are combined during maturation through the thymus. Therefore, mice thymectomized at the time of anti-CD4 treatment could not reject their grafts as anti-CD4 decreased the number of CD4+ T cells and these cells could not be recovered without the thymus (Table 1, group 11). Even when thymectomy was postponed until 2 weeks after anti-CD4 treatment (Table 1, group 12), the majority of grafts survived indefinitely. Four weeks between anti-CD4 treatment and thymectomy was enough time to recover the number of CD4 T cells to reject the grafts (Table 1, group 13). Recent thymic emigrants are not necessary to reject the grafts because mice thymectomized 4 weeks before grafting were able to reject the grafts (Table 1, group 10), suggesting that memory cells may be sufficient to induce graft rejection.
These observations suggest that the thymus is important to maintain the operational tolerance. These results indicate the importance of using alloantigens and anti-CD4 to induce donor-specific operational tolerance.
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Acknowledgments
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This work was supported by Foundation of Total Health Promotion and The Mother and Child Health Foundation.
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References
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Abstract
Introduction
