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Keywords:

  • ESC;
  • Stem cell;
  • Transplantation;
  • Immune privilege;
  • Regulatory T cells;
  • MHC

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We have previously reported that ESC-derived tissues are subject to some level of immune privilege, which might facilitate induction of immune tolerance. Herein, we further demonstrate that fully allogeneic ESC-derived tissues are accepted with a regimen of coreceptor blockade even in recipients known to be relatively resistant to such a tolerizing protocol. Moreover, ESC-derived tissues could be spontaneously accepted across a class I major histocompatibility complex disparity. We further show that CD4+FoxP3+ regulatory T cells (Treg) appear to be essential for this natural “privileged” state as their ablation with an anti-CD25 mAb results in rejection of ESC-derived tissue. This same treatment exposes activation of macrophages and effector CD8+ T cells, suggesting that these cells are subject to regulatory T cell control. Thus, spontaneous acceptance of ESC-derived tissues mimics the acquired immune privilege induced by coreceptor blockade and is determined by Treg-mediated suppression. STEM CELLS 2010;28:1905–1914


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The unique capacity of ESC to self-renew and differentiate into multiple cell types have made them attractive candidates for use in regenerative medicine (reviewed in [1, 2]). Nevertheless, derivatives of ESC are immunogenic and graft rejection of ESC-derived tissues remains a critical hurdle for their success in cell replacement therapy (CRT). Although mouse and human ESC express low levels of class I and class II major histocompatibility complex (MHC) antigens [3, 4], expression of MHC-I increases when ESC are differentiated into embryoid bodies (EB) and increases even further in more differentiated tissues [4]. MHC-I expression can also be upregulated when ESC-derived tissues are exposed to interferon-γ (IFN-γ), often present in rejecting grafts [3–5]. Even if all MHC loci are matched, disparities at minor histocompatibility (mH) loci are sufficient to provoke rejection to ESC-derived tissues [6, 7].

Despite being immunogenic, ESC and their differentiated derivatives display a level of natural immune privilege in vivo [6–11]. We have previously demonstrated that composite tissues, such as EB, survive indefinitely in fully allogeneic CBA/Ca recipients treated with monoclonal antibodies (mAbs) specific for the T-cell coreceptors, CD4 and CD8 [6]. In contrast, coreceptor blockade alone with CD4 and CD8 mAb usually fails to tolerize to skin grafts differing across an MHC mismatch. Corbascio and coworkers have also shown that a cocktail of costimulation blocking reagents, including anti-CD40L, anti-LFA-1, and CTLA4-Ig, is sufficient to induce tolerance to human ESC transplanted into the testis of C57BL/6 mice [12]. Therefore, in common with tissues derived from the early fetus [13, 14], ESC-derived tissues appear to offer a less susceptible target for rejection than conventional allografts, possibly because of an inherent level of immune privilege.

Certain tissues (the anterior chamber of the eye, central nervous system, testes, and placenta), once thought sequestered from the immune system, are known to display levels of natural immune privilege [5, 15]. Prolonged allograft survival has been reported when grafts are placed in these anatomical sites, where local regulatory mechanisms are now thought to predominate over stimulatory responses. Similarly, the seeming resistance of cancers to immune attack might be attributed, at least in part, to active T-cell regulatory mechanisms that suppress antitumor immunity.

Tissues with large mucosal surfaces are constantly exposed to foreign antigens or organisms. Suppression mediated by regulatory T cells (Treg) is considered a necessary mechanism to limit inappropriate immune activation [16]. The two most-studied subsets of Treg are natural thymus-derived Treg (nTreg) and transforming growth factor (TGF)-β-induced Treg (iTreg). Both nTreg and iTreg express the transcription factor forkhead box protein three (FoxP3) [17–19] necessary for their function [20]. Treg can exert their suppressive functions through physical contact with immune cells and/or production of immunosuppressive cytokines such as IL-10 and TGF-β [21]. After coculture with Treg in vitro, human monocytes or macrophages display features typical of alternatively activated macrophages (AAM) such as an upregulation of CD206 (mannose receptor [MR]), enhanced phagocytic capacity, increased production of the chemokine CCL18, and reduced expression of human leukocyte antigen (HLA)-DR. These macrophages are anti-inflammatory and are known to be capable of mediating tumor survival [22]. Moreover, Treg can also suppress effector activity of tumor-specific CD8+ T-cells [23]. In a model of murine colon carcinoma, CD8+ T-cells expanded to the same extent and produced similar levels of IFN-γ in the presence or absence of tumor-specific Treg [23]. However, these Treg abrogated CD8+ T-cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8+ T-cells, as measured by percentage of cell lysis.

Previous experience in our laboratory revealed that recipients of the “black” background (such as C57Bl/6, C57Bl/10, or B10.Br) were more resistant to coreceptor blockade [24]. We investigated if the inherent privilege of ESC-derived tissues overrides that property, permitting coreceptor blockade to allow successful engraftment in C57Bl/10 recipients. To clarify the mechanisms through which ESC-derived tissues harness natural privilege, we transplanted under the kidney capsule of CBA/Carshalton (or CBA/Ca) recipients EB from CBK ESC [H-2k, Kb] differing by just a single transgenic MHC class I allele. Some of the grafts escape immune damage by CD8+ T cells reactive against the mismatched class I MHC determinant, Kb, and spontaneously survive in CBA/Ca recipients [H-2k] without the need for immunosuppression. As Treg play a pivotal role in therapeutic transplantation tolerance [25–27], we hypothesized that they might be responsible for suppressing reactive immune cells from rejecting ESC-derived tissues on antigen recognition. We sought to determine whether their inactivation by a short course of anti-CD25 mAb, PC61, would restore graft rejection.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

ESC Derivation

All mice were bred and maintained at the University of Oxford, and experimental work was carried out under Home Office regulations with local ethical committee approval. The CBK ESC line (ESF166) was derived from male CBK mice, and was generated by Dr. Paul Fairchild in collaboration with Prof. Sir Richard Gardner [6]. ESCs were maintained in ES medium consisting of Dulbecco's modified Eagle's medium (Cambrex Bio Science Verviers, Belgium), 15% v/v fetal calf serum (FCS) (Gibco, US), 50 μg/ml penicillin and streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol.

Preparation of EB for Transplantation

ESC were passaged twice on gelatin in ES medium supplemented with 1,000 U/ml of recombinant leukemia inhibitory factor (rLIF). They were allowed to differentiate into EB by growing in suspension culture in the absence of rLIF at 37°C for 14 days.

Administration of mAb

Depleting mAb specific for CD4 (clones YTA 3.1.2 and YTS 191.2) and CD8 (clones YTS 156.7 and YTS 169.4), or nondepleting mAb specific for CD4 (clone YTS 177.9) and CD8 (clone YTS 105.18) were injected i.p. (1 mg) on days 0, 2, and 4 after transplantation. mAb specific for CD25 (clone PC61) [28] was injected i.p. (1 mg) on days 6, 4, and 2 before transplantation.

Transplantation

Transplantation of EBs was carried out as detailed previously [29]. Briefly, two to four EBs were grafted under the kidney capsule, and groups of mice were sacrificed for analysis at specific time points. Graft acceptance was defined as an increase in diameter to >5 mm, vascularization and the lack of leukocyte infiltration and tissue damage, revealed by histology. The readout of transplantation was restricted by 28 days to minimize tumor-associated risks under Home Office regulations.

Immunohistochemistry

Samples were collected and frozen in Tissue-Tek OCT Compound embedding medium (Sakura Finetek, Torrance, CA) prior to sectioning. Slides of tissue sections (6-μm thick) were fixed in 4% paraformaldehyde and then blocked with 10% goat serum. Primary anti-mouse mAbs were used at 10 μg/ml for 1 hour at room temperature and secondary mAbs were used, where required. Slides were counter-stained with 500 μg/ml of DAPI (Vector Labs, US) for 5 minutes at room temperature in the dark. Slides were mounted with fluorescence mounting medium (Dako, US) and allowed to dry overnight at 4°C in the dark. Fluorescence was detected with the fluorescence microscope (Zeiss, Germany) and analyzed with Metamorph software (version 6.2.4).

Quantitative Polymerase Chain Reaction

Expression of the indicated genes was determined by quantitative polymerase chain reaction using the Taqman low-density array (Applied Biosystems, US) according to manufacturer's instructions and Taqman validated gene expression assays as detailed previously [6]. The relative gene expression level of each sample was expressed as an relative quantitation (RQ) value which represents the fold change in gene expression normalized to the housekeeping gene, Hprt.

Statistical Analysis

The data were expressed as arithmetic mean ± SD of triplicate determinations performed under the same conditions. Statistical analysis was performed using the student's t-test (*, p < .05, **, p < .01, ***, p < .001).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Coreceptor Blockade Enables Acceptance of ESC-Derived Tissues in Fully Allogeneic C57Bl/10 Recipients

Mice of the “Black” background are known to be resistant to the acquisition of skin-graft tolerance through coreceptor blockade even across mismatches for mH antigens [24]. When B10.Br [H-2k, C57Bl/10 minors] skin grafts were transplanted onto CBA/Ca [H-2k] recipients treated with anti-CD4 and anti-CD8 mAb, the grafts survived indefinitely (>39 days). In contrast, when CBA/Ca skin grafts were transplanted onto B10.Br recipients, the grafts were rejected within 20 days even if the recipients had received anti-CD4 and anti-CD8 mAb [21]. We have recently reported that ESC-derived tissues appear to be less immunogenic and are more susceptible to the induction of transplantation tolerance [6]. We show here that coreceptor blockade with anti-CD4 and anti-CD8 mAb enables CBA/Ca [H-2k] mice to accept C57Bl/10 [H-2b] EB under the kidney capsule, and remarkably, also enables acceptance of CBA/Ca EB in C57Bl/10 recipients (n = 10). The use of EBs as allografts is significant since, following implantation, they form teratomas-containing tissues and cell types from all three embryonic germ layers, suggesting that tolerance may be established to any cell types differentiated from ESC rather than specific tissues such as cardiomyocytes, whose immunogenicity is known to be low. These results suggest some inherent capacity of ESC-derived tissues to benefit from tolerizing protocols even in “resistant” strains.

MHC Class I Mismatched ESC-Derived Tissues Can Be Accepted Without Any Immunosuppression

We examined the impact of a single MHC class I difference by using EB generated by culturing ESC derived from the male CBK [H-2k, Kb] strain in suspension for 14 days, and then grafting these surrogate tissues under the kidney capsule of male or female CBA/Ca [H-2k] recipients. The EBs were allowed to grow and differentiate in vivo, and graft survival was measured 28 days after transplantation under Home Office regulations. Remarkably, the transplanted Kb-expressing EBs were still intact 3 weeks post-transplantation and approximately 50% of male recipients spontaneously accepted the EB (n > 100) after 4 weeks (Fig. 1A). By contrast, female recipients able to react to the male mH antigens in addition to the MHC class I determinants, rejected Kb-expressing EB more rapidly. Seventy-five percentage of female recipients had rejected the male EB within the first 3 weeks of transplantation (n = 8) and all recipients had rejected the EB by week 4 (n > 20). Histological analysis of serial sections of the accepted EB grafts from male recipients revealed a wide range of healthy differentiated tissues derived from all three embryonic germ layers, consistent with the pluripotency of parent ESC (Fig. 1B), whereas donor EB in female recipients failed to grow and showed signs of extensive tissue damage and necrosis (Fig. 1C). To show that spontaneous acceptance of EB across the MHC class I barrier could not be attributed to the lack of alloantigen expression, serial sections of EB from male and female recipients were stained with mAb specific for the Kb molecule (Fig. 1D–1E). Our results demonstrate that the accepted EB maintained expression of the Kb antigen on differentiation (Fig. 1D), suggesting that other forms of regulation had provided protection from rejection.

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Figure 1. ESC-derived tissues survive naturally across the class I major histocompatibility complex (MHC-I), Kb, barrier without the need for immunosuppression. (A): Kinetic studies on the % survival of ESF166 (CBK) embryoid body (EB) in male and female CBA/Ca recipients. Graft survival was examined at 14, 21, and 28 days after transplantation. (B, C): Histological analyses of accepted and rejected EB. EBs differentiated from ESF166 (CBK) were transplanted for 28 days under the kidney capsule of (B) male and (C) female CBA/Ca recipients. EBs fail to engraft in female recipients. The asterisk denotes an area of tissue damage within the rejecting EB (Scale bar = 200 μm). (D, E): Expression of the MHC class I determinant H-2Kb by EB grafted under the kidney capsule of CBA/Carshalton (or CBA/Ca) mice. EB differentiated from ESF166 (CBK) were transplanted for 28 days under the kidney capsule of (D) male and (E) female CBA/Ca recipients, respectively. Sections of EB were stained with mAb specific for the MHC class I determinant H-2Kb (B–E) are fully representative of multiple recipients in the same group (n = 5; Scale bar = 100 μm).

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Rejection of CBK EB in Female CBA/Ca Recipients Is Mediated by Effector CD8+ T Cells

Examination of EB grafts by immunohistochemistry demonstrated a significant CD8+ T-cell infiltrate in EB grafts from female recipients (Fig. 2A–2C). We wondered whether CD8+ T cells alone were sufficient to reject an EB graft without help from CD4+ T-cells. Therefore, we injected female CBA/Ca mice with depleting mAb specific for either CD4 or CD8 or a combination of the two, around the time of transplantation with the CBK EB (Fig. 2D). Four weeks after transplantation, we observed that ablation of CD8+ T cells had resulted in 100% graft survival (p < .005, n = 5), implicating CD8+ T cells as the principle effectors of rejection. However, when CD4+ T cells were depleted, graft rejection was delayed compared with untreated recipients. This suggests that CD8+ T cells are the main effector T cells involved in rejecting the Kb-expressing EB, but need help from CD4+ T cells for optimal rejection. It is unlikely that natural killer (NK) cells were involved as CD49b+ NK cells were absent from both accepted or rejected EB (Supporting Information Fig. 1).

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Figure 2. Kb-expressing ESC-derived tissues are rejected by CD8+ T cells. EB differentiated from ESF166 (CBK strain) were transplanted for 28 days under the kidney capsule of (A) female and (B) male CBA/Ca recipients, respectively. EB were stained with monoclonal antibodies (mAb) specific for CD8 (red). All micrographs are representative of multiple recipients in the same group (n = 3). Scale bar = 100 μm. (C): The average numbers of CD8+ cells in the grafts, calculated by averaged intensity per unit area using the Metamorph software. Results were expressed as mean ± SD of mean (n = 3). (D): CD8+ T cells alone are sufficient to mediate graft rejection. EB differentiated from ESF166 (CBK) were transplanted for 28 days under the kidney capsule of female CBA/Ca recipients (n = 5, unless specified). Phosphate buffered saline (PBS), depleting anti-CD4 or anti-CD8 mAb, or both mAb in combination were injected into recipients at 0, 2, and 4 days after transplantation.

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“Privileged” ESC-Derived Grafts Are Infiltrated by AAM

We were interested in determining how allogeneic ESC-derived tissues are spontaneously accepted while allogeneic skin grafts with equivalent histoincompatibility are rejected. ESC-derived tissue differs from conventional tissues by a paucity of contaminating hematopoietic-derived dendritic cells (DC) [6]. Consistent with this, when immature or mature DC derived from CBK ES cells were injected intravenously into CBA/Ca recipients, all CBK EB grafts were rejected (n = 8) compared with 50% rejection in the phosphate buffered saline (PBS) control group. ESC-derived tissues lack lymphatic drainage, and this too may minimize rejection through limiting host sensitization (Supporting Information Fig. 2).

We reasoned that, by comparing the infiltrates in accepted and rejecting tissues, it might be possible to determine which infiltrating cells correlate with acceptance of ESC-derived tissues. Serial sections of the accepted CBK EB grafts from male CBA/Ca recipients and rejecting CBK EB grafts from female CBA/Ca recipients were, therefore, costained with mAb specific for either F4/80 and MHC class II (Fig. 3A) or F4/80 and MR (Fig. 3B). A greater number of F4/80+ macrophages had infiltrated the rejecting EB grafts (Fig. 3C). Among them, 98% were MHC class II+, and lacked MR expression, indicating that the macrophages in the rejecting tissues were active and possibly inflammatory (Table 1). In contrast, among those F4/80+ macrophages found in the accepted EB grafts, 89% were MHC class II and 30% were MR+. These are considered as less active or resting macrophages.

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Figure 3. Macrophages which infiltrate into the accepted ESC-derived tissues are less active. Embryoid bodies (EB) differentiated from ESF166 (CBK) were transplanted under the kidney capsule of male and female CBA/Ca mice for 28 days. Serial sections were stained with mAb specific for the macrophage markers, (A) F4/80 (red) and MHC class II (green), (B) F4/80 (red) and MR (green). Nuclear staining by DAPI is overlaid in blue. (C): The average numbers of F4/80+ cells in three different views. Scale bar = 20 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MHC II, class II major histocompatibility complex; MR, mannose receptor.

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Table 1. % of MHC-II+ or 5D3+ cells in the F4/80+ population
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The expression of a panel of inflammatory and anti-inflammatory cytokine genes was studied to determine the quality of the inflammatory response associated with graft survival and/or rejection. The cytokine expression profile within syngeneic grafts was used as a control because there should be no adaptive immune response against self-antigens. As shown in Figure 4, the expression profile of proinflammatory cytokines Il-1β, Il-6, and Il-23 in the accepted grafts was similar to that of the syngeneic or the tolerized grafts. There was no expression of Il-5 mRNA in the accepted grafts, even though this could be detected in syngeneic grafts at day 28 or in tolerized grafts at day 10 after transplantation. Expression of Il-17 mRNA was detected at day 10 in the accepted grafts and day 14 in the syngeneic grafts. Moreover, there was no or very low expression of IFN-γ, Il-12p40, Il-4, or Il-9 mRNA in any cohort at days 10, 14, or 28 after transplantation (data not shown). In general, there was no statistically significant difference in expression of proinflammatory cytokines in the rejecting, accepted, syngeneic, or tolerized grafts.

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Figure 4. Accepted tissues express less inflammatory cytokines. Gene expression of cytokines in different types of grafts was measured using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Embryoid bodies (EBs) were grafted under the kidney capsule of CBA/Ca recipients for 10, 14, or 28 days. Total RNA was extracted from four types of EB: rejecting EB (CBK) derived from female CBA/Ca recipients; accepted EB derived from male CBA/Ca recipients; syngeneic EB derived from male CBA/Ca recipients; and tolerized EB derived from female CBA/Ca recipients treated with nondepleting anti-CD8 monoclonal antibodies. The level of gene expression of Il-1β, Il-5, Il-6, Il-10, Il-17, Il-23, or the house keeping gene Gapdh was normalized to expression of the Hprt transcript (a Student's t-test was carried out to compare gene expression with the rejecting group). Results were expressed as mean ± SD of mean (**, p < .005, n = 3). Abbreviations: RQ, relative quantitation; Gapdh, glyceraldehyde 3-phosphate dehydrogenase.

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Expression of the anti-inflammatory cytokine mRNA Il-10 was also measured in rejecting, accepted, syngeneic, and tolerized grafts (Fig. 4). Our results showed that Il-10 mRNA expression was significantly greater in the accepted (p < .005), syngeneic (p < .005), and tolerized grafts compared with the rejecting grafts at day 14 after transplantation. Gene expression of Il-10 could still be detected in the accepted grafts, but not in the rejecting, syngeneic, or tolerized grafts 4 weeks after transplantation.

Natural Acceptance of ESC-Derived Tissues Involves Treg-Mediated Immunosuppression

There is a large body of evidence demonstrating that IL-10 and TGF-β induce expression of FoxP3 and differentiation of CD4+Foxp3+ Treg from naive T cells on antigen recognition [30]. Our group has previously reported that ESC-derived tissues secrete high levels of TGF-β2, which can be important for de novo differentiation of Treg from naïve T cells [6]. In line with our previous study, TGF-β signaling was ongoing in the accepted EB grafts, as evidenced by expression of Smad2/3, a downstream target of TGF-β, which was found throughout the cytoplasm of all parenchymal cells (Supporting Information Fig. 3A). Moreover, nuclear expression of phospho-Smad2/3 was also observed in the accepted EB grafts (Supporting Information Fig. 3B). To investigate whether Treg are involved in suppressing the immune response against the accepted EB grafts, the PC61 antibody specific for CD25 was administered three times (1 mg each) in the week prior to transplantation. PC61 has been widely used by ourselves and others for the selective ablation of Treg function [28, 31, 32]. Accordingly, all PC61-treated recipients rejected the CBK EB grafts by 4 weeks post-transplantation (p < .05; Table 2). Histological analysis of serial sections from mAb-treated recipients showed signs of tissue damage (Fig. 5A), similar to those in grafts rejected by female recipients (Fig. 1C). Even though PC61 targets other cell types expressing CD25, the vigorous rejection of CBK EBs following its administration to recipients, demonstrates that sufficient CD8+ effector T cells are preserved so to be able to reject the grafts (Fig. 5D). These data strongly suggest, therefore, that Treg are important in protecting the CBK EB grafts in male CBA/Ca recipients from rejection.

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Figure 5. Ablation of Tregs with anti-CD25 monoclonal antibodies (mAb) induces rejection in normally tolerated grafts. Embryoid bodies (EBs) derived from ESF166 (CBK) were transplanted under the kidney capsule of male CBA/Ca mice treated with anti-CD25 and the grafts excised after 28 days. (A): EBs fail to survive in recipients treated with anti-CD25. Infiltration of activated macrophages (B, C) and CD8+ cells (D, E) was detected in anti-CD25-treated EB using immunohistochemistry. ESF166 (CBK) were transplanted for 28 days under the kidney capsule of male CBA/Ca mice treated with anti-CD25 monoclonal antibodies (mAb). Serial sections from EB stained with mAb specific for (B) F4/80 (red) and MHC class II (green) or (D) CD8 (red) and granzyme B (green). Nuclear staining by DAPI is overlaid in blue. All micrographs are fully representative of multiple recipients in each group (n = 3). Scale bar = 20 μm (B, D). The average number of (C) F4/80+ cells or (E) CD8+ cells in the accepted EB (no treatment) or EB derived from recipients treated with anti-CD25 mAb was counted and presented in the bar chart. The percentages of MHC class II+ cells among the F4/80+ population (C) or granzyme B (GranB+)-positive cells among the CD8+ population (E) are shown in parenthesis. Results are expressed as mean ± standard deviation of mean (p = .1, n = 3). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MHC II, class II major histocompatibility complex.

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Table 2. % Graft acceptance after PC61 treatment
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Treg exert suppressive effects on both innate and adaptive immune cells. Macrophages, in particular, might be one of their direct or indirect targets. To investigate whether Treg suppress activation of macrophages, serial sections of CBK EB excised from the PC61-treated male CBA/Ca recipients were stained with mAb specific for F4/80 and MHC class II (Fig. 5B). Ninety-one percentage of the F4/80+ macrophages were MHC class II+ (Fig. 5E), indicating that macrophages had become active and possibly inflammatory in the absence of Treg-mediated suppression.

Treg have also been shown to suppress tumor-specific CD8+ T cells by specifically suppressing their cytotoxicity rather than their ability to proliferate or to secrete cytokines such as IFN-γ and TNF-α [23]. Furthermore, CD8+ T cells have been shown to be the major effector cells responsible for graft rejection in this model (Fig. 2D). As CD8+ T-cell infiltration was observed in the privileged CBK EB transplanted into male CBA/Ca recipients (Fig. 2B), it is possible that the CD8+ T-cell cytotoxicity was suppressed by Treg operating in mice that had accepted transplanted tissues. To address this issue, serial sections of the EB from male CBA/Ca recipients, given PC61 mAb, as well as untreated controls, were costained with mAb specific for CD8 and granzyme B (Fig. 5D). Our results show that none of the CD8+ T cells in the accepted EB expressed the proapoptotic protein, granzyme B (Fig. 5E), whereas 94% of the infiltrated CD8+ T cells expressed granzyme B in the PC61-treated recipients. Treg may, therefore, contribute to protection against immune-mediated damage by suppressing the cytotoxicity of infiltrating CD8+ T cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Despite being immunogenic, mismatched ESC-derived tissues display an inherent level of immune privilege. We have previously reported the spontaneous acceptance of EB across a disparity of a single mH antigen, unlike tissues from conventional sources [6]. We now report the first evidence of natural survival of ESC-derived tissues in an MHC class I-mismatched donor-recipient combination, without the need for any immunosuppression (Fig. 1A, 1B). This study therefore defines, for the first time, the extent of immunological disparity between donor and recipient that may be accommodated in the context of CRT without recourse to immune intervention and investigates the mechanisms involved. By actively depleting Treg cells, we have demonstrated their capacity to influence the functional phenotype of infiltrating macrophages and CD8+ T cells, disarming them of their proinflammatory and cytolytic functions.

In this study, we compared the immune response against the “accepted” Kb-expressing allografts derived from male CBA/Ca recipients versus the “rejected” Kb-expressing allografts derived from female CBA/Ca recipients. In fact, we were restricted to studying graft rejection in female recipients because we were unable to tell which of the 50% of male recipients were going to accept or reject the graft, at least, within the first 3 weeks of transplantation. In the model studied here, the acceptance of mismatched EBs seems to be dependent on the activity of Treg, as ablation of Treg function with an anti-CD25 mAb exposed a capacity for rejection in a graft-host combination, where, CD8+ T cells were the principal effectors of rejection (Fig. 2D). This finding suggests that CD25+ regulatory T cells (previously identified as having a role in coreceptor antibody-induced acceptance of allografts), are also of critical importance in the “natural “acceptance of EB allografts.

Although our data strongly support a role for CD25+ Treg in preventing EB rejection, they do not identify exactly where and when these Treg operate, be this centrally in lymphoid tissue or in the graft, or both. In a previous publication using a T-cell receptor transgenic mouse lacking natural Treg (nTreg) [6], we demonstrated de novo induction of Treg (iTreg) within naturally accepted allogeneic grafts. In that study, we did not attempt to ablate Treg to prove that they were responsible for the natural privilege observed. In the present article, we have extended our previous findings to show the existence of natural privilege in conventional mice with a full repertoire of lymphocytes and their various subsets. In this situation, ablation of CD25+ Treg at the time of EB grafting abolished the natural privilege. In terms of potential mechanism for this effect, TGF-β is known to induce the differentiation of Foxp3+ iTreg from peripheral CD4+Foxp3 T cells through Smad2/3 signaling [33, 34].Certainly, the transplanted EB express abundant TGFβ and show evidence of TGFβ signaling through expression of downstream transcription factors, phospho-Smad2/3 (Supporting Information Fig. 3). Indeed, overexpression of TGF-β in pancreatic islets can expand iTreg cells and protect non-obese diabetic (NOD) mice from diabetes [35]. However, as there are no distinguishing markers to discriminate pre-existing thymic-derived nTreg from EB-induced iTreg, it was not possible to establish which type of Treg was instrumental in preventing rejection of CBK EB. To investigate how Treg contribute to immune privilege of ESC-derived tissues, we compared the types and quantities of infiltrating macrophages and effector CD8+ T-cells, reactive against the mismatched class I MHC, in “accepted” CBK EB from male CBA/Ca recipients and “rejected” CBK EB from female CBA/Ca recipients. We observed less infiltration of active F4/80+ MHC class IIlo macrophages in the privileged EB when compared with rejected grafts (Fig. 5E). Macrophages which infiltrated privileged grafts expressed less MHC class II, but expressed MR instead (Fig. 5D), a characteristic of AAM found in certain immunosuppressive microenvironments [22]. Recently, it has been reported that Treg may steer differentiation of monocytes/macrophages toward AAM [21]. It is, therefore, conceivable, that macrophages freed from the influence of Treg may regain proinflammatory activity. Furthermore, CD8+ T cells were shown to be incapable of killing their target cells unless Treg function had been ablated [23]. One of the mechanisms by which CD8+ T cells induce target cell death is via secretion of cytotoxic granules such as granzyme B in a contact-dependent manner [36]. CD8+ T cells deficient in granzyme B do not activate caspase-3 and therefore fail to kill their target cells [37]. In our study, the CD8+ T-cells which infiltrated accepted EB grafts (Fig. 2B) were deficient in granzyme B. However, after treatment with anti-CD25 mAb, 94% of the CD8+ T cells exhibited granzyme B expression (Fig. 5G), suggesting that Treg dampen the ability of CD8+ T cells to kill target cells. Furthermore, the number of F4/80+MHC-IIhi macrophages infiltrating the grafts was increased in Treg-ablated mice (Fig. 5D, 5E).

Although it is important to study privilege mechanisms in defined differentiated tissues, EB grafts represent a mixture of cell types differentiated from each of the three embryonic germ layers, which model the implantation of complex tissues. Given that mouse models of CRT have shown that tissues derived from ESC can be rejected [6, 7] even due to a single mH antigen mismatch between donor and recipient [6], it seems clear that enhancing the ability of a stem cell-derived graft to protect itself from immune damage is a necessary goal for broad application of stem cell replacement therapy. Our results suggest that amplification of Treg function offers a realizable target to achieve that goal. Long-term acceptance of fully allogeneic ESC-derived tissues via coreceptor blockade, for example, may exploit and reinforce the “innate” mechanism of privilege within the tissue.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

ESC-derived tissues can be naturally accepted across an MHC class I barrier without the use of immunosuppression. Treg were shown to mediate graft acceptance in this model. These findings suggest that Treg are needed for the manifestation of natural privilege, as they are for enabling therapeutic tolerance through coreceptor blockade.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by the Medical Research Council (U.K.). K.O.L. held a Dorothy Hodgkin Postgraduate Award. Additional support for A.S.B. and P.J.F. was provided by Geron Corporation (Menlo Park, California) and the Oxford Stem Cell Institute at the James Martin 21st Century School (Oxford, U.K.). A.S.B. is currently affiliated with the NIH COBRE in Tissue Repair & Stem Cell Biology, Boston University School of Medicine, Roger Williams Medical Center, Providence, RI. We thank Richard Stillion for assistance with histology.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_506_sm_suppfigure1.eps5502KSupporting Information Figure 1.
STEM_506_sm_suppfigure2.eps5066KSupporting Information Figure 2.
STEM_506_sm_suppfigure3.eps8658KSupporting Information Figure 3.

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