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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: Am J Transplant. 2012 Apr 14;12(6):1398–1408. doi: 10.1111/j.1600-6143.2012.04060.x

Dendritic cell therapies in transplantation revisited: Deletion of recipient DCs deters the effect of therapeutic DCs

Z Wang a,b,c, SJ Divito a,b, WJ Shufesky a,b, T Sumpter d, H Wang c, O A Tkacheva d, W Wang c, C Liu c, A T Larregina d,e, A E Morelli a,b,e
PMCID: PMC3365643  NIHMSID: NIHMS364017  PMID: 22500950

Abstract

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the allo-response and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

Keywords: Dendritic cells, Dendritic cell therapies, Transplantation, Immunosuppression, Tolerance

INTRODUCTION

Dendritic cells (DCs) are bone marrow (BM)-derived professional Ag-presenting cells (APCs) with capacity to induce immunity or tolerance (1). During the past 15 years, it has been demonstrated that in vitro-generated (donor- or recipient-derived) tolerogenic/immunosuppressive (IS) DCs, administered alone or with suboptimal doses of immunosuppressants, prolong survival of heart allografts in murine models (2–17). It is generally considered that the mechanism by which in vitro-generated ISDCs prolong heart allograft survival, is by interacting with anti-donor T cells, promoting deletion, immune-deviation, and/or regulation. However, since DCs pass Ag to other APCs via trogocytosis, nanotubes, cross-dressing, apoptotic cell-vesicles or exosomes (18–26), transfer of donor-Ag from i.v. injected ISDCs to recipient quiescent APCs expressing low levels of co-stimulatory molecules could represent an alternative and efficient means for restraining the anti-donor response, since a single ISDC can transfer donor-Ag to several recipient APCs. Such passage of donor-Ag could be highly relevant, considering that a small percentage of i.v. administered ISDCs migrate to lymphoid organs and once injected, they are short-lived or attacked by NK or CTLs(27–29).

Thus, considering that ISDCs dye shortly after i.v. infusion but are still capable of passing Ag to host APCs, we investigated the role of recipient APCs on the effect of ISDC-based therapies in organ transplantation, by transiently depleting recipient DCs at the time of the ISDC-therapy in a mouse model of cardiac transplantation. Surprisingly, our results indicate that, in contrast to the accepted paradigm, conventional CD11chigh DCs (cDCs) of the recipient, instead of the injected ISDCs, are critical for the donor-specific immune-suppression and prolongation of allograft survival that follows ISDC- therapy.

MATERIAL AND METHODS

Mice and reagents

C57BL/6 (B6), BALB/c, C3H and B6xBALB/c F1 mice were purchased from The Jackson Laboratory. B6.FVB-Tg (Itagx-DTR/eGFP)57Lan/J (CD11c-DTR-eGFP B6) and C.FVB-Tg (Itagx-DTR/eGFP)57Lan/J (CD11c-DTR-eGFP BALB/c) were provided by Dr. L. Falo (University of Pittsburgh). 1H3.1 and 2C RAG1KO B6 mice were bred in our animal facility. Studies were approved by the IACUC. GM-CSF and IL-4 were from PeproTech; 1α,25-(OH)2 vitamin D3 (VD3), LPS and DT from Sigma; and CD40 Ab (FGK45.5) from BioXCell. Abs were from BD-PharMingen, eBiosciences, Invitrogen or Jackson ImmunoResearch.

Generation of irradiation bone marrow (BM) chimeras

For generation of CD11c-DTR-eGFP B6 → wt B6 chimeras, 5–6 wk-old wt male B6 mice were γ-irradiated with 550 rads twice (6 hr apart). Two hr later, animals were i.v. injected with 107 BM cells from CD11c-DTR-eGFP B6 or wt B6 (control wt-B6 chimeras) mice. During the first wk, chimeras were kept in autoclaved cages and water with antibiotics (Sulfatrim). Chimeras were used 8 wk after BM transplant. The same procedure was followed for generation of CD11c-DTR-eGFP BALB/c → wt BALB/c chimeras. The reason for making CD11c-DTR BM chimeras is because the DTR is expressed also al low levels by non-hematopoietic cells (apparently in the central nervous system) of CD11c-DTR-eGFP mice, so these animals do not tolerate the repetitive administration of DT required to delete CD11chi DCs for more than 48 hr (30).

In vitro generation of DCs

BM cells from femurs and tibias were depleted of erythrocytes by hypotonic lysis. Erythroid cells, T and B lymphocytes, NK cells and granulocytes were removed by incubation with the Abs TER-119, CD3ε, B220, NK-1.1, Gr1, and IAb followed by rabbit complement (Cedarlane®). BM cells were cultured in RPMI-1640 with 10% FCS, glutamine, non-essential amino acids, sodium pyruvate, HEPES, 2-ME, antibiotics, and 1000 U/ml GM-CSF and 500 U/ml IL-4, either with addition of 10nM VD3 beginning on day 2 of culture (maturation-resistant DCs, MR-DCs) or not (immature DCs, Im-DCs). Medium, cytokines and VD3 were renewed every other day. MR-DCs and Im-DCs were purified from CD86+ cells by negative depletion (Dynabeads). To generate LPS-mature DCs, Im-DCs (day 6) were cultured with LPS (200 ng/ml) overnight.

Adoptive transfer of TCR transgenic (tg) T cells

1H3.1 CD4 and 2C CD8 T cells were purified from spleens and lymph nodes of TCRtg mice with CD4 or CD8 Dynabead negative isolation kits, stained with 7.5 μM Vybrant CFDA SE Cell Tracer (Invitrogen), and administered i.v. at 3×106 T cells per mouse.

Heart transplantation and ISDC-therapy

Recipients were left untreated, or injected (tail vein) with 5×106 MR-DCs, 7 days prior to heterotopic (abdomen) vascularized cardiac transplantation. In some experiments, ISDC-therapy was accompanied by rapamycin (1mg/kg body weight), administered i.p. for 10 consecutive days starting the day of the transplant (from day 0 to 9). For depletion of recipient DCs, BM chimeras (or control wt mice) were injected up to 4 times with DT (4ng/g body weight), every other day (on days -8, -6, -4 and -2 relative to transplantation), starting 1 day before injection of the ISDCs (on day -7).

Microscopic analysis of tissue sections

Paraffin-embedded sections of the grafts were processed for H&E. Graft fragments were snap-frozen, cryosectioned, fixed in 95% ethanol, and treated with 5% normal goat serum followed by the avidin/biotin blocking kit (Vector). Sections were stained with the following combinations of Abs: (i) purified CD4 Ab and biotin-CD8 Ab, followed by alexa-555-anti-rat IgG and Dylight-488-streptavidin; (ii) purified granzyme B Ab and biotin-CD8 Ab, followed by alexa-555-anti-rat IgG and Dylight-488-streptavidin; or (iii) purified anti-myeloperoxidase rabbit serum and biotin-F4/80 Ab, followed by Cy3-anti-rabbit IgG and Dylight-488-streptavidin.

For detection of 1H3.1 and 2C T cells in heart, liver and kidney, cryosections were labeled with alexa-647-CD4 or –CD8 Abs, and biotin-Thy1.1 Ab plus Cy2-streptavidin. Nuclei were stained with DAPI (Invitrogen). Slides were examined with a Zeiss Axiovert 135 microscope equipped with a CCD camera.

ELISPOT assays

For assessment of the direct pathway response, purified splenic T cells (enrichment columns, R&D Systems) from recipient CD11c-DTR-B6 chimeras untreated or i.v. injected with B6 (control) or donor-derived (BALB/c) MR-DCs (the latter ± DT injection), were incubated with CD3-depleted, γ-irradiated, splenic B6, BALB/c or C3H APCs (3×104 T cells + 2.5×105 APCs/well) in 96-well ELISPOT plates coated with IFN-γ Ab. To analyze the indirect pathway, purified recipient splenic T cells were incubated with CD3-depleted, γ-irradiated, splenic B6 APCs (3×105 T cells + 2.5×105 APCs/well) and sonicates (50μl/well) prepared from BALB/c, B6 or C3H splenocytes (from 2×107 cells/ml). ELISPOT plates were developed 36hr later (BD Biosciences).

Flow cytometric analysis

B6 and BALB/c MR-DCs were blocked with 10% normal goat serum and incubated (30min, 4°C) with PE-Cy5-CD11c Ab and one of the following PE-conjugated Abs specific for IAb, IAd, H2Kb, H2Kd, CD40, CD80, or CD86. For assessment of the kinetics of recipient DC depletion and recovery, splenocytes and lymph node cells from CD11c-DTR-B6 chimeras injected or not with DT were incubated with APC-CD11c, PE-Cy5-CD8α and PE-CD45RA Abs. To measure in vivo the lifespan of donor-Ag derived from: (i) donor (BALB/c) MR-DCs; (ii) recipient (B6) MR-DCs or Im-DCs, both pulsed with BALB/c IEα52-68 peptide); and (iii) B6xBALB/c F1 MR-DCs; CD11c-DTR-eGFP B6 chimeras or wt B6 mice (Thy.1.2) were injected i.v. with each of the DC subsets, and reconstituted with CFSE-labeled 1H3.1 CD4 T cells (Thy1.1) 1, 3, 5, or 7 days after DC administration. Three days after transfer of 1H3.1 cells, splenocytes were stained with PE-CD4 and PE-Cy5-Thy1.1 Abs.

For analysis of TCRtg T cells, splenocytes and lymph node cells from CD11c-DTR-B6 or control wt-B6 chimeras, reconstituted with CFSE-labeled 1H3.1 cells alone or with 2C cells and treated with DC therapy (± DT injection), were incubated with PE-annexin-V, PE-Cy5-CD62L, PE-Cy7-CD69, APC-Thy1.1, Pacific blue-CD4 and AlexaFluor-700-CD8. For detection of TCRtg T cell anergy/regulation, splenocytes were labeled with PE-Cy5-CD28, PE-Cy7-CD69, APC-Thy1.1, Pacific blue-CD4 and AlexaFluor-700-CD8 Abs. Next, cells were treated with cytofix/cytoperm solution and stained with PE-FoxP3 Ab.

Appropriate irrelevant Abs were used as controls. Cells were fixed in paraformaldehyde and data were acquired with FACSCalibur™ or BD™ LSR flow cytometers (BD Biosciences).

ELISA

Detection of IL-2, IL-10 and IFN-γ were performed by ELISA according to manufacturers’ instructions (BD Biosciences).

Statistical analysis

GraphPad Prism was used for statistical analyses. Results are expressed as mean ± SD, if one representative experiment is shown, or as mean ± SEM if data is averaged from more than one experiment. Comparison between two groups was performed by Student’s t-test. Graft survivals were compared by Kaplan-Meier analysis and the log-rank test. A “p” value < 0.05 was considered significant.

RESULTS

Recipient DCs are critical for the effect of donor ISDC-therapy in cardiac transplantation

To investigate the role of recipient DCs in ISDC-therapies in transplantation, recipient cDCs (CD11chigh) were depleted in CD11c-diphtheria toxin (DT) receptor (DTR)-eGFP B6 (CD11c-DTR-B6) BM chimeras, by DT-injection 1 day before and 1, 3 and 5 days after ISDC-therapy (Figure S1A). As prototypic donor-derived ISDCs, we used BALB/c maturation-resistant (MR)-DCs generated in vitro (Figure S1B), which exhibit low allostimulatory potential and, when administered i.v., prolong cardiac allograft survival (Figure 1A). Injection of BALB/c MRDC in CD11c-DTR-B6 chimeras triggered division of 1H3.1 T cells specific for IEα52-68 (BALB/c)-IAb (B6) (Figure S1C) only in not DC-depleted mice, indicating that cDCs are the only recipient professional APCs that present donor-Ag transferred from the injected MR-DCs since, in this model, plasmacytoid DCs are not depleted by DT injection (Figure S1D) (31,32).

Figure 1. Role of recipient cDCs in donor-derived ISDC-therapy in cardiac transplantation.

Figure 1

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(A) Survival of BALB/c hearts transplanted in CD11c-DTR-B6 chimeras, or control wt-B6 chimeras, transiently depleted of cDCs by DT-injection (or not, control) by the time of i.v. administration of BALB/c MR-DCs (or not, control). The number of recipients per group is between parentheses. (B) Microscopic analysis of allograft histology (H&E staining) and leukocyte infiltrate (immunofluorescence) 7 days after transplantation. MPO: myeloperoxidase. Results are representative of 3 mice per group. Bars show the average cell number per low powered field (LPF, 200X) of CD4 and CD8 T cells (mean ± SD). (C) Assessment by IFN-γ ELISPOTs of the indirect and direct pathway T cell responses in recipient spleens. Each group included 3 mice (mean ± SD).

To evaluate the role of recipient DCs in ISDC-therapy in cardiac transplantation, the following key parameters were considered: (i) the optimal effect is achieved when ISDCs are administered 7 days before transplantation (2–4), (ii) recipient APCs in lymphoid tissues are necessary for elicitation of acute rejection (33–35), and (iii) donor-derived ISDCs die within 3 days after i.v. administration (17). We confirmed in CD11c-DTR-B6 chimeras, that CD11chigh DCs are DT-depleted (Figure S2A) during the 3 day-window when donor-Ag from the i.v. injected ISDCs remains in the spleen (the latter assessed by proliferation of 1H3.1 T cells, Figure S2B). We also proved that after DT-administration, splenic CD11chigh DCs recover in number by the time of transplantation (day 0), so that they contribute to elicitation of rejection (Figure S2A).

Administration BALB/c MR-DCs in CD11c-DTR-B6 chimeras 7 days before transplantation prolonged (p<0.001) survival of BALB/c cardiac allografts (mean survival time [MST]=31±5 days), compared to untreated chimeras (MST=17±3 days, Figure 1A). In contrast, injection BALB/c MR-DCs in CD11c-DTR-B6 chimeras DT-injected on days 8, 6, 4, and 2 prior to transplantation, did not have effect (MST=13±5 days, Figure 1A). This result was not caused by the DT, since BALB/c MR-DCs also prolonged survival of BALB/c hearts in DT-injected wt-B6 chimeras (MST=41±27 days, Figure 1A). DT did not affect the tempo of rejection since without ISDC-therapy, DT-treated wt-B6 chimeras rejected BALB/c grafts with the same kinetics (MST=17±3 days) as CD11c-DTR-B6 chimeras not exposed to DT (MST=17±3 days). Similarly, transient DC-depletion before transplantation did not affect the kinetics of allograft rejection, since rejection in DC-depleted B6 chimeras was similar to that in DT-injected wt-B6 chimeras (MST=16±3 vs.17±3 days) (Figure 1A). Thus, the beneficial effect of donor MR-DCs requires recipient cDCs.

BALB/c hearts transplanted in CD11c-DTR-B6 chimeras treated with donor-MR-DCs exhibited less damage and leukocyte infiltration than allografts from chimeras treated with syngeneic MR-DCs, or from DC-depleted chimeras treated with donor-MR-DCs (Figure 1B). In spleen, donor-MR-DC injection decreased drastically (p≤0.01) the frequency of T cells recognizing donor-Ag via the direct (intact donor-MHC presented by donor APCs) or indirect (donor-peptides presented by recipient APCs) pathway (Figure 1C). This did not occur in DC-depleted recipients, suggesting that donor-derived ISDC-therapy requires recipient cDCs to restrain the indirect response as expected, but also to control the direct T cell response.

Recipient cDCs decrease the anti-donor T cell response induced by donor ISDC-therapy

Next, we investigated the effect of DC-deletion on the anti-donor response induced by donor ISDC-therapy. CD11c-DTR-B6 chimeras (Thy1.2) reconstituted with CFSE-labeled (Thy1.1) 1H3.1 CD4 T cells were i.v. injected the following day with BALB/c MR-DCs. Three days later, splenic 1H3.1 cells proliferated comparably to positive controls that also received agonistic CD40 Ab, but remained CD69neg/lo CD62Lhi and with increased apoptosis, signs of deficient activation (Figure 2A,B). Fourteen days after donor ISDC-therapy, the number and IFN-γ-secretion of splenic 1H3.1 cells decreased significantly (Figure 2B) due to deletion, since 1H3.1 cells were not detected in periphery. By contrast, 1H3.1 cell deletion did not occur in chimeras DT-injected at the time of ISDC-therapy (Figure 2B). Similar 1H3.1 cell deletion occurred 14 days after donor-derived MR-DC treatment in CD11c-DTR-B6 chimeras transplanted with BALB/c hearts 7 days after DC injection, but not when recipient DCs were depleted at the time of DC administration (Figure 2B). A relative increase in the percentage of splenic FoxP3+ 1H3.1 T cells occurred in CD11c-DTR-B6 chimeras 14 days after ISDC-therapy (Figure 2C). These data suggest that recipient cDCs mediate the preferential deletion of effector over regulatory T cells after ISDC-therapy.

Figure 2. Donor-ISDC-therapy promotes abortive activation of indirect CD4 T cells via recipient cDCs.

Figure 2

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(A) FACS-analysis of proliferation (CFSE-dilution), phenotype, and apoptosis (annexin-V binding) of CFSE-labeled (Thy1.1) 1H3.1 CD4 T cells adoptively transferred in CD11c-DTR-B6 chimeras (Thy1.2), that were i.v. injected the following day with syngeneic (B6, control) or donor (BALB/c) MR-DCs, the latter alone or in combination with agonistic CD40 Ab (i.p.) to promote 1H3.1 T cell activation (positive control), or DT (i.p.). FACS-analysis was done 3 days after DC injection. (B) Number of splenic 1H3.1 T cells (by FACS) and their IFN-γ-secretion in response to 24 hr ex vivo re-stimulation with IEα52-68 (by ELISA), assessed 3 and 14 days after B6 (control) or BALB/c MR-DCs therapy (the latter ± agonistic CD40 Ab, i.p.) in CD11c-DTR-B6 chimeras, depleted (or not, control) of recipient cDCs at the time of DC injection, and transplanted (Tx) or not with BALB/c hearts 7 days after ISDC-therapy. (C) Numbers of splenic FoxP3+ CD4 1H3.1 T cells from CD11c-DTR-B6 chimeras 14 days after ISDC-therapy under the same conditions as (B). NS: not significant. Results are representative of at least 3 mice per group (mean ± SD).

Role of immunosuppressants and mouse strain

Clinical application of ISDCs will likely require co-treatment with suboptimal doses of immunosuppressive drugs. Since the short lifespan of donor-derived ISDCs in vivo could be prolonged by the effect of immunosuppressants on recipient NK or CTLs, the probability of the ISDCs of interacting directly with donor-reactive T cells could increase, reducing their requirement of recipient cDCs. We tested this in CD11c-DTR-B6 chimeras treated with donor-derived MR-DCs plus a short-course of rapamycin (RAPA), which prolonged survival of transplanted BALB/c hearts compared to controls treated with MR-DCs alone (MST=50±8 vs. 31±5 days, p=0.0013) (Figure 3A and 1A). However, when recipients were DC-depleted at time of ISDC-therapy, allograft survival was similar to that of recipients treated with RAPA alone (MST=21±2 vs. 25±7 days, Figure 3A). Thus, even in combination with a broadly used immunosuppressant, the injected ISDCs prolonged allograft survival through recipient DCs. These findings were not strain-dependent, since transient DC-depletion in CD11c-DTR-BALB/c chimeras abolished the added beneficial effect of donor-derived MR-DCs plus a short course of RAPA on allograft survival (Figure 3B).

Figure 3. Role of pharmacological immunosuppression and strain combination on the effect of recipient cDCs in donor-derived ISDC-therapy.

Figure 3

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(A) Survival of BALB/c hearts transplanted (day 0) in CD11c-DTR-B6 chimeras transiently DC-depleted by DT (or not, control), at the time of i.v. administration of donor (BALB/c) MR-DCs (day -7). Recipients received a short-course of rapamycin (RAPA) from day 0 to 9 after transplantation. (B) Survival of cardiac allografts under the same conditions detailed in (A) but using wt B6 mice as heart donors and CD11c-DTR-BALB/c chimeras as recipients. (A,B) The number of recipients per group is between parenthesis.

Recipient cDCs and recipient-derived ISDC-therapy

Therapy with donor-derived ISDCs will be difficult to implement with deceased donors because in vitro-propagation of DCs requires days. More recently, recipient-derived ISDCs loaded with donor-Ag have been used to prolong heart allograft survival in murine models (11–14). However, it is unclear if donor- and recipient-derived ISDCs utilize a common mechanism via donor Ag-transfer to recipient cDCs, or if recipient ISDCs (unlike donor-ISDCs) mediate their immune-regulatory effects by themselves. We addressed this by DC-depletion at the time of recipient-derived MR-DC administration.

First, we assessed the survival of recipient-derived MRDCs pulsed with allo-Ag, one injected systemically. Wt B6 mice were i.v. injected with B6 MR-DCs loaded with the (BALB/c) IEα52-68 peptide or with control (not loaded) B6 MR-DCs. One, 3, 5 or 7 days later, animals receive CFSE-labeled 1H3.1 T cells and 1H3.1 cell division was analyzed by FACS. 1H3.1 cell proliferation was detected only when 1H3.1 cells were transferred 1 or 3 days after administration of recipient MR-DC pulsed with IEα52-68 (Figure S2C). The brief presentation of the donor peptide suggests that recipient-derived MR-DCs, once injected i.v., do not survive longer than 3 days. This finding was not restricted to recipient MR-DCs generated with VD3 or to short lifespan of the peptide, since similar results were detected following injection of recipient immature DCs (Im-DCs, not exposed to VD3) loaded with IEα52-68, or after infusion of B6 X BALB/c F1 MR-DCs which express endogenous IAb- IEα52-68 (Figure S2C).

Next, to test whether recipient MR-DCs pulsed with peptides and injected i.v. interact directly with T cells in vivo or transfer the added peptides to cDCs in lymphoid tissues. B6 MR-DCs loaded with SIYRL and IEα52-68 peptides were injected i.v. into CD11c-DTR-B6 chimeras (Thy1.2) reconstituted the day before with Thy1.1+ CFSE-labeled 1H3.1 CD4 and 2C CD8 T cells (3×106 of each,) specific for IAb-IEα52-68 and Kb- SIYRL, respectively. Three days after injection of the peptide-loaded B6 MR-DCs, splenic 1H3.1 cells (Figure 4A) and 2C cells (Figure S3A) proliferated with signs of deficient activation defined as CD69neg/lo CD62Lhi and increased percentage of apoptotic cells in contrast to 1H3.1 and 2C cells of positive controls treated i.p. with agonistic CD40 Ab. 1H3.1 and 2C T cell proliferation was comparable between host B6 mice treated with peptide-pulsed B6 MR-DCs and positive control animals that also received agonistic CD40 Ab (Figure 4B and S3B). DT injection 1 day before and 1 day after MR-DC administration decreased drastically proliferation of 1H3.1 and 2C in the spleen (Figure 4B and S3B). This effect was independent of the VD3 used for production of MR-DCs, since similar findings were obtained after injecting Im-DCs generated without added VD3 (Figure 4A and S3A). Importantly, DT had no effect on activation/proliferation of 1H3.1 and 2C cells in control B6 mice injected with peptide-pulsed B6 MR-DCs (Figure 4A and S3A).

Figure 4. Presentation by cDCs of Ags from i.v. administered recipient-derived ISDCs to CD4 T cells.

Figure 4

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(A) Assessment of proliferation (CFSE-dilution), apoptosis (annexin-V binding) and CD62L and CD69 expression in CFSE-labeled (Thy1.1) CD4 1H3.1 T cells in spleens of CD11c-DTR-B6 chimeras that were transiently depleted by DT, or not, of cDCs by the time of i.v. injection of B6 MR-DCs or immature DCs (Im-DCs), both pulsed with IEα52-68 (recognized by 1H3.1 cells). FACS- analysis was done 3 days after DC administration. CD11c-DTR-B6 chimeras injected with non-pulsed B6 MR-DCs or B6 MR-DC-IEα52-68 plus agonistic CD40 Ab (i.p.) were added as negative and positive controls of 1H3.1 cell activation, respectively. Control wt B6 mice, injected with MR-DC-IEα52-68 plus DT, were included to assess the effect of DT on 1H3.1 cell activation. Dot plots represent 3 mice per variable. (B) Numbers of 1H3.1 cells (3 days after ISDC administration) in spleen of CD11c-DTR-B6 chimeras depleted, or not, by DT of cDCs, at the time of injection of B6 MR-DCs or B6 immature (Im)-DCs, both pulsed with IEα52-68. Results were pooled from 3 mice per group.

To rule out that the systemically injected host-derived ISDCs promote T cell anergy through direct contact with the T cells, we repeated the experiments but challenging with LPS-mature B6 BMDCs pulsed with SIYRL and IEα52-68, 7 days after administration of host-derived peptide-loaded MR-DCs into CD11c-DTR-B6 chimeras depleted of cDCs by DT-injection (Figure 5). Three days after challenge, 1H3.1 and 2C cells exhibited similar proliferation/activation, percentages of FoxP3+ cells, and IL-2 and IFN-γ secretion as cells from chimeras treated with non-pulsed B6 MR-DCs and challenged in the same way (positive control) (Figure 5). Thus, without recipient cDCs, systemically injected host-derived ISDCs pulsed with allopeptides are unable to down-regulate T cell function by themselves via direct interaction with the T cells.

Figure 5. Assessment of T cell unresponsiveness in mice pre-treated with recipient-derived ISDCs pulsed with Ag.

Figure 5

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(A) CD11c-DTR-B6 chimeras reconstituted with CFSE-labeled (Thy1.1) CD4 1H3.1 and CD8 2C T cells were transiently depleted, or not, of cDCs at the time of i.v. administration of host-derived MR-DCs or immature DCs (Im-DCs), both pulsed with IEα52-68 and SIYRL. After 7 days, chimeras were challenged (or not, control) with LPS-mature B6 BMDCs pulsed with the same peptides. After 3 days, proliferation (CFSE -dilution), expression of FoxP3 and CD69 by 1H3.1 T cells, or CD28 and CD69 by 2C T cells was measured by FACS. (B) Three days after challenge, IFN-γ and IL-2 secretion by splenocytes in response to 24 hr re-stimulation with IEα52-68 or SIYRL added to the wells was assessed by ELISA. Results were pooled from 3 mice per group.

DISCUSSION

The results presented indicate that depletion of recipient cDCs by the time of ISDC-therapy prevents its beneficial effect on cardiac transplantation. Importantly, this phenomenon was independent of the mouse strain combination and it also occurred when the ISDC-therapy was combined with low-dose pharmacological immunosuppression which represents the most likely scenario for the potential clinical use of ISDC-therapies in transplantation.

Following ISDC-therapy, presentation of donor-derived peptides by the subpopulation of cDCs led to preferential deletion of indirect pathway CD4 effector T cells causing a relative increase in the percentage of FoxP3 CD4 T cells. Interestingly, a similar mechanism has been reported following in situ-targeting of quiescent cDCs(36–41).

Our findings agree with previous studies that demonstrated that i.v. administration of donor-derived ISDCs before cardiac transplantation decreases significantly the anti-donor response elicited via the direct pathway (intact donor MHC recognized by recipient T cells) (2–10). The high precursor frequency of direct pathway T cells plus their predominance in most in vitro assays of allo-reactivity led to the prevailing idea that the direct pathway is the dominant mechanism of allorecognition during acute rejection of cardiac allografts. It also led to the conclusion that systemically infused donor-derived ISDCs control the direct response by interacting physically with direct pathway T cells to delay/prevent acute rejection of heart allografts. However, we demonstrated that at least in our model, transient depletion of recipient DCs at the time of donor-derived ISDC-therapy prevents not only down-regulation of the indirect T cell response as expected, but also the direct pathway response elicited by the heart allografts. This finding suggests that in vivo, donor-derived ISDCs by themselves are unable to down-regulate the direct T cell response, unless recipient cDCs are present.

A possible explanation is that elicitation of the direct response requires help of indirect T cells, which are severely depleted and with an increased regulatory/effector T cell ratio following donor-ISDC-therapy. As demonstrated in the “3 cell-model” (22), a single recipient DC presenting donor intact MHC Ag and donor-peptides (both acquired from passenger leukocytes) function as a “bridging APC” where activated indirect CD4 T cells provide help to direct CD8 T cells interacting, simultaneously or not, on the same APC. This explains the observation that indirect CD4 T cells provide help for direct CD8 T cell for skin allograft rejection in mice (42). This interpretation and our results agree with previous findings in cardiac transplantation where: (i) elimination of the indirect pathway in recipients is followed by minimal direct T cell priming (43); (ii) T cells are more rapidly and preferentially primed through the indirect pathway (43,44); (iii) recipients unable to mount an indirect allo-response exhibit indefinite or prolonged allograft survival (33–35), and (iv) the indirect pathway is critical to achieve long-term allograft survival following costimulation blockade (45). In the field of DC therapies in cardiac transplantation, a decreased in the indirect T cell help to direct T cells may explain why administration of recipient-derived ISDCs pre-loaded with donor-Ag, which are supposed to down-regulate exclusively the indirect pathway, also decreases the direct T cell response and delays acute rejection of heart allografts in murine models (11–14).

Since generation of donor-derived ISDCs is not a feasible method with deceased donors, the use of recipient-derived ISDCs pulsed with donor-Ag is currently considered a more practical clinical approach. However, our results indicate that i.v. injected host-derived ISDCs pulsed with MHC-I or –II -restricted peptides, also mediate their suppressive effects on T cells through endogenous cDCs. Interestingly, it has been reported that following (positive) vaccination with syngeneic DCs loaded with a model peptide and injected s.c., approximately 2/3 of T cell priming is mediated by the endogenous DCs (46).

In summary, our results indicate that donor- and recipient-derived ISDC therapies mediate their beneficial effects and prolong cardiac allograft survival not by themselves, as classically assumed, but through quiescent cDCs of the recipient. This leads to the conclusion that DC therapies, at least in cardiac transplantation, restrain the anti-donor response by sharing a similar mechanism of indirect allorecognition previously described in much simpler donor-cell based therapies, including donor-specific transfusion (47,48) and donor apoptotic cell-based therapies (39,40). These findings have implications for ISDC therapies in transplantation, since the intrinsic immune-regulatory properties of the i.v. injected ISDCs (as long as they are not immunogenic) do not seem to be relevant in vivo, and the pro-tolerogenic properties of the recipient quiescent cDCs in lymphoid organs, which are critical for the success of the ISDC therapies, may be severely compromised during end-stage diseases that require organ transplantation.

Supplementary Material

Supp Fig S1-S3

Acknowledgments

We thank L.D. Falo for providing the B6 CD11c-DTR-eGFP mice and G. Chalasani for the 2C mice. Supported by grants from the National Institutes of Health R01 HL077545 (A.E.M), R01 AI077511 (A.T.L.), F30 DK082131 (S.J.D.), and funds from the T.E. Starzl Transplantation Institute.

Abbreviations

Ab

antibody

Ag

antigen

APCs

antigen-presenting cells

BM

bone marrow

cDCs

conventional DCs

DCs

dendritic cells

DT

diphtheria toxin

DTR

diphtheria toxin receptor

GM-CSF

granulocyte-macrophage colony stimulating factor

IL

interleukin

Im-DCs

immature DCs

ISDCs

immunosuppressive DCs

i.v

intravenously

eGFP

enhanced green fluorescent protein

LPS

lipopolysaccharide

MR-DCs

maturation-resistant DCs

MST

mean survival time

tg

transgenic

RAPA

rapamycin

VD3

vitamin D3

Footnotes

Disclosure

The authors of this manuscript have no conflict of interest to disclose as described by the American Journal of Transplantation

References

  • 1.Morelli AE, Thomson AW. Tolerogenic dendritic cells and the quest for transplant tolerance. Nat Rev Immunol. 2007;7(8):610–621. doi: 10.1038/nri2132. [DOI] [PubMed] [Google Scholar]
  • 2.Fu F, Li Y, Qian S, et al. Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86-) prolong cardiac allograft survival in nonimmunosuppressed recipients. Transplantation. 1996;62(5):659–665. doi: 10.1097/00007890-199609150-00021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Lu L, Li W, Fu F, et al. Blockade of the CD40-CD40 ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival. Transplantation. 1997;64(12):1808–1815. doi: 10.1097/00007890-199712270-00031. [DOI] [PubMed] [Google Scholar]
  • 4.Lutz MB, Suri RM, Niimi M, et al. Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo. Eur J Immunol. 2000;30(7):1813–1822. doi: 10.1002/1521-4141(200007)30:7<1813::AID-IMMU1813>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  • 5.Min WP, Gorczynski R, Huang X-Y, et al. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. J Immunol. 2000;164(1):161–167. doi: 10.4049/jimmunol.164.1.161. [DOI] [PubMed] [Google Scholar]
  • 6.Bonham CA, Peng L, Liang X, et al. Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-κB oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA-Ig. J Immunol. 2002;169(6):3382–3391. doi: 10.4049/jimmunol.169.6.3382. [DOI] [PubMed] [Google Scholar]
  • 7.DePaz HA, Oluwole OO, Adeyeri AO, et al. Immature rat myeloid dendritic cells generated in low-dose granulocyte macrophage-colony stimulating factor prolong donor-specific rat cardiac allograft survival. Transplantation. 2003;75(4):521–528. doi: 10.1097/01.TP.0000048380.84355.4A. [DOI] [PubMed] [Google Scholar]
  • 8.Lan YY, Wang Z, Raimondi G, et al. “Alternatively activated” dendritic cells preferentially secrete IL-10, expand Foxp3+CD4+ T cells, and induce long-term organ allograft survival in combination with CTLA4-Ig. J Immunol. 2006;177(9):5868–5877. doi: 10.4049/jimmunol.177.9.5868. [DOI] [PubMed] [Google Scholar]
  • 9.Emmer PM, van der Vlag J, Adema GJ, Hilbrands LB. Dendritic cells activated by lipopolysaccharide after dexamethasone treatment induce donor-specific allograft hyporesponsiveness. Transplantation. 2006;81(10):1451–1459. doi: 10.1097/01.tp.0000208801.51222.bd. [DOI] [PubMed] [Google Scholar]
  • 10.Zhang X, Li M, Lian D, et al. Generation of therapeutic dendritic cells and regulatory T cells for preventing allogeneic cardiac graft rejection. Clin Immunol. 2008;127(3):313–321. doi: 10.1016/j.clim.2008.01.013. [DOI] [PubMed] [Google Scholar]
  • 11.Garovillo M, Ayoola A, Depaz HA, et al. Induction of transplant tolerance with immunodominant allopeptide-pulsed host lymphoid and myeloid dendritic cells. Am J Transplant. 2001;1(2):129–137. [PubMed] [Google Scholar]
  • 12.Taner T, Hackstein H, Wang Z, Morelli AE, Thomson AW. Rapamycin-treated, alloantigen-pulsed host dendritic cells induce Ag-specific T cell regulation and prolong graft survival. Am J Transplant. 2005;5(2):228–236. doi: 10.1046/j.1600-6143.2004.00673.x. [DOI] [PubMed] [Google Scholar]
  • 13.Xu DL, Liu Y, Tan J-M, et al. Marked prolongation of murine cardiac allograft survival using recipient immature dendritic cells loaded with donor-derived apoptotic cells. Scand J Immunol. 2004;59(6):536–544. doi: 10.1111/j.1365-3083.2004.01427.x. [DOI] [PubMed] [Google Scholar]
  • 14.Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang Z, Thomson AW. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol. 2007;178(11):7018–7031. doi: 10.4049/jimmunol.178.11.7018. [DOI] [PubMed] [Google Scholar]
  • 15.Mirenda V, Berton I, Read J, et al. Modified dendritic cells coexpressing self and allogeneic major histocompatibility complex molecules: An efficient way to induce indirect pathway regulation. J Am Soc Nephrol. 2004;15(4):987–997. doi: 10.1097/01.asn.0000119575.98696.1d. [DOI] [PubMed] [Google Scholar]
  • 16.Sato K, Yamashita N, Yamashita N, Baba M, Matsuyama T. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse. Immunity. 2003;18(3):367–379. doi: 10.1016/s1074-7613(03)00055-4. [DOI] [PubMed] [Google Scholar]
  • 17.Divito JS, Wang Z, Shufesky WJ, et al. Endogenous dendritic cells mediate the effects of intravenous injected therapeutic immunosuppressive dendritic cells in transplantation. Blood. 2010;116(15):2694–2705. doi: 10.1182/blood-2009-10-251058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Inaba K, Turley S, Yamaide F, et al. Efficient presentation of phagocytosed cellular fragments on the major histocompatibiliy complex class II products of dendritic cells. J Exp Med. 1998;188(11):2163–2173. doi: 10.1084/jem.188.11.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bedford P, Gardner K, Knight SC. MHC class II molecules transferred between allogeneic dendritic cells stimulate primary lymphocyte reactions. Int Immunol. 1999;11(11):1739–1744. doi: 10.1093/intimm/11.11.1739. [DOI] [PubMed] [Google Scholar]
  • 20.Russo V, Zhou D, Sartirana P, et al. Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells. Blood. 2000;95(11):3473. [PubMed] [Google Scholar]
  • 21.Harshyne LA, Watkins SC, Gambotto A, Barratt-Boyes SM. Dendritic cells acquire antigens from live cells for cross-presentation to CTL. J Immunol. 2001;166(6):3717–3723. doi: 10.4049/jimmunol.166.6.3717. [DOI] [PubMed] [Google Scholar]
  • 22.Barroso Herrera O, Golshayan D, Tibbott R, et al. A novel pathway of alloantigen presentation by dendritic cells. J Immunol. 2004;173(8):4828–4837. doi: 10.4049/jimmunol.173.8.4828. [DOI] [PubMed] [Google Scholar]
  • 23.Wakim LM, Bevan MJ. Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection. Nature. 2011;471:629–632. doi: 10.1038/nature09863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Watkins SC, Salter RD. Functional connectivity between immune cells mediated by tunneling nanotubules. Immunity. 2005;23(3):309–318. doi: 10.1016/j.immuni.2005.08.009. [DOI] [PubMed] [Google Scholar]
  • 25.Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature. 1998;392(6671):86–89. doi: 10.1038/32183. [DOI] [PubMed] [Google Scholar]
  • 26.Montecalvo A, Shufesky WJ, Beer Stolz D, et al. Exosomes as a short-range mechanism to spread alloantigen between dendritic cells during T-cell allorecognition. J Immunol. 2008;180(5):3081–3090. doi: 10.4049/jimmunol.180.5.3081. [DOI] [PubMed] [Google Scholar]
  • 27.Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC. NK cells promote transplant tolerance by killing donor antigen-presenting cells. J Exp Med. 2006;203(8):1851–1858. doi: 10.1084/jem.20060603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Laffont S, Seillet C, Ortaldo J, Coudert JD, Guery J-C. Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells. Blood. 2008;112(3):661–671. doi: 10.1182/blood-2007-10-120089. [DOI] [PubMed] [Google Scholar]
  • 29.Laffont S, Coudert JD, Garidou L. CD8+ T-cell-mediated killing of donor dendritic cells prevents alloreactive T helper type 2 responses in vivo. Blood. 2006;108(7):2257–2264. doi: 10.1182/blood-2005-10-4059. [DOI] [PubMed] [Google Scholar]
  • 30.Zaft T, Sapoznikov A, Krauthgamer R, Littman DR, Jung S. CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naïve and memory CD8+ T cells. J Immunol. 2005;175(10):6428–6435. doi: 10.4049/jimmunol.175.10.6428. [DOI] [PubMed] [Google Scholar]
  • 31.Jung S, Unutmaz D, Wong P, et al. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity. 2002;17(2):211–220. doi: 10.1016/s1074-7613(02)00365-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sapoznikov A, Fisher JA, Zaft T, Krauthgamer R, Dzionek A, Jung S. Organ-dependent in vivo priming of naïve CD4+, but not CD8+, T cells by plasmacytoid dendritic cells. J Exp Med. 2007;204(8):1923–1933. doi: 10.1084/jem.20062373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Zheng XX, Sayegh MH, Zheng X-G, et al. The role of donor and recipient B7-1 (CD80) in allograft rejection. J Immunol. 1997;159 (3):1169–1173. [PubMed] [Google Scholar]
  • 34.Mandelbrot DA, Furukawa Y, McAdam AJ, et al. Expression of B7 molecules in recipient, not donor, mice determines the survival of cardiac allografts. J Immunol. 1999;163(7):3753–3757. [PubMed] [Google Scholar]
  • 35.Garrod KR, Liu F-C, Forrest LE, Parker I, Kang S-M, Cahalan MD. NK cell patrolling and elimination of donor-derived dendritic cells favor indirect alloreactivity. J Immunol. 2010;184(5):2329–2336. doi: 10.4049/jimmunol.0902748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hawiger D, Inaba K, Dorsett Y, et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med. 2001;194(6):769–779. doi: 10.1084/jem.194.6.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med. 2002;196(12):1627–1638. doi: 10.1084/jem.20021598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mahnke K, Qia Y, Knop J, Enk AH. Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood. 2003;101(12):4862–4869. doi: 10.1182/blood-2002-10-3229. [DOI] [PubMed] [Google Scholar]
  • 39.Wang Z, Larregina AT, Shufesky WJ, et al. Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells. Am J Transplant. 2006;6(6):1297–1311. doi: 10.1111/j.1600-6143.2006.01308.x. [DOI] [PubMed] [Google Scholar]
  • 40.Wang Z, Shufesky WJ, Montecalvo A, Divito SJ, Larregina AT, Morelli AE. In situ-targeting of dendritic cells with donor-derived apoptotic cells restrains indirect allorecognition and ameliorates allograft vasculopathy. PLoS ONE. 2009;4(3):e4940. doi: 10.1371/journal.pone.0004940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Tanriver Y, Ratnasothy K, Bucy RP, Lombardi G, Lechler R. Targeting MHC class I monomers to dendritic cells inhibits the indirect pathway of allorecognition and the production of IgG alloantibodies leading to long-term allograft survival. J Immunol. 2010;184(4):1757–1764. doi: 10.4049/jimmunol.0902987. [DOI] [PubMed] [Google Scholar]
  • 42.Lee RS, Grusby MJ, Glimcher LH, Winn HJ, Auchincloss H., Jr Indirect recognition by helper cells can induce donor-specific cytotoxic T lymphocytes in vivo. J Exp Med. 1994;179(3):865–872. doi: 10.1084/jem.179.3.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Chen Y, Demir Y, Valujskikh A, Heeger PS. The male minor transplantation antigen preferentially activates recipient CD4+ T cells through the indirect presentation pathway in vivo. J Immunol. 2003;171(12):6510–6518. doi: 10.4049/jimmunol.171.12.6510. [DOI] [PubMed] [Google Scholar]
  • 44.Brennan TV, Jaigirdar A, Hoang V, et al. Preferential priming of alloreactive T cells with indirect reactivity. Am J Transplant. 2009;9(4):709–718. doi: 10.1111/j.1600-6143.2009.02578.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Yamada A, Chandraker A, Laufer TM, Gerth AJ, Sayegh MH, Auchincloss H., Jr Recipient MHC class II expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade. J Immunol. 2001;167(10):5522–5526. doi: 10.4049/jimmunol.167.10.5522. [DOI] [PubMed] [Google Scholar]
  • 46.Kleindienst P, Brocker T. Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo. J Immunol. 2003;170(6):2817–2823. doi: 10.4049/jimmunol.170.6.2817. [DOI] [PubMed] [Google Scholar]
  • 47.Quezada SA, Fuller B, Jarvinen LZ, et al. Mechanisms of donor-specific transfusion tolerance: preemptive induction of clonal T-cell exhaustion via indirect presentation. Blood. 2003;102(5):1920–1926. doi: 10.1182/blood-2003-02-0586. [DOI] [PubMed] [Google Scholar]
  • 48.Kishimoto K, Yuan X, Auchincloss H, Sharpe AH, Mandelbrot DA, Sayegh MH. Mechanisms of action of donor-specific transfusion in inducing tolerance: Role of donor MHC molecules, donor co-stimulatory molecules, and indirect antigen presentation. J Am Soc Nephrol. 2004;15(9):2423–2428. doi: 10.1097/01.ASN.0000137883.20961.2D. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supp Fig S1-S3
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