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Original Basic Science—General

Plasmacytoid Dendritic Cell–driven Induction of Treg Is Strain Specific and Correlates With Spontaneous Acceptance of Kidney Allografts

Oh, Nicholas A. MD1,2; O’Shea, Thomas BS1,3; Ndishabandi, Dorothy K. BS1,3; Yuan, Qing MD1; Hong, Shuahuan PhD1; Gans, Jared MD1,4; Ge, Jifu MD1; Gibney, Sean BS1; Chase, Catharine BS1; Yang, Chao MD1; Rosales, Ivy MD3; Shinoda, Kazunobu MD1; Drew, Benjamin MD1; Kojima, Lisa BS1; Russell, Paul S. MD1; Madsen, Joren C. MD1,5; Colvin, Robert B. MD1,3; Alessandrini, Alessandro PhD1

Author Information
doi: 10.1097/TP.0000000000002867
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Abstract

INTRODUCTION

Mouse kidney allografts are spontaneously accepted in select donor-recipient strain combinations without any treatment.1 We have shown that DBA/2 to B6 spontaneously accepted kidney allografts developed localized aggregates of lymphocytes around the small arteries of the graft.2 These structures are shown to be consistently present in accepted kidney allografts and absent in rejected allografts.2 These structures contain prominent Foxp3+ regulatory T cells (Treg) and have hence been named Treg-rich organized lymphoid structures (TOLS). Depletion of Treg from recipients results in dissolution of TOLS and acute rejection of the graft.2

We hypothesize that dendritic cells, specifically plasmacytoid dendritic cells (pDCs), are involved in spontaneous acceptance of murine kidney allografts because of their known role in allo- and self-tolerance.3-5 In the periphery, pDCs have been shown to drive production of naturally occurring Treg, which are distinct from conventional dendritic cell–induced Treg by their greater production of interleukin (IL)-10.6 Hadeiba and Butcher7 have shown that c-c motif chemokine receptor 9 (CCR9) pDCs induce Foxp3+ Treg and prevent graft versus host disease. In addition, pDCs express Siglec-H on their cell surface and it has been shown that this endocytic receptor is capable of internalizing various proteins from the environment8 and inhibit T helper cell and antibody (Ab) responses in an antigen-specific manner.9

In this article, we show that pDCs isolated from an accepted kidney allografts not only express higher levels of Siglec-H when compared with pDCs from native kidneys, but are able to induce greater Foxp3 expression in vitro when compared with resident pDCs isolated from a native, untransplanted kidney. We further show that Foxp3 induction by pDCs in vitro correlated with strain combinations that led to kidney allograft survival. Foxp3 induction is dependent on pDC viability, immaturity, cell-cell contact, and class II but not class I differences at the H2-I-Ab locus and abrogated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) inhibition. Our findings suggest an explanation for the strain-specific spontaneous acceptance of kidney allografts observed in mice.

MATERIALS AND METHODS

Mice

The C57BL/6 (B6, H2b), DBA/2J (DBA/2, H2d), C3H/HeJ (C3H, H2k), B6.Foxp3-GFP, and B6.C-H2d/bByJ strains were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were maintained under pathogen-free conditions in filter-top cages with an automatic water system and were cared for according to methods approved by the American Association for the Accreditation of Laboratory Animal Care.

Histological and Immunopathological Features

Sagittal sections of allografts were fixed in formalin, and sections were stained for hematoxylin and eosin; Periodic acid-Schiff sections were scored according to Banff criteria. All arteries present in 2 full sagittal sections of the kidney were scored for endarteritis (mean, 16.5 ± 4.4; range, 11–25). Sections were stained for Foxp3 (JK-16S; eBiosciences, San Diego, CA) and plasmacytoid dendritic cell antigen-1 (PDCA-1) (BST2; Novus Biologicals). Light microscopic imaging was performed with a microscope (Eclipse 50i; Nikon, Tokyo, Japan) equipped with a digital camera (Spot RT KE; Diagnostics Instruments, Sterling Heights, MI). All figures from immunohistochemistry staining are representative of the tissue as a whole.

Isolation of CD4+CD25+ and CD4+CD25 T cells

CD4+CD25− and CD4+CD25+ T cells were isolated from splenocytes by magnetic cell sorting system utilizing the Mouse CD4+CD25+ Regulatory T cells Isolation Kit protocol (Miltenyi Biotec, Auburn, CA). Cells were resuspended in complete media (Roswell Park Memorial Institute [RPMI] media with 1% L-glutamine, 10% fetal bovine serum (FBS), 1% sodium pyruvate, 1% penicillin/streptomycin, 1% non–essential amino acids, and 0.65% β-mercaptoethanol).

Isolation of pDCs

pDCs were retrieved from femur and tibia bone marrow (BM) of DBA/2, B6, and C3H mice. pDCs were isolated using the Plasmacytoid Dendritic Cell Isolation Kit II by elimination of non-pDCs (negative selection) (Miltenyi Biotec). pDCs were also isolated from the kidneys of DBA/2 to B6 allogeneic allografts, B6 to B6 syngeneic allografts, and naive DBA/2 kidneys. Kidneys were procured, manually pulverized, resuspended in sterile RPMI and collagenase, and allowed to digest in a 42°C bath for 30 minutes. Cold RPMI was added afterward to stop the reaction. The solution was centrifuged and filtered to eliminate large debris. The pDCs were isolated from the resulting solution using the Plasmacytoid Dendritic Cell Isolation Kit II as used above.

In Vitro Culture of CD4+CD25 T Cells and pDC

CD4+ CD25 T cells (1.5 × 105) and 5.0 × 104 BM pDCs (3:1) were cultured in 96-well U-bottom polystyrene plates (Sigma-Aldrich Co., St Louis, MO) in the presence of recombinant IL-2 (final: 10 ng/1 mL) (R&D Systems, Minneapolis, MN) and platelet-derived transforming growth factor-β (TGF-β) (final: 10 ng/1 mL) (R&D Systems) at 37°C for 4 days. T-cell/pDC ratio and time points are considered optimal for induction (unpublished data). To analyze for proliferative changes during culture, T cells were stained with Cell Proliferation Dye eFluor 670 (eBiosciences) before culture at a 5 nmol/L concentration. After 4 days in culture, cells were analyzed for fluorescence-activated cell sorter or for in vivo studies by adoptive transfer. All groups in in vitro experiments were done in triplicate. Cell cultures for adoptive transfer were upscaled by a factor of 2 (ie, 3.0 × 105 CD4+ CD25 T cells and 1.0 × 105 BM pDCs) in the presence of doubled amount of IL-2 and TGF-β for 4 days incubation.

Flow Cytometry

Cells were collected at scheduled time points and stained with PerCP-Cy5.5-conjugated anti-CD3 (145-2C11; eBiosciences). Intracellular staining was performed utilizing the Foxp3 staining buffer set (eBiosciences) and stained with activated protein C–conjugated anti-Foxp3 (FJK-16s; eBiosciences). The purity of pDCs was confirmed by staining Ab of PerCP-eFluor 710 CCR9 (eBioCW-1.2; eBiosciences), Ab of FITC-conjugated B220 (RM4–5; BioLegend, San Diego, CA), and Ab of PE-conjugated CD317 (PDCA-1, BST2) (eBio129c eBiosciences). For negative controls, unstained cells or cells stained with each isotype-controlled mAb were utilized. All samples were analyzed on Accuri C6 FTM (BD Biosciences) or FACSverse (BD Biosciences) with FlowJo software (Tree Star). Figures from flow cytometry are representative of the triplicate samples.

Mixed Lymphocyte Reaction

CD4+CD45+ T cells were isolated from C57BL/6 mouse spleens as described above. Cells were then labeled with the Vybrant CFDA-SE Cell Tracer Kit at 1 µmol/L concentration (Life Technologies, Gaithersburg, MD). Labeled cells of 1.5 × 105 were plated in 96-well plates, cultured for 96 hours, and analyzed for carboxyfluorescein succinimidyl ester (CFSE) fluorescence by fluorescence-activated cell sorter (. For proliferation assays, cells were stimulated with anti-CD3/anti-CD28 Dynabeads (Life Technologies, Gaithersburg, MD) plated in a 1:1 ratio to labeled cells. To test whether T cells were sensitized to allogeneic antigens, allogeneic splenocytes irradiated at 2000 rads were added instead of beads at 5.0 × 105 cells/well. Labeled cells in culture media without any form of allogeneic stimulus (medium added) were utilized as a negative control. To analyze for suppressive capacity, induced Treg (iTreg) cultured as described above were added to mixed lymphocyte reactions (MLRs) at the time of labeled cell plating. Briefly, cell cultures containing iTreg, CD4+CD25 T cells, and allogeneic pDCs were washed twice with complete media (to remove cytokines from culture). They were resuspended in complete media and transferred to wells containing CFDA-labeled splenic B6 CD4+CD25 T cells. Suppressive capacity was determined as diminished proliferative response to anti-CD3/anti-CD28 stimulus or allogeneic irradiated splenocyte stimulus. Active/inactive kinase inhibitors (1 µmol/L) were added at the start of the in vitro induction assay. MEK inhibitors, U0126 (active) and U0124 (inactive), Glycogen synthase kinase-3-beta (GSK-3B) inhibitors BIO (active) and MeBIO (inactive), and the NFκB inhibitor, IT901, were purchased from Techne Corporation (Minneapolis, MN).

Transwell Assay

BM pDCs (5.0 × 104) were plated on a 6.5 mm transwell insert. Membrane pore size was sufficiently small to impede pDC migration. CD4+ CD25 T cells (1.5 × 105) were plated in the bottom of each well of a 24-well plate and filled with 1 mL of growth media with recombinant IL-2 (final: 10 ng/1 mL) (R&D Systems) and platelet-derived TGF-β (final: 10 ng/1 mL) (R&D Systems). The transwell inserts were placed, and the assay was incubated at 37°C for 4 days. After incubation, flow cytometry analysis was performed as detailed above.

Adoptive Cell Transfer

Cell cultures from 2 groups (DBA pDCS + B6 CD4+CD25 T cells, DBA pDCS + B6 CD4+CD25- T-cells + IL-2/TGF-β) were centrifuged to remove cytokines and dead cells as described above. They were resuspended in RPMI and then injected intravenously into the recipient through the tail vein or the penile vein. Mice were anesthetized before injection by Tribromoethanol. Injections occurred at 2 weeks, 1 week, or both 2 weeks and 1 week before heterotopic heart transplant.

Study Approval and Ethics Statement

All surgical procedures and pre-/postoperative care of animals were performed in accordance with methods approved by the American Association for the Accreditation of Laboratory Animals and approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.

Heterotopic Heart Transplantation

DBA/2 to B6 heterotopic murine heart transplantation was performed according to our previously described microsurgical technique.10 In brief, the aorta and pulmonary arteries of DBA/2J heart grafts were anastomosed to the B6-recipient infrarenal aorta and inferior vena cava, respectively, in an end-to-side fashion. Cold ischemic times of <25 minutes were maintained throughout. The survival of grafts was monitored daily by abdominal palpation (scoring 1–3) until the cessation of cardiac contraction.

Kidney Transplantation

Kidney transplantation was performed in the same way described in a previous report.2 In brief, kidney allografts were prepared with the cuff of aorta and inferior vena cava. Anastomoses were performed in an end-to-side manner. The ureter was anastomosed to the urinary bladder. Bilateral nephrectomy was also simultaneously performed.

Statistical Analysis

Results are given as the mean ± 95% confidence interval. Variables among groups were compared using Student t test, with P < 0.05 considered significant. Allograft survival curves were constructed by the Kaplan and Meier method, and comparisons were performed using the log-rank test. These analyses were performed with Prism ver.5 (GraphPad Software Inc., CA).

RESULTS

pDC Population and Phenotype in Normal Kidneys and Hearts

Flow cytometry of CD11c+ cells isolated from native DBA/2 kidneys contains CD11c+PDCA+B220+ cell populations which are not detectable in hearts from the same strain (Figure 1A). Similar organ differences are seen in the BALB/c strain, whose kidneys are also spontaneously accepted in B6 mice, but not hearts, long term. Normal kidney pDCs have consistently low expression of the tolerogenic marker Siglec-H,9 whereas BM pDCs are Siglec-Hhi (Figure 1B).

FIGURE 1
FIGURE 1:
Plasmacytoid dendritic cell (pDC) populations and phenotypes in normal murine kidney and hearts. A, CD11c+ cells were isolated from naive kidneys and heart from DBA/2J and BALB/cBy mice and costained for plasmacytoid dendritic cell antigen-1 (PDCA-1) and B220 and analyzed by flow cytometry. We observe the presence of pDCs in the kidney but fail to detect a resident population in the heart. B, Siglec-H expression is increased in allogeneic transplanted, murine kidney pDCs. Siglec-H levels were measured in various kidney samples using flow cytometry. Bone marrow (BM) pDCs (blue) express Siglec-H, as described in the literature. Interestingly, Siglec-H in native kidneys and syngeneic transplants have low expression of Siglec-H, whereas accepted allogeneic kidney pDCs demonstrate Siglec-H+ phenotype.

pDC Phenotype in Kidney Allografts

Immunohistochemical analysis of DBA/2 to B6 spontaneously accepted kidney allografts at 6 weeks post-transplantation reveals aggregates of lymphocytes around arteries that are rich in CD3+/Foxp3-positive cells (TOLS) (Figure 2A) and PDCA-1+ cells, a specific marker for pDCs in mice (Figure 2B). In contrast, B6 to DBA/2 transplanted kidneys, which are rejected with a survival time of 8 days (Figure 2C), had diffuse infiltration of leukocytes and lymphocytes (Figure 2D) and significantly decreased pDCs and no TOLS formation (Figure 2E). In accepted kidney allografts, pDCs are Siglec-Hhi (Figure 1B) and enriched in TOLS as early as 1 week after transplantation (Figure 2F). Costaining with Siglec-H and Foxp3 demonstrate frequent “capping” of Siglec-H between pDC and Foxp3+ cells, suggesting a cellular interaction between pDCs positive for the tolerogenic Siglec-H molecule and Foxp3+ Treg (Figure 2G). Phosphorylated ERK is present within TOLS of spontaneously accepted kidneys, suggesting the activation of MEK/ERK signaling pathways in Foxp3+ cells (Figure 2H).

FIGURE 2
FIGURE 2:
Plasmacytoid dendritic cell (pDC) phenotype in kidney allografts. A, Representative Foxp3 stain view of Treg-rich organized lymphocyte structures (TOLS) located around arterioles within spontaneously accepted DBA/2J to B6 kidney allografts (×200). B, Plasmacytoid dendritic cell antigen-1 (PDCA-1) stain of a separate arteriole within the same allograft showing pDCs within the TOLS (×200). C, Kidney allografts are spontaneously accepted in a DBA/2J to B6 strain combination while rejected in a B6 to DBA/2J strain combination. D, Representative hematoxylin and eosin stain view of rejected B6 to DBA/2J kidney allografts on posttransplant day 9. Rejected allografts demonstrate an increase in polymorphonuclear leukocytes and lymphocytes consistent with acute rejection (×400). E, PDCA-1 stain view of the same allograft demonstrating significantly decreased pDCs, observed in all rejecting allografts (×400). F, Siglec-H (arrows) staining within TOLS of accepted DBA/2 kidney allografts at 1 and 6 wk post-transplantation (×400). G, Siglec-H (brown) and Foxp3 (blue) costaining of an accepted DBA/2 allograft kidney 53 d post-transplantation shows a number of Siglec-H+ cells proximate to Foxp3+ cells (×400). Localized magnification of the staining is included to show clearer cell-cell proximity (arrows, ×600). H, Immunohistochemical staining of phospho-ERK reveals the presence of phosphorylated ERK proteins in cells making up TOLS in spontaneously accepted kidneys indicating an activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways (×400). I, Isolated CD45+CD11c+Siglec-H+PDCA-1+ cells from accepted DBA/2J to B6 kidney allografts, posttransplant wk 1 and 6. We observed an increase in the population of I-Ab+ pDCs from wk 1 to 6, whereas the population of I-Ad+ pDCs in the same allograft was unchanged. J, Further analysis of pDCs from accepted DBA/2J to B6 kidney allografts demonstrates a population of I-Ab+ pDCs expressing both I-Ab and I-Ad on posttransplant wk 6. Tx, transplantation.

At 1 week post-transplant, both donor and recipient pDCs are found in the allograft, as judged by single staining for either I-Ad or I-Ab. An average of 10% was donor derived and 47% recipient derived (Figure 2I). At 6 weeks post-transplant, an average of 86% of the pDCs were from the recipient (Figure 2I). The majority, if not all, of the pDCs that displayed I-Ad (donor) were positive for Ab (recipient) (Figure 2J) suggesting “cross-dressing” of recipient pDC. No detectable cross-dressing was observed at 1 week (Figure 2J).

Functional Activity of pDCs From Accepted Kidney Allografts

pDCs isolated from native DBA/2 kidneys, syngeneic B6 kidneys, and accepted DBA/2 allografts from B6 recipients on days 31 and 52 post-transplant were tested for their ability to induce Foxp3+ expression in vitro in CD4+CD25 T cells. pDCs from accepted DBA/2 kidneys induced more T cells expressing Foxp3 than pDC from normal DBA/2 kidneys or syngeneic B6 kidneys (mean, 3.36% ± 0.07% versus 1.09% ± 0.23% versus 0.80% ± 0.09%, respectively) (Figure 3). There was no significant difference in Foxp3 induction by pDCs from accepted posttransplants days 31 and 52 (3.35% ± 0.43% versus 3.36% ± 0.07%, respectively) (Figure 3).

FIGURE 3
FIGURE 3:
Functional activity of plasmacytoid dendritic cells (pDCs) from accepted kidney allografts. Isolated pDCs from naive DBA/2 kidney, syngeneic B6 to B6 kidney, and spontaneously accepted DBA/2 to B6 kidney were cocultured with B6 T CD4+CD25 T cells and interleukin (IL)-2/transforming growth factor (TGF)-β. After a 4-d incubation period, Foxp3 expression was induced in naive B6 T cells in greater levels by pDCs from spontaneously accepted allografts when compared with pDCs from naive and syngeneic kidney. There was no difference between pDCs isolated from allografts at different posttransplant days.

Strain-specific Differences in Foxp3 Induction Potency

We hypothesized that pDCs from donor mouse strains whose kidneys were spontaneously accepted would be more effective in promoting Foxp3 induction than strains whose kidneys are rejected. pDCs were isolated from BM to maximize the number of conditions we could include in each assay. BM-derived pDCs derived from naive DBA/2 mice were positive for pDC markers PDCA-1 and B220 as well as the pDC immaturity marker CCR9 (Figure 4A).4 When cocultured with CD4+CD25 naive B6 T cells in the presence of IL-2 and TGF-β, these cells induced substantial Foxp3 expression, as measured by the percent of intracellular staining for Foxp3 among all CD3+ cells (Figure 4B and C). This expression was greater than that in the absence of pDCs (mean, 27.2% versus 1.75%; P < 0.0001) or in cells cocultured with syngeneic pDCs (mean, 2.53%; P < 0.0001) (Figure 4B and C). In contrast, BM-derived pDCs from B6 mice were unable to induce Foxp3 expression in DBA/2 naive T cells when cocultured for 4 days with IL-2 and TGF-β (Figure 4D), and although BM-derived pDCs from C3H mice could induce Treg in a dose-dependent manner, DBA/2 BM pDCs induced significantly more Foxp3+ cells than C3H at any cell ratio (P < 0.01) (Figure 4E). For instance, DBA/2 BM pDCs at a pDC:T-cell culture ratio of 1:15 induced significantly greater Foxp3 expression than C3H BM pDCs at a pDC:T-cell ratio of 1:3 (20% versus 13%, respectively, P < 0.02). Thus, the ability of BM-derived pDC to induce Foxp3+ varied with strain. The pDC potency correlated with the strain whose kidney allografts were spontaneously accepted (Figure 4E).

FIGURE 4
FIGURE 4:
Strain-specific differences in Foxp3+ induction. A, Fluorescence-activated cell sorter (FACS) analysis of DBA/2 bone marrow (BM) plasmacytoid dendritic cells (pDCs) isolated by magnetic bead separation. Cells were gated on FSC vs SSC to exclude dead cell debris. DBA/2 BM pDCs were B220, PDCA-1, and CCR9 positive but CD103 negative. B, After 4 d of culture with IL-2 and TGF-β, splenic-derived CD4+CD25 B6 T cells show little Foxp3 expression in CD3+ cells. Including BM-derived DBA/2 pDCs in the in vitro culture significantly increased Foxp3 expression compared with T cells cultured with IL-2 and TGF-β (25.6% vs 1.65%, P < 0.001) even with the addition of syngeneic B6 BM pDCs (mean, 25.6% vs 2.86%; P < 0.001). Induction was ablated by irradiation of pDCs with 2000 rads before coculture (P < 0.001). C, Foxp3 induction among CD4+CD25 B6 T-cell cultures utilizing various culture combinations. D, Foxp3+ expression among CD3+ cells after occurred in allogenic strain combinations that correlate with spontaneous kidney allograft acceptance. E, Separate 4-d cultures of 150 000 splenic-derived CD4+CD25 B6 T cells with IL-2 and TGF-β with 0 (circle), 10 000, 25 000, or 50 000 BM pDCs from DBA/2 (square) or C3H (triangle) mice (P < 0.01). The experiments were performed in triplicates and what is shown is representative of the data. FSC, forward scatter; IL, interleukin; PDCA-1, plasmacytoid dendritic cell antigen-1; SSC, side scatter; TGF, transforming growth factor.

Requirements for Foxp3 Induction by pDC

The induction of Foxp3 required the presence of viable pDCs, as irradiated DBA/2 BM pDCs lost their viability to induce Foxp3 (Figure 4B and C). Furthermore, because of the capping of Siglec-H, we tested whether cell-cell contact was necessary for induction of Foxp3. Using Transwell migration assays, we placed DBA/2 BM pDCs and B6 CD4+CD25- T cells on opposite sides of the membrane. We found that T cells were not induced to express Foxp3+ (Figure 5). Interestingly, Luminex analyses of the supernatant from the in vitro FoxP3 induction assay at day 4 revealed no detectable differences in the levels of IL-4, 6, 10, and 17a, indicating that these cytokines are unlikely to play a role in Fox3 induction by pDCs, at least at day 4 (data not shown).

FIGURE 5
FIGURE 5:
Cell-cell contact requirement for plasmacytoid dendritic cell (pDC)–mediated Foxp3 induction. Transwell experiment was performed using DBA/2 pDCs and naive B6 T cells. CD3+ cells did not increase expression of Foxp3, suggesting the requirement for cell-cell contact in pDC-mediated Foxp3 induction.

The induction of Foxp3 by pDC is dependent on MEK/ERK, GSK-3β, and NFκB pathways. Previous studies demonstrated that the MEK/ERK, GSK-3β, and NFκB signaling pathways are activated during the induction of Foxp3 expression.11-14 Therefore, we tested whether inhibition of these pathways would influence pDC ability to induce Foxp3 expression. We found that inhibition of MEK1/2 and NFκB results in the suppression of Foxp3 induction in vitro (Figure 6A). Studies have also shown that Akt activation results in the inhibition of Treg induction in peripheral T cells.15-17 One of the downstream kinases negatively regulated by Akt is GSK-3β. We next asked if direct inhibition of GSK-3β, mimicking Akt activation, would result in the suppression of Foxp3 induction. In Figure 6B, we show that treatment of the in vitro Foxp3 induction assay with BIO, a specific inhibitor of GSK-3β, results in the complete reduction of Foxp3 expression.

FIGURE 6
FIGURE 6:
Foxp3 induction by plasmacytoid dendritic cells (pDCs) dependent on MEK/ERK and NFκB pathways. A, Inhibition of MEK/ERK and NFκB signaling pathways result in the suppression of Foxp3 expression in naive T cells. U0126 (active MEK1/2 inhibitor) and U0124 (inactive counterpart control) were each used at 1 µmol/L; IT901 (inhibitor of the NFκB pathway) was used at 1 µmol/L. Data presented are representative of 2 independent experiments done in triplicates. B, Inhibition of GSK-3b signaling pathway results in the suppression of Foxp3 expression in naive T cells. BIO (active GSK-3b inhibitor) and Me-BIO (inactive counterpart control) were each used at 1 µmol/L. Data presented are representative of 2 independent experiments done in triplicates. GSK-3b, Glycogen synthase kinase-3-beta; IL, interleukin; MEK/ERK, Mitogen-activated protein kinase/Extracellular signal-regulated kinase; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; pDC, plasmacytoid dendritic cell; TGF, transforming growth factor.

Maturation of pDCs inhibits their ability to induce Foxp3. To test whether in vitro maturation of pDCs could prevent Foxp3 induction, pDCs were treated for 24 hours with toll-like receptor 9 agonist CpG ODN 1668 (Invivogen, San Diego, CA) and then cultured together with B6 CD4+CD25 T cells and IL-2/TGF-β for 4 days. Culturing with CpG stimulated these immature pDCs to upregulate expression of the maturity markers CD80 and CD86: from 0.25% to 2.92% for CD80 and from 4.45% to 20.6% for CD86 (Figure 7A). Compared with untreated fresh and 24-hour untreated BM pDCs, CpG-treated pDCs failed to induce Foxp3 (mean, 1.3% Foxp3+ for CpG pDCs treated versus 15.6%, for untreated 24-h pDCs; P < 0.001) (Figure 7B).

FIGURE 7
FIGURE 7:
Immaturity requirement for plasmacytoid dendritic cell (pDC)–mediated Foxp3 induction. A, DBA/2 bone marrow (BM) pDCs were cocultured for 24 h with and without CpG oligodeoxynucleotide and isolated by column separation. Cells were analyzed by fluorescence-activated cell sorter (FACS), gated on forward scatter (FSC) vs side scatter (SSC) to exclude dead cell debris and stained for CD80 and CD86. DBA/2 BM pDCs cocultured with CpG for 24 h were 20.6% CD86+ compared with 4.5% without CpG. There was no significant difference in levels of CD80 positivity between BM pDCs cocultured 24 h with and without CpG. B, DBA/2 BM pDCs cocultured with CpG for 24 h failed to induce Foxp3 expression in T cells in the presence of interleukin (IL)-2/transforming growth factor (TGF)-β compared with 24 h aged BM pDCs (P < 0.01).

Class II, but not class I, alloantigens are sufficient to promote pDC-mediated Foxp3 induction in vitro. Because syngeneic pDCs induced little or no Foxp3 induction, we postulated that alloantigens promoted Foxp3 expression and sought to identify the nature of the allogeneic stimulus. We first tested whether a major histocompatibility complex (MHC) disparity was sufficient, using B6.C (H2d/bByJ [H2d]) pDCs that differ from B6 mice at the MHC locus but not at the minor antigen level. B6.C pDCs induced Foxp3 in B6 CD4+CD25 naive T cells equally to DBA/2 pDCs (mean, 15.5% versus 15.5%; P = 0.97) (Figure 8A). Irradiated pDCs of B6.C H2d/bByJ lost the ability to upregulate Foxp3 expression, thus suggesting the importance of MHC in this induction (data not shown).

FIGURE 8
FIGURE 8:
Class II alloantigens are sufficient to promote plasmacytoid dendritic cell (pDC)–mediated Foxp3 induction. A, B6.C-H2d/bByJ bone marrow (BM)–derived pDCs (congenic with B6 mice except for the expression of H2d) induced Foxp3 expression to a similar level as DBA/2J BM–derived pDCs (mean, 19.8% Foxp3+ vs 16.6%; P = 0.10). DBA/2 BM pDCs had significantly reduced capacity to induce Foxp3 among B6.C-H2d/bByJ T cells compared with B6 T cells (16.6% vs 4.89%, P < 0.001). Error bars represent 95% confidence intervals. B, bm12 BM–derived pDCs (congenic with B6 except for MHC class II disparity) induced a similar induction of CD3+Foxp3+ cells (gated on the CD4+ population of cells) as DBA/2 BM pDCs when cocultured for 4 d with naive B6 T cells and interleukin (IL)-2/transforming growth factor (TGF)-β. C, bm1 pDCs (congenic with B6 except for major histocompatibility complex class I disparity) induced little expression of Foxp3, similar to the syngeneic pDC/naive T-cell culture. D, BM pDCs from B6.NOD-(D17Mit21-D17Mit10)LtJ (which express H2d except for a g7 mutation at the Ab locus of the MHC class II molecule) induce less CD4+CD3+Foxp3+ cells than H2d DBA/2 BM pDCs when cocultured with naive B6 T cells and IL-2/TGF-β.

To separate the effects of class I or class II disparities, pDCs from bm1 (H2bm1) and bm12 (H2bm12) mice were used. bm12 mice differ from B6 in genetics at only the H2-I-Ab locus of their MHC class II molecules, whereas bm1 mice differ from B6 in their class I genetics. pDCs from bm12 mice induced a relatively similar CD4+CD3+Foxp3+ Treg population as DBA/2J pDCs (mean 15.0% versus 18.4%) (Figure 8B), whereas pDCs from bm1 were unable to induce CD3/Foxp3 positivity from CD4+ naive T cells (mean, 0.8% versus 16.1%) (Figure 8C). These data suggest that a mismatched MHC class II, but not class I, is necessary for pDCs ability to produce Treg from naive B6 T cells. To further investigate the role of the MHC class II mismatching, we used pDCs from B6.NOD-(D17Mit21-D17Mit10)/LtJ (NOD) mice which have H2d MHC class II genetics at all loci, except for a g7 mutation at the H2-I-Ab locus. Coculturing BM B6.NOD pDCs with CD4+ naive B6 T cells with IL-2 and TGF-β shows that B6.NOD pDCs induced a smaller CD4+ CD3+ Foxp3+ population to that observed with fully H2d DBA/2 BM pDCs (Figure 8D). This suggests that certain mismatching MHC class II molecules, specifically at the H2-I-Ab locus, can alter pDCs ability to induce Treg in vitro. Thus, MHC-II but not MHC-I alloantigens are sufficient for in vitro pDC Foxp3 induction.

In Vitro Function of Foxp3+ Cells Induced by pDC

To assess the function of Foxp3+ T cells induced by DBA/2 pDCs, B6 CD4+Foxp3+ cells, induced by coculture with DBA/2 BM pDCs in the presence of IL-2/TGF-β, were tested for their ability to inhibit proliferation of T cells from sensitized recipients (B6 recipients that rejected DBA/2 skin or heart allografts). Coculture of T cells from sensitized recipients activated with either irradiated DBA/2 splenocytes or anti-CD3/anti-CD28 beads resulted in the proliferation of T cells. Addition of Foxp3+ T cells induced by DBA/2 BM pDCs significantly suppressed proliferation caused either by DBA/2 splenocytes (mean, 53.7% versus 8.01% proliferating cells; P < 0.001) or anti-CD3/anti-CD28 beads (mean, 95.4% versus 34.0% proliferating cells; P < 0.002) (Figure 9A and B). This suppression was significantly greater than that seen in controls, including cultures of pDCs with T cells, pDCs with cytokines, and T cells with cytokines (Figure 9A and B).

FIGURE 9
FIGURE 9:
Functionality of Foxp3+ cells induced by plasmacytoid dendritic cells (pDCs) in vitro. A, carboxyfluorescein succinimidyl ester (CFSE) staining of CD4+CD25 responders showing proliferation as measured by CFSE dilution after 4 d in the presence of only medium added or DBA/2 pDCs, B6 T CD4+CD25 T cells and interleukin (IL)-2/transforming growth factor (TGF)-β from a previous 4-d culture. Suppression occurred via the pDC/regulatory T cell (Treg) induction coculture in response to stimulus including medium (no stimulus), anti-CD3/anti-CD28 beads, and irradiated DBA/2 splenocytes. Histograms represent the percentage of proliferating CFSE stained responders after 4 d after stimulus with medium, anti-CD3/anti-CD28 beads, or irradiated DBA/2 splenocytes. Suppression did not occur with addition of other 4-d cultures, including B6 T CD4+CD25 T cells cultured with DBA/2 pDCs and DBA/2 pDCs cultured with IL-2/TGF-β. Similar suppression occurred with freshly isolated splenic-derived CD4+CD25+ T cells cultured in a 1:1 T cell Responders:Tcell suppressor (Treg) ratio. B, Bar plots represent proliferating cell populations from the suppression assay including conditions of pDCs with T cells, pDCs with cytokines, and T cells with cytokine. BM, bone marrow.

In Vivo Activity of Foxp3+ Cells Induced by pDC

To assess for the in vivo suppressive capacity of pDC-induced Foxp3+ cells, we transferred 1.0 × 106 cells after culturing DBA BM pDCs and B6 CD4+CD25 T cells in the presence IL-2/TGF-β for 4 days in vitro into B6 mice at various times before DBA to B6 heterotopic heart transplantation. Intravenous injection of these cells, after IL-2/TGF-β were washed away, significantly prolonged the survival of heterotopic DBA cardiac allografts as assessed by palpation score (n = 7, median survival time [MST] = 11, P < 0.001) (Figure 10). Cardiac allografts had an MST of 6 days post-transplant without any cell injection (n = 6). Transfer of cells that were incubated in vitro for 4 days without the addition of IL-2/TGF-β led to no significant prolongation (n = 3, MST = 7, P = 0.37). Prolongation also occurred with Foxp3+ cell injection at 1 week before transplantation (n = 3, MST = 13, P < 0.001). Two injections of cultured cells containing iTreg at 2 weeks and 1 week before transplant prolonged allograft survival by 17 days (n = 4, MST = 24, P < 0.001).

FIGURE 10
FIGURE 10:
Activity of plasmacytoid dendritic cell (pDC) induced Foxp3+ cells in vivo. No cell injection lead to rejection around 6 d after transplant. Injecting cultures (DBA pDCs + B6 CD4+CD25 T cells) where cytokines were not added, and thus, there was no Foxp3+ induction lead to no prolongation (P = 0.37). Injecting cell cultures (DBA pDCs + B6 CD4+CD25 T cells + interleukin [IL]-2/transforming growth factor [TGF]-β) producing Foxp3+ cells prolong allograft survival by a mean of 5 d (median survival time [MST], 11 d) at 2 wk before injection (P < 0.001). Prolongation also occurred with injection at 1 wk before transplantation (P < 0.001). Injecting cells 2 and 1 wk before transplant prolong allograft survival by 17 d (P < 0.001).

DISCUSSION

Regulatory or tolerogenic dendritic cells, which include pDCs, have been shown to prolong allograft survival in murine and nonhuman primate models, but the mechanism of these therapies remains unknown.18,19 Meanwhile, Foxp3+ Treg are widely known to suppress alloimmune reactions in vitro and in vivo and hence are of specific interest in cellular therapy.20-22 We sought evidence to test our hypothesis that pDCs are responsible for the expansion of Foxp3+ Treg in TOLS in vivo and that these Treg mediate spontaneous acceptance of some kidney allografts. First, we demonstrated the presence of pDCs in murine kidneys, and not hearts, which corroborates our observation of spontaneous acceptance in kidney allografts and rejection in heart allografts. We showed that pDCs and Foxp3+ Treg, which are both tolerogenic immune cell types, coexist in TOLS of spontaneously accepted kidney allografts. Immunohistochemistry and flow cytometric analyses show that unlike pDCs from native kidneys, pDCs isolated from spontaneously accepted kidneys allografts are Siglec-Hhi and specifically found in TOLS. The increased expression of Siglec-H is consistent with its ability to contribute to tolerance, as previously described.9,23 However, it is unclear the role that increased expression of Siglec-H plays in the antigen presentation process of pDCs and necessity for Treg induction is undetermined in this study and will be the subject of future experiments. Furthermore, pDCs isolated from accepted DBA/2 kidney allografts were able to induce greater Foxp3 expression in naive B6 T cells in vitro than pDCs isolated from native kidneys. From these data, we postulated that pDCs from certain mouse strains would be better in driving Treg induction and those strains would correlate with the spontaneous acceptance of kidney allografts. Using BM-derived pDCs, we set up an in vitro assay to assess whether the relative induction of Treg by pDCs from various strains corresponded to strain combinations that have been previously reported to manifest spontaneous kidney allograft acceptance.1,24 For example, significantly greater expansion of B6 Foxp3+ Treg was caused by DBA/2 pDCs (H2d) compared with C3H (H2k) pDCs; DBA/2 kidneys are accepted by B6, while C3H are not. This result is compatible with the hypothesis that pDCs are relevant to local Foxp3 expansion in the graft. Foxp3+ induction in naive B6 T cells by pDCs was only observed between allogeneic strains; isogeneic pDCs were inactive. We show that the induction of Treg in vitro by pDCs is dependent on the activation of MEK/ERK, GSK-3β, and NFκB signaling pathways. While we acknowledge that BM pDCs may behave differently when isolated from kidney pDCs from accepted allografts, we found that they both display pDC markers Siglec-H and PDCA-1, which may indicate a shared phenotype. Further, BM pDCs provide a larger yield that is required for our in vitro experiments.

MHC alloantigen was sufficient for Foxp3+ induction as shown by the ability of H2d congenic B6.C H2d/bByJ BM pDCs to induce Foxp3 expression in B6 CD4+CD25 T cells. Foxp3+ induction was observed with only class II disparity (bm12 pDC and B6 T cells) and not class I disparity (bm1 pDC and B6 T cells). The reduced in vitro induction of Foxp3 by B6.NOD pDCs, which are H2d except for a g7 mutation at the H2-I-Ab locus, demonstrates the importance of the H2-I-Ab locus. This result is not unexpected because the responding T cells are CD4+ and recognize class II antigens. These results do not rule out other triggers for Foxp3 induction (other class I disparities or non-MHC differences); however, they do show that a single class II antigen difference is sufficient. We further show that these data support the conclusions from our previous studies that the ability of pDCs to induce Foxp3+ cells is predominantly an allogeneic response and strain dependent (Table 1).

TABLE 1
TABLE 1:
Donor/Recipient strain combinations and formation of TOLS

We report here that pDCs immaturity is critical in their ability to induce Treg. There has been controversy about whether immature or mature pDCs carry a tolerogenic potential. Previous ex vivo induction experiments utilizing splenic murine or peripherally derived human pDCs have utilized CpG- or lipopolysacchride-matured (ie, increased CD80/86 expression) pDCs to produce CD4+CD25+ Foxp3+ Treg from CD4+CD25 T cells an indoleamine 2,3 dioxygenase (IDO)- and contact-dependent mechanism.25-27 IDO is additionally implicated in pDC-induced Treg production in the setting of tumors and HIV infection.28,29 Importantly, IDO expression is crucial in spontaneous murine kidney allograft acceptance.30 The present study clearly demonstrates that CpG-matured BM pDCs failed to induce Foxp3 expression achieved by utilizing freshly isolated or unmanipulated, 24-hour untreated BM-derived, CD80/86 low, CCR9+ pDCs thus implying immaturity. Irradiation may have eliminated expression of IDO among BM pDCs and thus prevented induction. It may also have deterred other contact-dependent methods of pDC-mediated Treg induction, such as stimulation with inducible T-cell costimulator-ligand expression.3,31 The differences between these findings and previously published works may be associated with the importance of the specific MHC mismatching which appears to control induction in this setting.

This study demonstrates that the level of in vitro induction of functional Treg by pDCs is strain specific and dependent on allogeneic differences. These findings parallel the spontaneous acceptance of kidney allografts that we observe in our mouse transplant models and may explain known organ-specific and strain-specific differences in tolerance induction. While increased pDC and FoxP3+ T-cell populations in TOLS of spontaneously accepted kidneys suggest that pDCs play an important role in this process, we have not yet established direct evidence of in vivo FoxP3+ induction in T cells by pDCs. However, our findings have laid the groundwork for future studies that aim to elucidate the mechanisms of Foxp3+ T-cell induction by pDCs in vivo. Furthermore, optimizing and stabilizing pDC-mediated Treg induction both in vitro and in vivo may provide a future cellular therapy for allograft acceptance.

ACKNOWLEDGMENTS

We would like to thank Dr A. B. Cosimi for his invaluable input regarding the manuscript.

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