SEARCH

SEARCH BY CITATION

Keywords:

  • Depletion of Foxp3+ Tregs;
  • DEREG mice;
  • donor alloantigen-specific tolerance;
  • kidney allograft infiltrating Foxp3+ Tregs;
  • spontaneous kidney allograft tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Foxp3+ regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3+ Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3+ Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H-2d) to C57BL/6 (H-2b) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3+ Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3+ Tregs (K-Tregs) expressed elevated levels of TGF-β, IL-10, interferon gamma (IFN-γ), the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3). These K-Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen-specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3+ Tregs in the maintenance of spontaneously induced kidney allograft tolerance.


Abbreviations
Blimp-1

B lymphocyte-induced maturation protein-1

Ccr4

C–C chemokine receptor type 4

CXCL

chemokine ligand

Cxcr3

chemokine receptor 3

DAPI

4′,6-diamidino-2-phenylindole

DEREG

DEpletion of REGulatory T cells

DLN

draining lymph node

DT

diphtheria toxin

DTR-eGFP

diphtheria toxin receptor and enhanced green fluorescent protein

GFP

green fluorescent protein

H&E

hematoxylin and eosin

IFN-γ

interferon gamma

K-Tregs

kidney allograft infiltrating Tregs

LN

lymph node

MST

median survival time

MyD88

myeloid differentiation primary response gene 88

PAS

periodic acid-Schiff

PB

peripheral blood

PTx

posttransplantation

RFP

red fluorescent protein

SP-Tregs

Tregs from spleen of tolerant mice

Th

T helper

Tr

induced Treg

Tregs

regulatory T cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Donor-specific tolerance of solid organ allografts arises spontaneously in various animal models [1-3] and has been well documented in treated patient populations [4, 5]. A number of means have been utilized to achieve transplant tolerance, including costimulatory blockade [6, 7], mixed chimerism [8] and cell therapeutic strategies including transfer of regulatory T cells (Tregs) [9]. Tregs comprise a broad range of cells but the most well-characterized population comprises the Foxp3+ Tregs (Foxp3+CD4+CD25+), T cells derived from the thymus and involved in suppression of autoimmune responses in the periphery [10]. Similar Foxp3+ Tregs can be induced in the periphery from non-Foxp3+ T cell precursors, and other subsets secreting suppressive cytokines such as TGF-β and IL-10 but not expressing Foxp3 have also been identified [11]. Transplant tolerance has been achieved using Tregs in animal models in a variety of situations [12-15]. In clinical transplantation, the functional significance of Tregs is less clear. FOXP3+ has been associated with rejection of both liver and kidney transplants [16, 17] though FOXP3+ may be induced transiently in activated T cells without regulatory function or graft infiltration with FOXP3+ Tregs may reflect a protective response that has failed to control rejection.

Recently, the role of Foxp3+ Tregs within the graft has been studied more closely and experiments have demonstrated that T effectors in skin transplants are suppressed by Tregs within the graft [18]. Further, naïve T cells in proximity to the graft acquire Foxp3, suggesting induction of Tregs by the graft [18]. These studies used hCD2-Foxp3 transgenic mice to deplete Foxp3+ Tregs because of concerns regarding systemic autoimmunity with the DEpletion of REGulatory T cells (DEREG) mice, which carry the diphtheria toxin receptor and enhanced green fluorescent protein (DTR-eGFP) transgene under Foxp3 promoter. However, the strain of DEREG mice used in the present studies develops autoimmunity only when Foxp3+ Tregs are depleted in neonatal mice and not in adult mice [19]. We have previously reported that spontaneous kidney transplant tolerance in the C56BL/6 to B10BR model was associated with increased Foxp3+ Tregs expression in kidney allografts [20]. In kidney transplants in the DBA/2 to C57BL/6 tolerance model, early depletion of Tregs leads to graft loss [21]. Moreover, tolerance observed in the BALB/c to C57BL/6 strain combination when inflammation is restricted by the absence of the myeloid differentiation primary response gene 88 (MyD88) signaling is dependent upon Tregs [22]. Although increased numbers of infiltrating Foxp3+ Tregs are observed in spontaneous murine kidney transplant tolerance in the C56BL/6 to B10BR model [20] and DBA/2 to C57BL/6 model [21], the functional significance of this Treg infiltrates as distinct from that of Tregs within the lymph node (LN) and its ability to mediate donor alloantigen-specific tolerance have not been determined. Similar expansion of graft infiltrating FOXP3+ Tregs accompanies clinical kidney and liver allograft rejection rather than graft acceptance [16, 17] and the apparent contradiction between the simultaneous presence of FOXP3+ Tregs within the graft in both rejection and tolerance can only be resolved with further functional and mechanistic studies.

This study evaluates the function of Foxp3+ Tregs in a model of spontaneous mouse kidney allograft tolerance (DBA/2 [H-2d] to C57BL/6 [H-2b]) and characterizes the tissue-specific Tregs, including assessment of their role in maintaining kidney allograft tolerance, and their ability to transfer dominant tolerance to donor strain skin allografts and to maintain memory to donor-specific allograft in vivo while concurrent third-party allografts are rejected.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Animals

C57BL/6, DBA/2, Rag−/− (H-2b), B10BR (H-2k), CBA (H-2k) and C3H/He5 (H-2k) mice were obtained from the Animal Resource Centre (Perth, Australia). Breeding pairs of Foxp3RFP(red fluorescent protein), Foxp3GFP(green fluorescent protein) and DEREG mice (all on C57BL/6 background) were provided by Professor Richard Flavell (Yale University, CT), Professor Alex Rudensky (University of Washington, WA) and Professor Tim Sparwasser (Hannover Medical School, Germany), respectively. Male mice aged 10–14 weeks were used in all experiments. Experiments were conducted using established guidelines for animal care and approved by the Animal Ethics Committee of Western Sydney Local Health District.

Kidney transplantation

DBA/2 mouse kidneys were transplanted into C57BL/6 background mice as previously reported [20]. Briefly, the donor kidney was removed and the ureter was dissected close to the bladder. After removal of the left recipient kidney, the aorta and vena cava of the donor kidney were anastomosed end-to-side with the recipient aorta and vena cava. The end of the donor ureter was tied and implanted into the recipient bladder by pulling the suture through the back wall to the front. The ureter was fixed at the ureter implant site by suturing periureteral tissue to the back wall of the bladder. Excess ureter was cut at the anterior surface of the bladder, and after retraction of the ureter, the defect was closed. The remaining native kidney was removed at 4 days posttransplantation (PTx). Kidney transplantation was performed on three groups on the C57BL/6 background mice (C57BL/6 [n = 15], Foxp3RFP [n = 10], Foxp3GFP [n = 11], unmanipulated DEREG [n = 3]), DEREG mice with diphtheria toxin (DT; n = 8) and Foxp3RFP mice with DT (n = 7). Kidney graft rejection was indicated by significant morbidity and death and confirmed by histology/elevated serum creatinine [20].

Skin transplantation

Full-thickness tail skin grafts from DBA/2, Foxp3GFP and B10BR, CBA and C3H/He5 mice were placed on graft beds prepared on Rag−/− mice 2 days after adoptive transfer of kidney allograft infiltrating Foxp3 Tregs (K-Tregs) or control Tregs. Grafts were covered with protective bandages for 7 days. Rejection and acceptance were assessed as previously described [22-24].

Depletion of Tregs in DEREG mice

DEREG mice, which carry a DTR-eGFP transgene under the control of an additional Foxp3 promoter, were used for specific depletion of Tregs [25]. DEREG and Foxp3GFP mice received 8 ng/g DT (Calbiochem, Darmstadt, Germany) intraperitoneally for 3 days prior to transplant. DT also was injected into DEREG at 8, 20 and 50 ng/g without further transplantation. Flow cytometry was employed to assess the depletion of Tregs (GFP+) in peripheral blood (PB) of DEREG mice at Day 4 after DT administration by quantitation of the GFP+ proportion of total CD4+ T cells.

Assessment of kidney function

Serum creatinine was measured using the VITROS 250/350/950 System (Ortho-Clinical Diagnostics, Rochester, NY) by the Clinical Biochemistry Department of the Children's Hospital at Westmead, Sydney, Australia.

Isolation of allograft infiltrating cells

Kidney allografts were recovered from Foxp3RFP and Foxp3GFP recipients at Days 7, 14 and ≥69 PTx. Then, allograft infiltrating lymphocytes were isolated by Percoll density gradient centrifugation (Sigma–Aldrich, Castle Hill, NSW, Australia) after perfusion as reported previously [22].

Cell sorting and flow-cytometric analysis

K-Tregs were sorted from allograft infiltrating lymphocytes by flow cytometry (FACS Vantage-Diva; BD Biosciences, San Jose, CA) based on expression of CD4, the fluorescent Foxp3 fusion protein and the congenic marker CD45.2 as described [26], using an LSRII-cytometer (BD Biosciences) and Diva software (BD Biosciences). Data were analyzed using FlowJo 7.6.2 (TreeStar Inc., Ashland, Oregon). PB, spleen, LN, draining lymph node (DLN) and allograft infiltrating lymphocytes from transplanted or control mice were collected at specific time points for flow-cytometric analysis [26]. Antibodies comprised of fluorescein isothiocyanate-conjugated, phycoerythrin-conjugated and pacific-blue-conjugated anti-mouse CD4, allophycocyanin-conjugated anti-mouse CD25, allophycocyanin-conjugated anti-mouse CD45.1, phycoerythrin-conjugated anti-mouse CD45.2 (BD Biosciences) and phycoerythrin-conjugated anti-mouse Foxp3 (eBioscience, San Diego, CA).

Adoptive transfer of K-Tregs and challenge with effector T cells

Sorted K-Tregs (CD4+GFP+; CD45.2+) from each individual kidney allograft (1.2 × 104–3.2 × 104) and sorted spleen Tregs from the tolerant mice (SP-Tregs; 1.2 × 104–3.2 × 104) at Day 14 PTx were adoptively transferred intravenously into individual Rag−/− mice. Skin transplantation was performed on Rag−/− mice at Day 2 after adoptive transfer. At Day >100 PTx, these mice were challenged with 1 × 105 effector T cells (CD4+GFP CD45.1/2+). Flow cytometry confirmed the presence of both Foxp3+ Tregs and effector T cells.

Histological examination of grafts

Kidney and skin grafts were fixed in 10% formalin and embedded in paraffin before staining with periodic acid-Schiff (PAS) or hematoxylin and eosin (H&E) [22, 26]. PAS staining was performed on 5-mm paraffin kidney sections to assess tubulitis, glomerulosclerosis and interstitial fibrosis [22]. Primary anti-mouse CD3 (17A2; eBioscience) was used for staining kidney allografts for infiltration by T cells, as described previously [27]. H&E was performed to confirm the skin graft rejection and acceptance [22, 23].

Fluorescence imaging of grafts

OCT Tissue Tek–embedded frozen sections (Sakura Finetek, Koto-ku, Tokyo, Japan) were used for enumeration of kidney K-Tregs. For detection of Foxp3-GFP+ Tregs, nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Life Technologies, Mulgrave, Victoria, Australia) [28] was combined with direct detection of GFP.

Cytokine cytometric bead array

The BD cytometric bead array mouse Th1/Th2/Th17 cytokine kit was used to detect IL-2, IL-4, IL-6, interferon gamma (IFN-γ), TNF, IL-17A and IL-10 serum protein by BD FACS Canto-II following the manufacturer's instructions (BD Bioscience). Data were analyzed by BD FCAP Array (BD Bioscience) [29].

Real time reverse transcription polymerase chain reaction

The expression of IL-10, TGF-β, IFN-γ, transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3) on sorted K-Tregs from Days 7–14 and ≥69 PTx mice was determined by TaqMan® Gene Expression Assay (Applied Biosystems, Invitrogen, Life Technologies, Mulgrave, Victoria, Australia) as described [26, 29].

Statistical analysis

The log-rank test was employed for comparison of survival data between groups. Analysis of the differences between multiple groups (Figure 4G and I) was performed using two-tailed Student's t-test with a Bonferroni correction for multiple analyses; p < 0.05 was considered significant for single comparisons, p < 0.025 for two comparisons, etc. Comparison of multiple groups for all other parameters measured utilized the Kruskal–Wallis one-way analysis of variance test. p < 0.05 was considered statistically significant. Data are represented as mean ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Spontaneous acceptance of kidney allografts in the DBA/2 to C57BL/6 strain is dependent upon Foxp3+ Tregs

Spontaneous acceptance of kidney allografts in the DBA/2 to C57BL/6 strain combination was confirmed (Figure 1). To assess the role of Tregs, DEREG mice were used for specific depletion of Tregs [25]. DT was given to DEREG mice for 3 days prior to transplant to delete Foxp3+ Tregs (Figure 1A). Treg-depleted DEREG mice rejected DBA/2 kidney allografts with a median survival time (MST) of 6.5 days (Figure 1B). In contrast, C57BL/6 background mice including C57BL/6, Foxp3RFP, Foxp3GFP and unmanipulated DEREG mice showed long-term tolerance with an MST >100 days (p < 0.0001) for DBA kidney allografts. Further, the control Foxp3GFP group that received DT administration showed long-term survival of DBA/2 allografts with MST >82 days (p < 0.001; Figure 1B), indicating that graft loss in the DEREG mice receiving DT was due to specific Treg deletion and not to a nonspecific effect of the toxin.

image

Figure 1. Kidney allograft tolerance is Foxp3+ Treg dependent. (A) Tregs (CD4+GFP+) were depleted from 10.1% to 0.6% of CD4+ cells in PB following treatment of DEREG mice with DT. (B) Specific deletion of Foxp3+ Tregs in DEREG mice leads to loss of tolerance in DBA/2 to C57BL/6 model. C57BL/6 background (C57BL/6/RFP/GFP) mice including C57BL/6 (n = 15) and Foxp3RFP (n = 10), Foxp3GFP (n = 11) and unmanipulated DEREG (n = 3) mice showed long-term tolerance to DBA/2 allografts with MST > 100 days (diamond; ***p < 0.0001). Following depletion of Foxp3+ Tregs, DEREG mice (n = 8) rejected DBA/2 allografts with MST = 6.5 days (squares). Control Foxp3GFP after DT injection (n = 7) showed long-term survival of DBA/2 allografts with MST > 82 days (triangle; **p < 0.001). (C–H) Histology of allografts (PAS). Rejecting (C, D and E) and tolerant kidney allografts (F, G and H) at Day 7 PTx. The kidney allografts were collected after perfusion. (I and J) GFP+ Tregs (green) are present in tolerant allografts but not in rejecting allografts at Day 7 PTx (blue DAPI nuclear counterstain). (K) Immunohistochemical staining of CD3+ T cells in rejecting kidney allografts and (L) tolerant kidney allograft at Day 7 PTx. DAPI, 40,6-diamidino-2-phenylindole; DEREG, Depletion of REGulatory T cells; DT, diphtheria toxin; GFP, green fluorescent protein; MST, median survival time; PB, peripheral blood; PAS, periodic acid-Schiff; PTx, posttransplantation; RFP, red fluorescent protein; Treg, regulatory T cell.

Download figure to PowerPoint

Tolerant kidney allografts have normal function in vivo with localized interstitial infiltrates of mononuclear cells including Tregs

Kidney allograft histology (Day 7 PTx) from Treg-depleted DEREG mice showed acute rejection with a heavy infiltrate of mononuclear cells (Figure 1C), severe tubulitis (Figure 1D) and monocyte accumulation (Figure 1E). In contrast, tolerant kidney allografts at matched time points had localized interstitial infiltrates of mononuclear cells (Figure 1F), no tubular damage (Figure 1G) and no glomerulitis (Figure 1H). GFP+ Tregs were found in tolerant allografts at Day 7 (Figure 1J), but not in rejecting allografts (Figure 1I). Rejecting kidney allografts showed massive CD3 T cell infiltration and accumulation in glomeruli and tubules (Figure 1K). T cells were few and localized in the interstitium in the tolerant kidneys (Figure 1L). Normal kidney morphology was found in controls including nontransplanted DBA/2 naïve kidney (Figure 2A and B), DEREG host kidney at Day 7 PTx (Figure 2C and D) and Foxp3GFP host kidney at Day >100 PTx (Figure 2E and F) after administration of DT. Nontransplanted DEREG kidney at Day 43 following administration of DT at 8 ng/g (the treatment dose), and the higher doses of 20 and 40 ng/g (data not shown) also appeared normal. Histological examination of long-term tolerant kidney allografts (Day >100 PTx) yielded similar results to Day 7 (Figure 2G and H). Further, GFP+ Tregs were observed in tolerant allografts at Day >100 PTx (Figure 2J) but not in control allografts (Figure 2I). This demonstrates that loss of Tregs breaks tolerance to kidney allografts in the DBA/2 to C57BL/6 model and that Foxp3+ Tregs play a key role in kidney allograft survival.

image

Figure 2. Foxp3+ Tregs in long-term surviving tolerant allografts. (A and B) Kidney histology from controls including DBA/2 naïve kidney, (C and D) DEREG host kidney at Day 7 PTx, (E and F) Foxp3GFP host kidney at Day >100 PTx after administration of DT and (G and H) long-term tolerant kidney allografts (Day >100 PTx; H&E). (I) The fluorescent detection of GFP+ Tregs in control Foxp3GFP host kidney at Day >100 PTx after administration of DT and (J) in tolerant allografts >100 days PTx. DAPI was used as the nuclear counterstain. The renal allografts and control kidneys were collected after perfusion. DAPI, 40,6-diamidino-2-phenylindole; DEREG, Depletion of REGulatory T cells; DT, diphtheria toxin; GFP, green fluorescent protein; H&E, hematoxylin and eosin; PTx, posttransplantation; Tregs, regulatory T cells.

Download figure to PowerPoint

To evaluate kidney function in these life-supporting grafts, serum creatinine was measured in each group at sequential time points. No differences in the level of serum creatinine (20–45 µmol/L) were detected between tolerant and control groups, including normal nontransplanted C57BL/6 strain mice (C57BL/6, DEREG and Foxp3GFP), control nontransplanted DEREG mice after administration of DT and tolerant groups at various time points (Days 7–14, 45 and >100 PTx; Table 1). However, rejecting mice showed impaired kidney function with a significantly higher serum creatinine (185 µmol/L) in the rejecting group than the control normal nontransplanted mice (p < 0.01) or control transplanted mice receiving DT (p < 0.05) (Table 1).

Table 1. Assessment of kidney function by measurement of serum creatinine
GroupKidney Tx1Number of miceDays PTxSerum creatinine (µmol/L)
  • DEREG, Depletion of REGulatory T cells; DT, diphtheria toxin; GFP, green fluorescent protein; PTx, posttransplantation.

  • 1

    Transplantation.

  • 2

    Nontransplanted C57BL/6, Foxp3GFP and DEREG mice.

  • 3

    7 days after DT injection.

  • 4

    p < 0.01 with normal non-Tx mice and p < 0.05 with DEREG + DT non-Tx group.

Normal non-Tx2No9N/A22.5 ± 4.6
DEREG + DTNo47320.3 ± 2.4
Foxp3GFP + DTYes67–1428.3 ± 6.3
Foxp3GFPYes64544.8 ± 23.4
C57BL/6Yes4>10028.0 ± 4.8
Foxp3GFP + DTYes3>10042.0 ± 9.2
DEREG + DTYes56–7185 ± 32.64

Inflammatory cytokines were increased in rejection and IL-10 was increased in tolerant recipients

We assessed the serum cytokines IL-2, IL-4, IL-6, TNF, IFN-γ, IL-10 and IL-17 in each group at Days 7–14 PTx and in long-term tolerant mice at Day >100 PTx (Figure 3). IL-6 (Figure 3A), IFN-γ (Figure 3B) and TNF (Figure 3C) were significantly increased in the rejecting mice compared with the tolerant and control groups, including nontransplanted mice and nontransplanted DEREG DT administered mice (p < 0.01). TNF was increased in all transplanted mice acutely and this was greatest in the rejecting group (Figure 3C). IL-10 (Figure 3D) was increased in long-term tolerant mice (Day >100 PTx) compared with control and rejecting mice. Serum IL-2 (<10 pg/mL; Figure 3E), IL-4 (<20 pg/mL; Figure 3F) and IL-17 (<20 pg/mL; Figure 3G) showed no significant differences between control, rejecting and tolerant groups. IL-10, as a major regulatory cytokine, was evaluated further by real-time reverse transcription polymerase chain reaction in K-Tregs.

image

Figure 3. Serum level of Th1/Th2/Th17 cytokine in rejecting and tolerant mice. (A) IL-6 and (B) IFN-γ were increased in rejecting mice (n = 3) at Day 7 PTx (Rej) compared with controls (Con; n = 6) including nontransplanted normal mice and nontransplanted DEREG DT-treated mice (**p < 0.01), tolerant mice (n = 7) at the matching time (Days 7–14; Tol) and long-term tolerant mice (n = 3; >100 days; Tol-L). (C) TNF was increased in all transplanted mice (*p < 0.05), and this increase was greatest in the rejecting group (**p < 0.01). (D) IL-10 was elevated in long-term tolerant mice compared with control and rejecting mice. (E) The levels of serum cytokines IL-2 (<10 pg/mL), (F) IL-4 (<20 pg/mL) and (G) IL-17 (<40 pg/mL) were low and did not differ between controls, rejecting and tolerant groups. DEREG, Depletion of REGulatory T cells; DT, diphtheria toxin; IFN-γ, interferon gamma; PTx, posttransplantation.

Download figure to PowerPoint

Increased Tregs in kidney allografts at early time points

Although Tregs play an important role in transplantation tolerance, the role of Tregs in the kidney allograft is controversial [9]. To determine whether the localized interstitial infiltrate of mononuclear cells in kidney allografts contained Tregs, kidney allograft infiltrating lymphocytes were isolated from Foxp3RFP and Foxp3GFP recipients [22]. Flow cytometry showed that increased proportions of Tregs (CD4+RFP+ and CD4+GFP+) in the kidney allograft infiltrate both at Day 7 (p < 0.05; Figure 4A and B) and Day 14 PTx (p < 0.05; Figure 4C and D) but not at Day 69 PTx (Figure 4E) compared with splenic Tregs in nontransplanted mice. Further, the proportion of splenic Tregs was increased in transplanted mice at Day 14 PTx (p < 0.05) but not at Day 7 PTx compared with nontransplanted control mice (Figure 4B and D). This suggested initial Treg expansion or recruitment in the allograft and then later in the spleen.

image

Figure 4. The expansion of Foxp3+ Tregs in kidney allografts and DLNs. (A and B) Foxp3+ Tregs were more numerous in infiltrating cells from kidney allografts (KTx) at Day 7 PTx (CD4+RFP+/CD4+; 36.2 ± 4.7%; n = 3) compared with splenic Tregs in nontransplanted (SP/N; 20.5 ± 2.1%; n = 3; *p < 0.05) and transplanted mice (SP/Tx; 25.4 ± 2.0%; n = 3). (C and D) Foxp3+ Tregs (CD4+GFP+/CD4+) were more frequent, both among allograft-infiltrating cells (17.8 ± 1.8%; n = 5; *p < 0.05) and in the spleens of transplanted mice (18.1 ± 1.5%; n = 5; *p < 0.05) at Day 14 PTx compared with splenic Tregs of nontransplanted mice (11.5 ± 1.4%; n = 5). (E) The percentage of Foxp3+ Tregs at Day 69 PTx did not differ between kidney infiltrate, and spleen of transplanted or nontransplanted mice (n = 2). (F) Tregs (Foxp3+GFP+/CD4+) were highest in the DLN (22.3%; DLN/Tx) compared with PB (15.2%; PB/Tx) and control LN (15.1%; LN/Tx) at Day 7 PTx in transplanted Foxp3GFP mice, and PB (7.2%; PB/N) and LN (16.1%; LN/N) in a representative un-transplanted mouse (G). Tregs (Foxp3+GFP+/CD4+) were significantly higher in DLN (20.6 ± 0.9%; n = 3) than PB (9.9 ± 3.4%; **p < 0.01; n = 6) and control LN (12.9 ± 2.4%; **p < 0.01; n = 6) at Day >84 PTx, and PB (7.2 ± 1.6%; **p < 0.01; n = 6) and LN (13.9 ± 3.1%; n = 2) in nontransplanted mice. The proportion of Foxp3+GFP+/CD4+ was higher in LN of nontransplanted mice (13.9 ± 3.1%; n = 2; *p < 0.05) and LN of tolerant mice (12.9 ± 2.4%; **p < 0.01; n = 6) than PB (7.2 ± 1.6%; n = 6) of nontransplanted mice. (H and I) Tregs in PB, LN and DLN were predominantly CD25+Foxp3+ in nontransplanted mice, transplanted mice at Day 7 and >84 PTx. There were no differences in the proportion of CD25+Foxp3, Foxp3+CD25 in CD4+ T cells at Day 7 PTx (H) and Day ≥84 PTx (I). DLN, draining lymph node; GFP, green fluorescent protein; LN, lymph node; PB, peripheral blood; PTx, posttransplantation; Tregs, regulatory T cells; tx, transplantation.

Download figure to PowerPoint

Treg expansion in the DLN at early and later time points

To assess Tregs in the DLN, the proportions of GFP+Foxp3+, CD25+Foxp3+ and CD25Foxp3+CD4+ T cells at intervals PTx were determined. The proportion of Foxp3+GFP+ Tregs was the highest in the DLN compared with PB and control LN at Day 7 PTx in transplanted and nontransplanted Foxp3GFP mice (Figure 4F). Further, the proportion of Foxp3+GFP+ Tregs was significantly higher in DLN than PB (p < 0.01) and control LN (p < 0.01) in transplanted Foxp3GFP mice surviving greater than 84 days, and PB (p < 0.01) and LN (p < 0.05) of nontransplanted Foxp3GFP mice (Figure 4G). These Tregs were predominantly CD25+Foxp3+ (Figure 4H), with no differences in the proportions of CD25Foxp3+ and CD25+Foxp3 among CD4+ T cells (Figure 4H and I). These data demonstrate that Treg expansion in the DLN, and locally in the allograft, occurs in kidney allograft tolerance.

K-Tregs express elevated levels of TGF-β, IL-10, IFN-γ, Blimp-1 and Cxcr3

Next, K-Tregs from kidney allografts in tolerant reporter mice were isolated and sorted based on Foxp3 fluorescence and CD4 expression. K-Tregs from early time points (Days 7–14 PTx) had significantly higher expression of transcripts for IL-10 (p < 0.05), IFN-γ (p < 0.05), TGF-β, Blimp-1 (p < 0.05) and Cxcr3 (p < 0.05) than naïve Tregs (Figure 5A). K-Tregs from later time points (Day >69 PTx) had significantly higher expression of TGF-β (p < 0.05) compared with naïve Tregs (Figure 5B). In contrast, the expression of cytokines, Blimp-1 and Cxcr3 was similar between splenic Tregs from transplanted mice and naïve mice at both time points (Figure 5A and B).

image

Figure 5. Assessment of immune profile of kidney allograft Foxp3+ Tregs. (A) K-Tregs from Days 7 to 14 PTx mice (n = 6) expressed high levels of TGF-β, IL-10 (*p < 0.05), IFN-γ (*p < 0.05), Blimp-1 (*p < 0.05) and Cxcr3 (*p < 0.05), while (B) K-Tregs from mice at Day ≥69 PTx (n = 3) expressed high levels of TGF-β (*p < 0.05) compared with naïve Tregs from the spleen of nontransplanted mice (n = 6) and Tregs from the spleen (SP) of transplanted mice (n = 6). Blimp-1, B lymphocyte-induced maturation protein-1; Cxcr3, chemokine receptor 3; IFN-γ, interferon gamma; K-Tregs, kidney allograft infiltrating Tregs; PTx, posttransplantation; Tregs, regulatory T cells.

Download figure to PowerPoint

K-Tregs mediate donor alloantigen-specific tolerance to skin allografts

We further assessed K-Tregs functionally for the ability to mediate dominant tolerance. Infiltrating CD4+GFP+ cells were isolated and sorted from surviving kidney allografts and spleen at Day 14 PTx and adoptively transferred into Rag−/− mice, which were subsequently transplanted with DBA/2 skin allografts, third-party B10BR, CBA or C3H/He5 skin allografts and syngeneic grafts. All skin allografts survived >100 days after adoptive transfer of K-Tregs or SP-Tregs (Figure 6A). These mice were then challenged with Foxp3 negative CD4+ effector T cells (CD4+GFP). The donor-strain DBA/2 skin allografts remained intact with MST >100 days following challenge while all third-party skin grafts were rejected with MST 21 days (p < 0.001; Figure 6B). Rag−/− mice who reconstituted with SP-Tregs rejected DBA/2 skin allografts with MST 27 days (p < 0.05) and third-party grafts with MST 21.5 days (p < 0.05), respectively, after challenge with CD4+GFP cells (Figure 6B). Control RAG1−/− mice with DBA/2 grafts and no Tregs given CD4+GFP cells rejected these allografts with MST = 32 (p < 0.05) (Figure 6B). Control syngeneic skin grafts and allografts on mice reconstituted with K-Tregs alone showed normal histology at Day 100 PTx (Figure 6C). Skin histology at both Day 37 (Day 137 PTx) and Day 100 (Day 200 PTx) after challenge in mice with K-Tregs revealed normal appearances (Figure 6D). However, mice without K-Tregs rejected skin allografts after challenge as shown histologically at Day 37 (Figure 6E). Rejected CBA, C3H/He5 and B10BR third-party skin allografts (Figure 6F) had heavy cellular infiltrates and destruction of the skin structures. DBA/2 skin allografts on Rag−/− mice receiving SP-Tregs and challenged with CD4+GFP cells at Day 27 demonstrate cellular rejection (Figure 6G).

image

Figure 6. Kidney allograft-infiltrating Foxp3+ Tregs (K-Tregs) mediate donor-specific tolerance to skin allografts. (A) Syngeneic skin graft (Foxp3GFP; n = 5; white squares), DBA/2 skin allograft (n = 8; black upright triangle) and third-party allografts (B10BR, CBA and C3H/He5; n = 15; black-inverted triangle) survived on Rag−/− mice >100 days after adoptive transfer of K-Tregs of Day 14 PTx. Syngeneic skin graft (Foxp3GFP; n = 3; black diamond), DBA/2 skin allograft (n = 3; black circle) and third-party allografts (B10BR, CBA and C3H/He5; n = 6; letter x) survived on Rag−/− mice >100 days after adoptive transfer of Day 14 PTx spleen-Tregs from tolerant transplanted mice (SP-Tregs). (B) DBA/2 skin allografts (n = 6; white upright triangle) showed long-term survival with MST >100 days on Rag−/− mice reconstituted with K-Tregs after challenge with CD4+GFP cells, compared with third-party allografts with MST 21 days (n = 12; black circle; **p < 0.001). However, DBA/2 skin allografts (n = 3; black-inverted triangle) and third-party grafts (n = 6; letter x) were rejected with MST 27 days (*p < 0.05) and MST 21.5 days (*p < 0.05), respectively, on Rag−/− mice reconstituted with SP-Tregs after challenge with CD4+GFP cells. Rag−/− mice with CD4+GFP rejected DBA/2 skin allografts (MST = 31 days; n = 4; black diamond; *p < 0.05). (C) Histology of syngeneic grafts (Foxp3GFP) and DBA/2 allografts (H&E) on Rag−/− recipients with K-Tregs at Day 100 PTx, (D) Rag−/− mice receiving K-Tregs and challenged with CD4+GFP cells at Day 37 (Day >137 PTx) and Day 100 after challenge (Day >200 PTx), (E) on Rag−/− mice only reconstituted with CD4+GFP cells at Day 37 and (F) third-party B10BR, CBA and C3H/He5 allografts on Rag−/− recipients of K-Tregs and challenged with CD4+GFP cells at Day 20. (G) Syngeneic grafts and DBA/2 allografts on Rag−/− mice receiving SP-Tregs and challenged with CD4+GFP cells at Day 27. (H) The spleens of control mice reconstituted with K-Tregs showed only CD45.2+ after gating on CD4+ whereas mice reconstituted with both K-Tregs and CD4+GFP cells contained both CD45.2+ (52.6% and 60.6%, respectively) and CD45.2+CD45.1+ (46.7% and 38.6%, respectively) cells after gating on CD4+ T cells at Day 37 and 100 after challenge. GFP, green fluorescent protein; H&E, hematoxylin and eosin; MST, median survival time; PTx, posttransplantation; Tregs, regulatory T cells.

Download figure to PowerPoint

The extent of reconstitution of Treg populations and the relative number of adoptively transferred K-Tregs and CD4+GFPeffector CD4+ T cells were assessed using the CD45 congenic marker (CD45.1/CD45.2) in the recipient's spleen. Effector CD4+GFP cells were derived from CD45.1/.2 mice and K-Tregs from CD45.2 mice. CD4+ T cells from mice reconstituted with K-Tregs alone were exclusively CD45.2+, whereas total CD4+ T cells from mice with both effector T cells (CD45.1+CD45.2+) and K-Tregs (CD45.2+) are shown in their respective gates at a ratio of approximately 1:1 at Day 37 and then with an increased ratio of Tregs/effector cells by Day 100 (Figure 6H).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

We and other groups have identified a role for Foxp3+ Tregs in spontaneous kidney allograft tolerance across certain MHC-mismatched mouse models [2, 20, 21, 30, 31]. We had previously reported the increased numbers of Foxp3+ T cells in murine models of allograft tolerance [20]. In the present study, we detected early expansion of Foxp3+ Tregs in both DLNs and the transplanted kidney. Kidney transplants in the DBA-2 to C57BL/6 mismatch were rapidly rejected when Foxp3+ Tregs were depleted, both in our hands, and when a different C57BL/6-Foxp3-DTR strain was used by other authors [21]. Alloantigen-specific Tregs gravitate to sites of alloantigen expression [32], and recent work has elegantly demonstrated the co-existence within allograft infiltrates of effector cells capable of rejecting the graft, with regulatory cells holding them in check [18]. Collectively, these studies suggest that the increase in Tregs found in human rejecting kidneys is a response to rejection rather than a cause [33]. Therefore, we have sought to define more clearly the phenotype and functional role of the kidney infiltrating Tregs to assess whether they develop effector functions or allograft specificity.

It has recently been shown that Tregs can develop effector functions that match those of the effector T cell population that they are suppressing [11]. This has been demonstrated with Th1, Th2 and T follicular helper (Tfh)-related Tregs, which have in common the expression of the repressive transcription factor Blimp-1 in addition to Foxp3 [11, 34]. Cytokine production is one way in which effector Tregs mirror the effector population that they supress [11, 35]. Th1-driven immune responses are defined by IFN-γ including acute T cell-mediated allograft rejection [36]. However, IFN-γ is also a critical cytokine in tolerance induction [37-39], and the generation and function of allospecific Tregs during the development of operational tolerance to donor alloantigens in vivo [40]. The microenvironment of renal allografts in the early posttransplant period is rich in IFN-γ [20, 41]. Chemokines inducible by IFN-γ include chemokine ligand (CXCL) 9, CXCL10 (IP-10) and CXCL11 [42]. In this context, the differentiation of Tregs as well as Th1 effector cells to express the corresponding chemokine receptor Cxcr3 is likely to play an important role in promoting their co-localization within renal allografts. CXCR3+ peripherally circulating CD4+FOXP3+ Tregs have been found in patients to correlate with renal allograft function, again suggesting a role for CXCR3 in their recruitment into peripheral sites of inflammation by their ligand CXCL10 (IP-10) [43, 44]. Whereas the chemokine receptor Ccr4 [C–C chemokine receptor type 4] has been found to be essential for the migration of Tregs into sites of inflammation in some models [45], Ccr4-deficient mice did not show any defect in Treg recruitment nor in the development of spontaneous tolerance in the DBA/2 to C657BL/6 mouse kidney transplant model [21], implying that usage of alternative chemokine receptors, including Cxcr3, may be more important in this setting.

We found the high levels of IFN-γ in allograft K-Tregs at early but not at later time points. Further, splenic Tregs from transplanted mice did not express increased IFN-γ transcripts suggesting a key role for this cytokine in effector maturation but not necessarily in maintenance of allograft tolerance. We evaluated the Tregs in the kidney for evidence of a Th1-Treg effector phenotype and found expression of both Blimp-1 and Cxcr3 at early time points (Days 7–14 PTx), concordant with such a phenotype. Conversely, IL-10 appeared to play a role in the maintenance of established tolerance, as well as during the tolerance induction phase. Serum levels of IL-10 were higher in long-term tolerant mice compared with either tolerant mice at early times PTx or rejecting mice and high IL-10 was also found in tolerated grafts at early time points. IL-10 production either by induced Treg or by natural Tregs has been associated with mechanisms of tolerance in various settings [46], including organ-specific tolerance in the gut [47]. TGF-β was increased in K-Tregs at both early and late time points consistent with its role as a key regulatory cytokine. Both IL-10 and TGF-β are required for Tregs to mediate tolerance to allografts in a number of transplantation models [14, 48]. However, DBA/2 to C57BL/6 (H-2b) mouse spontaneous kidney allograft tolerance is TGF-β and not IL-10 dependent [31].

In addition to altering Treg phenotype, prior antigen exposure leads to Treg memory and greater potency in infectious models [49]. Tregs with prior activation or memory status have many features in common with classical memory T cells [50, 51]. We found that the Tregs infiltrating the kidneys had altered functional capacity and specificity consistent with a previously activated phenotype, including the expression of Blimp-1 and Cxcr3 suggestive of a Th1-suppressing Treg. We further demonstrated that K-Tregs within the kidney allograft are capable of transferring donor alloantigen-specific dominant tolerance to fully allogeneic skin grafts. The ability of a relatively modest number of K-Tregs to prevent rejection in this stringent model is consistent with previous findings that donor-specific Tregs are more potent and effective in limiting skin graft rejection than are polyclonal natural Tregs [32].

K-Tregs are undoubtedly donor alloantigen-specific, yet it remains to be determined whether they represent an expanded pool of natural Tregs with T cell receptors cross-reactive for alloantigen, or allospecific T cells in which a regulatory phenotype has been induced following alloantigen exposure [52]. Tregs in our studies are depleted pretransplant and this is followed by subsequent rejection, suggesting that induced Tregs are not a major component of the regulatory cells active during tolerance induction, or that natural Tregs are required for a short-term inhibition of rejection in order for peripherally induced allospecific Tregs to develop. Though DT may potentially suppress the development of induced Tregs at the time of transplant, Tregs can still be induced later, following the pretransplant DT administration. Therefore, the likely source of the Tregs is expansion of cross-reactive natural Tregs. Induced and natural Tregs may play different roles in the suppression of immune responses [52] and further studies of the K-Treg population may delineate these.

In conclusion, Tregs are required to maintain kidney allograft tolerance and are expanded in the allograft and DLN of tolerant allografts. They appear to arise from cross-reactive natural Tregs and demonstrate features of effector Tregs with the expression of TGF-β and IL-10, IFN-γ, Blimp-1 and Cxcr3 and the suppression of inflammatory cytokines. Further, the K-Tregs within the kidney allograft are capable of transferring dominant, donor alloantigen-specific tolerance. These studies provide evidence that Tregs have the capacity to promote allograft tolerance in kidney transplantation. Well-characterized allograft infiltrating Tregs suggest the potential for developing the specificity and potency of allospecific Tregs for future therapeutic use.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

This study was supported by National Health and Medical Research Council of Australia (NHMRC) (Grants 1029205, 1029601, 512246) and NHMRC Training Fellowship APP1013185 (to MH). We thank Dr. Xin Wang and flow units of Centenary Institute for sorting, and Dr. Laurence Cantrill for fluorescent imaging.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

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

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  • 1
    Skoskiewicz M, Chase C, Winn HJ, Russell PS. Kidney transplants between mice of graded immunogenetic diversity. Transplant Proc 1973; 5: 721725.
  • 2
    Russell PS, Chase CM, Colvin RB, Plate JM. Kidney transplants in mice. An analysis of the immune status of mice bearing long-term, H-2 incompatible transplants. J Exp Med 1978; 147: 14491468.
  • 3
    Lu L, Rudert WA, Qian S, et al. Growth of donor-derived dendritic cells from the bone marrow of murine liver allograft recipients in response to granulocyte/macrophage colony-stimulating factor. J Exp Med 1995; 182: 379387.
  • 4
    Alexander SI, Smith N, Hu M, et al. Chimerism and tolerance in a recipient of a deceased-donor liver transplant. N Engl J Med 2008; 358: 369374.
  • 5
    Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 2008; 358: 353361.
  • 6
    Azuma H, Chandraker A, Nadeau K, et al. Blockade of T-cell costimulation prevents development of experimental chronic renal allograft rejection. Proc Natl Acad Sci USA 1996; 93: 1243912444.
  • 7
    Kinnear G, Jones ND, Wood KJ. Costimulation blockade: Current perspectives and implications for therapy. Transplantation 2013; 95: 527535.
  • 8
    Spitzer TR, Sykes M, Tolkoff-Rubin N, et al. Long-term follow-up of recipients of combined human leukocyte antigen-matched bone marrow and kidney transplantation for multiple myeloma with end-stage renal disease. Transplantation 2011; 91: 672676.
  • 9
    Wood KJ, Bushell A, Hester J. Regulatory immune cells in transplantation. Nat Rev Immunol 2012; 12: 417430.
  • 10
    Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004; 22: 531562.
  • 11
    Cretney E, Kallies A, Nutt SL. Differentiation and function of Foxp3(+) effector regulatory T cells. Trends Immunol 2013; 34: 7480.
  • 12
    Lee MK, Moore DJ, Jarrett BP, et al. Promotion of allograft survival by CD4+ CD25+ regulatory T cells: Evidence for in vivo inhibition of effector cell proliferation. J Immunol 2004; 172: 65396544.
  • 13
    Cobbold SP, Castejon R, Adams E, et al. Induction of FOXP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants. J Immunol 2004; 172: 60036010.
  • 14
    Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol 2001; 166: 37893796.
  • 15
    Kingsley CI, Karim M, Bushell AR, Wood KJ. CD25+ CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J Immunol 2002; 168: 10801086.
  • 16
    Muthukumar T, Dadhania D, Ding R, et al. Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N Engl J Med 2005; 353: 23422351.
  • 17
    Taubert R, Pischke S, Schlue J, et al. Enrichment of regulatory T cells in acutely rejected human liver allografts. Am J Transplant 2012; 12: 34253436.
  • 18
    Kendal AR, Chen Y, Regateiro FS, et al. Sustained suppression by Foxp3+ regulatory T cells is vital for infectious transplantation tolerance. J Exp Med 2011; 208: 20432053.
  • 19
    Lahl K, Loddenkemper C, Drouin C, et al. Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 2007; 204: 5763.
  • 20
    Wang C, Cordoba S, Hu M, et al. Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments. Transpl Immunol 2011; 24: 149156.
  • 21
    Miyajima M, Chase CM, Alessandrini A, et al. Early acceptance of renal allografts in mice is dependent on Foxp3(+) cells. Am J Pathol 2011; 178: 16351645.
  • 22
    Wu H, Noordmans GA, O'Brien MR, et al. Absence of MyD88 signaling induces donor-specific kidney allograft tolerance. J Am Soc Nephrol 2012; 23: 17011716.
  • 23
    Watson D, Zhang GY, Sartor M, Alexander SI.Pruning” of alloreactive CD4+ T cells using 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester prolongs skin allograft survival. J Immunol 2004; 173: 65746582.
  • 24
    Cunningham EC, Tay SS, Wang C, et al. Gene therapy for tolerance: High-level expression of donor major histocompatibility complex in the liver overcomes naïve and memory alloresponses to skin grafts. Transplantation 2013; 95: 7077.
  • 25
    Lahl K, Sparwasser T. In vivo depletion of FoxP3+ Tregs using the DEREG mouse model. Methods Mol Biol 2011; 707: 157172.
  • 26
    Hu M, Watson D, Zhang GY, et al. Long-term cardiac allograft survival across an MHC mismatch after “pruning” of alloreactive CD4 T cells. J Immunol 2008; 180: 65936603.
  • 27
    Hu M, Wu J, Zhang GY, et al. Selective depletion of alloreactive T cells leads to long-term islet allograft survival across a major histocompatibility complex mismatch in diabetic mice. Cell Transplant 2012 [Epub ahead of print].
  • 28
    Zheng G, Lyons JG, Tan TK, et al. Disruption of E-cadherin by matrix metalloproteinase directly mediates epithelial-mesenchymal transition downstream of transforming growth factor-beta1 in renal tubular epithelial cells. Am J Pathol 2009; 175: 580591.
  • 29
    Polhill T, Zhang GY, Hu M, et al. IL-2/IL-2Ab complexes induce regulatory T cell expansion and protect against proteinuric CKD. J Am Soc Nephrol 2012; 23: 13031308.
  • 30
    Inoue K, Niesen N, Albini B, Milgrom F. Studies on immunological tolerance induced in mice by kidney allografts. Int Arch Allergy Appl Immunol 1991; 96: 358361.
  • 31
    Bickerstaff AA, Wang JJ, Pelletier RP, Orosz CG. Murine renal allografts: Spontaneous acceptance is associated with regulated T cell-mediated immunity. J Immunol 2001; 167: 48214827.
  • 32
    Sagoo P, Lombardi G, Lechler RI. Relevance of regulatory T cell promotion of donor-specific tolerance in solid organ transplantation. Front Immunol 2012; 3: 184.
  • 33
    Wood KJ, Ushigome H, Karim M, Bushell A, Hori S, Sakaguchi S. Regulatory cells in transplantation. Novartis Found Symp 2003; 252: 177188, discussion 188–193, 203–110.
  • 34
    Cretney E, Xin A, Shi W, et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat Immunol 2011; 12: 304311.
  • 35
    Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 2010; 28: 445489.
  • 36
    Wiseman AC, Pietra BA, Kelly BP, Rayat GR, Rizeq M, Gill RG. Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection. J Immunol 2001; 167: 54575463.
  • 37
    Konieczny BT, Dai Z, Elwood ET, et al. IFN-gamma is critical for long-term allograft survival induced by blocking the CD28 and CD40 ligand T cell costimulation pathways. J Immunol 1998; 160: 20592064.
  • 38
    Kishimoto K, Sandner S, Imitola J, et al. Th1 cytokines, programmed cell death, and alloreactive T cell clone size in transplant tolerance. J Clin Invest 2002; 109: 14711479.
  • 39
    Hassan AT, Dai Z, Konieczny BT, et al. Regulation of alloantigen-mediated T-cell proliferation by endogenous interferon-gamma: Implications for long-term allograft acceptance. Transplantation 1999; 68: 124129.
  • 40
    Sawitzki B, Kingsley CI, Oliveira V, Karim M, Herber M, Wood KJ. IFN-gamma production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J Exp Med 2005; 201: 19251935.
  • 41
    Sharland A, Shastry S, Wang C, et al. Kinetics of intragraft cytokine expression, cellular infiltration, and cell death in rejection of renal allografts compared with acceptance of liver allografts in a rat model: Early activation and apoptosis is associated with liver graft acceptance. Transplantation 1998; 65: 13701377.
  • 42
    Curbishley SM, Eksteen B, Gladue RP, Lalor P, Adams DH. CXCR 3 activation promotes lymphocyte transendothelial migration across human hepatic endothelium under fluid flow. Am J Pathol 2005; 167: 887899.
  • 43
    Hoerning A, Kohler S, Jun C, et al. Peripherally circulating CD4(+) FOXP3(+) CXCR3(+) T regulatory cells correlate with renal allograft function. Scand J Immunol 2012; 76: 320328.
  • 44
    Hoerning A, Koss K, Datta D, et al. Subsets of human CD4(+) regulatory T cells express the peripheral homing receptor CXCR3. Eur J Immunol 2011; 41: 22912302.
  • 45
    Faustino L, da Fonseca DM, Takenaka MC, et al. Regulatory T cells migrate to airways via CCR4 and attenuate the severity of airway allergic inflammation. J Immunol 2013; 190: 26142621.
  • 46
    Saraiva M, O'Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 2010; 10: 170181.
  • 47
    Kamanaka M, Kim ST, Wan YY, et al. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 2006; 25: 941952.
  • 48
    Walsh PT, Taylor DK, Turka LA. Tregs and transplantation tolerance. J Clin Invest 2004; 114: 13981403.
  • 49
    Rosenblum MD, Gratz IK, Paw JS, Lee K, Marshak-Rothstein A, Abbas AK. Response to self antigen imprints regulatory memory in tissues. Nature 2011; 480: 538542.
  • 50
    Kurtulus S, Tripathi P, Opferman JT, Hildeman DA. Contracting the ‘mus cells’—Does down-sizing suit us for diving into the memory pool? Immunol Rev 2010; 236: 5467.
  • 51
    Akbar AN, Vukmanovic-Stejic M, Taams LS, Macallan DC. The dynamic co-evolution of memory and regulatory CD4+ T cells in the periphery. Nat Rev Immunol 2007; 7: 231237.
  • 52
    Sakaguchi S, Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T. Regulatory T cells: How do they suppress immune responses? Int Immunol 2009; 21: 11051111.