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* Department of Gene and Cell Medicine, Recanati/Miller Transplantation Institute, and Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Although many studies have been conducted to define the cellular and molecular mechanisms by which Treg develop (17, 18, 19), there is little knowledge about the anatomic compartments where Foxp3-expressing Treg are activated, expanded, or display suppressor function for tolerization to ensue. Distinct lymphoid and nonlymphoid compartments may be differentially involved in the development of Treg, because the lymphoid environment plays essential and diverse roles in either rejection or tolerance (20). Lymphoid architecture also plays an important role in Treg development and differentiation, because differences in lymphoid origin have been used to classify two distinct subsets of Treg cells: natural (thymus) vs adaptive (periphery), according to structural site of generation (21). Naturally occurring CD4+CD25+ Treg are generated as a result of multiple selection events during T cell development within the thymus (18, 22). Evidence for extrathymic CD4+CD25+ T cell development supports an alternative de novo pathway for the origin of Treg in the periphery (23), and indicates that CD4+CD25+ Treg generated in the periphery are probably different from thymically produced CD25+CD4+ Treg (21). Therefore, it is likely that particular anatomic sites provide specific milieus that allow tolerization to occur by permitting suppressor Treg to be activated, expanded, or function in the periphery. Thus, defining the Foxp3-expressing CD4+CD25+ T cell population in distinct anatomic compartments may elucidate the precise sites where tolerization takes place.
We previously demonstrated that CD62L-mediated lymph node (LN) homing is necessary for the induction of anti-CD2 plus anti-CD3 mAb-induced tolerance, because coadministration of anti-CD62L mAbs prevents both LN homing and tolerance, in both nonvascularized and vascularized cardiac transplant models (24). Adoptive transfer of CD62L–/– T cells and the use of CD62L–/– recipients provided complementary genetic evidence to confirm the importance of CD62L and LN occupancy by T cells in tolerization. In this report, we further characterized these observations by studying the CD4+CD25+ Treg population in distinct anatomic domains after treatment with two different tolerization protocols (3, 4), and after manipulating T cell LN homing with anti-CD62L mAb and the sphingosine 1-phosophate receptor modulator FTY720 (reviewed in Ref.25). Our results demonstrate that alloantigen-specific Treg occupancy in the LNs of tolerant animals is necessary for immunological tolerance to vascularized cardiac allografts. Alloantigen-specific Treg were expanded in recipient LNs, but not other peripheral sites, under both tolerization protocols. Anti-CD62L mAbs prevented T cell LN homing, resulting in abrogation of both Treg expansion in the LNs or other lymphoid areas and graft survival, whereas FTY720-driven T cell LN sequestration restored both LN Treg expansion and graft survival. Together, the results suggest an essential role of the LN for the development and function of CD4+CD25+ Treg in tolerant animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BALB/c (H-2d), C57BL/6 (H-2b), and CBA (H-2k) mice, 8–10 wk of age, were purchased from The Jackson Laboratory, and used as donors, recipients, or third-party controls. All mice were housed in a specific pathogen-free facility in microisolator cages. All experiments were performed with age- and sex-matched mice in accordance with the Institutional Animal Care and Utilization Committee-approved criteria and protocols.
Reagents
The 145-2C11 hamster anti-murine CD3
hybridoma was a gift from J. Bluestone (University of California, San Francisco, CA), and the 12-15 rat IgG1 anti-murine CD2 hybridoma was a gift from P. Altevogt (Immunology and Genetics Institute, Heidelberg, Germany). The MEL-14 rat IgG2a anti-murine CD62L hybridoma was purchased from the American Type Culture Collection. The hybridomas were grown in culture, and supernatants were purified over protein G or A columns (Amersham Biosciences). For cell surface phenotype analysis, cells were stained with CyChrome-anti-CD4, FITC-anti-CD25, allophycocyanin-anti-CD62L, biotin-anti-CD45RB, PE-anti-CD44, or PE-anti-CD69 (BD Pharmingen). Anti-CD40L (MR-1) was from Bender MedSystems. FTY720 was a gift from V. Brinkman (Novartis, Basel, Switzerland) and was dissolved in water at 0.1 mg/ml.
Vascularized cardiac transplantation
BALB/c hearts were transplanted as fully vascularized heterotopic grafts into C57BL/6 as described (26). Recipients received i.v. injections of different Abs in 0.5 ml of PBS at the indicated times. For the anti-CD2/anti-CD3 tolerization protocol, recipients received 100 µg of anti-CD2 mAbs on days 0 and 1, and 100 µg of anti-CD3 mAbs on days 2, 3, 4, 5, and 10 with respect to transplantation. Anti-CD62L was administered at 100 µg on days 0 and 1. For the donor-specific transfusion (DST) and anti-CD40L mAb protocol, recipients received DST (107 donor splenocytes i.v. 7 days before transplant) and 250 µg of anti-CD40L mAbs on days –7, –4, 0, and +4. One group of mice received no additional treatment, whereas the other group received 100 µg of anti-CD62L mAbs on days –7 and –6. FTY720 was administered daily via oral gavage. Graft function was monitored every other day by abdominal palpation, and rejection was defined as complete cessation of a palpable beat and was confirmed by direct visualization at laparotomy. Recipients with grafts surviving >100 days were defined as tolerant and were used for in vitro experiments. Untreated recipients rejected allograft at days 8–10 and were used as controls. Anti-CD62L mAb-treated animals rejected at 28–39 days.
Cell preparations
Mice were sacrificed at the indicated times, and LNs, spleens, and cardiac allografts were removed and gently dissociated into single-cell suspensions. RBCs were removed by Tris-NH4Cl lysis. Cells were placed in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino acids, and 2 x 10–5 M 2-ME). Lymphocytes were isolated from peripheral blood using Lympholyte Mammal density separation medium (Cedarlane). Homogenized heart tissue was treated with 5% Collagenase Type II (Worthington) for 30 min at 37°C before lymphocyte isolation.
Purification of cell subsets
CD4+ T cell subsets were isolated from LN and spleen using the Mouse T Cell CD4 Subset Column kit (R&D Systems) according to the manufacturer’s protocol, and the purity of the enriched CD4+ cells ranged between 85 and 90%. Afterward, the enriched CD4+ cells were stained with CyChrome-anti-CD4 mAbs and FITC-anti-CD25 mAbs, and sorted into CD4+CD25+ cells and CD4+CD25– cells using MoFlo (DakoCytomation). The purity of the sorted cells was >98%.
MLR and suppressor assay
A total of 2 x 105 responder CD4+CD25+ T cells from different groups was cocultured in triplicate with 5 x 105 1500-rad gamma-irradiated BALB/c splenocytes that had been T cell depleted by negative selection using Mouse Pan T Dynabeads according to the manufacturer’s protocol (Dynal). Cells were cultured in 96-well plates for 3 days at 37°C in a humidified atmosphere of 5% CO2. Eighteen hours before the termination of the culture, the wells were pulsed with 1 µCi of [3H]thymidine, and incorporation was quantified with a scintillation counter. Results are expressed as stimulation index (SI), determined from mean of triplicate determinations ± SEM. Alternatively, CD4–CD25+ or CD4+CD25– T cells from tolerant animals were also assessed for T cell function in MLR. The experiment was conducted as described above. To test the suppressive properties of the CD4+CD25+ T cells from tolerant animals, 4 x 104 freshly isolated responder CD4+CD25– T cells from naive mice were stimulated with 1 x 105 irradiated allogeneic T-depleted splenocytes for 3 days, along with 2 x 104 CD4+CD25+ T cells from different groups. The experiment was conducted as described above.
Flow cytometry
Cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C. Flow cytometric analysis was performed on LSR II (BD Biosciences) and analyzed with FlowJo (Tree Star). Results are expressed as percentage of cells staining above background, and mAbs were titered at regular intervals during the course of these studies to ensure that saturating concentrations were used.
Real-time PCR
Total cellular RNA was extracted from 5 x 105 CD4+CD25+ T cells using TRIzol reagent (Invitrogen Life Technologies) digested with RNase-free DNase I (Invitrogen Life Technologies), and reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen). Foxp3 random primers (Invitrogen Life Technologies) according to the manufacturer’s protocol, and mRNA levels were quantified by real-time PCR using QuantiTect SYBR Green PCR kit (Qiagen) with the LightCycler (Roche). PCR consisted of a 15-min 95°C denaturation step followed by 45 cycles of 15 s at 94°C, 20 s at 56°C, and 15 s at 72°C. Primers were as follows: Foxp3, 5'-CCC AGG AAA GAC AGC AAC CTT-3' and 5'-TTC TCA CAA CCA GGC CAC TTG-3'; cyclophilin A, 5'-AGG GTG GTG ACT TTA CAC GC-3' and 5'-ATC CAG CCA TTC AGT CTT GG-3'. Normalized values for Foxp3 mRNA expression were calculated as the relative quantity of Foxp3 divided by the relative quantity of cyclophilin A. All samples were run in triplicate. For CCR2, CCR5, and CCR7, total RNA was extracted using the RNeasy Maxi kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed from 2 µg of RNA. Quantitative real-time PCR was conducted in duplicate from 25 ng of cDNA with 0.4 µM each primer in a 30-µl final reaction volume of 1x SYBR Green PCR Master Mix (Applied Biosystems). PCR cycling conditions were as follows: 50°C for 2 min, 95°C for 15 min and 40 cycles of 95°C for 15 s, and 60°C for 1 min. Relative expression levels were calculated as 2(Ct ubiquitin RNA – Ct gene) (for details see ABI PRISM 7700, User Bulletin No. 2) using ubiquitin RNA as an endogenous control. The following were used: CCR2-forward, GTT ACC TCA GTT CAT CCA; CCR2-reverse, CAA GGC TCA CCA TCA TCG TAG TC; CCR5-forward, TTG CAA ACG GTG TTC AAT TTT C; CCR5-reverse, TCT CCT GTG GAT CGG GTA TAG AC; CCR7-forward, CAC GCT GAG ATG CTC ACT GG; CCR7-reverse, CCA TCT GGG CCA CTT GGA; ubiquitin-forward, TGG CTA TTA ATT ATT CGG TCT GCA T; ubiquitin-reverse, GCA AGT GGC TAG AGT GCA GAG TAA.
RT-PCR
Total cellular RNA was extracted from 5 x 105 cells using TRIzol reagent (Invitrogen Life Technologies) digested with RNase-free DNase I (Invitrogen Life Technologies), and reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen) and random primers (Invitrogen Life Technologies) according to the manufacturer’s protocol. Foxp3 mRNA levels were quantified by RT-PCR (Applied Biosystems). PCR consisted of a 15-min 95°C denaturation step followed by 35 cycles of 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C. Foxp3 primers were as follows: 5'-CCC AGG AAA GAC AGC AAC CTT-3' and 5'-TTC TCA CAA CCA GGC CAC TTG-3'. All samples were run in triplicate.
Histology
For B220 and Thy1.2, LNs and spleens were harvested, subdivided, frozen directly in OCT (Fisher), and stored at –80°C in preparation for immunological studies. Sections of 8 µm were cut using a Leica 1900CM cryomicrotome, fixed, and mounted with Gel/Mount (Biomeda) in polylysine-coated slides. FITC-anti-B220 and biotinylated anti-Thy1.2 were purchased from BD Pharmingen and were developed by HRP-conjugated rabbit anti-FITC (DakoCytomation), and by alkaline phosphatase-conjugated streptavidin (Zymed). For H&E sections, LNs, spleens, and grafts were fixed, embedded in paraffin, and cut into 10-µm-thick sections using a Finesse Microtome (Shandon).
Immunofluorescent microscopy
Fresh tissue was harvested, subdivided, frozen directly in OCT (Fisher), and stored at –80°C. Sections of 8 µm were cut using a Leica 1900CM cryomicrotome. Endogenous peroxidase, FcRs, and biotin binding sites were blocked as previously described (27). Biotinylated rat anti-mouse CD4 (H129.19) and biotinylated rat IgG2a, were purchased from BD Pharmingen. Anti-FOXP3 rabbit anti-sera were a gift from Novus Biologicals. The Cy3-conjugated donkey anti-rabbit IgG, and 7-amino-4-methylcoumarin-3-acetic acid-streptavidin were purchased from Jackson ImmunoResearch. All slides were mounted with Vectashield (Vector Laboratories) to preserve fluorescence. Images were acquired using a Leica DMRA2 fluorescence microscope and a digital Hamamatsu close circuit device camera. Separate green, red, and blue images were collected and analyzed with Openlab software (Improvision). Captured image layers were sliced to standard density values, and total cell numbers per square millimeter of tissue were measured as single independent intensity objects with the Openlab cell measurement module. Twelve-bit grayscale objects with an area <0.01 U were ignored. Final image processing was performed using Volocity software (Improvision).
Statistical analysis
For graft survival, one-way ANOVA was performed. For CD4+CD25+ T cell growth, t test and F test were performed to compare variances. For cell proliferation, one-way ANOVA was performed at each time point. The group effects were all significant at p 
0.05. To examine individual differences, comparison between every pair of groups was performed. For Foxp3+ T cell counts and chemokine receptor gene expression, one-way ANOVA and Dunnett test were performed, to examine individual differences compared with the naive control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We have previously shown that lymphocyte LN homing and localization are required for anti-CD2 plus anti-CD3 mAb-induced tolerance to nonvascularized and vascularized heart allografts (24). We next investigated whether this finding held true using another well-defined tolerizing regimen, DST plus anti-CD40L mAb (4). BALB/c donor vascularized cardiac grafts were transplanted into C57BL/6 recipients, which received either anti-CD2 plus anti-CD3 mAbs, DST plus anti-CD40L mAbs, or were left untreated. Both tolerance protocols induce long-term graft survival with normal histology in vascularized allografts (3, 28) (Fig. 1A). Third-party CBA (H-2k), but not donor strain BALB/c (H-2d), second-set allografts placed >60 days following the initial transplant were rejected (Refs.29 and 30 , and data not shown), demonstrating donor alloantigen-specific tolerance. Administration of anti-CD62L mAb prevented allograft survival in animals given DST plus anti-CD40L mAb (Fig. 1B). Coadministration of anti-CD62L mAb decreased mean survival time from >70.6 ± 13.2 to 28.0 ± 6.8 days in the anti-CD2 plus anti-CD3 mAb-treated group, and from >88.2 ± 1.8 to 39.6 ± 0.9 days in the DST plus anti-CD40L mAb-treated group (Fig. 1B). These results demonstrate that both the anti-CD2 plus anti-CD3 mAb and the DST plus anti-CD40L mAb tolerogenic regimens require CD62L-dependent LN homing to induce prolonged survival and tolerance in a vascularized cardiac allograft model. Furthermore, administration of FTY720 to anti-CD62L mAb plus tolerogen-treated mice (Fig. 1C) restored graft survival with normal allograft histology, and supports the hypothesis that LN occupancy is required for tolerance.
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To further determine the significant role of distinct anatomic compartments during tolerance and acute rejection, we examined the structure of lymphoid organs and grafts of the tolerized anti-CD2 plus anti-CD3 mAb-treated, rejecting anti-CD62L mAb-treated, tolerized FTY720-treated, and rejecting untreated animals (Fig. 2). Allografts, spleens, and LNs (inguinal, axillary, and paraortic; n = 3 in each group) were harvested from tolerant recipients at >100 days following transplantation, from rejecting recipients on days 8–10 at the time of acute rejection, and from naive controls. Tissue sections were stained with H&E for light microscopy, and for Thy1.2 and B220 for staining of T and B cells, respectively. The allografts from the acutely rejecting and anti-CD62L mAb-treated recipients have significant interstitial infiltrates with severe myocardial necrosis and disrupted architecture, consistent with graft failure due to acute cellular rejection. In contrast, the allografts from the tolerogen-treated and FTY720-treated groups contain minimal or no cellular infiltrates, with no evidence of vascular intimal hyperplasia. In untreated and rejecting anti-CD62L mAb-treated mice, the LNs are expanded with disrupted germinal center architecture, with scattered T and B cell populations forming dispersed groups of large cells, indicative of an unmodified rejection response. On the contrary, the LN architecture in mice treated with the tolerogenic regimen or FTY720 is similar to that of naive mice, with preserved structure containing small lymphocytes and discernible T cell areas with little dispersion of the B cell population. Furthermore, FTY720 treatment alone in naive or transplanted animals had no effect in tissue architecture (data not shown). Compared with naive mice, the spleen of the acutely rejecting groups maintains organized architecture, with well-defined white and red splenic pulp areas. In addition, the splenic white pulp is expanded containing markedly enlarged lymphocytes and germinal centers. In contrast, the splenic architecture of the mice treated with the tolerogenic regimens is disorganized, with a significant decrease in size of the splenic white pulp. These results suggest not only that the systemic lymphoid organs to which lymphocytes migrate are important determinants of rejection vs tolerance, but also that the histologic organization of microdomains are key factors regulating immune responses.
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The immunohistochemistry showed differences in lymphoid structure and organization among the rejecting and tolerant groups. To characterize the relationship of lymphoid compartment occupancy and structure during tolerization, we further examined whether these alterations were associated with a shift in the distribution of CD4+CD25+ T cells in the blood, LN, spleen, and grafts of tolerant and rejecting mice. Total mononuclear cells (TMN), CD4+ T cells, and CD4+CD25+ T cells were examined in various lymphoid and nonlymphoid organs of the tolerized anti-CD2 plus anti-CD3 mAbs, and DST plus anti-CD40L mAb-treated mice; and compared with untreated rejecting mice at different time points. Fig. 3A shows that the TMN cell number decreases in the LN of rejecting mice, whereas there is a progressive increase of LN cells in the tolerant mice. In contrast, the number of mononuclear cells increases sharply in the spleens of the rejecting recipients while increasing only slightly in the tolerogen-treated group. Therefore, the tolerogen-treated group has a higher number and percentage of total lymphocytes in the LN compared with the rejecting group. As expected, the graft has an increase in mononuclear cells in the rejecting animals, whereas the tolerant group does not. The blood TMN counts do not show significant differences among any of the treatment groups. Paralleling the results of the TMN cells, the total CD4+ T cell population decreases in the LN and graft, but increases over time in the spleen and blood during rejection. Conversely, during the establishment of tolerance, the CD4+ T cells increase in the LN and graft, but remain the same in the spleen and blood. Focusing further on the putative Treg subset, the CD4+CD25+ T cell population in the rejecting animals is low in all compartments, and decreases further as rejection ensues. CD4+CD25+ T cells increase in the LNs of tolerized recipients, which is significant by day 30, and persists for >100 days, indicating that it takes time for the CD4+CD25+ T cells to develop. There is also a less significant increase in the CD4+CD25+ T cells in the grafts of tolerant recipients, accompanied by an overall decrease in these cells in the spleen. More detailed analysis of these cells in Fig. 3B shows that the CD4+CD25+ T cell population increases dramatically in the LNs of tolerant animals, eventually constituting 
50% of the total CD4+ T cell population, compared with 5–10% of CD4+ LN cells in naive or rejecting mice. The CD4+CD25+ T cell population also increases in the grafts of tolerant mice, although less dramatically, constituting 20% of the total CD4+ T cell population. Fig. 3B also shows an increase in the CD4–CD25+ LN cell population to similar levels (21–23%) in both the tolerant and rejecting groups, indicating that other cell subtypes are also activated. As expected, the activated CD4–CD25+ cell population increases in the graft during rejection, when compared with tolerant or naive animals.
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Because the results in Fig. 3 suggest that Treg might be generated and/or expanded in the LNs, it was important to further characterize these T cells and, in particular, to distinguish them from activated effector T cells. Because the CD25 molecule is a surface marker for either activated or suppressor CD4+ T cells, we therefore analyzed in parallel additional cell surface markers expressed on CD4+CD25+ T cells that delineate the regulatory phenotype. Activated T cells express high levels of CD44, CD69, and CD25, and low levels of CD45RB; naive T cells express low levels of CD44 and CD69 and high levels of CD62L and CD45RB; memory T cells express low levels of CD45RB and high levels of CD44; and Treg express high levels of CD25 and low levels of CD45RB (31, 32, 33, 34, 35). T cell activation is followed by CD69 up-regulation and gated CD4+CD25+ T cells from the LNs of rejecting mice show markedly increased CD69 expression compared with groups receiving the tolerogenic regimen or naive animals (Fig. 4A, first panel). CD62L mediates homing of leukocytes to the LNs through binding to ligands on high endothelial venules, and it is shed after T cell activation. Fig. 4A (second panel) indicates that LN CD4+CD25+ T cells from tolerant and naive animals continue to express high levels of CD62L, whereas LN CD4+CD25+ T cells from rejecting animals have down-modulated CD62L. CD45RBhigh is expressed in Ag-inexperienced T cells, whereas CD45RBlow is expressed in Ag-experienced T cells (activated/memory), and transition from the naive to the Ag-experienced state is accompanied by down-regulation of CD45RB. CD45RB was expressed at high levels on most CD4+CD25+ T cells from naive mice and at low levels on most CD4+CD25+ T cells from both the tolerant and rejecting animals (Fig. 4A, third panel). CD44 is also a marker for T cell activation, but in contrast with CD69, it was highly expressed on CD4+CD25+ T cells from both tolerant and rejecting animals (Fig. 4A, fourth panel). Taken together, these results indicate that the expanded CD4+CD25+ T cell population in the LNs of both groups of tolerogen-treated animals express a distinct phenotype compared with the CD4+CD25+ T cells in the rejecting group. The results suggest that the majority of the CD4+CD25+ T cells from all groups are Ag experienced (CD44highCD45RBlow), but that T cells from the tolerogen-treated groups are only partially activated (CD62LhighCD69low) in comparison to T cells from the rejecting group, which are more fully activated (CD62LlowCD69high).
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The Foxp3+CD4+CD25+ LN T cells are anergic and alloantigen-specific suppressors
The results demonstrate differences in CD4+CD25+ T cell distribution in distinct lymphoid and nonlymphoid compartments, and phenotypic variations within the LNs of anti-CD2 plus anti-CD3 mAbs or DST plus anti-CD40L mAb-treated recipients, compared with rejecting control or naive mice. In vitro proliferation assays were next performed to prove that CD4+CD25+ T cells possess regulatory, suppression function. The results show that CD4+CD25+ T cells from tolerant LNs exhibited 4-fold more potent suppression of the MLR when compared with tolerant splenic CD4+CD25+ T cells, or when compared with CD4+CD25+ LN or splenic T cells from the naive or rejecting groups (Fig. 5A). Further characterization of the CD4+CD25+ T cells shows that the suppressive activity is alloantigen specific when evaluated >100 days after transplantation (Fig. 5B). Interestingly, at earlier time points, CD4+CD25+ Treg suppressive activity is alloantigen specific for LN-derived, but not splenic Treg. This indicates that Treg may expand first in the LN, and then migrate to different lymphoid and nonlymphoid organs, because Fig. 3A shows a significant increase in CD4+CD25+ LN T cells by day 30. Additionally, we analyzed the MLR response of freshly isolated Treg, along with CD4+CD25– and CD4–CD25+ T cells from tolerant LNs (Fig. 5C). These latter cells constitute 15 and 22%, respectively, of the total LN population from tolerant animals (Fig. 3B). The results demonstrate that, as expected, the CD4+CD25+ T cells are anergic, whereas isolated CD4+CD25– and CD4–CD25+ (mostly CD8+, not shown) T cells from the tolerant animals are alloreactive. When these latter subsets are stimulated in the presence of the CD4+CD25+Foxp3+ T cells, the proliferative response is suppressed (Fig. 5C). These results demonstrate that the CD4+CD25+ Treg actively suppress alloreactive T cells, which have not been deleted, in the tolerant state.
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Because we previously demonstrated that T cell LN occupancy is required to induce alloantigen-specific tolerance (24), we hypothesized that Treg development within the LNs of tolerant animals is necessary for tolerance to ensue. We administered anti-CD62L mAb, which alters the distribution of T cells among secondary lymphoid organs (36) by inhibiting LN homing, alters the secondary lymphoid organ architecture (Fig. 2), and prevented allograft survival in animals receiving either anti-CD2 plus anti-CD3 or DST plus anti-CD40L mAbs (Fig. 1B). Because anti-CD62L mAb administration prevented tolerance in both groups, we examined whether this treatment also prevented the development or accumulation of Treg within the LN. The results in Fig. 6A when compared with those in Fig. 3B show that the CD4+ T cell population is markedly reduced in the LN, with a simultaneous decrease in CD4+CD25+ T cells, after treatment with anti-CD62L mAbs. Remarkably, whereas the total CD4+ T cell population increases in the spleen, there is no concomitant increase in the CD4+CD25+ splenic T cell population, suggesting that Treg failed to develop or expand in this compartment. The rejected grafts also accumulated CD4+ T cells and activated CD25+ cells. Taken together, the results indicate that after anti-CD62L mAb treatment, CD4+ T cells fail to enter or accumulate in the LNs. Furthermore, CD4+CD25+ Treg are absent in recipient LNs and other peripheral lymphoid and nonlymphoid sites, even though tolerogenic treatment had been administered, and rejection rapidly ensues. Phenotypic analyses of the CD4+CD25+ T cells within the LNs of anti-CD62L mAb-treated recipients reveal an activated CD44highCD45RBlowCD62LlowCD69high phenotype, rather than a regulatory CD62LhighCD69low one (Fig. 6B). Further evidence for the absence of Treg development in the periphery of these recipients is the relative lack of Foxp3 expression in the CD4+CD25+ T cells from LNs and spleens, measured by both fluorescent immunohistochemistry and quantitative real-time RT-PCR (Fig. 6, C and D).
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To further address the requirement for Foxp3-expressing CD4+CD25+ Treg within the LNs of tolerant animals, we hypothesized that if CD4+ T cells could be driven back into the LNs, this could restore the presence of an expanded Treg population in the LN and graft survival. FTY720 is a sphingosine lipid with agonist and antagonist properties on sphingosine 1-phosphate receptors, that drives T cells into LNs in a CD62L-independent manner (24, 37). When FTY720 was administered to rejecting anti-CD62L-treated animals, the CD4+CD25+ T cell population was restored to the LNs of FTY720-treated animals (Fig. 7A compared with Fig. 6A). Analysis of cell surface phenotype (Fig. 7B) and Foxp3 expression (C and D) of the CD4+CD25+ T cell populations demonstrated that these cells are CD44highCD45RBlowCD62LhighCD69low and Foxp3+, suggesting that CD4+CD25+ Treg developed and/or expanded in the LNs of tolerized animals, and are necessary for the induction and maintenance of tolerance.
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To address the mechanisms by which Treg may develop within the LNs of tolerant animals, we studied the chemokine receptor expression in the CD4+CD25+ T cells from the distinct animal groups. Table I shows that higher levels of the LN homing receptor CCR7 are expressed in the LN CD4+CD25+ T cells after treatment with DST plus anti-CD40L, when compared with naive or rejecting mice. Addition of anti-CD62L, which abrogates tolerance, resulted in a significant decrease in the LN CD4+CD25+ T CCR7 levels; an increase in the CCR5, expressed in activated cells; and no significant differences in CCR2 levels. Furthermore, addition of FTY720 re-established high levels of CCR7 RNA in LN CD4+CD25+ T cells, increased CCR2, and decreased CCR5 in a fashion similar to the tolerant group. Together, the data suggest active recruitment of T cells to the LNs during tolerance via CCR7 and CCR2 but not CCR5. Further genetic evidence supporting these results is shown in Fig. 8, where CCR2 knockout or paucity of LN T cells mice, lacking the expression of CCR7 ligands CCL21 and CCL19, rejected their allografts in both untreated recipients or after treatment with DST plus CD40L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Several laboratories, including our own (38, 39, 40, 41, 42), have reported the induction of transplantation tolerance using a variety of distinct approaches that interfere with signals 1, 2, and/or 3. The mechanisms that prevent graft rejection following these therapies have not been fully characterized, although evidence for anergy, ignorance, deletion, immune deviation, and immune regulation has been reported (43, 44, 45, 46, 47). Recent studies have also implicated Treg in tolerance induction (7, 48) and have shown that CD4+CD25+ Treg can also develop in the periphery through thymus-independent pathways (48, 49). To further address the role of Treg in tolerance, we induced tolerance to vascularized cardiac transplants by using two distinct tolerant protocols, and characterized the CD4+CD25+ T cell population in distinct anatomic compartments at different time points. In the anti-CD40L plus DST model, other investigators have provided evidence that anergy, activation-induced cell death, immune deviation, and Treg contribute to the induction and maintenance of tolerance (29). In the anti-CD2 plus anti-CD3 mAb model, graft survival and tolerance were related to mechanisms that included partial T cell activation, anergy, immune deviation, and partial T cell depletion (3, 30). In this report, we now provide evidence that both of these tolerance protocols also require the activity of Foxp3+CD4+CD25+ Treg and that these cells must be localized to the LNs but not the spleen.
Evidence from a variety of autoimmune studies suggests that the LN is a critical site for Ag presentation that determines either priming or tolerization (21). These models suggest that natural Treg expand in the pancreatic LN as a consequence of exposure to self-Ag, and failure to generate CD4+CD25+ T cells correlates with accelerated diabetes progression in the nonobese diabetic mouse (50, 51). The increase in CD4+CD25+ T cells occurs in the draining LN, but not the spleen or other peripheral LNs (6), supporting the idea that distinct anatomic sites play different roles in autoimmunity. Transplant studies are significantly different from the autoimmune model in many respects. With regard to the role of secondary lymphoid organs, recent reports demonstrated that alloantigen is presented not in a localized fashion, but systemically in all recipient lymphoid organs very soon after allografting (52, 53). As a result, multiple lymphoid tissues participate simultaneously in alloantigen presentation, which makes it more difficult to define the role of each secondary lymphoid organ and its microdomains in transplant rejection or tolerance. Our results here show at the structural level, that the LN architecture in the tolerized animals is preserved, and is similar to that of naive mice, whereas in the spleen the architecture is significantly altered (Fig. 2). Conversely, the architecture in the spleen of the acutely rejecting untreated mice is preserved, but is disorganized in the LNs. Thus, as in autoimmune models, the lymphoid organs and their anatomic structure where lymphocytes encounter and interact with alloantigen under the influence of systemic immunosuppression are critical determinants for the development of either immunity or tolerance. In particular, LNs are important for actively supporting tolerization.
Secondary lymphoid tissues have a highly organized architecture, regulating complex T cell-APC interactions (54, 55) that determine the development of either rejection or tolerance (20). Lakkis et al. (20) reported that asplenic Hox11–/– mice reject cardiac and skin allografts, whereas splenectomized aly/aly mice that lack all secondary lymphoid organs do not reject either skin or vascularized heart allografts. These results were interpreted as showing that secondary lymphoid organs are required for priming and initiation of an alloresponse, and indicate that the spleen is not absolutely necessary for rejection, as long as other lymphoid tissues are intact. Aly/aly mice reject heart but not skin grafts, arguing that it is the draining lymphoid tissues that are responsible for proper Ag presentation and T cell priming; and that the designation of "draining" is dependent on the type of allograft, its anatomic location, and the surgical manipulation required for its placement. In contrast, Zhou et al. (56) showed that splenectomized lymphotoxin-
and lymphotoxin-
receptor gene knockout mice, that also lack secondary lymphoid organs, reject skin and heart allografts, demonstrating that secondary lymphoid organs are not always necessary for initiating the alloimmune response. These contradictory results on the role of secondary lymphoid organs in transplant rejection may relate to the use of genetically altered mice, which have immunologic and developmental differences due to both gene knockout and background strain variations. We also suggest that experimental manipulations not only cause differences in T cell priming that lead to graft rejection, but also cause differences in T cell priming that lead to Treg development, with subsequent effects on suppressive and tolerogenic responses to alloantigen. Therefore, the study of lymphoid organs and domains that are responsible for priming and rejection must now be coupled to studies that analyze these same domains for the induction or maintenance of tolerance (57). This conclusion is supported by a recent report that shows that autoimmune diabetes is the result of the balance between effector and regulatory T cells in the pancreatic LN, and that evaluation of only a single T cell subset in the LN may lead to incomplete conclusions (58).
Finally, the mechanisms by which Treg accumulate or develop in the LN of tolerant animals may be related to chemokine compartmentalization. Table I shows that CCR7 is up-regulated in LN CD4+CD25+ T cells during tolerance, but not during rejection, which suggests that CD4+ T cells may migrate to the LNs where they further develop during tolerance. Therefore, it is possible that under the cover of immunosuppressive therapy such as anti-CD2 plus anti-CD3 mAb or DST plus anti-CD40L mAb, CD4+ T cells home to the LNs, where altered TCR signal transduction, partial T cell activation, and/or low-affinity Ag binding in the context of persistent allo- and tissue-specific Ag exposure, favor the development of Treg. Furthermore, chemokine-dependent LN T cell migration may represent a mechanism directly responsible for preventing CD4+ T cell-mediated rejection. Höpken et al. (59) recently described a key role for CCR7 in alloreactive T cell priming within the LNs, and specific localization of Treg within the lymphoid compartment may interfere with successful effector T cell priming that leads to graft rejection. This implies that anti-CD62L-treated T cells become primed to alloantigen, yet are not subject to tolerogenic influences because of their inability to home to the LNs, whereas FTY720 induces homing and colocalization of both effector and Treg cells to the LN.
We conclude that, whereas alloantigen can prime effector T cells either in the LNs or the spleen to initiate an immune response that leads to graft rejection, Treg can only be activated and/or expanded in an alloantigen-dependent manner in the LNs under systemic tolerization protocols. Our results reveal the location where thymic-derived CD4+CD25+ Treg may expand, and/or where CD4+ T cells may develop de novo into alloantigen-specific Treg in the peripheral lymphoid environment. CD4+CD25+ T cells localized near the high endothelial venule conduits within the LN microenvironment may help to maintain a tolerant environment by regulating multiple interactions between naive T cells with APC. This finding may help to unmask the factors that promote the differentiation and activation of regulatory suppressor cells and allow the generation of extrathymic Treg for the control of immune responses.
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1 This work was supported by National Institutes of Health Grants R01 AI41428, AI62765, and AI44929 (to J.S.B.). 
2 Address correspondence and reprint requests to Dr. Jonathan S. Bromberg or Dr. Jordi C. Ochando, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1104, New York, NY 10029-6574. E-mail address: jon.bromberg{at}msnyuhealth.org or jordi.ochando{at}mssm.edu
3 Current address: Bristol-Myers Squibb, Princeton, NJ 08543.
4 Abbreviations used in this paper: Treg, T regulatory cell; LN, lymph node; DST, donor-specific transfusion; SI, stimulation index; TMN, total mononuclear cells.
Received for publication November 15, 2004. Accepted for publication March 22, 2005.
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), or 1 x 107 donor splenocytes i.v. 7 days before transplant, and 250 µg of anti-CD40L mAbs on days –7, –4, 0, and +4 (
). Control rejecting mice received hamster Ig in PBS (
) (n = 6 for each group). Rejection of cardiac allografts was determined by cessation of heartbeat. B, Mice were treated as in A, along with 100 µg of anti-CD62L mAb administered on days 0 and 1 in the anti-CD2 plus anti-CD3 mAbs group, or on days –7 and –6 in the DST plus anti-CD40L mAb group. C, Mice were treated as in B plus FTY720 administered orally via gavage at 0.1 mg/kg/day. Representative allograft images of H&E staining of the above groups are shown. Tolerant animals >100 days; untreated control recipients on day 10 (time of acute rejection); rejecting anti-CD62L mAb-treated animals, 28.0 ± 6.8 days in the anti-CD2 plus anti-CD3 mAbs, and 39.6 ± 0.9 days in the DST plus anti-CD40L mAb-treated group; and tolerant FTY720-treated animals >100 days. Magnification, x100.
), and DST plus anti-CD40L (
). Histograms are representative of three independent studies. B, Foxp3 expression is up-regulated in the LNs of tolerant animals (>100 days) as measured by real-time RT-PCR from freshly isolated CD4+CD25+ T cells, when compared with naive or rejecting mice. Results representative of three independent studies are shown (*, p < 0.05; **, p < 0.01 by unpaired Student’s t test compared with naive control). C, Fluorescent immunohistochemistry of LN and spleen from different groups, showing CD4+ T cells (CD4–7-amino-4-methylcoumarin-3-acetic acid, blue) and Foxp3-expressing cells (Foxp3-Cy3, red) as merged (pink) images. The optical thickness of the image is 8 µm. Magnification, x400. Numbers represent number of Foxp3+CD4+ cells per square millimeter (*, p < 0.01 by Dunnett test compared with naive control). D, Foxp3 expression of CD4+CD25+ T cells over time measured by RT-PCR.
). CD4+CD25– effector T cells were cocultured at indicated ratios with CD4+CD25+ suppressor T cells and irradiated BALB/c T cell-depleted splenocytes. Results are expressed as SI, determined from mean of triplicate determinations ± SEM. Three sets of experiments were performed at each time point. B, Alloantigen specificity of the CD4+CD25+ T cells from tolerant animals at different indicated time points. Freshly isolated LN and splenic CD4+CD25+ T cells from naive and tolerant animals were cultured with naive CD4+CD25– (ratio 1:4), and with either alloantigen-specific BALB/c or third-party CBA T cell-depleted stimulator splenocytes. Results are expressed as SI, determined from mean of triplicate determinations ± SEM (**, p < 0.01 by one-way ANOVA). C, The CD4+CD25+ T cell population inhibits in vitro proliferation of other alloreactive populations. CD4+CD25+, CD4+CD25–, and CD4–CD25+ T cells from >100-day tolerant animals (2 x 105) were cultured with alloantigen-specific BALB/c T cell-depleted stimulator splenocytes (5 x 105). MLR results are expressed as SI, determined from mean of triplicate determinations ± SEM (**, p < 0.01 by one-way ANOVA).
