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The Journal of Immunology, 2007, 179, 2808 -2814
Copyright © 2007 by The American Association of Immunologists, Inc.
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CD101 Surface Expression Discriminates Potency Among Murine FoxP3+ Regulatory T Cells1

Irina Fernandez*, Robert Zeiser{dagger}, Holger Karsunky*, Neeraja Kambham*, Andreas Beilhack{dagger}, Kalle Soderstrom*, Robert S. Negrin{dagger} and Edgar Engleman2,*

* Department of Pathology, and {dagger} Division of Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25–30% of CD4+CD25+ Treg and ~20% of conventional memory T cells. CD101high Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101low Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101high Treg, compared with CD101low Treg. Moreover, treatment with CD101high Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101high Treg all of the in vivo suppressor activity was contained within the CD62Lhigh subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Studies show CD4+CD25+ regulatory T cells (Treg)3 are critical for the induction and maintenance of tolerance toward self and foreign Ags (1, 2, 3). Based on their beneficial effects in animal models of autoimmunity (4), allograft rejection (5), and acute graft-vs-host disease (GVHD) (3, 6), Treg are promising candidates for clinical immunomodulatory therapy. A major issue in the characterization of Treg is the heterogeneity of suppressor function within the CD25-expressing CD4 T cell population. The most specific marker for Treg is FoxP3 (7, 8), which encodes a forkhead-winged-helix transcription factor designated Scurfin (9). However, FoxP3 is located in the nucleus, and identification of Treg based on FoxP3 expression requires cell permeabilization, which precludes isolation of viable cells. Other Treg markers have been described (10, 11, 12, 13, 14), but none distinguishes these cells from conventional T cells (Tconv) or delineates the potency of suppressor activity within activated Treg.

CD101 (previously known as V7) is a 1021 aa type I transmembrane glycoprotein containing seven Ig-like type IgV domains with seven glycosylation sites in the extracellular portion and a short cytoplasmic tail with several serine-threonine phosphorylation consensus sites (15, 16). CD101 is expressed on monocytes, granulocytes, dendritic cells (DCs), and activated T cells (16, 17, 18).

A mouse mAb directed against human CD101 inhibited the proliferative response of T cells to allogeneic cells or immobilized anti-CD3 Ab, suggesting that engagement of CD101 can antagonize TCR/CD3-mediated lymphocyte activation (17). Mechanistic studies showed that anti-human CD101 inhibits tyrosine phosphorylation of TCR/CD3-associated substrates, blocks translocation of NF-AT to the nucleus, and inhibits transcription of IL-2 (19, 20). Moreover, human peripheral CD8+ T cells that express CD101 and CD103 ({alpha}E integrin) display suppressive function after activation with intraepithelial cells (21) or placental trophoblasts (22). In addition to its role on T cells, triggering CD101 on human cutaneous DC inhibits T cell proliferation via IL-10 production, suggesting that the CD101 molecule may be involved in tolerance induction (23).

In light of these findings we generated a mAb against murine CD101 and investigated its expression on Tconv and Treg. The results show that CD101 cell surface expression is highly correlated with functional suppressor activity within CD4+CD25+ Treg both in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Ab generation

A rabbit mAb against murine CD101 was generated in collaboration with Epitomics using a full-length mouse CD101 cDNA as immunogen. The 3100 bp murine CD101 cDNA sequence was cloned into pcDNA3.1V5His (Invitrogen Life Technologies). The CD101 plasmid was transfected into a rabbit cell line against which rabbits were immunized. B cells were isolated and fused with the rabbit myeloma line 240E-w, an improved version of the original 240E (24), to generate the hybridomas. The hybridomas were screened for specific Ag recognition by FACS using a 293T CD101 transfectant. Twelve positive clones were identified and, after subcloning by limiting dilution, three subclones were chosen. A hybridoma, designated B6, was used to produce the rabbit mAb used in this study. The CD101 mAb was labeled with Alexa Fluor 488 using the Invitrogen Life Technologies protein labeling kit.

The following Abs were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2) and fluorochrome-conjugated Abs to CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61), Thy-1.1 (H1S51), Thy-1.2 (53-2.1), CD44 (IM7), CD62 ligand (CD62L, MEL-14), glucocorticoid-induced TNFR (GITR, DTA-1), CTLA-4 (UC10-4B9), TGF-β1 (catalog no. 555053; BD Biosciences), FoxP3 (FJK-16), CD127 (A7R34), and CD69 (H1.2F3). All Abs except anti-CD101 were purchased from BD Pharmingen or eBioscience. Analytical flow cytometry was performed on an LSRII (BD Biosciences).

Mice

FVB/N (H-2kq, Thy-1.1), C57BL/6 (H-2kb, Thy-1.1 or Thy-1.2) mice and BALB/c mice (H-2kd, Thy-1.2) were purchased from The Jackson Laboratory or Charles River Breeding Laboratory. Mice were used between 6 and 12 wk of age. Heterozygous luciferase transgenic (luc+) offspring of the transgenic founder line FVB-L2G85 (25) were used as indicated for transplantation experiments. To generate luc+ C57BL/6 animals, FVB-L2G85 mice were backcrossed into the C57BL/6 background, and luc+ C57BL/6 animals were used for the transplant experiments after 10 generations. All animal protocols were approved by the Committee on Use and Care of Laboratory Animals at Stanford University.

Cell isolation and sorting

For cell sorting, single-cell suspensions from peripheral lymph nodes and spleens were enriched for CD25+ cells after sequential staining with anti-CD25 PE (BD Pharmingen) and anti-PE magnetic beads using the AutoMACS system (Miltenyi Biotec). CD25+ cells were then stained with anti-CD4 allophycocyanin and anti-CD101 Alexa Fluor 488 and sorted on a FACSVantage SE (BD Biosciences) for the CD25high population (25–30% of the enriched CD25+ cells). Re-analysis of the sorted cells indicated that the CD4+CD25high population was >96% Foxp3+. The resultant CD4+CD25highCD101high and CD4+CD25highCD101low populations were each >95% pure. For MLRs, CD4+CD25– T cells were isolated from C57BL/6 splenocytes, which had been depleted of CD25+ cells using magnetic beads (Miltenyi Biotec) yielding a purity of ~90%. For Tconv (CD4+/CD8+), single-cell suspensions from peripheral lymph nodes and spleens were enriched for CD4+ and CD8+ T cells using anti-CD4 and anti-CD8 Ab-conjugated magnetic beads (Miltenyi Biotec).

MLR analysis

To evaluate Treg activity in vitro, 2 x 105 CFSE-labeled T cells (CD4+CD25–H-2kb+ Thy-1.2+) were incubated for 48 h with 2 x 105 gamma-irradiated (30 Gy) splenocytes (H-2kd+) in the presence of CD101high Treg (CD4+CD25highH-2kb+Thy-1.1+), CD101low Treg, or medium alone. For cell proliferation analysis CD4+CD25– T cells (1 x 107/ml) were resuspended in PBS and stained with Vybrant CFDA SE Tracer kit (Molecular Probes). Cultures were performed in triplicate in 96-well flat-bottom plates in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen Life Technologies), and 5 µg/ml 2-ME (Sigma-Aldrich). Cell division was monitored on the basis of CFSE dilution by flow cytometry.

GVHD model

GVHD was induced as previously described (6). Briefly, BALB/c recipients were given 5 x 106 T cell-depleted bone marrow cells after lethal irradiation with 800 cGy on day 0. To induce GVHD, 1 x 106 CD4+/CD8+ T cells (Tconv) from luc+ C57BL/6 donors were injected into the BALB/c on day 2. To prevent GVHD, 2.5 x 105 Treg were injected on day 0 resulting in a 1:4 ratio of Treg to Tconv. Mice were maintained on antibiotic water (sulfomethoxazole-trimethoprim; Schein Pharmaceutical) for the first 30 days. For histology, mice were euthanized 17 days after cell transfer. H&E staining of OCT-embedded tissue sections was performed according to standard protocols. Histologic GVHD scoring was performed by a pathologist (N.K.) who received coded OCT-embedded tissue samples of liver and small and large bowel and was blinded to the treatment groups. Evaluation of the stained tissue sections was performed on a Nikon microscope (Eclipse, TE 300).

TNF-{alpha} and IFN-{gamma} ELISA

Serum was collected from BALB/c recipients on days 8 and 17 after transplantation. ELISAs were performed according to the manufacturer’s instructions (R&D Systems). Briefly, samples were diluted 1/2 to 1/5, and TNF-{alpha} or IFN-{gamma} was captured by the specific primary mAb precoated on the microplate and detected by HRP-labeled secondary mAbs. Plates were read at 450 nm using a microplate reader (model Spectra Max 190; Bio-Rad). Recombinant murine TNF-{alpha} and IFN-{gamma} were used as standards. Samples and standards were run in duplicate, and the sensitivity of the assays was 16–20 pg/ml for each cytokine, depending on the sample dilution.

Statistical analysis

Differences in proliferation of Tconv by CFSE dilution or as photons/second/mouse and serum cytokine levels were analyzed using the two-tailed Student’s t test. Differences in animal survival (Kaplan-Meier survival curves) were analyzed by log-rank test. A value for p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD101 surface expression on lymphoid cells

To investigate CD101 surface expression on different cell populations and tissues, we generated a rabbit mAb against the murine CD101 molecule and conjugated it to Alexa Fluor 488 as described in Materials and Methods. In initial studies we stained splenic T cells and thymocytes from naive C57BL/6 mice with this Ab and found that CD101 expression was higher on splenic CD44+CD62L– memory T cells as compared with T cells with a naive (CD44lowCD62L+) phenotype (Fig. 1A). Among CD4+CD25+ T cells, 25–30% were CD101 positive, compared with <10% of CD4+CD25– T cells (Fig. 1B). A similar CD101 expression pattern was observed on single positive thymocytes (Fig. 1B). Intracellular staining for FoxP3 confirmed that 25% of FoxP3+ CD4+CD25+ T cells express CD101 (Fig. 1C). CD101 was also expressed by most DC, macrophages, and monocytes (data not shown), which is consistent with findings from the human system (17). Thus, expression of CD101 within the T cell compartment is mainly restricted to CD4+CD25+ Treg and Tconv with a memory phenotype. CD101 expression on T cells did not increase upon stimulation of the cells with allogeneic APCs (data not shown) making it unlikely that CD101 is simply a marker of activated Treg.


Figure 1
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  		FIGURE 1. Surface expression of CD101 on mouse lymphoid cells. Spleen cell suspensions and thymocytes were prepared from 10-wk-old C57BL/6 mice and stained with rabbit anti-CD101 Alexa Fluor 488 Ab. A, CD101 expression on memory (CD44+CD62L–) and naive (CD44lowCD62L+) spleen T cells. B, CD101 expression on CD4+CD25+ or CD4+CD25– thymocytes (CD4+CD3+) or spleen T cells (gated, left) is shown in the histograms (right). C, CD101 expression on FoxP3+CD4+CD25+ spleen Treg. The staining with a rabbit IgG isotype control (gray-filled histogram) is shown.

 
CD101 surface expression is predictive of Treg activity in vitro

To investigate whether Treg activity correlates with CD101 surface expression, we sorted CD4+CD25highCD101high and CD4+CD25highCD101low cells from the spleens of 10-wk-old mice and analyzed each population for their suppressor activity in the MLR (Fig. 2, A and B). CD4+CD25highCD101high Treg (H-2kb) displayed significantly greater suppressive activity as compared with CD4+CD25highCD101low Treg (H-2kb) (p < 0.05) based on inhibition of proliferation of CD4+CD25– T cells (H-2kb) in response to irradiated H-2kd stimulator cells (Fig. 2B). Suppression by both populations was dose-dependent and was not affected when CD101 was cross-linked with a secondary Ab (data not shown). This ~3-fold difference was observed at 2:1, 1:1 and 1:2 Treg to CD4+CD25– T cell ratios (Fig. 2B).


Figure 2
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  		FIGURE 2. CD101 surface expression is predictive of Treg activity, in vitro. A, Sorting of CD4+CD25+ Treg and subsequent isolation of CD101low (25.6%) and CD101high (24.2%) cells within this population. B, Proliferation of CD4+CD25– T cells in the MLR was measured by CFSE dilution. Different ratios of Treg and CD4+CD25– T cells are depicted. *, p < 0.05. C, Expression of the indicated surface markers on CD101high (gray-filled histogram) and CD101low Treg (black line histogram) is depicted. Staining with an isotype control Ab (gray-lined histogram) is shown.

 
We sorted CD4+CD25highCD101high and CD4+CD25high CD101low lymphocytes from the spleens of 10-wk-old C57BL/6 mice and analyzed each population for the expression of Treg markers. Both subpopulations displayed similarly high expression levels of GITR and CTLA-4 (Fig. 2C), which have been described as expressed on and functionally involved in Treg activity (10, 11, 26). Both populations were also similar with respect to expression of surface TGF-β1 and intracellular FoxP3 and IL-10 (Fig. 2C). CD127 was recently reported as a useful negative marker for human Treg (13, 14) and consistent with this finding we observed that both CD101high and CD101low Treg subsets had reduced CD127 expression as compared with CD4+CD25– T cells (Fig. 2C). CD101high Treg expressed high levels of CD44 and CD69, indicative of their mature activated phenotype (Fig. 2C).

CD101high Treg included CD62Llow as well CD62Lhigh populations (Fig. 3A), which could limit the ability of these cells to function in vivo because CD62L is required for homing to lymph nodes where Treg interaction with DC and Tconv takes place (27, 28). To rule out the possibility that CD62L expression affects the activity of CD101high Treg in vitro, we studied FACS sorted CD4+CD25+CD101highCD62Llow and CD4+CD25+CD101highCD62Lhigh Treg (Fig. 3A). Comparison of Treg suppressor activity against alloantigen driven expansion of CFSE-labeled CD4+CD25– T cells in vitro demonstrated no differences between these two subsets (Fig. 3B). These results are in accord with previously published data indicating no difference in Treg activity of CD62Llow and CD62Lhigh Treg in vitro where migration is dispensable for suppressor function (27). However our data extend these findings because we enriched for CD101high Treg before CD62L gating. In contrast to the CD101high Treg population, CD101low Treg were mainly CD62Lhigh (Fig. 3A).


Figure 3
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  		FIGURE 3. Inhibition of the MLR by CD62Llow and CD62Lhigh subpopulations of CD4+CD25highCD101high Treg. A, Expression of CD62L in CD4+CD25highCD101high (gray-filled histogram) and CD4+CD25highCD101low (dotted line histogram). Sorting of CD4+CD25highCD101high cells and subsequent isolation of CD62Llow and CD62Lhigh cells within this population (black arrows) is shown. Staining with an isotype control Ab (gray-lined histogram) is shown. B, Response of CD4+CD25– T cells to allogeneic stimulators in the presence of CD62Llow or CD62Lhigh Treg cells was measured by CFSE dilution. The results show the percentages of proliferating cells in the presence of different ratios of Treg to CD4+CD25– T cells. C, Expression of CD62L on whole Treg not divided according to CD101 surface expression.

 
CD101 expression correlates with potent Treg activity in a lethal GVHD model

To evaluate the in vivo activity of CD101high and CD101low Treg we used a previously described model of GVHD in which Treg were shown to be capable of suppressing CD4+/CD8+ T cells (Tconv) expansion (3, 6). We chose this model because expansion of CD4+ and CD8+ T cells from luc+ C57BL/6 (H-2kb) animals can be visualized in MHC class I and class II mismatched BALB/c (H-2kd) recipients using in vivo bioluminescence imaging. CD101high and CD101low Treg were isolated from normal C57BL/6 mice under the same conditions used for the in vitro studies and injected at a dose of 2.5 x 105 cells per animal together with 5 x 106 T cell-depleted bone marrow (TCD-BM) cells on day 0. To induce GVHD we administered 1 x 106 C57BL/6 Tconv to the bone marrow transplantation (BMT) recipients on day 2. Mice that received T cell-depleted bone marrow plus Tconv displayed vigorous Tconv expansion (Fig. 4A, left column) in contrast to animals that received Treg in addition to Tconv, either from CD101low or CD101high Treg subpopulations (Fig. 4A, middle and right column). By day 8, signals from luc+ Tconv projected over GVHD target organs such as skin, gastrointestinal tract, and liver (data not shown). Consistent with our in vitro findings, CD101high Treg were significantly more effective than CD101low Treg (p < 0.05) in preventing Tconv expansion, in vivo (Fig. 4B). CD101low Treg had measurable suppressor activity as evidenced by reduced T cell expansion in comparison to animals receiving Tconv alone (Fig. 4B).


Figure 4
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  		FIGURE 4. CD101high Treg reduce expansion of Tconv, production of proinflammatory cytokines, and mortality in a model of GVHD. GVHD was induced as described in Materials and Methods and five animals per group were treated as indicated with luc+ Tconv, Tconv plus CD101high Treg, or Tconv plus CD101low Treg. A, Photon emission pattern is shown from Tconv at days 6, 12, and 16 after BMT. Data are from one representative experiment with five animals per group. Results show effect on proliferation of luc+ Tconv (left) of co-transferred Treg, either CD101low Treg (middle) or CD101high Treg (right). B, Quantification of total body photon emission from luc+ T cells at serial time points after BMT shows reduction of signal intensity in animals receiving Tconv and CD101high Treg as compared with Tconv plus CD101low Treg. Data are pooled from two representative experiments. *, p < 0.05. C, Survival of BMT recipients is greater in animals receiving Tconv and CD101high Treg compared with animals receiving Tconv and CD101low Treg. Survival data from two independent experiments are combined. *, p < 0.05. D and E, Serum was collected from BMT recipients on days 8 and 17 after transplantation and assayed for IFN-{gamma} (D) or TNF-{alpha} (E) as indicated for five animals each. *, p < 0.05.

 
Mice given T cell-depleted bone marrow alone appeared healthy, and 100% of the animals survived for at least 80 days (Fig. 4C). Mice that received Tconv along with T cell-depleted bone marrow developed signs of severe GVHD, including reduced activity, hunched posture, ruffled fur, diarrhea, and weight loss and all animals died <45 days after transplantation. Treg (either CD101high or CD101low) reduced the incidence of GVHD-related death after BMT, but there was significantly greater reduction within the group that received CD101high Treg compared with animals that received CD101low Treg (p < 0.05) (Fig. 4C).

To evaluate the effect of Treg on production of proinflammatory cytokines we assayed serum on days 8 and 17 for IFN-{gamma} and TNF-{alpha}. The highest levels of these cytokines were detected on day 8 after BMT in animals that received Tconv without Treg (Fig. 4, D and E). IFN-{gamma} and TNF-{alpha} were significantly lower in animals that received CD101high Treg and Tconv compared with animals receiving CD101low Treg and Tconv (Fig. 4, D and E).

Assessment of target organ histopathology by an experienced pathologist (N.K.) who was blinded to the treatment groups demonstrated a significantly lower GVHD score in mice that received Tconv plus CD101high Treg as compared with Tconv plus CD101low Treg or Tconv alone (p < 0.05) (Fig. 5). Target organ damage in the mice treated with CD101high Treg was markedly suppressed, whereas damage in the CD101low Treg-treated group tended to be almost as severe as in the group that received only Tconv.


Figure 5
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  		FIGURE 5. Treatment with CD101high Treg reduces end organ damage in recipients of allogeneic bone marrow and Tconv. A, The cumulative histopathology score for the different experimental groups on day 17 is shown. Analysis included the large bowel, small bowel, and liver from BMT recipients. Coded tissue samples were scored by a pathologist blinded to the treatment groups as described in Materials and Methods. *, p < 0.05. B, Representative large bowel sections from the groups T cell-depleted bone marrow (TCD-BM) only (i), T cell-depleted bone marrow and Tconv (ii), T cell-depleted bone marrow and Tconv and CD101low Treg (iii), and T cell-depleted bone marrow and Tconv and CD101high Treg (iv). Black arrows indicate crypt abscesses.

 
To further delineate the relative contribution of the CD62Llow as compared with the CD62Lhigh subset within the CD101high Treg population, we transferred Tconv plus CD101highCD62Llow Treg or Tconv plus CD101highCD62Lhigh Treg to BMT recipients (Fig. 6A). Bioluminescence imaging of the expanding luc+ Tconv demonstrated that CD101high CD62Llow cells had little or no detectable in vivo activity, whereas their CD101highCD62Lhigh counterparts were highly effective in protecting against GVHD (Fig. 6, A and B). This translated into significantly improved survival of BMT recipients receiving Tconv plus CD101highCD62Lhigh Treg as compared with animals receiving Tconv plus CD101highCD62Llow Treg (p < 0.05) (Fig. 6C). Of interest, only a single recipient of CD101highCD62Lhigh cells died during the experiment and this animal showed no signs of GVHD. The surface expression of CD69 and CD44 was not significantly different on CD101high CD62Llow Treg and CD101high CD62Lhigh Treg before transfer (Fig. 6D).


Figure 6
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  		FIGURE 6. CD62L expression is critical for CD101high Treg function in vivo. GVHD was induced as described in Materials and Methods and five animals per group were treated as indicated with luc+ Tconv, Tconv plus CD101high CD62Llow Treg, or Tconv plus CD101highCD62Lhigh Treg. A, Photon emission pattern from the indicated groups at days 6 and 12 after BMT. Data are from five animals per group. B, Quantification of total body photon emission from luc+ T cells at serial time points after BMT. *, p < 0.05. C, Percentage of survival of BMT recipients in animals receiving Tconv plus CD101highCD62Lhigh Treg compared with animals receiving Tconv plus CD101highCD62Llow Treg. *, p < 0.05. D, Expression of CD69 and CD44 was analyzed by FACS on the transferred CD101highCD62Llow Treg and CD101highCD62Lhigh Treg.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cell surface markers that have the potential to distinguish functional subpopulations of CD4+CD25+ Treg from other cells are under intense investigation. Although CD127 negativity correlates with the degree of suppressor activity within human Treg (13, 14), a positive marker could be useful for isolation of the population with the highest biological activity within the Treg compartment. We have previously described the transmembrane glycoprotein CD101 as a negative regulator of T cell activation in human cells (20). Generation of a monoclonal anti-mouse CD101 Ab allowed us to characterize murine T cell populations for CD101 surface expression. Our initial studies revealed that CD101 is expressed on ~25% of CD4+CD25+FoxP3+ cells. Further investigation disclosed a direct association between the expression of CD101 and in vitro and in vivo suppressor potency within CD4+CD25+FoxP3+ cells that were otherwise phenotypically similar.

CD101 expression was found on T cells that constitutively express several surface markers characteristic of memory T cells, which is consistent with previous studies of Treg (29). Importantly, alloactivation with MHC mismatched APCs did not increase CD101 expression on Treg, making it unlikely that CD101 is a marker that distinguishes activated from nonactivated Treg. GITR and CTLA-4 are both expressed on Treg (10, 11, 26); however, these molecules are also expressed on conventional effector T cells, limiting their use as Treg markers (30, 31). Given its pattern of expression on T cells, CD101 is probably best used in combination with other Treg markers, such as CD25 or GITR, to identify maximally potent Treg.

To probe Treg activity in vivo we used an aggressive GVHD model in which Treg were adoptively transferred to BMT recipients that also received conventional CD4+ and CD8+ T cells. The superior potency of CD101high Treg observed in vitro in the MLR was also seen in vivo. CD101high Treg limited expansion of allogeneic T cells, inhibited production of proinflammatory cytokines and reduced the incidence and severity of GVHD. In another GVHD model where Treg prevent GVHD, we found that Treg home to and expand in secondary lymphoid organs after BMT (32). This process is dependent on inflammatory stimuli and interaction with DC (33). Because Treg expansion is essential for suppressor function in this GVHD model, it is possible that CD4+CD25+FoxP3+ cells that lacked CD101 were only weakly suppressive because they failed to expand in vivo. However, based on our in vitro data in which equal numbers of CD101high and CD101low Treg were used and showed differential suppressor activity, it is unlikely that a difference in the rate of cellular expansion alone explains the difference in the suppressive activity of these populations seen in vivo.

Phenotypic analysis indicated that the CD101high Treg subset contains distinct subpopulations of CD62L high and low cells and that CD69 and CD44 expression does not differ markedly between these subpopulations. Previous studies have demonstrated that CD44 is present on highly suppressive Treg (34), which is consistent with our observation that CD44 expression is higher on CD101high as compared with CD101low Treg. Our data showed that the CD62L high and low subpopulations within the CD101high Treg population are equally suppressive in vitro. However, these subpopulations are not equal in their capacity to prevent GVHD as indicated by the observation that all of the in vivo suppressor activity is confined to the CD62Lhigh subpopulation of CD101high cells. This observation is consistent with previous studies showing that Treg capable of inhibiting GVHD are all CD62Lhigh (27, 28), and likely reflects the critical role of CD62L in homing of Treg to lymph nodes where they interact with DC and Tconv.

The ligand for CD101 is unknown. However, two recent studies of gene clusters associated with autoimmune type 1 diabetes susceptibility in the NOD mouse (Idd loci) identified CD101 as a candidate gene involved in the development of diabetes (35, 36). Genetic polymorphisms that lead to an altered amino acid structure of the CD101 protein could ultimately lead to numeric (37) or functional deficits of the Treg pool as has been reported for autoimmune type 1 diabetes. Further studies with CD101-deficient animals are warranted to determine whether CD101 has relevance for Treg ontogeny, expansion or function. CD101 is also expressed on a subset of human FoxP3+ Treg (our unpublished data), suggesting that it may be useful for isolation and diagnostic monitoring of Treg.

In conclusion, our findings indicate that the Treg population consists of at least two subsets with differential suppressor activity. Furthermore, the results demonstrate the utility of CD101 as a marker of potent Treg in vitro and in vivo.


   Acknowledgments
 
We thank Lorna Tolentino and Jolley Beth Lim for cell sorting, Paula Colmenero and Ines Mende for helpful discussion, and Claudia Benike for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by Grants RO1 CA0800065, P01 HL075462, and RO1 AR051748 from the National Institutes of Health and by Dr. Mildred-Scheel-Stiftung, Germany (to R.Z.). Back

2 Address correspondence and reprint requests to Dr. Edgar Engleman, Department of Pathology, Stanford Blood Center, 3373 Hillview Avenue, Palo Alto, CA 94304. E-mail address: edengleman{at}stanford.edu Back

3 Abbreviations used in this paper: Treg, CD4+CD25+ regulatory T cell; GITR, glucocorticoid-induced TNFR; DC, dendritic cell; GVHD, graft-vs-host disease; CD62L, CD62 ligand; BMT, bone marrow transplantation. Back

Received for publication June 19, 2007. Accepted for publication June 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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