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-Dependent Mechanism
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* Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; and
National Food Research Institute, Tsukuba, Ibaraki, Japan
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on the surface after activation, and exert regulatory function by the membrane-bound TGF- in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25-, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF- to the active form. In the CD4+CD45RBhigh-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+LAP+ and CD25+LAP- cells, but also by CD25-LAP+ cells. CD4+CD25-LAP+ T cells were part of the CD45RBlow cell fraction. CD4+CD25-LAP-CD45RBlow cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25-LAP+ cells was abrogated in vivo by anti-TGF- mAb. These results identify a new TGF--dependent regulatory CD4+ T cell phenotype that is CD25- and LAP+. |
Introduction |
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mAb (3, 4) or anti-IL-10R mAb (5) can reverse the regulatory function of CD4+CD45RBlow and/or CD4+CD25+ T cells in the CD4+CD45RBhigh-reconstituted immunodeficient mouse model of colitis, and anti-TGF- polyclonal Ab and/or anti-IL-10 mAb can reverse the inhibitory effect of allergen-specific CD4+CD45RBlow T cells at the effector phase in a mouse model of allergic asthma (6). Our group has shown that suppression by CD4+CD25+ T cells can be partially reversed by soluble TGF-RII-Fc chimera and/or soluble IL-10R-Fc chimera in vitro (7). Also, suppression by human thymus CD4+CD25+ regulatory T cells is partially blocked by anti-TGF- (8). These in vivo and in vitro data indicate that the regulatory function of CD4+CD25+ T cells appears to be mediated in part by cytokines, although this issue remains controversial (2, 9).
To address this apparent discrepancy, viz., no detectable cytokine secretion despite cytokine-dependent suppression by CD4+CD25+ T cells, Nakamura et al. (10) showed that CD4+CD25+ T cells express TGF- on their surface and mediate their suppressive function by presenting surface TGF- to a receptor(s) on target cells by cell-to-cell contact. They used a chicken anti-TGF- polyclonal Ab and a mouse anti-human latency-associated peptide (LAP)3 mAb (27232.11) for staining. LAP is the amino-terminal domain of TGF- precursor peptide; it remains noncovalently associated with TGF- peptide after cleavage and forms the latent TGF- complex (11). In vitro, CD4+CD25+ T cells become positive for surface TGF- or LAP, as measured by these Abs, only after strong stimulation (10). Preactivated human thymus CD4+CD25+ regulatory T cells also express membrane-bound TGF-, which is important for their regulatory function (8).
Although CD25+ expression is well correlated with the regulatory activity in vitro, CD4+CD25+ T cells are not the only regulatory CD4+ T cells in vivo. In the model of colitis induced by CD4+CD45RBhigh T cells in immunodeficient mice, CD4+CD25-CD45RBlow T cells have been reported to protect against the disease, although they may be less efficient than the same number of CD4+CD25+ cells (4, 12). In a diabetes model of islet -cell-specific TCR-transgenic x RAG-/- mice, CD4+DX5+cells from normal NOD mice, which were shown not to be NKT cells, have regulatory activity regardless of CD25 expression (13). In a rat model of diabetes, in addition to CD4+CD25+ T cells, CD4+CD25-CD45RC- T cells also suppress disease onset (14). In allogeneic transplantation models in mice and rats, both CD4+CD25+ and CD4+CD25- T cells from tolerant mice have transferable suppressive activity against graft rejection (15, 16).
Here we demonstrate that LAP expression, as measured by a biotinylated goat anti-LAP polyclonal Ab, identifies a regulatory T cell population in CD4+CD25- T cells, and that the action of these regulatory T cells is TGF- dependent.
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Materials and Methods |
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Female BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Eight- to 11-wk-old mice were used for in vitro experiments. Male BALB/c mice and male SCID mice purchased from Taconic Farms (Germantown, NY) were used for colitis experiments under virus Ab-free conditions in the animal resource facility of Harvard Medical School. There were no phenotypic differences between the two BALB/c strains.
Reagents for flow cytometry and cell isolation
Affinity-purified biotinylated goat anti-LAP polyclonal Ab, biotinylated normal goat IgG, normal goat IgG, mouse anti-LAP mAb 27232.11, and recombinant human LAP were purchased from R&D Systems (Minneapolis, MN). Goat anti-thrombospondin-1 (anti-TSP-1) and -2 polyclonal Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit anti-TSP polyclonal Ab was obtained from NeoMarkers (Fremont, CA). Anti-CD16/CD32 (FcBlock), anti-CD3 (145-2C11), FITC-anti-CD4 (GK1.5), PerCP-Cy5.5-anti-CD4 (RM4-5), CyChrome-anti-CD4 (RM4-5), PE-anti-CD25 (PC61), FITC-anti-CD45RB (16A), PE-CTLA-4 (9H10), streptavidin (SA)-PE, and SA-allophycocyanin were purchased from BD PharMingen (San Diego, CA). SA-Tricolor was obtained from Caltag Laboratories (Burlingame, CA). FITC-donkey anti-chicken IgY polyclonal Ab and PE-F(ab')2 donkey anti-goat IgG polyclonal Ab were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). 7-Amino-actinomycin D (7-AAD) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-PE microbeads, anti-FITC microbeads, and SA microbeads were obtained from Miltenyi Biotec (Auburn, CA). Hybridomas for neutralizing anti-TGF- mAb (1D11, mouse IgG1) and anti-KLH mAb (SOL, mouse IgG1) as control Ab were obtained from American Type Culture Collection (Manassas, VA). The Abs were purified from ascites from nude mice by ammonium precipitation, followed by T-GEL absorbent (Pierce, Rockford, IL).
Cell purification
Spleen cells were prepared by mechanical disruption, followed by lysis of RBC with ACK buffer. The T cell fraction was first enriched by T cell enrichment columns (R&D Systems). For sorting of CD4 cells by CD25 and LAP, T cells were stained with biotinylated anti-LAP, then with SA-Tricolor, PE-anti-CD25, and FITC-anti-CD4. CD25+ and/or LAP+ cells were positively selected on an LS MACS column using anti-PE microbeads, which can bind both PE and Tricolor. The CD25+ and/or LAP+ fraction was further sorted into CD4+CD25+LAP+, CD4+CD25+LAP-, and CD4+CD25-LAP+ cells using a FACSVantage SE (BD Biosciences, Mountain View, CA). The CD25-LAP- flow-through fraction was also sorted into CD4+CD25-LAP- cells by FACS. In SCID colitis Experiment I, CD4+CD25-CD45RBhigh cells were sorted from the T cell fraction by FACS after staining with CyChrome-anti-CD4, PE-anti-CD25, and FITC-anti-CD45RB. In SCID colitis Experiments II and III, the T cell fraction was stained with FITC-anti-CD25 after incubation with FcBlock, and CD25+ cells were depleted by a BS or CS MACS column using anti-FITC microbeads. Small portions were taken before and after the depletion of CD25+ cells to confirm the depletion efficiency by staining with PE-anti-CD4 and 7-AAD. The remaining CD25- T fraction was then labeled with biotinylated anti-LAP followed by SA microbeads. An aliquot was checked for LAP and CD45RB expression by staining with FITC-anti-CD45RB, PE-anti-CD4, and SA-Tricolor. The labeled cells were enriched into LAP+ and LAP- fractions on an LS MACS column. Both the LAP+ fraction and the LAP- fraction were stained with FITC-anti-CD45RB, PE-anti-CD4, and SA-Tricolor, then sorted into CD4+LAP+CD45RBlow cells from the LAP+ fraction or into CD4+LAP-CD45RBhigh and CD4+LAP-CD45RBlow cells from the LAP- fraction, respectively, by FACS. Exact or typical purities are shown in Figs. 2 and 5.
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The presence of autofluorescent cells may result in misinterpretation of FACS staining. This problem was minimized by staining total spleen with 7-AAD after staining with Abs, and 7-AAD+ dead cells and autofluorescent cells were excluded in the FL3 channel in a FACSort equipped with argon and 632-nm diode lasers or using FACSCalibur (BD Biosciences). When cells were stained with four colors of Abs or stained intracellularly, the T cell-enriched fraction from the T cell enrichment columns (R&D Systems), rather than whole spleen cells, was used, since this T enrichment step removes almost all autofluorescent cells (not shown), and dead cells were excluded by the forward and side scatter profile. All staining was performed after blocking Fc receptor with FcBlock. Cells were first stained with biotinylated anti-LAP to exclude the possibility of nonspecific interaction with other Abs and then were stained with SA-allophycocyanin and other fluorochrome-labeled Abs, except for double staining with anti-TSP. For TSP staining, cells were first stained with goat anti-TSP, followed by PE-anti-goat IgG, then the cells were blocked with an excess of purified goat IgG. After this step, cells were stained with biotinylated anti-LAP and with other Abs as described above. CTLA-4 was stained intracellularly using the Cytofix/Cytoperm kit (BD PharMingen) after surface staining as described above.
In vitro culture
DMEM (BioWhittaker, Walkersville, MD) containing 10% FCS (BioWhittaker), antibacterial/micotics mixture (Invitrogen, Carlsbad, CA), 5 mM HEPES, 1 mM sodium pyruvate, and 50 µM 2-ME was used for in vitro culture. For the proliferation assay, Thy-1-depleted APCs were prepared from BALB/c spleen cells on an LD MACS column with anti-Thy-1 microbeads and were irradiated with 3000 rad. The presence of in vitro regulatory activity of CD25+LAP+, CD25+LAP-, and CD25-LAP+ cells was tested by mixing these cell populations with 5 x 104 CD4+CD25-LAP- cells at ratios of 1/8 and 1/2. These mixtures or 2.5 x 104 cells being tested for regulatory function were stimulated with soluble anti-CD3 (1 µg/ml) plus 5 x 104 APCs for 72 h in round-bottomed, 96-well plates. The cells were pulsed with 1 µCi of [3H]thymidine for the last 12 h, and thymidine incorporation was measured with a liquid scintillation counter. For the cytokine ELISA, 1 x 105 purified cells were stimulated with plate-bound anti-CD3 (10 µg/ml) in round-bottomed, 96-well plates for 40 h, and the supernatant was collected. When TGF- was measured, serum-free medium (X-vivo20; BioWhittaker) supplemented with 50 µM 2-ME was used instead of FCS-containing DMEM.
ELISA
IL-2, IL-4, IL-10, and IFN-
were measured with Ab sets from BD PharMingen as described previously (7). TGF- was measured by the TGF-1 Emax ImmunoAssay System (Promega, Madison, WI) with or without acidification according to the manufacturer’s instruction.
Induction of colitis
The well-characterized CD4+CD45RBhigh-induced SCID colitis model was used (17). In brief, the indicated number of sorted (see above) CD4+CD25-CD45RBhigh cells (colitis experiment I) or CD4+CD25-LAP-CD45RBhigh cells (colitis experiments II and III) from 9-wk-old male BALB/c mice were injected into 9-wk-old male SCID mice i.p. on day 0. Various cell fractions, sorted as described above, were injected at the same time to test for regulatory function. In colitis Experiment III, anti-TGF- mAb (1D11) or control mAb (SOL) was injected i.p. weekly (2.5 mg/mouse) starting on day -1. Animals were weighed for up to 60 days as an indicator of colitis. For mice that died during the experiments, the weight measured just before death was used unless or until the average weight of its group was lower than that of the dead animal.
Statistics
Weight changes in the colitis experiments are presented as the mean of changes from the initial weights ± SEM. Data on day 60 were analyzed using one-way ANOVA, followed by Tukey multiple comparisons. An associated p < 0.05 was considered significant.
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Results |
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Nakamura et al. (10) showed that a small fraction (
15%) of CD4+CD25+ cells, but not of CD4+CD25- cells, stained with biotinylated chicken anti-TGF- Ab or mouse LAP mAb, and that the percentage of positive cells increased after activation in vitro. However, both Abs stained CD4+CD25+ cells brightly only after stimulation in vitro (10) and thus could not be used to purify unstimulated LAP+ cells. To overcome this problem, we used an affinity-purified biotinylated goat anti-LAP Ab for staining that brightly stained a small fraction of murine CD4+ cells without stimulation (Fig. 1A). The addition of a 500-fold excess of recombinant human LAP diminished staining by the biotinylated goat anti-LAP Ab to a similar level as control biotinylated goat IgG (Fig. 1A). In addition, all CD4+LAP+ cells, both CD25+ and CD25-, were weakly positive with an unlabeled form of chicken anti-TGF- Ab, followed by FITC-anti-chicken IgY (not shown), whereas CD4+LAP- cells were uniformly negative with this Ab. Hereafter, we refer to these goat anti-LAP-stained cells as LAP+ cells unless otherwise noted. CD4+LAP+ cells existed in both CD25+ and CD25- populations, consisting of 1 and 3–5% of CD4+ cells, respectively (Fig. 1A).
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peptide, and its association with mature TGF- confers latency to TGF- activity, we asked whether these LAP+ cells have only latent TGF- without biological activity or whether the LAP+ cells have a TGF- activation mechanism(s) that would be a prerequisite for their being TGF--dependent regulatory T cells. TSP-1 is known to activate latent TGF- either alone (18) or in combination with urokinase (19). We thus checked for TSP expression and found that almost all CD4+LAP+ cells expressed TSP (Fig. 1B). Dead cells and autofluorescent cells were excluded by gating in the FL3 channel after 7-AAD staining, so this double-positive population was not from autofluorescent cells, was not the result of nonspecific binding of the Abs to dead cells, and was not secondary to an artificial interaction between biotinylated goat anti-LAP Ab and goat anti-TSP Ab, as control Abs did not produce such staining patterns (not shown). The same staining pattern was also seen with rabbit anti-TSP Ab, followed by anti-rabbit IgG-PE (not shown). These results demonstrate that CD4+LAP+ cells have both latent TGF- and the TGF- activation molecule TSP on their surface, and thus have the potential to activate TGF- by themselves. In addition to CD25, the CD45RBlow phenotype is another marker for regulatory CD4+ T cells. This marker is important especially in vivo, since CD4+CD25-CD45RBlow cells also have a regulatory function in the colitis model of CD4+CD45RBhigh-transferred immunodeficient mice, although they seem to have a weaker regulatory function than CD4+CD25+ cells (4, 12). Thus, we checked CD45RB expression in the four fractions defined by CD25 and LAP expression (Fig. 1C). As reported previously (1), CD4+CD25+LAP+ and CD4+CD25+LAP- cells were exclusively CD45RBlow, while CD4+CD25-LAP+ cells were mostly CD45RBlow, but contained a small amount (up to 10% in the lymphocyte gate) of cells with extremely high expression of CD45RB (CD45RBhigh++). However, we eventually noted that these CD4+CD25-LAP+CD45RBhigh++ cells were not T cells, since they were CD3-, B220+, Ly6G/C+ (unpublished observation), corresponding to the recently reported murine type I IFN-producing cells (IPCs) or plasmacytoid cells (20, 21, 22, 23). CD4+CD25-LAP- cells contained both CD45RBlow cells and CD45RBhigh cells.
CTLA-4 has been shown to be important in the regulatory function of CD4+CD25+ T cells in vitro and in vivo (4, 24). Thus, we checked CTLA-4 expression by intracellular staining along with surface LAP staining. As reported, CD4+CD25+ cells as a group expressed more CTLA-4 than did CD4+CD25- cells (not shown). In the CD4+CD25+ fraction, the expression of CTLA-4 was greater for CD25+LAP+ cells than for CD25+LAP- cells (Fig. 1D). CTLA-4 expression was much less for CD25-LAP+ cells and was negative for CD25-LAP- cells (Fig. 1D).
Cytokine production from CD4+ cells sorted by CD25 and LAP expression
Since the LAP molecule is closely connected to TGF-, we checked the production of TGF- and other cytokines from CD4+ cells sorted by CD25 and LAP expression in vitro. Spleen cells from BALB/c mice were sorted into four fractions: CD4+CD25+LAP+, CD4+CD25+LAP-, CD4+CD25-LAP+, and CD4+CD25-LAP- cells. Typical staining profiles and purities were shown in Fig. 2A. The cells were stimulated with plate-bound anti-CD3 for 40 h. This time point was chosen because incubation for >48 h resulted in the death of CD4+CD25+LAP+ cells and significant, but lower, loss of viability in CD4+CD25-LAP+ cells (not shown). The production of total TGF- was consistently higher in CD4+CD25+LAP+ cells and CD4+CD25-LAP+ cells than in CD4+CD25+LAP- cells and CD4+CD25-LAP- cells at this early time point (Fig. 2B). CD4+CD25+LAP- cells also produced larger amount of TGF- than did CD4+CD25-LAP- cells, and the amount increased with culture hours up to 72 h (data not shown), reflecting the good viability of this fraction. The amount of free active TGF-, measured without acidification, was below the detection limit (<15 pg/ml) in all samples. CD4+CD25+LAP+ and CD4+CD25-LAP+ cells also produce high levels of IL-10 compared with LAP- fractions. Although there was a tendency for CD25-LAP+ cells to produce more IL-10 than CD25+LAP+ cells, this was not seen in all experiments. CD4+CD25-LAP+ cells also produced IL-2, IL-4, and IFN- consistent with their CD45RBlow memory phenotype.
In vitro regulatory activity of CD4+ cells sorted by CD25 and LAP
Since TGF- is one of the strongest immunosuppressive factors and has been shown to be involved in CD4+CD45RBlow and/or CD4+ CD25+ regulatory function in vitro (7, 10) and in vivo (3, 4, 6), we investigated whether the expression of LAP correlated with regulatory function in vitro. Splenic CD4+ cells sorted by CD25 and LAP expression were added to cultures of 5 x 104 CD4+CD25-LAP- cells in the indicated numbers and stimulated with soluble anti-CD3 (1 µg/ml) plus irradiated Thy-1-depleted APCs for 72 h. CD4+CD25+LAP+ and CD4+CD25+LAP- cells equally suppressed proliferation of CD4+CD25-LAP- cells (Fig. 3). However, CD4+CD25-LAP+ cells did not suppress CD4+CD25-LAP- cells.
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Although LAP expression did not correlate with regulatory function in the coculture assay, in vitro assays do not always reflect function in vivo. We observed massive cell death of LAP+ cells and loss of bright LAP expression during in vitro culture (not shown), which might explain the inability to demonstrate in vitro suppression by CD25-LAP+ cells. Thus, we tested the regulatory activity of the various cell populations in vivo in the well-described colitis model in which immunodeficient mice are injected with CD4+CD45RBhigh cells from wild-type mice. SCID mice were injected with CD4+CD25-CD45RBhigh cells (2 x 105) plus CD4 cells (2 x 105) sorted by CD25 and LAP expression (except for 8 x 104 cells from the CD4+CD25+LAP+ fraction because of low cell recovery). Both CD4+CD25+LAP+ cells and CD4+CD25+LAP- cells inhibited weight loss in the CD4+CD45RBhigh-induced colitis (p < 0.01; Fig. 4), consistent with the results of the in vitro coculture assay. However, as opposed to the in vitro results, CD4+CD25-LAP+ cells were as potent as both of the CD25+ cell populations in inhibiting weight loss (p < 0.01). CD4+CD25-LAP- cells, which contained the both CD45RBhigh and CD45RBlow fractions, had a tendency to ameliorate the weight loss to some extent, but this did not reach significance (p > 0.05).
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CD4+CD25-CD45RBlow cells also have been found to regulate CD4+CD45RBhigh-induced colitis, although perhaps not as well as CD4+CD25+ cells (4, 12). A majority of CD4+CD25-LAP+ cells were CD45RBlow, but up to 10% of CD4+CD25-LAP+ cells in the lymphocyte gate showed a CD45RBhigh++ phenotype (Fig. 1B). These CD4+CD25-LAP+CD45RBhigh++ cells were not T cells, but correspond to recently reported (20, 21, 22, 23) murine IPCs (our unpublished observations). Since it has been shown that IPCs can differentiate into DC2 in human (25), it is thus possible that these cells could possess regulatory function. Thus, we asked whether CD4+CD25-LAP+CD45RBlow T cells, which excluded CD45RBhigh++ IPCs, were truly regulatory, and if so, whether they were the sole regulatory fraction in CD4+CD25-CD45RBlow T cells. We injected 4 x 105 CD4+CD25-LAP-CD45RBhigh cells along with either CD4+CD25-LAP+CD45RBlow cells or CD4+CD25-LAP-CD45RBlow cells (1 x 105) from BALB/c mice into SCID mice. Profiles of the cells during purification and after sorting are shown in Fig. 5A. After the transfer, changes in weight were monitored. CD4+CD25-LAP+CD45RBlow cells reversed the weight loss induced by CD4+CD25-LAP-CD45RBhigh cells (p < 0.01; Fig. 5B). CD4+CD25-LAP-CD45RBlow cells, however, did not significantly protect the mice from weight loss (p > 0.05). Thus, LAP+ T cells are a major regulatory fraction in CD4+CD25-CD45RBlow T cells in the colitis model.
Regulatory function of CD4+CD25-LAP+CD45RBlow cells is TGF- dependent
We then investigated whether the regulatory function of CD4+CD25-LAP+ cells is TGF- dependent. SCID mice were coinjected with 3.6 x 105 CD4+CD25-LAP-CD45RBhigh cells and 8.4 x 104 CD4+CD25-LAP+CD45RBlow cells from BALB/c mice. The mice were treated with anti-TGF- mAb (1D11) or control mAb (SOL) weekly i.p. starting 1 day before the injection of cells. Anti-TGF- had a small worsening effect on the colitis induced with CD4+CD25-LAP-CD45RBhigh cells (Fig. 6, 
; p < 0.05 vs the control mAb-treated group (
) on day 57, but not significantly different on other days). Anti-TGF- inhibited the protection by CD4+CD25-LAP+CD45RBlow cells (Fig. 6, 
; p < 0.01 vs CD4+CD25-LAP+CD45RBlow-coinjected and control mAb-treated group (
)), and the weight loss was the same as that in the mice injected only with CD4+CD25-LAP-CD45RBhigh cells and treated with anti-TGF- (Fig. 6, ; not significant at any time point).
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1 (LAP+ cells) regardless of whether these cells are CD25- or CD25+; furthermore, we show that CD25-LAP+ T cell regulation is inhibitable with concomitant administration of anti-TGF-. These data strongly suggest that the dominant regulatory cells affecting CD4+CD45RBhigh-induced colitis express TGF- either on the surface or in the secretion pattern.
A notable feature of CD4+LAP+ cells that we defined by the biotinylated goat anti-LAP Ab is the positivity for TSP. TSP can convert latent TGF- to the active form in a cell-free system (18) and is important in TGF- activation in certain cell culture systems (26, 27). Recently, TSP-deficient mice were reported to show autoimmune disease-like inflammation in several organs similar to that seen in TGF-1-deficient mice, and this inflammation was abrogated by treatment with a TSP-derived peptide that could activate TGF-1 (28). Thus, TSP is also important in TGF- activation in vivo. It is thought that TSP changes the conformation of latent TGF- so that TGF- peptide can be recognized by a TGF- receptor(s) (29) rather than removing LAP from active TGF- peptide by proteolysis as other proteinases do. Thus, it is possible that that TSP is also involved in the regulatory function of CD4+LAP+ cells. The human leukemia T cell line MOLT16 produces TGF- (30), and we have found that this MOLT16 line is LAP+ and TSP+ and presents active TGF- to other cells only by cell-to-cell contact (unpublished observations). We are also using this cell line as a model of LAP+ T cells to clarify TGF- activation and presentation.
Although the surface phenotype, cytokine production, and in vitro suppressive function of CD4+CD25+LAP+, CD4+CD25+LAP-, and CD4+CD25-LAP+ cells are different from one another, it is possible that these regulatory cells change their phenotype depending on the activation state and/or milieu, and CD4+CD25-LAP+ cells derive from CD4+CD25+ cells and represent different stages of the same population. For example, some CD4+CD25+ cells become CD25- when they are transferred alone into immunodeficient mice (12, 31), and CD4+CD25+ cells need the presence of CD4+CD25- cells to maintain CD25 expression (12). The possible conversion among the four fractions is best determined in a congenic mouse system in which the origin of cells can be distinguished by a congenic marker, and the degree to which pathogenic or regulatory T cells exist in the four fractions after the disease can be measured by functional analysis in secondary transfer experiments. One difference between the CD25+-derived CD25- regulatory cells studied previously and those studied here is that the former manifest suppressive activity in vitro (31), whereas our CD25- cells do not. One explanation for this is that the CD4+CD25-LAP+ cells in fresh spleen cells, as opposed to phenotypically similar cells described previously, produce IL-2 (Fig. 2B) and therefore make responder cells resistant to TGF--mediated suppression. Another explanation is that we observed massive cell death of LAP+ cells and loss of bright LAP expression during in vitro culture. However, it should be noted that these cells act as protective cells in vivo in CD4+CD45RBhigh-induced colitis.
Anti-TGF- treatment inhibited the in vivo regulatory activity of CD4+CD25-LAP+ cells, highlighting the importance of TGF- for the regulatory activity. At present we do not know at which stage of the disease process TGF- acts to regulate CD4+CD45RBhigh-induced colitis.
Both TGF- and IL-10 have been reported to be important cytokines in a number of systems in which regulatory cells have been studied (32, 33, 34). Although anti-TGF- abrogated the protection by CD4+CD25-LAP+ T cells, in some systems TGF- exerts its regulatory function in coordination with IL-10 in vitro (35, 36) and in vivo (37, 38); thus, IL-10 could also be involved in the in vivo suppressive effect of CD4+CD25-LAP+ T cells that we observed.
In summary, we have shown that LAP staining by a biotinylated goat anti-LAP Ab identifies a TGF--dependent regulatory cell in the in vivo colitis model. Our findings identify a new regulatory T cell phenotype that is CD25- and LAP+.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Howard L. Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 730, Boston, MA 02115. E-mail address: hweiner{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: LAP, latency-associated peptide; 7-AAD, 7-amino-actinomycin D; DC, dendritic cell; IPC, IFN-producing cell; SA, streptavidin; TSP, thrombospondin.
Received for publication September 12, 2002. Accepted for publication December 24, 2002.
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References |
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  N. Sasaki, T. Yamashita, M. Takeda, M. Shinohara, K. Nakajima, H. Tawa, T. Usui, and K.-i. Hirata Oral Anti-CD3 Antibody Treatment Induces Regulatory T Cells and Inhibits the Development of Atherosclerosis in Mice Circulation, November 17, 2009; 120(20): 1996 - 2005. [Abstract] [Full Text] [PDF]
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 H. Wu, E. Center, G. Tsokos, and H. Weiner Suppression of murine SLE by oral anti-CD3: inducible CD4+CD25-LAP+ regulatory T cells control the expansion of IL-17+ follicular helper T cells Lupus, June 1, 2009; 18(7): 586 - 596. [Abstract] [PDF]
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 H. Y. Wu, F. J. Quintana, and H. L. Weiner Nasal Anti-CD3 Antibody Ameliorates Lupus by Inducing an IL-10-Secreting CD4+CD25-LAP+ Regulatory T Cell and Is Associated with Down-Regulation of IL-17+CD4+ICOS+CXCR5+ Follicular Helper T Cells J. Immunol., November 1, 2008; 181(9): 6038 - 6050. [Abstract] [Full Text] [PDF]
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M.-L. Chen, B.-S. Yan, Y. Bando, V. K. Kuchroo, and H. L. Weiner Latency-Associated Peptide Identifies a Novel CD4+CD25+ Regulatory T Cell Subset with TGF{beta}-Mediated Function and Enhanced Suppression of Experimental Autoimmune Encephalomyelitis J. Immunol., June 1, 2008; 180(11): 7327 - 7337. [Abstract] [Full Text] [PDF]
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Y. A. Taher, B. C. A. M. van Esch, G. A. Hofman, P. A. J. Henricks, and A. J. M. van Oosterhout 1{alpha},25-Dihydroxyvitamin D3 Potentiates the Beneficial Effects of Allergen Immunotherapy in a Mouse Model of Allergic Asthma: Role for IL-10 and TGF-{beta} J. Immunol., April 15, 2008; 180(8): 5211 - 5221. [Abstract] [Full Text] [PDF]
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R. A. Derks, E. Jankowska-Gan, Q. Xu, and W. J. Burlingham Dendritic Cell Type Determines the Mechanism of Bystander Suppression by Adaptive T Regulatory Cells Specific for the Minor Antigen HA-1 J. Immunol., September 15, 2007; 179(6): 3443 - 3451. [Abstract] [Full Text] [PDF]
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J. A. Kapp, K. Honjo, L. M. Kapp, K. Goldsmith, and R. P. Bucy Antigen, in the Presence of TGF-beta, Induces Up-Regulation of FoxP3gfp+ in CD4+ TCR Transgenic T Cells That Mediate Linked Suppression of CD8+ T Cell Responses J. Immunol., August 15, 2007; 179(4): 2105 - 2114. [Abstract] [Full Text] [PDF]
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 H. Ishikawa, H. Ochi, M.-L. Chen, D. Frenkel, R. Maron, and H. L. Weiner Inhibition of Autoimmune Diabetes by Oral Administration of Anti-CD3 Monoclonal Antibody Diabetes, August 1, 2007; 56(8): 2103 - 2109. [Abstract] [Full Text] [PDF]
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Y. Futagami, S. Sugita, J. Vega, K. Ishida, H. Takase, K. Maruyama, H. Aburatani, and M. Mochizuki Role of Thrombospondin-1 in T Cell Response to Ocular Pigment Epithelial Cells J. Immunol., June 1, 2007; 178(11): 6994 - 7005. [Abstract] [Full Text] [PDF]
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R. Gandhi, D. E. Anderson, and H. L. Weiner Cutting Edge: Immature Human Dendritic Cells Express Latency-Associated Peptide and Inhibit T Cell Activation in a TGF-beta-Dependent Manner J. Immunol., April 1, 2007; 178(7): 4017 - 4021. [Abstract] [Full Text] [PDF]
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 A. Toda and C. A. Piccirillo Development and function of naturally occurring CD4+CD25+ regulatory T cells J. Leukoc. Biol., September 1, 2006; 80(3): 458 - 470. [Abstract] [Full Text] [PDF]
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 A. Metwali, T. Setiawan, A. M. Blum, J. Urban, D. E. Elliott, L. Hang, and J. V. Weinstock Induction of CD8+ regulatory T cells in the intestine by Heligmosomoides polygyrus infection. Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G253 - G259. [Abstract] [Full Text] [PDF]
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M. Veldhoen, H. Moncrieffe, R. J. Hocking, C. J. Atkins, and B. Stockinger Modulation of Dendritic Cell Function by Naive and Regulatory CD4+ T Cells J. Immunol., May 15, 2006; 176(10): 6202 - 6210. [Abstract] [Full Text] [PDF]
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C. Baecher-Allan, E. Wolf, and D. A. Hafler MHC class II expression identifies functionally distinct human regulatory T cells. J. Immunol., April 15, 2006; 176(8): 4622 - 4631. [Abstract] [Full Text] [PDF]
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 X. Zhang, J. Reddy, H. Ochi, D. Frenkel, V. K. Kuchroo, and H. L. Weiner Recovery from experimental allergic encephalomyelitis is TGF-{beta} dependent and associated with increases in CD4+LAP+ and CD4+CD25+ T cells Int. Immunol., April 1, 2006; 18(4): 495 - 503. [Abstract] [Full Text] [PDF]
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 W. Wang, X. R. Huang, A. G. Li, F. Liu, J.-H. Li, L. D. Truong, X. J. Wang, and H. Y. Lan Signaling Mechanism of TGF-{beta}1 in Prevention of Renal Inflammation: Role of Smad7 J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1371 - 1383. [Abstract] [Full Text] [PDF]
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 S. M. Pop, C. P. Wong, D. A. Culton, S. H. Clarke, and R. Tisch Single cell analysis shows decreasing FoxP3 and TGF{beta}1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes J. Exp. Med., April 18, 2005; 201(8): 1333 - 1346. [Abstract] [Full Text] [PDF]
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J. Shimizu, R. Iida, Y. Sato, E. Moriizumi, A. Nishikawa, and Y. Ishida Cross-Linking of CD45 on Suppressive/Regulatory T Cells Leads to the Abrogation of Their Suppressive Activity In Vitro J. Immunol., April 1, 2005; 174(7): 4090 - 4097. [Abstract] [Full Text] [PDF]
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C. Di Giacinto, M. Marinaro, M. Sanchez, W. Strober, and M. Boirivant Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-{beta}-Bearing Regulatory Cells J. Immunol., March 15, 2005; 174(6): 3237 - 3246. [Abstract] [Full Text] [PDF]
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L. Fahlen, S. Read, L. Gorelik, S. D. Hurst, R. L. Coffman, R. A. Flavell, and F. Powrie T cells that cannot respond to TGF-{beta} escape control by CD4+CD25+ regulatory T cells J. Exp. Med., March 7, 2005; 201(5): 737 - 746. [Abstract] [Full Text] [PDF]
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R. K. Gregg, R. Jain, S. J. Schoenleber, R. Divekar, J. J. Bell, H.-H. Lee, P. Yu, and H. Zaghouani A Sudden Decline in Active Membrane-Bound TGF-{beta} Impairs Both T Regulatory Cell Function and Protection against Autoimmune Diabetes J. Immunol., December 15, 2004; 173(12): 7308 - 7316. [Abstract] [Full Text] [PDF]
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S. Makita, T. Kanai, S. Oshima, K. Uraushihara, T. Totsuka, T. Sawada, T. Nakamura, K. Koganei, T. Fukushima, and M. Watanabe CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells J. Immunol., September 1, 2004; 173(5): 3119 - 3130. [Abstract] [Full Text] [PDF]
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C. Schramm, S. Huber, M. Protschka, P. Czochra, J. Burg, E. Schmitt, A. W. Lohse, P. R. Galle, and M. Blessing TGF{beta} regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo Int. Immunol., September 1, 2004; 16(9): 1241 - 1249. [Abstract] [Full Text] [PDF]
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A. M. Blum, A. Metwali, D. E. Elliott, D. J. Berg, and J. V. Weinstock CD4+ T cells from IL-10-deficient mice transfer susceptibility to NSAID-induced Rag colitis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G320 - G325. [Abstract] [Full Text] [PDF]
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S. M. Wahl, J. Swisher, N. McCartney-Francis, and W. Chen TGF-{beta}: the perpetrator of immune suppression by regulatory T cells and suicidal T cells J. Leukoc. Biol., July 1, 2004; 76(1): 15 - 24. [Abstract] [Full Text] [PDF]
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 T. L. Sumpter and D. S. Wilkes Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1129 - L1139. [Abstract] [Full Text] [PDF]
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J. R. Torrealba, M. Katayama, J. H. Fechner Jr., E. Jankowska-Gan, S. Kusaka, Q. Xu, J. M. Schultz, T. D. Oberley, H. Hu, M. M. Hamawy, et al. Metastable Tolerance to Rhesus Monkey Renal Transplants Is Correlated with Allograft TGF-{beta}1+CD4+ T Regulatory Cell Infiltrates J. Immunol., May 1, 2004; 172(9): 5753 - 5764. [Abstract] [Full Text] [PDF]
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X. Zhang, D. N. Koldzic, L. Izikson, J. Reddy, R. F. Nazareno, S. Sakaguchi, V. K. Kuchroo, and H. L. Weiner IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells Int. Immunol., February 1, 2004; 16(2): 249 - 256. [Abstract] [Full Text] [PDF]
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L. A. Stephens, A. N. Barclay, and D. Mason Phenotypic characterization of regulatory CD4+CD25+ T cells in rats Int. Immunol., February 1, 2004; 16(2): 365 - 375. [Abstract] [Full Text] [PDF]
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K. Nakamura, A. Kitani, I. Fuss, A. Pedersen, N. Harada, H. Nawata, and W. Strober TGF-{beta}1 Plays an Important Role in the Mechanism of CD4+CD25+ Regulatory T Cell Activity in Both Humans and Mice J. Immunol., January 15, 2004; 172(2): 834 - 842. [Abstract] [Full Text] [PDF]
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 J V Weinstock, R Summers, and D E Elliott Helminths and harmony Gut, January 1, 2004; 53(1): 7 - 9. [Full Text]
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 L. Cosmi, F. Liotta, E. Lazzeri, M. Francalanci, R. Angeli, B. Mazzinghi, V. Santarlasci, R. Manetti, V. Vanini, P. Romagnani, et al. Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes Blood, December 1, 2003; 102(12): 4107 - 4114. [Abstract] [Full Text] [PDF]
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D. A. Horwitz, S. G. Zheng, and J. D. Gray The role of the combination of IL-2 and TGF-{beta} or IL-10 in the generation and function of CD4+ CD25+ and CD8+regulatory T cell subsets J. Leukoc. Biol., October 1, 2003; 74(4): 471 - 478. [Abstract] [Full Text] [PDF]
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C. Asseman, S. Read, and F. Powrie Colitogenic Th1 Cells Are Present in the Antigen-Experienced T Cell Pool in Normal Mice: Control by CD4+ Regulatory T Cells and IL-10 J. Immunol., July 15, 2003; 171(2): 971 - 978. [Abstract] [Full Text] [PDF]
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