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Suppress a Stimulatory Graft-versus-Host Disease with a Lupus-Like Syndrome1
Departments of
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Medicine and
Pathology, Division of Rheumatology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
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induced both CD4+ and CD8+ cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF--treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF--conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity. |
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-chain of the IL-2R, comprise a unique lineage of thymus-derived regulatory T cells that have a potent contact-dependent mechanism of action (4, 5). These cells have been called "natural" regulatory T cells (6). Other T cells down-regulate immune responses by producing immunosuppressive cytokines. Some produce predominantly IL-10 (7) and others produce predominantly TGF- (8).
Our laboratory has made substantial progress in identifying cytokines involved in the generation ex vivo of three human regulatory T cell subsets from peripheral blood lymphocytes. Initially, we reported that CD8+ cells activated in the presence of IL-2 and TGF- develop potent suppressive activity (9, 10). Subsequently, we learned that naive CD4+ cells activated by alloantigens in the presence of TGF- become potent contact-dependent, cytokine-independent suppressor cells with properties similar, if not identical, to natural CD4+CD25+ regulatory T cells. Like their murine counterpart, they had potent inhibitory effects on CD8+ cells (11, 12). Most recently, we have induced CD4+CD25- cells stimulated with low-dose staphylococcus enterotoxin B in the presence of IL-2 and TGF- to develop strong TGF--dependent suppressor cell activity (13).
The objective of the present study was to determine the in vivo suppressive effects of regulatory T cells generated ex vivo. For this purpose, we selected a mouse model of an autoimmune syndrome that mimics systemic lupus erythematosus, the prototype of human autoimmune diseases. As described by others, the injection of parental DBA/2 (D2) T cells into (DBA/2 x C57BL/6)F1 mice rapidly results in a stimulatory graft-vs-host disease (sGVHD)3 (14, 15, 16, 17). Lymphoid hyperplasia, polyclonal B cell activation and anti-dsDNA Ab production occur within 2 wk, and an immune complex glomerulonephritis develops within 4–6 wk. We have learned that a single injection of T cells alloactivated with IL-2 and TGF- essentially prevented the development of the disease and doubled the survival of mice with established disease.
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Materials and Methods |
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Female DBA/2 (D2, H-2d), C57BL/6 (B6, H-2b), C3H/He (H-2k), and (DBA/2 x C57BL/6)F1 (DBF1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). T cells were prepared from spleen cells by collecting nylon wool column nonadherent cells (18). In some experiments, CD4+ cells were prepared by complement (Low-Tox; Cedarlane Laboratories, Hornby, Ontario, Canada) lysis of T cells stained with Abs to CD8, CD24, and I-Ad (BD PharMingen, San Diego, CA).
Conditioning of T cells with TGF- (TTGF-)
Nylon wool nonadherent T-enriched cells (1.5 x 106/ml) were stimulated with similar numbers of irradiated (2000 rad) B6 nylon adherent, non-T cells with or without TGF-1 (0.1 - 10 ng/ml) and low-dose rhuIL-2 (10 U/ml; R&D Systems, Minneapolis, MN) in AIM V (Invitrogen, Carlsbad, CA) serum-free medium for 5–6 days in 24-well plates (BD Biosciences Labware, Franklin Lakes, NJ). After centrifugation through Ficoll-Hypaque (Atlanta Biologicals, Norcross, GA) to remove dead cells, the cells were counted and injected into mice in the numbers indicated. The phenotype of the cells was determined by staining with anti-CD25 (BD PharMingen). Intracellular CTLA-4 (BD PharMingen) staining was performed using Fix and Perm (Caltag Laboratories, Burlingame, CA).
In vivo survival study
Five million D2 T cells that had been conditioned with 1 ng/ml TGF- (TTGF-), without TGF- (Tcon), or fresh (nonprimed) T cells were stained with CFSE (Molecular Probes, Eugene, OR) and transferred to syngeneic mice. Mice were sacrificed 1 day later to determine baseline numbers of each subset that had migrated to the spleen and lymph nodes and that were circulating in peripheral blood. Other animals were sacrificed at days 7 and 14 to the amount of cell division by changes in the mean fluorescence intensity. Absolute values for splenic CFSE-labeled T cells were determined by counting the spleen cells, staining them with anti-CD3, and determining the percentage of CFSE-labeled T cells.
Assessment of T regulatory activity in vitro
Suppressive activity by CD4+ and CD8+ cells conditioned with TGF- was assessed by their ability to inhibit T cell proliferation in vitro. Splenic T cells from D2 mice were cultured with irradiated splenic B6 non-T cells with or without TGF-1 (1 ng/ml) in AIM V serum-free medium for 6 days. CD4 or CD8 cells were isolated by cell sorting (purity >98%). The sorted cells were added to 1 x 105 CFSE-labeled T responder cells in ratios of 1:5–1:20 along with 1 x 105 irradiated B6 stimulator cells. In some experiments, 10 U/ml IL-2 was added to the cultures. The cells were cultured for 5–6 days in RPMI 1640 medium (Invitrogen) with 10% FBS (HyClone, Logan UT), were counted, and intensity of CFSE was assessed by flow cytometry using a FACStarPlus (BD Biosciences). The effect of conditioned CD4+ cells was determined by staining the cells with anti-CD8-PE and measuring the CFSE intensity of the CD8 cells. Similarly, with conditioned CD8 cells, the CFSE intensity of CD4+ cells was determined.
Induction of GVHD and adoptive transfers
A chronic sGVHD with a lupus-like syndrome was induced in DBF1 mice as described previously (17) by injecting 80 x 106 D2 spleen cells or 20 x 106 T cells into the tail vein. Other groups received this number of D2 cells plus 0.05–20 x 106 TTGF-, Tcon., or 0.5 ml of HBSS. In most experiments, there were six mice per group.
Assessment of the sGVHD
Before transfer and weekly thereafter, the animals were bled and serum IgG and anti-dsDNA autoantibodies were measured by an ELISA (19). All samples tested for anti-dsDNA were performed at the same time. Serum was diluted 1/400 or 1/800 for anti-DNA and 1/40,000 for measuring IgG. Proteinuria was measured using Albustix reagent strips (Bayer, Elkart, IN). Mice were sacrificed 2, 8, 10, and 12 wk after transfer of parental T cells for assessment of lymphoid hyperplasia and immune complex glomerulonephritis. The total number and phenotype of the spleen cells were determined from single-cell suspensions. The cells were stained with FITC-anti-H-2b, PE-anti-H-2d (BD PharMingen) and single-positive anti-H-2d cells considered to be parental D2 cells. Additional staining with anti-CD4-, anti-CD8-, or anti-CD19-conjugated biotin plus streptavidin-CyChrome (BD PharMingen) was used to determine the percentages of donor and F1 CD4+ and CD8+ T cells.
Histology and immunofluorescence
Kidneys were fixed in 10% buffered Formalin or frozen. H&E, and trichrome slides were prepared by standard techniques. The slides were numbered and examined by a pathologist who was unaware of the experimental data. Histopathologic evidence of glomerulonephritis was assessed using a semiquantitative (1–4+) scoring scale as follows: no evidence of endocapillary proliferation, extracapillary proliferation (crescents) or scarring = 0; 1–25% of glomeruli showed endocapillary or extracapillary proliferation or scarring = 1+; 26–50% or glomeruli showed proliferation or scarring = 2+; 51–75% of glomeruli showed proliferation or scarring = 3+; and 76–100% of glomeruli showed proliferation or scarring = 4+. Cryostat sections of frozen kidney tissue were examined for deposits of IgG using a standard procedure (20). Sections were incubated with fluorescein-labeled goat F(ab')2 IgG antiserum to mouse IgG. The sections were read blindly by the same investigator, grading the intensity of fluorescence from 0 to 4+.
Statistical analysis
Statistical comparison between groups of mice was performed by t test and Wilcoxon test survival curves with the log rank test using GraphPad PRISM software (GraphPad, San Diego, CA).
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induce alloactivated mouse CD4+ and CD8+ cells to become suppressor cells and enhance their viability in vitro and in vivo
Our first objective was to generate mouse T suppressor cells using our methods developed for human T cells (9, 10, 11, 13). Previously, we reported that costimulatory effects of IL-2 and TGF- on alloactivated human CD4+ cells up-regulated expression of CD25 and CTLA-4, markers displayed by regulatory T cells. We also documented increased expansion and resistance to apoptosis (11). Although human T cells produce sufficient IL-2 to overcome the inhibitory effects of TGF-, exogenous IL-2 was needed for costimulatory effects on mouse T cells. As shown in Fig. 1A, the addition of IL-2 (10 U/ml) to TGF- significantly altered CD25 and CTLA-4 expression by D2 CD4+ cells. Although peak expression of CD25 by control alloactivated CD4+ cells had occurred by day 3 or 4, the combination of IL-2 and TGF- further increased the percentages of cells displaying this marker on days 5 and 6. The combination of IL-2 and TGF- significantly increased expression of CTLA-4.
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(TTGF-) did not expand in primary culture, we considered that the principal costimulatory effects were due to protection of these cells from apoptosis. D2 T cells were stimulated with C57BL/6 (B6) alloantigens with IL-2 and TGF- for 4 days and restimulated without TGF- for an additional 6 days. Although TTGF- cells decreased by 30–45% in primary cultures, their number remained stable following restimulation (Fig. 1B). By contrast, T cells primed without TGF- (Tcon cells) increased initially following restimulation, but then rapidly decreased due to activation-induced apoptosis. Annexin staining revealed that >90% of Tcon had undergone apoptosis by day 6, whereas approximately one-half of TTGF- were viable at this time.
T cells primed with IL-2 and TGF- also had a survival advantage in vivo. A pilot experiment revealed that 15 million CFSE-labeled D2 TTGF- cells transferred to syngeneic mice underwent several cell divisions and comprised 10% of splenic T cells at 2 wk. However, they were no longer detectable at 1 mo because of dilution of CFSE staining (data not shown). Further studies with CFSE-labeled T cell subsets revealed that TTGF- and Tcon cells divide following adoptive transfer, but the numbers of Tcon cells rapidly fall and have almost disappeared at 2 wk (Fig. 1C). By contrast, the numbers of TTGF- cells, unlike the control, unprimed cells decreased much more slowly. The decrease of Tcon cells could not be attributed to dilution of CFSE by cell division since the mean fluorescence intensity of these cells was only slightly less than that of TTGF- cells. In other studies not shown, the mean fluorescence intensity of the three populations of CFSE-labeled T cells found in the lymph nodes and the blood was similar to those found in the spleen.
Suppressive activity of CD4+ and CD8+ T cells primed with IL-2 and TGF-
To determine the functional properties of TTGF- cells, they were sorted into CD4+ and CD8+ subsets and each was tested for an ability to suppress the proliferation of syngeneic alloactivated T cells. This was evaluated by documenting inhibitory effects on CFSE-labeled syngeneic responder T cells. The addition of CD4TGF- to responder T cells in only a 1:20 ratio markedly suppressed the activation of CD8+ cells. Most of the remaining CD8+ cells after 5 days of culture were small, resting T cells (see scatter profile, Fig. 2A, upper panel). By contrast, in cultures with a similar ratio of CD4con to responder cells, most CD8+ cells had become lymphoblasts and had divided several times (Fig. 2A, lower panel). Similar findings were observed with TGF--conditioned CD8+ cells gating on CFSE-labeled CD4+ cells (results not shown).
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conditioned CD4+ and CD8+ cells. Both CD4TGF- and CD8TGF- cells markedly suppressed T cell activation in a ratio of 1:10 or 1:20. In sharp contrast, CD4con and CD8con cells lacked suppressive activity. Thus, both CD4+ and CD8+ cells alloactivated in the presence of TGF- acquire very potent suppressive activities (Fig. 2B).
The addition of neutralizing anti-TGF- or anti-IL-10 Abs to cultures containing CD4TGF- and CD8TGF- and responder cells revealed that suppression was partially mediated by TGF-, but not by IL-10. The experiment shown in Fig. 2B also shows that anti-TGF- reduced, but did not eliminate suppression by CD4+ and CD8+ cells. In five separate experiments, at least 50% reversal of the suppression was observed in all studies with CD4TGF-. The effects were more variable with CD8TGF- cells. In one experiment, anti-TGF- had minimal effects and in two others <50% of the activity was abolished. These studies suggest that the suppressive effects are being mediated by at least two populations of regulatory T cells. One produces immunosuppressive levels of TGF- and the other has effects that are independent of TGF- and IL-10.
TTGF- cells can prevent the sGVHD with a lupus-like syndrome
As reported previously, the transfer of parental D2 T cells to DBF1 mice caused polyclonal B cell activation, anti-dsDNA autoantibodies, and proteinuria (14). These pathologic effects were abolished by priming D2 T cells with B6 alloantigens in the presence of IL-2 and TGF- (Fig. 3A). Moreover, adding these TTGF- cells to parental D2 splenic cells in a 1:4 ratio markedly suppressed, if not prevented the lupus-like syndrome. Examination of sera of F1 mice 2 wk following the adoptive transfer of D2 cells revealed markedly elevated IgG levels (>9 mg/ml) characteristic of polyclonal B cell activation. By contrast, mice that received TTGF- cells with D2 cells had IgG levels similar to those of control normal mice (Fig. 3B). Similarly, levels of anti-dsDNA Abs in mice that received T TGF- cells were much lower than mice that were injected with D2 T cells only or D2 T cells with Tcon cells (Fig. 3B). Repeat bleedings at 4 wk showed no evidence of polyclonal B cell activation in mice that had received TTGF- cells, and anti-dsDNA Ab production was also markedly suppressed compared with mice that received Tcon. By 8 wk, the urine of Tcon mice contained >1.5 g/dl protein, whereas mice that had received D2 T cells with TTGF- cells demonstrated only modest proteinuria (Fig. 3B). Finally, this suppressive effect of TTGF- cells was Ag specific. D2 T cells alloactivated with third-party alloantigens (C3H/He, H2k) in the presence of IL-2 and TGF- had no suppressive effects on the development of the lupus-like syndrome (Fig. 3C).
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-conditioned cells showed near normal glomeruli (0–1+ severity). In addition, glomerular IgG deposition was 3–4+ in the group that received D2 cells, 4+ in those that received D2 cells and Tcon, and 0–1+ in those that received D2 cells and TTGF-. A representative example of this pathology in each group is shown in Fig. 4.
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with parental T cells markedly inhibited the expansion of both host and donor lymphoid cells in F1 mice. Examination of the spleens from both groups of control mice sacrificed between 8 and 10 wk after transfer showed the characteristic lymphoid hyperplasia. Both T cells and B cells were increased and this effect was enhanced in mice that also received Tcon. By contrast, the addition of TTGF- to parental D2 T cells almost completely abolished the splenomegaly and the increase in T and B cells (Fig. 5). The absolute numbers of donor T cells in these mice were 3-fold less although the percentage of donor T cells in F1 mice that received TTGF- cells was only modestly decreased (Table I).
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Three experiments have documented that regulatory T cells generated ex vivo can ameliorate disease in mice that had already developed anti-dsDNA Abs. In the first experiment, 20 x 106 TTGF- or Tcon cells were transferred to groups of six F1 mice 2 wk after the injection of D2 parental spleen cells. Although Tcon cells enhanced anti-DNA autoantibody titers and accelerated the onset and magnitude of proteinuria over the next few weeks, TTGF- cells had the opposite effect and prolonged the life of mice with sGVHD. All mice from both control groups were dead at week 19, whereas three of six mice that had received TTGF- remained alive at that time (results not shown). At week 19, the spleen of a mouse that had just succumbed to sGVHD was characteristically enlarged in contrast to a sacrificed TTGF- which was normal (Fig. 6A).
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doubled the survival of the animals (Fig. 6D). A third experiment with six mice per group compared the effect of 0.5 and 0.05 million cells with 5 million cells. Again the five million dose doubled survival and a similar effect was observed in three of six animals that received 10-fold fewer cells. The protective effect was lost when the dose was decreased to 50,000 cells (Fig. 6E). |
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profoundly alters their functional activity and survival. This treatment abolished their capacity to cause a lupus-like syndrome when injected into F1 hybrid mice and prevented other T cells from developing this stimulatory chronic GVHD disease. In addition, these treated T cells inhibited the progression of established disease. A single dose of parental T cells that had been primed with TGF- (TTGF-) doubled the survival of F1 mice that had already developed anti-dsDNA autoantibodies. Unlike most alloactivated T cells that undergo apoptosis within 2 wk after secondary stimulation, the numbers of those alloactivated with TGF- declined much more gradually in vitro. Subsequent in vivo studies have revealed a 2- to 3-fold increase in the numbers of splenic CD4+CD25+ cells in D2 mice 1 mo after they received a single injection of 10 million TTGF- cells. The functional properties of these cells are currently under investigation.
The present results follow our observations that IL-2 and TGF- can induce human CD8+ cells to develop suppressive activity (9, 10), generate CD4+CD25+ cytokine-independent regulatory T cells (11), or induce CD4+CD25- cells to become TGF--producing CD4+ cells (13). In this study, treatment of alloactivated splenic T cells from DBA/2 mice with this combination of cytokines induced both CD4+ and CD8+ cells to develop potent suppressive activity and protected these cells from activation-induced apoptosis. These regulatory T cells were probably a mixture of several T regulatory cell subsets since their activity was only partially blocked by anti-TGF-. Consistent with studies of positively selected CD4+CD25+ regulating T cells, the suppression was Ag specific (21).
TTGF- cells rendered parental T cells tolerant to recipient histocompatibility Ags. They inhibited the expansion of both donor and recipient lymphocytes. As a consequence, polyclonal B cell activation was prevented, anti-dsDNA autoantibodies were markedly reduced, and clinically significant glomerulonephritis was averted. Thus, the characteristic lymphoid hyperplasia and immune injury induced by donor T cells transferred to F1 mice was prevented.
Significantly, a single injection of five million TTGF- cells transferred after the animals had developed anti-dsDNA Abs decreased the level of these autoantibodies, delayed and reduced proteinuria, and doubled the survival of the animals. Even a 10-fold lower dose had some protective effects. TGF- was responsible for these therapeutic effects since the transfer of control T cells alloactivated with IL-2 in the absence of this cytokine increased anti-dsDNA autoantibodies and the severity of immune complex glomerulonephritis. Thus, regulatory T cells generated ex vivo cannot only prevent immune-related tissue injury, but also can alter the course of established disease.
Others have previously reported that anergic, positively selected CD4+CD25+ T cells have a protective role in transplantation tolerance (22, 23, 24, 25, 26), and one group has recently confirmed our observation that IL-2 and TGF- enhance the growth of CD4+CD25+ regulatory cells and protect them from activation-induced cell death (23). Treatment of positively selected CD4+CD25+ cells with these cytokines enabled them to prevent acute GVHD (23). This group has subsequently shown that CD4+CD25- cells alloactivated ex vivo in the presence of TGF- and IL-10 can also prevent acute GVHD (27).
The CD4+ regulatory T cells generated in this study were probably a heterogeneous mixture of cytokine-independent and -dependent suppressor cells since their activity was only partially blocked by anti-TGF-. As described above, certain CD4+CD25+ T cells that coexpress CTLA-4 comprise a thymus-derived subset that has a contact-dependent, cytokine-independent mechanism of action (4, 5, 6). However, we have recently shown that conventional CD4+CD25- cells activated with a superantigen in the presence of TGF- express both CD25 and CTLA-4 and secrete immunosuppressive levels of this cytokine (13). At the present time there are no specific markers to distinguish natural, thymus-derived suppressor cells from subsets induced in the periphery.
There is accumulating evidence that the combination of IL-2 and TGF- support the growth, differentiation, and survival of several populations of regulatory T cells (28). TGF-, by itself, has well-known inhibitory effects on T cell function (29) and inhibits the differentiation of Th1 and Th2 cells (30, 31). The positive effects of TGF- are dependent upon a sufficient amount of IL-2 to overcome its inhibitory effects. In our studies with human T cells, we had observed that the addition of graded amounts of neutralizing anti-IL-2 Abs to allostimulated CD4+ T cells first abolished the costimulatory properties of TGF- and then brought out immunosuppressive effects of this cytokine. (S.G.Z. and D.A.H., unpublished observation). Although allostimulated human T cells produce enough IL-2 for the costimulatory effects of TGF- and suppressor cell differentiation (11), we needed to add IL-2 to mouse T cells to develop CD25+CTLA-4+ cells with potent suppressive activity. Others have also reported that the combination of IL-2 and TGF- protected T cells from apoptosis (32, 33, 34, 35), and this may occur by up-regulation of the antiapoptotic protein Bcl-xL (36). CD4+CD25+ cells regulatory T cells display IL-2R -chains (CD122) in addition to -chains (37, 38) and gene deletion studies have revealed an essential role for IL-2 and CD122 in the thymic development and growth of these regulatory T cells (39).
It is remarkable that the adoptive transfer of small numbers of regulatory T cells has such prolonged suppressive effects in vivo. Previously, we observed that the addition of <1% of TGF--primed CD4+CD25+ cells marked inhibited the activation of CD8+ T cells in vitro (11). This effect could be explained by the ability of these cells to transfer suppressive activity to other T cells through a phenomenon called "infectious tolerance" (40). Others have shown that natural CD4+CD25+ suppressor cells can induce conventional T cells to develop suppressive activity through infectious tolerance (41, 42). Recent studies in our laboratory have revealed that human CD4+CD25+ suppressor cells generated ex vivo with TGF- also have this effect (S.G.Z and D.A.H., unpublished observations). Thus, the combination of IL-2 and TGF- not only promotes the growth and survival of CD4+ regulatory T cells, but also enables these cells to transfer this function to other T cells.
The ability to induce several T cell subsets with IL-2 and TGF- ex vivo to develop potent suppressive effects has the potential to be used as an adoptive immunotherapy for certain autoimmune diseases. In systemic lupus erythematosus, impaired lymphocyte production of IL-2 and TGF- in systemic lupus erythematosus (43, 44, 45) presumably contribute to defective T regulatory cell activity in this disorder. Although these two cytokines are too toxic to administer in vivo, their use to generate regulatory T cells ex vivo offers a novel therapeutic approach. Moreover, the adoptive transfer of alloantigen-specific regulatory T cells generated ex vivo has the potential to block organ graft rejection by inducing immune tolerance. Although immunosuppressive agents currently in use have generally blocked acute graft rejection, they are less successful in preventing chronic rejection. Moreover, these agents have serious toxic side effects. Harnessing the body’s own immune system to block organ graft rejection has the potential to be more effective and safer than currently used therapies. Moreover, this approach avoids the problems caused by the immunogenicity of biologicals and gene therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Horwitz, Division of Rheumatology and Immunology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 711, Los Angeles, CA 90033. E-mail address: dhorwitz{at}usc.edu
3 Abbreviations used in this paper: sGVHD, stimulatory graft-vs-host disease; TTGF-, T cells allostimulated with IL-2 and TGF-; Tcon, T cells allostimulated with IL-2 only; GVHD, graft-vs-host disease; Treg, T regulatory cell.
Received for publication July 23, 2003. Accepted for publication October 21, 2003.
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, Animals injected with only HBSS; 
, animals that received 80 million fresh D2 spleen cells; 
, animals that received 20 million D2 T cells alloactivated in the presence of IL-2 for 6 days; •, animals that received 20 million D2 T cells alloactivated in the presence of IL-2 and TGF-
) or 20 (
) million TTGF-
). A, Spleen weights of a normal 25-wk-old DBF1 mouse and mice that were sacrificed 19 wk after receiving D2 cells (GVHD). One had received only D2 cells (GVHD) and one received syngeneic T cells primed with TGF-