The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.
Right arrow Articles by Kwon, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.
Right arrow Articles by Kwon, B.
The Journal of Immunology, 2006, 176: 5223-5231.
Copyright © 2006 by The American Association of Immunologists

Conversion of Alloantigen-Specific CD8+ T Cell Anergy to CD8+ T Cell Priming through In Vivo Ligation of Glucocorticoid-Induced TNF Receptor1

Juyang Kim*, Woon S. Choi*, Hyun Kang*, Hye J. Kim*, Jae-Hee Suh{dagger}, Shimon Sakaguchi{ddagger} and Byungsuk Kwon*,2

* Immunomodulation Research Center (IRC) and Department of Biological Science, University of Ulsan, Ulsan, Republic of Korea; {dagger} Department of Pathology and Biomedical Research Center, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Republic of Korea; and {ddagger} Department of Experimental Pathology, Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced TNF receptor (GITR) in a murine model of systemic lupus erythematosus-like chronic graft-vs-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD toward aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cell anergy. Functionally active donor CD8+ T cells produced high levels of IFN-{gamma} and had an elevated CTL activity against host Ags. In in vitro MLR, anergic responder CD8+ T cells were generated, and DTA-1 stimulated the activation of these anergic CD8+ T cells. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, were responsible for the DTA-1-mediated conversion of cGVHD to aGVHD. These results indicate that donor CD8+ T cell anergy is a restriction factor in the development of aGVHD and that in vivo ligation of GITR prevents CD8+ T cell anergy by activating donor CD8+ T cells that otherwise become anergic. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD vs aGVHD and as a target for therapeutic intervention in a variety of related diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral T cells can be activated in response to invading foreign Ags through the engagement of the TCR with a MHC-antigenic peptide complex. In the absence of costimulation, however, peripheral T cell anergy is induced such that Ag-specific T cells enter into a state of hyporesponsiveness to subsequent Ag encounter. Conversely, enforcing signaling through costimulatory receptors (e.g., CD28, CD40, OX40, or 4-1BB) simultaneously with TCR engagement prevents T cell anergy and reverses established anergy in vivo ( 1, 2, 3, 4, 5).

In the murine parent-into-unirradiated F1 graft-vs-host (GVHD)3 model, the activity of donor CD8+ T cells plays a critical role in determining the development of acute GVHD (aGVHD) vs chronic graft-vs-host (cGVHD). Under conditions in which the CTL activity of donor CD8+ T cells is suppressed, aGVHD is converted to cGVHD ( 6, 7, 8, 9). In contrast, it is not clear that enforcing the graft’s cytotoxicity against the host can induce the conversion of cGVHD to aGVHD. In cGVHD, in contrast to aGVHD, donor CD8+ T cells are poorly engrafted into the host, and the engrafted CD8+ T cells have no cytotoxicity against host cells ( 10). This might indicate that donor CD8+ T cells quickly fall into anergy after transplantation into cGVHD recipients.

Glucocorticoid-induced TNF receptor (GITR), a member of the TNF receptor superfamily, is constitutively expressed on naive T cells and is up-regulated after activation ( 11, 12). GITR expression is more prominent on CD4+CD25+ regulatory T (Treg) cells, where GITR signals abrogate the suppressive function ( 11, 12). However, recent studies have demonstrated that GITR functions as a costimulatory molecule for conventional CD25–CD4+ and CD8+ T cells in vitro ( 13, 14, 15, 16, 17) and in vivo ( 18), and that GITR-deficient Treg cells have suppressive activity equal to that of wild-type Treg cells ( 14). The in vivo role of GITR remains largely unknown. In this study, we present evidence that GITR engagement can prevent CD8+ T cell anergy in cGVHD. These findings reveal a novel mechanism for the effect of GITR in the stimulation of T cell response in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female DBA/2 (H-2d) and BDF1 ((C57BL/6 x DBA/2)F1) (H-2b/d) mice, 6–8 wk of age, were purchased from Charles River Laboratories. All mice were maintained in pathogen-free conditions. These studies were approved by the institutional animal care committee.

Abs and reagents

The anti-GITR mAb (DTA-1) was described previously ( 11) and purified from ascites. Control rat IgG was purchased from Sigma-Aldrich. DTA-1 was conjugated with FITC, according to a standard method. The following FITC-, PE-, PerCP-, or biotin-conjugated mAbs to mouse cell surface molecules were purchased from BD Biosciences Pharmingen: CD3, CD4, CD8, CD25, CD49d, CD62L, B220, H-2Kb, CCR5, and IFN-{gamma}. Streptavidin-Cy (CyChrome), -FITC, and -PE were also purchased form BD Biosciences Pharmingen. HRP-conjugated rat anti-mouse IgG1 was purchased from Southern Biotechnology Associates.

Induction of cGVHD

Single-cell suspensions in PBS were prepared from the spleens and lymph nodes of normal DBA/2 parental donors, filtered through a sterile mesh (BD Falcon), and washed. After the erythrocytes were lysed in hemolysis buffer (144 mM NH4Cl and 17 mM Tris-HCl; pH 7.2), the remaining cells were resuspended at 8 x 107 cells/0.2 ml in PBS. cGVHD was induced by transfer of 8 x 107 of DBA/2 parental cells into the tail vein of normal, unirradiated BDF1 mice. Immediately thereafter, 200-1000 µg of DTA-1 or control Ig was administered i.p. In some experiments, CD4+ T cells, CD8+ T cells, or Treg cells were removed from DBA/2 spleen/lymph node cells by anti-CD4-, anti-CD8-, or anti-CD25-conjugated magnetic beads (Miltenyi Biotec), respectively. FACS analysis showed that >90% of the target cells were deleted. The remaining cells (8 x 107) were transferred into BDF1 mice to induce cGVHD.

T cells and APCs

Total T cells, CD4+ T cells, or CD8+ T cells were purified from the spleens and lymph nodes of naive DBA/2 mice or BDF1 mice with cGVHD, using anti-CD3-, anti-CD4-, or anti-CD8-conjugated microbeads (Miltenyi Biotec), respectively. The resulting cells were >90% pure. APCs were isolated from naive BDF1 spleens by two methods. In the first method, APCs were isolated from BDF1 spleens by removing T cells, using anti-CD3-conjugated microbeads. These APCs were used for coculture with naive DBA/2 T cells. In the second method, BDF1 splenocytes were incubated in 10-cm plates at 37°C for 2 h to remove nonadherent lymphocytes. The adherent cells were further incubated overnight at 37°C. These APCs were irradiated (30 Gy) before being used to stimulate T cells of DBA/2 origin.

GITR expression and T cell proliferation in in vitro MLR

T cell cultures were set up in 24-well plates containing 1 ml of RPMI 1640 medium supplemented with 10% FCS. Purified T cells of naive DBA/2 mice were plated at 1 x 106 cells/ml, along with 5 x 106 cells/ml BDF1 APCs. Cells were harvested at various time points to examine GITR expression. In some experiments, 2 x 105 cells/ml T cells and 1 x 106 cells/ml irradiated BDF1 APCs were cocultured in 96-well plates to test whether DTA-1 could block the interactions of GITR/GITR ligand in in vitro MLR. Soluble DTA-1 or isotype-matched Ig (5 µg/ml) was added at 0 h, or cultures were done in DTA-1 or control Ig-coated wells. Proliferation on day 3 was assessed in triplicate by pulsing with 1 µCi of [3H]thymidine added during the final 12 h.

Flow cytometry

The spleen of cGVHD mice was harvested on the indicated days after parental cell transfer. After lysis of the erythrocytes, the splenocytes were preincubated in a blocking buffer (PBS containing 2.4G2 mAb/0.2% BSA/0.1% sodium azide), and then incubated with the relevant mAbs for 30 min at 4°C. Finally, they were washed twice with staining buffer (PBS containing 0.2% BSA/0.1% sodium azide) and analyzed by FACScan (BD Biosciences Pharmingen). Staining for cells obtained from in vitro MLR was done as described above. For the measurement of IFN-{gamma}-producing T cells, splenocytes were isolated from cGVHD mice and were cultured in the presence of PMA (20 ng/ml) and ionomycin (200 ng/ml) for 4 h. Protein secretion was blocked by 1 µg/ml Golgiplug (BD Biosciences Pharmingen) added during the culture. Intracellular IFN-{gamma} staining was performed, according to the manufacturer’s protocol (BD Biosciences Pharmingen).

Proliferation assay for cGVHD mouse T cells

CD8+ T cells were purified from the spleen of control Ig- or DTA-1-treated cGVHD mice 14 days after disease induction, using anti-CD8-microbeads. The purified CD8+ T cells were stained with anti-H-2Kb to count donor CD8+ T cells. T cells containing equal numbers of donor CD8+ T cells (1 x 105) were used as responder cells in the presence of various concentrations of irradiated BDF1 APCs. Proliferation on day 3 was assessed in triplicate by pulsing with 1 µCi of [3H]thymidine added during the final 12 h. CD8+ T cells isolated from naive DBA/2 mice were used as positive controls. To analyze cytokines secreted by donor T cells, CD8+ T cells and APCs (1:5) were cocultured for 2 days, and culture supernatant was collected.

Immune responses to OVA

BDF1 mice were immunized by s.c. injection with 50 µg of chicken OVA (Sigma-Aldrich) in CFA (Sigma-Aldrich) 14 days after cGVHD induction. Ten days later, splenocytes were harvested and stimulated in the presence of OVA (10 µg/ml). Proliferation assay was done as described above.

ELISA

Mice were bled from the tail vein, and serum titers of anti-DNA IgG1 were assessed by ELISA. In brief, plates (96-well) were incubated overnight at 4°C with 100 µl of salmon sperm DNA (Sigma-Aldrich) at a concentration of 10 µg/ml. After blocking with 2% BSA, the plates were incubated with 100 µl of serially diluted serum samples for 1 h at room temperature. Plates were washed three times with PBS containing 0.1% Tween 20, HRP-conjugated anti-mouse IgG1 was added to each well, and the plates were kept at room temperature for 1 h. Plates were washed again with the same solution, and color was developed in 100 µl of the 3,5,3',5'-tetramethylbenzidne substrate for 15 min (Pierce), and stopped by adding 100 µl of 0.2 N H2SO4. The plates were then read at 450 nm with a Wallac Vector 1420 Multilabel Counter (EG & G Wallac). OD values from separate experiments were normalized to cGVHD mouse sera used in every assay (arbitrarily defined as 100 U). IL-2 and IFN-{gamma} were measured from culture supernatant, using an ELISA kit (Endogen), according to the manufacturer’s protocol.

Per-cell-based CTL assay

CTL assays were modified as described previously ( 5). Splenocytes from control Ig- or DTA-1-cGVHD mice were stained with anti-H-2Kb and anti-CD8 to count donor CD8+ T cells. Splenocytes containing equal numbers of donor CD8+ T cells were used as effector cells to compare the cytotoxicities of single cells between experimental groups. P815 cells (1 x 105) were used as a target cell.

Generation of anergic CD8+ T cells in in vitro MLR

DBA/2 CD8+ T cells (1 x 106) and BDF1 APCs (5 x 106) purified by MACS were cultured in 24-well plates for 5 days. After removal of apoptotic CD8+ T cells, using Ficoll-Paque Plus, according the manufacturer’s manual (Amersham Biosciences), CD8+ T cells were purified by MACS. The purified CD8+ T cells were used for a proliferation assay to assess their proliferative capacity. Cells (2 x 105/ml) were stimulated by anti-CD3 (0.5 µg/ml)/anti-CD28 (1 µg/ml) in the presence or absence of human IL-2 (1000 U/ml). Proliferation on day 3 was assessed in triplicate by pulsing with 1 µCi of [3H]thymidine added during the final 12 h. CD8+ T cells isolated from naive DBA/2 mice were used as positive controls. In some experiments, 5 µg/ml DTA-1 was added to the MLR culture to examine whether DTA-1 prevented the generation of anergic CD8+ T cells.

Histopathology and immunohistochemistry of the kidney

Kidneys and intestines were collected and immediately immersed in 10% neutral buffered Formalin. The Formalin-fixed tissue was embedded in paraffin, and 4-µm sections were stained with H&E and evaluated by light microscopy. The glomeruli were assessed by scoring each glomerulus as having no inflammation, segmental inflammation, or global involvement of inflammation. At least 170 glomeruli were counted for each specimen. A semiquantitative scoring system was used to assess the following abnormalities associated with kidney disease: tubular damage, including tubular epithelial cell dropout, thyroidization, and tubular atrophy; and interstitial damage, revealed by perivascular lymphocytic cuffing. The scoring system had 4 denotations, from 0 (normal) to 3 (severe). For immunohistochemistry, kidneys were embedded in optimal cutting temperature compound (Sakura Finetek) and snap-frozen in liquid nitrogen. Sections (8-µm) were air-dried, fixed with acetone, and stained with FITC-conjugated anti-mouse IgG (BD Biosciences Pharmingen). Fluorescence was examined by confocal microscopy (Olympus).

Pathological scoring of GVHD

Formalin-fixed ear skin, liver, and distal small and large intestine were embedded in paraffin, and 5-µm-thick sections were stained with H&E for histological examination. Slides were coded and examined in a blinded fashion by one individual (J.-H.S.), using a semiquantitative system for abnormalities known to be associated with GVHD ( 19, 20).

Statistical analysis

The Student’s t test was used to determine the statistical significance of differences between experimental groups. The log-rank test was used to compare survival between experimental groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Up-regulation of GITR in alloreactive CD4+ and CD8+ T cells

To assess whether allostimulation of CD4+ and CD8+ T cells results in up-regulation of GITR expression in cGVHD (DBA/2->BDF1), we analyzed GITR cell surface expression on alloreactive donor CD4+ and CD8+ T cells after stimulation with host APCs. In vitro allostimulation with host APCs substantially induced GITR expression in both responder CD4+ and CD8+ T cells on day 1 (Fig. 1). GITR expression continued to increase until reaching a peak on day 5 and decreased thereafter. The in vivo kinetics of GITR expression on donor CD4+ and CD8+ T cells showed a similar pattern as shown in vitro (data not shown). Therefore, we conclude that alloreactive CD4+ and CD8+ T cells had increased GITR expression in cGVHD.


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1. GITR expression in in vitro MLR. Purified DBA/2 CD4+ T cells or CD8+ T cells (1 x 106 cells/ml) were cocultured with BDF1 APCs (5 x 106 cells/ml) in 48-well plates. After the indicated days, cells were stained with anti-CD4 or anti-CD8 plus anti-GITR and analyzed by FACS. Thick line, anti-GITR; thin line, isotype-matched control.

 
DTA-1 inhibits cGVHD

To investigate the role of GITR in the development of cGVHD, we assessed the effect of the stimulatory anti-GITR mAb (DTA-1) on cGVHD. First, we wanted to confirm that DTA-1 had a stimulatory effect on alloreactive T cells in in vitro MLR. As shown in Fig. 2A, both soluble and coated DTA-1 had a similar potency to stimulate proliferation of responder DBA/2 T cells. Because DTA-1 does not have any effects on stimulator APCs ( 18), and soluble DTA-1 stimulated rather than inhibited proliferation of responder T cells in in vitro MLR, DTA-1 was not likely to block the interactions between GITR on T cells and GITR ligand on APCs in vivo. Furthermore, DTA-1 treatment in vivo induced the expression of the chemokine receptor CCR5 and up-regulation of the adhesion molecule and late activation marker VLA-4 on CD4+ and CD8+ T cells of the mesenteric lymph node 3 wk after cGVHD induction (Fig. 2B).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 2. DTA-1 activates alloreactive T cells. A, Purified DBA/2 T cells (1 x 105) were cocultured with BDF1 APCs (5 x 105) in 96-well plates. Soluble DTA-1 or isotype-matched Ig (5 µg/ml) was added at 0 h, or cultures were done in DTA-1 or control Ig-coated wells. Proliferation on day 3 was assessed in triplicate by pulsing with 1 µCi of [3H]thymidine added during the final 12 h. Data are shown as mean ± SEM. ***, p < 0.001 between the control Ig-treated groups (either coated or soluble) and the DTA-1-treated groups (either coated or soluble). B, cGVHD was induced by transferring 8 x 107 DBA/2 spleen/lymph node cells into BDF1 mice. Immediately thereafter, DTA-1 or control Ig (500 µg/mouse) was injected. Three weeks after disease induction, lymphocytes were harvested from the recipient mesenteric lymph node and stained with anti-CD4, anti-CD8, or an isotype control in conjunction with anti-CCR5 or anti-VLA-4. The expression of CCR5 and VLA-4 was analyzed in the gate of CD4+ or CD8+ T cells.

 
A single administration of DTA-1 at the time of disease induction significantly blocked the production of anti-DNA IgG1, a predominant Ig isotype in cGVHD ( 21). A decrease in anti-DNA IgG1 levels was observed as early as week 2 and maintained thereafter (Fig. 3A). Glomerulonephritis, the primary cause of mortality in mice afflicted with cGVHD, was also significantly inhibited by DTA-1 (Fig. 3B). The kidneys of the control Ig-treated cGVHD mice demonstrated severe glomerulonephritis involving 60% of total glomeruli (20% with global sclerosis and 40% with segmental sclerosis) (Fig. 3C). Histological sections from the control Ig-treated cGVHD mice also showed severe tubular damage (Fig. 3D), prominent perivascular inflammatory cell infiltrate (Fig. 3E), and fibrosis (not shown). In contrast, the general kidney pathology was greatly reduced in DTA-1-treated animals (Fig. 3, C–E). Consistent with the histological examination, IgG deposition in the kidney was significantly decreased by DTA-1 (Fig. 3B).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. DTA-1 inhibits cGVHD. A, Serum samples were collected every 2 wk after disease induction and assayed in duplicate for the presence of IgG1 anti-DNA autoantibody by ELISA. The OD of duplicate samples for each mouse was measured, using serially diluted serum samples. Arbitrary units (AU) are shown as mean ± SEM (n = 10 per group) of 10-fold dilution of each sample. B, Representative kidney sections for H&E staining (top columns) and IgG immunostaining (bottom columns). Kidneys were harvested at 12 wk after disease induction. C–E, H&E-stained kidney sections from mice, at 12 wk of disease, were scored for kidney pathology. C, Glomerulonephritis: {blacksquare}, no sclerosis; {cjs2108}, segmental sclerosis; {square}, global sclerosis. D, Tubular damage. E, Perivascular lymphocytic cuffing. **, p < 0.01 between the two groups. Data shown are representative of more than three independent experiments.

 
DTA-1 converts cGVHD to aGVHD

The profile of recipient splenocytes provides an indication of the type of GVHD that occurs. For example, the injection of C57BL/6 splenocytes into immunocompetent BDF1 mice results in the engraftment of a high number of parental cells and the elimination of host lymphocytes, manifesting aGVHD, whereas the injection of DBA/2 splenocytes results in a chronic progressive disease with little parental cell engraftment, characteristics of cGVHD ( 22). Treatment of DTA-1 led to a marked reduction in the number of total splenocytes (Table I). This lymphopenic phenotype was due to the elimination of host B cells, host CD4+ T cells, and host CD8+ T cells. Because this splenocyte profile is typically found in aGVHD mice, this result suggested the possibility that DTA-1 converted cGVHD to aGVHD. Indeed, we obtained additional evidence supporting this hypothesis. First, most of the DTA-1-treated mice showed clinical signs of aGVHD, as manifested by profuse diarrhea and hunched posture. There was a significant decrease in the average body weight of the DTA-1-treated mouse group (Table II). Third, donor cells were almost completely engrafted into the host spleen by week 2 after DTA-1 treatment (Table II). In all experiments, the engraftment of donor cells correlated with the incidence of lymphopenia in the DTA-1-treated mouse spleen. The lymphopenia induced by DTA-1 resulted in a decreased immune response to OVA, a third party Ag (Table II). Finally, in accordance with the clinical signs, there was a significant degree of damage in the small and large intestines (major target organs of aGVHD) of the DTA-1-treated mice (Table II). Although the DTA-1-treated animals showed no clinical manifestation of cutaneous GVHD such as ear erosion, tail scaling, and alopecia, erosions, or ulcers in hair-bearing areas, histological examination demonstrated that DTA-1 did induce skin destruction (Table II). Puzzlingly, there was no difference in the severity of liver damage between the two groups of animals (Table II). Taken together, these results clearly indicate the conversion of cGVHD to aGVHD by DTA-1. This phenotypic change also provides an explanation for the inhibitory effect of DTA-1 on cGVHD and the hyporesponsiveness to the third party Ag.


View this table:
[in this window]
[in a new window]
  		Table I. DTA-1 induces lymphopenia and a sustained activation of donor CD8+ T cellsa

 

View this table:
[in this window]
[in a new window]
  		Table II. DTA-1 converts cGVHD to aGVHD

 
DTA-1 converts cGVHD to aGVHD by preventing donor CD8+ T cell anergy

aGVHD is characterized by donor anti-host CTLs that eliminate activated autoreactive host B cells. In contrast, donor CD8+ T cells have no cytotoxicity against host cells in cGVHD, suggesting that the CTL activity of donor CD8+ T cells is a restriction factor in the development of aGVHD ( 10). Therefore, we hypothesized that the conversion of cGVHD to aGVHD by DTA-1 results from the prevention of donor CD8+ T cell hyporesponsiveness and the subsequent activation of alloreactive CD8+ T cells. First, we monitored the fate of donor CD8+ T cells after DTA-1 treatment. DTA-1 induced a sustained activation of donor CD8+ T cells (Table I). In sharp contrast, the percentage of activated donor CD8+ T cells of the control Ig-treated cGVHD mice continued to be decreased since 1 wk after parental cell transfer, when compared with those of the DTA-1-treated cGVHD mice. Although the activation pattern of donor CD4+ T cells was similar between the control Ig-treated and DTA-1-treated groups, the DTA-1-treated group showed a more activated phenotype with lower levels of CD62L expression (Table I). This result suggests that DTA-1 activates both donor CD4+ and CD8+ T cells in cGVHD. Next, we examined whether donor CD8+ T cells become anergic in cGVHD and whether DTA-1 prevents anergy of donor CD8+ T cells. An exo vivo MLR assay showed that donor CD8+ T cells isolated from 2-wk cGVHD mice treated with control Ig proliferated poorly and did not secrete IL-2 and IFN-{gamma}, when restimulated with the recipient APCs (Fig. 4A). In contrast, naive DBA/2 CD8+ T cells were able to vigorously proliferate and secrete IL-2 and IFN-{gamma} by stimulation with BDF1 APCs. We next compared the proliferative capacity of donor CD8+ T cells isolated from control Ig-treated and DTA-1-treated cGVGD mice 2 wk after disease induction. In contrast to donor CD8+ T cells of the control Ig-treated cGVGD mice, CD8+ T cells of the DTA-1-treated cGVHD mice showed vigorous proliferation to allostimulation (Fig. 4B), suggesting that DTA-1 prevented donor CD8+ T cells from becoming anergic. Results from functional studies demonstrated increased cytotoxicity against alloantigens of donor CD8+ T cells from the DTA-1-treated group at the total cell level (data not shown), and a per-cell-based cytotoxicity assay, which reflects more precisely the qualitative change of donor CD8+ T cells, also showed increased levels of cytotoxicity (Fig. 4C). In agreement with others ( 10, 23, 24), the control Ig-treated group showed a minimal level of cytotoxicity (Fig. 4C). Consistently, the percentage of IFN-{gamma}-producing donor CD8+ T cells of the DTA-1-treated mice was significantly higher than those of the control Ig-treated group (Fig. 4D). There was a comparable percentage of IFN-{gamma}-producing donor CD4+ T cells between the two experimental groups (Fig. 4D). Presumably, this discrepancy between CD4+ and CD8+ T cells was due to a greater extent of apoptosis of IFN-{gamma}-producing, activated donor CD4+ T cells induced by DTA-1 as compared with donor CD8+ T cells (Fig. 4E). This observation is consistent with data obtained from an aGVHD model ( 18). Collectively, the results from these studies suggest that, as cGVHD progresses, the engrafted donor CD8+ T cells become anergic, and that GITR stimulation prevents the induction of donor CD8+ T cell anergy. These data also imply that effector donor CTLs, not CD4+ T cells, are mainly responsible for the development of DTA-1-mediated aGVHD.


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 4. DTA-1 prevents CD8+ T cell anergy. A, Two weeks after disease induction, CD8+ T cells were purified from spleens, using anti-CD8-conjugated microbeads. After counting the number of donor CD8+ T cells by staining the purified cells with anti-H-2Kb, the purified CD8+ T cells containing 1 x 105 of donor CD8+ T cells were stimulated in the presence of various concentrations of enriched BDF1 APCs. Equal numbers of CD8+ T cells isolated from naive DBA/2 mice were cultured similarly as positive control. Proliferative activity was determined by [3H]thymidine incorporation on day 3. The data are the mean ± SEM of triplicate wells. Production of IL-2 and IFN-{gamma} was measured from 48-h culture supernatant. ND, not detectable. Data shown are representative of at least three independent experiments (n = 3–5 per group). B, Proliferation of donor CD8+ T cells was compared between control Ig-treated and DTA-1-treated cGVHD mice. Experiments were performed similarly as described in A. C, A per-cell-based cytotoxicity assay. Splenocytes were harvested 2 wk after disease induction. After counting the number of donor CD8+ T cells, equivalent numbers of donor CD8+ T cells were set up in a conventional 4-h 51Cr-release assay with EL4 cells as a target cell. Data shown are representative of at least three independent experiments. D, Percentage of IFN-{gamma}-producing donor CD4+ and CD8+ T cells. Splenocytes were harvested 2 wk after disease induction and stimulated with PMA/ionomycin for 12 h, then stained with anti-H-2Kb plus anti-CD8 combined with intracellular staining for IFN-{gamma}. The figures in the FACS plot boxes are the mean ± SEM for the percentage of IFN-{gamma}-producing cells (n = 5 per group). ***, p < 0.001 between the two groups. E, Percentage of apoptotic donor CD4+ and CD8+ T cells. Splenocytes were harvested 6 days after disease induction and stained with anti-H-2Kb plus anti-CD4 or anti-CD8 combined with annexin V staining. Data are the mean ± SEM (n = 4 per group).

 
To more clearly characterize donor CD8+ T cells that have the ability to fall into anergy in cGVHD, we set up an in vitro MLR system to generate anergic CD8+ T cells. We cocultured purified DBA/2 CD8+ T cells with T cell-depleted BDF1 APCs. We found that CD8+ T cells were divided into apoptosis-resistant CD8high T cells and CD8int T cells that underwent apoptosis during culture (Fig. 5, A and B). Whereas the CD8int T cells expressed low levels of CD62L (activation phenotype), the CD8high T cells expressed high levels of CD62Lhigh (naive phenotype). Because most donor CD8+ T cells were eliminated in cGVHD recipients and a small number of residual donor CD8+ T cells displayed a naive phenotype (Table I), this result suggested that the CD8high T cells might be anergic CD8+ T cells. Indeed, the CD8high T cells poorly proliferated in response to anti-CD3/anti-CD28 mAbs compared with naive CD8+ T cells (Fig. 5C). In contrast, treatment of anti-CD3/anti-CD28 was shown to induce a vigorous proliferation of CD8high T cells from MLR culture treated with DTA-1 (Fig. 5D). Because IL-2 recovered the proliferation of CD8high T cells together with TCR stimulation (Fig. 5D), a characteristic of anergic T cells ( 25), we concluded that anergic CD8+ T cells can be generated in in vitro MLR and that addition of DTA-1 prevents CD8+ T cells from becoming anergic.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. Effects of DTA-1 on the generation of anergic CD8+ T cells in in vitro MLR. Purified DBA/2 CD8+ T cells (1 x 106) and T cell-depleted BDF1 APCs (5 x 106) were cocultured without or with control Ig or DTA-1 (5 µg/ml). A and B, Apoptosis-resistant CD8high T cells show a naive phenotype. A, Cultured cells from 5-day MLR were triple-stained for CD8, annexin V, and CD62L. B, Percentage of annexin V+ T cells were calculated in the two populations of CD8int and CD8high T cells. C and D, DTA-1 prevents CD8+ T cell anergy. The data are the mean ± SEM of triplicate wells. C, Live CD8+ T cells (2 x 105) purified from 5-day MLR were stimulated in the presence of anti-CD3 (0.5 µg/ml)/anti-CD28 (1 µg/ml), and proliferative activity was determined by [3H]thymidine incorporation on day 3. CD8+ T cells purified from naive DBA/2 mice were used as positive control. D, Similar experiments were performed for live CD8+ T cells purified from 5-day MLR culture treated with control Ig or DTA-1.

 
Donor CD8+ T cells, but not CD4+ T cells, are required for the effect of DTA-1 on the conversion of cGVHD to aGVHD

To directly address the involvement of donor CD8+ T cells in the conversion of cGVHD to aGVHD by DTA-1, cGVHD was induced by transferring parental spleen/lymph node cells depleted of CD8+ T cells. In this model, donor CD4+ T cells are sufficient to induce cGVHD and donor B cells do not play a role in disease induction ( 26). Elimination of donor CD8+ T cells completely abrogated the effects of DTA-1 on deletion of total splenocytes and B cells and donor cell engraftment, two important parameters indicating aGVHD (Table III). Consistent with this result, other signs of aGVHD induced by DTA-1, such as loss of body weight, intestinal damage, and abolishment of autoantibody production, did not take place in the absence of donor CD8+ T cells (data not shown). It was clear from these results that, even though donor CD4+ T cells were sufficient for the development of cGVHD, the activation of donor CD4+ T cells by DTA-1 alone was not capable of shifting cGVHD toward aGVHD as well as inhibiting cGVHD. These results suggest that the conversion of cGVHD to aGVHD requires alloreactive effector CD8+ T cells that are generated by DTA-1.


View this table:
[in this window]
[in a new window]
  		Table III. Requirement of donor CD8+ T cells in the DTA-1-mediated conversion of cGVHD to aGVHD

 
Next, we examined the ability of donor CD8+ T cells to induce the DTA-1-mediated conversion of cGVHD to aGVHD independently of donor CD4+ T cells. Deletion of donor CD4+ T cells had no effect on the major changes mediated by DTA-1; typical parameters of aGVHD, including elimination of splenocytes and B cells and donor cell engraftment, were observed in the DTA-1-treated mice 3 wk after transfer of parental cells depleted of CD4+ T cells (Table III). Overall, our observations indicate that donor CD8+ T cells activated by DTA-1 play a major role in shifting cGVHD toward aGVHD.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The main finding of this study is a novel function of GITR in the pathogenesis of GVHD. Our results demonstrate that GITR signals can directly regulate the capacity of donor CD8+ T cells to respond while they are in the process of becoming tolerant. This finding is of particular interest due to its tremendous implications for the manipulation of the T cell response in clinical situations such as cancer and transplant rejection. Our results also add another important dimension to the pathogenesis of GVHD, and indicate that a divergence point between cGVHD and aGVHD is determined by whether donor CD8+ T cells become either tolerant or activated. GITR may play a central role in this process.

Studies using various systems for the parent-into-unirradiated F1 GVHD have shown that inocula of donor cells that preferentially act through alloreactive CD8+ T cells cause symptoms of aGVHD, whereas donor cell inocula that preferentially act through alloreactive CD4+ T cells cause symptoms of cGVHD ( 25, 27, 28, 29). Therefore, the development of aGVHD or cGVHD minimally requires a disparity of MHC class I or class II, respectively. When donor cells differ from the host by both MHC class I and class II, the donor genetic background is critical in determining cGVHD vs aGVHD. The best known example is that C57BL/6 donor cells that have a tendency for the CTL response cause aGVHD in the unirradiated BDF1 host, whereas DBA/2 splenocytes, which have a tendency for the CD4+ T cell response, but no CTL response, cause cGVGD. Accordingly, aGVHD can be driven toward cGVHD by the inactivation of perforin, an important CTL effector molecule ( 7), or neutralizing TNF-{alpha}, which is critical for anti-host CTL generation ( 6). Conversely, IL-12 can drive cGVHD to aGVHD by inducing donor CTL’s activation ( 30).

The transfer of alloantigen-specific TCR transgenic CD8+ T cells into Ag-bearing hosts results in the development of anergy in the residual population ( 31, 32, 33), as observed in our parental-into-F1 cGVHD model. The use of anti-4-1BB mAb can restore proliferation and effector functions of anergic alloreactive CD8+ T cells in this model ( 5). In the parent-into-F1 cGVHD model, however, there is a distinguishable difference in the mechanism of action of 4-1BB and GITR. 4-1BB signals inhibit cGVHD by eliminating pathogenic donor CD4+ T cells ( 21), whereas GITR signals do so by shifting cGVHD toward aGVHD. This difference may be derived from their relative potency in costimulating donor CD4+ and CD8+ T cells in cGVHD. Because stimulation of 4-1BB ( 21) can trigger activation-induced cell death (AICD) of donor CD4+ T cells more strongly than stimulation of GITR (Fig. 4E), it appears that 4-1BB is superior to GITR in costimulating CD4+ T cells. In contrast, GITR seems to be a more potent costimulator for donor CD8+ T cells than 4-1BB ( 21), with regard to the phenomena that follow the increase in donor CTL activity, such as the deletion of B cells and donor cell engraftment. However, in the OT-1 T cell anergy model, GITR only weakly stimulated the clonal expansion of transferred OT-1 cells (our unpublished data), in contrast to 4-1BB stimulation ( 5). Therefore, it is tempting to draw a conclusion that the two receptors play distinct roles in the two functions of CD8+ T cells: GITR in the cytotoxicity and 4-1BB in the expansion of CD8+ T cells. This interpretation is consistent with the observation that 4-1BB is involved in the clonal expansion of CD8+ T cells rather than in the development of cytotoxicity ( 34).

Our in vitro MLR seems to reflect the in vivo situation of DBA/2->BDF1 cGVHD (Table I and Fig. 5). A majority of DBA/2 CD8+ T cells underwent AICD after allostimulation, and a small population of T cells having resistance to apoptosis became anergic. It is commonly observed that allogeneic CD8+ T cells extensively proliferate initially in unconditioned recipients, followed by a state of unresponsiveness in which a small number of residual CD8+ T cells become anergic ( 31, 32). In our cGVHD, donor CD8+ T cells also were able to proliferate initially (our unpublished data). Therefore, a state of anergy may be the natural fate of T cells that escape AICD after sustained allostimulation occur ( 32, 33). DTA-1 could not prevent AICD of donor CD8+ T cells (Fig. 4E; Ref. 35), and DTA-1 generated donor CD8+ T cells that showed a sustained activated phenotype (Table I). This observation may suggest that these T cells share some property with anergic T cells despite a state of activation–resistance to AICD and a long-term survival. Therefore, it is possible that DTA-1 can activate donor CD8+ T cells that are destined to be anergic.

Muriglan et al. ( 18) have shown that GITR stimulation induces AICD of donor CD4+ T cells, whereas it enhances activation of donor CD8+ T cells. In cGVHD, we also obtained evidence that GITR activation preferentially induces apoptosis of CD4+ T cells (Fig. 4E). Unlike aGVHD, however, GITR stimulation did not block the development of cGVHD in the absence of donor CD8+ T cells (data not shown). This and another observation that GITR-activated donor CD4+ T cells exacerbate cGVHD (our unpublished data) but ameliorate aGVHD ( 18) indicate that GITR activation may delete Th1 cells that have the potential to mediate aGVHD but not Th2 cells mediating cGVHD ( 24, 29). Therefore, we believe that donor CD8+ T cells are the main driving forces to induce aGVHD by giving damage to target organs, and to inhibit cGVHD by killing host lymphocytes.

GITR signals can regulate the T cell response by depressing the immunosuppressive activity of Treg cells ( 11, 12). However, our data, which is consistent with others ( 13, 14, 15, 16, 17, 18), indicate that DTA-1 could increase the activity of CD8+ T cells in the absence of donor CD4+ T cells (Table III). Furthermore, specific depletion of donor Treg cells had no effect of DTA-1 on the activation of donor CD8+ T cells (our unpublished data). Thus, it seems that direct activation of alloreactive donor CD8+ T cells by DTA-1 provides an explanation for the DTA-1-mediated conversion of cGVHD to aGVHD. Because host Treg cells have been shown to regulate the development of cGVHD in a MHC-matched cGVHD model ( 36), however, we cannot exclude the possibility that host Treg cells or CD4+ T cells are involved in the DTA-1-mediated conversion of cGVHD to aGVHD. Ongoing research to clarify this issue is currently underway in our laboratory.

The overall effect of in vivo GITR stimulation requires careful interpretation because GITR stimulation can have a differential effect on donor and host Treg cells, CD4+ T cells, CD8+ T cells, and other cells. Our data on cGVHD suggest that GITR stimulation provides a novel tool for dissecting the immunological mechanism determining the development of cGVHD vs aGVHD.


   Acknowledgments
 
We thank Immunomodulation Research Center members for their assistance in performing the research.


   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 work was supported by grants from the Science Research Center Fund to the IRC from Korea Science and Engineering Foundation and the Korean Ministry of Science and Technology, a Xenotransplantation Research Center grant from the Korean Ministry of Science and Technology, and a grant from University of Ulsan (2005-0274). Back

2 Address correspondence and reprint requests to Dr. Byungsuk Kwon, The IRC, University of Ulsan, San29, Mukeo-dong, Nam-ku, Ulsan, Ulsan 680-749, Republic of Korea. E-mail address: bkwon{at}mail.ulsan.ac.kr Back

3 Abbreviations used in this paper: GVHD, graft-vs-host disease; aGVGD, acute GVHD; cGVHD, chronic GVHD; GITR, glucocorticoid-induced TNF receptor; Treg, CD4+CD25+ regulatory T; int, intermediate; AICD, activation-induced cell death. Back

Received for publication August 31, 2005. Accepted for publication February 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Harding, F. A., J. G. McArthur, J. A. Cross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signaling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature 256: 607-609.
  2. Boussinotis, B. A., G. J. Freeman, G. Gray, J. Gribben, L. M. Nadler. 1993. B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance. J. Exp. Med. 178: 1753-1763. [Abstract/Free Full Text]
  3. Sotomayor, E. M., I. Borrello, E. Tubb, F. M. Rattis, H. Bien, Z. Lu, S. Fein, S. Schoenberger, H. I. Levitsky. 1999. Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40. Nat. Med. 5: 780-789. [Medline]
  4. Banasal-Pakala, P., A. G.-H. Jember, M. Croft. 2001. Signaling through OX40 (CD134) breaks peripheral T-cell tolerance. Nat. Med. 7: 907-912. [Medline]
  5. Wilcox, R. A., K. Tamada, D. B. Flies, Z. Zhu, A. I. Chapoval, B. R. Blazar, W. M. Kast, L. Chen. 2004. Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo. Blood 103: 177-184. [Abstract/Free Full Text]
  6. Via, C. S., A. Shustov, V. Rus, T. Lang, P. Nguyen, F. D. Finkelman. 2001. In vivo neutralization of TNF-{alpha} promotes humoral autoimmunity by preventing the induction of CTL. J. Immunol. 167: 6281-6286.
  7. Shustov, A., I. Luzina, P. Nguyen, J. C. Papadimitriou, B. Handwerger, K. B. Elkon, C. S. Via. 2000. Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity. J. Clin. Invest. 106: 39-47.
  8. Via, C. S., F. D. Finkelman. 1993. Critical role of interleukin-2 in the development of acute graft-versus-host disease. Int. Immunol. 5: 565-572. [Abstract/Free Full Text]
  9. Via, C. S., P. Nguyen, A. Shustov, J. Drappa, K. B. Elkon. 1996. A major role of the Fas pathway in acute graft-versus-host disease. J. Immunol. 157: 5387-5393. [Abstract]
  10. Via, C. S., S. O. Sharrow, G. M. Shearer. 1987. Role of cytotoxic T lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-versus-host disease. J. Immunol. 139: 1840-1849. [Abstract]
  11. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi. 2002. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3: 135-142. [Medline]
  12. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. C. Byrne. 2002. CD4+CD25+ immunoregualtory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16: 311-323. [Medline]
  13. Tone, M., Y. Tone, E. Adams, S. F. Yates, M. R. Frewin, S. P. Cobbold, H. Waldmann. 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Nat’l. Aca. Sci. USA 100: 15059-15064. [Abstract/Free Full Text]
  14. Ronchetti, S., O. Zollo, S. Bruscoli, M. Agostini, R. Bianchini, G. Nocentini, E. Ayroldi, C. Riccardi. 2004. Frontline: GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulation. Eur. J. Immunol. 34: 613-622. [Medline]
  15. Kohm, A. P., J. S. Williams, S. D. Miller. 2004. Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis. J. Immunol. 172: 4686-4690. [Abstract/Free Full Text]
  16. Ji, H.-b., G. Liao, W. A. Faubion, A. C. Abadia-Molina, C. Cozzo, F. S. Laroux, A. Caton, C. Terhorst. 2004. Cutting edge: the natural ligand for glucocorticoid-induced TNF receptor-related protein abrogates regulatory T cell suppression. J. Immunol. 172: 5823-5827. [Abstract/Free Full Text]
  17. Kanamaru, F., P. Youngnak, M. Hashiguchi, T. Nishioka, T. Takahashi, S. Sakaguchi, I. Ishikawa, M. Azuma. 2004. Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J. Immunol. 172: 7306-7314. [Abstract/Free Full Text]
  18. Muriglan, S. J., T. Ramirez-Montagut, O. Alpdogan, T. W. van Huystee, J. M. Eng, V. M. Hubbard, A. A. Kochman, K. H. Tjoe, C. Riccardi, P. P. Pandolfi, et al 2004. GITR activation induces an opposite effect on alloreactive CD4+ and CD8+ T cells in graft-versus-host disease. J. Exp. Med. 200: 149-157. [Abstract/Free Full Text]
  19. Anderson, B. E., J. Yan, H. Doyle, M. Mamula, M. J. Shlomchik, W. D. Shlomchik. 2003. Memory CD4+ T cells do not induce graft-versus-host disease. J. Clin. Invest. 112: 101-108. [Medline]
  20. Cooke, K. R., W. Krenger, T. R. Martin, L. Kobzik, J. Brewer, R. Simmons, J. M. Crawford, M. R. M. van den Brink, J. L. M. Ferrara. 1998. Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation. Blood 92: 2571-2580. [Abstract/Free Full Text]
  21. Kim, J., W. S. Choi, S. La, J.-H. Suh, B.-S. Kim, H. R. Cho, B. S. Kwon, B. Kwon. 2005. 4-1BB (CD137) stimulation inhibits chronic graft-versus-host-disease by inducing activation-induced cell death of donor CD4+ T cells. Blood 105: 2206-2213. [Abstract/Free Full Text]
  22. Murphy, W. J.. 2000. Revisiting graft-versus-host disease models of autoimmunity: new insights in immune regulatory processes. J. Clin. Invest. 106: 745-747. [Medline]
  23. Pals, S. T., T. Radaszkiesicz, E. Gleichmann. 1984. Allosuppressor- and allohelper-T cells in acute and chronic graft-vs-host disease. IV. Activation of donor allosuppressor cells is confined to acute GVHD. J. Immunol. 132: 1669-1678. [Abstract]
  24. Rus, V., A. Svetic, P. Nguyen, W. C. Gause, C. S. Via. 1995. Kinetics of Th1 and Th2 cytokine production during the early course of acute and chronic murine graft-versus-host disease. J. Immunol. 155: 2396-2406. [Abstract]
  25. Redmond, W. L., L. A. Sherman. 2005. Peripheral tolerance of CD8 T lymphocytes. Immunity 22: 275-284. [Medline]
  26. Morris, S. C., R. L. Cheek, P. L. Cohen, R. A. Eisenberg. 1990. Autoantibodies in chronic graft versus host result from cognate T-B interactions. J. Exp. Med. 171: 503-517. [Abstract/Free Full Text]
  27. Rolink, A. G., W. G. J. van der Meer, C. J. M. Melief, E. Gleichmann. 1983. Intra-H-2 and T-cell requirements for the induction of maximal positive and negative allogeneic effects in vitro. Eur. J. Immunol. 13: 191-197. [Medline]
  28. Goldman, M., P. Druet, E. Gleichman. 1991. Th2 cells in systemic autoimmunity: insights from allogeneic diseases and chemically induced autoimmunity. Immunol. Today 12: 223-227. [Medline]
  29. De Wit, D., M. Van Mechelen, C. Zanin, J.-M. Doutrelepont, T. Velu, C. Gerard, D. Abramowicz, J.-P. Scheerlinck, P. De Baetselier, J. Urbain. 1993. Preferential activation of Th2 cells in chronic graft-versus-host reaction. J. Immunol. 150: 361-366. [Abstract]
  30. Via, C. S., V. Rus, M. K. Gately, R. D. Finkelman. 1994. IL-12 stimulates the development of acute graft-versus-host disease in mice that normally would develop chronic, autoimmune graft-versus-host disease. J. Immunol. 153: 4040-4047. [Abstract]
  31. Gonzalez, M., S. A. Quezada, B. R. Blazar, A. Panoskaltsis-Mortari, A. Y. Rudensky, R. J. Noelle. 2002. The balance between donor T cell anergy and suppression versus lethal graft-versus-host disease is determined by host conditioning. J. Immunol. 169: 5581-5589. [Abstract/Free Full Text]
  32. Zhang, L., R. G. Miller, L. Zhang. 1996. Characterization of apoptosis-resistant antigen-specific T cells in vivo. J. Exp. Med. 183: 2065-2073. [Abstract/Free Full Text]
  33. Dey, B., Y. G. Yang, F. Preffer, A. Shimizu, K. Swenson, D. Dombkowki, M. Sykes. 1999. The fate of donor T-cell receptor transgenic T cells with known host antigen specificity in a graft-versus-host disease model. Transplantation 68: 141-149. [Medline]
  34. Cooper, D., P. Bansal-Pakala, M. Croft. 2002. 4-1BB (CD137) controls the clonal expansion and survival of CD8+ T cells in vivo but does not contribute to the development of cytotoxicity. Eur. J. Immunol. 32: 521-529. [Medline]
  35. Esparza, E. M., R. H. Arch. 2005. Glucocorticoid-induced TNF receptor functions as a costimulatory receptor that promotes survival in early phase of T cell activation. J. Immunol. 174: 7869-7874. [Abstract/Free Full Text]
  36. Anderson, B. E., J. M. McNiff, C. Matte, I Athanasiadis, W. D. Shlomchik, M. J. Shlomchik. 2004. Recipient CD4+ T cells that survive irradiation regulated chronic graft-versus-host disease. Blood 104: 1565-1573. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Ronchetti, G. Nocentini, R. Bianchini, L. T. Krausz, G. Migliorati, and C. Riccardi
Glucocorticoid-Induced TNFR-Related Protein Lowers the Threshold of CD28 Costimulation in CD8+ T Cells
J. Immunol., November 1, 2007; 179(9): 5916 - 5926.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Kim, H. J. Kim, K. Park, J. Kim, H.-J. Choi, H. Yagita, S. H. Nam, H. R. Cho, and B. Kwon
Costimulatory molecule-targeted immunotherapy of cutaneous graft-versus-host disease
Blood, July 15, 2007; 110(2): 776 - 782.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Tuyaerts, S. Van Meirvenne, A. Bonehill, C. Heirman, J. Corthals, H. Waldmann, K. Breckpot, K. Thielemans, and J. L. Aerts
Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+CD25+ regulatory T cells
J. Leukoc. Biol., July 1, 2007; 82(1): 93 - 105.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, J.
Right arrow Articles by Kwon, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, J.
Right arrow Articles by Kwon, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS