Reversal of Spontaneous Autoimmune Insulitis in Nonobese Diabetic Mice by Soluble Lymphotoxin Receptor

Qiang Wua, Benoît Salomona,b, Min Chena, Yang Wanga, Lisa M. Hoffmana, Jeffrey A. Bluestonea,b, and Yang-Xin Fua
a Committee on Immunology and the Department of Pathology, University of Chicago, Chicago, Illinois 60637
b Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637

Correspondence to: Yang-Xin Fu, Department of Pathology and Immunology, University of Chicago, 5841 S. Maryland, Rm. J541, MC3083, Chicago, IL 60637. Tel:773-702-0929 Fax:773-834-8940 E-mail:yfu{at}midway.uchicago.edu.


  Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

One striking feature of spontaneous autoimmune diabetes is the prototypic formation of lymphoid follicular structures within the pancreas. Lymphotoxin (LT) has been shown to play an important role in the formation of lymphoid follicles in the spleen. To explore the potential role of LT-mediated microenvironment in the pathogenesis of insulin-dependent diabetes mellitus (IDDM), an LTß receptor–immunoglobulin fusion protein (LTßR–Ig) was administered to nonobese diabetic mice. Early treatment with LTßR–Ig prevented insulitis and IDDM, suggesting that LT plays a critical role in the insulitis development. LTßR–Ig treatment at a late stage of the disease also dramatically reversed insulitis and prevented diabetes. Moreover, LTßR–Ig treatment prevented the development of IDDM by diabetogenic T cells in an adoptive transfer model. Thus, LTßR–Ig can disassemble the well established lymphoid microenvironment in the islets, which is required for the development and progression of IDDM.

Key Words: adhesion molecule, autoimmune diabetes, insulitis, lymphotoxin, lymphotoxin ß receptor


  Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Insulin-dependent diabetes mellitus (IDDM) is a T cell–mediated autoimmune disease in which the insulin-secreting ß cells are selectively destroyed after weeks or months of insulitis. Much of our current knowledge about the complex pathogenesis of IDDM derives from the studies of nonobese diabetic (NOD) mice (1) (2) (3) (4) (5). In NOD mice, infiltration of autoreactive T cells into the islets is essential for the development of IDDM. Studies performed in these animals have revealed that the influx of T cells into the pancreas is associated with an increased expression of adhesion molecules. However, factors that upregulate adhesion molecules in the pancreas have not been identified. Lymph node–like structures with de novo formation of lymphoid follicles are gradually established within the islets over 2–3 mo, with initial cellular infiltration starting at 3–4 wk of age (1) (3) (4) (6) (7). While the formation of lymphoid follicular structures is a prototypic feature of chronic progressive inflammation (6) (7) (8), the molecular mechanisms by which the lymphoid follicles are formed and the role of these follicles in the pathogenesis of IDDM have not been well defined.

The interaction between membrane lymphotoxin (LT) and its receptor is essential for the development and maintenance of the lymphoid microenvironment (9) (10) (11) (12) (13) (14) (15). LT{alpha}-/- mice and wild-type mice treated with LTß receptor–immunoglobulin fusion protein (LTßR–Ig) exhibit altered lymphoid microenvironment. This effect is likely caused by a failure to induce lymphoid tissue chemokines and adhesion molecules (12) (13) (14) (15). These results imply that LT may be a master cytokine responsible for the formation of lymphoid structures in chronic inflammation and autoimmune diseases, such as IDDM. Here, we report that administration of LTßR–Ig reverses the formation of lymphoid follicles, prevents ß cell destruction by autoreactive T cells, and forestalls the development of IDDM.


  Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Reagents and Mice.
The generation and production of recombinant LTßR–Ig has been described previously (15) (16). The murine LTßR–human Ig in culture supernatants of BHK/VP16 (15) or Chinese hamster ovary cells (16) was purified on a protein A column. No difference could be found between the two preparations. Human Ig was obtained from Biogen, Inc. or Sigma-Aldrich. Female NOD mice were purchased from The Jackson Laboratory and maintained under specific pathogen–free conditions at the University of Chicago. Antibodies to vascular cell adhesion molecule (VCAM), peripheral lymph node addressin (PNAd; MECA 79), mucosal addressin cell adhesion molecule 1 (MAdCAM-1; MECA367), B220, CD4 (GK1.5), CD8 (3.155), and CD11c (N418) were purchased from PharMingen.

LTßR–Ig Treatment and Measurement of Blood Glucose.
NOD mice were given 100 µg of LTßR–Ig or human Ig intraperitoneally once per week for 3 wk or as indicated. The glucose concentration in blood obtained from a tail vein was measured using SureStep® strips (Johnson and Johnson). Diabetes was monitored by levels of blood glucose. Animals were considered diabetic after two consecutive measurements of >=250 mg/dl of glucose.

Adoptive Transfer.
Recipient NOD mice (7–9 wk) were irradiated with 6 Gy (199 rads/min) using a 137Cs irradiator. 2 h later, the mice were injected intravenously with 2 x 107 splenocytes from diabetic NOD mice in 0.2 ml of PBS, followed by intraperitoneal injection with 100 µg of LTßR–Ig, anti–MAdCAM-1, or control human Ig.

Histology and Immunohistochemistry.
Pancreatic tissue was collected 10 d after the last LTßR–Ig treatment. Hematoxylin and eosin staining was performed on 6-µm sections of 10% formalin-fixed tissue. Infiltration into islets was examined microscopically and counted by a third party pathologist. Sections were prepared on multiple levels, and 20–40 randomly chosen islets per mouse were semiquantitatively classified according to the severity of insulitis: moderate and severe insulitis are defined to be less or more than half of the structure was infiltrated, respectively. Additionally, some of the pancreata and spleen were frozen at –70°C in OCT compound for immunohistology. 6-µm cryostat sections were incubated with rat antibodies to CD3 (L363–29B), VCAM, PNAd (MECA 79), MAdCAM-1 (MECA367), B220, CD4 (GK1.5), CD8 (3.155), hamster antibody to CD11c (N418), or species- and isotype-specific nonreactive control mAbs overnight at 4°C. The next day, room temperature incubation with biotinylated rabbit anti–rat or goat anti–hamster secondary antibody (Vector Laboratories) for 30 min was followed by a 1-h incubation with streptavidin–horseradish peroxidase complex (Vector Laboratories).


  Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Prevention of Diabetes by the Administration of LTßR–Ig.
Typically, 65–75% of untreated NOD mice in our colony develop IDDM by 25 wk. To study the role of membrane LT at an early stage of the disease, 3–4-wk-old NOD female mice were treated weekly with LTßR–Ig (100 µg/wk) for 3 wk. Only one of the NOD mice treated with LTßR–Ig developed IDDM by 32 wk, while 67% of the NOD mice treated with control Ig developed IDDM by 26 wk (Fig 1 A). To study whether LTßR–Ig treatment reduced IDDM after the initial phase, female NOD mice were treated with the soluble receptor at 6–7 wk of age, a time when many islets are infiltrated with autoreactive T cells. Treatment with LTßR–Ig at this time similarly prevented the development of IDDM, while the majority of control mice developed the disease by 25 wk (Fig 1 B). These results suggest that LT is essential for the early development of IDDM.





View larger version (46K):
[in this window]
[in a new window]
 
Figure 1. LTßR–Ig treatment prevents diabetes in young NOD mice. Young NOD mice (n = 10) 3–4 (A) and 6–7 wk of age (B), were treated intraperitoneally with LTßR–Ig or control human Ig for 3 wk. (C) Young CD28-/- NOD mice (n = 5 per group) of 3 wk of age were treated intraperitoneally with LTßR–Ig or PBS for 4 wk. Incidence of diabetes was evaluated at weekly intervals.

Previous studies have shown that CD4+CD25+ regulatory cells control spontaneous autoimmune diabetes, even at late stages, by limiting the destructive infiltrate in the islets (17). We previously reported that both B7-/- mice and CD28-/- NOD mice exhibited severe insulitis and accelerated disease as a consequence of a reduced number of these suppressor cells (17) (18). To study whether LT was functioning by altering this regulatory T cell subset, CD28-/- NOD mice, deficient in CD4+CD25+ T cells, were treated weekly with LTßR–Ig or control Ig for 4 wk starting at 3 wk of age and examined for disease incidence. All of the control mice developed IDDM by 11 wk of age, similar to the time course observed in previous experiments. In contrast, there was a delay in disease onset in the LTßR–Ig-treated mice. In fact, some of the mice were free of IDDM for an additional 10–13 wk (Fig 1 C). Similar retarded diabetes was found in CD28-/- NOD mice that were only treated twice with the fusion protein beginning at the ages of 4 and 5 wk. However, the number of CD4+CD25+ T cells in the spleens of LTßR–Ig-treated mice remained comparable with that in the control group (data not shown). Therefore, these results suggest a critical role for LT in the development of IDDM in this accelerated model (Fig 1 C), which is independent of CD28 and cannot be attributed to the CD4+CD25+ T cell pathway.

Reversal of Islet Destruction by Preexisting Diabetic T Cells Using LTßR–Ig.
By 10 wk, most islets in NOD mice show severe infiltration by autoreactive T cells, accompanied by early signs of islet cell destruction. Very few reagents have been shown to block the development of IDDM at this late stage (5) (19). To determine whether the effect of LTßR–Ig could prevent the development of IDDM at this late phase, 10-wk-old NOD mice were similarly treated weekly with LTßR–Ig over 3 wk. None of the LTßR–Ig-treated NOD mice became diabetic, whereas most of control NOD mice developed IDDM by 25 wk (Fig 2 A). To study whether LTßR–Ig treatment just retarded the development of IDDM for a few more days or had a prolonged protection, we extended our observation up to 38 wk and found that none of the LTßR–Ig-treated NOD mice became diabetic. More astonishingly, two doses of LTßR–Ig blocked the development of diabetes in 85% of prediabetic NOD mice (11/13) treated as late as 14 wk of age, a time point when some NOD mice (two mice from each group) were already diabetic, with nearly all the islets attacked by autoreactive T cells (Fig 2 B). Only 15% (2/13) of prediabetic mice treated with LTßR–Ig developed IDDM at 20 wk of age, whereas most of the control Ig–treated group (63%) developed IDDM by 18 wk of age. These findings suggest that LT also plays an important role in the late phases of the disease. However, treatment failed to reverse IDDM in the mice that were already diabetic at the time of treatment.





View larger version (43K):
[in this window]
[in a new window]
 
Figure 2. LTßR–Ig treatment blocks autoreactive T cell–mediated islet destruction. (A) NOD (10 wk of age) female mice (n = 10) were given weekly intraperitoneal injection of 100 µg of LTßR–Ig or human Ig for 3 wk. (B) NOD (14 wk of age) female mice were given weekly intraperitoneal injection of 100 µg of LTßR–Ig (n = 13) or human Ig (n = 8) for only 2 wk. Blood glucose levels were checked weekly after the end of treatment (A and B). (C) Sublethally irradiated NOD female mice (10 mice per group at 7–9 wk of age) were injected intravenously with 2 x 107 splenocytes from mice with diabetes and treated with a single intraperitoneal injection of 100 µg of LTßR–Ig or human Ig. Blood glucose levels were checked weekly starting 1 wk after adoptive transfer.

The transfer of splenocytes from diabetic NOD mice into irradiated NOD recipient results in diabetes within a few weeks, due to acute infiltration of donor autoreactive T cells into islets. To further address whether the administration of LTßR–Ig can prevent diabetogenic T cells from destroying islet cells, splenocytes from diabetic NOD mice were transferred into irradiated NOD recipients treated with a single dose of LTßR–Ig. The development of IDDM in these irradiated mice was prevented (Fig 2 C). These results support a model wherein the use of LTßR–Ig can prevent the development of IDDM mediated by preexisting autoreactive T cells in the circulation.

Prevention and Resolution of Insulitis after LTßR–Ig Therapy.
One striking feature of IDDM in NOD mice is the prototypic formation of lymphoid follicular structures within the pancreas. The profound blockade of different stages of IDDM by LTßR–Ig treatment may be attributed to the reduction or even reversal of LT-mediated lymphoid structures in the pancreas. To test this, 3–4- or 10-wk-old NOD mice were treated weekly with LTßR–Ig and control Ig for 3 wk. 10 d after the end of treatment, the pancreata were collected for hematoxylin and eosin staining. As seen in Fig 3, the number with destructive insulitis and its severity was greatly reduced in both treated groups of mice during the inductive phase (Fig 3 A). The percentage of islets in an early treated group of mice displaying overall infiltration with formation of lymphoid structures (28.4%) was considerably lower than that observed in the control mice (70.1%; Fig 3 B). In NOD mice treated at the late stage (10 wk), only 15% of islets developed insulitis, and 3% of those showed the formation of lymphoid structure inside ß cells by 14 wk. In contrast, 82% of the corresponding control group demonstrated insulitis and 69% exhibited severe insulitis (Fig 3 B). The data also demonstrated that the number and scope of infiltrating cells in LTßR–Ig-treated mice at the age of 14 wk was reduced far below the level of untreated mice at the age of 8–9 wk (before the treatment). These results indicate that LTßR–Ig treatment not only prevents but also resolves the formation of lymphoid structures in the pancreas. The data also reveal that the infiltration of inflammatory cells and the development of lymphoid follicles may be a dynamic process and can be reversed once the influx of cells is blocked.




View larger version (166K):
[in this window]
[in a new window]
 
Figure 3. Reversal of insulitis by LTßR–Ig treatment. (a) 4- (A and B) or 10-wk-old (C and D) NOD mice were treated with control Ig (A and C) or LTßR–Ig (B and D) for 3 wk. The pancreata were collected 10 d after the end of treatment, and the sections were stained by hematoxylin and eosin. Representative fields (most common fields) are shown. (b) Individual islets from four mice per group were scored for insulitis at 8 or 14 wk, respectively.


  Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Our study demonstrates that the treatment of NOD mice with LTßR–Ig can be effective at blocking both the initiation and effector phases of diabetes. More importantly, such treatment reverses insulitis and prevents the formation of lymphoid follicles, even when insulitis is well established and diabetogenic T cells are clearly present. Such treatment at either early or late stage has prolonged the protection, suggesting that short-term treatment with LTßR–Ig could significantly alter the course of IDDM. Several possible mechanisms may be associated with this treatment. First, as both LT and LIGHT bind LTßR in vitro, these effects may arise from blockage of LIGHT signaling. LIGHT is a costimulatory molecule through its receptor, herpes virus entry mediator, that has been shown to promote T cell proliferation and IFN-{gamma} production (20). Its biological activity in vivo, however, has not been well defined. Further dissection of the role of LIGHT is difficult due to the lack of reagents that effectively block LIGHT activity in vivo. Anti-LTß antibody has an extremely short half-life of 1 d, while the half-life of LTßR–Ig is 7 d (16). Second, LT may be a critical cytokine for the development of the CD4+CD25+ T cells that regulate autoimmunity. Although LTßR–Ig inhibited the development of the disease in the CD4+CD25+ cell–reduced CD28 knockout NOD mice, there was no increase in the number of regulatory T cells in the spleen during or after treatment. There was also no increase of CD4+CD25+ cells in wild-type NOD mice after treatment. Thus, it appears unlikely that the effects of LTßR–Ig treatment are mediated through CD4+CD25+ T cells.

The most likely mechanism by which LTßR–Ig prevents the development of IDDM is through LT-mediated expression of adhesion molecules and chemokines and the formation of lymphoid tissues (9) (10) (11) (12) (13) (14) (15). Compared with other adhesion molecules, such as VCAM-1 and PNAd, the expression of MAdCAM-1 is more closely associated with the development of insulitis. In fact, treatment of NOD mice with anti–MAdCAM-1 antibody (MECA367) alone can reduce insulitis and prevent the development of IDDM (21). Interestingly, both LTßR–Ig and anti-LTß antibody can very efficiently block the expression of MAdCAM-1 in the spleen (12) (13). We found that the expression of MAdCAM-1 in the pancreas and spleen was readily reduced after the administration of LTßR–Ig. To directly test whether blocking MAdCAM-1 can prevent the migration of autoreactive T cells, NOD mice that received T cells from diabetic mice were treated with anti–MAdCAM-1 (MECA367) antibody. Such treatment could prevent or retard the development of IDDM (data not shown). Together, these data suggest that LT-mediated adhesion molecules, such as MAdCAM-1, are at least partially responsible for the development of IDDM. It is possible that the expression of MAdCAM-1 and other adhesion molecules is required for the formation of insulitis or lymphoid follicles in the pancreas. It has been proposed recently that the expression of membrane LT is required for the formation of large lymphoid follicles in the islets of B lymphocyte chemokine–mediated transgenic mice (8). We have previously showed that the migration of dendritic cells into the spleen is impaired in LTßR–Ig-treated mice (15). It is possible that the chemokines and adhesion molecules reduced by LTßR–Ig treatment slow the influx of inflammatory cells into the pancreas. In this study, we demonstrated that LTßR–Ig reduced the formation of lymphoid follicles and even reversed severe insulitis in NOD mice. Taken together, these data suggest that LT-mediated microenvironment is essential for the development of lymphoid structure that is required for the development of IDDM.

The generation of a genetic defect of the LT gene in NOD mice is another potential model that could be used to address the role of LT in the development of IDDM. Several disadvantages to this model exist, however. First, intercrossing of the NOD mice with LT{alpha}-/- mice to retain a defect in both LT and the MHC loci is an impractical task, as the locus of LT is very close to that of MHC class II, which is a key genetic locus for the development of IDDM. Second, LT{alpha}-/- mice lack organized lymph nodes, which may be critical for the development of IDDM by itself. Such compound defects may complicate the interpretation of the results. On the other hand, the use of soluble receptor has several advantages. We can correlate different phases of the disease with the timing of administration of fusion protein. Long-term impact after the termination of treatment can be readily assessed.

Resolution of severe insulitis by LTßR–Ig treatment suggests that the migration of inflammatory cells into the islets is a dynamic process in which these cells may constantly move in and out of the target tissue. Interference with this dynamic process may reduce inflammation, which prevents further activation of autoreactive T cells and tissue damage. Interestingly, alteration of the LT-mediated microenvironment by LTßR–Ig is a transient effect that ends once the treatment is terminated. For example, the expression of splenic MAdCAM-1 and the development of B cell follicles was gradually restored 4–5 wk after the termination of LTßR–Ig treatment. Thus, the observation that short-term LTßR–Ig treatment has a profound and prolonged impact on islet destruction may provide a new avenue for the study of pathogenesis and for the treatment of this disease or other autoimmune diseases. In the future, it will be interesting to explore whether LTßR–Ig treatment may prevent islet cell rejection by autoreactive or alloreactive T cells.


  Footnotes

Q. Wu and B. Salomon contributed equally to this study. Drs. Bluestone and Fu should both be considered senior authors of this article.


  Acknowledgements
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

We thank Jennifer Arcella, Lesley Rhee, and Shihong Li for their expert technical support. We also thank the National Cell Culture Center for the generation of LTßR–Ig using its bioreactor.

This research was supported in part by Biogen, Inc., National Institutes of Health grants, and Juvenile Diabetes Foundation International (1-2000-875). Y.-X. Fu is a recipient of a clinical investigator award (AI01431). J.A. Bluestone, B. Salomon, and some of the research were supported by the Juvenile Diabetes Foundation as part of the JDFI Islet Transplant Center at University of Chicago and University of Minnesota.

Submitted: 14 November 2000
Revised: 8 March 2001
Accepted: 20 March 2001


  References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

  1. Tisch, R., and McDevitt, H. 1996. Insulin-dependent diabetes mellitus. Cell. 85:291-297[Medline].

  2. Salomon, B., and Bluestone, J.A. 2001. Importance of co-stimulatory molecules in autoimmunity and transplantation. Annu. Rev. Immunol. 19:225-252[Medline].

  3. Delovitch, T.L., and Singh, B. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity. 7:727-738[Medline].

  4. Bach, J.F., and Mathis, D. 1997. The NOD mouse. Res. Immunol. 148:285-286[Medline].

  5. Atkinson, M.A., and Leiter, E.H. 1999. The NOD mouse model of type 1 diabetes: as good as it gets? Nat. Med. 5:601-604[Medline].

  6. Green, E.A., and Flavell, R.A. 1999. Tumor necrosis factor-alpha and the progression of diabetes in non-obese diabetic mice. Immunol. Rev. 169:11-22[Medline].

  7. Ludewig, B., Odermatt, B., Landmann, S., Hengartner, H., and Zinkernagel, R.M. 1998. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J. Exp. Med. 188:1493-1501[Abstract/Free Full Text].

  8. Luther, S.A., Lopez, T., Bai, W., Hanahan, D., and Cyster, J.G. 2000. BLC expression in pancreatic islets causes B cell recruitment and lymphotoxin-dependent lymphoid neogenesis. Immunity. 12:471-481[Medline].

  9. Fu, Y.X., Molina, H., Matsumoto, M., Huang, G.M., Min, J.J., and Chaplin, D.D. 1997. Lymphotoxin-{alpha} (LT{alpha}) supports development of splenic follicular structure that is required for IgG responses. J. Exp. Med. 185:2111-2120[Abstract/Free Full Text].

  10. Fu, Y.-X., and Chaplin, D.D. 1999. Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17:399-433[Medline].

  11. Iizuka, K., Chaplin, D.D., Wang, Y., Wu, Q., Pegg, L.E., Yokoyama, W.M., and Fu, Y.X. 1999. Requirement for membrane lymphotoxin in natural killer cell development. Proc. Natl. Acad. Sci. USA. 96:6336-6340[Abstract/Free Full Text].

  12. Mackay, F., Majeau, G.R., Lawton, P., Hochman, P.S., and Browning, J.L. 1997. Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice. Eur. J. Immunol. 27:2033-2042[Medline].

  13. Mackay, F., and Browning, J.L. 1998. Turning off follicular dendritic cells. Nature. 395:26-27[Medline].

  14. Ngo, V., Korner, H., Gunn, M., Schmidt, K., Riminton, D.S., Cooper, M., Browning, J., Sedgwick, J., and Cyster, J.G. 1999. Lymphotoxin {alpha}/ß and tumor necrosis factor are required for stromal cells expression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403-412[Abstract/Free Full Text].

  15. Wu, Q., Wang, Y., Wang, J., Hedgeman, E.O., Browning, J.L., and Fu, Y.X. 1999. The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues. J. Exp. Med. 190:629-638[Abstract/Free Full Text].

  16. Browning, J.L., Sizing, I.D., Lawton, P., Bourdon, P.R., Rennert, P.D., Majeau, G.R., Ambrose, C.M., Hession, C., Miatkowski, K., and Griffiths, D.A. et al. 1997. Characterization of lymphotoxin-alpha-beta complexes on the surface of mouse lymphocytes. J. Immunol. 159:3288-3298[Abstract].

  17. Salomon, B., Lenschow, D.J., Rhee, L., Ashourian, N., Singh, B., Sharpe, A., and Bluestone, J.A. 2000. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity. 12:431-440[Medline].

  18. Lenschow, D.J., Herold, K.C., Rhee, L., Patel, B., Koons, A., Qin, H.Y., Fuchs, E., Singh, B., Thompson, C.B., and Bluestone, J.A. 1996. CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity. 5:285-293[Medline].

  19. Chatenoud, L., Primo, J., and Bach, J.F. 1997. CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice. J. Immunol. 158:2947-2954[Abstract].

  20. Tamada, K., Shimozaki, K., Chapoval, A.I., Zhu, G., Sica, G., Flies, D., Boone, T., Hsu, H., Fu, Y.X., and Nagata, S. et al. 2000. Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway. Nat. Med. 6:283-289[Medline].

  21. Michie, S.A., Sytwu, H.K., McDevitt, J.O., and Yang, X.D. 1998. The roles of alpha 4-integrins in the development of insulin-dependent diabetes mellitus. Curr. Top. Microbiol. Immunol. 231:65-83[Medline].