Antisense Oligonucleotides Down-Regulating Costimulation
Confer Diabetes-Preventive Properties to Nonobese Diabetic
Mouse Dendritic Cells1
Jennifer Machen,* Jo Harnaha,* Robert Lakomy,* Alexis Styche,* Massimo Trucco,*†and
Phenotypically “immature” dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune
suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic imma-
turity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell
surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides
targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection
of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells
proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4?CD25?T cells in NOD recip-
ients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4?CD25?T cells compared
with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative
regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimu-
latory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of
regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as
a potential therapeutic option for type 1 diabetes. The Journal of Immunology, 2004, 173: 4331–4341.
lular level, the ? cells are subjected to cytokine-induced impair-
ment by the actions of infiltrating macrophages and T cells with
subsequent T cell-mediated destruction (1, 2). There is no question
that defects in T cell selection at the central level in the thymus and
impaired peripheral regulation of ? cell Ag-specific T cells under-
lie the etiopathogenesis of T1DM (1, 2). Dendritic cells (DC) have
proven to be integral participants in the initiation and propagation
of T1DM at multiple levels including the regulation of diabetes
onset (3–5, 6–8). DC are the primary APCs of the immune system
and as such, they control the activation of naive T cells (4, 9–11).
For full activation of naive CD4?T lymphocytes to occur, two
signals are required. The first is the presentation of the Ag to the
TCR in the context of class II MHC on DC. This will cause the
responding T cell to up-regulate the CD154 molecule (CD40 li-
gand) to its cell surface, thereby activating the initiation of the
second signal. In this process of coactivation, CD154 will interact
with the CD40 molecule at the surface of the APC resulting in the
ype 1 diabetes mellitus (T1DM),3a disorder of glucose
homeostasis, is the consequence of the autoimmune tar-
geting of pancreatic insulin-producing ? cells. At the cel-
up-regulation of CD80 and CD86 at the cell surface of the APC.
Immediately thereafter, CD80 and CD86, acting as the second sig-
nal, in the process of costimulation, will engage the CD28 mole-
cule on the T cell resulting in its full activation. In the absence of
the interactions between CD80, CD86, and CD28, the T cell will
either enter a state of functional silence, termed anergy, or will be
primed for apoptosis, perhaps in a CD95-CD95L (Fas-FasL)-de-
pendent manner (12–14). Converging lines of evidence indicate
that the phenotype of the DC cell surface can play an important
role in tolerance to self-Ags and can be manipulated to promote
allogeneic as well as autoimmune hyporesponsiveness (11, 15).
The first use of DC to prevent T1DM in NOD mice was docu-
mented by Clare-Salzler et al. (8) who demonstrated that transfer
of pancreatic lymph node DC derived from 8- to 20 wk-old NOD
mice into prediabetic NOD mice conferred significant protection
from T1DM, insulitis, and adoptive transfer of T1DM. The authors
suggested that acquisition of islet Ags by DC during insulitis may
have resulted in them acquiring a phenotype, once in the pancreatic
lymph nodes, that was able to result in the stimulation of regula-
tory immune cells which attenuated the insulitic process. Interest-
ingly, while transfer of DC isolated from nonpancreatic lymph
nodes to NOD mice was unable to affect T1DM incidence, transfer
of these DC pulsed with sonicated islets did confer protection (8).
More recently, Morel and colleagues (16, 17) have shown prolon-
gation of a diabetes-free state in NOD recipients of bone marrow-
derived syngeneic DC. Other methods of generating diabetes-sup-
pressive DC include vitamin D receptor ligands, Ag pulsing, and
IFN-? treatment (18–20).
NOD DC exhibit strong immunostimulatory capacity, underlied
by hyperactivation of NF-?B (21–23). Therefore, we proposed and
very recently showed that inhibition of NF-?B using short, double-
stranded transcriptional decoys could render NOD DC less immu-
nostimulatory and that administration of these engineered DC into
*Diabetes Institute,†Division of Immunogenetics, Department of Pediatrics, and‡De-
partment of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Received for publication October 9, 2003. Accepted for publication July 26, 2004.
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.
1This work was supported by National Institutes of Health Awards DK61328 and
DK60183 (to N.G.).
2Address correspondence and reprint requests to Dr. Nick Giannoukakis, Diabetes
Institute, University of Pittsburgh School of Medicine, Rangos Research Center, 3460
Fifth Avenue, Pittsburgh, PA 15213. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: T1DM, type 1 diabetes mellitus; DC, dendritic
cell; AS-ODN, antisense oligonucleotide.
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc.0022-1767/04/$02.00
NOD prediabetic mice could prevent the development of diabetes
(24). Attractive as this approach is, we nevertheless do have con-
cerns that are under investigation that NF-?B blockade may inter-
fere with functions crucial to DCsurvivalinvivothatmayimpacton
the persistence of the immunosuppressive effect of these DC in NOD
mice. Hence, as a second, and complementary approach, we decided
to engineer DC in a way where the expression of only the costimu-
latory molecules CD40, CD80, and CD86 would be suppressed at the
cell surface. We based our rationale on the many studies demonstrat-
ing the effectiveness of CD80/CD86-CD28 blockade in generating
immune hyporesponsiveness to alloantigens and in preventing auto-
our approach limits the cell population that is targeted, because the
treatment is performed ex vivo and does not involve systemic dis-
semination of a protein which, in the instance of CTLA4-Ig and
anti-CD40L have exhibited nonspecific and toxic effects (53, 54).
DCs with a mixture of antisense oligonucleotides (AS-ODN) target-
ing the CD40, CD80, and CD86 transcripts confers specific suppres-
sion of the respective cell surface proteins. We further demonstrate
that a single injection of these engineered DC into syngeneic predi-
abetic female NOD mice significantly delays the incidence of T1DM
without affecting the response of T cells from diabetes-free DC
recipients to alloantigen. Furthermore, there was no evidence of
insulitis in the diabetes-free recipients. In NOD-scid recipients, we
show that ODN-treated NOD DC administration in cotransfer with T
cells promotes an increased prevalence of CD4?CD25?CD62L?T
cells. The use of AS technology specifically targeting the transcripts
of key DC cell surface proteins involved in T cell activation and
regulation could be a useful technique to study DC:T cell interactions
promoting immunoregulatory cell networks and as a potential means
of T1DM cell therapy.
Materials and Methods
Female C57BL/6, NOD/LtJ (H2g7), NOD-scid, and C3H/HeJ (H2k) mice
were purchased from The Jackson Laboratory (Bar Harbor, ME) and
housed under pathogen-free conditions. The ?-actin-GFP transgenic mouse
was bred on the C57BL/6 background and was propagated in our mouse
colony. All animal experimentation was conducted in compliance with the
Animal Research Care Committee of the Children’s Hospital of Pittsburgh.
Abs to immune cells were purchased from BD Biosciences (San Diego,
CA) and were used as the direct FITC, PE, CyChrome, or allophycocyanin
fluorochrome conjugates. The clones used were as follows: CD40 (clone
3/23), CD11c (clone HL3), CD86 (clone GL1), CD80 (clone 16-10A1),
CD4 (clone RM4-5), CD25 (clone 7D4), and CD62L (MEL-14). Anti-
insulin and anti-glucagon Abs were purchased from DakoCytomation
(Carpinteria, CA). Isotype- and species-matched irrelevant monoclonal or
polyclonal Abs (where appropriate) were used as controls. The NIT-1 cell
line was obtained from American Type Culture Collection (CRL-2055;
Manassas, VA) and propagated as described by the repository. Phospho-
rothioate-modified ODNs were synthesized by the University of Pittsburgh
DNA Synthesis Facility and HPLC purified. Cell culture reagents (serum
and media) were purchased from Invitrogen Life Technologies (Gaithers-
burg, MD). Beadlyte multiplex fluorescence cytokine detection kits were
purchased from Upstate Biotechnology (Lake Placid, NY) and fluores-
cence-based proliferation as well as phagocytosis probes from Molecular
Probes (Eugene, OR). Recombinant murine cytokines and immune cell
enrichment columns were purchased from R&D Systems (Indianapolis,
IN). All other biochemicals were purchased from Sigma-Aldrich (St.
DC propagation and treatment with AS-ODN
DC were propagated from bone marrow progenitors of 5- to 8-wk-old
female C57BL/6 or NOD mice in GM-CSF and IL-4 as outlined by Ma et
al. (24) and originally described in Fu et al. (55, 56). Briefly, bone marrow
was obtained from the femurs and tibiae of female NOD mice. The RBC
were lysed using a commercially available reagent (Red Blood Cell Lysing
Buffer; Sigma-Aldrich) and the bone marrow cells were plated in 24-well
multiwell plates at 2 ? 106cells/ml in R-10 medium (RPMI 1640/10%
heat-inactivated FBS/50 ?M 2-ME/1% sodium pyruvate/1% nonessential
amino acids/1% penicillin-streptomycin solution (Invitrogen Life Technol-
ogies) with the addition of 4 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D
Systems). Two days later, the nonadherent cells were removed and a 1:1
volume of conditioned medium, fresh R-10 medium, and cytokines were
added to the adherent cells. Three days later, the loosely adherent cells
were gently agitated and harvested. By FACS analysis, ?85% of these
cells are routinely DC with class II MHC, CD11c, CD80, CD40, and CD86
positivity (57). The AS-ODN mixture consisted of phosphorothioate-mod-
ified ODNs each targeting the 5? end of the CD40, CD80, and CD86 pri-
mary transcripts. The sequences are: CD40 AS-ODN, 5?-CAC AGC CGA
GGC AAA GAC ACC ATG CAG GGC A-3?; CD80 AS-ODN, 5?-GGG
AAA GCC AGG AAT CTA GAG CCA ATG GA-3?; CD86 AS-ODN,
5?-TGG GTG CTT CCG TAA GTT CTG GAA CAC GTC-3?. The AS-
ODN were HPLC purified and resuspended in PBS. DC were treated
18–24 h in 10% heat-inactivated FBS/RPMI 1640 with a mixture of 3.3
?M CD40 AS-ODN, 3.3 ?M CD80 AS-ODN, and 3.3 ?M CD86 AS-
ODN (the mixture is collectively referred to as AS-ODN in this study). The
cells were then washed extensively in PBS and subsequently used in cul-
ture or in vivo. Ag uptake and processing capacity were assessed using the
Vybrant Phagocytosis Assay reagent as described by the manufacturer
(Molecular Probes). Uptake of fluorescent bioparticles was measured in a
fluorescence microplate reader at 480 nm excitation/520 nm emission (Vic-
tor2; PerkinElmer Instruments, Boston, MA).
Flow cytometry analysis
All the FACS analyses were performed in a FACSVantage SE flow cy-
tometer with FACSDiva version 2.2.1, capable of eight-color, multiparam-
eter discrimination (BD Biosciences). In every FACS analysis, cells were
stained with propidium iodide to exclude dead cells. The initial cell pop-
ulations were selected based on forward and side scatter properties specific
for DC or T cells. By forward and side scatter, we excluded debris and
clumped cells and by propidium iodide we excluded dead cells from all
analyses. The initial gate was set around the remaining cells. In T cell
populations, wherever we aimed at discriminating putative T regulatory
cells, we gated CD4?cells and analyzed this population for CD25 and
CD62L positivity. Results were recorded as dot plots in QuadStat analyses.
In vitro phenotype of DC treated with AS-ODN and Ag loading
AS-ODN-treated DC were stimulated with 25 ?g/ml LPS (Sigma-Aldrich)
or 50 ng/ml recombinant murine CD40L (R&D Systems) for 18–24 h. The
culture supernatant was collected and assayed for NO production using the
Griess assay and profiled for cytokine secretion using the Beadlyte multi-
plex assay system (Upstate Biotechnology) in a Luminex Fluorescence
Analyser (Luminex, Austin, TX). In parallel, the cells were stained with
fluorescence-conjugated Abs against CD40, CD80, and CD86. T cell pro-
liferation was measured in cocultures of irradiated splenocytes and spleen-
isolated and column-enriched (R&D Systems) T cells from 5- to 8-wk-old,
diabetes-free, diabetic female NOD or T cells from the spleen of C3H/HeJ
females (5–10 wk of age). Proliferation was measured after 5 days in cul-
ture using the CyQuant fluorescence reagent (Molecular Probes). Cytokine
production was measured in the culture supernatants of the cocultures at 5
days using the Beadlyte-Luminex assay. All assays were performed in
triplicate on at least two different occasions.
Examination of DC phenotype following in vivo transfer of
CFSE-labeled or GFP-positive (GFP?) DC into
We first labeled control and AS-ODN-treated NOD DC (from 7-wk-old
female donors) with CFSE (Molecular Probes) as directed by the manu-
facturer. Cells (2 ? 106) were injected i.p. into age-matched female recip-
ients. At weekly intervals, for 3 wk, we harvested the spleens from indi-
vidual recipients and examined the levels of CD80 and CD86 in recovered
CFSE?cells by FACS using Abs for CD80 and CD86 (BD Biosciences).
As a complementary approach, we obtained DC from the bone marrow
progenitors of GFP transgenic mice. In these mice, the GFP transgene is
under the control of the chicken ?-actin promoter. These mice were gen-
erated on a C57BL/6 background. They express GFP in almost all tissues
including monocytes and DC. The DC were treated ex vivo with AS-ODN
30. Steurer, W., P. W. Nickerson, A. W. Steele, J. Steiger, X. X. Zheng, and
T. B. Strom. 1995. Ex vivo coating of islet cell allografts with murine CTLA4/Fc
promotes graft tolerance. J. Immunol. 155:1165.
31. Woodward, J. E., A. L. Bayer, K. D. Chavin, M. L. Blue, and P. Baliga. 1998.
T-cell alterations in cardiac allograft recipients after B7 (CD80 and CD86) block-
ade. Transplantation 66:14.
32. Chahine, A. A., M. Yu, M. McKernan, C. Stoeckert, P. S. Linsley, and H. T. Lau.
1994. Local CTLA4Ig synergizes with one-dose anti-LFA-1 to achieve long-term
acceptance of pancreatic islet allografts. Transplant. Proc. 26:3296.
33. Lenschow, D. J., Y. Zeng, K. S. Hathcock, L. A. Zuckerman, G. Freeman,
J. R. Thistlethwaite, G. S. Gray, R. J. Hodes, and J. A. Bluestone. 1995. Inhibition
of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibod-
ies. Transplantation 60:1171.
34. Azuma, H., A. Chandraker, K. Nadeau, W. W. Hancock, C. B. Carpenter,
N. L. Tilney, and M. H. Sayegh. 1996. Blockade of T-cell costimulation prevents
development of experimental chronic renal allograft rejection. Proc. Natl. Acad.
Sci. USA 93:12439.
35. Bluestone, J. A. 1996. Costimulation and its role in organ transplantation. Clin.
36. Bolling, S. F., H. Lin, R. Q. Wei, and L. A. Turka. 1996. Preventing allograft
rejection with CTLA4Ig: effect of donor-specific transfusion route or timing.
J. Heart Lung Transplant. 15:928.
37. Cabrian, K. M., K. K. Berry, W. W. Shuford, R. S. Mittler, J. N. Rodgers, and
P. S. Linsley. 1996. Suppression of T-cell-dependent immune responses in mon-
keys by CTLA4Ig. Transplant. Proc. 28:3261.
38. Gribben, J. G., E. C. Guinan, V. A. Boussiotis, X. Y. Ke, L. Linsley, C. Sieff,
G. S. Gray, G. J. Freeman, and L. M. Nadler. 1996. Complete blockade of B7
family-mediated costimulation is necessary to induce human alloantigen-specific
anergy: a method to ameliorate graft- versus-host disease and extend the donor
pool. Blood 87:4887.
39. Judge, T. A., A. Tang, L. M. Spain, J. Deans-Gratiot, M. H. Sayegh, and L. A. Turka.
1996. The in vivo mechanism of action of CTLA4Ig. J. Immunol. 156:2294.
40. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix,
C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley,
K. J. Winn, and T. C. Pearson. 1996. Long-term acceptance of skin and cardiac
allografts after blocking CD40 and CD28 pathways. Nature 381:434.
41. Daikh, D., D. Wofsy, and J. B. Imboden. 1997. The CD28–B7 costimulatory
pathway and its role in autoimmune disease. J. Leukocyte Biol. 62:156.
42. Gainer, A. L., G. S. Korbutt, R. V. Rajotte, G. L. Warnock, and J. F. Elliott. 1997.
Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet
allograft survival. Transplantation 63:1017.
43. Hale, D. A., R. Gottschalk, T. Maki, and A. P. Monaco. 1997. Use of CTLA4-Ig
in combination with conventional immunosuppressive agents to prolong allograft
survival. Transplantation 64:897.
44. Hayashi, S., Y. Namii, T. Kozima, T. Kobayashi, I. Yokoyama, I. Saito,
H. Hamada, S. Ohtsuka, K. Uchida, and H. Takagi. 1997. Effect of CTLA4-Ig
gene transfer using adenoviral vector in organ and cell transplantation. Trans-
plant. Proc. 29:2212.
45. Levisetti, M. G., P. A. Padrid, G. L. Szot, N. Mittal, S. M. Meehan, C. L. Wardrip,
G. S. Gray, D. S. Bruce, J. R. Thistlethwaite, Jr., and J. A. Bluestone. 1997. Immu-
nosuppressive effects of human CTLA4Ig in a non-human primate model of alloge-
neic pancreatic islet transplantation. J. Immunol. 159:5187.
46. Roy-Chaudhury, P., P. W. Nickerson, R. C. Manfro, X. X. Zheng, J. Steiger,
Y. S. Li, and T. B. Strom. 1997. CTLA4Ig attenuates accelerated rejection (pre-
sensitization) in the mouse islet allograft model. Transplantation 64:172.
47. Sun, H., V. Subbotin, C. Chen, A. Aitouche, L. A. Valdivia, M. H. Sayegh,
P. S. Linsley, J. J. Fung, T. E. Starzl, and A. S. Rao. 1997. Prevention of chronic
rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and
anti-CD40 ligand monoclonal antibody. Transplantation 64:1838.
48. Tu, Y., A. Rehman, and M. W. Flye. 1997. CTLA4-Ig treatment prolongs rat
orthotopic liver graft survival. Transplant. Proc. 29:1036.
49. Weber, C. J., M. K. Hagler, J. T. Chryssochoos, J. A. Kapp, G. S. Korbutt,
R. V. Rajotte, and P. S. Linsley. 1997. CTLA4-Ig prolongs survival of microen-
capsulated neonatal porcine islet xenografts in diabetic NOD mice. Cell Trans-
50. Yi-qun, Z., K. Lorre, M. de Boer, and J. L. Ceuppens. 1997. B7-blocking agents,
alone or in combination with cyclosporin A, induce antigen-specific anergy of
human memory T cells. J. Immunol. 158:4734.
51. Gainer, A. L., W. L. Suarez-Pinzon, W. P. Min, C. Hancock-Friesen, G. S. Korbutt,
R. V. Rajotte, A. Rabinovitch, G. L. Warnock, and J. F. Elliott. 1998. Prolongation
Fas ligand or a combination of the two. Transplant. Proc. 30:534.
52. Guo, Z., D. Mital, Y. Y. Mo, Y. Tian, J. Shen, A. S. Chong, P. Foster, H. Sankary,
L. McChesney, S. C. Jensik, and J. W. Williams. 1998. Effect of gene gun-
mediated CTLA4IG and Fas ligand gene transfection on concordant xenogeneic
islet graft rejection. Transplant. Proc. 30:589.
53. Hong, J. C., and B. D. Kahan. 2000. Immunosuppressive agents in organ trans-
plantation: past, present, and future. Semin. Nephrol. 20:108.
54. Wekerle, T., P. Blaha, F. Langer, M. Schmid, and F. Muehlbacher. 2002. Tol-
erance through bone marrow transplantation with costimulation blockade. Trans-
plant Immunol. 9:125.
55. Fu, F., Y. Li, S. Qian, L. Lu, F. D. Chambers, T. E. Starzl, J. J. Fung, and
A. W. Thomson. 1997. Costimulatory molecule-deficient dendritic cell progeni-
tors induce T cell hyporesponsiveness in vitro and prolong the survival of vas-
cularized cardiac allografts. Transplant. Proc. 29:1310.
56. Fu, F., Y. Li, S. Qian, L. Lu, F. Chambers, T. E. Starzl, J. J. Fung, and
A. W. Thomson. 1996. Costimulatory molecule-deficient dendritic cell progeni-
tors (MHC class II?, CD80dim, CD86?) prolong cardiac allograft survival in
nonimmunosuppressed recipients. Transplantation 62:659.
57. Lu, L., D. McCaslin, T. E. Starzl, and A. W. Thomson. 1995. Bone marrow-
derived dendritic cell progenitors (NLDC 145?, MHC class II?, B7-1dim, B7-2?)
induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. Trans-
58. Hamaguchi, K., H. R. Gaskins, and E. H. Leiter. 1991. NIT-1, a pancreatic ?-cell
line established from a transgenic NOD/Lt mouse. Diabetes 40:842.
59. Reid, B. D., H. Y. Qin, S. Prange, E. Lee-Chan, Q. Yu, J. F. Elliott, and B. Singh.
1997. Modulation and detection of IDDM by membrane associated antigens from
the islet ? cell line NIT. J. Autoimmun. 10:27.
60. Shimizu, J., O. Kanagawa, and E. R. Unanue. 1993. Presentation of ?-cell antigens
to CD4?and CD8?T cells of non-obese diabetic mice. J. Immunol. 151:1723.
61. Giannoukakis, N., C. A. Bonham, S. Qian, Z. Chen, L. Peng, J. Harnaha, W. Li,
A. W. Thomson, J. J. Fung, P. D. Robbins, and L. Lu. 2000. Prolongation of
cardiac allograft survival using dendritic cells treated with NF-?B decoy oligode-
oxyribonucleotides. Mol. Ther. 1:430.
62. Cameron, M. J., G. A. Arreaza, and T. L. Delovitch. 1997. Cytokine- and co-
stimulation-mediated therapy of IDDM. Crit. Rev. Immunol. 17:537.
63. Cameron, M. J., G. A. Arreaza, P. Zucker, S. W. Chensue, R. M. Strieter,
S. Chakrabarti, and T. L. Delovitch. 1997. IL-4 prevents insulitis and insulin-
dependent diabetes mellitus in nonobese diabetic mice by potentiation of regu-
latory T helper-2 cell function. J. Immunol. 159:4686.
64. Sharif, S., G. A. Arreaza, P. Zucker, and T. L. Delovitch. 2002. Regulatory
natural killer T cells protect against spontaneous and recurrent type 1 diabetes.
Ann. NY Acad. Sci. 958:77.
65. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa,
A. Cumano, and A. Bandeira. 2001. CD25?CD4?T cells regulate the expansion
of peripheral CD4 T cells through the production of IL-10. J. Immunol. 166:3008.
66. Hara, M., C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell,
P. J. Morris, F. Powrie, and K. J. Wood. 2001. IL-10 is required for regulatory T
cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789.
67. Jonuleit, H., E. Schmitt, M. Stassen, A. Tuettenberg, J. Knop, and A. H. Enk. 2001.
Identification and functional characterization of human CD4?CD25?T cells with
regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285.
68. Chatenoud, L., B. Salomon, and J. A. Bluestone. 2001. Suppressor T cells-they’re
back and critical for regulation of autoimmunity! Immunol. Rev. 182:149.
69. Bach, J. F., and L. Chatenoud. 2001. Tolerance to islet autoantigens in type 1
diabetes. Annu. Rev. Immunol. 19:131.
70. Shevach, E. M., R. S. McHugh, A. M. Thornton, C. Piccirillo, K. Natarajan, and
D. H. Margulies. 2001. Control of autoimmunity by regulatory T cells. Adv. Exp.
Med. Biol. 490:21.
71. Shevach, E. M., R. S. McHugh, C. A. Piccirillo, and A. M. Thornton. 2001. Control
of T-cell activation by CD4?CD25?suppressor T cells. Immunol. Rev. 182:58.
72. McHugh, R. S., E. M. Shevach, and A. M. Thornton. 2001. Control of organ-
specific autoimmunity by immunoregulatory CD4?CD25?T cells. Microbes In-
73. Thornton, A. M., and E. M. Shevach. 2000. Suppressor effector function of
74. Feili-Hariri, M., and P. A. Morel. 2001. Phenotypic and functional characteristics of
BM-derived DC from NOD and non-diabetes-prone strains. Clin. Immunol. 98:133.
75. Tarbell, K. V., S. Yamazaki, K. Olson, P. Toy, and R. M. Steinman. 2004.
CD25?CD4?T cells, expanded with dendritic cells presenting a single autoan-
tigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 199:1467.
76. Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, and
R. M. Steinman. 2003. Direct expansion of functional CD25?CD4?regulatory
T cells by antigen-processing dendritic cells. J. Exp. Med. 198:235.
77. Ruedl, C., P. Koebel, M. Bachmann, M. Hess, and K. Karjalainen. 2000. Ana-
tomical origin of dendritic cells determines their life span in peripheral lymph
nodes. J. Immunol. 165:4910.
78. Lee, W. C., C. Zhong, S. Qian, Y. Wan, J. Gauldie, Z. Mi, P. D. Robbins,
A. W. Thomson, and L. Lu. 1998. Phenotype, function, and in vivo migration and
survival of allogeneic dendritic cell progenitors genetically engineered to express
TGF-?. Transplantation 66:1810.
79. Wang, B., Y. B. Geng, and C. R. Wang. 2001. CD1-restricted NK T cells protect
nonobese diabetic mice from developing diabetes. J. Exp. Med. 194:313.
80. Laloux, V., L. Beaudoin, D. Jeske, C. Carnaud, and A. Lehuen. 2001. NK T
cell-induced protection against diabetes in V?14-J?281 transgenic nonobese di-
abetic mice is associated with a Th2 shift circumscribed regionally to the islets
and functionally to islet autoantigen. J. Immunol. 166:3749.
81. Herman, A. E., G. J. Freeman, D. Mathis, and C. Benoist. 2004. CD4?CD25?T
regulatory cells dependent on ICOS promote regulation of effector cells in the
prediabetic lesion. J. Exp. Med. 199:1479.
82. Tang, Q., K. J. Henriksen, M. Bi, E. B. Finger, G. Szot, J. Ye, E. L. Masteller,
H. McDevitt, M. Bonyhadi, and J. A. Bluestone. 2004. In vitro-expanded antigen-
specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199:1455.
83. Tang, Q., K. J. Henriksen, E. K. Boden, A. J. Tooley, J. Ye, S. K. Subudhi,
X. X. Zheng, T. B. Strom, and J. A. Bluestone. 2003. Cutting edge: CD28 con-
trols peripheral homeostasis of CD4?CD25?regulatory T cells. J. Immunol.
84. Adorini, L. 2003. Tolerogenic dendritic cells induced by vitamin D receptor
ligands enhance regulatory T cells inhibiting autoimmune diabetes. Ann. NY
Acad. Sci. 987:258.
85. Bottino, R., P. Lemarchand, M. Trucco, and N. Giannoukakis. 2003. Gene- and
cell-based therapeutics for type I diabetes mellitus. Gene Ther. 10:875.
86. Trucco, M., P. D. Robbins, A. W. Thomson, and N. Giannoukakis. 2002. Gene
therapy strategies to prevent autoimmune disorders. Curr. Gene Ther. 2:341.
4341The Journal of Immunology