A GMCSF-neuroantigen fusion protein is a
potent tolerogen in experimental
autoimmune encephalomyelitis (EAE) that is
associated with efficient targeting of
neuroantigen to APC
J. Lori Blanchfield and Mark D. Mannie1
The Department of Microbiology and Immunology, East Carolina University, Brody School of Medicine, Greenville, North
RECEIVED JULY 31, 2009; REVISED OCTOBER 8, 2009; ACCEPTED OCTOBER 24, 2009. DOI: 10.1189/jlb.0709520
Cytokine-NAg fusion proteins represent an emerging
platform for specific targeting of self-antigen to particu-
lar APC subsets as a means to achieve antigen-specific
immunological tolerance. This study focused on cyto-
kine-NAg fusion proteins that targeted NAg to myeloid
APC. Fusion proteins contained GM-CSF or the soluble
extracellular domain of M-CSF as the N-terminal do-
main and the encephalitogenic 69–87 peptide of MBP
as the C-terminal domain. GMCSF-NAg and MCSF-NAg
fusion proteins were ?1000-fold and 32-fold more po-
tent than NAg in stimulating antigenic proliferation of
MBP-specific T cells, respectively. The potentiated anti-
genic responses required cytokine-NAg covalent link-
age and receptor-mediated uptake. That is, the re-
spective cytokines did not potentiate antigenic re-
sponses when cytokine and NAg were added as
separate molecules, and the potentiated responses
were inhibited specifically by the respective free cyto-
kine. Cytokine-dependent targeting of NAg was specific
for particular subsets of APC. GMCSF-NAg and MCSF-
NAg targeted NAg to DC and macrophages; con-
versely, IL4-NAg and IL2-NAg fusion proteins, respec-
tively, induced an ?1000-fold enhancement in NAg re-
activity in the presence of B cell and T cell APC.
GMCSF-NAg significantly attenuated severity of EAE
when treatment was completed before encephalito-
genic challenge or alternatively, when treatment was
initiated after onset of EAE. MCSF-NAg also had signif-
icant tolerogenic activity, but GMCSF-NAg was sub-
stantially more efficacious as a tolerogen. Covalent
GMCSF-NAg linkage was required for prevention and
treatment of EAE. In conclusion, GMCSF-NAg was
highly effective for targeting NAg to myeloid APC and
was a potent, antigen-specific tolerogen in EAE. J.
Leukoc. Biol. 87: 511–523; 2010.
Therapeutic advancements for treatment of multiple sclerosis
are often based on nonantigen-specific approaches that may
inhibit pathogenic and adaptive immunity [1–3]. Therapies,
such as those based on IFN-?, mAb, or anti-metabolic/prolifer-
ative drugs, have a generalized effect on the immune system.
The uncomfortable paradigm is that efficacy of a drug for
multiple sclerosis may be commensurate with the severity of
side-effects, such that the degree of benefit for multiple sclero-
sis may be directly due to the ability of the drug to compro-
mise the immune system.
Antigen-specific therapy represents an important alternative
[4–6]. Antigen-specific approaches are based on the concept
of using immunological tolerance to alleviate an autoimmune
disease state. Several antigen-specific strategies have been de-
veloped by use of EAE as an immune-regulatory model of mul-
tiple sclerosis. These therapies include oral tolerance [7, 8],
high dose tolerance [9, 10], deletional tolerance , or im-
mune deviation [12, 13] and are based on the use of altered
peptide ligands [14, 15], antibody-antigen fusion proteins [16,
17], synthetic peptides, recombinant self-antigens [18, 19],
and DNA vaccines [20, 21], among others [22, 23]. Disadvan-
tages of antigen-specific approaches include the possibility of
anaphylaxis, inadvertent sensitization, and worsening disease
[24, 25]. These disadvantages are coupled with current uncer-
tainty of which self-antigens should be targeted to alleviate
multiple sclerosis. Nonetheless, antigen-specific therapies offer
the best hope of inhibiting pathogenic autoimmune responses
1. Correspondence: Department of Microbiology and Immunology, Brody
School of Medicine, 600 Moye Blvd., Greenville, NC, 27834, USA. E-mail:
Abbreviations: DC?dendritic cell, DCreg?regulatory DC, DHFR?dihydrofolate
reductase, EAE?experimental autoimmune encephalomyelitis, GPMBP?
guinea pig myelin basic protein, IL1RA-NAg?IL-1R antagonist-NAg,
MBP?myelin basic protein, MOG?myelin oligodendrocyte glycoprotein,
NAg?neuroantigen (the dominant encephalitogenic region of GPMBP and
the GP69-88 synthetic peptide), PAP?pulmonary alveolar proteinosis, Treg
cell?regulatory T cell
0741-5400/10/0087-511 © Society for Leukocyte Biology
Volume 87, March 2010
Journal of Leukocyte Biology 511
Epub ahead of print January 22, 2010 - doi:10.1189/jlb.0709520
Copyright 2010 by The Society for Leukocyte Biology.
without compromising adaptive immunity. Antigen-specific
therapies that reliably re-establish or re-enforce tolerance to
self-antigens may prove to be a safe, effective means to transi-
tion from a strategy of disease management to strategies that
have a curative potential.
Cytokine-NAg fusion proteins represent an alternative anti-
gen-specific approach for induction of tolerance [26–29]. Fu-
sion proteins comprised of IL-2 fused to the encephalitogenic
determinant of MBP (IL2-NAg) were effective for treatment
and prevention of EAE . Likewise, a fusion protein consist-
ing of the encephalitogenic peptide fused to the cytokine do-
main of IL-16 (NAg-IL16) was also highly effective for prophy-
laxis and treatment of EAE .
The postulate was that the cytokine domain would target the
covalently tethered NAg to a particular APC subset to achieve
substantially enhanced presentation of NAg by that APC sub-
set. Indeed, the IL2-NAg fusion protein was 1000-fold more
potent as an antigen than NAg in the presence of rat
CD25?MHCII?T cell blasts . For IL2-NAg and NAg-IL16
fusion proteins, the covalent cytokine-NAg link was critical for
effectiveness in modulating EAE. An IFN?-NAg fusion protein
was also tolerogenic . Pretreatment with IFN?-NAg attenu-
ated a subsequent bout of EAE, but in contrast to IL2-NAg
and NAg-IL16, the covalent linkage between IFN-? and the
NAg was not necessary for prevention of EAE. Rather, separate
injections of IFN-? and NAg at adjacent sites were as effective
as the IFN?-NAg fusion protein, whereas administration of
IFN-? alone or NAg alone was without effect. Thus, tolero-
genic mechanisms associated with cytokine-NAg fusion pro-
teins correlated with a strict cytokine-mediated targeting of
particular APC subsets (IL2-NAg) or appeared to involve a
more generalized action on APC (IFN-?).
The purpose of the current study was to test cytokine-NAg
fusion proteins that targeted NAg to DC and macrophage
APC. These APC have been implicated as critical APC for in-
duction and maintenance of self-tolerance [30–39]. For exam-
ple, DC have pivotal roles in negative thymic selection, expan-
sion of Treg cells, and elimination of autoimmune effector T
cells. The main question was whether the tolerogenic activity
of DC and perhaps other myeloid APC could be harnessed by
cytokine-NAg fusion proteins to specifically regulate patho-
genic T cells in vivo. GM-CSF and M-CSF were chosen for this
study because the two cytokines represent the key cytokines
driving the differentiation, survival, and growth of myeloid-
derived APC [40, 41]. GM-CSF and M-CSF exhibit substantial
overlap in biological function, but each also has a unique
spectrum of activity. GM-CSF production is largely activation-
dependent, and GM-CSF in turn activates key immunogenic
and regulatory pathways of the immune system. In contrast,
M-CSF is constitutively produced by many cell types, is nor-
mally in the circulation, and is necessary for the homeostatic
maintenance of nonactivated, quiescent macrophages. This
study revealed that GMCSF-NAg and MCSF-NAg fusion pro-
teins targeted NAg for preferential antigen presentation by
APC such as DC and macrophages rather than T cell or B cell
APC. GMCSF-NAg, and to a lesser extent, MCSF-NAg were also
highly effective tolerogens that could prevent or treat severe
MATERIALS AND METHODS
Structure and purification of recombinant proteins
Two GM-CSF-based fusion proteins (GMCSF-NAg and GM-CSF) were used
in this study. The GMCSF-NAg fusion protein contained the mature rat
GM-CSF domain as the N terminus and the major encephalitogenic 69–87
determinant of GPMBP (Y-G-S-L-P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H; i.e., the
NAg) together with six additional H residues as the C terminus. The
numbering system for GPMBP was based on Accession Number P25188
(www.ncbi.nlm.nih.gov). We also expressed a GM-CSF fusion protein that
lacked the encephalitogenic peptide but was otherwise identical to GMCSF-
NAg. This fusion protein was comprised of the mature rat GM-CSF domain
as the N terminus fused directly without a linker to a seven his-tag C termi-
nus. The rat GM-CSF domain of both fusion proteins was based on a par-
tial rat mRNA sequence (NCBI U00620; www.ncbi.nlm.nih.gov), which en-
coded the mature rat cytokine. The signal sequence of mouse GM-CSF was
inserted by standard PCR cloning procedures to ensure processing and secre-
tion of the fusion protein. Thus, both GM-CSF fusion genes contained an
AGC-CTC sequence encoding a S15–L16sequence of mouse GM-CSF, whereas
the rat GM-CSF gene had an AGT-TTC sequence encoding a S15-F16sequence.
This 1 aa sequence difference in the signal sequence was of no consequence,
as the mouse GM-CSF signal sequence supported efficient expression of rat
GM-CSF fusion proteins in baculovirus expression systems.
Two M-CSF fusion proteins (MCSF-NAg and M-CSF) were also used in
this study based on the rat M-CSF sequence (NCBI NP_076471; www.ncbi.
nlm.nih.gov). The N-terminal domain of both fusion proteins contained
the native rat 33-aa M-CSF signal sequence and the 220-aa N-terminal do-
main that forms a secreted, biologically active homodimer. These proteins
lacked the pro-peptide, transmembrane, and cytoplasmic domains of full-
length M-CSF. The MCSF-NAg and M-CSF fusion proteins had the same
C-terminal domains as the GMCSF-NAg and GM-CSF fusion proteins, respec-
Several additional cytokine-NAg fusion proteins were used in this study,
including IL1RA-NAg, IL2-NAg, IL4-NAg, IL10-NAg, IL13-NAg [27–29],
IFN?-NAg , and IL6-NAg. These fusion proteins contained the respec-
tive rat cytokine as the N-terminal domain linked to a C-terminal domain
that included the 73–87 encephalitogenic peptide (P-Q-K-S-Q-R-S-Q-D-E-N-
P-V-V-H). All of these fusion proteins had a C-terminal his-tag to facilitate
purification. An additional fusion protein, NAgIL-16, was comprised of an
N-terminal his-tag, the 69–87 encephalitogenic peptide of GPMBP, and the
rat 118-aa IL-16 cytokine C terminus .
Recombinant proteins expressed by use of baculovirus expression sys-
tems were purified by two consecutive affinity chromatography steps [29,
42]. Expression supernatants containing the GM-CSF- or M-CSF-based fu-
sion proteins were concentrated by ultrafiltration and were purified initially
by affinity chromatography based on binding to a single-chain (scFv) anti-
6his antibody immobilized on a chitin resin. After elution, the fusion pro-
teins were subjected to the final affinity chromatography step on Ni-NTA
agarose columns (Qiagen, Valencia, CA, USA). Proteins were then concen-
trated and diafiltrated in Amicon Ultra-15 centrifugal filter devices. Protein
quantity was assessed by the bicinchoninic acid protein assay (Pierce, Rock-
ford, IL, USA) and by absorbance at 280 nm. Purity was assessed by SDS-
Animals and reagents
Lewis rats were housed at East Carolina University Brody School of Medi-
cine (Greenville, NC, USA). Animal care and use were performed in accor-
dance with approved animal use protocols and institutional guidelines of
the East Carolina University Institutional Animal Care and Use Committee.
Injections were administered to Lewis rats anesthetized by isoflurane (Ab-
bott Laboratories, Chicago, IL, USA). GPMBP was purified from spinal
cords (Rockland, Gilbertsville, PA, USA). Synthetic GP69-88 peptide (Y-G-S-
L-P-Q-K-S-Q-R-S-Q-D-E-N-P-V-V-H-F) was obtained from Quality Controlled
Biologicals, Inc. (Hopkinton, MA, USA). B cell hybridoma supernatants
containing the OX-6 anti-I-A (RT1B) IgG1, OX-33 anti-CD45 (B cell form)
IgG1, OX-8 anti-CD8-? IgG1, OX-1 anti-CD45 (rat leukocytes) IgG1, and
Journal of Leukocyte Biology
Volume 87, March 2010
hibiting an ongoing inflammatory autoimmune disease. Over-
all, GMCSF-“self antigen” fusion proteins may represent an
important tool for manipulating the immune response and
causing antigen-specific immunological tolerance.
J. L. B. and M. D. M. contributed to the conception, design,
performance, and composition of this article.
This study was supported by research grants from the National
Multiple Sclerosis Society and the Brody Brothers Endowment
1. Goodin, D. S., Cohen, B. A., O’Connor, P., Kappos, L., Stevens, J. C.
(2008) Assessment: the use of natalizumab (Tysabri) for the treatment of
multiple sclerosis (an evidence-based review): report of the Therapeutics
and Technology Assessment Subcommittee of the American Academy of
Neurology. Neurology 71, 766–773.
2. Kleinschnitz, C., Meuth, S. G., Wiendl, P. H. (2008) The trials and errors
in MS therapy. Int. MS J. 15, 79–90.
3. Linker, R. A., Kieseier, B. C., Gold, R. (2008) Identification and develop-
ment of new therapeutics for multiple sclerosis. Trends Pharmacol. Sci. 29,
4. Lutterotti, A., Sospedra, M., Martin, R. (2008) Antigen-specific thera-
pies in MS—current concepts and novel approaches. J. Neurol. Sci.
5. Miller, S. D., Turley, D. M., Podojil, J. R. (2007) Antigen-specific toler-
ance strategies for the prevention and treatment of autoimmune disease.
Nat. Rev. Immunol. 7, 665–677.
6. Steinman, L. (2007) Antigen-specific therapy of multiple sclerosis: the
long-sought magic bullet. Neurotherapeutics 4, 661–665.
7. Miyamoto, K., Kingsley, C. I., Zhang, X., Jabs, C., Izikson, L., Sobel, R. A.,
Weiner, H. L., Kuchroo, V. K., Sharpe, A. H. (2005) The ICOS molecule
plays a crucial role in the development of mucosal tolerance. J. Immunol.
8. Song, F., Guan, Z., Gienapp, I. E., Shawler, T., Benson, J., Whitacre, C. C.
(2006) The thymus plays a role in oral tolerance in experimental autoim-
mune encephalomyelitis. J. Immunol. 177, 1500–1509.
9. Weishaupt, A., Jander, S., Bruck, W., Kuhlmann, T., Stienekemeier, M.,
Hartung, T., Toyka, K. V., Stoll, G., Gold, R. (2000) Molecular mecha-
nisms of high-dose antigen therapy in experimental autoimmune enceph-
alomyelitis: rapid induction of Th1-type cytokines and inducible nitric
oxide synthase. J. Immunol. 165, 7157–7163.
10. Minguela, A., Pastor, S., Mi, W., Richardson, J. A., Ward, E. S. (2007)
Feedback regulation of murine autoimmunity via dominant anti-inflam-
matory effects of interferon ?. J. Immunol. 178, 134–144.
11. Chan, J., Ban, E. J., Chun, K. H., Wang, S., Backstrom, B. T., Bernard,
C. C., Toh, B. H., Alderuccio, F. (2008) Transplantation of bone marrow
transduced to express self-antigen establishes deletional tolerance and
permanently remits autoimmune disease. J. Immunol. 181, 7571–7580.
12. Sewell, D., Qing, Z., Reinke, E., Elliot, D., Weinstock, J., Sandor, M.,
Fabry, Z. (2003) Immunomodulation of experimental autoimmune en-
cephalomyelitis by helminth ova immunization. Int. Immunol. 15, 59–69.
13. Chen, Y., Hancock, W. W., Marks, R., Gonnella, P., Weiner, H. L. (1998)
Mechanisms of recovery from experimental autoimmune encephalomyeli-
tis: T cell deletion and immune deviation in myelin basic protein T cell
receptor transgenic mice. J. Neuroimmunol. 82, 149–159.
14. Nicholson, L. B., Greer, J. M., Sobel, R. A., Lees, M. B., Kuchroo, V. K.
(1995) An altered peptide ligand mediates immune deviation and pre-
vents autoimmune encephalomyelitis. Immunity 3, 397–405.
15. Young, D. A., Lowe, L. D., Booth, S. S., Whitters, M. J., Nicholson, L.,
Kuchroo, V. K., Collins, M. (2000) IL-4, IL-10, IL-13, and TGF-? from an
altered peptide ligand-specific Th2 cell clone down-regulate adoptive
transfer of experimental autoimmune encephalomyelitis. J. Immunol. 164,
16. Saoudi, A., Simmonds, S., Huitinga, I., Mason, D. (1995) Prevention of
experimental allergic encephalomyelitis in rats by targeting autoantigen
to B cells: evidence that the protective mechanism depends on changes
in the cytokine response and migratory properties of the autoantigen-
specific T cells. J. Exp. Med. 182, 335–344.
17. Yu, P., Gregg, R. K., Bell, J. J., Ellis, J. S., Divekar, R., Lee, H. H., Jain, R.,
Waldner, H., Hardaway, J. C., Collins, M., Kuchroo, V. K., Zaghouani, H.
(2005) Specific T regulatory cells display broad suppressive functions
against experimental allergic encephalomyelitis upon activation with cog-
nate antigen. J. Immunol. 174, 6772–6780.
18. Zhong, M. C., Kerlero de Rosbo, N., Ben-Nun, A. (2002) Multiantigen/
multiepitope-directed immune-specific suppression of “complex autoim-
mune encephalomyelitis” by a novel protein product of a synthetic gene.
J. Clin. Invest. 110, 81–90.
19. Elliott, E. A., Cofiell, R., Wilkins, J. A., Raine, C. S., Matis, L. A., Mueller,
J. P. (1997) Immune tolerance mediated by recombinant proteolipid pro-
tein prevents experimental autoimmune encephalomyelitis. J. Neuroimmu-
nol. 79, 1–11.
20. Ho, P. P., Fontoura, P., Platten, M., Sobel, R. A., DeVoss, J. J., Lee, L. Y.,
Kidd, B. A., Tomooka, B. H., Capers, J., Agrawal, A., Gupta, R., Zernik, J.,
Yee, M. K., Lee, B. J., Garren, H., Robinson, W. H., Steinman, L. (2005)
A suppressive oligodeoxynucleotide enhances the efficacy of myelin cock-
tail/IL-4-tolerizing DNA vaccination and treats autoimmune disease. J.
Immunol. 175, 6226–6234.
21. Garren, H., Ruiz, P. J., Watkins, T. A., Fontoura, P., Nguyen, L. T., Est-
line, E. R., Hirschberg, D. L., Steinman, L. (2001) Combination of gene
delivery and DNA vaccination to protect from and reverse Th1 autoim-
mune disease via deviation to the Th2 pathway. Immunity 15, 15–22.
22. Margot, C. D., Ford, M. L., Evavold, B. D. (2005) Amelioration of estab-
lished experimental autoimmune encephalomyelitis by an MHC anchor-
substituted variant of proteolipid protein 139–151. J. Immunol. 174, 3352–
23. Turley, D. M., Miller, S. D. (2007) Peripheral tolerance induction using
ethylenecarbodiimide-fixed APCs uses both direct and indirect mecha-
nisms of antigen presentation for prevention of experimental autoim-
mune encephalomyelitis. J. Immunol. 178, 2212–2220.
24. McDevitt, H. (2004) Specific antigen vaccination to treat autoimmune
disease. Proc. Natl. Acad. Sci. USA 101 (Suppl. 2), 14627–14630.
25. Pedotti, R., Mitchell, D., Wedemeyer, J., Karpuj, M., Chabas, D., Hattab,
E. M., Tsai, M., Galli, S. J., Steinman, L. (2001) An unexpected version of
horror autotoxicus: anaphylactic shock to a self-peptide. Nat. Immunol. 2,
26. Mannie, M. D., Abbott, D. J., Blanchfield, J. L. (2009) Experimental auto-
immune encephalomyelitis in Lewis rats: IFN-? acts as a tolerogenic adju-
vant for induction of neuroantigen-dependent tolerance. J. Immunol. 182,
27. Mannie, M. D., Abbott, D. J. (2007) A fusion protein consisting of IL-16
and the encephalitogenic peptide of myelin basic protein constitutes an
antigen-specific tolerogenic vaccine that inhibits experimental autoim-
mune encephalomyelitis. J. Immunol. 179, 1458–1465.
28. Mannie, M. D., Clayson, B. A., Buskirk, E. J., DeVine, J. L., Hernandez,
J. J., Abbott, D. J. (2007) IL-2/neuroantigen fusion proteins as antigen-
specific tolerogens in experimental autoimmune encephalomyelitis
(EAE): correlation of T cell-mediated antigen presentation and tolerance
induction. J. Immunol. 178, 2835–2843.
29. Mannie, M. D., Devine, J. L., Clayson, B. A., Lewis, L. T., Abbott, D. J.
(2007) Cytokine-neuroantigen fusion proteins: new tools for modulation
of myelin basic protein (MBP)-specific T cell responses in experimental
autoimmune encephalomyelitis. J. Immunol. Methods 319, 118–132.
30. Belkaid, Y., Oldenhove, G. (2008) Tuning microenvironments: induction
of regulatory T cells by dendritic cells. Immunity 29, 362–371.
31. Goubier, A., Dubois, B., Gheit, H., Joubert, G., Villard-Truc, F., Asselin-
Paturel, C., Trinchieri, G., Kaiserlian, D. (2008) Plasmacytoid dendritic
cells mediate oral tolerance. Immunity 29, 464–475.
32. Guan, Y., Yu, S., Zhao, Z., Ciric, B., Zhang, G. X., Rostami, A. (2007) An-
tigen presenting cells treated in vitro by macrophage colony-stimulating
factor and autoantigen protect mice from autoimmunity. J. Neuroimmunol.
33. Huang, Y. M., Yang, J. S., Xu, L. Y., Link, H., Xiao, B. G. (2000) Autoan-
tigen-pulsed dendritic cells induce tolerance to experimental allergic en-
cephalomyelitis (EAE) in Lewis rats. Clin. Exp. Immunol. 122, 437–444.
34. Khoury, S. J., Gallon, L., Chen, W., Betres, K., Russell, M. E., Hancock,
W. W., Carpenter, C. B., Sayegh, M. H., Weiner, H. L. (1995) Mecha-
nisms of acquired thymic tolerance in experimental autoimmune enceph-
alomyelitis: thymic dendritic-enriched cells induce specific peripheral T
cell unresponsiveness in vivo. J. Exp. Med. 182, 357–366.
35. Li, H., Zhang, G. X., Chen, Y., Xu, H., Fitzgerald, D. C., Zhao, Z., Ros-
tami, A. (2008) CD11c?CD11b? dendritic cells play an important role
in intravenous tolerance and the suppression of experimental autoim-
mune encephalomyelitis. J. Immunol. 181, 2483–2493.
36. Miyake, Y., Asano, K., Kaise, H., Uemura, M., Nakayama, M., Tanaka, M.
(2007) Critical role of macrophages in the marginal zone in the suppres-
sion of immune responses to apoptotic cell-associated antigens. J. Clin.
Invest. 117, 2268–2278.
37. Cools, N., Ponsaerts, P., Van Tendeloo, V. F., Berneman, Z. N. (2007)
Balancing between immunity and tolerance: an interplay between den-
dritic cells, regulatory T cells, and effector T cells. J. Leukoc. Biol. 82,
38. Yamazaki, S., Dudziak, D., Heidkamp, G. F., Fiorese, C., Bonito, A. J., In-
aba, K., Nussenzweig, M. C., Steinman, R. M. (2008) CD8? CD205?
Journal of Leukocyte Biology
Volume 87, March 2010
splenic dendritic cells are specialized to induce Foxp3? regulatory T
cells. J. Immunol. 181, 6923–6933.
39. Yamazaki, S., Steinman, R. M. (2009) Dendritic cells as controllers of an-
tigen-specific Foxp3? regulatory T cells. J. Dermatol. Sci. 54, 69–75.
40. Hamilton, J. A. (2008) Colony-stimulating factors in inflammation and
autoimmunity. Nat. Rev. Immunol. 8, 533–544.
41. Conti, L., Gessani, S. (2008) GM-CSF in the generation of dendritic cells
from human blood monocyte precursors: recent advances. Immunobiology
42. Blank, K., Lindner, P., Diefenbach, B., Pluckthun, A. (2002) Self-immobi-
lizing recombinant antibody fragments for immunoaffinity chromatog-
raphy: generic, parallel, and scalable protein purification. Protein Expr.
Purif. 24, 313–322.
43. Mannie, M. D., Norris, M. S. (2001) MHC class-II-restricted antigen pre-
sentation by myelin basic protein-specific CD4? T cells causes prolonged
desensitization and outgrowth of CD4– responders. Cell. Immunol. 212,
44. Patel, D. M., Arnold, P. Y., White, G. A., Nardella, J. P., Mannie, M. D.
(1999) Class II MHC/peptide complexes are released from APC and are
acquired by T cell responders during specific antigen recognition. J. Im-
munol. 163, 5201–5210.
45. Mannie, M. D., Fraser, D. J., McConnell, T. J. (2003) IL-4 responsive
CD4? T cells specific for myelin basic protein: IL-2 confers a prolonged
postactivation refractory phase. Immunol. Cell Biol. 81, 8–19.
46. Woollett, G. R., Barclay, A. N., Puklavec, M., Williams, A. F. (1985) Mo-
lecular and antigenic heterogeneity of the rat leukocyte-common antigen
from thymocytes and T and B lymphocytes. Eur. J. Immunol. 15, 168–173.
47. Patel, D. M., Dudek, R. W., Mannie, M. D. (2001) Intercellular ex-
change of class II MHC complexes: ultrastructural localization and
functional presentation of adsorbed I-A/peptide complexes. Cell. Im-
munol. 214, 21–34.
48. Chen, T. T., Tao, M. H., Levy, R. (1994) Idiotype-cytokine fusion pro-
teins as cancer vaccines. Relative efficacy of IL-2, IL-4, and granulocyte-
macrophage colony-stimulating factor. J. Immunol. 153, 4775–4787.
49. Tao, M. H., Levy, R. (1993) Idiotype/granulocyte-macrophage colony-
stimulating factor fusion protein as a vaccine for B-cell lymphoma. Nature
50. Schwegler, C., Dorn-Beineke, A., Nittka, S., Stocking, C., Neumaier, M.
(2005) Monoclonal anti-idiotype antibody 6G6.C4 fused to GM-CSF is
capable of breaking tolerance to carcinoembryonic antigen (CEA) in
CEA-transgenic mice. Cancer Res. 65, 1925–1933.
51. Wortham, C., Grinberg, L., Kaslow, D. C., Briles, D. E., McDaniel, L. S.,
Lees, A., Flora, M., Snapper, C. M., Mond, J. J. (1998) Enhanced protec-
tive antibody responses to PspA after intranasal or subcutaneous injec-
tions of PspA genetically fused to granulocyte-macrophage colony-stimu-
lating factor or interleukin-2. Infect. Immun. 66, 1513–1520.
52. Tso, C. L., Zisman, A., Pantuck, A., Calilliw, R., Hernandez, J. M., Paik,
S., Nguyen, D., Gitlitz, B., Shintaku, P. I., de Kernion, J., Figlin, R., Bell-
degrun, A. (2001) Induction of G250-targeted and T-cell-mediated antitu-
mor activity against renal cell carcinoma using a chimeric fusion protein
consisting of G250 and granulocyte/monocyte-colony stimulating factor.
Cancer Res. 61, 7925–7933.
53. Rodriguez, D., Rodriguez, J. R., Llorente, M., Vazquez, I., Lucas, P.,
Esteban, M., Martinez, A. C., del Real, G. (1999) A human immunodefi-
ciency virus type 1 Env-granulocyte-macrophage colony-stimulating factor
fusion protein enhances the cellular immune response to Env in a vac-
cinia virus-based vaccine. J. Gen. Virol. 80, 217–223.
54. Picca, C. C., Larkin III, J., Boesteanu, A., Lerman, M. A., Rankin, A. L.,
Caton, A. J. (2006) Role of TCR specificity in CD4? CD25? regulatory
T-cell selection. Immunol. Rev. 212, 74–85.
55. Larkin III, J., Rankin, A. L., Picca, C. C., Riley, M. P., Jenks, S. A., Sant,
A. J., Caton, A. J. (2008) CD4?CD25? regulatory T cell repertoire for-
mation shaped by differential presentation of peptides from a self-anti-
gen. J. Immunol. 180, 2149–2157.
56. Cunningham, M. W. (2003) Autoimmunity and molecular mimicry in the
pathogenesis of post-streptococcal heart disease. Front. Biosci. 8, s533–
57. Guilherme, L., Ramasawmy, R., Kalil, J. (2007) Rheumatic fever and
rheumatic heart disease: genetics and pathogenesis. Scand. J. Immunol. 66,
58. Ioachimescu, O. C., Kavuru, M. S. (2006) Pulmonary alveolar proteinosis.
Chron. Respir. Dis. 3, 149–159.
59. Pette, M., Fujita, K., Kitze, B., Whitaker, J. N., Albert, E., Kappos, L.,
Wekerle, H. (1990) Myelin basic protein-specific T lymphocyte lines from
MS patients and healthy individuals. Neurology 40, 1770–1776.
60. McQualter, J. L., Darwiche, R., Ewing, C., Onuki, M., Kay, T. W., Hamil-
ton, J. A., Reid, H. H., Bernard, C. C. (2001) Granulocyte macrophage
colony-stimulating factor: a new putative therapeutic target in multiple
sclerosis. J. Exp. Med. 194, 873–882.
61. Ponomarev, E. D., Shriver, L. P., Maresz, K., Pedras-Vasconcelos, J., Ver-
thelyi, D., Dittel, B. N. (2007) GM-CSF production by autoreactive T cells
is required for the activation of microglial cells and the onset of experi-
mental autoimmune encephalomyelitis. J. Immunol. 178, 39–48.
62. Aloisi, F., De Simone, R., Columba-Cabezas, S., Penna, G., Adorini, L.
(2000) Functional maturation of adult mouse resting microglia into an
APC is promoted by granulocyte-macrophage colony-stimulating factor
and interaction with Th1 cells. J. Immunol. 164, 1705–1712.
63. King, I. L., Dickendesher, T. L., Segal, B. M. (2009) Circulating Ly-6C?
myeloid precursors migrate to the CNS and play a pathogenic role dur-
ing autoimmune demyelinating disease. Blood 113, 3190–3197.
64. Marusic, S., Miyashiro, J. S., Douhan III, J., Konz, R. F., Xuan, D., Pelker,
J. W., Ling, V., Leonard, J. P., Jacobs, K. A. (2002) Local delivery of gran-
ulocyte macrophage colony-stimulating factor by retrovirally transduced
antigen-specific T cells leads to severe, chronic experimental autoim-
mune encephalomyelitis in mice. Neurosci. Lett. 332, 185–189.
65. Kroenke, M. A., Carlson, T. J., Andjelkovic, A. V., Segal, B. M. (2008) IL-
12- and IL-23-modulated T cells induce distinct types of EAE based on
histology, CNS chemokine profile, and response to cytokine inhibition. J.
Exp. Med. 205, 1535–1541.
66. Ganesh, B. B., Cheatem, D. M., Sheng, J. R., Vasu, C., Prabhakar, B. S.
(2009) GM-CSF-induced CD11c?CD8a–dendritic cells facilitate Foxp3?
and IL-10? regulatory T cell expansion resulting in suppression of auto-
immune thyroiditis. Int. Immunol. 21, 269–282.
67. Gaudreau, S., Guindi, C., Menard, M., Besin, G., Dupuis, G., Amrani, A.
(2007) Granulocyte-macrophage colony-stimulating factor prevents diabe-
tes development in NOD mice by inducing tolerogenic dendritic cells
that sustain the suppressive function of CD4?CD25? regulatory T cells.
J. Immunol. 179, 3638–3647.
68. Sheng, J. R., Li, L., Ganesh, B. B., Vasu, C., Prabhakar, B. S., Meriggioli,
M. N. (2006) Suppression of experimental autoimmune myasthenia gra-
vis by granulocyte-macrophage colony-stimulating factor is associated with
an expansion of FoxP3? regulatory T cells. J. Immunol. 177, 5296–5306.
69. Sheng, J. R., Li, L. C., Ganesh, B. B., Prabhakar, B. S., Meriggioli, M. N.
(2008) Regulatory T cells induced by GM-CSF suppress ongoing experi-
mental myasthenia gravis. Clin. Immunol. 128, 172–180.
70. Kared, H., Leforban, B., Montandon, R., Renand, A., Layseca Espinosa,
E., Chatenoud, L., Rosenstein, Y., Schneider, E., Dy, M., Zavala, F. (2008)
Role of GM-CSF in tolerance induction by mobilized hematopoietic pro-
genitors. Blood 112, 2575–2578.
71. Filipazzi, P., Valenti, R., Huber, V., Pilla, L., Canese, P., Iero, M., Castelli,
C., Mariani, L., Parmiani, G., Rivoltini, L. (2007) Identification of a new
subset of myeloid suppressor cells in peripheral blood of melanoma pa-
tients with modulation by a granulocyte-macrophage colony-stimulation
factor-based antitumor vaccine. J. Clin. Oncol. 25, 2546–2553.
72. Serafini, P., Carbley, R., Noonan, K. A., Tan, G., Bronte, V., Borrello, I.
(2004) High-dose granulocyte-macrophage colony-stimulating factor-pro-
ducing vaccines impair the immune response through the recruitment of
myeloid suppressor cells. Cancer Res. 64, 6337–6343.
73. Sotomayor, E. M., Fu, Y. X., Lopez-Cepero, M., Herbert, L., Jimenez, J. J.,
Albarracin, C., Lopez, D. M. (1991) Role of tumor-derived cytokines on
the immune system of mice bearing a mammary adenocarcinoma. II.
Down-regulation of macrophage-mediated cytotoxicity by tumor-derived
granulocyte-macrophage colony-stimulating factor. J. Immunol. 147, 2816–
74. Tang, H., Guo, Z., Zhang, M., Wang, J., Chen, G., Cao, X. (2006) Endo-
thelial stroma programs hematopoietic stem cells to differentiate into
regulatory dendritic cells through IL-10. Blood 108, 1189–1197.
75. Zhang, M., Tang, H., Guo, Z., An, H., Zhu, X., Song, W., Guo, J., Huang,
X., Chen, T., Wang, J., Cao, X. (2004) Splenic stroma drives mature den-
dritic cells to differentiate into regulatory dendritic cells. Nat. Immunol. 5,
76. Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M.,
Ravetch, J. V., Steinman, R. M., Nussenzweig, M. C. (2001) Dendritic cells
induce peripheral T cell unresponsiveness under steady state conditions
in vivo. J. Exp. Med. 194, 769–779.
77. Witmer-Pack, M. D., Swiggard, W. J., Mirza, A., Inaba, K., Steinman, R. M.
(1995) Tissue distribution of the DEC-205 protein that is detected by the
monoclonal antibody NLDC-145. II. Expression in situ in lymphoid and
nonlymphoid tissues. Cell. Immunol. 163, 157–162.
78. Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M. C.,
Steinman, R. M. (2002) Efficient targeting of protein antigen to the den-
dritic cell receptor DEC-205 in the steady state leads to antigen presenta-
tion on major histocompatibility complex class I products and peripheral
CD8? T cell tolerance. J. Exp. Med. 196, 1627–1638.
79. Trumpfheller, C., Finke, J. S., Lopez, C. B., Moran, T. M., Moltedo, B.,
Soares, H., Huang, Y., Schlesinger, S. J., Park, C. G., Nussenzweig, M. C.,
Granelli-Piperno, A., Steinman, R. M. (2006) Intensified and protective
CD4? T cell immunity in mice with anti-dendritic cell HIV gag fusion
antibody vaccine. J. Exp. Med. 203, 607–617.
80. Hawiger, D., Masilamani, R. F., Bettelli, E., Kuchroo, V. K., Nussenzweig,
M. C. (2004) Immunological unresponsiveness characterized by increased
expression of CD5 on peripheral T cells induced by dendritic cells in
vivo. Immunity 20, 695–705.
rodent ? T cells ? cytokines ? tolerogenic vaccine ? DC
Blanchfield and Mannie
Tolerogenic GM-CSF-based vaccines
Volume 87, March 2010
Journal of Leukocyte Biology 523