ASSOCIATE EDITORS
K. Frank Austen
Harvard Medical School, Boston, Massachusetts, USA

Tasuku Honjo
Kyoto University, Kyoto, Japan

Fritz Melchers
University of Basel, Basel, Switzerland

Hidde Ploegh
Massachusetts Institute of Technology, Massachusetts, USA

Kenneth M. Murphy
Washington University, St. Louis, Missouri, USA


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CONTRIBUTORS
Hisashi Arase
Department of Immunochemistry, Research Institute for Microbial Diseases, and Laboratory
of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Suita,
Osaka, Japan
Ameya Champhekar
Department of Microbiology, Immunology, and Molecular Genetics, University of
California Los Angeles, Los Angeles, California, USA
Huai-Chia Chuang
Immunology Research Center, National Health Research Institutes, Zhunan, and Research
and Development Center for Immunology, China Medical University, Taichung,
Taiwan, ROC

Marie-Dominique Filippi
Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s
Research Foundation, and University of Cincinnati College of Medicine, Cincinnati,
Ohio, USA
Hidetoshi Inoko
GenoDive Pharma Inc., Atsugi, Kanagawa, Japan
Alain Lamarre
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS), INRSInstitut Armand-Frappier, Laval, Quebec, Canada
Pascal Lapierre
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS), INRSInstitut Armand-Frappier, Laval, Quebec, Canada
Satoko Morishima
Division of Hematology, Fujita Health University School of Medicine, Toyoake, Aichi,
Japan
Yasuo Morishima
Division of Epidemiology and Prevention, Aiichi Cancer Center Research Institute,
Chikusa-ku, Nagoya, Japan
Armstrong Murira
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS),
INRS-Institut Armand-Frappier, Laval, Quebec, Canada
Ellen V. Rothenberg
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena,
California, USA
Takehiko Sasazuki
Institute for Advanced Study, Kyushu University, Higashi-ku, Fukuoka, Japan

Advances in Immunology, Volume 129
ISSN 0065-2776
/>
#


2016 Elsevier Inc.
All rights reserved.

ix


x

Contributors

Luis J. Sigal
Thomas Jefferson University, Department of Microbiology and Immunology, Philadelphia,
Pennsylvania, USA
Tse-Hua Tan
Immunology Research Center, National Health Research Institutes, Zhunan; Research and
Development Center for Immunology, China Medical University, Taichung, Taiwan,
ROC, and Department of Pathology & Immunology, Baylor College of Medicine, Houston,
Texas, USA
Jonas Ungerba¨ck
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena,
California, USA, and Department of Clinical and Experimental Medicine, Experimental
Hematopoiesis Unit, Faculty of Health Sciences, Link€
oping University, Link€
oping, Sweden
Xiaohong Wang
Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas,
USA


CHAPTER ONE


Rheumatoid Rescue of Misfolded
Cellular Proteins by MHC Class II
Molecules: A New Hypothesis for
Autoimmune Diseases
Hisashi Arase*,†,1
*Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Suita,
Osaka, Japan
†
Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Suita,
Osaka, Japan
1
Corresponding author: e-mail address:

Contents
1. Introduction
2. Transport of ER-Misfolded Proteins to the Cell Surface by MHC Class II Molecules
2.1 MHC Class II Molecules Induce Cell Surface Expression of Misfolded MHC Class I
Molecules
2.2 MHC Class II Molecules Function as a Molecular Chaperon to Transport
Misfolded Cellular Protein to the Cell Surface
3. Function of Protein Antigens Presented by MHC Class II Molecules
3.1 MHC Class II Molecules Present Protein Antigens to B Cells
3.2 Misfolded Cellular Proteins Rescued from Protein Degradation by MHC Class II
Molecules Might Be Pathogenic
3.3 Aberrant MHC Class II Expression on Autoimmune-Diseased Tissues
4. Misfolded Proteins Presented on MHC Class II Molecules Are Targets for
Autoantibodies in Autoimmune Diseases
4.1 IgG Heavy Chain Presented on MHC Class II Molecules Is a Specific Target for
Autoantibodies in RA

4.2 β2-Glycoprotein I Associated with MHC Class II Molecules Is a Specific Target
for Autoantibodies in Antiphospholipid Syndrome
5. Susceptibility to Autoimmune Diseases Is Associated with the Affinity of Misfolded
Proteins for MHC Class II Molecules
5.1 MHC Class II Alleles and Autoimmune Disease Susceptibility
5.2 Autoantibody Binding to Misfolded Protein/MHC Class II Complex Is
Associated with Autoimmune Disease Susceptibility
6. Involvement of Misfolded Protein–MHC Class II Molecule Complexes in
Autoantibody-Mediated Pathogenicity
6.1 Pathogenesis of Autoantibodies in RA and APS
6.2 B Cell Removal Are Effective Treatment for Autoimmune Diseases

Advances in Immunology, Volume 129
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2016 Elsevier Inc.
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7. Misfolded Cellular Proteins Rescued from Degradation by MHC Class II Molecules
May Abrogate Immune Tolerance
7.1 Misfolded Proteins Associated with MHC Class II Molecules as “Nonself”Antigens
7.2 Misfolded Protein Complexed with MHC Class II Molecules as Primary
Autoantigens for Autoantibodies
8. Misfolded Proteins Presented on MHC Class II Molecules as a Therapeutic Target for
Autoimmune Diseases
9. Concluding Remarks
Acknowledgments
References

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Abstract
Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and
thus are not transported outside cells. However, misfolded proteins in the endoplasmic
reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by
MHC class II molecules without being processed to peptides. Studies on the misfolded
proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in
autoimmune diseases. Furthermore, a strong correlation has been observed between
autoantibody binding to misfolded proteins associated with MHC class II molecules
and the autoimmune disease susceptibility conferred by each MHC class II allele. These
new insights into MHC class II molecules suggest that misfolded proteins rescued from
protein degradation by MHC class II molecules are recognized as “neo-self” antigens by
immune system and are involved in autoimmune diseases as autoantibody targets.

1. INTRODUCTION
Specific major histocompatibility complex (MHC) class II alleles affect
susceptibility to many autoimmune diseases. Recent genome-wide association studies have confirmed that the MHC class II loci are the genes most
strongly associated with susceptibility to many autoimmune diseases, including rheumatoid arthritis (RA). Because MHC class II molecules present peptide antigens to helper T cells, an abnormal helper T cell response has been
considered to be the main cause of MHC class II gene-associated autoimmune diseases. However, the pathogenic peptide antigens associated with
the autoimmune disease susceptibility conferred by each MHC class II allele
have not yet been identified; therefore, it remains unclear how the MHC
class II gene controls susceptibility to autoimmune diseases. On the other


A New Hypothesis for Autoimmune Diseases

3


hand, our recent analyses of MHC class II molecules have revealed that proteins misfolded in the endoplasmic reticulum (ER) are transported to the cell
surface without being processed to peptides upon association with MHC
class II molecules ( Jiang et al., 2013). Furthermore, misfolded proteins presented on MHC class II molecules appear to be involved in autoimmune disease susceptibility as specific targets for autoantibodies ( Jin et al., 2014;
Tanimura et al., 2015). These novel functions of MHC class II molecules
provide new insights into the molecular mechanism underlying autoimmune diseases that will help us answer questions regarding why autoantibodies against autoantigens are produced in patients with autoimmune
diseases and why MHC class II genes are strongly associated with susceptibility to many autoimmune diseases.

2. TRANSPORT OF ER-MISFOLDED PROTEINS TO THE
CELL SURFACE BY MHC CLASS II MOLECULES
2.1 MHC Class II Molecules Induce Cell Surface Expression
of Misfolded MHC Class I Molecules
The MHC class I molecule comprises a trimolecular complex that includes a
heavy chain, β2-microglobulin, and a peptide. MHC class I molecules are
not expressed on the cell surface in the absence of β2-microglobulin or transporter associated with antigen processing (TAP), the latter of which transports proteasome-derived peptides to the ER where they are acquired by
MHC class I molecules, indicating that both β2-microglobulin and peptide,
are required for the cell surface expression of MHC class I molecules. On the
other hand, some unique monoclonal antibodies (mAbs) that are specific for
human MHC class I molecules lacking β2-microglobuin and peptide antigens, such as HC10 and L31, have been identified (Giacomini et al., 1997;
Stam, Spits, & Ploegh, 1986). These mAbs do not recognize normal MHC
class I molecules associated with β2-microglobulin and peptide antigens
(Sibilio et al., 2005). Interestingly, the epitopes recognized by these mAbs
are localized on the α1- and α2-domains of MHC class I, which are not
located within the β2-microglobulin-binding region, suggesting that these
mAbs recognize a certain unique conformation of MHC class I molecules
that is induced in the absence of β2-microglobulin and peptide antigens
(Arosa, Santos, & Powis, 2007). These incomplete MHC class
I molecules fail to achieve a correct conformation and are not expressed
on the cell surface. However, certain cells, such as B cell lines, are recognized



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Hisashi Arase

by these mAbs specific for unusual or misfolded MHC class I molecules lacking β2-microglobulin and peptides. This phenomenon suggests the existence of a molecular chaperon that transports misfolded MHC class
I molecules lacking β2-microglobulin and peptides to the cell surface.
Expression cloning to identify molecules that would permit expression of
MHC class I proteins on the cell surface unexpectedly revealed that MHC
class II molecules induce the cell surface expression of unusual or misfolded
MHC class I molecules ( Jiang et al., 2013). Upon further analysis, some
MHC class II alleles induced misfolded MHC class I expression on the cell
surface but others did not, and this difference depended on the amino acid
residues present within the peptide-binding groove of the MHC class II
molecule. Furthermore, the MHC class II-induced expression of misfolded
MHC class I molecules was almost completely blocked by a peptide covalently attached to MHC class II molecules. Indeed, some MHC class
II-positive cells express misfolded MHC class I molecules that are recognized by HC10 or L31 mAbs, and the direct association of MHC class
I molecules with MHC class II molecules is detectable in these cells. Therefore, MHC class II molecules appear to be involved in the expression of
misfolded MHC class I molecules on the cell surface (Fig. 1).

Figure 1 Misfolded major histocompatibility complex (MHC) class I expression facilitated by MHC class II molecules. MHC class I molecules normally are expressed in association with β2-microglobulin and peptide antigens (right). In the absence of peptide
antigens or β2-microglobulin, MHC class I molecules are not folded correctly and are
not expressed on the cell surface. However, when the misfolded MHC class
I molecule is associated with an MHC class II molecule in the endoplasmic reticulum,
it is directly transported to the cell surface by the MHC class II molecule without undergoing peptide processing (middle).


A New Hypothesis for Autoimmune Diseases

5


2.2 MHC Class II Molecules Function as a Molecular Chaperon
to Transport Misfolded Cellular Protein to the Cell Surface
The cell surface expression of misfolded MHC class I molecules by MHC
class II molecules raised the possibility that other misfolded proteins might
also be transported to the cell surface by MHC class II molecules. Hen egg
lysozyme (HEL) is a well-characterized secreted protein, the correct folding
of which requires S–S bonds (Ohkuri, Ueda, Tsurumaru, & Imoto, 2001).
When a mutant HEL in which two cysteine residues were substituted with
alanine was co-expressed with MHC class II molecules, the mutant HEL
protein, which was neither secreted nor expressed on the cell surface, was
induced on the cell surface in the presence of MHC class II molecules
( Jiang et al., 2013). Furthermore, the full-length HEL protein was
co-precipitated with MHC class II molecules. These findings, together with
the analyses of MHC class I molecules, suggest that ER-misfolded proteins
are transported to the cell surface by MHC class II molecules upon association with their peptide-binding grooves. In other words, MHC class II
molecules function as a molecular chaperon to transport ER-misfolded proteins to the cell surface.
The presentation of whole proteins by MHC class II molecules may seem
unusual because it is widely accepted that MHC class II molecules present short
peptide antigens. However, several papers have reported the association of
large proteins with the peptide-binding grooves of MHC class II molecules
(Aichinger et al., 1997; Anderson, Swier, Arneson, & Miller, 1993; Busch,
Cloutier, Sekaly, & Hammerling, 1996; Lechler, Aichinger, & Lightstone,
1996). When MHC class II molecules were expressed with an invariant chain
lacking the endosomal localization signal, the invariant chain was directly transported to the cell surface in association with MHC class II molecules (Anderson
et al., 1993). In addition, association of MHC class II molecules with large proteins was observed in the absence of invariant chain (Aichinger et al., 1997;
Busch et al., 1996). Therefore, the transport of ER-misfolded proteins to
the cell surface is an intrinsic function of MHC class II molecules. In addition,
antigens captured by the endocytic pathway form large molecular complexes
with MHC class II molecules in antigen-presenting cells (Castellino,

Zappacosta, Coligan, & Germain, 1998). These observations suggest that
MHC class II molecules exhibit the capacity to present large molecular antigens
derived not only from misfolded proteins in the ER but also from antigens captured by the endocytic pathway. However, the immunological functions of
large proteins associated with MHC class II molecules were not extensively
analyzed; therefore, these functions remained unclear.


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Hisashi Arase

3. FUNCTION OF PROTEIN ANTIGENS PRESENTED BY
MHC CLASS II MOLECULES
3.1 MHC Class II Molecules Present Protein Antigens to
B Cells
The presentation of whole proteins instead of peptides by MHC class II molecules suggests that MHC class II molecules might be involved in an as yet
unknown immune response. B cells expressing a high-affinity antigen receptor can be stimulated with soluble antigens. However, these antigens must be
associated with certain cell surface molecules to stimulate B cells with lowaffinity antigen receptors, such as those expressed on naı¨ve B cells
(Batista & Harwood, 2009; Qi, Egen, Huang, & Germain, 2006). The
presentation of whole proteins by MHC class II molecules suggests that these
proteins might be involved in B cell activation. Indeed, B cells expressing a
low-affinity antigen receptor against HEL protein can be stimulated with
HEL protein presented on MHC class II molecules but not by soluble
HEL protein alone ( Jiang et al., 2013). This indicates that MHC class II molecules might be directly involved in the antigen-specific B cell response.

3.2 Misfolded Cellular Proteins Rescued from Protein
Degradation by MHC Class II Molecules Might Be
Pathogenic
The invariant chain, which is associated with newly synthesized MHC class
II molecules, transports MHC class II molecules to endolysosomal compartments, where they acquire peptide antigens (Germain, 2011). However, the

affinities of MHC class II molecules for the invariant chain are known to
differ in an MHC class II allele-dependent manner (Davenport et al.,
1995). It is possible that MHC class II molecules will preferentially associate
with misfolded proteins rather than the invariant chain if the former has a
stronger affinity for MHC class II molecules than the latter. Indeed, the efficiency of the invariant chain to block the association of misfolded proteins
with MHC class II molecules differs in an allele-dependent manner, as
described above ( Jiang et al., 2013; Jin et al., 2014; Tanimura et al.,
2015). As most misfolded proteins do not possess a lysosomal targeting signal, MHC class II molecules associated with misfolded proteins instead of
invariant chain are directly transported to the cell surface without going
to the endolysosomal compartments.


A New Hypothesis for Autoimmune Diseases

7

The folding of a newly synthesized protein is a complex process that constitutively generates significant amounts of misfolded proteins. In certain
types of cells, more than half of all newly synthesized proteins are folded
incorrectly (Meusser, Hirsch, Jarosch, & Sommer, 2005). However, these
newly synthesized misfolded proteins typically are promptly degraded in
the cells through various pathways such as ER-associated degradation
(ERAD) and therefore not transported outside the cells (Meusser et al.,
2005). Accordingly, immune cells are not exposed to these misfolded proteins. Whereas both the primary structures and conformations of antigens are
involved in antibody recognition, only the primary structures of antigens are
involved in T cell recognition because T cell receptor recognizes short peptide antigens presented on MHC molecules. Therefore, unlike T cells, it is
possible that some B cells do not acquire tolerance to misfolded cellular proteins. If these misfolded cellular proteins are rescued from protein degradation by MHC class II molecules and subsequently transported extracellular,
B cells might recognize these proteins as “neo-self”-antigens and initiate an
antibody response (Fig. 2).

3.3 Aberrant MHC Class II Expression on AutoimmuneDiseased Tissues

More than 30 years ago, it was reported that autoimmune-diseased tissues
aberrantly expressed MHC class II molecules (Bottazzo, Pujol-Borrell,
Hanafusa, & Feldmann, 1983). Unlike normal thyroid tissues, tissues from
patients with Graves’ disease or Hashimoto’s thyroiditis aberrantly express
MHC class II molecules. Similar aberrant MHC class II expression was reported
in various tissues affected by autoimmune diseases such as RA, type I diabetes,
primary biliary cirrhosis, and psoriasis (Ballardini et al., 1984; Feldmann et al.,
1988; Gottlieb et al., 1986). Because particular MHC class II alleles are associated with autoimmune disease susceptibility, this aberrant MHC class II expression in autoimmune-diseased tissues was considered to be involved in the
pathogenicity of autoimmune diseases. Indeed, nonimmune cells, such as
endothelial cells, strongly express MHC class II molecules in response to stimulation from cytokines such as IFN-γ ( Jaffe et al., 1989; Pober et al., 1983;
Todd, Pujol-Borrell, Hammond, Bottazzo, & Feldmann, 1985). However,
these nonimmune cells do not express costimulatory molecules required for
the induction of T cell responses, such as CD80 or CD86. T cells that recognize
antigens presented on MHC class II molecules in the absence of costimulatory
signals are likely to become anergic (Appleman & Boussiotis, 2003). Therefore,
aberrant MHC class II expression on nonimmune cells has been considered a


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Hisashi Arase

Figure 2 Rheumatoid rescue of misfolded proteins by major histocompatibility complex (MHC) class II molecules. In steady state, MHC class II expression is restricted to specific immune cells such as dendritic cells and B cells. However, MHC class II molecules are
expressed on most cells following stimulation with certain cytokines such as IFN-γ,
which is produced in response to infection or inflammation. The invariant chain associates with nascent MHC class II molecules and blocks the association of MHC class II
molecules with endoplasmic reticulum (ER)-misfolded proteins. However, the affinities
of various MHC class II molecules for the invariant chain differ due to allelic polymorphism of MHC class II genes. If the avidity of an MHC class II molecule for a misfolded
protein is higher than that for the invariant chain, it is possible that misfolded proteins,
rather than the invariant chain, will bind to MHC class II molecules. As the misfolded
proteins do not contain an endolysosomal-targeting signal, they are transported

directly to the cell surface by MHC class II molecules. Thus, MHC class II molecules function as a molecular chaperon to rescue ER-misfolded proteins from protein degradation.
Because immune cells normally are not exposed to misfolded proteins and may therefore be intolerant to them, misfolded proteins rescued by MHC class II molecules may be
recognized as “neo-self”-antigens and thus induce autoantibody production. In this
way, misfolded proteins rescued from protein degradation by MHC class II molecules
may be involved in the pathogenesis of autoimmune diseases as autoantibody targets.

consequence of the inflammation elicited by autoimmunity rather than a cause
of autoimmune disease, and the pathophysiological function of this aberrant
MHC class II expression in autoimmune-diseased tissues has not been extensively analyzed. Unlike T cells, B cells do not require costimulatory signals to
respond to antigens, although they require T cell help for Ig class switching.


A New Hypothesis for Autoimmune Diseases

9

Given this difference between T cells and B cells, it is possible that misfolded
proteins rescued by aberrantly expressed MHC class II molecules can stimulate
B cells to produce autoantibodies.

4. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II
MOLECULES ARE TARGETS FOR AUTOANTIBODIES IN
AUTOIMMUNE DISEASES
4.1 IgG Heavy Chain Presented on MHC Class II Molecules
Is a Specific Target for Autoantibodies in RA
Disease-specific autoantibodies are produced in many autoimmune disorders.
Some of these autoantibodies are directly involved in the pathogenicity of
autoimmunity. Therefore, it is important to identify the target molecules recognized by autoantibodies in order to understand the pathogenicity of autoimmune diseases. If misfolded proteins rescued from protein degradation by
MHC class II molecules are targets for autoimmune diseases, autoantibodies
may recognize these proteins presented on MHC class II molecules.

Rheumatoid factor (RF) is a well-known autoantibody discovered
approximately 75 years ago. RF is specific for denatured, but not native,
IgG and is detected in approximately 80% of patients with RA (Dorner,
Egerer, Feist, & Burmester, 2004). Because RF titers are well correlated with
the clinical symptoms of RA, RF remains an important diagnostic indicator
of RA. However, the natural target antigens that induce RF production
remain undefined. In addition, it remains unclear why most patients with
RA are RF positive.
Antibodies comprise a heavy chain and a light chain; the heavy chain is
not secreted or expressed on the cell surface in the absence of the light chain.
However, the heavy chain alone can be expressed well on the cell surface in
the presence of MHC class II molecules ( Jin et al., 2014). Furthermore, IgG
presented on MHC class II molecules is recognized by autoantibodies from
patients with RA. More importantly, the IgG heavy chain presented on
MHC class II molecules was recognized by autoantibodies from patients
with RA but not by those from non-RA patients, including those positive
for RF. This suggests that the IgG heavy chain presented on MHC class II
molecules is more specific for autoantibodies from RA patients when compared with traditional RF detected by immobilized IgG. This finding implicates the IgG heavy chain, when presented on MHC class II molecules, as a
major target for RA autoantibodies.


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Hisashi Arase

4.2 β2-Glycoprotein I Associated with MHC Class II Molecules Is
a Specific Target for Autoantibodies in Antiphospholipid
Syndrome
Antiphospholipid syndrome (APS) is an autoimmune disorder associated
with thrombosis and pregnancy complications (Wilson et al., 1999).

Although autoantibodies associated with APS were initially characterized
by reactivity to phospholipids such as cardiolipin, recent analyses have revealed that these autoantibodies are directed mainly against the phospholipidassociated β2-glycoprotein I (Bas de Laat, Derksen, & de Groot, 2004; Galli,
Barbui, Zwaal, Comfurius, & Bevers, 1993; McNeil, Simpson,
Chesterman, & Krilis, 1990). β2-glycoprotein I forms a circular structure
in sera that is linearized upon binding to phospholipids, thus exposing cryptic autoantibody epitopes on β2-glycoprotein I (Agar et al., 2010; de Laat,
Derksen, van Lummel, Pennings, & de Groot, 2006). However, it has
remained unclear whether phospholipid-bound β2-glycoprotein I is a natural target for autoantibodies and is involved in the pathogenesis of APS. In
an analysis of the association between β2-glycoprotein I and MHC class II
molecules, which was similar to that described earlier for the IgG heavy
chain, intact β2-glycoprotein I was also found to be presented on MHC
class II molecules on the cell surface. Furthermore, β2-glycoprotein
I presented on MHC class II molecules was recognized by APS autoantibodies (Tanimura et al., 2015). Anti-β2-glycoprotein I Ab and anticardiolipin Ab titers are used clinically to diagnose APS, although some
patients with clinical manifestations of APS do not have detectable these
Abs. On the other hand, more than 80% of patients express autoantibodies
against β2-glycoprotein I presented on MHC class II molecules (Tanimura
et al., 2015). This suggests that β2-glycoprotein I, when presented on MHC
class II molecules, is a major target antigen for autoantibodies in patients with
APS (Fig. 3).
The presence of autoantibodies against autoantigens associated with MHC
class II molecules suggests that these autoantigens associate with MHC class II
molecules in certain tissues. Indeed, complexes of the IgG heavy chain or β2glycoprotein I with MHC class II molecules were detected on synovial membranes from patients with RA or uterine decidual tissues from patients with
APS, respectively. Similar to the analyses of autoantibodies from patients with
RA and APS, autoantibodies associated with other autoimmune diseases also
specifically recognize autoantigens complexed with MHC class II molecules
(Hui Jin, Ryosuke Hiwa, Satoko Morikami, Noriko Arase, & Hisashi Arase,
unpublished observation). Therefore, complexes of misfolded proteins with


A New Hypothesis for Autoimmune Diseases


11

Figure 3 Recognition of β2-glycoprotein I (β2GPI) presented on MHC class II molecules
by antiphospholipid autoantibodies. Native β2GPI forms a circular structure in sera that
is not recognized by antiphospholipid autoantibodies. When associated with a phospholipid such as cardiolipin, β2GPI forms a linear structure that appears to expose cryptic autoantibody epitopes. However, autoantibodies from some APS patients do not
recognize phospholipid-associated β2GPI. On the other hand, β2GPI is also expressed
on cell surfaces in association with MHC class II molecules. In addition, more than
80% of APS patients possess autoantibodies against β2GPI presented on MHC class II
molecules, suggesting that β2GPI presented on MHC class II molecules may be a major
target antigen for autoantibodies in antiphospholipid syndrome.

MHC class II molecules appear to be major targets of autoantibodies in many
autoimmune diseases.

5. SUSCEPTIBILITY TO AUTOIMMUNE DISEASES IS
ASSOCIATED WITH THE AFFINITY OF MISFOLDED
PROTEINS FOR MHC CLASS II MOLECULES
5.1 MHC Class II Alleles and Autoimmune Disease
Susceptibility
Particular MHC class II gene alleles are strongly associated with susceptibility to many autoimmune diseases. Extensive analyses of RA susceptibility
according to HLA-DR alleles have suggested that specific amino acid residues in the peptide-binding groove of HLA-DR are associated with susceptibility to RA (Raychaudhuri et al., 2012). Therefore, certain peptide
antigens are thought to be involved in autoimmune diseases. However, peptide antigens that could explain the susceptibility to autoimmune diseases
conferred by each MHC class II allele have not been identified, and therefore, the molecular mechanism underlying the control exerted by particular


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Hisashi Arase

MHC class II alleles over susceptibility to autoimmune diseases remains

unknown (Raychaudhuri et al., 2012).

5.2 Autoantibody Binding to Misfolded Protein/MHC Class II
Complex Is Associated with Autoimmune Disease
Susceptibility
In an analysis of autoantibody binding to IgG heavy chains presented on
HLA-DR molecules encoded by various alleles, a strong correlation was
observed between autoantibody binding to IgG heavy chains presented
on HLA-DR and the RA susceptibility conferred by each HLA-DR allele
( Jin et al., 2014). The invariant chain only partially blocks the association of
IgG heavy chains with RA-susceptible HLA-DR, but strongly blocks the
association of IgG heavy chains with RA-resistant HLA-DR. Autoantibodies fail to bind IgG heavy chains presented on RA-resistant HLA-DR
in the presence of the invariant chain. Thus, the IgG heavy chain is the first
molecule associated with the susceptibility to RA conferred by each HLADR allele. Because IgG heavy chain associated with MHC class II molecules
comprises a specific RA autoantibody target, it is possible that the IgG heavy
chain–MHC class II molecule complex is involved directly in RA pathogenicity as an autoantibody target.
Similarly, the presentation of self-antigens on MHC class II molecules
encoded by disease-susceptible alleles has been observed in APS
(Tanimura et al., 2015). APS-susceptible HLA-DR efficiently presents
β2-glycoprotein I, and autoantibodies preferentially bind to this complex
even in the presence of the invariant chain. Therefore, differences in autoimmune disease susceptibility among the different MHC class II alleles might
be explained by different efficiencies of autoantigen presentation by MHC
class II molecules.

6. INVOLVEMENT OF MISFOLDED PROTEIN–MHC CLASS
II MOLECULE COMPLEXES IN AUTOANTIBODYMEDIATED PATHOGENICITY
6.1 Pathogenesis of Autoantibodies in RA and APS
Autoantibodies that are produced in most autoimmune diseases are involved
in the pathogenicity of some of these diseases. For example, myasthenia
gravis, Graves’ disease, and pemphigus are representative autoimmune diseases caused by autoantibodies that exert blocking, activating, and destructive functions, respectively. In addition, the adoptive transfer of serum IgG

from RA patients to mice lacking FcγRIIB, an inhibitory Fc receptor, has


A New Hypothesis for Autoimmune Diseases

13

been shown to induce arthritis, suggesting that autoantibodies play an important role in the pathogenesis of RA, although the target antigens for these
pathogenic autoantibodies remain undefined (Petkova et al., 2006). Autoantibodies also play a crucial role in some RA mouse models such as K/BxN
mice and the type II collagen-induced arthritis model. In K/BxN mice, autoantibodies against glucose-6-phosphate isomerase are responsible for arthritis
development (Matsumoto et al., 2002; Matsumoto, Staub, Benoist, & Mathis,
1999). The adoptive transfer of anti-glucose-6-phosphate isomerase autoantibodies from K/BxN mice to healthy mice induces arthritis. Similarly, antitype II collagen autoantibodies induced via immunization with type II collagen directly mediate arthritis. Similar to the autoantibodies in K/BxN mice,
these anti-type II collagen autoantibodies induce arthritis when transferred to
healthy mice (Griffiths & Remmers, 2001). Therefore, autoantibodies appear
to play an important role in the pathogenesis of arthritis not only in RA mouse
models but also in patients with RA.
APS autoantibodies are directed mainly against the serum lipoprotein
β2-glycoprotein I and are thought to be involved in the pathogenesis of
APS. However, it remains unclear how these autoantibodies against
β2-glycoprotein I induce thrombosis or pregnancy complications, as the
protein is not expressed on the surface of healthy blood vascular endothelial
cells. In addition, it remains unknown why some patients mainly exhibit
thrombosis and others predominantly develop pregnancy complications,
despite detecting similar autoantibodies in both groups of patients. Endothelial cells strongly express MHC class II molecules upon IFN-γ stimulation
( Jaffe et al., 1989; Pober et al., 1983). Indeed, aberrant MHC class II expression has been observed in uterine decidual tissues from patients with APS.
More importantly, complexes of β2-glycoprotein I with MHC class II molecules have been detected in uterine decidual tissues from patients with APS
(Tanimura et al., 2015). These observations suggest that aberrantly expressed
MHC class II molecules on endothelial cells could be targeted by autoantibodies, possibly leading to thrombosis in peripheral vessels or the uterus.
Therefore, in addition to the presence of autoantibodies, aberrant MHC
class II expression might play an important role in autoantibody-mediated

pathogenesis.

6.2 B Cell Removal Are Effective Treatment for Autoimmune
Diseases
The removal of B cells via the administration of an anti-CD20 mAb
(rituximab) has been clinically approved as an effective treatment for RA
( Jacobi & Dorner, 2010). Anti-CD20 mAb treatment is also effective for


14

Hisashi Arase

other autoimmune diseases such as APS (Erkan, Vega, Ramon, Kozora, &
Lockshin, 2013), systemic lupus erythematosus (Anolik et al., 2004), myasthenia gravis (Sieb, 2014), Graves’ disease (Heemstra et al., 2008), and pemphigus (Ahmed, Spigelman, Cavacini, & Posner, 2006; Joly et al., 2007).
Furthermore, anti-B lymphocyte stimulator (BlyS) mAb (Belimumab) that
decreases autoantibody producing B cells is effective for some autoimmune
diseases such as SLE (Navarra et al., 2011). Because B cells are involved in
antibody production as well as antigen presentation and cytokine secretion,
B cell depletion may affect various aspects in autoimmunity. B cell depletion
by anti-CD20 mAb has been reported to correlate with a decrease in autoantibody levels as well as clinical manifestation, suggesting that autoantibody
producing B cells play an important role in the pathogenicity of autoimmune diseases (Cambridge et al., 2006; Thurlings et al., 2008). However,
it is difficult to directly test the pathogenicity of human autoantibodies from
autoimmune patients in mice because of species differences in autoantigens.
Therefore, the pathogenicity of human autoantibodies has been demonstrated only in the context of some autoimmune diseases such as Graves’ disease, myasthenia gravis, and pemphigus. As autoantigen–MHC class II
molecule complexes are targeted by autoantibodies, mice expressing both
human autoantigens and human MHC class II molecules would be useful
for testing the pathogenesis of human autoantibodies.

7. MISFOLDED CELLULAR PROTEINS RESCUED FROM

DEGRADATION BY MHC CLASS II MOLECULES MAY
ABROGATE IMMUNE TOLERANCE
7.1 Misfolded Proteins Associated with MHC Class II
Molecules as “Nonself”-Antigens
As described above, misfolded proteins, when complexed with MHC class II
molecules, are specific targets for the autoantibodies produced in autoimmune diseases. In addition, a strong association has been observed between
autoantibody binding to IgG heavy chains presented on MHC class II molecules and the RA susceptibility conferred by each HLA-DR allele. These
observations suggest that self-antigens presented on MHC class II molecules
are involved in the pathogenicity of autoimmune diseases by serving as targets for autoantibodies. In cases involving aberrantly induced or increased
MHC class II expression in response to infection or inflammation in which
ER-misfolded proteins have a stronger affinity for MHC class II molecules
than the invariant chain, misfolded cellular proteins associate with MHC
class II molecules and are subsequently transported to the cell surface.


A New Hypothesis for Autoimmune Diseases

15

The prompt degradation of ER-misfolded proteins through various cellular
mechanisms in steady state (Meusser et al., 2005) prevents the exposure of
these proteins to immune cells. Therefore, ER-misfolded proteins aberrantly transported to the cell surface by MHC class II molecules might appear
as “neo-self ”-antigens and induce an abnormal immune response (Fig. 2).
Indeed, XBP-1, a transcription factor induced by the unfolded protein
response, was cloned originally from plasma cells in synovial membranes of
patients with RA (Iwakoshi et al., 2003), suggesting high levels of unfolded
protein production in these cells. Autoantibodies against citrullinated proteins are also generated in RA. Interestingly, citrullination is known to cause
protein misfolding (Tarcsa et al., 1996). Therefore, citrullination-induced
conformational changes in proteins might augment the association of autoantigens with MHC class II molecules. Furthermore, MHC class II expression is strongly increased in the synovial membranes of patients with RA
(Feldmann et al., 1988; Klareskog, Forsum, Scheynius, Kabelitz, &

Wigzell, 1982). Because plasma cells expressing low levels of MHC class
II molecules produce large amounts of IgG, misfolded IgG heavy chains
might associate with MHC class II molecules encoded by RA-susceptible
alleles in certain conditions that induce upregulated MHC class II expression
on plasma cells; the resulting complexes could induce production of autoantibody against IgG. Similarly, β2-glycoprotein I is mainly produced in
hepatocytes, which do not express MHC class II molecules in steady state.
If APS-susceptible MHC class II molecules that preferentially bind to β2glycoprotein I are expressed on hepatocytes in response to inflammation
or infection, β2-glycoprotein I will associate with these molecules and thus
trigger autoantibody production.
Certain misfolded proteins may associate constitutively with MHC
class II molecules on B cells or dendritic cells. However, autoantibodies
against these misfolded proteins are not produced in steady state; immune
cells are exposed constitutively to these complexes, a process that appears
to have induced tolerance. Indeed, transgenic mice expressing MHC class
II molecules on pancreatic β cells did not exhibit β cell autoimmunity
(Lo et al., 1988; Sarvetnick, Liggitt, Pitts, Hansen, & Stewart, 1988).
Because MHC class II expression is not inducible in these transgenic mice,
even if certain β cell-specific misfolded proteins are presented on MHC
class II molecules, the proteins associated with MHC class II molecules
are always presented to immune cells and may not be recognized as
“neo-self ”-antigens. Analyses of mice harboring an inducible MHC class
II transgene might provide valuable information about the function of aberrantly expressed MHC class II molecules.


16

Hisashi Arase

The CIITA transcription factor is involved in the expression of both MHC
class II molecules and the invariant chain (Reith, LeibundGut-Landmann, &

Waldburger, 2005). Therefore, the invariant chain is expressed in most
MHC class II-expressing cells, where it blocks the binding of ER-misfolded
proteins to MHC class II molecules. However, MHC class II and invariant
chain gene transcription is regulated differentially, and thus the expression of
these molecules is not always equivalent (Paul et al., 2011). In addition, the
affinity of MHC class II molecules for the invariant chain differs depending
on the encoding MHC class II allele (Patil et al., 2001). Therefore, the amounts
of misfolded proteins, invariant chain, and MHC class II molecules, as well as
the MHC class II allele, seem to determine the efficiency of the association
between misfolded proteins and MHC class II molecules.

7.2 Misfolded Protein Complexed with MHC Class II Molecules
as Primary Autoantigens for Autoantibodies
It remains unclear how autoantibodies specific for autoantigens presented on
MHC class II molecules are produced. Given the presence of specific autoantibodies against these complexes, it is likely that the complexes themselves
induce autoantibody production. However, it is uncertain whether the selfantigens presented on MHC class II molecules initiate local antibody
responses in nonlymphoid tissues because the germinal center usually is
required for antibody responses (Klein & Dalla-Favera, 2008). On the other
hand, cell surface MHC class II molecules are known to be released from
cells as exosomes (Thery, Ostrowski, & Segura, 2009) and have been
detected as such in serum (Almqvist, Lonnqvist, Hultkrantz, Rask, &
Telemo, 2008; Karlsson et al., 2001; Taylor, Akyol, & Gercel-Taylor,
2006). Therefore, when self-antigens are expressed in complex with
MHC class II molecules in certain tissues, the complexes may be released
from the cells as exosomes, which might subsequently induce the production of specific antibodies against the complexes in lymphoid tissues.
Most autoantibodies are detected using self-antigens immobilized on plates
or microbeads, suggesting that MHC class II molecules might not be required
for autoantibody recognition. However, protein immobilization causes significant conformational changes. For example, RF does not bind native IgG in
sera but does bind immobilized IgG. Similarly, autoantibodies against β2glycoprotein I do not bind native β2-glycoprotein I in sera but do bind to
β2-glycoprotein I when immobilized on negatively charged plates. In addition, most autoantibodies can detect target antigens when assayed by Western

blot analysis, indicating that they recognize denatured forms of autoantigens.


A New Hypothesis for Autoimmune Diseases

17

These observations are compatible with the fact that autoantibodies are
directed against misfolded proteins presented on MHC class II molecules.
Autoantibodies from most patients with APS recognize β2-glycoprotein
I presented on MHC class II molecules, whereas only some patients
possess autoantibodies against plate-bound β2-glycoprotein I. Therefore,
β2-glycoprotein I presented on MHC class II molecules seems to be the primary target antigen for APS autoantibodies. Most autoantibodies are the
result of a somatic hypermutation process that increases the affinity for autoantigens (Rajewsky, 1996). Because somatic hypermutation is not observed
in naı¨ve B cells, the antigen specificities of autoantibodies that are
engineered in vitro to restore the codons present in the germline Ig gene will
provide information about the original antigens that stimulated naı¨ve B cells
to produce autoantibodies. A recent analysis indicated that germlinereverted autoantibodies from pemphigus patients did not recognize the
autoantigen desmoglein-3, suggesting that this autoantigen did not induce
autoantibody production (Di Zenzo et al., 2012). However, some
germline-reverted autoantibodies still recognize autoantigens when presented on MHC class II molecules (Hui Jin & Hisashi Arase, unpublished
observation). Therefore, autoantigens presented on MHC class II molecules
might be the primary target antigens that induced autoantibody production.
Although most autoantibodies are directed against denatured autoantigens,
some autoantibodies recognize native autoantigens. Because epitope spreading affects antibody diversity, it is possible that autoantibodies raised against
misfolded proteins presented on MHC class II molecules might have
acquired reactivity against native autoantigens through epitope spreading.
The molecular mimicry of autoantigens by microbial antigens is also
involved in the production of some autoantibodies (Munz, Lunemann,
Getts, & Miller, 2009). Considering that the antigenicity of misfolded autoantigens is more similar to that of native autoantigens than of microbial antigens, misfolded proteins presented on MHC class II molecules may possibly

be involved in the production of autoantibodies against native autoantigens.

8. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II
MOLECULES AS A THERAPEUTIC TARGET FOR
AUTOIMMUNE DISEASES
As misfolded proteins aberrantly rescued from protein degradation by
MHC class II molecules might be involved in the pathogenesis of autoimmune diseases, blocking the association of misfolded proteins with MHC


18

Hisashi Arase

class II molecules would be a good candidate treatment for autoimmune diseases. The aberrant MHC class II expression observed in autoimmunediseased tissues seems to result from stimulation by cytokines such as
IFN-γ. Blocking the MHC class II expression induced by cytokine stimulation would therefore effectively treat autoimmune diseases (Miller,
Maher, & Young, 2009). HMG-CoA reductase inhibitors, or statins, are
among the drugs used to reduce serum cholesterol levels. Statins have been
reported to reduce inflammation in some autoimmune diseases such as RA,
APS, and Sj€
ogren’s syndrome (Greenwood, Steinman, & Zamvil, 2006;
Khattri & Zandman-Goddard, 2013). Although the exact mechanisms
remain unclear, statins appear to reduce IFN-γ-induced MHC class II
expression on human endothelial cells in vitro (Kwak, Mulhaupt, Myit, &
Mach, 2000; Youssef et al., 2002). The anti-inflammatory function of statins
might also include inhibiting the association of misfolded proteins with
MHC class II molecules. The development of a strong and specific inhibitor
with which to block the association of misfolded proteins with aberrantly
expressed MHC class II molecules represents a new target in autoimmune
disease therapy.


9. CONCLUDING REMARKS
The MHC class II locus is the gene most strongly associated with susceptibility to many autoimmune diseases. Extensive analyses of misfolded proteins
aberrantly rescued from protein degradation by MHC class II molecules have
not only revealed that these proteins are specific targets for autoantibodies but
have also suggested that autoantibody binding to these proteins may explain the
autoimmune disease susceptibility conferred by certain MHC class II alleles.
Therefore, aberrant MHC class II expression on certain tissues or cells might
induce autoimmune disease. An understanding the molecular factors that
induce aberrant MHC class II expression would be quite important to an understanding of the causes of autoimmune diseases. On the other hand, the physiological functions of the aberrantly expressed MHC class II molecules on
nonimmune cells remain unclear. If aberrant MHC class II expression on nonimmune cells is solely responsible for diseases involvement, the pathway by
which MHC class II is expressed on nonimmune cells will be lost in the course
of evolution. Although MHC class II molecules expressed on nonimmune cells
cannot evoke helper T cell responses because of the lack of costimulatory molecule expression, these aberrantly expressed MHC class II molecules might
confer certain benefits to maintain homeostasis. Further analyses of the


A New Hypothesis for Autoimmune Diseases

19

misfolded proteins rescued from protein degradation and transported to the cell
surface by MHC class II molecules will reveal currently unknown mechanisms
of immunity in both normal and disease situations.

ACKNOWLEDGMENTS
We thank Prof. Lewis L. Lanier for critical reading of our manuscript. This work was partially
supported by JSPS KAKENHI Grant Numbers (15K15131, 15H02545, 26117714 and
24115005), the Practical Research Project for Allergic Diseases and Immunology from
Japan Agency for Medical Research and development, AMED, The Naito Foundation,
The Tokyo Biochemical Research Foundation, The Uehara Memorial Foundation and

Terumo Life Science Foundation.

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