Glycobiology, 2016, vol. 26, no.

10, 1029–1040
doi: 10.1093/glycob/cww062
Advance Access Publication Date: 28 May 2016
Review

Review

Carbohydrates as T-cell antigens with

implications in health and disease
Lina Sun, Dustin R Middleton, Paeton L Wantuch, Ahmet Ozdilek,

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and Fikri Y Avci1
Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, and Complex Carbohydrate
Research Center, University of Georgia, Athens, GA 30602, USA
To whom correspondence should be addressed: Tel: +1-706-542-3831; Fax: +1-706-542-4412; e-mail: avci@uga.edu
1

Received 16 April 2016; Revised 11 May 2016; Accepted 23 May 2016

Abstract
Glycosylation is arguably the most ubiquitous post-translational modification on proteins in micro-
bial and mammalian cells. During the past few years, there has been intensive research demonstrat-
ing that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and
modulate adaptive immune responses. We now know that carbohydrates can be directly recognized
by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of
carbohydrate antigens takes place via their presentation by major histocompatibility complex path-
ways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell
antigens modulating adaptive immune responses. Through discussion of glycan-containing anti-
gens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based gly-
coconjugate vaccines, we will illustrate the key molecular and cellular interactions between
carbohydrate antigens and T cells and the implications of these interactions in health and disease.

Key words: adaptive immunity, glycoconjugate, glycolipid, glycopeptide, glycoprotein

Introduction
As the major cell population of the adaptive immune system, T cells
recognize foreign antigens that are processed and presented by
major histocompatibility complex (MHC) molecules on antigen-
presenting cells (APCs). Glycosylation is a common modification of
proteins and lipids, usually localized on bacterial and viral surfaces.
Also, numerous protein or lipid-associated glycans have been identi-
fied as tumor-associated antigens. Traditional views held that carbo-
hydrates were T-cell-independent antigens. However, more recent
research has demonstrated that carbohydrates interact with the
adaptive immune system through participation in T-cell epitopes or
direct recognition by T cells as reviewed here. Thus, understanding
T-cell recognition of the carbohydrate-containing antigens is import-
ant in revealing the impact of carbohydrates on adaptive immunity
with implications to health and disease.
Recent review articles by others and us reviewed what roles car-
bohydrates play in regulating adaptive immune responses (Cobb
and Kasper 2005a; Baum and Crocker 2009; Avci et al. 2010;
Rabinovich et al. 2012; Avci et al. 2013; Wolfert and Boons 2013).
This review will thoroughly summarize the effects of carbohydrates
on T-cell responses and serve as an update on recent developments
and discoveries. The processing and presentation of carbohydrate
antigens in the APCs and how they interact with MHC proteins and
T-cell receptors (TCRs) will be discussed as well as biomedical
implications of these immune interactions.
Glycopeptides and glycoproteins
The long-standing belief has been that carbohydrates are T-cell-
independent antigens due to their inability to stimulate T-cell
responses in their pure forms. A wealth of studies using synthesized
glycopeptides or naturally glycosylated immunogenic glycopeptides
generated from processing glycoproteins revealed that they can bind
to MHC molecules and that glycan portions specifically influence
T-cell recognition of the peptides they are bound to (Table I). The
effects of glycosylation on MHC binding of peptides and stimulation

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1030 L Sun et al.

Table I. T-cell responses induced by carbohydrate antigens

Carbohydrate antigens Sources Presentation Responding cells Cytokines Representative publications

Glycoproteins/glycopeptides
Type II collagen Host protein MHCII CD4+ T cells Corthay et al. (1998),
Michaelsson et al. (1994)
Apa Mtb MHCI/MHCII CD8+/CD4+ T cells IL-2, IFN-γ TNF-α Nandakumar et al. (2013)
LprG M. Leprae Mtb MHCII CD4+ T cells Sieling et al. (2008)
Miscellaneous glycopeptides Multiple MHCI/MHCII CD8+/CD4+ T cells Ishioka et al. (1992),
Haurum et al. (1994)
Glycoconjugates
GBSIII CPS Streptococcus MHCII CD4+ T cells IL-2, IL-4 Avci et al. (2011, 2012)
Sp14 CPS S. pneumoniae MHCII CD4+ T cells Lai and Schreiber (2009)
MCPS CPS N. meningitidis MHCII CD4+ T cells Muthukkumar and Stein
2004

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ZPSs
PSA B. fragilis MHCII CD4+ T cells IL-10 Cobb et al. (2004), Johnson
et al. (2015b)
IFN-γ Wang et al. (2006),
Mazmanian et al. (2005)
CP5/CP8 CPS S. aureus MHCII CD4+ T cells IFN-γ Tzianabos et al. (2001)
Sp1 CPS S. pneumoniae MHCII CD4+ T cells Velez et al. (2009)
Glycolipids
αGalCer Sponge-derived glycolipid CD1d iNKT cells IL-4, IFN-γ Kronenberg et al. (2005),
Avci et al. (2013)
Glycosphingolipids B. fragilis CD1d iNKT cells An et al. (2014)

of T-cell responses are multiple and depend on the chemical struc-
ture of the carbohydrate moiety, its occupation site on the peptide,
as well as on the structure of the peptide as discussed below. The
current understanding of how MHC molecules bind peptides differs
between class I and II (MHCI, MHCII) molecules. Due to a closed
binding groove, MHCI can accommodate peptides of 8–9 amino
acids in length that originate from cytosolic or nuclear locations.
MHCII, on the other hand, can accommodate larger peptides (i.e.,
>10 amino acids), as the binding groove is open and allows for
greater flexibility (Neefjes et al. 2011).
Glycopeptides
In a multitude of studies, the effect of glycosylation on MHCI/II
binding of peptides and their T-cell recognition have been evaluated
by using natural or synthetic glycopeptides. These studies, although
not concurrent, could be categorized as the impact of carbohydrate
structure and the site of glycosylation on antigen presentation and
T-cell recognition as discussed here.
Utilizing synthetic glycopeptides and T-cell hybridomas, the spe-
cificity of the carbohydrate moiety as an antigenic determinant
recognized by T cells has been extensively investigated. The MHCII-
binding hen egg-white lysozyme peptide [HEL(52–61)] conjugated
with the disaccharide galactobiose through internal Ser56 residue
via covalent coupling was used as an immunogen to produce T-cell
hybridomas (Deck et al. 1995). Those hybridomas that recognized
galactobiose on Ser56 did not recognize a different disaccharide cel-
lobiose on Ser56. In addition, peptides containing galactobiose on a
different residue or galactobiose coupled to a different peptide even
from the same protein did not stimulate the T-cell hybridomas
(Deck et al. 1995). Similar results obtained from other studies test-
ing T-cell hybridomas against glycopeptides containing the tumor-
associated carbohydrate antigens (TACAs) also demonstrated the
presence of glycopeptide-specific T-cell responses (Galli-Stampino
et al. 1997; Jensen et al. 1997; Gad et al. 2002). A clone of the
CD4+ T-cell hybridoma was able to distinguish between glycopep-
tides containing α-D-GlcNAc or α-D-GalNAc (Jensen et al. 1997).
In another study, a panel of tumor-associated glycopeptides with
varying carbohydrates were used to investigate the effects of glycan
groups on MHC binding and immunogenicity. Among all the glyco-
peptides, monosaccharide-coupled ones had higher binding affinity
to MHCII than those with more complex oligosaccharides (Galli-
Stampino et al. 1997). Furthermore, T cells primed with simple gly-
copeptides (i.e., low degree of glycosylation) did not cross-react with
other glycopeptides, while T cells specific for complex glycopeptides
(i.e., high degree of glycosylation) showed strong cross-reactivity
(Galli-Stampino et al. 1997). In addition to CD4+ T cells, CD8+
cytotoxic T lymphocytes (CTLs) have been shown to recognize gly-
can structures (Abdel-Motal et al. 1996). CTL lines were generated
by immunizing mice with glycopeptides coupling di- or trisacchar-
ides to peptides known to bind MHCI proteins. In an antigen-
specific CTL killing assay, CTLs treated with glycopeptides killed
target cells more efficiently than those treated with nonglycosy-
lated peptides. Meanwhile, the CTL activity was inhibited by treat-
ment with a monoclonal antibody to the glycan portion of the
glycopeptide (Abdel-Motal et al. 1996), strongly indicating T-cell
specificity to the carbohydrate moiety. These carbohydrate-specific
CTLs were MHCI unrestricted and could kill the target cells
presenting the same carbohydrate on a different MHCI allele
(Abdel-Motal et al. 1996).
In addition to the glycan structure, the glycosylation site is also
important for T-cell recognition. In one early study (Ishioka et al.
1992), it was reported that a single N-acetylglucosamine (GlcNAc)
conjugated with a known T-cell peptide (OVA 323–339) via cova-
lent coupling elicited MHCII-dependent, glycopeptide-specific T-cell
stimulation. When residues away from the core MHCII-binding
region on the peptide were glycosylated by GlcNAc, this modifica-
tion had little to no effect on the MHCII-binding capacity or T-cell
stimulation of the peptide (Ishioka et al. 1992). In another study,
when a glycan was coupled to the amino acids that function as
Carbohydrates as T-cell antigens
MHCII anchor residues, MHCII binding and T-cell stimulation
were completely abolished (Jensen et al. 1996). In this study, each of
the 10 positions in the peptide of mouse hemoglobin-derived deca-
peptide Hb (67–76), VITAFNEGLK, which binds to MHCII, was
substituted with serine or threonine and O-glycosylated with the
tumor-associated carbohydrate Tn (α-D-N-acetyl-galactosamine, or
α-D-GalNAc). If the amino acid side chain was oriented towards the
binding groove of the MHCII, glycopeptide binding was completely
lost (Jensen et al. 1996). Yet, when a glycan was linked to an amino
acid pointing away from the binding groove of the MHCII, the
binding was maintained and the carbohydrate became the T-cell
stimulating epitope (Deck et al. 1999). Based on these independent
observations, the T-cell stimulation of glycopeptides was retained
only when the glycosylation sites were outside the core MHCII-
binding region. Interestingly, unlike the disruption of MHCII
binding with glycosylation in the binding region, a peptide with
O-glycosylation within its MHCI binding region was still able to
bind MHCI and elicit a strong glycopeptide-specific CD8+ CTL
response (Haurum et al. 1994). In a parallel study, CTL clones spe-
cific for a GlcNAc-modified glycopeptide cross-reacted with another
glycopeptide where the GlcNAc substitution was on a neighboring
residue, indicating that the GlcNAc on the glycopeptide can interact
directly with TCR (Haurum et al. 1995). Subsequently, human
MHCI protein, H-2Db, was cocrystallized with the glycopeptide
composed of a synthetic analog of Sendai virus nucleoprotein pep-
tide (FAPSNYPAL) and a GlcNAc on the internal serine residue
(Figure 1A) (Glithero et al. 1999). This MHCI–glycopeptide com-
plex was then modeled with an existing TCR crystal structure. This
model suggested that the glycan was solvent exposed and available
for direct recognition by the TCR (Glithero et al. 1999). The central
region of the putative TCR binding site was dominated by extensive
exposure to the carbohydrate. A separate crystallography study
also demonstrated that glycan position, linker length and carbohy-
drate size are all important factors for T-cell recognition (Speir
et al. 1999).
Peptides are essential components of T-cell epitopes associated
with MHC binding and T-cell recognition. The amino acid residues
Fig. 1. Crystal structures of MHCI and CD1d proteins complexed with a glyco-
peptide and a glycolipid. (A) Human MHCI allele H-2Db (PDBid: 1QLF) (pink)
bound with glycopeptide K3G (Glithero et al. 1999), a synthetic analog of
Sendai virus nucleoprotein peptide 324-332 (blue) where the O-GlcNAc resi-
due is depicted as a combination of van der Waals spheres and bonds in
orthogonal view. The K3G peptide is altered from the original peptide to con-
tain an O-GlcNAc residue at the P4 position of the peptide. (B) Human CD1d
(PDBid: 1ZT4) (α1-α3 domains) bound with glycolipid αGalCer depicted as a
combination of van der Waals spheres and bonds in orthogonal view (Koch
et al. 2005). This figure is available in black and white in print and in color at
Glycobiology online.
1031
on the peptides directly contribute to the recognition of
glycopeptide-specific T-cell hybridomas by the TCR (Deck et al.
1999). Using a glycopeptide that conjugated the disaccharide gala-
biose [Galα(l-4)Galβ] to the amino terminus of a T-cell peptide
determinant from HEL(52–61), the researchers analyzed the effects
of peptide residues on T-cell recognition of glycopeptides. T-cell
hybridomas against the glycopeptide did not respond to its analog
with alanine substitutions at two critical T-cell-contacting resi-
dues, although this did not affect MHCII-binding activity. In add-
ition, T-cell recognition was shown to be lost when the amino
terminus of the glycopeptide was elongated (Harding et al. 1993).
Collectively, peptide glycosylation influences MHCI/II binding
and T-cell recognition, depending upon carbohydrate structure, size,
complexity and its orientation with the peptide.
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Glycoproteins
Glycosylation-dependent epitope recognition by T cells takes place
following natural exposure to glycoproteins. Bee venom allergen
phospholipase A2 (PLA), a glycosylated protein, induces a
carbohydrate-specific CD4+ T-cell response (Dudler et al. 1995).
PLA enriched CD4+ T-cell clones respond only to native PLA pro-
teins and not to their carbohydrate moiety, nonglycosylated var-
iants, or to the mixture of carbohydrate and nonglycosylated
proteins (Dudler et al. 1995). One critical piece of evidence support-
ing direct T-cell recognition of carbohydrates resulted from the ana-
lysis of TCRs from T-cell hybridomas recognizing glycans on
peptides derived from host antigen type II collagen (Corthay et al.
1998). TCR DNA sequencing was performed and analysis of gene
segment usage and amino acid conservation in the complementarity-
determining region 3 (CDR3) indicated their TCR usage was
strikingly different from other T-cell hybridomas with even slight
differences in specificity (Corthay et al. 1998). Importantly, glycosy-
lation of type II collagen plays a critical role in collagen-induced
arthritis (Michaelsson et al. 1994; Corthay et al. 1998; Backlund
et al. 2002; Holm et al. 2002). Elimination of the carbohydrates sig-
nificantly decreases the incidence of arthritis, delays its onset and
reduces severity (Michaelsson et al. 1994).
Although glycoproteins appear to be less common in prokaryotic
organisms compared to eukaryotic cells, it is now widely accepted
that prokaryotes are also capable of expressing glycosylated proteins
(Upreti et al. 2003). Glycoproteins on the surface of bacteria are
involved in maintaining their structural integrity, pathogenicity,
antigenicity, host-pathogen interactions and evasion of immunity
(Upreti et al. 2003; Nothaft and Szymanski 2013; Cuccui and Wren
2014). A 45–47 kDa secretory and cell surface glycoprotein,
alanine-proline-rich antigen (Apa), expressed by Mycobacterium
tuberculosis (Mtb) and Mycobacterium bovis, was shown to induce
glycan-specific T-cell responses (Horn et al. 1999). Nonglycosylated
Apa expressed in Escherichia coli showed decreased CD4+ T-cell
stimulation compared to the native antigen isolated from Mtb both
in vivo and in vitro (Horn et al. 1999). Subsequently, CD4+ T-cell
hybridomas were generated specifically recognizing Apa glycopep-
tides (Nandakumar et al. 2013). These observations demonstrated
that glycosylation is critical for T-cell activation of Apa during infec-
tion. A lipoglycoprotein (LprG) from bacterial Mycobacterium
leprae and Mtb mediates CD4+ T-cell activation through processing
within APCs and through MHCII presentation. Reducing the glyco-
sylation of the LprG protein by expression either in E. coli, or in
Mycobacterium smegmatis followed by glycosidase treatment
impaired the ability to activate T cells (Sieling et al. 2008) (Table I).
1032 L Sun et al.

Processing and presentation of glycoproteins in APCs
Unlike most other immune cells recognizing intact antigens, T cells
can only recognize antigens that are processed and presented by
MHC pathways on APCs. Processing and presentation of protein
antigens in APCs have been largely studied. Exogenous proteins are
processed into short peptides in APCs for MHCII presentation to
CD4+ T cells, while endogenous proteins are processed for MHCI
presentation to CD8+ T cells, respectively (Neefjes et al. 2011). For
glycoprotein antigens, the fate of glycans can be different, resulting
in potentially two different processed epitopes: (1) The glycan could
be removed entirely during processing, resulting in naked peptides.
In one example, T-cell hybridomas that were generated by glycopep-
tide immunization only recognized the deglycosylated peptide rather
than the glycopeptide, thus strongly supporting this scenario (Jensen
to MHCI molecules. This could be because the majority of the
MHCI binding peptides are derived from cytosolic proteins targeted
and degraded by the proteasome, whereas, N-glycans are removed
by a cytosolic N-glycanase before the glycoprotein interacts with the
proteasome (Werdelin et al. 2002).
These studies provide evidence that glycans on glycoproteins
participate as important epitope determinants in regulating T-cell
recognition and modulating immune responses. Identification and
characterization of microbial and mammalian glycoproteins and
understanding their immune activation mechanisms serve as power-
ful tools and platforms to combat infectious and autoimmune
diseases.

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et al. 1997). (2) The glycan group survives the antigen processing
and is left intact on the peptide fragment (Chicz et al. 1994; Vlad
et al. 2002; Werdelin et al. 2002). Investigation of the processing
and presentation of a tumor antigen MUC1 glycopeptide revealed
that complex carbohydrates on proteins were not removed during
processing and presentation by APCs. MUC1 glycoprotein was pro-
cessed into smaller peptides and presented via MHCII molecules
with intact glycans on dendritic cells (DCs) for T-cell stimulation
(Vlad et al. 2002) (Figure 2). O-glycosylation of MUC1 modulates
the protein processing in APCs by preventing proteolysis of the
Thr3-Ser4 peptide bond if either amino acid is glycosylated
(Hanisch et al. 2003). Hence, the O-linked glycans can alter
proteolytic processing or presentation of the MHCII-restricted
glycopeptides in a site-specific manner. Analysis of peptides eluted
from MHCI molecules revealed that MHCI-bound peptides carry
O-linked GlcNAc (O-βGlcNAc) residues (Haurum et al. 1999). So
far, however, N-linked carbohydrates have not been shown to bind
Glycolipids
Glycolipids are important for inducing natural-killer T (NKT) cell
responses. Alpha-galactosylceramide (α-GalCer) is a glycolipid anti-
gen known to be specific for invariant natural-killer T (iNKT) cells
when presented by the MHCI-like molecule CD1d (Naidenko et al.
1999; Gumperz et al. 2000). The crystal structures of glycosylcera-
mides with CD1d molecules demonstrated that the aliphatic lipid tails
fit into the CD1d binding groove, whereas its monosaccharide head
group extends above the surface of the lipid-binding groove and
thereby is exposed for recognition by the TCR of iNKT cells (Gadola
et al. 2002; Zajonc et al. 2003; Batuwangala et al. 2004; Koch et al.
2005) (Figure 1B). Compared to TCRs on CD8+ or CD4+ T cells
binding to conventional MHCI/II peptide complexes, iNKT TCRs
bind to glycosylceramides and CD1d complexes with higher affinities
and particularly long half-lives (Sidobre et al. 2002; Sim et al. 2003).
Activation of iNKT cells depends upon TCR recognition of cog-
nate antigens, resulting in an induction of NKT cell cytotoxicity and

Fig. 2. Overview of T-cell-dependent immune responses induced by glycoantigens. (A) Glycopeptides, either prepared synthetically or are products of glycopro-
tein processing by proteases in APCs, bind to MHCI or MHCII molecules and are presented to CD8+ or CD4+ T cells, respectively. Glycopeptide recognition by
TCR induces T cells to produce functional cytokines, such as IL-2 and IFN-γ. (B) Glycoconjugates prepared by conjugation of capsular polysaccharides and car-
rier proteins are processed by reactive oxygen species (ROS) and proteases in APCs generating glycanp-peptides. Binding of peptide portion of glycanp-peptide
to MHCII allows the presentation of the carbohydrate epitope to CD4+ T cells. Activation of carbohydrate-specific T cells (Tcarbs) results in production of cyto-
kines such as IL-4 and IL-2. (C) Extracellular ZPS (i.e., PSA) is processed into smaller molecular weight polysaccharides in the APCs by reactive nitrogen species
(RNS). The processed carbohydrate epitope is presented on the surface of the APCs for T-cell recognition. PSA induces CD4+ T cells to produce cytokines IFN-γ
and IL-10. (D) Glycolipids (i.e., α-GalCer), presented by the MHCI-like molecule CD1d, induce iNKT cells to produce cytokines IFN-γ and IL-4. This figure is avail-
able in black and white in print and in color at Glycobiology online.
Carbohydrates as T-cell antigens
direct killing of target tumor cells (Metelitsa et al. 2001). After rec-
ognition of αGalCer/CD1d complex, iNKT cells rapidly produce
cytokines such as interferon-γ (IFN-γ) and interleukin-4 (IL-4), IL-2,
tumor necrosis factor (TNF), IL-5, IL-13 and GM-CSF (Kronenberg
2005) (Figure 2). In vivo administration of αGalCer elicits profound
immunological consequences depending on CD1d-restricted iNKT
cells. Following αGalCer treatment, iNKT cells activate NK cells, B
cells and memory CD8+ and CD4+ T cells (Burdin et al. 1999;
Carnaud et al. 1999; Singh et al. 1999). iNKT cells recognize
αGalCer that is presented by DCs. Due to the complex interactions
and regulatory networks between APCs and iNKT cells, αGalCer
may skew the immune response of both iNKT and conventional T
helper cells towards either T helper 1 cell (Th1 cell) or T helper 2
cell (Th2 cell) phenotype (Chen et al. 1997; Mendiratta et al. 1997;
Burdin et al. 1999; Cui et al. 1999; Kitamura et al. 1999; Singh
et al. 1999). A recent study determined that the altered cytokine
responses are associated with the length of the sphingosine base in
the glycolipid resulting in differential binding to CD1d and TCR
affinity (Sullivan et al. 2010).
The ability to specifically activate iNKT cells makes αGalCer a
potential direct therapy for cancer and infectious diseases (Skold
and Behar 2003; Avci et al. 2013). α-GalCer was shown to induce
tumor regression in experimental animal models (Morita et al.
1995). The antitumor activities of α-GalCer and other glycosylcera-
mides are mediated by their presentation by CD1d to iNKT cells
(Cui et al. 1997, 1999). It was also reported that αGalCer functions
as an effective adjuvant to enhance immunogenicity of vaccine
antigens (Gonzalez-Aseguinolaza et al. 2002; Fujii et al. 2003; Silk
et al. 2004; Kopecky-Bromberg et al. 2009; Singh et al. 2014;
Venkataswamy et al. 2014). Semisynthetic carbohydrate-lipid vac-
cine conjugating Streptococcus pneumoniae capsule polysaccharides
to the αGalCer significantly protects against S. pneumoniae infection
in mice by promoting germinal center formation, carbohydrate-
specific long-lived memory B cells and high-affinity IgG antibodies
(Cavallari et al. 2014). Treatment with αGalCer to mice infected
with Mtb reduced the bacterial burden in the lungs, diminished tis-
sue injury and prolonged survival of mice following inoculation
with virulent Mtb (Chackerian et al. 2002). Moreover, glycosphin-
golipids, found in a handful of bacterial membranes, play a critical
role in initiating signaling cascades that facilitate survival of intes-
tinal symbiotic bacteria (An et al. 2011). An intestinal microbe,
Bacteroides fragilis, uses glycosphingolipids to modulate the homeo-
stasis of host iNKT cells. These bacterial glycosphingolipids nega-
tively regulate iNKT-cell proliferation in the colon during neonatal
development and protect the host against iNKT-cell-mediated colitis
challenges in a CD1d-dependent manner (An et al. 2014) (Table I).
In addition, mycobacterial lipids are processed and presented in
APCs to human T cells by CD1 molecules, leading to IFN-γ produc-
tion (Batuwangala et al. 2004; Kasmar et al. 2009; Kawasaki et al.
2014). It is noteworthy that in these studies, sialic acid-binding
immunoglobulin-type lectins (Siglecs) were shown to target the
mycobacterial antigens to APCs inducing robust T-cell activation.
Zwitterionic polysaccharides
The majority of bacterial polysaccharides are composed of neutral or
negatively charged monosaccharides. Studies of polysaccharides from
a variety of microbial species have shown their failure to activate
T cells and induce B cell isotype switching (Ovodov 2006; Avci et al.
2010, 2013). While mechanisms of endosomal oxidative processing
of these polysaccharides have been shown (Duan et al. 2008), the
1033
processed, non-zwitterionic oligosaccharides fail to bind MHCII, a
requirement for antigen presentation and T-cell activation. Due to
the inability of non-zwitterionic polysaccharides (ZPSs) to bind
MHCII, these polysaccharides are considered T-cell-independent
antigens (Barrett 1985; Avci et al. 2010, 2013).
A unique group of microbial polysaccharides have been recog-
nized that are comprised of alternating positive and negative charged
monosaccharides. These ZPSs have been identified in certain patho-
genic strains of Staphylococcus aureus type 5 and type 8 polysacchar-
ides (CP5 and CP8), S. pneumoniae type 1 polysaccharide (Sp1) and
B. fragilis polysaccharide A (PSA) (Pantosti et al. 1991; Tzianabos
et al. 1992; Kalka-Moll et al. 2001; Tzianabos et al. 2001; Avci et al.
2010, 2013). The most widely studied ZPS is PSA of Gram-negative
commensal B. fragilis. Unlike non-ZPSs, ZPSs, after processing by
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APCs, can associate with MHCII for surface presentation and
T-cell activation for a robust and carbohydrate-specific response
(Cobb et al. 2004; Duan et al. 2008; Groneck et al. 2009; Avci
et al. 2010, 2013).
The immune regulation by ZPSs
Numerous studies have demonstrated the roles of PSA in pathogen-
esis as well as the significance of PSA as an immunomodulatory mol-
ecule (Kalka-Moll et al. 2002; Cobb and Kasper 2005b; Mazmanian
and Kasper 2006; Stephen et al. 2010; Surana and Kasper 2012).
Early studies performed in rodent models reported that PSA con-
ferred protection against intra-abdominal abscess formation and
prevented Th1-mediated fibrosis, suggesting a immune regulatory
role of PSA (Stingele et al. 2004; Ruiz-Perez et al. 2005). In the inves-
tigations of the symbiotic relationship between host and intestinal
commensal bacteria, PSA has been found to stimulate a subset of T
cells with anti-inflammatory function. PSA is able to reduce inflam-
mation and down-regulate inflammatory bowel disease (Mazmanian
et al. 2005, 2008; Round and Mazmanian 2010) by inducing CD4+
T cells producing anti-inflammatory cytokine IL-10 (Mazmanian
et al. 2008). Similarly, exposure to PSA or adoptive transfer of PSA
responsive CD4+ T cells significantly prevents the onset of experi-
mental asthma in an IL-10-dependent fashion (Johnson et al. 2015a).
PSA was also shown to confer protection in the experimental
autoimmune encephalomyelitis model of multiple sclerosis that is
associated with enhanced numbers of Foxp3(+)T(reg) cells (Ochoa-
Reparaz et al. 2010; Dasgupta et al. 2014). In addition, PSA induces
differentiation of human peripheral CD4+ T cells into regulatory
T cells producing IL-10 that is required to exhibit nonspecific
bystander immunosuppression (Kreisman and Cobb 2011). Chemical
conversion to remove charged monosaccharides from ZPSs, altering
the zwitterionic state, eliminated T-cell stimulation and the protective
ability of the polysaccharides (Tzianabos et al. 1993; Tzianabos et al.
2000). Another recent study extended the characterization of PSA
responsive T cells (Johnson et al. 2015b). Immunization with PSA
induced a population of effector/memory CD4+ T cells producing
IL-10. By using next-generation sequencing of the αβTCR of PSA-
induced CD4+ T cells, the researchers demonstrated a lack of specific
variable β and joining region use. Moreover, PSA-induced T cells
have significantly higher proportions of zwitterionic amino acid
sequences containing both acidic (negatively charged) and basic
(positively charged) residues in their TCR β CDR3 region. It is
noteworthy that PSA is a ZPS, implying potential electrostatic
interactions between the PSA epitope and the CDR3 loop sequences
of the TCR of PSA-specific T cells (Johnson et al. 2015b). PSA
is also known to stimulate CD4+ helper 1 T cells (Th1 cells)
1034
producing IFN-γ, which is dependent on both Toll-like receptor 2
and antigen presentation (Wang et al. 2006). This observation
indicates a critical role for PSA in coordinating innate and adap-
tive immune responses. In addition, colonization of animals with
B. fragilis corrected the Th1/Th2 imbalances in germ-free mice
through PSA activation of CD4+ T cells to secrete IFN-γ (Mazmanian
et al. 2005). These studies not only show the significance of PSA as a
zwitterionic carbohydrate T-cell antigen, but also provide evidence of
a single molecule from a human intestinal symbiont affecting host
immune system maturation.
Two other ZPSs, CP5 and CP8 from S. aureus are reported to
facilitate intra-abdominal abscess formation in animal models, which
is associated with activated CD4+ T-cell responses induced by CP5
and CP8 (Tzianabos et al. 2001). Zwitterionic S. aureus capsular poly-
saccharides activate T cells to produce IFN-γ in a MHCII-dependent
manner. The presence of IFN-γ at the S. aureus infection site regulates
neutrophil recruitment in vivo (McLoughlin et al. 2008). Type I
pneumococcal polysaccharide (Sp1) also has been shown to activate
T cells in vivo and in vitro. APCs internalize and process Sp1 through
a nitric oxide-dependent mechanism. Processed Sp1 is presented asso-
ciated with MHCII to CD4+ T cells (Velez et al. 2009).
Processing and presentation of ZPSs
To date there is little known about the processing and presentation
of ZPSs as antigens to CD4+ T cells at the structural level. It is
known that APCs are required as well as MHCII molecules.
However, secondary and higher order structures of these polysac-
charides have been studied significantly less due to the flexibility of
their glycosidic bonds and the difficulty of attributing fixed struc-
tures (Cobb et al. 2004). Therefore, few ZPS molecules have identi-
fied secondary structures. Additionally, interactions between protein
complexes required for presentation are poorly studied and do not
have a defined structure. This section will focus on the signaling
pathways involved in presentation, MHCII binding of ZPSs and the
future directions of this growing field of study.
ZPSs could activate T cells through entry into an APC utilizing the
same endocytic pathway as protein antigens (Cobb et al. 2004). Once
the ZPS is taken into the endocytic pathway by APCs, an oxidative
burst occurs in which nitric oxide is produced (Cobb et al. 2004). This
step is crucial as it is responsible for processing the ZPS into a lower
molecular weight polysaccharide. ZPS containing endosomes then fuse
with the lysosomes. The MHCII proteins, HLA-DR, HLA-DM and li
(HLA-DR bound invariant chain), are assembled in the ER, trafficked
through the Golgi, and budded into exocytic vesicles (Watts 1997).
The endolysosomes fuse with these exocytic vesicles. This step forms
the MHCII compartment (MIIC), which also carries HLA-DR, HLA-
DM, LAMP-1 and the antigen (Cobb et al. 2004). Acidification acti-
vates proteases within the MIIC, which processes the ZPS and cleaves
the li leaving the CLIP peptide (cleaved form of li) in the binding
groove of MHCII (Shi et al. 1992, 1999). The acidic environment
allows HLA-DM to catalyze the exchange of CLIP for antigenic pep-
tides (Busch et al. 1998). Finally, the processed antigens are loaded
onto HLA-DR by HLA-DM and shuttled to the cell surface to be
recognized by αβTCRs. ZPS molecules then generate an immune
response through APC/T-cell recognition (Cobb et al. 2004) (Figure 3).
Mechanism of interactions between ZPSs and MHCIIs
The discovery that the endocytic pathway was utilized not only in
protein antigen presentation but also for glycoantigen presentation
caused a major shift in the known paradigm of antigen processing
L Sun et al.
and presentation. However, the mechanism of how antigens bind to
MHCII was still largely unknown. It is widely accepted that process-
ing yields a low molecular weight polysaccharide (about 10 kDa)
and that cleavage into smaller fragments was done through nitric
oxide (Cobb et al. 2004; Kreisman et al. 2007; Cobb and Kasper
2008). ZPS molecules need to be long enough to maintain a helical
conformation, but also small enough to bind MHCII. Using circular
dichroism, it was shown that the presence of helical structures is dir-
ectly related to MHCII binding (Kreisman et al. 2007). Additionally,
cleavage of li, in the endocytic pathway described above, is critical
for MHCII binding, as it makes the binding groove on MHCII avail-
able for the antigen (Cobb et al. 2004).
In 2008, work was published using PSA to model the interactions
between a ZPS and MHCII. This rigorous study revealed many
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insights about the characteristics of glycoantigen binding to HLA-
DR. First, the interactions between the two are at a 1:1 ratio and
show selectivity between DR1, DR2 and DR4. It was also shown that
binding is sensitive to ionic strength and pH (Cobb and Kasper
2008). Additionally, using competitive binding experiments, it was
shown that PSA, peptides and superantigens compete with each other,
which suggests that the binding domains may overlap (Cobb and
Kasper 2008). It was previously shown that HLA-DM plays a role in
PSA presentation (Cobb et al. 2004). It was also found that HLA-
DM increases binding rate, thus making it critical for cell surface
presentation and T-cell activation (Cobb and Kasper 2008). Lastly, it
was shown that the loss of the alternating charge motif, which is
characteristic of ZPS, results in the ablation of in vitro and in vivo
MHCII binding. However, this loss does not affect uptake into an
APC, vesicular trafficking or processing (Cobb and Kasper 2008).
This work was important for not only understanding MHCII
binding of ZPSs, but it was also the first characterization of carbo-
hydrate antigen presentation by MHCII proteins. Additionally, it
demonstrated the importance of acidic pH, self-peptide release and
catalysis by HLA-DM in achieving cell surface presentation and
T-cell recognition. While the field has come a long way in under-
standing the processing and presentation of these glycoantigens, the
structural aspect is still lacking. To date there have been a number
of ZPS structures solved, but there are many more to be character-
ized. Additionally, the structural relationships between the MHCIIs
and ZPSs need to be identified and understood.
Carbohydrate-based glycoconjugate vaccines
Carbohydrates are widely found on the surface of bacteria, fungi
and viruses. Naturally, targeting glycans of pathogens as vaccine
components has attracted considerable attention in the past decades.
A major breakthrough in vaccine development has been in glycocon-
jugate vaccines in which the carbohydrates are conjugated to carrier
proteins to evoke strong protective antibody responses against the
pathogens associated with the carbohydrate. Most carbohydrate-
based vaccines focus on eliciting humoral immunity to produce pro-
tective antibodies, which are addressed elsewhere (Li and Wang
2015; Vella and Pace 2015; Zimmermann and Lepenies 2015). The
glycoconjugates that induce T-cell responses to help B cell producing
protective antibodies are discussed in the following section.
Glycoconjugate vaccines eliciting T-cell-dependent
immune protection
The CPSs of major human pathogens, including S. pneumoniae,
Neisseria meningitidis and Haemophilus influenzae, significantly
Carbohydrates as T-cell antigens 1035

Extracellular glycoantigens
T-Cell Activation via
αβTCR Binding ZPS
+-+ -
Glycoconjugate

Membrane Fusion

End
and Presentation Processed

ocy
glycoantigens

to s
ROS/RNS/

is
MIIC Protease

Fusion
LAMP-1 +-+ -

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CLIP

Acidification Exocytic
Vesicle

Golgi
HLA-DM
Antigen Presenting Cell
Endoplasmic li
Reticulum HLA-DR

Fig. 3. Processing and presentation of glycoantigens for T-cell activation. Extracellular glycoantigen is endocytosed by the APC and processed into low molecu-
lar weight oligosaccharides or glycanp-peptide (processed glycan-peptide) by ROS, RNS and proteases. The processed glycoantigen containing endolysosome
fuses with the exocytic vesicle to form the MIIC vesicle for interaction with MHCII molecules HLA-DR, HLA-DM. After removal of CLIP and subsequent loading of
the antigen into HLA-DR by HLA-DM, glycoantigens are presented on the surface of the APC for recognition by the TCR. This figure is available in black and
white in print and in color at Glycobiology online.

contribute to their virulence mechanisms. CPSs support immune
evasion through increasing bacterial attachment to the host, inhibiting
complement activation and preventing phagocytosis (AlonsoDeVelasco
et al. 1995; Casadevall and Pirofski 2009). In addition, several bac-
terial pathogens can subvert immune response by displaying sialic
acid-based ligands that can be recognized by immunoreceptor
tyrosine-based inhibitory motifs-bearing Siglecs (Macauley et al.
2014). The surface localization and unique structures of CPSs make
them ideal candidates for vaccines designed to elicit a carbohydrate-
specific immune response to protect against these bacteria.
Most microbial polysaccharides, however, are poorly immuno-
genic. Pure, non-zwitterionic CPSs are T-cell-independent antigens and
cannot trigger T cell help to induce antibody class switching, affinity
maturation or create memory B and T cells (Coutinho and Möller
1973; Barrett 1985; Weintraub 2003). Pure polysaccharide vaccines
are used today and have been somewhat effective against a few sero-
types of S. pneumoniae, Salmonella typhi and Neisseria meningitides.
These vaccines, while having some success in adults, have been unsuc-
cessful in providing protection in infants, the elderly and immunocom-
promised individuals (Jones 2005; Avci et al. 2010, 2013).
Eliciting a T-cell-dependent immune response against CPSs can
be accomplished by coupling CPSs to a carrier protein to create a
glycoconjugate vaccine (Schneerson et al. 1980; Jennings and
Lugowski 1981; Beuvery et al. 1982). Immunizations with glycocon-
jugate vaccines such as Prevnar 13®, Menhibrix® and Pentacel®
have been effective at inducing a long-lasting protective capability
against encapsulated pathogens. Glycoconjugate vaccines induce a
CPS-specific adaptive immune response, IgM to IgG class switching,
B and T-cell memory, and significantly higher protection compared to
pure polysaccharide vaccines (Wessels et al. 1993; Guttormsen et al.
1998). Glycoconjugates containing CPSs from S. pneumoniae,
H. influenzae and N. meningitidis have been highly successful in
preventing these infections (Weintraub 2003; Jones 2005; Trotter et al.
2008; Avci et al. 2013). Although success has been observed, problems
with current glycoconjugate vaccines still persist. Glycoconjugates have
been poorly immunogenic in individuals with HIV and the elderly
(Pirofski 2001; Avci 2013). These problems illustrate the need for
efforts to enhance vaccine efficacy by developing structurally defined
glycoconjugate vaccines based on the mechanisms of their activation of
the immune system.
Glycoconjugate vaccine induces carbohydrate-specific
T-cell response
The traditional hypothesis for glycoconjugate vaccine activation of an
adaptive immune response proposes that helper CD4+ T cells recog-
nize a carrier protein-derived peptide (Mitchison 2004). According to
this hypothesis, the glycoconjugate binds to the surface of a B cell
that stimulates the production of antibodies to the CPS portion of the
glycoconjugate. The B cell processes the protein portion of the glyco-
conjugate and presents a peptide from the carrier protein in the con-
text of MHCII to the TCR of CD4+ T cells. Activation of the T cell
results in production of cytokine IL-4, which induces maturation of
the B cell and consequent production of antibodies specific for CPS,
immunoglobulin class switching from IgM to high-affinity IgG, and
production of memory B and T cells (Guttormsen et al. 1998; Avci
1036
et al. 2010). This hypothesis was originally based on the assumption
that only protein antigens can be presented to and recognized by
T cells. The traditional mechanism assumes that the strong covalent
link between the CPS and carrier proteins is broken during endoso-
mal degradation of the glycoconjugate and does not consider whether
T cells can recognize non-zwitterionic carbohydrates linked to a
peptide whose binding to MHCII allows presentation of the carbohy-
drate by the APC to T cells (Kalka-Moll et al. 2002; Trotter et al.
2008; Avci 2013; Avci et al. 2013).
The mechanisms of glycoconjugate vaccine activation of an
adaptive immune response have recently been uncovered that shifts
the paradigm of peptide-centric T-cell recognition (Avci et al. 2011).
One of the most comprehensive study of glycoconjugate vaccine
activation mechanisms to date, Avci et al. demonstrated that, upon
uptake by APCs, glycoconjugate vaccines are involved in a depoly-
merization reaction that processes the glycoconjugate into a smaller
molecular weight processed glycan-peptide conjugate (glycanp-
peptide) (Avci et al. 2011). This glycanp-peptide is then displayed
on the surface of the APC in association with MHCII. MHCII-
deficient APCs showed no processed glycanp-peptide presentation.
These findings suggest that the carrier protein-derived peptide por-
tion of the glycoconjugate binds to MHCII, leaving the hydrophilic
glycan exposed on the APC surface to be recognized by the TCR of
CD4+ T cells (Figure 3). This study also shows that glycoconjugate
immunization induces a subset of CD4+ T cells, termed Tcarbs, that
recognize the carbohydrate portion of the glycoconjugate. A series
of immunization experiments revealed that stimulation of Tcarbs by
their carbohydrate epitopes recruits T-cell help for B cell IgM to IgG
class switching (Avci et al. 2011).
Through immunization, in vitro CD4+ T-cell expansion and lim-
iting dilution experiments, two Tcarb clones were isolated. One
clone recognized GBSIII epitope in the context of the I-Ed and the
other with the I-Ad. The CD4+ T-cell clones secreted both IL-2 and
IL-4, but not IFN-γ, in the presence of glycoconjugates other than
the proteins alone (Avci et al. 2011, 2012). The knowledge gained
from these mechanistic studies was used to synthesize a new glyco-
conjugate vaccine enriching the glycan epitope to create a much
more robust carbohydrate-specific IgG response and protein from
GBS challenge (Avci et al. 2011, 2012).
Two other reports along with our mechanistic study on GBSIII
glycoconjugates suggest the generalizability of Tcarb-mediated
immune responses induced by glycoconjugate vaccines. In one report
based on confocal microscopy images, the carbohydrate component
of a pneumococcal glycoconjugate vaccine was shown to localize on
the APC surface (Lai and Schreiber 2009). The microscopy images
depicting APC surface co-localization of MHCII and the glycan por-
tion of the conjugate vaccine suggest that the glycan portion of the
pneumococcal conjugate is presented on the APC surface in the con-
text of MHCII molecule. In the second study, immunization with
meningococcal polysaccharide-tetanus toxoid conjugate was shown
to induce carbohydrate reactive T cells (Muthukkumar and Stein
2004). Here, the glycoconjugate and not the carrier protein specific-
ally stimulated some of the T-cell clones generated from glycoconju-
gate vaccine immunization.
Conjugation technologies
Current conjugation techniques involve the activation of proteins or
polysaccharides to create reactive functional groups such as alde-
hydes, esters, thiols or hydrazides (Costantino et al. 2011; Avci et al.
2012; Avci 2013). These strategies often lead to heterogeneous
L Sun et al.
structures of various molecular weights and linkages that form
matrix-like structures (Bardotti et al. 2008). A current strategy-
protein glycan coupling technology utilizes the oligosyltransferase
enzyme pglB of Campylobacter jejuni to enzymatically transfer a
polysaccharide to a recombinantly expressed carrier protein. With
this technology, glycans, coupling enzymes and proteins are synthe-
sized in the E. coli glycoconjugate factory (Kowarik et al. 2006;
Ihssen et al. 2010). Studies are underway that could extend the use-
fulness of this method to generate O-linked glycoconjugates through
an enzymatic coupling process (Faridmoayer et al. 2007; Naegeli
and Aebi 2015; Ravenscroft et al. 2016). The major drawbacks to
this method are the limited oligosaccharides that can be used as a
substrate for pglB and the inability to conjugate the polysaccharide
to peptides or polypeptides (Terra et al. 2012; Avci 2013; Berti and
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Adamo 2013). Recently, researchers have demonstrated the conjuga-
tion of polysaccharide to protein via copper-free click chemistry
reactions, conferring the ability to perform site-selective conjugation
of the oligosaccharides (Agard et al. 2006; Nilo et al. 2014;
Stefanetti, Hu, et al. 2015; Stefanetti, Saul, et al. 2015). This method
provides a structurally defined glycoconjugate that can be easily
characterized by analytical methods. A few drawbacks to this
approach are multiple steps, low yield and potential unwanted
immunological responses (Nilo et al. 2014). Novel approaches for
glycoconjugation are necessary for creating well-defined glycoconju-
gate vaccines that lack heterogeneity and batch-to-batch variability.
These strategies, along with the mechanistic insights of glycoconju-
gate vaccine activation of adaptive immunity, provide an outline for
the development of highly immunogenic vaccines against pathogenic
bacterial, parasitic and viral surface glycans.
Conclusion
The discovery of carbohydrate-specific T cells has brought a
new perspective to the concept of immune responses against carbo-
hydrate antigens formerly considered as T-cell-independent antigens.
A number of studies using glycosylated immunogenic peptides and
natural glycoproteins demonstrated that, as one of the most ubiqui-
tous modifications, glycosylation modulates T-cell immune responses
through either participating in the TCR epitopes or by T-cell recogni-
tion. Glycosylation influences T-cell recognition in multiple ways,
such as the impact of glycan’s structure, charge-state, complexity and
size, or the site of glycosylation on the peptide to which the glycan is
bound.
Carbohydrate recognition by T cells has important implications
in health and disease. Glycosylation of host protein type II collagen
correlates with the autoimmune disease collagen-induced rheuma-
toid arthritis. The glycosylation of bacterial proteins (i.e., Apa and
LprG) is important for their immunogenicity and for T-cell antigeni-
city during infection. A major roadblock in cancer immunotherapies
is to target cancer cells without harming healthy cells. As tumor
markers, TACA antigen-based vaccines could be effective and are
promising in inducing carbohydrate-specific antibodies and T-cell
responses leading to cancer prevention and/or therapy. Endogenous
and microbial glycolipids are important immunogenic antigens that
induce iNKT-cell responses when presented by the MHCI-like mol-
ecule CD1d with implications in cancers, infectious diseases and
immune system development. The ZPS of Gram-negative commensal
B. fragilis, PSA, has been extensively studied for its immunoregula-
tory function with implications in inflammatory and autoimmune
diseases as well as healthy immune system maturation. Unlike ZPSs,
most other bacterial CPSs are either negatively charged or neutral
Carbohydrates as T-cell antigens
and therefore need to be bound to carrier proteins to induce T-cell
responses. Glycoconjugate vaccines composed of bacterial CPSs
covalently conjugated to carrier proteins are of great importance in
eliciting strong protective humoral immune responses against patho-
genic bacteria. Current glycoconjugate vaccine technology is based
on trial and error and does not utilize carbohydrate presentation by
APCs as a screening criterion for immunogens. Demonstrating that
Tcarb stimulation is essential for carbohydrate-specific adaptive
immune responses may provide the immunological knowledge to
design and synthesize a new generation of vaccines that are substan-
tially more immunogenic than traditional vaccines. This task utilizes
the structural identification of glycans presented by the MHCII
molecules on the surface of the APCs as T-cell epitopes and charac-
terization of these key interactions between the TCR and MHCII
bound glycan epitopes.
Glycoimmunology is an emerging and rapidly growing field
(Baum and Crocker 2009). The continuous advancement of analyt-
ical tools enables the detection and characterization of carbohydrate
moieties in their natural environments and concentrations. We are
now better equipped to demystify key molecular and cellular inter-
actions between carbohydrate antigens and molecules and cells of
the immune system. Current and future research at the interface of
glycosciences and immunology will play a critical role in addressing
fundamental questions about host-pathogen interactions and explor-
ing treatment of and protection from infectious diseases, cancers
and autoimmune diseases.
Funding
This work was supported by funding from the National Institute of Health
grants: AI-115451-01, 1R01AI123383-01A1.
Acknowledgements
We thank Ms. Karen Howard for her excellent editorial assistance.
Conflict of interest statement
None declared.
Abbreviations
α-GalCer, alpha-galactosylceramide; CDR3, complementarity-determining
region 3; CTL, cytotoxic T lymphocyte; DCs, dendritic cells; IFN-γ, interferon-
γ; IL-4, interleukin-4 iNKT, invariant natural-killer T; LprG, lipoglycoprotein;
MHC, major histocompatibility complex; MIIC, MHCII compartment Mtb,
Mycobacterium tuberculosis; NKT, natural-killer T; PLA, phospholipase A2;
PSA, polysaccharide A; Siglecs, sialic acid-binding immunoglobulin-type lec-
tins; Sp1, S. pneumonia type 1 polysaccharide; TACAs, tumor-associated
carbohydrate antigens; TCRs, T-cell receptors; Th1 cell, T helper 1 cell;
TNF, tumor necrosis factor
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