Antigen Processing and Presentation

1. 501732
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2. Introduction
 A century ago the composite word ‘antigen’ referred to ‘that which
generates antibodies’. This tautology developed before the concept of a
‘receptor’, before the definition of macromolecular proteins, and before
it was known that antibodies actually bind to antigens. The antigen–
antibody tautology remains central to our concepts of immunity and to
the tautology of ‘self ’ and ‘non-self ’.
 At a molecular level, an ‘antigen’ can be defined today as any molecule
recognized by (i.e., binding specifically to) the antigen-binding domain of
an ‘antigen receptor’ (antibody or T-cell receptor – TCR).
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3.  The antigen bound by a particular antigen receptor is sometimes called
its cognate antigen. The concept of a cognate antigen is useful when
identifying the molecular rules governing antigen–antigen receptor
interactions, for predicting antigens from sequence and structural
information, for designing subunit and genetic vaccines, and for
understanding what the immunologist means by such terms as ‘self’ and
‘nonself’.
 In this lecture we shall focus on the antigens regarding their processing
and presentation by specialized cells in order for the immune system to
be activated or ‘sensitized’ so that the body can counter the attack.
 In order for us to elaborate antigen processing and presentation we
should focalize on the antigen recognition process later on.
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4. concepts
 Endogenous Ags: antigens synthesized within cells, including self and non-self
protein----class Ⅰ MHC molecules.
 Exogenous Ags: antigens comes from outside the cells, including self and nonself protein----class Ⅱ MHC molecules.
 Antigen processing: the conversion of native proteins to peptides which can
combine with MHC molecules.
 Antigen presentation: the course of formation and display of peptide-MHC
complexes on the surface of APCs and the course of peptide-MHC complexes
recognition by T cells.
 Ag capturing ----Endocytosis (internalization)
Phagocytosis, Pinocytosis, Receptor-mediated endocytosis
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6. Antigens and Antigen Presenting
Cells (APCs)
 Before we can explain the mechanisms of antigen processing, presentation and recognition by the
immune system we should focus on the antigen itself.
 Antigens could be peptides, proteins, nucleic acids, polysaccharides, lipids, or small chemicals.
That does not evoke any problem if the cell acting is a B cell because a humoral immune
response will take place ; nonetheless when T cells are acting this will be a problem given that
most of them recognize peptide antigens which will elicit cell-mediated immune response.
 T cells are specific for amino acid sequences of peptides while B cells recognize conformational
determinants of antigens, even proteins, in their native tertiary (folded) configuration. The antigen
receptors of T cells recognize very few residues even within a single peptide, and different T cells
can distinguish peptides that differ even at single amino acid residues.
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7.  Antigen Presenting Cells (APCs/accessory cell) are cells that display foreign antigen complexes
with major histocompatibility complex (MHC) on their surfaces. T-cells may recognize these
complexes using their T-cell receptors (TCRs). These cells process antigens and present them to
T-cells.
 T cells cannot recognise, and therefore react to, 'free' antigen. T cells can only 'see' antigen that
has been processed and presented by cells via an MHC molecule. Most cells in the body can
present antigen to CD8+ T cells via MHC class I molecules and, thus, act as "APCs"; however, the
term is often limited to those specialized cells that can prime T cells (i.e., activate a T cell that has
not been exposed to antigen, naive T cell). These cells, in general, express MHC class II as well
as MHC class I molecules, and can stimulate CD4+ ("helper") cells as well as CD8+ ("cytotoxic") T
cells, respectively.
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8.  T cells recognize and respond to foreign peptide
antigens only when the antigens are attached to the
surfaces of APCs, whereas B cells and secreted
antibodies bind soluble antigens in body fluids as well
as exposed cell surface antigens. This is because T cells
can recognize only peptides bound to and displayed by
MHC molecules, and MHC molecules are integral
membrane proteins expressed on APCs.
 T cells from anyone individual recognize foreign
peptide antigens only when these peptides are bound to
and displayed by the MHC molecules of that
individual. This feature of antigen recognition by T
cells, called self MHC restriction, can be demonstrated
in experimental situations in which T lymphocytes
from one individual are mixed with APCs from
another individual.
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9.  Dendritic cells are the most effective APCs for activating
naive CD4+ and CD8+ T cells, and therefore for
initiating T cell responses. Macrophages present antigens
to differentiated (effector) CD4+T cells in the effector
phase of cell-mediated immunity, and B lymphocytes
present antigens to helper T cells during humoral
immune responses. Dendritic cells, macrophages, and B
lymphocytes express class II MHC molecules and
costimulators, and are, therefore, capable of activating
CD4+ T lymphocytes. For this reason, these three cell
types have been called professional APCs; however, this
term is sometimes used to refer only to dendritic cells
because this is the only cell type whose principal
function is to capture and present antigens, and the only
APC capable of initiating T cell responses.
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10. PHAGOCYTES
Mononuclear cells
Macrophage
Press
Dendritic cells
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11. Types of Dendritic Cells
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12. Fibroblast
 A non-professional APC does not constitutively express
the Major Histocompatibility Complex class II (MHC
class II) proteins required for interaction with naive T
cells; these are expressed only upon stimulation of the
non-professional APC by certain cytokines such as IFN- γ.
Non-professional APCs include:
o Fibroblasts (skin)
o Thymic epithelial cells
o Thyroid epithelial cells
Glial (Neuroglial) Cells
o Glial cells (brain)
o Pancreatic beta cells
o Vascular endothelial cells
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15. 15

16. Major Histocompatibility Complex (MHC)
Two groups of MHC genes: Structurally and functionally distinct
1. Class I
recognition by CD8+ T cells
2. Class II
recognition by CD4+ T cells
• HLA molecules are responsible for the compatibility of the tissues of genetically different
individuals and for the rejection of transplant
• MHC genes are codominantly expressed in each individual
• Monozygotic twins have the same histocompatibility molecules on their cells
• MHC genes are the most polymorphic genes present in the genome! (Up to 250 alleles
identified for some loci)
MHC expression
 Class I On all nucleated cells (no MHC on red blood cells, weak expression on cells in brain)
Class II Found on antigen presenting cells
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17. MHC class I molecule
1. Heavy chain
α1, α2 domain:
polymorphic sites α3
domain: binding of
CD8
2. β-2 microglobulin
3. Peptide
Ig Domain
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18. MHC class II molecule
1. α chain
α1: polymorphic sites
α2: binding of CD4
2. β chain
β1: polymorphic sites
β2: binding of CD4
3. Peptide
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19. Different MHC molecules have different
groove structure.
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20. Peptides in MHC Grooves
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21. Elaboration of MHC restriction
Because alloreactive T cells distinguish between self and nonself MHC
molecules, it is tempting to think that MHC restriction mediates
self/nonself discrimination. However, it is clear that allospecific antibodies
also distinguish self from nonself MHC molecules. MHC restriction may
function in two opposing ways.
 First, antigen processing increases the complexity of pathogen antigens by
exposing epitopes not available on the surface of pathogens. MHC
molecules are ‘merely’ the mechanism for presenting processed epitopes
to T cells. MHC polymorphism further increases the size of the species’
antigenic universe.
 Second, the requirement that T cells do not respond unless activated by
co-stimulation from the APC is enforced by anchoring MHC molecules
on the APC. This second mechanism thus minimizes autoimmunity. In
this view, the experimental observations of genetic MHC restriction and
alloreactivity are by-products of the polymorphism of MHC molecules.
 The agents that raise
 The agents that raise
endosomal and
endosomal and
lysosomal pH, or
lysosomal pH, or
directly inhibit
directly inhibit
endosomal proteases,
endosomal proteases,
block class II-restricted
block class II-restricted
but not class I-restricted
but not class I-restricted
antigen presentation,
antigen presentation,
whereas inhibitors of
whereas inhibitors of
ubiquitination or
ubiquitination or
proteasomes selectively
proteasomes selectively
block class I-restricted
block class I-restricted
antigen presentation.
antigen presentation.
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23. Peptide binding by MHC class I and II
molecules. Class I molecules are usually closed
at both ends. The peptide termini must interact
with terminal sockets. Peptides that are too long
must be cleaved (arrows) prior to entry into the
binding site. The clefts of class II molecules are
open at the ends, permitting the binding of long
peptides.
MHC restriction carries out two critical functions. First, by
presenting processed peptides derived from within proteins and
pathogens, MHC molecules sample a broader antigenic
landscape than antibodies, whose epitopes are surface oriented.
Second, naïve T cells respond to cognate epitopes only when
presented by an activated APC (B) but not when the APC is
resting (A). Experimentally observed MHC restriction results
when an activated APC cannot present the proper
MHC/peptide pair (C).
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24. T cells do not recognize native antigens
Y
Y
Y
Y
Y
Y
YY
Cross-linking of
surface membrane Ig
YYY Y
Y Y Y Y
B
B B
B B BB
B B
Proliferation and
antibody production
T
T
No proliferation
No cytokine release
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Y
Y

25. Antigens must be processed in order
to be recognised by T cells
T
Y
Soluble
native Ag
Cell surface
native Ag
Soluble
peptides
of Ag
APC
No T cell
response
No T cell
response
Cell surface peptides of
Ag presented by cells that
express MHC molecules
Cell surface
peptides
of Ag
ANTIGEN
PROCESSING
No T cell
response
No T cell
response
T cell
response
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26. Provision of additional stimuli to the T cell beyond
those initiated by recognition of peptide-MHC
complexes by the T cell antigen receptor
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28. Y
The site of pathogen replication or mechanism of antigen
uptake determines the antigen processing pathway used
Y
EXTRACELLULAR OR
ENDOSOMAL REPLICATION
Vesicular Compartment
Contiguous with extracellular fluid
Exogenous processing
(Streptococcal, tumor antigens)
INTRACELLULAR REPLICATION
Cytosolic compartment
Endogenous processing
(Viral, tumor antigens )
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31. 2. Foreign proteins or into the rough
1. Antigens are transportedself-proteins within the cytosol are broken down into fragments that are
endoplasmic reticulumaction of(Transporter which appears as a cylinder composed of a stacked array
antigens by the by TAP proteasomes
Associated with Antigen two outer rings, each ring being composed of seven subunits. Three of the
of two inner and Presentation). Interestingly,
the TAP1 and TAP2 genes are next to the genes proteolysis. A larger, 1500-kD proteasome is likely to be
seven subunits are the catalytic sites for
encoding LMP-2 and LMP-7 in the MHC, and the
most important for generating class I-binding peptides and is composed of the 700-kD structure
synthesis of the TAP protein is also stimulated by
plus
IFN-y. several additional subunits that regulate proteolytic activity. Two catalytic subunits present in
many 1500-kD proteasomes, called LMP-2 and LMP-7, are encoded by genes in the MHC, and
3. Antigens combine with MHC
are particularly
class I molecules. important for generating class I-binding peptides. Cytokine IFN-y treatment
3
2
increases production of LMP-2 and LMP-7.
MHC class I
4. The MHC class I/antigen
Protein
complex is transported to the
Golgi apparatus, packaged
into a vesicle, and
transported to the plasma
membrane.
5. Foreign antigens combined
with MHC class I molecules
stimulate cell destruction.
6. Self-antigens combined with
MHC class I molecules do
not stimulate cell destruction.
molecule
1
Protein
fragments
(antigens)
4
Membrane Lumen
5
Rough
endoplasmic
reticulum
Golgi
apparatus
Foreign
antigen
Self-antigen
6
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32. Antigen processing for MHC class I. Two chief pathways for antigen process
intersect within the cytosol. Most endogenous antigens are synthesized on cytosolic
ribosomes, processed by proteasomes, and enter the ER through the TAP
(Transporter associated protein)transporter. A minor set of antigens are processed
within the ER from proteins secreted into the ER. Professional antigen-presenting
cells transfer endocytosed antigens into the cytosol for processing.
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33. TAP (Transporter Associated with Antigen Presentation)
Transport associated protein -TAP is responsible
for the peptide transport from cytoplasm to ER.
•Proteins are degraded to peptide in proteasome.
•The peptides are picked up by TAP proteins
and transported from the cytosol into the RER
where they assemble with
–the transmembrane polypeptide and beta-2
microglobulin.
–this trimolecular complex then moves through
the Golgi apparatus and is inserted in the plasma
membrane
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35. The functions of class II MHC-associated invariant chains and HLA-OM. Class II molecules with bound
invariant chain, or CLIP, are transported into vesicles, where the CLIP is removed by the action of OM.
Antigenic peptides generated in the vesicles are then able to bind to the class II molecules. Another class
II-like protein, called HLA-DO, may regulate the OM-catalyzed removal of CLIP. CIIV. class II vesicle.
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36. 2. The unprocessed extracellular fragments to fuses with vesicles produced by the Golgi apparatus that
antigen is containing the processed antigen
1. 3.The vesiclebroken down into
antigen is ingested by
form processedand is within a
contain MHC class II molecules. proteins are
endocytosis antigens. Internalized A class II-rich subset of late endosomes that plays an important role in
vesicle. presentation. late endosomes and
degraded enzymatically inIn macrophages and human B cells, it is called the MHC class II compartment, or
antigen
lysosomes to generate peptidescells, are able to
MIIC. (In some mouse B that a similar organelle containing class II molecules has been identified and
Vesicle
bindnamed peptide-binding clefts of class IIhas a characteristic multilamellar appearance by electron microscopy.
to the the class II vesicle.) The MIIC MHC
containing
MHC class II
molecules. The degradationall the components required for peptide-class II association, including the enzymes
Importantly, it contains of protein antigens in
molecules
vesicles is an active process mediated by proteases
that degrade protein antigens, the class II molecules, the I i (or invariant chain-derived peptides), and a
that molecule called optima. leukocyte antigen DM (HLA-DM). Within the 2
have acidic pH human
MIIC, the I i dissociates from class II
MHC molecules by the combined action ofproteolytic 1
enzymes and the HLA-DM molecule, and antigenic
3
Vesicle
peptides are then able to bind to the available peptide-binding clefts of the class II molecules. Because the I i
containing
blocks access to the peptide-binding cleft of a class II MHC molecule, it must processed before complexes
be removed
4. The MHC class II/antigen complex
antigen
isof peptide and class plasma
transported to the II molecules can form. The same proteolytic enzymes, such as cathepsin S, that generate
Unprocessed
membrane.
4
antigen
peptides from internalized proteins also act on the I i, degrading it and leaving only a 24-amino acid remnant
5. The displayed MHC classinvariant chain peptide (CLIP).The processed antigen and the MHC class II
called class II-associated II/antigen
complex can stimulate immune cells.
molecule combine.
5
MHC class II
molecule
Processed
antigen
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37. Two pathways for loading antigens onto class II MHC molecules. Autophagy and endocytosis
transfer cytosolic and external antigens, respectively, into the endosomes. Nascent class II molecules
are chaperoned to the endosomes from the Golgi by the invariant chain. The DM molecules
catalyze the exchange of antigenic peptides for invariant chain. Mature class II molecules recycling
from the cell surface can acquire peptides in a DM-independent manner. Antigens binding initially
as polypeptides are trimmed into oligopeptides in the endosomes and at the surface.
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38. B lymphocyte as APC
Unlike DC and macrophages, B cells are not phagocytic or macropinocytic, and do not express multiple
types of antigen receptors. Their capacity to endocytose antigen is restricted almost exclusively to those
captured through their cell surface receptor, the BCR. While this makes B cells less efficient than other
APC at presenting most antigens, it also makes them the most focused. The BCR consists of an mIg, which
provides the antigen recognition component to the receptor, noncovalently associated to an Igα:Ig β
heterodimer. This dimer provides the signaling module to the receptor, as it contains an immunoreceptor
tyrosine-based activation (ITAM) motif required for signal transduction that upon antigen engagement
triggers internalization of the receptor–antigen complex and initiates a signaling cascade leading to B-cell
activation. Ig-mediated endocytosis allows B cells to concentrate in their endosomal compartments minute
amounts of antigen due to the high specificity of their mIg molecules. It is easy to imagine how this process
operates on soluble antigens, but less so for antigens associated with cellular membranes. However, it has
recently been demonstrated that B cells can indeed endocytose antigens ‘ripped’ from cell surfaces, so
BCR-mediated endocytosis can account for presentation of even cell-associated antigens without the need
to invoke a phagocytic mechanism for antigen capture
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39. Like all lymphocytes, B-lymphocytes circulate back and
forth between the blood and the lymphoid system of the
body. B-lymphocytes are able to capture and present
peptide epitopes from exogenous antigens to effector T4lymphocytes.
The MHC-II molecules bind peptide epitopes from
exogenous antigens and place them on the surface of the
B-lymphocytes. Here the MHC-II/peptide complexes
can be recognized by complementary shaped T-cell
receptors (TCRs) and CD4 molecules on an effector T4lymphocytes.
This interaction eventually triggers the effector T4lymphocyte to produce and secrete various cytokines that
enable that B-lymphocyte to proliferate and differentiate
into antibody-secreting plasma cells.
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40. Activated B cells as APC
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41. B Lymphocyte
T Lymphocyte
B Lymphocyte
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42. T Cell Surveillance for Foreign Antigens
 The class I and class II pathways of antigen presentation sample available proteins for display to T cells.
 Most of these proteins are self proteins. Foreign proteins are relatively rare; these may be derived from
infectious microbes, other foreign antigens that are introduced into the body, and tumors.
 T cells survey all the displayed peptides for the presence of these rare foreign peptides and respond to the
foreign antigens.
 Self peptides do not stimulate T cell responses, either because T cells with receptors for these peptides were
deleted during their maturation in the thymus or the cells have been rendered inactive by recognition of the
self antigen.
 MHC molecules sample both the extracellular space and the cytosol of nucleated cells, and this is important
because microbes may reside in both locations.
 Even though peptides derived from foreign (e.g., microbial) antigens may not be abundant, these foreign
antigens are recognized by the immune system because of the exquisite sensitivity of T cells.
 In addition, infectious microbes stimulate the expression of costimulators on APCs that enhance T cell
responses, thus ensuring that T cells will be activated when microbes are present.
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43. Nature of T Cell Responses
 The presentation of vesicular versus cytosolic proteins by the class II or class
I MHC pathways, respectively, determines which subsets of T cells will
respond to antigens found in these two pools of proteins.
 The unique specificity of T cells for cell-bound antigen is essential for the
functions of T lymphocytes, which are largely mediated by interactions
requiring direct cell-cell contact and by cytokines that act at short distances.
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44. Immunogenicity of Protein Antigens
MHC molecules determine the immunogenicity of protein antigens in two related ways:
 The epitopes of complex proteins that elicit the strongest T cell responses are the peptides that are
generated by proteolysis in APCs and bind most avidly to MHC molecules. If an individual is immunized
with a multideterminant protein antigen, in many instances the majority of the responding T cells are
specific for one or a few linear amino acid sequences of the antigen. These are called the
immunodominant epitopes or determinants. The proteases involved in antigen processing produce a
variety of peptides from natural proteins, and only some of these peptides possess the characteristics that
enable them to bind to the MHC molecules present in each individual.
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45.  The expression of particular class II MHC alleles in an individual determines the
ability of that individual to respond to particular antigens. The phenomenon of
genetically controlled immune responsiveness. We now know that the immune
response (lr) genes that control antibody responses are the class II MHC structural
genes. They influence immune responsiveness because various allelic class II MHC
molecules differ in their ability to bind different antigenic peptides and therefore to
stimulate specific helper T cells.
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46. Presentation Of Lipid Antigens By CD1 Molecules
Unconventional T Cells
Some of these T cells do not follow the rule of MHC-peptide
recognition.
 An exception to the rule that T cells can see only peptides is the recognition of
lipid and glycolipid antigens by a numerically rare population of T cells called NKT cells.
 These lymphocytes have many unusual properties, including the expression of
markers that are characteristic of both T cells and NK cells, and the limited
diversity of their antigen receptors. NK-T cells recognize lipids and glycolipids
displayed by the class I-like "non-classical" MHC molecule called CD I. There are
several CD1 proteins expressed in humans and mice.
 Although their intracellular traffic pathways differ in subtle ways, all the CDI
molecules bind and display lipids by a unique pathway.
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47. 1.
Newly synthesized CD1 molecules pick up cellular lipids and carry these to
the cell surface.
2.
From here, the CD1-lipid complexes are endocytosed into endosomes or
lysosomes, where lipids that have been ingested from the external
environment are captured and the new CD1-lipid complexes are returned to
the cell surface.
3.
Thus, CD1 molecules acquired endocytosed lipid antigens during recycling
and present these antigens without apparent processing. The NK-T cells that
recognize the lipid antigens may play a role in defense against microbes,
especially mycobacteria (which are rich in lipid components).
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48. T cell
bacteria
TCR
CD1
CD1
re-expression
internalization
Transportation
to cell surface
CD1
GPI
??
phagocytosis
Degradation
Vesicle maturation/
Fusion
bacterial glycolipid
+
CD1
ENDOSOMES/MIC
TGN
Glycosylation
CD1
HC
??
+
calnexin
Golgi
+
b2m
ER
endogenous glycosylphosphatidyl
inositol (GPI)
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49. γδ T cells
A minor T cell population in the peripheral blood and lymphoid organs of human
expresses an alternative TCR made up of γ and δ chains. The high number of
gamma/delta-expressing T cells also found in the epithelial lining layer (skin and guts)
suggests that they form a first line of defense against invading pathogens. It is thought
that they may form part of the early innate immune response to pathogens. Unlike αβ
T cells, T-cell antigen receptors composed of polypeptide chains (TCRs) can directly
recognize antigens in the form of intact proteins or non-peptide compounds. About 5
% of peripheral blood T cells bear TCRs, most of which recognize non-peptide phosp
horylated antigens.
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50. Unconventional T Cells
Some of these T cells do not follow the rule of MHC-peptide
recognition.
 NKT cells recognize CD1 presented Ag
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51. Intracellular trafficking of CD1a and CD1b
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53. References:
- Abbas A., A. Lichtman & S. Pillai, Cellular and
MolecularImmunology, Sixth edition, Saunders- Elsevier,
2007.
Rich R. R., Fleisher T. A., Shearer W. T., Schroeder Jr.
H. W., Frew A. J., and Weyand C. M., Clinical
Immunology: Principles and Practice, third edition, 2008
Websites
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