Immunology

Block 1 Review 1
Topic 3- Antigen Capture and Presentation to Lymphocytes
Questions to consider
1. How do the rare lymphocytes specific for any microbial antigen find that microbe, especially considering that microbes
may enter anywhere in the body?
2. How does the immune system produce the effector cells and molecules best able to eradicate a particular type of
infection, such as antibodies against extracellular microbes & CTLs to kill infected cells with microbes in their cytoplasm?
Answer: The answer to both questions is that the immune system has developed a highly specialized system for
capturing and displaying antigens to lymphocytes (-_-).
Initiation of adaptive Immune responses
First signal of activation is the recognition of antigen
B-lymphocytes (antibody receptors) recognize proteins, polysaccharides, lipids, nucleic acids, and small chemicals
(haptins) in soluble or cell surface-associated form.
Most T lymphocytes can see only peptide fragments of protein antigens, and only when specialized peptide display
molecules on host cells present these peptides.
Membrane bound antibodies do not require presenting. They phagocytose the antigen and present it to T cell
MHC restriction
**MHC restrictionThe majority of T lymphocytes recognize peptide antigens that are bound to
and displayed by major histocompatibility complex (MHC) molecules of antigen-presenting
cells (APCs).
MHC Molecule:
Genetic locus whose products function as the peptide display molecules of the immune system.
MHC genes are all on the same chromosome (See specifics below).
High degree of polymorphisms
Responsible for acceptance or rejection of a tissue graft or organ.
Compatibility is difficult to find because the MHC molecules vary between individuals.
**TCR has dual specificityrecognizes peptide antigen residues & polymorphic MHC molecule residues.
Peptides bind to MHC molecule pockets by anchor residues.
The capture and display of microbial antigens
Microbes enter through an epithelium captured by APCs resident in the
epithelium (In subepithelium, dendritic cell capture the antigen with their long
processes) When DC encounters with microbe, maturation of APC
occurstravels by lymph vessel to nearest lymph node present the antigen to
a nave t cell.
If antigens in the tissue reach the bloodstream, it will reach the spleen where it will
be captured by resident APC.
Antigen presentation
Antigens enter captured mainly by DCs peripheral lymphoid
organsimmune responses initiated.
DC most effective "professional" APCs to initiate clonal expansion and
effector cell differentiation.
DCs present protein antigens to Naive T lymphocytes that have CD28 receptor.
DCs are good at presenting antigen and providing co-stimulators B7-1 and B7-2 second signal.
Differentiated effector T cells need to see antigen again, it is presented by APCs such as Macrophages at the site of
infection, to activate the effector functions of T cells in humoral/cell-mediated immune responses.
TH1 CD 1 secretes IFN gamma cytokine that is going to activate the macrophagestheir activation leads to the
killing of those microbes.
Dendritic Cells (DC)
Immature dendritic cells reside in epithelia, such as the skin, and form a network of cells with interdigitating processes,
which is going to ensure that they will capture any microbe that may reside in your body.
All of the interfaces between the body and the external environment are lined by epithelia (physical barrier).

Immunology Block 1 Review 2

The epithelia and sub-epithelial tissues contain a network of dendritic cells; the same cells are present in the T cell-rich
areas of peripheral lymphoid organs and, in smaller numbers, in most other organs.
TNF and IL1 result in inflammation
Immature dendritic cells
Epidermal dendritic cells in skin Langerhans cells"immature," because they are
inefficient at stimulating T lymphocytes no encounter with antigen yet.
***IMP! The capture and presentation of antigens by dendritic cells
1. Dendritic cells made in the bone marrow.
2. In the epithelium Immature DCs express pattern recognition receptors (PRR)
that bind microbes
3. PRRs capture & phagocytose microbial antigens
4. Simultaneously, as microbes bind to TLR it binds to other sensors on DC as well as
in epithelial cells and resident macrophages in the tissue, which results in production
of inflammatory cytokines such as TNF and IL-1
5. These cytokines activate the DCs (induces expression of MHC and costimulators,
changes in phenotype & function)
6. Dendritic cells stop expressing their cell adhesion molecule (loss of adhesiveness)
leave epithelium.
7. Receptors expressed in the DC (such as CCR7) attract the DC to the lymph nodes,
which contains and liberates the chemokines to which the CCR7 receptor responds.
8. The activated DC cell migrates through lymphatic vessels to the lymph nodes.
9. The dendritic cells present antigens to naive T lymphocytes in the lymph nodes.
10. T cells inspect the DC for antigens and eventually move into the circulation if they dont find the correct antigen.
11. If they find the correct one, they differentiate and proliferate into effector cells.
12. Effector t cells then leave lymphoid organ and travel to the site of infection where macrophages present the same
antigen to them.
Important point on maturation! relevant to step 5!
During migration the DCs mature; at different stages of maturation they may express different membrane proteins.
Change their behavior to stop phagocytosis and start expressing immune stimulatory molecules. Mature DC need to
express two things:
Signal #1 up regulate MHC because they need to bind the peptide and present antigen to the TCR.
Signal # 2 up regulate levels of B7 co-stimulators
Mature dendritic cells reside in the T cell-rich areas (paracortex) of lymph nodes.
Mature Dendritic Cells
Immature DC (cells designed to capture antigens) During migration dendritic cells mature into APCs capable of
stimulating T lymphocytes. Involves increased synthesis and stable expression of:
MHC molecules
Co-stimulators (B7-1 and B7-2), which are required for full T cell responses.
Dendritic cells bearing captured antigen and naive T cells poised to recognize antigens come together in lymph nodes.
Remarkably efficient process; estimated that a T cell response begins in the nearby lymph nodes within 12 to 18 hrs.
**IMP!!!** What happens if T-cell receives signal 1 and not signal 2?
T cells become anergic state of unresponsiveness to antigen.
Different types of APCs serve distinct functions in T-cell dependent immune responses
+
1. Different types of dendritic cells direct the differentiation of nave helper CD4 T cells (MHC class II) by the different
cytokines that they are exposed to into distinct populations that function in defense against different types of microbes.
2. Macrophageabundant in all tissues In cell-mediated immune reactions, they phagocytose microbes and display
the antigens to nave t cells but mainly to effector T cells, which activate the macrophages to kill the microbes.
3. B-lymphocytes ingest protein antigens and display them to helper T cells within lymphoid tissues; this is important for
the development of humoral immune responses.
4.All nucleated cells can present antigens derived from microbes in the cytoplasm to CTLs (CD8 MHC class I)
All nucleated cells present MHC class 1 molecule. Important because every cell in your body can theoretically be formed
into cancer cell. Because they have the ability to present antigens to CTL, they will kill the infected cell.

Immunology Block 1 Review 3

Who expresses and recognizes what?

MHC class 1 expressed on all nucleated cells recognized by cytotoxic lymphocyte (CTL, CD8+)
MHC class 2 expressed ONLY on antigen presenting cells recognized by T Helper cells (TH CD4)
Costimulator molecules (B71 B72) Expressed ONLY on antigen presenting cells! recognized by both T cells.
***IMP!***Cross-presentation of antigen
Since antigen presenting cells are the only ones that express the costimulator molecule, cross presentation needs to
occur in order for T cells to get that second signal and differentiate into an effector cell.
Cross-presentation is when the APC engulfs the whole infected cell or the tumor cell and processes the antigen; APC can
then present the antigen plus B7 co-stimulator (B7-1 or B7-2, just know B7)
Viruses may infect any type of host cells and these infected cells may not produce all of the signals needed to initiate
T cell activation (MHC class 1 and costimulators).
They do express MHC class 1 but not costimulators
Costimulators are only present in antigen presenting cells!!
+
**Naive CD8 cells respond to these intracellular antigens using the mechanism where dendritic cells ingest the
+
infected cells and display the antigens present in the infected cells for recognition by CD8 T lymphocytes.
In most cases, this cross-presentation (cross-priming) applies to CTLs recognizing class I MHC-associated antigens.
The same cross-presenting APC may display class II MHC-associated antigens from the microbe for recognition
+
by CD4 helper T cells.
Cross-presentation of microbial antigens from infected cells by professional APCs
Cells infected with intracellular microbes, such as viruses, are ingested
(captured) by professional APCs, and the antigens of the infectious microbes are
broken down and presented in association with the MHC molecules of the APCs.
T cells recognize the microbial antigens and co-stimulators expressed on the
APCs, and the T cells are activated.
The structure and function of MHC molecules
MHC molecules are membrane proteins on APCs that display peptide antigens for recognition by T lymphocytes.
Genetic locusprincipal determinant of acceptance or rejection of tissue grafts exchanged between individuals.
****Individuals identical at their MHC locus (inbred animals and identical twins) accept grafts from one another, and
individuals that differ at their MHC loci reject such grafts.
MHC restriction of T cells the physiologic function of MHC molecules is to display peptides derived from protein
antigens to be recognized by the TCR on antigen-specific T lymphocytes.
Some of these polymorphic MHC residues contribute to variations in the peptide-binding cleft (top or floor) and thus in
the ability of different MHC molecules to bind peptides.
The genes of the MHC locus
Map of the human MHC (HLA complex) illustrating the major genes encoding molecules involved in immune responses.
Sizes of genes and intervening DNA segments are not shown to scale. HLA,
human leukocyte antigen; LT, lymphotoxin; TAP, transporter associated with
antigen processing; TNF, tumor necrosis factor.
Three classes of genes present in chromosome 6:
**MHC Class 1 genes Right side of locusthree different class 1 genes on
each arm of chromosome 6.
HLA-A, HLA B and HLA C Each of these class one genes
makes alpha subunits.
Alpha subunit is expressed on chromosome 6; it is highly polymorphic.
MHC class 1 molecule has an alpha and a beta subunit:
HLA 1 gene (Chrom. 6) alpha subunit (highly polymorphic) made up of 3 chains
The beta 2 subunit of the MHC molecule comes from chromosome 15 one chain
**MHC Class 2 genes Left side of locus.
HLA DP, HLA DQ and HLA DR**
Two subunits make up a class two molecule Alpha and beta subunits (highly polymorphic)
Both are expressed from chromosome six.

Immunology Block 1 Review 4

****DR is important contributes to making your MHC more different for tissue graft,
Dm is part of class I pathway (Dont let the position in the genome confuse you)
Tap 1 and tap 2 are transporter proteins in the ER membrane. Bind peptides and are actively imported in the ER.
Class 3 genes are not peptides display molecules. They are multiple proteins that are involved in complement. C4, factor
B, C2. Important cytokines are present here. Lymphotoxin Beta (also called TNF B), TNF alpha and LT.
Human Leukocyte antigens (HLAs) or MHC
Human leukocyte antigens (HLAs)were discovered as leukocyte antigens
that were identified with specific antibodies.
In all species, the MHC locus contains two sets of highly polymorphic
genes, called the class I & class II MHC genes.
In addition to the polymorphic genes, the MHC locus contains many nonpolymorphic genes. Class II loci are shown on
the previous slide as single blocks but each consists of at least two genes encoding the alpha and beta subunits.
The class III MHC locus refers to genes that encode molecules other than peptide display molecules; this term is not used
commonly. There are also multiple class I-like genes and pseudogenes (not shown).
Its high level of polymorphism makes it hard to find a tissue match
The structure of Class I MHC molecules
Class
MHC I
Where is it
expressed?
What does it
present?
To who it presents
Chromosome
Composition
Peptide binding
cleft
Invariant
Break down of
genes

MHC II

All nucleated cells

Dendritic cells, monocytes/macrophages, Blymphocytes.

MHC molecules can display peptides derived from foreign microbial proteins as well as peptides from
that individuals own self-protein.
-MHC molecules are constantly presenting self-antigens, what activates them are foreign particles.
CD8
CD4
6 HLA alpha chain genes 15 b2microglobulin (b2m)
3 alpha chains non covalently attached to 1 Beta
chain b2-microglobulin (b2m).

- Amino terminal alpha 1 & 2 domains.

- Accommodates 8-11 amino acids that express
different polymorphisms.
Highly polymorphic Alpha 3 binds CD8

- Amino terminal alpha 1 and beta 1 domains.

- Accommodates 10-30 residues.

3 genes: HLA-A, HLA-B, and HLA-C

-You get 2 sets of these three genes, one from
mom and one from dad.
- If genes are heterozygous you will be able to
express 6 genes.

4 genes: HLA-DP, HLA-DQ, HLA-DRa, HLA-DRb

-One DP gene Alpha and Beta chain
-One DQ gene Alpha and Beta chain
-One DRa gene Alpha chain
-One or two DRb genes Beta 1 &/or Beta 2
* The DR subunit a + b1 and/or a +B2
*Get one allele from each parent
*Possible chain outcomes
** DR can inherit 1 or both from each 6, 7, 8

Two chains, alpha and beta

Non polymorphic B2 domain binds CD4+

Mom
DPab
DQab
DRab1**
DRab2**

Dad
DPab
DQab
DRab1 **
DRab2**

Immunology Block 1 Review 5

Thus, a heterozygous individual can inherit six or eight class II MHC alleles, three or four from each parent (one set
each of DP and DQ, and one or two of DR).
Although the two classes of molecules differ in subunit composition, they are very similar in overall structure. Compare
Features and functions of MHC genes
Co-dominantly expressed meanings that the alleles inherited from both
parents are expressed equally.
Highly polymorphic many different alleles are present among the
different individuals in the population. No two individuals in an outbred
population have exactly the same set of MHC genes and molecules.
MHC haplotype set of MHC alleles present on each chromosome & each
HLA haplotype has a numerical designation.
Ex: HLA-A2, HLA-B5, HLA-DR3, and so on.
Heterozygous individualshave two HLA haplotypes, one
from each chromosome.
HLA genes are pretty close together on chromosome 6, most of the time
you inherit them as a haplotype.
DP and DQ you only have a choice of one alpha and beta with the
difference being the presence of polymorphisms.
DR class two genes are the most important to match when you are
considering a tissue graft or organ transplant.
Difference in DR subunits can make individuals susceptible to autoimmune diseases.
CTL comes in contact with the infected cell and it recognizes class 1 (self-antigen presentation) that is why it
cannot directly attack it.
Co-stimulator molecule (B71 and B72) are expressed on APCs. Receptor for these costimulators on nave t cells
is CD28. Since CTLs only recognize infected cells (Not APCS) cross presentation needs to occurs!!
Every cell in your body could be infected by a virus and they must express antigen with class I.
Cells expressing MHC Class II are more limited. Includes dendritic cell, mononuclear phagocytes and b cells. B-cells must
interact with CD4 cells to receive the necessary signals so they can produce the correct antibodies.
Binding of peptides to MHC molecules
Anchor residues of the peptide hold it in the pockets in the cleft of the MHC molecule.
There are pockets in the floors of the peptide-binding clefts of most MHC molecules.
The side chains of amino acids in the peptide antigens fit into these MHC pockets and anchor the peptides in the cleft of
the MHC molecule.
Peptides that are anchored in the cleft by these side chains (also called anchor residues) contain some residues that bow
upward and are recognized by the antigen receptors of T cells.
***Peptide binding domain MHCI alpha one alpha 2
***Peptide binding domain MHC2 alpha one beta 2
Interaction of peptide antigens with MHC molecules
One MHC molecule One cleft one peptide at a time, but Each MHC molecule can present many different peptides.
MHC molecules bind only peptides and not other types of antigens.
MHC molecules acquire their peptide cargo during their biosynthesis and assembly inside cells.
5
A single T cell may need to see a peptide displayed by only as few as 0.1% to 1% of the approximately 10 MHC
molecules on the surface of an APC, so even rare MHC molecules displaying a peptide are enough to initiate an immune
response. (Dont remember numbers)

Immunology Block 1 Review 6

Any peptide that has anchor residues that will fit in those pockets, even though they are different, will be bound by those
MHC molecules.
MHC molecules present intracellular peptides to T cells
MHC molecules display peptides derived from microbes that are inside host cells, and this is why MHC-restricted T cells
recognize cell-associated microbes and are the mediators of immunity to intracellular microbes.
**Class I MHC molecules acquire peptides from cytosolic proteins.
**Class II molecules from proteins in intracellular vesicles.
Although MHC molecules are constantly displaying self-peptides, we do not develop autoimmune responses to selfantigens because T cells specific for self-antigens are either killed or
inactivated (Tolerance)
Two ways microbes can get in our cells:
- Can live there
- Brought to cytoplasm by endocytic vesicles.
Features of peptide binding to MHC molecules
MHC molecules can bind many peptides but display only one at a time.
Unlike B-cells, the TCR does not recognize lipids, carbs or nucleic acids.
KNOW THE FOURTH POINT. Peptides MHCs synthesized in the ER.
- Class I loaded in ER
- Class II loaded in endocytic vesicle.
An MHC that is empty will be degraded and wont make it to the surface.
Antigen processing
The two pathways of antigen processing involve different cellular organelles
and proteins and are designed to sample all of the proteins present in the
extracellular and intracellular environments.
The segregation of antigen-processing pathways also ensures that different classes of T lymphocytes recognize
antigens from different compartments.
Extracellular proteins that are internalized by specialized APCs are processed in vesicles and displayed by MHC II.
APCs may internalize extracellular microbes or microbial proteins by several mechanisms. Microbes bind:
- to surface receptors specific for microbial products.
- to Fc receptors that recognize antibodies.
- products of complement activation (CR1 and C3b) attached to the microbes.
B-cells internalize proteins that bind to the cells antigen receptors.
After internalization into APCs by these pathways, the microbial proteins enter acidic intracellular vesicles, called
endosomes, endocytic vesicles, or phagosomes, which may fuse with lysosomes.
The proteins are broken down by proteolytic enzymes, generating many peptides of varying lengths and sequences.
Proteins in the cytosol were synthesized in the very cell of any nucleated cell are cleaved into peptides by the
proteasome these are imported into the ER and are bound to the class one molecule.
Extracellular microbesbrought inside by the phagocytosis will enter the class 2 processing pathways.
The mannose receptor, scavenger receptorbind ligand stimulate phagocytosis.
Pathways of intracellular processing of protein antigen
APC- class II pathway: Endocytosed protein antigens peptides that bind
MHC class II CD4+ recognition.
Class I MHC pathway: Cytoplasmic proteinpeptide that binds MHC I
+
recognition by CD8 T cells.
The invariant chain (Ii DONT CONFUSE WITH INVARIANT
PORTION ON THE MOLECULES) has two important functions:
-directs the class 2 molecule transport from the ER and to the
endocytic vesicle
-CLIP (part of Ii) is already filling the peptide binding site, prevents
the binding of endogenous peptides (destined for class 1 pathway).

Immunology Block 1 Review 7

Features of the pathways of antigen processing (IMP)

TAP transporter associated with antigen processing.
T cell maturation thymic epithelium.
They are chosen for binding peptides in low or high affinity. Cells that are presenting
these are the thymic epithelium cells through the process of positive and negative
selection.
Know the source of the antigens.

The class II MHC pathway of processing of internalized vesicular (exogenous)

antigens
Protein antigens ingested by APCs into vesicles where they are degraded into
peptides.
Newly synthesized (In ER) MHCII carries a invariant chain (Ii) protein contains a sequence (class II invariant chain
peptide [CLIP]) binds tightly to the peptide- binding cleft so it is
occupied and unable to bind endogenous peptides in the ER. CLIP does
not get cleaved by proteases.
This "inaccessible" class II molecule is transported to the cell surface in
an exocytic vesicle, which fuses with an endosomal vesicle containing
peptides derived from ingested extracellular proteins.
HLA-DM facilitates the removal of CLIP and binding of the antigenic
peptide.
If the class II MHC is able to bind one of the peptides, the complex
becomes stable and is delivered to the cell surface and are recognized
by CD4+ T cells.
If the MHC molecule does not find a peptide, the empty molecule is
unstable and is degraded by proteases in the endosomes.
One antigen may generate many peptides, only one or two may bind the
MHC molecules present in the individual. Therefore, only these peptides
stimulate immune responses in that individual and these are the
immunodominant epitopes (recognized by the antibody) of the
antigen.
The class I MHC pathway of processing of cytosolic (endogenous)
antigens
Antigenic proteins may be produced in the cytoplasm from:
- virus living inside infected cells,

Immunology Block 1 Review 8

- phagocytosed microbes that may leak/be transported from vesicles to cytoplasm.
- from mutated or altered host genes, as in tumors.
All of these, as well as the cell's own nonfunctional cytoplasmic proteins are unfolded, ubiquitinated, and degraded in
proteasomes (proteolysis)
TAP (transporter associated with antigen processing) located in the ER membrane. TAP binds peptides from the
cytoplasm and actively pumps them across the ER membrane into the interior of the ER where they can be bound by a
MHC I.
MHC I complexes transported to cell surface and are recognized by CD8+ cells.
Viral strategies to evade the adaptive immune system
Viruses developed strategies to block the class I MHC antigen presentation.
These strategies include:
1. Removing new MHC molecules from the ERdont make it to surface.
st
2. Inhibiting the transcription of MHC genes dont make receptors in the 1 place.
3. Blocking peptide transport by TAP
+
By inhibiting the class I MHC pathway, viruses reduce presentation of their own antigens to CD8 T cells and are thus
able to evade the adaptive immune system.

Why did we have to learn all this?

Physiologic significance of MHC associated Antigen presentation
MHC restricition ensures that T cells see and respond only to cell-associated antigens.
By segregating the class I and class II pathways of antigen processing, the immune system can respond to extracellular
and intracellular microbes in different ways that are best able to combat these microbes.
The nature of the protective immune response to different microbes is optimized by linking several features of antigen
presentation and T cell recognition:
1) the pathways of processing of vesicular and cytosolic antigens,
2) the cellular expression of class II and class I MHC molecules,
3) the specificity of CD4 and CD8 co-receptors for class II and class I molecules, and
+
+
4) the functions of CD4 cells as helper cells and of CD8 cells as CTLs
CD8 recognizes alpha 3!
Cd4 recognizes beta 2!
The role of MHC-associated antigen presentation in the recognition of microbes by CD4 T cells
Protein of microbes endocytosed by macrophages
and B lymphocytes enter the class II MHC pathway.
+
As a result, these proteins are recognized by CD4 helper
T lymphocytes, whose functions are to activate
macrophages to destroy phagocytosed microbes and
activate B cells to produce antibodies against
extracellular microbes and toxins.
Cell mediated immunity helper t cell and macrophages
are going to kill the extracellular and phagocytsoed
microbes.
CD8+ T cells
Cell mediated immunity helper t cell and macrophages are going to kill the extracellular and phagocytsoed microbes
Protein antigens of microbes that live in the cytoplasm of infected cells enter the class I MHC pathway of antigen
+
processing. As a result, these proteins are recognized by CD8 cytotoxic T lymphocytes, whose function is to kill infected
cells.