23 Antigen Processing

Dr. M. N. Gupta

1. Objectives

•  Learning about cytosolic and vesicular compartments for antigen processing for different kinds of pathogens

•  To understand how these two different compartments process antigens and allow the presentation alongwith MHC molecules

•  To learn about complementary roles of dendritic cells, macrophages and B-cells in antigen processing

•  To learn how the immune system “tolerates” gut flora and food antigens

2. Concept Map

3. Description

We have talked of how antigenic fragments are presented alongwith MHC molecules to the T-cells. We skipped the details of how exactly the antigens are processed by different APCs and where exactly it all occurs. We will also have a look at how these antigenic fragments reach cell surfaces as MHC complex.

MHC Class II molecules are present on all APCs (dendritic cells, macrophages and B-cells). Cytokines regulate the expression of MHC class II molecules on APCs.

In MHC class II processing pathway (also called exogenous pathway), pathogens are broken down by proteases into antigenic fragments. These fragments associate with MHC class II molecules which are produced in the endoplasmic reticulum.

These complexes of antigenic peptides: MHC class II moleculesreach cell surface to be able to interact with CD4+Th cells.

Gut flora and food antigens do not provoke immune response under normal circumstances. Severe infections with pathogenic organisms result in overcoming competition with the gut flora.

Antigen processing is the degradation of antigen to create smaller fragments. This is followed by their association with MHC or MHC like molecules. The complex MHC: antigen peptide or a similar complex clonally selects the T-cells specifically.

If the degradation is blocked, presentation cannot take place. T-cell response in that case is not available. Different cells process antigens with varied efficiency and thus are able to stimulate T-cells to a varying level.

Broadly, B-cells use Ig receptor to bind antigen via clonal selection. The specific antigen is internalized and returned to the cell surface. There the processed antigen gets associated with MHC class II molecules (present on all B-cells). The B-cell now acts as the antigen presentation cell for Th2 cells.

Phagocytosis by mononuclear phagocytes opsonize particles of immune complexes via their Fc and C3 receptors. Processing and presentation to Th1 cells follows.

Immature dendritic cells phagocytocyse antigens using multiple receptors: Fc, C3, scavenger and lectin family. These dendritic cells after this migrate to lymph nodes and processed antigens is presented to T-cells there.

Let us recall that T-cell activation requires three signals for activation:

• Antigenic peptide alongwith a MHC molecule to form nonself and self molecules complex
• Co-stimulatory signals received via CD28
• Specific cytokines

The peptides are derived from pathogens which can be present in two parts of the cell. The cell can be viewed as consisting of cytosol which is “linked” to the nucleus via pores on the membrane of the nucleus.

The vesicular system, on the other hand, is “linked” to the extracellular fluids. This includes endoplasmic reticulum, golgi bodies, endosomes, lysosomes and some other vesicles.

Endoplasmic reticulum can generate vesicles which fuse with golgi membranes. The fused vesicle can reach outside the cell. This is how the movement of molecules/materials takes place from inside to outside.

In the reverse direction, cells endocytocyse molecules/ particles which end up in endosomes. From there, these go into lysosome wherein hydrolases break these down.

It was Elie Metchnikoff who in 1882 discovered phagocytosis. In Greek, it means “eating by cells”. In fact, this led him to propose this process as the immune response and that antibodies were not so important. His experimental system was starfish larvae and the finding was based upon the observation that cells moving around in the cavity of the larvae would ingest particles injected into them.

Phagocytic cells are the ones which are involved in the uptake of particles/ pathogens which end up in their endosomes. We have discussed important phagocytic cells in one of the early modules.

We have listed earlier the wide range of cells which can act as antigen presenting cells (APCs). The choice of APC is made depending upon the first encounter between the pathogen and the immune system.

In lymph nodes and spleen, interdigitating dendritic cells (IDCs) are the most important APCs for CD4 T –cells. IDCs, B-cells, macrophages all can express high level of MHC II molecule. However, IDCs for some reasons are more effective APCs.

For many polysaccharides, marginal zone macrophages act as APCs. This presentation can last for months or years. Antigens displayed on recirculating macrophages in medulla lasts only for days/weeks.

The seminal work of Ada and Nossal has shown very early that only <1% of the injected antigen elicited immune response. The rest is excreted after degradation. So, antigen processing is the rate limiting step in T-cell responses.

Viruses and some bacteria infect cytoplasm or the nuclear compartment. On the other hand, most of the pathogenic bacteria and eukaryotic parasites proliferate in endosomes and move into lysosomes.

Cytosolic pathogens are taken care of by Tc (CD8) cells. The pathogens infecting other compartments are dealt with by CD4 T-cells.

Microbial antigens (include leprosy and tuberculosis causing mycobacteria) are taken up in macrophages and proliferate there. There is another route by which pathogens or their products end up in intracellular vesicles. Bacteria secreting toxins etc along with their products enter cells by phagocytosis, endocytosis or macropinocytosis.

Theses internalization mechanisms are operated by APCs which include dendritic cells, macrophages and B-cells.

T-cells are MHC restricted. At the same time, there is a need to distinguish between antigenic peptides originating from cytosolic and vesicular compartments.

This is neatly achieved by involving MHC molecules. MHC class I molecules deal with cytosolic origin. In turn CD8 T cells (Tc cells) kill the presenting APC. MHC class II are associated with peptides sourced from vesicular system and are dealt with CD4 T-cells.

Macropinocytosis is carried out by immature dencritic cells in the infected tissues. These cells also use receptor mediated phagocytosis. Arising from bone marrow, these cells become localized in various tissues. Later, these cells migrate via lymph to the nearby lymph nodes to meet naïve lymphocytes.

For receptor mediated engulfment of pathogens, these cells use their receptors for common structural components of pathogens such as bacterial cell wall proteoglycans. This action is similar to that of macrophages, neutrophils. Intracellular degradation follows.

Macropinocytosis does not involve receptors, it just internalizes viruses and bacteria. Either way, the dendritic cells take up infecting pathogens while resident in tissues. Thus, activated they migrate to the lymph node and become APC for T-cells there.

Immature dendritic cells populate much of the surface eoithelia and organs such as heart and kidney. Activated dendritic cells secrete cytokines which enables these cells to infact participate in both innate and adaptive immunity.

A T-cell responds to the dendritic cell (as APC) in lymph node and become effector cell. If a T-cell runs into uninfected epithelial cell, it becomes anergic and that is a dead end for this T-cell. It no longer can become an effector cell.

The precursors of dendritic cells are myeloid cells in the bone marrow which migrate to peripheral tissues. These cells have low level of MHC molecules and do not yet have B7 co-stimulators. In this immature form, these cells cannot activate naïve T-cells.

Nevertheless these cells do have receptors which enable them to recognize common pathogens and thereby ingest them just like macrophages. Antigens can also be taken up by receptors such as DEC205 or nonspecifically by macropinocytosis.

When infection occurs, these immature cells get loaded with antigen, migrate to the lymph node. Once there, these lose the property of phagocytosis, start expressing B7.1 and B7.2 and high level of both MHC class I and II molecules, along with adhesion molecules ICAM-1, ICAM-2, LFA-1, LFA-3 as well as dendritic cell specific adhesion molecule DC-SIGN which can bind to ICAM-3 strongly.

An important example of such cells are Langerhan’s cells of the skin. Their large granules are called Birbeck granules.

Triggered by the infection, Langerhan’s cells migrate to the lymph nodes and interact with T-cells. Maturation of dendritic cells like Langerhan’s cells consists of their becoming efficient APC for T-cells. Their presence of body surface complements innate immunity as part of the first parameter.

The chemokine DC-CK secreted by dendritic cells in lymphoid tissues attracts naïve T-cells. Migration of immature dendritic cells to lymph nodes is a part of their life of these cells. Cells which reach end of their life span without encountering infection/antigen also travel to the lymphoid tissues. Without any co-stimulatory molecule and antigen, all these cells have are self antigens-which may be derived from any peripheral tissues. The result is tolerance.

The initiation of the recognition of the pathogen by dendritic cells involve nonclonotypic receptors. Dendritic cells play an important role in viral infections. They are ingested but not killed. Using protein synthesis capabilities of dendritic cells, viruses synthesize the protein coats. However, this does lead to the peptides from these appearing on dendritic cell surface along with MHC class I molecules.

Dendritic cells also act as APCs for bacterial and fungal pathogens. Dendritic cells also act as APCs for inhaled environmental allergens and alloantigens of transplanted organs. (We will discuss organ transplantation later in a separate module). The stimuli activating lymphatic lining encourage migration of dendritic cells to the nearby lymph node. Hence, dendritic cells play an important role in allograft rejection.

Thus dendritic cells are powerful and versatile APC. Many members of toll like receptors family are expressed by these cells. All these features widen the range of pathogens which dendritic cells can recognize.

We have just mentioned the example of viruses infecting cells like dendrites and synthesizing proteins. That takes place in the cytosol. However, the export of the proteins to cell surface is via endoplasmic reticulum. The peptide binding to MHC class I protein take place also in the endoplasmic reticulum.

There are two ATP binding cassette (ABC) proteins which are associated with endoplasmic reticulum membrane. These are called transporter associated with antigen processing-1 and -2 (TAP-1 and TAP-2) and occur together as a sdimer. The genes for TAP-1 and TAP-2 map within MHC gene and are inducible by interferons produced in response to viral infection.

Each TAP-1 and TAP-2 have one hydrophobic domain and one ATP binding domain.

There peptide transporters use energy via ATP hydrolysis. TAPs have preference for binding peptides consisting of about 8 amino acid with hydrophobic or basic amino acid at carboxyl end. MHC class I protein also have similar specificity!

Cytosolic protein degradation is carried out by proteasome mostly. A cylindrical complex of about 28 subunits present in four stacked rings (of 7 subunits each). The core constitutes the giant active site for proteolytic reaction.

Ubiquitin tagged proteins are hydrolysed by proteasome and peptides presented alongwith MHC class I molecules. Some of the proteosome’s constitutive subunits are replaced by interferon inducible subunits. This changes the specificity of the proteosome to hydrolyzing bonds with hydrophobic and basic residues. The resulting peptides are preferred by MHC class I proteins for association.

The degraded peptides are transported into endoplasmic reticulum by TAP for binding to MHC class I molecules. The reverse-called retrograde translocation-is also possible and allows hydrolysis of misfolded proteins in endoplasmic reticulum. The membrane and secretory proteins, the viral envelop glycoproteins arising in the endoplasmic reticulum are also degraded in cytosol. The N-linked glycoprotein not only gets deglycosylated, asparagines residues also get converted to aspartic acid.

MHC class I molecules have a residence time in endoplasmic reticulum and wait there till they bind antigenic peptides. The formation of MHC molecule-peptide complex involves the binding of α-chain of the MHC molecule with β-microglobulin and this is followed by binding of the peptide. Initially, MHC molecules are in partially folded state. The formation of the complex also requires few accessory chaperon proteins.

The formation of the peptide complex releases MHC class I molecule from the endoplasmic reticulum to start its journey to the cell surfaces. In humans, α-chains of MHC class I enter the endoplasmic reticulum and bind to chaperone Calnexin.

This chaperone is also responsible for association with numerous other molecules of the immune system such as T-cell receptor, MHC class II molecules and IgS.

The next step is β-microglobulin binding to the α-chain. The α: β2 microglobulin dimer dissociates in partrially folded form and binds to a complex of proteins which includes Calreticulin. A TAP-associated protein tapasin is also a part of this complex.

Tapasin crosslinks TAP 1 and 2 and MHC class II. Another chaperone Erp57 which is functionally a protein disulphide isomerase participates in the peptide association of the complex.

Even Erp57 and calreticulin have a general role beyond this and act as quality control molecules in assembly of many glycoproteins in endoplasmic reticulum.

The binding of peptide to the partially folded dimer releases it from the complex of TAP, tapasin, calreticulin and Erp57. This completes the process and peptide: MHC class I complex can now move to the surface through golgi.

It may be noted that till the end, α:β2 microglobulin dimer remains in the partially folded state inspite of the involvement of chaperone molecules.

It is increasingly becoming clear that proteins manage multiple interactions by remaining in a disordered state which allows them to bind to multiple partners. So, the concept of what we call a folded state or native state is no longer rigid. It is likely that chaperones here are necessary to ensure that appropriate “partially folded” structure of the heterodimer is maintained before peptide is loaded.

Apart from epitope defining peptides, other peptides transported by TAP do not bind to MHC molecules in that cell and are cleared out of the endoplasmic reticulum by an ATP dependant (and TAP independent) mechanism back to cytosol.

In the uninfected cells, MHC Class I molecules spent sometime in endoplasmic reticulum to await infection. The ready availability of MHC class I molecules ensures that response to viral infections takes off swiftly.

In such uninfected cells, self origin peptides by MHC class I proteins ultimately reach cell surface to initiate tolerance.

Evasion mechanism of some viruses to avoid CD and T-cells is exemplified by herpes simplex virus. It prevents the transport of viral peptides by producing a TAP binding protein. On the other hand Adenoviruses produce a protein which retain MHC class I molecule in endoplasmic reticulum by binding to it.

The pathogens that replicate inside intracellular vesicles in macrophages are degraded to form peptide antigens differently. Protozoan parasite Leishmania and Mycobacteria are important examples of this class.

After macrophages activated, intravesicular proteases degrade proteins to produce peptide fragments that bind to MHC class II molecules. Extracellular pathogens and proteins internalized by endocytic vesicles follow the similar route. In both cases, CD4 T-cells recognize the peptides presented along with MHC class II molecules.

Proteins after endocytosis become part of endosomes. The endosomes gradually become increasingly acidic and finally fuse with lysosomes. The particulates entering the cell by either phagocytosis or macropinocytosis also follow this path.

Some drugs such as Chloroquine can make endosome interior less acidic and prevent antigen processing/presentation. Cathepsins B, D, S, L are acid proteases, S and L seems to be more critical.

Unactivated macrophages lack both MHC class II molecules and B7 molecules. Infecting pathogens result in their expressing MHC class II and B7 molecules. Some foreign proteins which do not provoke sufficient immune response do so when mixed with some bacterial adjuvants. The latter induce the co-stimulatory activity.

This design ensures that macrophages can discharge their functions: scavenge dead or dying cells or destroying pathogens which do not require adaptive immune mechanism. For all such activities, macrophages do not need co-stimulatory activity for T-cells. Kupffer cells and macrophages of spleenic red pulp are prime scavengers for cleaning dead cells from blood everyday.

B-cells as APCs

Macrophages are more efficient at taking up particulate matter as compared to soluble antigens. Immature dendritic cells, however, can take up soluble anbtigens from extracellular fluids by macropinocytosis.

B-cells complement these by specifically binding soluble antigens through the Ig receptors. We have often referred to this. Let us look at the details of what happens inside the B-cells.

B-cells express high levels of MHC class II molecules constitutively. The result is high levels of antigen presentation in the form of MHC class II antigenic peptide complexes.

This in turn leads to their interaction with CD4 T-cells. This activates naïve CD4T cells who in turn drive the B-cell differentiation and response further.

This T-cell interaction requires presence of co-stimulators on B-cells. Just like dendritic cells and macrophages, B-cells are induced to express co-stimulatory molecules like B7.1 and B7.2. Historically, B7.1 was identified as a molecule expressed in response to the presence of microbial polysaccharides.

Many soluble antigens such as HEW lysozyme, ovalbumin and lysozyme are able to provoke B-cell response only when bacterial adjuvants are used in vitro. This is understandable as immune system must have evolved to take care of microbial infections and not injections of soluble antigens.

We had discussed earlier that B-cells (and other APCs) in the absence of co-stimulating signals fail to activate naïve T-cells. Instead, they turn T-cells into anergic or unresponsive state. These mechanisms seem to be additional safety features so that immune response is not initiated against self-antigens.

Inside B-cells, the antigen is hydrolysed in intracellular vescicles. It is here that it binds to MHC class II molecules. These vescicles as such are transported to the cell surface.

It appears that B-cell specificity is based upon the quantitative criterion of how good has been the internalization of the antigen. Apparently, even non-specific antigens are internalized, albeit very poorly by B-cells. The corresponding antigenic fragments are also presented but the cell surface density of antigenic fragments: MHC complexes is very poor.

Intermediate Video

It is necessary to keep in mind that much of our knowledge regarding B-cell responses was based upon in vitro experiments or injecting soluble antigens into experimental animals. This includes the fundamental extensive work by Landsteiner. While it taught us much, in a way it also kept us away from realizing that B-cell mechanisms are tailored to factor in that soluble antigens are not large group of antigens during natural infections.Inset toxins, anti-coagulants produced by blood sucking insects, snake venom and allergens are examples of important but small class of soluble antigens. Tissue dendritic cells perhaps act as APCs for naïve T-cells in case of such antigens.

While dendritic cells resident in tissues lack specificity of B-cells and hence cannot concentrate these antigens, their initial encounter with the naïve T-cell specific to the antigen (derived fragment) is higher than B-cells.

Once the T-cell has stayed in the lymphoid tissue upon antigen presentation by a dendritic cell, it can encounter specific B-cell there with a greater probability.

Thus the roles of various APCs: dendritic cells, macrophages and B-cells is complementary as far as activating naïve T-cells is concerned. Dendritic cells cover a wide range as far as variety of antigens is concerned.

Macrophages act as APCs only for those pathogens which they cannot destroy on their own. So, they do not seek T-cell help unless necessary.

B-cells are more specific towards antigens which they can recognize via clonal selection. Only dencritic cells which are non-phagocytic lymphoid resident express co-stimulators constitutively. Macrophages and B-cells have inducible co-stimulatory molecules.

The three classes of APCs differ in their antigen uptake mechanism, MHC class II expression and the type of antigens which they cater to. Among these, they seem to cover all possibilities.

Oral Tolerance

Foreign antigens ingested as food do not provoke immune response. As these are not self antigens present in thymus and bone marrow, the corresponding lymphocytes are not eliminated that is, negatively selected for destruction.

Experiments with fed ovalbumin show that even along with adjuvants, the antibody response is veryu slow to develop. This suppression mechanism is specific as any antigen injected produces antibody response by the animal.

Hence it appears that antigens taken up by oral route involve peripheral tolerance. This is known as oral tolerance.

Before trying to understand oral tolerance, it is necessary to consider bacterial infections of the gut. We have a symbiotic bacteria (about > 400 specie), much of these in the colon ilieum.

Competitively, these keep away pathogenic bacteria colonizing the gut. Some situations such as breaks in mucosal lining of the gut, this non pathogenic strains of bacteria (E.coli is an important example) crosses over to blood and initiate systemic infection. Thus, lining of the gut is responsible for ensuring that the relationship with the gut flora remains symbiotic.

Infact animals which are guotobiotic (delivered and kept into germ free environments) have poorly developed immune responses. So, in some ways gut flora is necessary to keep adaptive immune responses to a desired level but the immune responses do not eliminate gut flora.

Nevertheless gut is often invaded by pathogenic microbes. Enteric bacteria (Salmonella, Yevsinia. Shigella, Listeria) protozoa (Entamoeba histolytica, cryptosporidium) and many viruses infect through gut.

Inflammatory response involving cytokines and chemokines induce adaptive immune responses. Dendritic cells and other APCs initiate clonal selection of the lymphocytes.

Some pathogens enter enterocytes, the cells lining intestine. This leads to signals in the form of released cytokines and chemokines: chemokines IL-8, MCP-1, MIP-1α, MIP-1β, RANTEs.

Stressed enterocytes also start expression of non-classical MHC such as MIC-A and MIC-B which bind to γ- δ T-cells. These kill infected mucosal cells and this initiates repair and recovery of the mucosa.

Other pathogens such as polio, retroviruses are transported by M-cells and are able to establish systemic infection. Enteric bacteria (S.typhii and S.typhimurium) also follow this route and bacteria reaches lymphoid tissues.

In the absence of the inflammatory response, the foreign antigens seem to induce tolerance in mucosal immune system. So, while pathogen cause inflammation, food antigens do not.

Multiple hypothesis have been given to further explain oral tolerance. The γ- δ T-cells which have extensive presence in the mucosa are probably involved in developing oral tolerance.

Some regulatory T-cells such as Th3 and TR1 also seem to play a role. These cells seem to suppress antigen specific responses when challenges with antigens occur.

We have so far discussed types of T-cells and now role of APCs in activating various types of T-cells. While Th and Tc are the two major T-cell classes, we also referred to the role of some other minot T-cells in the context of antigen processing and presentation.

This sets the stage for discussing how various cells co-operate with each other.

We also saw the curious case of antigens which we ingest, “process” during digestion and yet how “oral tolerance” prevents them provoking immune response. Having to eat is the process associated with mammals. Hence “oral tolerance” must have evolved very early.

Summary

•  Cytosolic and vesicular compartments for antigen processing
•  Active transport of antigens processed in cytosolic compartment
•  Role of endocytic vesicles in antigen processing
•  Dendritic cells, macrophages and B-cells as APCs
•  Oral tolerance