96 Specific (Adaptive) Immune Response – Role of MHC Class 1 in Activating Cyotoxic T cells

Roles of Major Histocompatibility Complex (MHC) Class I molecules in Adaptive (Specific) and Innate (Non-Specific) Immune Responses

MHC or HLA – what’s the difference?

The Major Histocompatibility Complex (MHC) was initially discovered in the 1940s as a genetic locus that code for cell surface self MHC proteins and are associated with the rejection of transplanted organs in mice.  MHC are now known as self-markers.

In the 1950s, the same genetic system was described in humans when examining the rejection of donated WBCs, in which the recipient’s antibodies were binding to antigens on donated leukocytes and targeting them for destruction.  These leukocyte cell surface antigens were called Human Leukocyte Antigens (HLA) and it was found that the HLA genes were responsible for the human version of the MHC genes discovered in mice.  Therefore, the terms MHC and HLA are used interchangeably in humans, and as discussed below, MHC (HLA) genes are expressed by most human cell types, not just leukocytes.

Human Major Histocompatibility Complex (MHC) Genes – Unique Inheritance

The MHC (HLA) gene locus is on human chromosome 6 and contains over 160 genes coding for glycoproteins involved in innate and adaptive immune responses, in addition to gene expression regulation.  This MHC gene locus is divided into three coding regions: MHC class I, class II, and class III.  The MHC Class I (MHC I) coding region contains the HLA 1 (MHC I) genes and the MHC Class II (MHC II) coding region contains the HLA II (MHC II) genes.

MHC Alleles – Polymorphic

MHC genes are highly polymorphic and there are estimated to be over 19,000 unique HLA class I alleles, and over 26,000 alleles of other HLA genes.  By inheriting a diverse set of 3-6 MHC I alleles, and 3-12 MHC II alleles, aside from identical twins, each person has a unique set of MHC I and MHC II genes and therefore a unique set of cell surface MHC proteins.

MHC Class I – Production and Location

MHC I are synthesized by all nucleated cells and are even present on anuclear platelets.  However, MHC Class I are absent in red blood cells (RBCs).  That being said, RBCs are known to have other self marker cell surface proteins, such as the ABO group and Rh antigen.

*Clinical Side Note:  For successful organ transplants and blood donations, it is necessary to closely match the MHC self markers and blood type (ABO group and Rh antigen) between recipients and donors.

MHC Class I proteins are comprised of 4 polypeptides, one of which is polymorphic (the alpha chain ‘fingerprint’).  Once synthesized, MHC 1 polypeptides bind to each other and fold to become transmembrane proteins.

MHC Class I – Polypeptide Folding in RER and Complex Assembly with Cleaved Proteins

Once produced and assembled in the rough endoplasmic reticulum (RER), the MHC I wait in the RER membrane to bind to peptides that are prepared by the proteasomes in the cytosol.  Proteasomes are cylindrical protein complexes that are responsible for cellular protein turnover.  Proteins that are old, dysfunctional, no longer required, or misfolded are targeted for degradation by being tagged with ubiquitin and sent to the proteasome, which uses its inner proteases to cleave these targeted proteins into peptides.

First Role of MHC Class I: Presentation of Self Antigens

Cells routinely display their own synthesized peptides (endogenous peptides) on the surface of their cells using MHC Class I molecules.  These peptides are created by proteasomes as part of the normal turnover of old proteins, though it is theorized that newly synthesized proteins are sometimes used as well.  The peptides selected by the cell are translocated from the cytosol into the membranous RER using a transporter protein.  Once in the RER, the peptides are coupled with MHC I molecules and sent to the Golgi apparatus, where the peptide-bound MHC I molecules are packaged in vesicles and sent to the plasma membrane for presentation to the immune system.  This process allows the immune system to monitor the proteins being synthesized in the cell.  The endogenous (self) peptides displayed by MHC 1 molecules are used as self-antigens for self identification and assist immunosurveillance by CD8+ T cells and Natural Killer lymphocytes (NK cells).

This process allows for the immune system to “peek” inside cells and determine the health status of the cell and find any ‘hiding’ infecting pathogens or other abnormalities, as explored in the next paragraph.

Second Role of MHC Class I: Presentation of Non-Self Antigens to CD8+ Cytotoxic T cells

Several types of pathogens may be causing infection and damage in the body, including various viruses, bacteria, protozoa, helminths and fungi (e.g., yeast).  While some pathogens remain extracellular, many are able to gain entry into cells and in doing so profit by being able to utilize cellular resources as well as evade WBCs, antibodies, and complement proteins.

As a defense mechanism, when cells are infected, the host cell’s cytoplasmic proteases and other lytic enzymes are often able to cleave the pathogen’s proteins.  These non-self peptides are displayed on cell surfaces by MHC Class I molecules.  Once recognized by CD8+ T cells, an adaptive immune response will be launched which will target the pathogen for destruction.

Additionally, human cells have developed other ways for an exogenous (non-self) antigen to be delivered to MHC 1 molecules for display.

For example, instead of accumulating pathogen by viral injection, cells may endocytose (or phagocytose) cell debris or apoptotic bodies that house non-self antigens.  This material is brought into the cell in endosomes or phagosomes.  Once inside the cell, lysosomes will fuse with these endosomes or phagosomes and the lysosome’s lytic enzymes will cleave the non-self proteins to create non-self antigen fragments.  Alternatively, proteases can also cleave these extracellular components.  Once cleaved, the non-self peptides are transported into the RER to be coupled with MHC Class I molecules, packaged by the Golgi apparatus into vesicles, and sent for display on the cell surface.

It is estimated that MHC I molecules can present approximately 10-100 million different peptides, which makes them extraordinarily equipped for their role of antigen presentation.

As mentioned earlier, once the MHC I molecules are displaying peptides on the cell surface of their host cell, CD8+ T cells will be able to check these peptides to determine the health status of the cell.  Typically, CD8+ T cells will ignore cells that display self-antigens, and will only launch an adaptive immune response against cells that display non-self antigens.  Should the cell be infected and non-self antigens are displayed, CD8+ T cells will launch a cell-mediated adaptive immune response.  Likewise cancerous cells that are displaying abnormal antigens, will also stimulate CD8+ (cytotoxic) T cells to launch an attack leading to the elimination of aberrant cells.

CD8+T cell Destruction of Cells that are Displaying Non-Self Antigens:  Overview

Once CD8+ T cells have detected non-self antigens, they will proliferate (undergo rapid mitosis) and the daughter CD8+ T cells produced will circulate and help to destroy cells that are displaying the same non-self antigens.  In this manner, cytotoxic T cells are particularly well suited to combating virus infections.  At times, CD8+ T cells can also effectively destroy other pathogens and cancerous cells in a similar fashion.

Cytotoxic CD8+ T cells will destroy infected and abnormal cells using different strategies.  Firstly, CD8+ T cells work similarly to NK cells by releasing perforin, granzyme proteases and granulysin.  Perforin and granulysin proteins are able to create portals in the targeted cell, making it leak in water, which can lead to osmotic lysis (cytolysis).  Additionally, the granzyme proteases can travel into these portals and stimulate the targeted cell to induce apoptosis.  Secondly, CD8+ T cells secrete cytokines, such as lymphotoxins that can kill cancer cells and lymphokines that stimulate WBC recruitment and activation, as well as inflammation.

How MHC 1 and Non-Self Antigens Trigger the CD8+ T-cell mediated Adaptive Immune System – Steps Involved:

In order to describe the process by which MHC I is able to trigger an effective CD8+ T cell immune response, let’s consider the steps involved in an infection with a common cold virus (e.g. rhinovirus or adenovirus).

1. Virus exposure: Host cells in the upper respiratory tract are exposed to rhinovirus and infected.
2. Virus Infection: The rhinovirus injects itself into host cells.
3. Viral peptides: Some rhinovirus proteins are enzymatically cleaved by proteases and are transported into the membranous RER to be packaged with MHC Class I complex proteins.
4. MHC I display of viral antigens: Complexes of non-self antigens (e.g., rhinovirus antigens) bound to MHC Class I molecules are then sent to the Golgi body, and then to the cell surface via transport vesicles.
5. CD8+ T cell co-receptors: It should be noted that CD8+ T cells require two co-receptors (CD8 and TCR) to bind to the MHCI-antigen complex.
   1. a) The Cluster of Differentiation 8 (CD8) transmembrane glycoprotein co-receptor on CD8+ T cell helps T cells to bind to MHC I molecules on host cells.
   2. b) The other co-receptor, T cell receptor (TCR) is used to bind the antigen peptide displayed by the MHC I molecule.
      • It should be noted that TCRs are coded from a unique region of DNA that is able to undergo DNA rearrangements. This ability to create millions of uniquely sequenced TCRs is beneficial as the immune system must be able to potentially recognize millions of non-self antigens. This genetic rearrangement of TCR occurs only once in each T cell.  Therefore, each T cell produces only one version of the TCR gene product, and every T cell is different from every other T cell, because each T cell displays a different version of TCR. The creation of each T cell occurs in the bone marrow.
      • The next step in CD8+ T cell maturation occurs in the thymus.
      • Within the thymus, it is important for CD8+ T cells to go through positive selection and negative selection.
      • Positive selection ensures that T cells are able to bind to MHC self-antigen complexes with low affinity. T cells that can’t bind these MHC self-antigen complexes at all, are triggered to go through apoptosis.
      • Negative selection ensures that T cells do not bind MHC self-antigen complexes too strongly. If this occurs, the T cell is stimulated to go through apoptosis. If these T cells did not go through apoptosis, autoimmunity could develop.
   3. CD8+ T cell bind to MHC-I displaying non-self antigen: CD8+ (Cytotoxic) T lymphocytes with T cell receptors (TCRs) that successfully bind the displayed non-self antigen will be stimulated to launch an adaptive immune response specifically against the rhinovirus, which involves the clonal expansion (proliferation) of the cytotoxic CD8+ T cells with the TCR that binds rhinovirus antigens.  Additionally stimulated CD8+ T cells can release a range of cytokines, some of which are anti-viral, anti-cancer, some that recruit and activate WBCs and inflammation.
   4. CD8+ T cell mitosis (cell division): CD8+ T cell with rhinovirus-antigen-binding-TCR proliferates (goes through several rounds of mitosis, also known as cell division) to produce many daughter CD8+ T cells that have this same TCR, and that have 2 different roles:  cytotoxic CD8+ T cells and cytotoxic memory CD8+ T cells.

• Cytotoxic CD8+ T cells embark on a ‘search and destroy’ mission – they travel bodily fluids and destroy the pathogen and any infected cells. To do this, the TCR on the cytotoxic T cell will first bind to the specific non-self antigen on the pathogen and/or displayed by pathogen infected cells.  Once bound, the cytotoxic T cell can kill the pathogen or infected cell through 4 different mechanisms.  Firstly, CD8+ T cells can release perforin and granulysin which create portals in the targeted cells, causing them to leak water, which will cause swelling and osmotic lysis (cytolysis).  Secondly, CD8+ T cells can release granzyme proteases which will enter the targeted cells through the portals created by perforin and induce apoptosis.  Lastly, cytotoxic T cells can kill targeted cells by releasing lymphotoxins and/or stimulating death receptors (e.g., FAS or TRAIL) on the targetted cell to induce apoptosis.
• Memory CD8+ T cells also express the specific TCR that recognizes the non-self antigen of the pathogen, and these daughter cells will continue to replenish themselves, persisting in the body and giving “memory” of the non-self antigen for many years, which serves to speed up the adaptive immune response upon second exposure to the same non-self antigen.

• Note:  Regulatory T cells (Tregs) are produced in the bone marrow and mature in the thymus or peripherally. Tregs are thought to prevent inappropriate or excessive responses to self-antigens or harmless antigens from the environment, food, or normal microbiota. Regulatory T cells are able to diminish the production and activity levels of activated CD8+ T cells and CD4+ T cells and are thought to help prevent autoimmune responses. Additionally, Treg cells play a role in reducing inflammation and stimulating repair of tissue damage that has occurred as a result of pathogens.
• Note: CD8+ T cells can also destroy cancerous cells, if cancer cells display abnormal antigens that can be bound by the TCRs of CD8 + T cells.
• Note: Activated CD4+ Helper T cells/lymphocytes release cytokines such as interleukin-2 which enhance CD8+ T cell survival and activity.  As this cytokine is released from a lymphocyte it is sometimes called a lymphokine, and is a member of the interleukin family of cytokines.

Third Role of MHC Class I: Absence, NK Alert, Stimulation of the Innate Immune Response

Some viruses (and possibly other pathogens) are able to disrupt the MHC I presentation pathway, preventing the presentation of antigens on the cell surface.  Additionally, some cancerous cells also down-regulate MHC I.  Both of these conditions lead to aberrant cells that aren’t able to display non-self or abnormal antigens, and are able to evade T cell detection.  This could potentially lead to further damage and infiltration by both virally- infected cells and cancerous cells.  Thankfully, NK cells are able to recognize the absence of MHC I on cells and target them for destruction.  NK cells contain granules in their cytoplasm which contain perforin, granzyme proteases, granulysin, and other proteins.  In a similar fashion to active cytotoxic CD8+ T cells, NK cells destroy transformed cells by secreting perforin and granulysin proteins that create portals in aberrant cells.  Perforin portals make the targeted cells leaky, which leads to an influx of water, swelling, and cytolysis.  At the same, the granzyme proteases can travel through these portals and induce apoptosis in the targeted cell.

*It should be noted, that NK cell activation requires both the absence of MHC I and the presence of stress ligands.  Thankfully, infected cells and cancerous cells that lack MHC I often display stress ligands or abnormal ligands, which lead to NK cell activation against those infected and abnormal cells resulting in their destruction.

**Side note:  Antibody opsonization of non-self antigens on infected or cancerous cells can lead to Antibody-Dependent Cell-mediated Cytotoxicity (ADCC).  In ADCC, antibodies use their Fab ends to bind the non-self antigens, and then NK cells can bind to the exposed Fc ends (opposite side) of antibodies allowing NK cells to attach to the aberrant cell.  NK cells can then destroy the abnormal cells through release of perforin, granzyme proteases and granulysin. At the same time, NK cells will release cytokines attracting and recruiting powerful WBC phagocytes, macrophages, dendritic cells and neutrophils to assist in this innate immune response.

As described in the above paragraph, NK cells are part of the innate (non-specific) immune system and are able to kill cells that display stress/abnormal ligands and do not have the “self” markers of MHC I.  This innate response is much faster in defending the body against pathogens and cancerous cells than the adaptive (specific) immune response that is launched by T and B cells.

As noted above, the absence of MHC I is not enough to activate NK cells.  Erythrocytes express very few MHC I molecules and of course are not attacked by NK cells.  Not only must there by an absence of MHC I, there also must be the presence of an activating signal (e.g. a stress ligand) for a NK cell to target a cell for destruction.  So, although RBCs lack the inhibitory signal (MHC I), they also lack an activating signal (stress ligand), and will therefore not be attacked by NK cells.

Summary:

Roles of Major Histocompatibility Complex (MHC) Class I Molecules

Adaptive (Specific) Immune Response

• Present endogenous (self) antigens to CD8+ T cells for immune surveillance.
• Present non-self antigens from intracellular pathogens (e.g., viruses) to CD8+ cytotoxic T cells.
• Trigger CD8+ T cell-mediated destruction of infected or abnormal cells.
• Involved in clonal expansion and memory formation of CD8+ T cells.
• CD8+ T cells destroy infected cells via perforin, granzyme, and cytokines.

Innate (Non-Specific) Immune Response

• NK cells recognize and target cells lacking MHC I.
• NK cells destroy aberrant cells using perforin, granzyme, and granulysin.
• Absence of MHC I with presence of stress ligands activates NK cells.
• Antibody opsonization enhances NK cell-mediated ADCC (Antibody-Dependent Cell-mediated Cytotoxicity).

MHC vs. HLA

• MHC discovered in 1940s in mice, responsible for transplant rejection.
• Human version of MHC discovered in 1950s, termed Human Leukocyte Antigens (HLA).
• MHC and HLA used interchangeably in humans.
• MHC (HLA) genes expressed by most human cell types.

Human Major Histocompatibility Complex (MHC) Genes

• Located on chromosome 6, containing over 160 genes.
• Divided into three coding regions: MHC Class I, Class II, Class III.
• MHC Class I region contains HLA I genes.
• MHC Class II region contains HLA II genes.

MHC Alleles – Polymorphism

• Highly polymorphic, with over 19,000 unique HLA class I alleles.
• Individuals inherit 3-6 MHC I alleles and 3-12 MHC II alleles, leading to unique MHC profiles.

MHC Class I – Production and Location

• Synthesized by all nucleated cells, absent in RBCs.
• Comprised of 4 polypeptides, including polymorphic alpha chain.
• Assembled in the rough endoplasmic reticulum (RER), bind peptides from proteasomes, and presented on the cell surface.

Clinical Note

• Successful organ transplants and blood donations require matching MHC markers and blood type (ABO group and Rh antigen).

MHC Class I – Roles in Immune Responses

1. Presentation of Self Antigens

1. • Display endogenous peptides for immunosurveillance by CD8+ T cells and NK cells.

2. Presentation of Non-Self Antigens to CD8+ Cytotoxic T cells

1. • Display peptides from intracellular pathogens for CD8+ T cell activation and destruction of infected cells.

3. Absence, NK Alert, Stimulation of the Innate Immune Response

1. • NK cells target and destroy cells lacking MHC I and displaying stress ligands.

Example of How MHC 1 and Non-Self Antigens Trigger the CD8+ T-cell Mediated Adaptive Immune System: Steps Involved

1. Virus exposure (Example Rhinovirus)
   • Host cells in the upper respiratory tract are exposed to rhinovirus and infected.
2. Virus Infection
   • Rhinovirus injects itself into host cells.
3. Viral peptides
   • Rhinovirus proteins are cleaved by proteases and transported into the RER to be packaged with MHC Class I complex proteins.
4. MHC I display of viral antigens
   • Complexes of non-self antigens bound to MHC Class I molecules are sent to the Golgi body and then to the cell surface via transport vesicles.
5. CD8+ T cell co-receptors
   • CD8+ T cells require two co-receptors (CD8 and TCR) to bind to the MHC I-antigen complex.
     • CD8 transmembrane glycoprotein co-receptor helps T cells bind to MHC I molecules.
     • T cell receptor (TCR) binds the antigen peptide displayed by MHC I.  Complimentary match must occur between specific TCR and antigen for binding to occur.
6. TCR coding
   • TCRs undergo DNA rearrangements, allowing for millions of unique TCRs. Each T cell displays a different version of TCR.
7. CD8+ T cell maturation
   • Occurs in the thymus, involving:
     • Positive selection: T cells must bind to MHC self-antigen complexes with low affinity or undergo apoptosis.
     • Negative selection: T cells must not bind MHC self-antigen complexes too strongly or undergo apoptosis to prevent autoimmunity.
8. CD8+ T cell binding to MHC-I displaying non-self antigen
   • CD8+ T cells with TCRs that bind non-self antigens launch an adaptive immune response, involving clonal expansion and cytokine release.
9. CD8+ T cell mitosis
   • CD8+ T cells with antigen-binding TCR proliferate to produce cytotoxic CD8+ T cells and cytotoxic memory CD8+ T cells.
10. Cytotoxic CD8+ T cells
    • Embark on a ‘search and destroy’ mission, binding to specific non-self antigens and killing the pathogen or infected or cancerous cells by releasing perforin, granulysin, granzyme proteases, lymphotoxins and/or by stimulating death receptors (e.g., FAS and TRAIL).
11. Memory CD8+ T cells
    • Persist in the body, providing long-term memory of the non-self antigen for faster adaptive immune response upon re-exposure.
12. Regulatory T cells (Tregs)
    • Prevent inappropriate or excessive immune responses and reduce inflammation.
13. CD8+ T cells and cancer
    • Can destroy cancerous cells if they display abnormal antigens.
14. CD4+ Helper T cells
    • Release cytokines like interleukin-2, enhancing CD8+ T cell survival and activity.