Describe the role of the microenvironment (bone marrow) in the development of B cells (lympopoeisis of B cells).
B-cell development in the bone marrow requires:
1. ADHESION to a stromal cell (eg. fibroblast, macrophage or epithelial cell)
2. Soluble factor "stem-cell factor" (SCF) and other soluble factors such as IL-7 for proliferation are secreted by stromal cells in the bone marrow, and these are required for the B-cell to rearrange its genes to develop its B-cell receptor/antibodies.
An immature B-cell with receptor leaves the bone marrow to the lymphoid tissue.
What is/are the microenvironements for T-cell development?
They start out as pluripotent stem cells in the bone marrow, then differentiate to become lymphoid stem cells, still within the bone marrow.
Some of the lymphoid/lymphoblast cells enter the thymus. There, these T-cell-committed lymphoblasts receive signals by thymic microenvironment, especially THYMIC CORTICAL EPITHELIAL CELLS, which release soluble factors and facilitate cell-to-cell contact to facilitate the development of mature T cells.
They undergo gene rearrangement to become either CD4+ helper cells or CD8+ killer cells in the thymus.
B-cells and T-cells both must undergo gene-rearrangement to develop their respective receptors.
What is the structural difference in the antigens recognised by B-cell receptors, ie., antibodies and TCRs ie., T-cell receptors?
B-cell receptors recognise & bind to antigens in their native state (intact, usually extracellular) in general to neutralise them, while T-cell receptors recognise denatured antigens in the form of peptide proteins, usually those that have gained entry into cells, been digested & presented via the MHC system by dendritic or other antigen-presenting cells (APCs).
In a B-cell receptor, aka immunoglobulin or antibody, what are the structural features?
2. Two long heavy chains & two short light chains in Y-shape
3. Base of the Y comprised of two heavy chains only - this is the CONSTANT REGION that defines specificity of class of antigen eg. IgE, IgG, IgD, etc.
4. Top 'v" part of Y comprised of both heavy chains and both light chains (one each on each arm) - this is the VARIABLE REGION (V-segments) that binds to epitopes on native antigen
5. Each heavy chain and light chain gene contains multiple copies of three different types of gene segments for the variable regions:
Heavy chain has 44 Variable (V) gene segments, 27 Diversity (D) gene segments & 6 Joining (J) gene segments. The light chains also possess numerous V and J gene segments, but do not have D gene segments.
What are the features of the T-cell receptor?
1. Not soluble
2. Release cytokines, which are soluble
3. Only two chains: Alpha & Beta, two segments each
4. Variable segments (V-alpha & V-beta) bind to denatured peptide antigen presented by MHC
5. Constant region (C-alpha & C-beta) inserts into plasma membrane of cell
6. Contain multiple V, D and J gene segments in their beta chains (and V and J gene segments in their alpha chains) that are rearranged during the development of the lymphocyte to provide that cell with a unique antigen receptor.
What is VDJ recombination & why is it important in development of lymphocyte receptors?
V(D)J recombination is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) & T-cell receptors (TCR) production of the immune system.
V(D)J recombination takes place in the primary lymphoid tissue (the bone marrow for B cells, and Thymus for T cells).
V(D)J recombination nearly randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens from bacteria, viruses, parasites, dysfunctional cells such as tumor cells and pollens.
Explain how positive selection and central tolerance work in the maturation of T-cells in the thymus.
1. A large repertoire of receptors, generated from gene rearrangement, occur during thymocyte development.
2. Positive selection acts on this repertoire to ensure T cells are selected that are beneficial to the host.
3. As T cells recognised antigen in association with self-MHC, only those cells that have the potential to recognise self-MHC will be selected.
4. However, thymocytes that react strongly with self-MHC are deleted: CENTRAL TOLERANCE - to prevent autoimmunity.
Explain how double-negative T-cells in the thymus "decide" to become CD4+ helper cells that bind to antigen presented by MHC Class 2 or CD8+ killer cells that bind only to antigen presented by MHC Class 1?
Cortical epithelium in the thymus mediates positive selection of thymocytes.
The double-negative T-cells are exposed to MHC Class I & MHC Class II expression in the thymus, which will trigger recognition by some T-cell-receptors and not others.
These will further be selected depending on their affinity and ability to detect self-antigen presented by MHC:
95% of developing thymocytes die because they have high or low affinithy:
LOW: Cells die by neglect;a s they don’t bind to MHC, they will be useless to the host when cells get infected
APPROPRIATE: Cells will be rescued; binding is not to weak nor to strong. Cells encouraged to survive & exit into periphery forming a major part of defence system.
HIGH: Cells die by apoptosis; as they bind too strongly to self protein, if allowed to exit into the periphery would likely to cause harm. NEGATIVE SELECTION
What is Central Tolerance?
Central tolerance is the second process of T-cell maturation, which occurs to allow for self-tolerance. Central tolerance is the mechanism by which newly developing T cells are rendered non-reactive to self.
T-cells undergo both positive and negative selection to produce T cells that recognize self-major histocompatibility complex (MHC) molecules but do not recognize self-peptides. T cell tolerance is induced in the thymus
Positive selection occurs in the thymic cortex. This process is primarily mediated by thymic epithelial cells, which are rich in surface MHC molecules.
If a maturing T cell is able to bind to a surface MHC molecule in the thymus it is saved from programmed cell death; those cells failing to recognize MHC on thymic epithelial cells die.
Thus, positive selection ensures that T cells only recognize antigen in association with MHC. This is important because one of the primary functions of T cells is to identify and respond to infected host cells as opposed to extracellular pathogens.
The process of positive selection also determines whether a T cell ultimately becomes a CD4+ cell or a CD8+ cell: prior to positive selection, all thymocytes are double positive (CD4+CD8+) i.e. bear both co-receptors. During positive selection they are transformed into either CD4+CD8- or CD8+CD4- T cells depending on whether they recognize MHC II or MHC I, respectively.
T cells may also undergo negative selection in the cortex, at the cortico-medullary junction, and the medulla (mediated in the medulla predominately by medulary thymic epithelial cells and dendritic cells. The thymic cells display "self" antigens to developing T-cells and signal those "self-reactive" T-cells to die via programed cell death (apoptosis) and thereby deleted from the T cell repertoire. This process is highly dependent on the expression of tissue specific antigens.
This clonal deletion of T cells in the thymus cannot eliminate every potentially self-reactive T cell; T cells that recognize proteins only found at other sites in the body or only at certain times of development (e.g. after puberty) must be inactivated in the periphery. In addition, many self reactive T cells may not have sufficient affinity (binding strength) for the self antigen to be deleted in the thymus.
Describe the structure & function of MHC molecules.
MHC molecules present antigen peptides to T-lymphocytes in lymph nodes to trigger proliferation of specialised T cells that can fight infection.
MHC molecules are produced inside either the affected cell or APCs, but they translocate toward the cell membrane to display antigen peptides on the cell surface.
What are the functional differences between MHC Class 1 & MHC Class 2 molecules?
MHC Class I molecules, expressed by all nucleated cells, present antigen peptides from antigen that has infiltrated or replicated inside the cell cytoplasm but is then degraded via proteosomes. Any nucleated cell infected by a virus would be able to express MHC I molecules.
MHC Class II molecules, expressed by specialist antigen-presenting cells that typically detect fight bacteria in extracellular fluid -- dendritic cells, macrophages and B cells -- express peptide fragments from antigen that the DCs or macrophages have phagocytosed.
What is the structure of MHC I molecules & how do they work?
MHC Class I molecules are synthesized and assembled in the ER of infected cells using chaperone proteins.
They have three alpha subunits and one betamicroglobulin. The MHC groove binds peptides of ~nine amino acids, transported into the ER via TAP (transporters associated with antigen-processing) molecules, then the MHC I: peptide complex moves together via Golgia apparatus to cell surface, where it's recognised by CD8+ Killer T cells.
What are the roles of the Invariant Chain (Ii) chaperone protein & CLIP peptide in how MHC Class 2 molecules work?
MHC Class II molecules are synthesized and assembled in the Golgi apparatus of APCs with the help of INVARIANT CHAIN (Ii) chaperone, which uses its invariant chain to block the groove from binding to any endogenous peptides.
The Ii-chain degrades to leave only the groove-binding CLIP peptide, which is replaced when the groove binds with a protein fragment that has been endocytosed from the extracellular fluid and lysed in a lysosome inside the APC.
What happens after a protein fragment (antigen peptide) that has been endocytosed from the extracellular fluid (eg., like bacteria) and lysed inside the APC is bound by an MHC Class 2 molecule?
The MHC II: peptide complex is transported via the Golgi secretory pathway to the APC's cell surface to be recognised by CD4+ Helper T cells.
What are the similarities in how extracellular (eg., bacteria, parasites) and intracellular (eg., viruses) antigens are presented?
Presentation of extracellular and intracellular antigens to T- lymphocytes require MHC molecules.
B cells detect whole antigen -- not antigen fragments -- usually on the surface of a pathogen and therefore don't require MHC presentation to be able to proliferate & mount an immune response.
Presentation by MHC molecules of both extracellular and intracellular antigens requires processing of antigen proteins into peptide fragments, binding to MHCs that are synthesized and assembled in cellular compartment and transported via the Golgi apparatus to the cell surface for detection by T-lymphocytes.
What are the differences in how extracellular and intracellular antigens are presented?
Extracellular antigens are presented at the surface of ANTIGEN-PRESENTING CELLS (APCs) such as dendritic cells, macrophages (both derived from monocytes) and B-cells by MHC Class II molecules (2 alpha + 2 beta) following endocytosis of antigen from extracellular fluid & lysosomal digestion by lysosomes.
Presentation of extracellular antigens, mainly bacteria, by MHC Class II molecules is recognised ONLY by CD4+ Helper T cells (Th1 & Th2), and this basically sets up B cells to differentiate and produce specific antibodies and/or increase macrophage activity.
Presentation of intracellular antigens, mainly viruses, by MHC Class I molecules is recognised ONLY by CD8+ Killer T cells, and causes them to set in motion programmed cell death of infected cell via perforin/granzyme/caspasde cascade, Fas/Fas-ligand interaction & production of cytotoxic cytokines Tumour Necrotic Factors (TNF-alpha & TNF-beta).
Describe the various antigen-presenting cells & compare their functions.
Dendritic cells (DCs):
"Professional APCs" and the "link" between innate &
adaptive immune systems. Derived from circulating monocytes, immature DCs migrate to EPITHELIAL SURFACES (like in the gut, respiratory tract, mucosal tissue) where they encounter & ingest foreign antigen in the extracellular fluid (often bacteria). Then they migrate via afferent lymph to paracortex of lymph nodes, where they process (endocytose, lyse, load) and present this antigen to naive T-cells via MHC II.
- also derived from circulating monocytes, but unlike DCs, they present ingested extracellular-antigen via MHC II to DIFFERENTIATED EFFECTOR T cells (TH-1). Macrophages are made "angry" or turned into "HULK" by Interferon-gamma (IFN-gamma) released by CD4+ Th 1 cells.
- recognise whole antigen, usually on the surface of an extracellular pathogen. Also express both MHC I & MHC II cells; especially important due to use of MHC II presentation of endocytosed & lysed extracellular-antigen peptides to DIFFERENTIATED EFFECTOR T-cells (TH-2), similar to macrophages. B cells are induced to mature into PLASMA CELLS that produce antibodies (receptors aka immunoglobin) into the extracellular fluid (as opposed to into vesicles) under direction of cytokines, especially Interleukin 4, released by CD4+ Helper T cell Type 2.
Discuss how MHC genes influence immune responses, in terms of resistance to infection and susceptibility to immune-mediated disease.
Diversity in individuals' MHC genes means that a population will have MHC presentation of many different types of peptide fragments.
This means that many different types of antigens can be detected, even in cases of viral mutation; ie., a population cannot be easily wiped out simply because everybody possessed the same MHC molecules that presented only the same peptide fragments to the same T-lymphocytes.
Small gene pool, such as interbreeding of dogs, for example, can increase disease susceptibility because the repertoire of antigen-peptides for presentation is limited to only a few MHC molecules.
Small gene pool / limited MHC genes can also reduce effect of vaccination, while certain MHC molecules can increase risk of autoimmune disease - a larger gene pool and more MHC molecules can mitigate this disadvantage.