Lecture 21: Cell-Mediated Immunity and Lymphocyte Ontogeny Flashcards
A 60 year old man presented to his GP for a repeat prescription of his usual medications. On examination, he was found to have palpable lymph nodes (up to 2cm in size) in his cervical, axillary and inguinal regions. The spleen was palpable 4cm below the costal margin. On further questioning, he told his GP that he first noticed some lymph nodes in his neck about 6 months ago, but they had not changed in size or troubled him. He felt well in himself. A full blood count showed a lymphocytosis of 15 x 109/L and the presence of smear cells.
Diagnose
B-cell chronic lymphocytic leukemia (B-CLL) affects B lymphocytes. Film shows not a lot of neutrophils, mostly lymphocytes, smear cells (broken fragile lymphocytes when blood is smeared).
It is usually i_ndolent (slow growing_)- just observe.
If a_ggressive,_ RFC chemotherapy:
- R is rituximab (anti-CD20 Mab) (monoclonal antibodies target B cell surface antigen to activate complement to kill them)
- F is fludarabine (purine analog) (inhibit DNA replication, less specific)
- C is cyclophosphamide (inhibit DNA replication, alkylating agent)
What is B-CLL?
B-cell chronic lymphocytic leukemia (B-CLL) affects B lymphocytes. Film shows not a lot of neutrophils, mostly lymphocytes, smear cells (broken fragile lymphocytes when blood is smeared).
It is usually indolent (slow growing). If aggressive, RFC chemotherapy:
- R is rituximab (anti-CD20 Mab) (monoclonal antibodies target B cell surface antigen to activate complement to kill them)
- F is fludarabine (purine analog) (inhibit DNA replication, less specific)
- C is cyclophosphamide (inhibit DNA replication, alkylating agent)
Describe Skin Graft Rejection
Skin Graft Rejection
Allograft is tissue that is surgically transplanted from one person to another. Autograft is tissue that is surgically transplanted within person’s own body.
- In the first graft, a_llograft is rejected_ after period of time.
- It is rejected because of cell-mediated CD8 T cells response, recognising HLA antigen difference on the graft (vascularise well so not because of vascular failure)).
- In the second graft, autograft is rejected after shorter period of time.
- It is rejected because of antibody-mediated response, against HLA antigen leftover from first graft (does not vascularise well)
What does Cell Mediated Immuinty mean?
- Cell-mediated immunity (CMI) was originally coined to describe localized reactions to organisms, usually intracellular pathogens, mediated by lymphocytes and phagocytes rather than by antibodies (humoral immunity).*
- Now it is used in a more general sense of any response, in which antibodies play a lesser role.*
Describe T cell clonal activation (CD8)
Secondary lymphoid organs contain antigen-sensitive CD8 T cells, each cell bearing a set of _TCRαβ receptors th_rough which it can recognize outside world. These are cytotoxic T lymphocyte precursors (CTLP, i.e. develop into cytotoxic T cells).
- Foreign antigens (e.g. cells that have become infected with a particular virus, or perhaps cells from an incompatible transplanted graft) come into contact with these T cells.
- Those with TCR receptors that can bind most strongly with foreign antigenic determinants presented to them in the peptide-binding groove of class I MHC surface molecules are selected and activated to respond.
- Interaction with antigen provides an activation signal and further signals are provided by co-stimulator interactions and cytokines such as IL-2 and IFNγ from TH cells.
- CTLP then proliferate and differentiate to form cytotoxic effector cells, which function by seeking out and destroy any cells, which bear recognizable foreign determinants.
- Also form memory cells
What are the Killing Mechanisms of CD8 T cells?
Cytotoxic T cells require direct physical contact with their targets to exert their cytotoxic effector function.
They kill targets by three mechanisms.
- They insert a complex of molecules called perforin into target cell membrane. These assemble into _doughnut-shaped hole_s in membrane in a similar way to the complement membrane-attack complex.
- They release enzymes that d_igest target cell membrane._
- They release cytokines (TNFα, TNFβ, IFNγ) that bind to receptors on target cell, inducing apoptosis (programmed cell death)
Describe lymphocyte ontogeny (Lymphocyte ontogeny- life paths)
Primary lymphoid organs are bone marrow and thymus. They are the sites in which B and T cells develop.
From there, mature antigen-sensitive lymphocytes are exported to secondary lymphoid organs, where they respond to antigens.
- Therefore we have B-cell precursors in the Bone marrow
- and T-cell precursors in the thymus
Every _antigen-sensitive lymphocyt_e in secondary lymphoid organs is committed to only one particular shape of antigen-binding site.
- Mechanisms are in place to ensure this commitment occurs in primary organs during B and T cell ontogeny.
To generate antigen receptor repertoire diversity in bone marrow and thymus, lymphocytes undergo a series of genetic rearrangements in immunoglobulin genes (in bone marrow) and in T cell receptor genes (in thymus) that are unique to them.
What does ontogeny mean?
Ontogeny or development of the Immune System
Describe B cell Ontogeny (the different stages of B cells)
B Cell Ontogeny
B cells mature in bone marrow through:
Initially there are u_ncommited stem cell_s in the bone marrow. These can give rise of a large number of different B cells.
-
Pre-B cell stage
- (only h_eavy chain gene rearranged_),
-
Immature B cell stage
- (h_eavy and light chain genes rearranged_, start expressing sIgM)
-
Mature, antigen-sensitive B cell stage
- (sIgM and sIgD expression).
Censorship of potentially self-reactive B cells can occur at the immature B cell stage, although other mechanisms for maintaining self-tolerance also exist.
Each Mature B cell only has 1 antigen specificity. therefore somewhere along this ontogeny, there is a selection for the particular heavy and light chain region- this is called becoming committed to a particular receptor. This occurs by genetic rearrangement within the immunoglobulin genes.
Describe the Immunoglobulin gene rearrangements in bone marrow
There are three sets of Immunoglobulin genes, which are a heavy chain locus, a κ light chain locus and λ light chain locus.
The sequence of the gene rearrangement events occurring during B cell ontogeny in the bone marrow is:
- Germ line DNA
-
Heavy chain rearrangement of V, D and J exons (Constant region does not rearrange- blue) to produce cytoplasmic μ heavy chains (pre-B cell stage).
- Relatively random process
- You end up with V, D and J exons that codes for the variable region of the heavy chain
- Light chain rearrangement, initially κ, and then λ if κ rearrangements fail to produce a translatable sequence.
- Expression of surface IgM (immature B cell stage).
- Test for sIgM receptor binding to self-antigens in the bone marrow.
- If no self-recognition, then expression of surface IgD and export to secondary lymphoid organs (mature B cell stage).
Heavy chains are defined by their _________ regions
Constant
How do we insure the B cells don’t bind to self-antigens?
Describe Central Tolerance of B cells
Ig gene rearrangements occur i_ndependently of antigen_, therefore some receptors may be able to recognize self-antigens.
During B cell ontogeny in bone marrow, receptors on immature B cells are tested for binding to common self-antigens in bone marrow.
- If they bind with high affinity, those B cells are deleted.
- This means that o_nly B cells_ with receptors that do not recognize these self-antigens are permitted to mature and leave bone marrow to join B cell repertoire.
This process of immunological censorship leads immune tolerance to some self-antigens.
Describe Class Switching of individual Mature B cells
Mature individual B cells can switch immunoglobulin classes [(e.g. IgM, IgG) by changing constant regions and retains variable region] one or more times during its life, while retaining the same antigen-binding specificity.
The order of switching is therefore defined by the order of heavy chain constant regions on the chromosome.
This is controlled by signalls from the T cells
The memory cells lost the abiltiy to make IgM so when these are triggered, they’ll make IgG (but retain the same antigen-specificity)
State 8 important points around B cell Ontogeny and Immunoglobulin Genes
- Development in bone marrow (1o lymphoid organ)
- Multiple V, D and J exons recombine -> Variable (V) region
- Recombination i_ndependent of antigen_
- Variable region diversity arises from this
- B cells become committed to one VH and one VL
- Single c_onstant region_ exons for each class (μ, γ, α, δ, ε)
- Negative selection to r_emove strong self-reactivity_
- Class switching retains V region, changing C region
Describe the changes in T Lymphocyte Ontogeny as we age
In thymus, there are interdigitating, antigen-presenting cells as well as a network of thymic epithelial cell types are involved in the intrathymic ‘education’ of T lymphocytes.
As we grow older, the thymus shrinks, which results in less T cells being produced (so we rely on previously produced T cells)
- In this process, T cells express receptors for antigens and are selected on the basis of suitability of those receptors.
- Only those T cells with appropriate receptors are allowed to mature fully and then are exported from the thymus to secondary lymphoid organs.
Describe the TCR Gene Rearrangements in the Thymus
TCR genes are part of immunoglobulin superfamily of genes and undergo rearrangement during T cell development in a manner analogous to immunoglobulin genes.
There are _four genetic loci c_oding for TCR polypeptides, which are α, β, γ and δ. TCRs are heterodimers of either α and β chains or γ and δ chains.
- Most T cells in body have TCRαβ (α and β loci are used for receptors)
-
Few percent of T cells have TCRγδ, which are found in larger numbers in gut mucosa and skin (than in the blood).
- Their physiological role is uncertain but they may play a role in early defence against pathogens as they respond to “heat shock proteins” which may be expressed by invading micro-organisms in response to stress.
Describe T cell Ontogeny (Positive and Negative Selection in the Thymus)
During T cell development within the thymus, stem cells move from the cortex into medulla and become progressively more mature.
- Each thymocyte will _rearrange either its TCRαβ or TCRγδ g_enes during the break in the thymus, and each mature thymocyte will express only αβ or γδ TCR.
- Cortical thymocytes express both CD4 and CD8 accessory molecules (‘double positive’ CD4/CD8 cells). Subsequent maturation leads to these cells becoming ‘single positive’ medullary thymocytes which are either CD4 or CD8.
Like B cells, T cells therefore undergo s_election processe_s during development. However, there are additional requirements to the selection of T cell lineage:
- Effective T cell receptor gene rearrangement,
-
Positive selection for recognition of self MHC,
- Recognise our own HLA
-
Negative selection to eliminate strongly auto-reactive cells,
- Death for cells that bind to self-peptides
- Differentiation of CD4 and CD8 subpopulations.
- Export to secondary lymphoid organs as mature T cells
Firstly, T cells must express TCR which recognizes self-MHC molecules with low affinity.
- Therefore, when they are exported to secondary lymphoid organs, they will be able to _recognize self-MHC i_n association with foreign epitopes with higher affinity.
- The self-MHC molecules responsible for this selection reside on thymic epithelial cells of the cortex.
Secondly, T cells must not recognize self-MHC plus self-peptide so strongly that they will be self-reactive in absence of foreign antigens (i.e. negative selection).
- The self-MHC molecules responsible for this selection reside on the interdigitating reticular cells of the medulla.
- This selection process probably occurs after thymocytes have down-regulated expression of either CD4 or CD8 (i.e. during ‘single positive’ stage of thymocyte differentiation).
