VME7153 Lecture 12 Notes PDF

VMED 7153 Lecture 12 Notes Lecture 12: Lymphocyte development T cell Overview/background: Mature T lymphocytes arise from the thymus after bone marrow derived precursors undergo stringent selection and maturation processes. T lymphocytes express T Cell Receptor (T...

VMED 7153 Lecture 12 Notes Lecture 12: Lymphocyte development T cell Overview/background: Mature T lymphocytes arise from the thymus after bone marrow derived precursors undergo stringent selection and maturation processes. T lymphocytes express T Cell Receptor (TCR) on their cell surface. Most T cells express the αβTCR on their surfaces. TCR do not recognize antigens directly. It does so by binding to processed antigenic peptides presented on the Major Histocompatibility Complex (MHC) molecules (we say T cells are MHC-restricted). MHC molecules are of two types: class I and II. T cells expressing CD8 are capable of biding to respond to antigens presented in MHC- I, while T cells expressing CD4 will recognize antigens presented in MHC-II. MHC restriction: CD4- MHC-II CD8 -MHC-I TCR receptor Each receptor is composed of 2 proteins – either an α paired with a β protein, or γ paired with a δ protein. Each α, β, γ, and δ protein has a constant and a variable region (figure on the side). The constant region is attached to the cell via a cytoplasmic tail which extends through the cell membrane into the T-cell cytoplasm. All TCRs are associated with CD3 and zeta chains, these are signal transducing proteins. T cells recognize antigens through their TCR, in the context of MHC presentation. The TRC/MHC + peptide biding is stabilized by co- receptors (CD4 or CD8, figure below). The CD4 molecule consists of a single elongate chain, while CD8 may be composed of a shorter α and β chain or an alternative form consisting of two α chains (the CD8 homodimer). DOI: 10.1007/s12045-018-0605-3 T cells use their TCR to recognize a wide range of peptides. The TCR chains are encoded by a limited number of genes, and these genes must generate a diversity of receptors able to recognize a multitude of foreign antigens present in an animal’s environment and that the animal could potentially be exposed. Virtually, every generated T cell has a unique TCR and each individual should develop a vast ‘repertoire’ of different TCRs. This extraordinary diversity is created at the molecular level by rearranging the series of genes that encode different areas (pieces) of the complete TCR chains. 1 VMED 7153 Lecture 12 Notes The TCR alpha chain has a variable domain (Vα), a joining region (Jα), a constant domain (Cα). The TCR β chain is slightly more complex, with Vβ and Cβ domains inked by a joining region (Jβ) and diversity region (Dβ). For the α chain there are several possible Vα genes (Vα1, Vα2…to Vαn) and J region genes (Jα1, Jα2… to Jαn), but only a single constant region gene (Cα). The number of genes encoding the various segments of the TCR chains is in the order of 100 Vα genes, 75 Jα genes, 75 Vβ genes, 6 Jβ1 genes (humans). Genes encoding elements of the TCR δ chain are integrated with the α chain genes. Immature T cells may utilize the δ chain genes to form receptors, but in most species, there is a predominance of α chain utilization. Regardless, TCR genes that encode the α, β, γ, and δ proteins are assembled by mixing and matching gene segments How progenitors become mature T cells? Briefly: T cell development involves a series of stages as the cell moves from the cortex (to where it first enters the thymus) to the medulla (from where it is released to populate the secondary lymphoid tissues). Thymic maturation is a very wasteful process, as some 98% of immature T cells that enter the thymus do not leave. These cells die by apoptosis and the cellular debris is phagocytosed by macrophages. Steps: o Precursor migration: Precursors of T cells, called thymocyte progenitors, migrate from the bone marrow to the thymus. o Double negative phase: Initially, these progenitors do not express CD4 or CD8 receptors, making them double negative. o Recombination: The enzymes RAG-1, RAG-2, and TdT facilitate recombination of the different gene segments to form the TCR chain. Specifically, this is for the rearrangement of the V(D)J regions of the TCR genes. o Gamma-delta T cells: If the γ (gamma) and δ (delta) chains are formed, the resulting γδ T cell can enter the circulation. These cells play roles in both innate and adaptive immunity. o Beta chain formation: If the β chain forms, it associates with a pre-TCR α (pre-Tα) chain and the CD3 complex. This forms the pre-TCR. o Double positive phase: Formation of a functional pre-TCR leads to cell proliferation and the expression of both CD4 and CD8 receptors. These cells are now double positive and are ready for α-chain rearrangement. o Alpha chain rearrangement: The α-chain undergoes recombination and can then complex with the β-chain and CD3 to form a complete TCR complex. o Positive selection: This occurs in the cortex of the thymus. Only a small percentage (around 5%) of the double positive cells will survive this process. Following positive selection, cells will commit to either CD4 or CD8 expression based on their specificity for MHC I or MHC II, respectively. Immature T cell must “create” a TCR capable of interacting with peptide antigen presented to that T cell by an MHC molecule. Within the thymic cortex are numerous thymic epithelial cells. These display surface membrane MHC class I and II molecules that contain peptide 2 VMED 7153 Lecture 12 Notes fragments of self-antigens and present a wide range of normal tissue antigens. Each immature T cell must ‘test out’ its TCR to ensure that it can interact with the MHC– peptide complex, but only by having a moderate-affinity interaction. Cells that have such a functional TCR ‘pass the test’ of positive selection and are allowed to move on to the second examination. Cells that fail to interact by having produced a TCR with no or low affinity for peptide-MHC, die by ‘neglect’. 9. Negative selection in the medulla (exclusion of cells that recognize auto-antigens, T cell central tolerance). Positively selected T cells then move into the thymus medulla where they undertake their “second exam”, called ‘negative selection’. In this test the immature T cell meets a thymic dendritic cell displaying MHC class I or II molecules containing peptides derived from a wide array of self-proteins. Within the thymus there is expression of peptides derived from tissues such as the brain, endocrine pancreas and joint ( due to the AIRE gene expression). Many thousands of such peptides from non-thymic tissue antigens may be expressed. In this test a T cell must prove that its TCR is incapable of responding to self- antigens with high affinity. Cells that do not carry high-affinity autoreactive TCRs will pass and become mature naïve T cell. Cells that fail (i.e. are potentially autoreactive) will be instructed to undergo apoptosis. These two selection processes take about 3 weeks to complete, and it is very wasteful. Only 2% of double-positive thymocytes graduate and become single positive T cells (CD4 or CD8). Single positive T-cells that have graduated are known as “naïve” T-cells, and they leave the thymus and start circulating. These cells express homing molecules allowing them to cross high endothelial venules (High endothelial venules (HEVs) are blood vessels especially adapted for lymphocyte trafficking which are normally found in secondary lymphoid organs such as lymph nodes (LN) and Peyer's patches.). They travel through different lymph nodes searching for their antigen. Only after recognition of their antigen and activation, these cells acquire receptors that would allow them to migrate to peripheral sites (peripheral sites are all tissues, but primary and secondary lymphoid organs). B cell development As for all haemopoietic and immune cells, B lymphocytes are derived from bone marrow stem cells. B cells undergo a series of maturation events involving interactions with stromal cells that supply stimulatory cytokines. B-cell maturation is not as well characterized as the thymic development of T cells. Some stages of maturation likely occur within the bone marrow, but in some domestic animal species the final stages of B-cell maturation are thought to occur in extramedullary locations such as the ileal Peyer’s patch (e.g. in ruminants) or, in the bursa of Fabricius in birds. The B Cell Receptor, the membrane bound version of an antibody, include signal transducing molecules, called Ig alpha and Ig beta. The fundamental principles for the generation of B cell receptor diversity are very similar to those described for the TCR. The diversity is initially achieved by the recombination of different variable (V), diversity (D) and joining (J) regions to form a B cell receptor (BCR). 3 VMED 7153 Lecture 12 Notes Selection of different V-D-J segments involves a complex of enzymes that cleaves and ligates the DNA. The complex is collectively known as the V(D)J recombinase and includes two important enzymes encoded by the recombination activating genes RAG-1 and RAG-2. The immunoglobulin light chain genes include Kappa or a (Cκ) or a Lambda (Cλ) chains, and only one is used to express a BRC (meaning light chains can be either kappa or lambda). Light chains have V and J segments (with no D region) Rearrangement starts with the heavy chain, and if the recombination is successful (expression of pre-BCR), the lymphocyte will proliferate. Daughter cells will then start light chain recombination. There are species differences in the arrangement of heavy chain constant region genes, but most have a single Cμ, Cδ and Cε gene, with separate gene segments encoding constant regions for those immunoglobulins with subclasses (IgA and IgG). These are arranged in the order Cμ, Cδ, Cγ, Cε and Cα, meaning the first heavy chain constant region to be expressed is that of IgM. Later, during an immune response when mature B cells become activated, these cells may undergo the process called class switching. During this process the B cell express different constant regions, going from IgM to IgG, IgE and IgA. B cells can further increase diversity in their BCRs by the processes of alternative junctional recombination and somatic mutation. Junctional alternatives are simply variable boundary recombination between V–J or V– D–J segments (for light and heavy chains, respectively), which join at different points in their sequences. Somatic mutation refers to single nucleotide substitutions (point mutations) in a gene sequence that again would result in a different sequence and BCR protein. These two additional processes expand repertoire of possible receptors (and consequently possible antibodies) and the range of antigens they can recognize. The earliest form of B cell recognized (the pre-B cell) is a precursor that produces the μ heavy chain within the cytoplasm. In the next stage of development, complete IgM monomers are synthesized, and the immature B cell displays these on its surface membrane. Immature bone marrow B cells undergo a process similar to the negative selection of developing intrathymic T cells. BCR that bind to self-antigens undego deletion (by apoptosis). Alternatively, the B cell may undergo further gene rearrangements within the light chain and express an edited BCR. If this new receptor is still self-reactive, more editions may take place, or the cell may be eliminated. This process is known as receptor editing. The final step in development is the co-expression of surface membrane IgM and IgD molecules by naïve B lymphocytes. At this stage, the naïve B cells become part of the re-circulating and secondary lymphoid tissue B-cell pool. 4 VMED 7153 Lecture 12 Notes Clinical Correlation Severe combined immunodeficiency (SCID) is recognized in both dogs and horses and because children may be afflicted by the same disorder, the animal diseases have received much research attention. Canine SCID is recognized in the Bassett hound, Cardigan Welsh corgi and Jack Russell terrier and the genetic basis for the disease is different in each breed. SCID in the Jack Russell terrier involves a distinct mutation in the DNA protein kinase gene involved in the recombination of VDJ regions in the formation of TCRs and BCRs. Failure to develop these receptors inhibits the production of any adaptive immune response. The clinical consequences of SCID involve bacterial and viral infection after loss of maternal immunity. Affected animals are lymphopenic and have hypoplasia of lymphoid tissue. They have lack of serum IgG and IgA antibodies. Experimentally, this disease can be treated by approaches such as bone marrow transplantation, transplantation of heterologous stem cells following partial myeloablation by irradiation, or gene therapy in which affected dogs are given a retrovirus vector containing the faulty canine gene. SCID is also well described in Arab horses, where the disease also reflects a small base pair deletion in the DNA-dependent protein kinase involved in the VDJ recombination event that forms TCRs and BCRs. A single Angus calf with lymphoid tissue hypoplasia, low serum IgG (of maternal origin) and absent serum IgM and IgA was also suggested to have a form of SCID. Pigs have also been shown to display SCID, and pigs and mice with SCID have been used as models for the human disease. 5 VMED 7153 Lecture 12 Notes 6