Immunology Exam 2 PDF, Winter 2024-2025

MICRD 0520/ 0576/ 0599/ 1521 Lecture 7 Winter 2024 - 2025 Dr. Volin Antigen processing and presentation – HLA molecules Objectives: 1....

MICRD 0520/ 0576/ 0599/ 1521 Lecture 7 Winter 2024 - 2025 Dr. Volin Antigen processing and presentation – HLA molecules Objectives: 1. Describe the major histocompatibility complex and human leukocyte antigens with regard to basic structure, genetic polymorphism, and distribution in different cell types. 2. Describe the two major pathways of antigen processing and presentation and state the result of antigen presentation by each pathway. 3. List and describe the cell types involved in antigen processing and presentation via the two major pathways. I. Overview of this lecture: In contrast to B cells that recognize antigens found on the outside of a cell, T lymphocytes recognize antigens derived from inside a host cell. To accomplish this, T cells must recognize antigens that are “processed” by the cell in which they are found. Antigens are processed and presented to T cells by 2 major means. The exogenous antigen processing pathway denatures protein and glycoprotein antigens derived from either endosomes or phagosomes and degrades them to small polypeptides. Polypeptides derived from the exogenous antigen processing pathway are loaded into HLA class II molecules in the cell and transported to the surface of the cell for presentation to CD4+ T helper cells in order to activate them. The endogenous antigen processing pathway uses polypeptides derived from the cell’s own protein synthesis machinery (endoplasmic reticulum and ribosomes). These polypeptides are loaded into HLA class I molecules and transported to the cell surface for presentation to CD8+ cytotoxic T cells in order to activate them. Once activated, CD8+ cytotoxic T lymphocytes kill the cell that presented the antigen. Immune responses from T cells, therefore, are said to be “MHC-restricted.” or “HLA-restricted”. II. The Major Histocompatibility Complex (MHC) and human leukocyte antigens (HLA): A. Nearly every vertebrate species contains MHC genes and their products. These molecules were originally discovered because of their role in tissue transplantation rejection (transplant seen as foreign and killed by immune system) or tolerance (transplant accepted as self and no attack by immune system). Hence, they were initially known to play a pivotal role in the discrimination between foreignness and self. B. MHC class I and class II genes and their products, the HLA class I and II molecules, respectively, are located on chromosome 6. 1. The MHC is referred to as a “complex” of genes because they are tightly or closely linked and inherited as a block or unit. L7-1 Copyright Michael Volin at Midwestern University 2024  2. Each person possesses two sets of HLA molecules inherited as a block from each parent. The MHC exhibit genetic polymorphism – in that each gene for an HLA molecule can have different multiple stable forms or alleles within a species (e.g. humans). This means that when transcribed and translated into an HLA molecule, the overall structure of the resulting HLA molecule remains the same but the amino acid sequence will vary somewhat. More will be covered on this in the lecture covering transplantation and tumor immunology. C. Basic structure of HLA molecules. HLA class I molecule HLA class II molecule Peptide binding groove Peptide binding groove s 2 s s 1 1 s 1 s s s s s CD8 binding site 3 s s s 2 m CD4 binding site 2 s s 2 Cytoplasmic tails Cytoplasmic tail D. HLA molecules are very similar to immunoglobulin molecules in that they possess domain structures and can recognize and bind to antigenic epitopes. They belong to what is known as the immunoglobulin supergene family. 1. HLA class I molecules are transmembrane glycoproteins possessing a cytoplasmic tail. It is a monomer consisting of three domains (1, 2, and 3) and a very closely associated but not covalently linked molecule called 2-microglobulin (2m). a. A deep grove or cleft is formed between the 1 and 2 domains. This is the binding site for processed polypeptides – usually 8-9 amino acids in length. Like the immunoglobulin hypervariable region, this region contains a significant degree of amino acid sequence heterogeneity. b. The 3 subunit binds the CD8 co-receptor on T cells (covered in a future lecture). c. 2 microglobulin is critical to the shape of the HLA class I molecule because probably due to a role conformational integrity. L7-2 Copyright Michael Volin at Midwestern University 2024  d. The cytoplasmic tail links to intracellular signaling pathways so that once appropriate binding with a T cell occurs, signals are transduced internally in the antigen processing cell. 2. HLA class II molecules are also transmembrane glycoproteins. They are composed of a closely associated dimer of two molecules – an  chain and a  chain – each possessing a cytoplasmic tail. Its overall structure is not terribly different than that of HLA class I molecules. a. A grove or cleft is formed between the 1 and 1 domains. It functions as the binding site for polypeptides derived from endosomes or phagosomes – usually 12-17 amino acids in length. b. The 2 domain binds the CD4 co-receptor on T helper cells. This will be covered in the next lecture. c. The cytoplasmic tails link to intracellular signaling pathways so that once appropriate binding with a T cell occurs; signals are transduced internally in the antigen processing cell. III. The exogenous antigen processing pathway (a.k.a., the endosomal pathway). Now that we have covered the basic structure of the major molecules involved with T cell recognition of foreignness, we can proceed to the process itself. A. After phagocytosis or endocytosis and killing of bacteria, viruses, etc. (refer to previous lecture material on phagocytosis), their component antigens are denatured and degraded. Some segments of protein antigens are easily degraded to single amino acids and sugar molecules while other segments are more resistant to degradation. They remain as small polypeptide fragments in the endosome or phagolysosome. B. While breakdown of ingested antigens is occurring, HLA class II molecules are synthesized in the endoplasmic reticulum with a third protein called the invariant chain (Ii). The Ii is a molecular chaperone for HLA class II molecules and serves two purposes: 1. It stabilizes unbound HLA class II molecules while they are being synthesized and 2. It blocks incidental binding of endogenous self-antigens to the HLA-D molecules while they are being transported to the endosome C. Vesicles containing HLA class II molecule plus the Ii will migrate through the cytoplasm so that they can be fused with the phagolysosome or endosome that contains the processed exogenous antigens. After degradation of Ii in the proteolytic environment of the phagolysosome or endosome a small peptide remnant of the Ii termed “CLIP” remains in the peptide binding groove until a process called “peptide exchange” can occur. L7-3 Copyright Michael Volin at Midwestern University 2024 D. Peptide exchange – Intracellular protein, HLA-DM, catalyzes the “peptide exchange” process by promoting the dissociation of the CLIP protein from the HLA class II molecule. Once the CLIP is removed, HLA class II molecules are permitted to bind the foreign antigenic peptide fragments generated by endocytosis or phagocytosis. The antigenic peptides fit specifically into a small peptide binding groove” formed between the 1 and 1 subunits of the class II HLA molecule. The remaining polypeptides from the original antigen - usually 12 to 20 amino acids in length - are then loaded into the HLA class II peptide binding groove. Exogenous antigen Presentation to CD4 Peptide exchange Processing in endosome Clip Late endosome Ii Golgi Ii Synthesis of individual MHC class II chains and assembly with invariant chain (Ii) Endoplasmic reticulum E. The resulting HLA class II-foreign polypeptide complex is transported to the cell surface where it can interact with CD4+ T helper cells. F. The ability to process exogenous antigen and present it to CD4+ T helper cells is dependent on the ability to express MHC class II gene products (i.e., HLA class II molecules). L7-4 Copyright Michael Volin at Midwestern University 2024  1. HLA class II molecules are found constitutively (under all conditions) on special antigen-presenting cells (APC) such as dendritic cells of the spleen and lymph nodes, thymic epithelial cells and B cells. 2. HLA class II expression can be induced on several other cell types in response in response to inflammatory stimuli. These cells include tissue-based macrophages and endothelial cells. IV. The endogenous antigen processing pathway (a.k.a. “the cytosolic pathway”) A. Processing of endogenous antigen (the cytosolic processing pathway) -some intracellular antigens are not derived from endocytosis or phagocytosis of pathogens but actually originate or are synthesized within the host cell cytosol. Hence, they are “endogenous” in origin. Examples would include proteins made by viruses and certain tumors. Virus Presentation to CD8 Golgi Proteasome Endogenous viral protein TAP-1 and -2 Ubiquitin Viral nucleic acid Synthesis of MHC class I chains and 2m and assembly with caparones Nucleus Endoplasmic reticulum B. Endogenously generated foreign antigens are degraded and subjected to the continuous turnover of all normal host proteins. They are targeted for proteolysis by having a small protein called ubiquitin attached to them. Such protein-ubiquitin conjugates can be degraded by proteasomes that are cylindrical particles described as "a tunnel of degradative enzymes". Interestingly, in 2004 the Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hersko and Irwin Rose for their work on ubiquitin. L7-5 Copyright Michael Volin at Midwestern University 2024  C. The resulting degraded peptide fragments (8-10 amino acids in length) are transported to the rough endoplasmic reticulum by a couple of transporter molecules associated with antigen processing called TAP-1 and TAP-2. In the ER, they bind to the cleft formed between HLA class I 1 and 2 domains. D. Loading of peptide fragments into the HLA class I binding grove induces a conformational change in HLA class I that, in turn, induces release of protein chaperones. The chaperones hold the HLA class I molecule in a partially folded state and strengthens binding of ß2- microglobulin. They are no longer needed when proteasome -generated peptides are bound to HLA class I. E. HLA class I - peptide complexes are transported via the Golgi’s exocytic pathway to the cell surface where they can be recognized and activate CD8+ cytotoxic T lymphocytes (CTL). Upon activation, the CTL will be induced to kill the cell that presented the antigenic peptides. F. HLA class I molecules are constitutively expressed on all nucleated cells. However, their expression can be upregulated by certain signals. Summary and review: 1. HLA molecules play a pivotal role in the immune response to intracellular antigens. 2. Unlike B cells, T cells can only recognize and respond to antigenic peptides bound to HLA molecules on the surface of a cell that has processed that antigen. Hence, T cell responses are “MHC- restricted” or “HLA-restricted”. 3. CD4+ T helper cells recognized HLA class II molecules plus process peptides derived from the endosome or phagolysosome whereas CD8+ cytotoxic T cells recognize HLA class I molecules generated from the cytosol. 4. HLA class I molecules are constitutively expressed on all nucleated cells, but their expression can be upregulated by certain signals. HLA class II molecules are constitutively expressed only on thymic epithelial cells, dendritic macrophages of the spleen and lymph node, and on B cells. HLA class II expression can be induced on other tissue macrophages and endothelial cells. Recommended reading: Coico R. 2021. Immunology: A Short Course. 8th Ed. Chapter 3, Chapter 6 pages 80-81, and Chapter 8, pgs 123-128. Parham, P. 2022. The Immune System. 5th Ed. Chapter 5, pgs. 136-158. Doan , Lievano, Viselli, and Swanson-Mungerson. 2021. Lippincott Illustrated Reviews: Immunology 3rd Ed. Chapter 5, Chapter 6 pgs. 69-70, and Chapter 10 pgs. 127-132. L7-6 Copyright Michael Volin at Midwestern University 2024 MICRD 0520/ 0576/ 0599/ 1521 Lectures 8 Winter 2024-2025 Dr. Volin The biology of T lymphocytes and T helper cell activation Objectives: 1. Briefly describe the ontogeny of a T lymphocyte from a bone marrow stem cell to a mature T helper lymphocyte or T cytotoxic lymphocyte. 2. Diagrammatically depict the T cell antigen receptor (TCR) with regard to major features and structures and describe its role in the “immunological synapse”. 3. State the activation requirements for a naïve T helper cell and. I. Overview of this lecture: T lymphocytes (a.k.a. T cells) are a population of cells critical to the regulation of immune responses. Like B lymphocytes, they arise from bone marrow stem cells. In contrast to B cells, they do not leave the bone marrow as mature cells but must undergo additional maturation and a selection process in the thymus. Most T cell precursors die in the thymus during the selection process. Upon exiting the thymus, T cells will be mature but still considered to be naïve T helper lymphocytes or cytotoxic T lymphocytes. These mature T lymphocytes are “HLA-restricted”. This means they recognize foreign antigens only if they are presented to them in association with a self HLA molecule. T helper cells possess the CD4 co-receptor whereas cytotoxic T cells possess the CD8 co-receptor. Upon activation by an antigen-presenting cell, naïve T helper cells will be stimulated to progress to either effector cells or memory cells. T helper cells may possess a given phenotype based on their cytokine production pattern. Naïve T helper cells upon activation are Th0 cells and will progress to either of three phenotypes – Th1, Th2, or Th17. Th1 cells positively influence cellular immune responses whereas Th2 cells up-regulate antibody responses (a.k.a., humoral immunity). Th17 cells are involved in autoimmune responses. Th1, Th2, and Th17 cytokines tend to be counter- regulatory, in that Th1 responses may inhibit Th2 and Th17 responses and visa-versa. Cytotoxic T lymphocytes are HLA-class I restricted and upon appropriate activation will kill the cell presenting HLA-class I plus processed peptide. Other cytotoxic cells may use similar mechanisms, but they may recognize antigen in a non-HLA-restricted fashion. II. Ontogeny of T lymphocytes. A. The thymus is an absolute requirement for the maturation and differentiation of mature T cells. This is evidenced by the fact that children born without a thymus (e.g. DiGeorge’s syndrome) do not have mature T cells. T cell differentiation occurs throughout life but begins to taper off subsequent to puberty as the thymus decreases in size (a phenomenon termed “thymic involution”). L8 -1 Copyright Michael Volin at Midwestern University 2024 B. The thymus is the site of education of a T cell. It is where the T cell 1. Acquires the T cell antigen receptor or TCR, hence its antigen specificity. 2. Learns tolerance of self; and, 3. Learns self-HLA restriction (recognition of foreign peptide only in association with self HLA molecules). Hence, the thymus is the primary lymphoid organ for the development of T cells, analogous to the bone marrow as the primary organ for the development of B cells. C. The steps in T cell maturation are each characterized by the anatomical compartment in which they take place and the types of cell surface molecules that are acquired by the cells involved. The steps are as follows: 1. A common lymphoid progenitor stem cell leaves the bone marrow. It has no T cell characteristics at this point and could not be distinguished from a pro-B cell except that it can home from the bone marrow to the thymus. This process is mediated by soluble chemotactic cytokines (“chemokines”) and by cell adhesion molecules. 2. The precursor stem cells enter the thymus at the cortical medullary junction and then migrate to the cortex. At this time, they express neither CD4 nor CD8 and are called double negative (DN) cells. They next begin to express the genes for RAG-1 and L8 -2 Copyright Michael Volin at Midwestern University 2024  RAG-2 (recombinase-activating genes 1 and 2) which are responsible for randomly rearranging the genes that encode the ,  and ,  chains of the T cell antigen receptor (TCR). The decision whether to be a  or  T cell occurs at this DN stage. The DN cells that become  T cells rearrange functional  and  chains and express them along with CD3 and the  (zeta chain) on their surface. These  T cells leave the thymus and migrate to epithelial cites around the body. The DN that productively rearrange the  chain will express it on its surface with an invariant molecule pre-T along with CD3 and . Together the  chain, pre-T, CD3 and  make up the pre-T cell receptor and the cell is now called a pre-T cell. 3. Pre-T cells continue to differentiate within the thymic cortex. The functioning of the pre-T cell receptor results in intracellular signals that stop further rearrangement of the  chain and initiate rearrangement of the  chain. Additionally, the cell begins to proliferate and express both CD4 and CD8. At this point the thymocyte expresses both  and  chains of its unique TCR, CD3, CD4 and CD8 and is called a double positive cell. 4. As maturing double positive thymocytes filter through the thymic cortex they must pass through a mesh of stromal cells, the most important of which are the thymic epithelial cells and the interdigitating thymic dendritic cells. These double positive CD4+CD8+ thymocytes then undergo selection, which is mediated by interactions with HLA molecules on the stromal cells and CD4 and CD8 molecules on the thymocytes. It is divided into two phases: a. Positive selection – Thymic epithelium are among the few cells that express both classes of HLA molecules (class I and class II) constitutively (all the time). Using their newly expressed / TCR and CD4 and CD8 co-receptors, thymocytes must bind with a certain critical affinity to HLA molecules on thymic epithelium. If the affinity of binding is too high or too low, the cell is not selected and must undergo apoptosis and die. Those that bind “just right” survive and expand in number. These cells are now “educated” to self HLA molecules and will only respond to self HLA plus processed antigen (self or foreign). Hence, they are now considered to be “HLA-restricted” (a.k.a. “MHC-restricted”). b. Negative selection occurs during the transition from the thymic cortex to the thymic medulla when encountering the interdigitating dendritic cells at the corticomedullary junction and later when they interact with the medullary epithelial cells. Because T cells that survive positive selection are potentially able to respond to peptides derived from both self and foreign molecules loaded into MHC, allowing these cells to leave the thymus and enter the circulation could result in autoimmunity. If these developing T cells respond to MHC plus self-peptide at this stage, the cells die through central tolerance mechanisms. The process of negative selection “weeds out” self-reactive cells through apoptosis or by inducing T cell anergy. The surviving cells are said to be self-tolerant. L8 -3 Copyright Michael Volin at Midwestern University 2024  Central tolerance Stem cells THYMUS APOPTOSIS Self-reactive T cell T cell receptor MHC/self peptide Dendritic cell c. It is estimated that