Immunology Lecture 1

Immunology - Lecture 2Adaptive Immune System Molecules of the Adaptive Immunity 6Adaptive Cells and Organs 7Generation of Immune Diversity Lymphocyte Antigen Receptors - 8

WednesdayFriday Generation of Immune Diversity Lymphocyte Antigen Receptors - 8Lymphocyte Development- 9 Lymphocyte Activation 10Lymphocyte Effector Functions - 11The Notorious CDsCD markers (clusters of differentiation)

Based on groups (clusters) of monoclonal antibodies which recognize a particular surface cell marker

Surface protein isolated from leukocytes immunized mice tested antibody

Different antibodies recognized same differentiation antigens= antibodies grouped into clusters of differentiation (CD)

Surface molecules were called differentiation antigens

ICAM-1 = CD54Siglec-1 = CD169integrin beta chain b 2 = CD19

>300 CD Designations

Not just markers, but functional moleculeshttp://biology.berkeley.edu/crl/flow_cytometry_basic.html

Flow Cytometry: Assessing Immune Cells and Their Responses

From BD BiosciencesImportant technology that is used in the clinic and research to assess the incidence of immune cells as well as their level of responses.Automated instruments that assess the properties of single cells, one cell at a time-Size, granularity, DNA content, expression of intracellular and surface molecules-Often relies on fluorescent probes (usually flourochrome-conjugated antibodies) to enable assessment

3LymphocytesAll originate from HSC in bone marrow that are directed down the lymphoid lineage pathway

Named after the place they mature or undergo education before circulating in the blood

Non-activated cells are small with little cytoplasm but upon activation their cytoplasm and size is increased

4LymphocytesThymus-derived cells = T cells (CD3+ TCR+)CD4+ T cellsCD8+ T cells

Bone-marrow-derived cells = B cells (BCR+ CD19+)B cells BCR+ cells Plasma cells terminally differentiated B cells producing antibody and no longer displaying it on surface

Natural Killer Cells (CD3- BCR- CD56+)Granular appearance due to perforin and granzyme in cytolytic granulesDevelop in BM no education (Innate)

Cryo-electron microscopy reconstruction of a perforin poreLaw et al. Nature 2010. 447451 Granzyme - a family of serine proteases stored within lysosomal-granules of natural killer (NK) cells and cytotoxic lymphocytes (CTLs). Critical for the induction of target cell apoptosis, granzymes cleave intracellular substrates, triggering many apoptotic pathways to ensure target cells die. Granzymes also play a significant role in the immune defense against viruses, tumors and intracellular bacteria. More recent research demonstrated that granzymes have an extracellular role resulting in the destruction of tissues and vascular integrity that implicates granzymes in a number of inflammatory and age-related diseases.

The PRF1 gene provides instructions for making a protein called perforin. This protein is found in immune cells (lymphocytes) called T cells and natural killer (NK) cells, which destroy other cells. Perforin is involved in the process of cell destruction (cytolysis) and the regulation of the immune system.Perforin is a major component of structures called cytolytic granules within T cells and NK cells. One of the main ways in which T cells and NK cells destroy other cells is to transport and secrete these cytolytic granules, which contain cell-killing proteins, onto the membranes of the target cells. Perforin helps create a channel through the membrane, allowing cytolytic proteins to enter the cell and trigger it to self-destruct.5

In the gray area between innate and adaptive immune systems.

NK CellsGranular lymphocytesNo expression of TCRsNo expression of BCRs

Express receptors for:1. Stress molecules (KARs) 2. MHC class I molecules (KIRS)

NK T cellsExpress low levels of TCRs with limited repertoiresNatural Killer (NK) and Natural killer T (NKT) cells 6T Cell Receptor (TCR)T cells each T cell expresses a unique, epitope specific cell surface receptor

Heterodimers of two polypeptide chains

Lack the capacity to initiate signaling to the nucleus capacity and rely on CD3 to transmit signals

Unlike Ab can not bind soluble Antigens. See peptides in context of MHC

CD37Major Histocompatibility Complex (MHC)Tightly linked cluster of genes in all mammalsCalled Human Leukocyte Antigen (HLA) complexGene products (3 classes (I, II, III) play a role with important roles in the discrimination between self, nonself, and bad self.Central to T cell immune responses as they present Ag on structures enabling T cells to see Ag presenting structuresAn individuals set of MHC molecules influences the repertoire of Ag to which individual T cells can respondMay have a role in susceptibility to disease and in development of immunityOn Chromosome 6 8

MHC Class I, II, and IIILocated on Chrom 6; Human Leukocyte Antigen

http://nfs.unipv.it/nfs/minf/dispense/immunology/index.htmlMHC I 45KDa molecule one chain with 3 Ig domains also B2 immunoglobulin (soluble IgG) covalently associated to support MHCI stabilityMHC I on all nucleated cells what CD8+ T cells and NK cells are looking for help in virus cancer removal.3 loci in human A, B, C, MHCI fold to form a cleft between the a1 and a2 domains and non-convalently bind 8-9 AA peptides

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MHC Class I, II, and IIILocated on Chrom 6; Human Leukocyte Antigen

MHC II - are heterodimers of a and beta chainsHumans have 3 loci with an a,b region in eachA and beta chain noncovalently associate and Peptide grove formed can accomidate 18-20 AA

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http://www.tcells.org/scientific/abTCR/

http://nfs.unipv.it/nfs/minf/dispense/immunology/index.htmlT cells Respond to Peptide Ag Presented via MHC

Peptide recognition results in activation of signaling pathways and transcription factors regulating T cell functionsBrownlie and Zamoyska. Nature Reviews Immunology 13, 257-269 (April 2013)T cell receptor (TCR) signal transduction is initiated by the recognition of cognate peptideMHC molecules. The first molecule to be recruited to the TCRCD3 complex is the SRC family kinase (SFK) member LCK, which phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 chain, CD3 chain, CD3 chains and the -chains. Phosphorylation of the ITAMs enables the recruitment of ZAP70 (-chain associated protein kinase of 70 kDa), its phosphorylation by LCK and its activation. Activated ZAP70 phosphorylates four key tyrosine residues on linker for activation of T cells (LAT), which recruits numerous signalling molecules to form a multiprotein complex, termed the LAT signalosome. Important molecules that constitute this complex include phospholipase C1 (PLC1), growth factor receptor-bound protein 2 (GRB2), GRB2-related adaptor protein GADS, SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), adhesion- and degranulation-promoting adaptor protein (ADAP), interleukin-2-inducible T cell kinase (ITK), NCK1 and VAV1. The LAT signalosome propagates signal branching to three major signalling pathways, the Ca2+, the mitogen-activated protein kinase (MAPK) kinase and the nuclear factor-B (NF-B) signalling pathways, leading to the mobilization of transcription factors that are critical for gene expression and essential for T cell growth and differentiation. Signals initiated from the TCR also result in actin reorganization and the activation of integrins by inside-out signalling. AP1, activator protein 1; DAG, diacylglycerol; InsP3, inositol-1,4,5-trisphosphate; NFAT, nuclear factor of activated T cells; PKC, protein kinase C; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; RASGRP1, RAS guanyl-releasing protein 1; SKAP55, SRC kinase-associated phosphoprotein of 55 kDa.

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CD8+ T cells

Comprise one third of all T cellsRecognize antigen in complex with Class I MHC moleculesPotent cytotoxic functionsCytotoxic T cellsCD8 locks in on MHCI

Often called Cytotoxic T cells (CTL) dues to their ability to lyse cells. Critical for removal of cells that are infected with intracellular bacteria, virus, cancer13

CD4+ T cells

Comprise two thirds of all T cellsRecognize antigen in complex with Class II MHC moleculesProvide helper function to adaptive and innate immune cellsPoor direct cytotoxicityAmple production of modulatory cytokines

CD4 molecule help T cells see MHCII, locking mechanism, Thus CD4+ T cells are MHCII restricted

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Antigen Capture and Presentation 15

2 Types of MHC

MHC class I expressedon all nucleated cells

MHC class II expressed on APCs

Two subsets of T cells interact differently with MHC:

CD4+ T cells only interact with peptides bound to MHC class II

CD8+ T cells only interact with peptides bound to MHC class I17

Generation TCR diversity

1. Gene recombination in LC and HC chain generation and junctional diversity

2. A developing T cell randomly produces a unique light-heavy chain combination with unique specificity

The theoretical number of possible combinations produced within the body may be estimated to be the product of several possible light chains and several possible heavy chains

Estimated that 1 to 5 million epitope-binding combinations are possible for TCRs18Lymphoid tissues and organs - Primary Lymphoid organs

Primary Lymphoid organs Place where T and B cells learn to see self from non-self

Thymus: Bi-lobed organ wherepro-thymocytes from bone marrow turn into T cells

T cells acquire CD4, CD8, TCRSelf reactive cells removed

https://embryology.med.unsw.edu.au/embryology

Thymic education of T cellsThymic education: Many are "admitted;' but few "graduate:'

''Freshman"thymocytes are called double positive (DP) because they express both CD4and CD8 molecules in addition to T-cell receptors (TCRs). Positive selection:Thymocytes that recognize MHC class I (using CD8) or MHC class II (CD4)pass their first exam and are promoted; those that do not do this die. Negativeselection: Thymocytes that show strong interaction with MHC or peptide-MHCcombinations fail and meet an apoptotic death. Those few cells that survive thenegative selection are destined to "graduate" from the thymus as T cells.20

Presentation of Intracellular Antigens

Presentation of Extracellular Antigens

B cellsMature in the bone marrowExpress membrane bound antibody (Ig) on surfaceRecognize soluble AgNo MHC restriction

When Ab on the surface ofa B cell productively binds Ag for the first time, the B cell begins to divide rapidly to become plasma cell

Plasma cell secretes antibody; Small amount of membrane Ab; life span of a few days

Plasma cell Antatomically distinguishable from lymphocytes increased cytoplasm, golgi and ER reflecting their high protein output.24Antibodies or ImmunoglobulinsSynthesized by B lymphocytes (B cells)Synthesized and secreted by plasma cells terminally differentiated B cells Antibody is an immunoglobulin molecule with specificity for an epitope of an antigenAntibodies facilitate cells and molecules in the immune system to identify and interact with antigens.Soluble antibodies are components of humoral (soluble) immune responsesCan bind free Ag Ag is not MHC restricted25Antibodies, Immunoglobulin domains and Variable domains

http://www.blopig.com/blog/2013/07/high-resolution-antibody-modelling/The immunoglobulin domain is a type of protein domain that consists of a 2-layer sandwich of between 7 and 9 antiparallel -strands arranged in two -sheets with a Greek key topology, consisting of about 80 amino acids. The backbone switches repeatedly between the two -sheets.

Members of the immunoglobulin superfamily are found in hundreds of proteins of different functions. Examples include antibodies, the giant muscle kinase titin, and receptor tyrosine kinases. Immunoglobulin-like domains may be involved in proteinprotein and proteinligand interactions.[3]26

Immunoglobulin (Ig)

Basic structure: -4 polypeptide chains2 Heavy Chains2 Light Chains

-Ig Monomer can be more linked together via Fc region

Variable domains on HC And LC form epitopeBinding domain

Epitope = part of Ag

Fc - RegionThe immunoglobulin domain is a type of protein domain that consists of a 2-layer sandwich of between 7 and 9 antiparallel -strands arranged in two -sheets with a Greek key topology, consisting of about 80 amino acids. The backbone switches repeatedly between the two -sheets.

Fc (Fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.27

Ig Monomers

Contain 2 identical light chains and two identical heavy chains

Binding site for each monomer is identical

Diversity generated by:Gene recombination in LC and HC chain generation

Different pairings of heavy and light chains Property/FunctionIgGIgAIgMIgEIgDHeavy chaingamedHighest concentration in serumActivates complement

Crosses placenta

Present on B cell membrane

Binds to Fc receptors on phagocytes

Involved in mucosal immunity

Mast cell degranulation

Properties and Biological Activities of Immunoglobulin Isotypes

IgEIgAIgGIgDIgMPrimary Lymphoid organs - B CellsBone Marrow: lymphocytic lineages that become B cells stay and undergo differentiation here

If early IgM on surface recognizes self they undergo apoptotic death

30Cardiovascular system is responsible for circulating the soluble and cellular components of the immune system

Collection/filtration in the spleenLymphoid tissues and organs Lymphatic/circulatory system

Lymphoid tissues and organs SpleenSpleen Largest lymphoid organClears particulate from bloodConcentration of Ag and microbesLots of T cells and B cells (making antibody) Lots of Macrophages dead cell removal

Places of leukocyte accumulation and lymph filtration

Lymphoid tissues and organs Lymph nodes

Afferent lymphatic vessels enterthe cortical portion of the lymph node.leukocyte- and debris-rich lymphpercolates through the body of the lymphnode where it encounters phagocyticcells (macrophages and dendritic cells)that remove dead and dying cells,cellular debris, and microorganisms fromthe lymph.

The "scrubbed' lymph exitsthe lymph node via an efferent lymphaticvessel. The vessels of the cardiovascularsystem transport leukocytes to and fromthe lymph node.

33Extensive capillary network that drains the tissues and collects lymph

Lymph = watery clear fluid that contains leukocytes and cell debris

Drainage system to remove cellular debris and microbes from the bodys tissues to the lymph nodes

Lymphoid tissues and organs - Lymphatics

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