SOPHOMORE DENTAL/OPTOMETRY MICROBIOLOGY SECTION: IMMUNOLOGY

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1 SOPHOMORE DENTAL/OPTOMETRY MICROBIOLOGY SECTION: IMMUNOLOGY LYMPHOCYTE ACTIVATION Lecturer: Louis B. Justement, Ph.D Objectives: To review the structure of the T and B cell antigen receptor complexes, what they recognize and the nature of the signals that are transduced leading to lymphocyte activation. To become familiar with the additional receptors and ligands involved in promoting lymphocyte activation and differentiation. Reading Assignment: Kuby Immunology 6 th edition Chapter 10, pp and 11, pp LYMPHOCYTE ACTIVATION The activation of lymphocytes requires two distinct signals the first signal is provided by binding of antigen to the antigen receptor and the second signal is provided through costimulatory or accessory receptors this ensures specificity and selectivity of the immune response against foreign antigens The response of lymphocytes to antigens and second signals consists of several steps: Synthesis of new proteins: shortly after the encounter with antigen, lymphocytes transcribe genes that were previously silent, which synthesize proteins including cytokines and their receptors, costimulatory molecules and proteins involved in cell division Cellular proliferation: in response to antigen and other factors, antigen-specific lymphocytes enter the cell cycle and begin to proliferate to expand the number of antigen-specific cells (i.e. clonal expansion) Differentiation into effector cells: Progeny of the antigen-stimulated lymphocytes differentiate into effector cells that are responsible for elimination of foreign antigen. Homeostasis decline of immune response: after the immune response has run its course, the system returns to its basal state due to death of most of the antigenspecific lymphocyte progeny via apoptosis or programmed cell death decline occurs in conjunction with the clearance of foreign antigen

2 Differentiation into memory cells: some of the progeny of antigen-stimulated cells do not differentiate into effector cells but become functionally quiescent memory cells that are capable of surviving for an extended period of time LYMPHOCYTE ANTIGEN RECEPTORS B Cell Antigen Receptor (BCR): The BCR consists of two heavy chains and two light chains that are disulfide bonded to form a heterotetrameric structure Both the heavy and light chains contain variable domains at the amino terminus that exhibit sequence hypervariability within three CDRs The pairing of heavy and light chain variable domains forms the antigen binding site and each BCR molecule has the ability to bind two antigenic molecules All antibody molecules produced by a particular B cell have identical VH and VL regions and thus the same antigen specificity Antibody molecules can be displayed on the surface of the B cell or secreted into the surrounding environment or both The membrane-bound form of antibody is referred to as membrane immunoglobulin or the B cell antigen receptor (BCR) Mature, resting B cells express two isoforms of membrane immunoglobulin - membrane IgM and membrane IgD both isoforms of mig have the same VH and VL regions and therefore recognize the same antigen Importantly, the BCR can recognize many forms of antigen and antigen recognition is NOT restricted by other lymphoid surface molecules such as MHC Class I or II The membrane-bound forms of IgM and IgD possess a hydrophobic transmembrane domain, which contains charged amino acids, and a cytoplasmic tail that is only 3 amino acids long BCR Complex (CD79a/b): The BCR is non-covalently associated with one heterodimeric complexe - the heterodimer consists of one Igα (CD79a) subunit disulfide bonded to one Igβ (CD79b) subunit The Igα/Igβ heterodimer serves both a transport as well as a signal transducer function The cytoplasmic domains of both Igα and Igβ contain ITAMs that are important for signal transduction in response to binding of antigen to the BCR The Igα/Igβ heterodimer serves to recruit PTKs to the BCR complex - association of PTKs with the ITAMs of Igα and Igβ promotes activation of multiple signaling pathways that regulate the B cell response to antigen

3 T Cell Antigen Receptor (TCR): The TCR complex is comprised of an antigen recognition structure and a transport/transducer component The antigen recognition structure or TCR is comprised of either a disulfide-linked αβ or γδ heterodimer Each chain has two immunoglobulin domains containing an intrachain disulfide bond The amino-terminal domain in both chains exhibits marked sequence variation and is termed that variable domain whereas the membrane-proximal domain in more conserved and is therefore called the constant domain TCR variable domains contain three hypervariable domains or complimentarity determining regions (CDRs) The TCR exhibits dual specificity recognizing both polymorphic regions of either MHC class I or II as well as peptides that are bound to either class I or class II thus it is important to understand that the TCR recognizes antigen in the context of a particular self MHC class I or II molecule The transmembrane domain of the TCR polypeptides contain positively charged amino acids that mediate non-covalent interactions with associated transport/transducer elements TCR Complex (CD3): The TCR is non-covalently associated with the CD3 complex, which serves both a transport and signal transduction function CD3 is a complex of 5 invariant polypeptide chains that associate to form three dimers: a heterodimer of gamma and epsilon a heterodimer of epsilon and delta a homodimer of two zeta chains or occasionally a zeta/eta heterodimer The zeta and eta polypeptides are encoded by the same gene and differ at the carboxyterminal end due to alternative RNA splicing The cytoplasmic tails of the CD3 complex proteins contain one (γ, δ, ε) or more (ζ, π) motifs called the immunoreceptor tyrosine-based activation motif (ITAM), which has the consensus sequence D/E-X 7 -D/E-X 2 -Y-X 2 -L/I-X 7 -Y-X 2 -L/I The ITAMs are important for recruitment and activation of protein tyrosine kinases (PTK) that initiate signal transduction in response to binding of antigen TCR Accessory Molecules (CD4 and CD8): T cells can be divided into two subpopulations based on their expression of CD4 or CD8

4 CD4+ T cells recognize antigen that is bound to MHC Class II molecules and function primarily as helper T cells CD8+ T cells recognize antigen that is bound to MHC Class I and function primarily as cytotoxic T cells The extracellular domains of CD4 and CD8 exhibit the ability to bind to non-polymorphic regions in the membrane-proximal domains of MHC Class II (β2 domain) and Class I (α3 domain), respectively Binding of CD4 and CD8 is thought to increase the avidity of the TCR interaction with MHC/peptide and thus provide a stronger signal to the T cell CD4 and CD8 also serve an important signal transduction role during T cell activation as they are associated with the Src family protein tyrosine kinase Lck, which phosphorylates the ITAM motifs on the CD3 complex polypeptides Adhesion Molecules: The affinity of TCRs for peptide-mhc is low to moderate (K d 10-4 to 10-7 ) unlike the BCR, which binds to antigen with moderate to strong affinity (K d 10-6 to ) Additional molecules strengthen the interaction between T cells and antigen-presenting cells or target cells including CD2, and LFA-1, which bind to LFA-3 and ICAM-1, respectively Adhesion receptor-dependent contact between a T cell and APC allows the T cell to scan the APC for peptide-mhc complexes TCR-mediated activation also results in upregulation of adhesion molecules expression to promote further T cell activation via cytokines and other co-stimulatory receptors TCR AND BCR MEDIATED SIGNALING Compartmentalization of Function Within Receptor Subunits: Both the T and B cell express multimeric receptor complexes that contain antigen recognition polypeptides (migm or migd on the B cell, or αβ, γδ on the T cell) as well as polypeptides that transduce signal to the interior of the cell (CD3 on the T cell, Igα/Igβ on the B cell) Dephosphorylation of Key Src Protein Tyrosine Kinases by CD45: The transmembrane protein tyrosine phosphatase CD45 functions to maintain Src family PTKs in an active state so that they can initiate signal transduction in response to antigen binding and cross-linking of either the TCR or BCR Initiation of Signal Transduction by Membrane-Associated Src PTKs:

5 The Src PTKs Lck and Fyn in the T cell and Lyn in the B cell mediate phosphorylation of the ITAMs in signal transducer subunits thereby promoting their ability to recruit intracellular signaling proteins Assembly of Large Signaling Complexes Based on Protein Tyrosine Phosphorylation: Phosphorylation of the ITAMs in CD3 and Iga/Igb promotes recruitment of the PTKs (ZAP 70 in the T cell and Syk in the B cell) that phosphorylate adapter proteins and downstream enzymes thereby propagating the signal initiated in response to antigen binding Activation of Specific downstream Signaling Pathways: The initial activation of PTKs leads to the subsequent production of second messengers and the activation of serine-threonine kinases and phosphatases. Activation of Syk and ZAP-70 leads to activation of PLCgamma, which hydrolyzes PIP2 to produce the second messengers IP3 and DAG. IP3 causes Ca2+ levels in the cell to rise leading to activation of the serine phosphatase calcineurin. DAG activates the serine protein kinase PKC. Additional small molecular weight G proteins are activated by Syk and ZAP-70 which mediate activation of the serine-threonine MAP kinases (Erk, Jnk and P38). Changes in gene expression: The activation of intermediate serine-threonine kinases and phosphatases promotes activation of transcriptional regulators that translocate to the nucleus and promote gene transcription. Calcineurin promotes activation of NF-AT, PKC promotes activation of NF-kB and the MAP kinases mediate activation of Fos and Jun which comprise the AP-1 complex. These transcription factors work in concert to regulate gene transcription most importantly upregulation of IL-2 and IL-2R expression BCR Coreceptors: Signals transduced via the BCR complex are modulated by coreceptors, which either potentiate or attenuate the signal Coreceptors provide contextual information to the B cell concerning its location, the type of antigen and the state of the immune response In general, coreceptors are transmembrane glycoproteins that are phosphorylated on cytoplasmic tyrosine residues in response to BCR-dependent signaling Tyrosine phosphorylation of coreceptors creates phosphotyrosine motifs that function as docking sites for intracellular effector proteins that contain SH2 or PTB domains When coreceptors are brought into close proximity to the BCR complex, their associated

6 intracellular effector proteins have the ability to modulate signal transduction via the BCR complex CD19: Present in the membrane as part of a multimeric complex including CD21, CD81 and Leu-13 CD19 is recruited to the BCR via CD21 CD21 binds to the C3d complement component thus complement-coated antigen is bound by the BCR and CD21, the physical bridge between the BCR and CD21 serves to recruit CD19 to the BCR complex Tyrosine phosphorylation of CD19 promotes the recruitment of effector proteins that enhance BCR signal transduction CD19 increases the initial B cell response to bacterial pathogens CD22: Constitutively associated with the BCR complex is resting B cells Phosphorylation of CD22 promotes the recruitment of inhibitory effector proteins (the SHP-1 protein tyrosine phosphatase), which attenuate BCR-dependent signaling CD22 may be physically removed from the B cell surface or sequestered in lymphoid-rich regions thereby allowing the B cell to respond more effectively to antigenic stimulation CD22 therefore, may be important for preventing B cells from responding to antigen unless they are located in the appropriate immunological environment Other Coreceptors: FcγRIIb, PIR-B CO-STIMULATORY SIGNALS T Cell Co-stimulation: Optimal T cell activation is dependent on two distinct signals that promote proliferation and differentiation into effector cells: The initial signal is generated by the interaction between peptide-mhc and the TCR The second antigen-nonspecific co-stimulatory signal is provided primarily by the interaction between CD28 on the T cell with members of the B7 family on the APC There are two related forms of B7, B7-1 and B7-2 both are members of the Ig superfamily Both B7-1 and B7-2 are constitutively expressed on dendritic cells and are inducibly expressed on macrophages and B cells. The ligands are CD28 and CTLA-4 (CD152) and are expressed on T cells CD28 and CTLA-4 act antagonistically whereas CD28 is expressed on both resting and

7 activated T cells, CTLA-4 is inducibly expressed in response to T cell activation Signals delivered through CD28 promote T cell proliferation and differentiation they also prevent the development of clonal anergy a state of antigen-specific nonresponsiveness CTLA-4 is required to maintain homeostasis of the immune response and to promote resolution of the immune response Only professional APC (i.e. dendritic cells, macrophages and B cells) are able to present antigen in the context of MHC Class II and to deliver the co-stimulatory signal because dendritic cells constitutively express high levels of MHC class I and II as well as high levels of B7-1 and B7-2 they are very potent activators of naïve, memory and effector T cells B Cell Co-stimulation: The response to T-dependent soluble antigens requires two signals to induce full activation of the B cell: The first signal is delivered by binding of antigen to the BCR The second signal is delivered by CD40 ligand expressed on the T cell, which interacts with CD40 on the B cell Binding of antigen to the BCR is followed by internalization, processing and presentation in the context of MHC Class II Signals delivered via the BCR complex lead to upregulation of MHC Class II expression as well as B7 expression to enhance presentation of antigen to T cells Once a TH cell recognizes processed antigenic peptide in the context of MHC Class II on the B cell, the two cells form a T-B conjugate The formation of the T-B conjugate promotes upregulation of CD40L on the T cell, which then binds CD40 on the B cell thereby providing to co-stimulatory signal additionally, the T-B conjugate promotes the directional release of cytokines from the TH cell to the B cell CD40-dependent signals are essential for B cell proliferation, isotype switching, somatic mutation and memory cell formation