ORIGINS OF IMMUNE ORIGINS OF IMMUNE RESPONSERESPONSE

Mehtap KAÇAR KOÇAK MD. PhD. Mehtap KAÇAR KOÇAK MD. PhD. 20092009

Points to be discussedPoints to be discussedPoints to be discussedPoints to be discussed Monoclonal antibodiesMonoclonal antibodies CD (Cluster Determinants) classificationCD (Cluster Determinants) classification Origin and subsets of B cellsOrigin and subsets of B cells Origin and subsets of T cellsOrigin and subsets of T cells NK cellsNK cells Cytokines and chemokinesCytokines and chemokines Cell adhesion moleculesCell adhesion molecules Patterns and mechanisms of cell migrationPatterns and mechanisms of cell migration Immunoglobulins and gImmunoglobulins and generation of diversityeneration of diversity

CD (CLUSTER DETERMINANTS) CD (CLUSTER DETERMINANTS) CLASSIFICATIONCLASSIFICATION

CD (CLUSTER DETERMINANTS) CD (CLUSTER DETERMINANTS) CLASSIFICATIONCLASSIFICATION

Based on the identification of single epitopes by Based on the identification of single epitopes by monoclonal antibodiesmonoclonal antibodies

Involves mainly differentiation antigens of cells Involves mainly differentiation antigens of cells and cell receptorsand cell receptors

Other CD markers include various proteins, Other CD markers include various proteins, enzymes, complex lipids, adhesion moleculesenzymes, complex lipids, adhesion molecules, , cell receptorscell receptors etc. etc.

Most, but not all, CD markers are at cell surfaceMost, but not all, CD markers are at cell surface CD markers cover mainly hemopoetic cellsCD markers cover mainly hemopoetic cells Actual number of CD markers is about 250.Actual number of CD markers is about 250.

EXAMPLES OF CD MARKERSEXAMPLES OF CD MARKERSEXAMPLES OF CD MARKERSEXAMPLES OF CD MARKERS

CD3CD3 TCR signalling complex:TCR signalling complex: T cellsT cells CD4CD4 MHC class II receptor:MHC class II receptor: T cellsT cells CD8CD8 MHC class I receptor:MHC class I receptor: T cellsT cells CD10CD10 neutral endopeptidase:neutral endopeptidase: ALL cellsALL cells CD19CD19 co-receptor subunit:co-receptor subunit: B cellsB cells CD45CD45 LCA LCA (tyrosine phosphatase):(tyrosine phosphatase): leukocytesleukocytes CD62LCD62L L-selectin: L-selectin: T cells, mono-, T cells, mono-,

granulocytesgranulocytes CD247CD247 zeta chain of TCR :zeta chain of TCR : T cells, NK cellsT cells, NK cells

MARKERS AND SUBSETS OF MARKERS AND SUBSETS OF B CELLSB CELLS

MARKERS AND SUBSETS OF MARKERS AND SUBSETS OF B CELLSB CELLS

CD markers: CD19, CD20, CD21, CD22, CD35, CD markers: CD19, CD20, CD21, CD22, CD35,

CD40, CD72, CD80, CD86CD40, CD72, CD80, CD86 B1 cells: B1a (CD5B1 cells: B1a (CD5++) and B1b (CD5) and B1b (CD5--)) B1 cells: comprise about 20% B cells in blood B1 cells: comprise about 20% B cells in blood

and spleen of healthy people, secrete IgM onlyand spleen of healthy people, secrete IgM only B2 cells: majority of fully competent B cellsB2 cells: majority of fully competent B cells

CYTOKINES – KEY CONCEPTSCYTOKINES – KEY CONCEPTSCYTOKINES – KEY CONCEPTSCYTOKINES – KEY CONCEPTS

Cytokines have pleiotropic effects – they Cytokines have pleiotropic effects – they often have more than one receptoroften have more than one receptor

Cytokines can be redundant – their Cytokines can be redundant – their receptors often share subunitsreceptors often share subunits

Cytokines can have specific and unique Cytokines can have specific and unique functions – their receptors have ligand functions – their receptors have ligand specific subunits as wellspecific subunits as well

CYTOKINES – KEY CONCEPTSCYTOKINES – KEY CONCEPTS -2 -2CYTOKINES – KEY CONCEPTSCYTOKINES – KEY CONCEPTS -2 -2

Many immunologically relevant cytokines Many immunologically relevant cytokines are made by non-lymphoid cellsare made by non-lymphoid cells

Interleukins – may also act on Interleukins – may also act on nonhematopoetic cellsnonhematopoetic cells

Most of them act in autocrine or paracrine Most of them act in autocrine or paracrine fashionfashion

CYTOKINESCYTOKINESSIGNAL TRANSFERRING MOLECULESSIGNAL TRANSFERRING MOLECULES

CYTOKINESCYTOKINESSIGNAL TRANSFERRING MOLECULESSIGNAL TRANSFERRING MOLECULES

Interleukins directing other cells to divide and differentiateInterferons type I (alpha/beta), type 2-gammaColony stimulating factors (CSF) directing bone marrow stem cellsChemokines directing cell movementOther TNF, TNF, TGF – involved in inflammation,

cytotoxicity and immunosuppression respectively

Main properties of some interleukinsMain properties of some interleukins

IL-1 – proinflammatory, pleotropicIL-1 – proinflammatory, pleotropic IL-2 – growth factor for T, B and NK cellsIL-2 – growth factor for T, B and NK cells IL-4 – maturation and differentiation of B IL-4 – maturation and differentiation of B

cellscells IL-5 – maturation and differentiation of IL-5 – maturation and differentiation of

eosinophilseosinophils IL-6 – proinflammatory, differentiating IL-6 – proinflammatory, differentiating

agent for B cellsagent for B cells

Main properties of some interleukins-2Main properties of some interleukins-2

IL-10 – immunosuppressiveIL-10 – immunosuppressive IL-12 – strong activator of cellular IL-12 – strong activator of cellular

immune responseimmune response IL-15 – maturation of NK cells in bone IL-15 – maturation of NK cells in bone

marrowmarrow IL-17 – proinflammatory, pleotropicIL-17 – proinflammatory, pleotropic IL-18 – stimulates of interferon-IL-18 – stimulates of interferon-γγ

production by NK and T cellsproduction by NK and T cells

CHEMOKINES – CHEMOTACTIC CHEMOKINES – CHEMOTACTIC CYTOKINESCYTOKINES

CHEMOKINES – CHEMOTACTIC CHEMOKINES – CHEMOTACTIC CYTOKINESCYTOKINES

Ch. are small, soluble heparin-binding Ch. are small, soluble heparin-binding important proteins that regulate important proteins that regulate leukocyte traffickingleukocyte trafficking

Some of them are strategically located Some of them are strategically located on vascular endothelium, participating in on vascular endothelium, participating in the adhesion cascadethe adhesion cascade

They are divided into four families They are divided into four families defined by a cysteine motif – CXC, CC, defined by a cysteine motif – CXC, CC, C and CX3C (C is cysteine, X - any C and CX3C (C is cysteine, X - any aminoacid residue)aminoacid residue)

CHEMOKINES – CHEMOTACTIC CHEMOKINES – CHEMOTACTIC CYTOKINESCYTOKINES - 2 - 2

Different leukocyte subsets bear Different leukocyte subsets bear alternative sets of receptors to respond to alternative sets of receptors to respond to chemokinechemokine

SLC – secondary lymphoid ch. is located SLC – secondary lymphoid ch. is located on HEV, attracts T lymphocytes bearing on HEV, attracts T lymphocytes bearing SLC receptor CCR7SLC receptor CCR7

EXAMPLES OF CHEMOKINESEXAMPLES OF CHEMOKINES

Interleukin-8 (IL-8) – from monocytesInterleukin-8 (IL-8) – from monocytes RANTES – from T cellsRANTES – from T cells Eotaxin – from monocytesEotaxin – from monocytes MCP-1 – from monocytes, epitheliaMCP-1 – from monocytes, epithelia MIP-1alpha – from T cells, white cellsMIP-1alpha – from T cells, white cells IP-10IP-10 – from monocytes – from monocytes

CELL ADHESION MOLECULES (CAM)CELL ADHESION MOLECULES (CAM)CELL ADHESION MOLECULES (CAM)CELL ADHESION MOLECULES (CAM)

Integrins:Integrins:adhesion to endothelium and extracellular matrix adhesion to endothelium and extracellular matrix (VLA-1 to 6, LFA-1, LPAM, CR3, CR4)(VLA-1 to 6, LFA-1, LPAM, CR3, CR4)

CAM of the immunoglobulin supergene family:CAM of the immunoglobulin supergene family:various (ICAM-1-3, VCAM-1, PECAM-1, NCAM, CEA)various (ICAM-1-3, VCAM-1, PECAM-1, NCAM, CEA)

Selectins: Selectins: molecules on leucocytes and endothelium which bind molecules on leucocytes and endothelium which bind to carbohydrate (E, P, L-selectins, )to carbohydrate (E, P, L-selectins, )

CELL ADHESION MOLECULES (CAM)CELL ADHESION MOLECULES (CAM)-2-2

Cadherins: Cadherins: bind to catenins, cytoskeleton elements in bind to catenins, cytoskeleton elements in calcium dependent manner (E, N,T-calcium dependent manner (E, N,T-cadherins)cadherins)

CD44 and it variants:CD44 and it variants:cell hyaluronate receptor involved in cell-cell hyaluronate receptor involved in cell-to-cell and cell-to-matrix interactionsto-cell and cell-to-matrix interactions

CD4 Th1 CD4 Th1 andand Th2 Th2 T cells: T cells: profilprofile ofe of produ producedced cytokin cytokineses

Th1Th1 Th2Th2IILL-2-2 IL-4IL-4

IFN-gammaIFN-gamma IL-5IL-5

TNF-alphaTNF-alpha IL-6IL-6

LT-alphaLT-alpha IL-10IL-10

IL-13IL-13

REGULATORY (SUPRESSOR) T CELLS REGULATORY (SUPRESSOR) T CELLS (Treg)(Treg)

REGULATORY (SUPRESSOR) T CELLS REGULATORY (SUPRESSOR) T CELLS (Treg)(Treg)

Th2 cells – secrete IL-4Th2 cells – secrete IL-4 Th3 cells – secrete TGF-betaTh3 cells – secrete TGF-beta Tr1 cells – secrete IL-10Tr1 cells – secrete IL-10 CD4CD4++, CD25, CD25++, Foxp3 , Foxp3 T cellsT cells CD8CD8++, CD28, CD28-- T cells T cells Some cytotoxic T cellsSome cytotoxic T cells Some Some TCR TCR gamma/delta T cells gamma/delta T cells

CYTOTOXIC T CELL SUBSETSCYTOTOXIC T CELL SUBSETS(Tc)(Tc)

CYTOTOXIC T CELL SUBSETSCYTOTOXIC T CELL SUBSETS(Tc)(Tc)

TCR aTCR alpha/beta CD8lpha/beta CD8++ T cells T cells TCR aTCR alpha/beta CD4lpha/beta CD4++ Th1 cells Th1 cells TCR gTCR gamma/delta T cells (CD3amma/delta T cells (CD3++, CD4, CD4--, ,

CD8CD8--, CD16, CD16++)) NKT (NT) cells (CD3NKT (NT) cells (CD3++, CD4, CD4-+-+, CD8, CD8- -

CD56CD56++)) NK cells (CD2NK cells (CD2++, CD3, CD3--, CD4, CD4--, CD8, CD8--, CD56, CD56++

CD16CD16++))

FEATURES OF NK CELLSFEATURES OF NK CELLSFEATURES OF NK CELLSFEATURES OF NK CELLS

Belong to so called large granular Belong to so called large granular lymphocytes (LGL)lymphocytes (LGL)

Comprise about 10% lymphocytes in human Comprise about 10% lymphocytes in human bloodblood

Show spontaneous cytotoxic activity against Show spontaneous cytotoxic activity against infected and tumor cellsinfected and tumor cells

FEATURES OF NK CELLSFEATURES OF NK CELLS -2 -2FEATURES OF NK CELLSFEATURES OF NK CELLS -2 -2

Their cytotoxicity is inhibited by conventional Their cytotoxicity is inhibited by conventional MHC antigen expression on target cellsMHC antigen expression on target cells

Incidence of tumor formation is lower in Incidence of tumor formation is lower in individuals with high NK cell contentindividuals with high NK cell content

Beige mice lack NK cells – high incidence of Beige mice lack NK cells – high incidence of tumorstumors

T CELL ACTIVATION-EARLY STEPST CELL ACTIVATION-EARLY STEPS

Formation of immunological synapse Formation of immunological synapse – lymphocyte polarization, adhesion – lymphocyte polarization, adhesion to APC, maturation of synapseto APC, maturation of synapse

Microdomains (lipid rafts) – regions of Microdomains (lipid rafts) – regions of cell membranes rich in lipids: contain cell membranes rich in lipids: contain several proteins able for fast signal several proteins able for fast signal transduction, kinases from Src transduction, kinases from Src family, and otherfamily, and other

T CELL ACTIVATION-EARLY STEPS-2T CELL ACTIVATION-EARLY STEPS-2

Lymphocyte activation leads to Lymphocyte activation leads to microdomain grouping- so called microdomain grouping- so called supramolecular activation clusters-SMACsupramolecular activation clusters-SMAC

First (Ag-TCR) and second signal (CD28-First (Ag-TCR) and second signal (CD28-CD80, CD86, CD58-CD2) concept; naive CD80, CD86, CD58-CD2) concept; naive lymphocytes need 2 signals, activated lymphocytes need 2 signals, activated cells – only the first onecells – only the first one

Involves transduction of signals Involves transduction of signals from both T cell receptor and CD28from both T cell receptor and CD28

CD4 bound lck kinases become activated by CD4 bound lck kinases become activated by CD45 phosphataseCD45 phosphatase

ITAM domains of CD3 (zeta chains) become ITAM domains of CD3 (zeta chains) become phosphorylated by lckphosphorylated by lck

INTRACELLULAR SIGNALING INTRACELLULAR SIGNALING

IN T CELL ACTIVATIONIN T CELL ACTIVATION

INTRACELLULAR SIGNALING INTRACELLULAR SIGNALING

IN T CELL ACTIVATIONIN T CELL ACTIVATION

ITAMs associate with other kinases such as ITAMs associate with other kinases such as ZAP-70 and fynZAP-70 and fyn

Fyn activates phopholipase C (PLC) which Fyn activates phopholipase C (PLC) which cause release of intracellular calcium cause release of intracellular calcium (calcium flux)(calcium flux)

Calcium binds to calcineurin and activates Calcium binds to calcineurin and activates transcription factors (NF-AT, NF-kappa B, transcription factors (NF-AT, NF-kappa B, AP-1)AP-1)

INTRACELLULAR SIGNALING INTRACELLULAR SIGNALING

IN T CELL ACTIVATIONIN T CELL ACTIVATION

INTRACELLULAR SIGNALING INTRACELLULAR SIGNALING

IN T CELL ACTIVATIONIN T CELL ACTIVATION

SIGNALING IN B CELL ACTIVATIONSIGNALING IN B CELL ACTIVATIONSIGNALING IN B CELL ACTIVATIONSIGNALING IN B CELL ACTIVATION

Tyrosine kinases lck, lyn ,fyn become Tyrosine kinases lck, lyn ,fyn become activated via Igactivated via Ig and Ig and Ig of B cell of B cell receptorreceptor

They phosphorylate BCR ITAM They phosphorylate BCR ITAM domainsdomains

These can then bind Syk, another These can then bind Syk, another kinase, which activates phospholipase kinase, which activates phospholipase C (PLC-C (PLC-))

SIGNALING IN B CELL ACTIVATIONSIGNALING IN B CELL ACTIVATION-2-2SIGNALING IN B CELL ACTIVATIONSIGNALING IN B CELL ACTIVATION-2-2

PLC and three other pathways (Ras, PLC and three other pathways (Ras, RhO, RhO, PI-3K) lead to induction of transcription PI-3K) lead to induction of transcription factors such as NF-AT, AP-1 and NF-factors such as NF-AT, AP-1 and NF-kappa Bkappa B

Co-stimulators:CD40, CD19/CD21, Co-stimulators:CD40, CD19/CD21, CD22,CD22, CD CD3232

WHAT ARE TRANSCRIPTION FACTORS?WHAT ARE TRANSCRIPTION FACTORS?

AnswerAnswer: transcription factors are : transcription factors are complex protein molecules residing complex protein molecules residing in cytoplasm, which after stimulation in cytoplasm, which after stimulation and assembly are able to enter cell and assembly are able to enter cell nucleus and induce several genes nucleus and induce several genes transcription.transcription.

LEUCOCYTE-ENDOTHELIAL CELL LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONSINTERACTIONS

LEUCOCYTE-ENDOTHELIAL CELL LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONSINTERACTIONS

Leucocytes interact with the vessel wall Leucocytes interact with the vessel wall in multistep fashion, using several in multistep fashion, using several leucocyte surface molecules that leucocyte surface molecules that recognize their counter-receptors on recognize their counter-receptors on endothelial cellsendothelial cells

The rolling and tethering of leucocytes The rolling and tethering of leucocytes on vessel wall is mediated by selectins on vessel wall is mediated by selectins

LEUCOCYTE-ENDOTHELIAL CELL LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONSINTERACTIONS -2 -2

LEUCOCYTE-ENDOTHELIAL CELL LEUCOCYTE-ENDOTHELIAL CELL INTERACTIONSINTERACTIONS -2 -2

Chemokines and their receptors are Chemokines and their receptors are needed to activate leucocyte integrinsneeded to activate leucocyte integrins

Only activated integrins are able to Only activated integrins are able to mediate firm adhesion between mediate firm adhesion between leucocytes and endotheliumleucocytes and endothelium

The transmigration of leucocytes into The transmigration of leucocytes into the tissues requires proteinases and the tissues requires proteinases and repair mechanismsrepair mechanisms

LYMPHOCYTE RECIRCULATIONLYMPHOCYTE RECIRCULATIONLYMPHOCYTE RECIRCULATIONLYMPHOCYTE RECIRCULATION

Lymphocytes recirculate continuously Lymphocytes recirculate continuously between blood and lymphoid organsbetween blood and lymphoid organs

80% of lymphocytes enter the lymph 80% of lymphocytes enter the lymph nodes via specialized vessels called nodes via specialized vessels called high endothelial high endothelial venules (HEV)venules (HEV)

The remaining lymphocytes enter the The remaining lymphocytes enter the lymph nodes together with dendritic lymph nodes together with dendritic cells and antigens via afferent cells and antigens via afferent lymphaticslymphatics

LYMPHOCYTE RECIRCULATIONLYMPHOCYTE RECIRCULATION - 2 - 2LYMPHOCYTE RECIRCULATIONLYMPHOCYTE RECIRCULATION - 2 - 2

Lymphocytes leave the lymph nodes via Lymphocytes leave the lymph nodes via efferent lymphaticsefferent lymphatics

Lymphocyte recirculation allows the Lymphocyte recirculation allows the lymphocytes to meet their cognate lymphocytes to meet their cognate antigens and other leukocyte subsets to antigens and other leukocyte subsets to evoke an efficient immune responseevoke an efficient immune response

IMMUNOGLOBULINS IMMUNOGLOBULINS KEY CONCEPTSKEY CONCEPTS

IsotypeIsotype:: antigenic differences between antigenic differences between

classes, classes, subclasses subclasses andand types types

AllotypeAllotype:: antigenic differences between Ig antigenic differences between Ig

constant domains of various individualsconstant domains of various individuals

IdiotypeIdiotype:: antigenic differences within variable antigenic differences within variable

domains reflecting antigen binding sitedomains reflecting antigen binding site

IMMUNOGLOBULINS IMMUNOGLOBULINS KEY CONCEPTSKEY CONCEPTS - 2 - 2

Isotype Isotype (class)(class) switchingswitching:: the change of produced Ig the change of produced Ig (from IgM to other Ig)(from IgM to other Ig),,

usually in secondaryusually in secondary immune responseimmune response

Polyclonal IgPolyclonal Ig: a mixture of Igs having either kappa or : a mixture of Igs having either kappa or lambda chains (3:1 ratio in humans)lambda chains (3:1 ratio in humans)

Monoclonal IgMonoclonal Ig:: either kappa or lambda light chaineither kappa or lambda light chain – – incidence in tumors such as myelomaincidence in tumors such as myeloma

Immunoglobulin classesImmunoglobulin classes

IgGIgG – the most abundant Ig. Exists in 4 – the most abundant Ig. Exists in 4 subclasses (IgG1, IgG2, IgG3, IgG4)subclasses (IgG1, IgG2, IgG3, IgG4)

IgAIgA – exists as serum and secretory Ig – exists as serum and secretory Ig present on mucosal surfaces, 2 subclasses present on mucosal surfaces, 2 subclasses (IgA1 and IgA2).(IgA1 and IgA2).

IgMIgM – present in bloodstream is composed – present in bloodstream is composed of 5 molecules forming pentamer. Protects of 5 molecules forming pentamer. Protects from sepsis. Produced mainly in spleen.from sepsis. Produced mainly in spleen.

Immunoglobulin classes - 2Immunoglobulin classes - 2

IgD IgD –with IgM forms antigen receptor –with IgM forms antigen receptor on B cells. In serum in trace on B cells. In serum in trace amounts.amounts.

IgEIgE – anti-parasitic. Participates in – anti-parasitic. Participates in allergic reactions. Very short lifetime allergic reactions. Very short lifetime when free, but stable when bound to when free, but stable when bound to cell surface such as mast cells.cell surface such as mast cells.

COMPARISONS OF T- AND B- COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENSRECEPTORS FOR ANTIGENS

similaritiessimilarities Members of Ig superfamilyMembers of Ig superfamily Heterodimeric antigen-binding siteHeterodimeric antigen-binding site Divided into variable and constant Divided into variable and constant

domainsdomains Variable domains constructed by V(D)J Variable domains constructed by V(D)J

rearrangementsrearrangements

COMPARISONS OF T- AND B- COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENSRECEPTORS FOR ANTIGENS

SimilaritiesSimilarities -2 -2 Nongermline-encoded N-nucleotide Nongermline-encoded N-nucleotide

additions at V(D)J junctionsadditions at V(D)J junctions Exhibit allelic exclusionExhibit allelic exclusion Mature T and B cells display receptors Mature T and B cells display receptors

of one and only one antigenic specificityof one and only one antigenic specificity Negative selection for receptors with Negative selection for receptors with

self-antigen specific.self-antigen specific.

COMPARISONS OF T- AND B- COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENSRECEPTORS FOR ANTIGENS

differencesdifferences Ig binds native antigen in solution; TCR binds Ig binds native antigen in solution; TCR binds

processed antigen when presented by APCprocessed antigen when presented by APC

Ig can be secreted; TCR is notIg can be secreted; TCR is not

Somatic mutation of Ig genes; TCR genes – Somatic mutation of Ig genes; TCR genes – nevernever

COMPARISONS OF T- AND B- COMPARISONS OF T- AND B- RECEPTORS FOR ANTIGENSRECEPTORS FOR ANTIGENS -2 -2

DifferencesDifferences - 2 - 2 Isotype Isotype class class switching of Ig genesswitching of Ig genes

(from IgM to IgG or IgA or IgE)(from IgM to IgG or IgA or IgE)

Positive selection of TCR for self-MHC Positive selection of TCR for self-MHC recognition (MHC restriction)recognition (MHC restriction)

FEATURES OF IMMUNOGLOBULIN FEATURES OF IMMUNOGLOBULIN SUPERFAMILYSUPERFAMILY

Large family of ancestrally related Large family of ancestrally related genes genes (probably >100)(probably >100)

Most products involved in immune Most products involved in immune system function or other cell – cell system function or other cell – cell interactionsinteractions

FEATURES OF IMMUNOGLOBULIN FEATURES OF IMMUNOGLOBULIN SUPERFAMILYSUPERFAMILY - 2 - 2

Proteins exhibit domain structure of ca 110 Proteins exhibit domain structure of ca 110 amino acids, usually translated from a single amino acids, usually translated from a single exon and with a single intradomain disulfide exon and with a single intradomain disulfide bond, helping to stabilize the structurebond, helping to stabilize the structure

Examples include MHC class I and class II Examples include MHC class I and class II molecules, TCR, cytokine receptors, some molecules, TCR, cytokine receptors, some cell adhesion molecules and otherscell adhesion molecules and others