Specific adaptive immunity(T a B lymfocytes, antibodies)

PharmDr. Jitka Palich Fucikova, PhDJitka.fucikova@lfmotol.cuni.cz

Content of lecture

Basic components of specific immunity

• Immunoglobulines and TCR receptor

• Functions of T cells

• Functions of B cells

• Development of T and B cells and the formation

of antigen specific repertoire

• Minor populations of cells

Specific immune system

• Evolutionary younger, adaptive, antigen specific mechanisms (vertebrates)

• Distinguis antigens via high specific molecules (antibodies, antigen specific T cell receptors)

• Development of immune response is slower compare to innate immunity, takes several

days to weeks

• Important feautures is immunological memory

• It gets active after recognition of antigen

• Humoral – based on antibodies

• Cellular – based on T cells (Tc and Th)

Components of immune system (innate and adaptive immunity)

Summary

• The immune system is involved in maintaining the integrity of the organism maintaining tolerance to self-tissues and the ability to react to dangerous stimuli

• Antigen = substances which induce an immune response

• Immune response work with both humoral and cellular mechanisms and adaptive and innate immunity to execute its functions

Contents of lecture

• Basic components of specific immunity

Immunoglobulines and TCR receptor

• Functions of T cells

• Functions of B cells

• Development of T and B cells and the formation

of antigen specific repertoire

• Particular subpopulations of cells

• Defects of T and B cells function

Immunoglobulin molecules

• Two heavy chains are joined by disulfide bonds – to every heavy chain there are two light chains joined by another difulfide bonds• N terminus domains of H and L chains are variable (VH and VL) – details of their structure are individual for distinct clones of B cells• Other domains are constant – similar for chains of same type

Variable region – N terminus(binding of antigen)

Constant region– C terminus

• Heavy chains consist of 4 structurally similar domains (consist of 110-120 AC)• Spatial structure of domains is formed by polypeptides chains stabilized by disulfide bonds• Individual domains are joined by short connecting polypeptide chain• Light chains consist of two polypeptide domains• Molecules of distinct subclases of immunoglobulines (IgM, IgA) are consist of several

monomer units – joint together by J chain

Immunoglobulin molecules

The immunoglobulin heavy-chain isotypes are distinquished by the structure of their constant region

• 2 types of light chains – κ a λ – differ in primary structure of constant domains and are coded by different genes on different chromosomes

• Gene segment encoding constant regions of different isotypes of heavy chains are organised on one chromosome – encode heavy chains – μ, δ, γ, α a ϵ - imunoglobulines formed by these isotypes of heavy chains are called IgM, IgD, IgG (IgG1-4), IgA (IgA1, IgA2), IgE

Characterization of Ig antibodies

• IgM a IgD Located on B cells – form BCR Secreted IgM – in pentamer form – first type of producted antibodies during immune

response

• IgG Various subclasses differ capabilities bind C1 Binds to Fc receptors of fagocytes IgG binds to protein A some stafylococci

• IgA Two forms– mucosal and serum mucosal – molecules form an important part of protection against microorganisms

• IgE Protect mucosal against multicellular parasites Is a major cause of allergic reactions High-affinity receptors for IgE are located on the cell surface of mast cells and

basophils

Evolution of Ig after birth

Age (years)Gestational age

Receptor of B lymphocytes (BCR) = surface immunoglobulin

• BCR receptor forms the surface immunoglobuline

and associated signaling molecules

• Recognizes the antigen in its native form

• Immunoglobulin – two H and two L chains – H

chains are transmembrane proteins

• Surface immunoglobulines are usually IgM and IgD

subclass

• 2 transmembrane proteins Igα (CD79α) a Igβ

(CD79β) are asociated with BCR, these are

connected with cytoplasmic protein-tyrosin

kinases Src

T cell receptor (TCR)

• TCR receptor (antigen recognition) is associated

with complex of CD3 proteins important for signal

transmission

• In the process of antigen recognition TCR

cooperate with CD4 and CD8 coreceptors

• TCR recognize only MHC proteins with antigen

peptides fragments

• For full activation – T cell must recognize the

antigen on the cell surface of APC, first contact

occurs via nonspecific adhesion molecules, for full

signal T lymphocyte must contact via CD28

receptor costimulatory molecules (CD80, CD86) on

APC

T cell receptor(TCR)

CD nomenclature

• Most of the surface molecules of leukocytes are marked with CD nomeclature

• Originally chaotic system named the newly discovered molecules - the inventory

system, when the newly discovered molecules receive serial numbers

• The system does not say anything about the structural similarity or function of

these molecules - molecules has many alternative names that are related to their

structure or function

CD3 – T cells

CD4 – helper T cells

CD8 – cytotoxic T cells

CD86 – costimulatory molecules on APC

CD19 – B lymphocytes

CD34 – progenitor cells

Flow cytometry cell detection based on CD nomeclature

Summary

• Immunolgobulines are products of plasma cells which are differentiated from B

lymphocytes during their life cycle

• Molecules of antibodies consist of two heavy and two light chains, both of them are

having variable and constant region

• Constant region of heavy chain determines the imunoglobuline isotypes - IgG, IgA, IgM,

IgD, IgE

• Imunoglobulines binds to Fc receptors with their constant region

• Variable region determine the specifity of antibody for antigen

Content of lecture

• Basic components of specific immunity

• Immunoglobulines and TCR receptor

Functions of T cells

• Functions of B cells

• Development of T and B cells and the formation

of antigen specific repertoire

• Minor populations of cells

Subpopulations of T lymphocytes

• According to TCR are divided into αβ or γδ

• αβ-TCR the majority system in mammals and is divided into those who bear CD4

or CD8 coreceptor:

• CD8 = precursors of cytotoxic T cells – coreceptor MHC gp I

• CD4 = precursors of helper T cells – coreceptor MHC gp II

• Th1 – IL-2 a IFN-γ

• Th2 – IL4, IL-5, Il-6 a IL-10

• Regulatory T cells

• Th17

Cytotoxic T cells CD8 (Tc)

• Function: recognize cells infected with viruses or other intracellular parasites detect abnormal cells - the tumor cells

• Activation of reaction via APC:• Tc must recognise complex MHC gp I with antigen peptide on the cell

surface of APC, equipped with appropriate adhesive and costimulatory molecules (CD80, CD86)

• After initial contact with APCs Tc will proliferate and differentiate into mature clone effector cytotoxic cells

Three types of cytotoxic mechanisms:

1)Cytotoxic granules in Tc cytoplasm - protein perforin and proteases granzymes - after detection of targets granules will migrate to the plasma membrane close to the contact of the two cells – granules will be merged with the membrane and release the contents into the space between cells - peforin (like the complement proteinC9 creates pores in the membrane

1)Tc carry Fas ligand (FasL) - binds to apoptotic receptor Fas (CD95) present onthe surface of many cells - activation of cell death

3) Production of cytokines lymphotoxin - acts like FasL – in a less specific way

Cytotoxic T cells CD8 (Tc)

Helper T cells

• Immune response Th1 - inflammatory response• Immune response Th2 – help to B cells

• Function:• cooperation with the MF and their stimulation into activated MF - producing

stronger bactericidal mechanisms – NO• Pathogenic microorganisms (mycobacteria) can well survive even after the ingestion

of macrophage phagosomes• MF infected with the parasite produces hydrolytic fragments of parasite proteins -

these are associated with MHC II gp - there is a recognition of TCR - CD4

Th lymphocytes:

LFA-1 – adhesive receptorCD28 – costimulatoryreceptorIFN-γ – activation of MF

IL-12

Macrophage:

ICAM-1 – adhesive moleculeCD86 – costimulatory moleculeIL-12 – diferenciační faktor Th1

Immune response Th1 - inflammatory response

MIGRATION

Immune response Th1 - inflammatory response

Granulom:•Chronic inflammation - for something that MF can not be removed when the long-term resistance of pathogens within the MF

•TBC (Mycobacterium tuberculosis)•Syfilis (Treponema pallidum)•Lepra (Mycobacterium leprae)

Immune response Th1 - inflammatory response - defects

Immune response Th2 – help to B cells

• Function:• cooperation with B cells that were pre-stimulated with antigen recognition - interaction

leads to a multiplication of clones of B cells and their differentiation into plasma cells producing antibodies

• Help of Th2 cells to B cells is based on the secretion of cytokines (IL-4, IL5, IL-6) and direct intercellular contact

• Help of Th2cells to B lymphocytes is initiated by interactions of costimulatory receptor CD40 on B cells with CD40 ligand on the surface of Th2 - CD40L (CD154)

Th2 cells activates two types of B lymphocytes:

1) Same antigen

•B lymphocytes detected by BCR same antigen as APC (which elicited Th2 clone) processed and

expressed on MHC II gp (same complex as the initiator DC) = ideal situation

•Th2 provides direct help to B cells that were stimulated when recognition of the same

antigen

•Unnecessary signal via a costimulatory receptor - CD28, adhesion molecules and TCR are

sufficient

2) Different antigen

•B cells are activated by other antigen than APC used to activate Th2 – bystander help

Immune response Th2 – help to B cells

Reciprocal regulation of Th1 vs activities Th2

• Some types of infections (extracellular) are optimally suppressed by antibodies function

• Some types of infection (intracellular) are optimally suppressed by proper activity of macrophages dependent on Th1 cells

Wrong choice answers can be fatal

• the concentration ratio of two cytokines decides of Th1 or Th2 cells from precursor CD4 T cells– IL-12 a IL-4• IL12 – MF – Th1• IL4 – bazofily a mastocyty – Th2

• Cytokines produced by Th1 cells also promote the development of Th1 and strongly inhibit the development of Th2 cells and conversely, IL-4 and IL-10 products Th2 promote formation of Th2 and inhibit the development of Th1

NK cell (natural killers)

• Function: kill tumor cells and virus-infected cells

• Morfology: large granular lymphocytes, unlike T and B lymphocytes

they don´t have antigen specific receptors

• Recognise cells with abnormally low expression of MHC gp I (tumor cells, virus-infected cells – to espace the immunosurveilance of CD8 T cells)

• Important activators of NK cells are interferons α and β produced by various cells after viral infection

• Regulatory functions – they produce cytokines IFN-γ, IL-3, M-CSF

• Cytotoxic mechanisms:• Similar to Tc• Cytotoxic granules– perforin a granzym

Receptors of NK cells

•Stimulatory receptors - recognize structures present on the surface of various cell types - the signals provided by these receptors activate NK cell to use their cytotoxic mechanismsFc receptor - CD16 - when NK cell meets cell opsonized with IgG antibody, it get bind to the receptor CD16 on their Fc portion, it leads to aggregation of receptor and transferring the signal that activates the cytotoxic mechanisms = ADCC - cytotoxic reactions dependent on antibodies

Receptors of NK cells

• Inhibitory receptors - recognize MHC gp I. - signals that the cell gets through these receptors are negative - inhibit the cytotoxic mechanisms

immunoglobulin receptors = KIR (killer inhibitor receptors)kinase receptors with C-lectin

• The resulting reaction depends on predominance of stimulatory or inhibitory receptors - if the cell has enough normal MHC gp I peptides bearing - outweigh the negative signals and NK cell is not activated

Summary

• T cells play an important role in the process of antigen-specific cell-mediated and humoral

immune response

• They are divided into several cell types based on the expression of surface molecules,

cytokine secretion and functional properties

• CD4 Th cells have regulatory and helper functions

• CD8 Tc cells have effector functions - predominantly cytotoxic

• T cells must recognise antigens in the complex with MHC class I and II on the cell surface

of APC

• Although NK cells belonging to the non-specific parts of the immune system, their

morphology and functions are very similar cytotoxic lymphocytes Tc

Content of lecture

• Basic components of specific immunity

• Immunoglobulines and TCR receptor

• Functions of T cells

Functions of B cells

• Development of T and B cells and the formation

of antigen specific repertoire

• Particular subpopulations of cells

• Defects of T and B cells function

The humoral immune response

• Function:

• Recognition of antigen by antigen specific receptor of B cells – leads to multiplication of B cells – resulting clones and differentiate into plasma cells producing antibodies

• B cells response to bacterial polysaccharides do not require peptide-specific T-cell help• B cells response based on Th2 activation

B cells response to bacterial polysaccharides do not require peptide-specific T-cell help

• Only low-affinity antibodies IgM are formed!!

• T independent antigens

• TI-1 – bacterial lipopolysacharide (LPS) – in higher concentrations

it binds to B cell receptor for LPS nonspecific stimulation

• TI-2 – polymers –microbial surface polysaccharides

(pneumococci) or polymeric proteins (flagellin - in flagellum) -

these polymeric antigens react simultaneously with a large number

BCR and cluster them

B cells response based on Th2 activation Primary phase of antibody response (in secondary lymphoid structures)

• Stimulation of B cells with antigen binding to the BCR

• APC phygocytize and present on MHC gp II antigen to Th cells

form antigen specific Th2 cells clones which recognise antigen

stimulated B cells and provide them with signals

which leads to multiplication, differentiation

into plasma cells and production of antibodies

• Part of the cells does not transform into plasma cells, but

convert into memory cells that can continue to develop

and initiate secondary phase response

• Antibodies form immunocomplexes with - these are trapped in the lymph nodes or other

lymphoid organs on the surface of follicular DC (FDC) in primary lymphoid follicles = antigen

presenting cells to B cells

Secondary phase of response

• After antigen recognition on FDC (folicullar DCs) in primary follicles and after receiving

the signal from the specific Th, B cells begin a new round of division and differentiation -

leads to intense mutations in V genes segments for H and L chains and it produces

mutant clones of B cells bearing BCR whose binding sites are different

• Intensive proliferation – massive cell death of B cells formation of secondary

lymfoid foliculles with germinal centre contains proliferative cells, FDC and Th = germinal

reaction – leads to sotmatic mutation – will give rise to antibodies with higher specifity

• Isotype switch – substitution of constant part of produced antibodies – IgG, IgA, IgE

B cells response based on Th2 activation

B cells response based on Th2 activation

Functions of antibodies• Neutralization:Antibodies prevents bacterial adherence and also inhibit activity of toxins, viruses and other

microorganisms by binding to their important epitopes for toxic activities

• Opsonization:Antibodies promotes phagocytosis by APC • Complement activation:Antibody activates complement, which enhances opsonization and lyses some bacteria

Summary

• Antibodies against specific antigens are produced by plasma cells

• Plasma cells are the final stage of differentiation of B cells

• T-independent antigens - are able to stimulate B cells directly, while the majority of

antigens, particularly proteins causes production of antibodies only if there is a

partnership of B lymphocytes with helper T cells

• In the first phase low affinity antibodies are formed

• In the secondary phase of immune reaction occurs intense somatic mutation of

immunoglobulin genes and selecting clones producing antibodies with high affinity for

the antigen and isotype rearrangements leading to the formation of other isotypes of

antibodies IgG, IgA, IgE

Content of lecture

• Basic components of specific immunity

• Immunoglobulines and TCR receptor

• Functions of T cells

• Functions of B cells

Development of T and B cells and the

formation of antigen specific repertoire

• Particular subpopulations of cells

Antigen specifity – clonal theory

1. A progenitor cell gives rise large number of lymphocytes with different specificity

2. Removal of potential autoreactive immature cells - by clonal deletion

3. Creates a palette of mature naive lymphocytes, which are prepared to recognize foreign antigenic structure

4. Proliferation and differentiation of activated specific T lymphocytes - creating a clone of effector cells

V D J

Rearrangement of genes encoding variable chains of BCR

• Genes encoding immunoglobulin chains (similar TCR) are composed of several types of segments = gene segments which are subject to the process of rearrangement

• Genes encoding the H chain are located in the gene complex containing several similar gene sections called V (variability), a group of 50 short sections D (diversity) and 9 short sections J (joining)

• Recombination process =processes occurring at the gene level in the differentiation process of B cells, accompanied by rearrangement of the genes for immunoglobulinesInside B lymphocytes progenitor:

• Excision of the sections between D and J segment (DJ rearrangement) - both chromosomes in parallel

• Excision of large sections between the V segment and the DJ segment (VD rearrangement)If successfully grouped - on the second chromosome rearrangement is stopped = allelic exclusion

Elimination of autoreactive clones of B lymphocytes

• Recombination process give rise to a rich repertoire of clones of B cells provided individually

specific BCR based on random processes - can also arise clones bearing autoreactive

receptors that produce autoreactive antibodies

• Most of them are eliminated in the stage of immature B cells - if a cell encounters at this

developmental stage of the antigen, which in this phase binds with sufficiently high affinity

to the BCR - cell gets a negative signal occurs apoptotic death

• Key steps in the evolution of B lymphocyte:

1) Completion of gene rearrangement for the H chain and surface expression of the pre-BCR

2) Successful completion of the rearrangement of genes for L chain and surface expression

of IgM (BCR)

3) Testing autoreactivity of immature B cells

4) The process of maturation of somatic and affinity maturation, which survive only cells

carrying the BCR with the highest affinity for the antigen

Life cycle of B lymphocytes

Life cycle of T lymphocytes

Life cycle of T lymphocytes

Summary

• The main feature of specific immunity is the ability to recognize different antigen

structure using BCR and TCR receptors or antibodies

• Huge clonal diversity of these receptors in humans is based on the unique processes

occurring during the development of B and T lymphocytes on the gene level

• Genes encoding the variable regions of immunoglobulins and TCR are composed of

groups of large gene segments called V, D and J

• Selected sections of V, D, J are with the help of enzymes recombinases randomly selected

and rearange into unique VDJ combinations

Content of lecture

• Basic components of specific immunity

• Immunoglobulines and TCR receptor

• Functions of T cells

• Functions of B cells

• Development of T and B cells and the formation

of antigen specific repertoire

Particular subpopulations of cells

Innate like lymphocyte

1) Intraepitelial T lymphocytes

•Mucosal and epidermis

•γδ and αβ lymphocytes

•Proportional representation is different for each species

•Differentiate out of thymus and the task is not sufficiently known - most likely play a

role in the first line of defense on a very exposed areas of body surface or regulate the

production of mucosal immunity with specific cytokines

2) NK-T cells

•TCR αβ – also other stimulatory and inhibitory receptors similar to NK cells

•distinguish complexes of CD1d molecules - with glycolipids of microbial origin

•Source of cytokines with regulatory functions