Specific adaptive immunity
(T a B lymfocytes, antibodies)
PharmDr. Jitka Palich Fucikova, PhD
Jitka.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
Variable region – N terminus
(binding of antigen)
Constant region– C terminus
• 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
Immunoglobulin molecules
• 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
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
Gestational age
Age (years)
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
kinases Src
with
cytoplasmic
protein-tyrosin
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
Cytotoxic T cells CD8 (Tc)
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 protein
C9 creates pores in the membrane
2) Tc carry Fas ligand (FasL) - binds to apoptotic receptor Fas (CD95) present on
the surface of many cells - activation of cell death
3) Production of cytokines lymphotoxin - acts like FasL – in a less specific way
Helper T cells
• Immune response Th1 - inflammatory response
• Immune response Th2 – help to B cells
Immune response Th1 - inflammatory response
• 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:
IL-12
Macrophage:
LFA-1 – adhesive receptor
ICAM-1 – adhesive molecule
CD28 – costimulatoryreceptor CD86 – costimulatory molecule
IFN-γ – activation of MF
IL-12 – diferenciační faktor Th1
Immune response Th1 - inflammatory response
MIGRATION
Immune response Th1 - inflammatory response - defects
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 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)
Immune response Th2 – help to B cells
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
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
mechanisms
Fc 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
B cells response based on Th2 activation
 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
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
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 immunoglobulines
Inside 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
V
D
J
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
• Precursors of T cells generated in the bone marrow are transferred to the thymus and are
called pro-thymocytes
• Thymus – firstly rearangement of genes for TCRβ - after the successful rearangement pre-TCR
occurs on the surface of thymocytes (composed of β chain and the CD3 complex) - followed
by rearrangement of the TCRα and the formation of the final TCR composed of α and β
chains
Negative selection = elimination of autoreactive cells
Positive selection = elimination of cells with broken TCR
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