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Study Materials, Module IB
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1. Phagocytosis
Phagocytosis - basic tool of the innate immunity. Professional phagocytes - neutrophils (elimination of
extracellular bacteria), monocytes, macrophages (elimination of intracellular parasites and apoptotic
bodies).
1. Passage of blood phagocytes through capillary wall into surrounding tissue (diapedesis,
extravasation)
Phagocytes bind to endothelium (interaction between selectins of endothelial cells and surface
carbohydrate structures such as sialyl-Lewis x antigen of phagocytes), which slows down their movement
in the circulation (rolling). Subsequently, phagocyte integrins bind to adhesion molecules of endothelial
cells (ICAM-1 or VCAM-1 depending on the type of phagocyte) and phagocytes pass through capillary
wall into surrounding tissue.
2. Chemotaxis
Phagocyte migration in the tissue is directed by concentration gradient of chemotactic factors (e.g. IL-8,
leucotrien B4, C5a and C3a complement fragments, bacterial peptides such as fMLP) produced at the site
of inflammation.
3. Recognition of target structures
Microorganisms have on their surface molecules (PAMPs = Pathogen Associated Molecular Patterns)
which are not present on cells of human body. The cells of innate immunity recognize these molecules
using receptors such as lectins (proteins binding carbohydrate structures), CD14 (receptor for bacterial
lipopolysaccharide), Toll-like receptors (binding viral DNA or RNA, bacterial flagellin, etc.).
4. Engulfment of target structures (ingestion)
Phagocytes extend pseudopods around the particle until it is completely enclosed in a vacuole
(phagosome).
5. Elimination of target structures
Phagosome fuses with lysosomes (phagolysosome). The pathogen is then destroyed by microbicidal
substances such as proteolytic enzymes, lactoferrin, defensins, reactive oxygen intermediates, nitric oxide,
etc.
Other cells of the innate immunity
Eosinophils take part in allergic reactions and defense against multicellular parasites (they release
substances impairing the parasite)
Basophils and mast cells play a crucial role in immune response against multicellular parasites and in
pathogenesis of allergic diseases. They express high affinity receptors for IgE (FcRI) and thereby become
coated with IgE antibodies (sensitized). The allergen or antigenic epitopes of parasite bind to Fab
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fragments of IgE (antigen binding site), which results in aggregation of Fc receptors and subsequent mast
cell degranulation. Substances released from mast cells support events leading to parasite clearance (e.g.
histamine with bronchoconstriction effects evokes coughing up of parasites living in the respiratory tract).
NK cells (natural killer cells) destroy virus-infected and malignant cells (mainly cells with low
expression of MHC molecules). They express both inhibitory (binding MHC molecules) and activating
receptors. If activating receptors are engaged, a „killing” instruction is issued to the NK cell, which is
normally overridden by an inhibitory signal triggered by inhibitory receptors after recognition of MHC
molecules. NK cells use cytotoxic mechanisms of T cells (perforins and granzymes) and antibody
dependent cell-mediated cytotoxicity (NK cell recognizes a cell coated with antibodies through Fc
receptors and subsequently releases cytotoxic substances from its granules).
Fig.1 Diapedesis of neutrophils and subsequent phagocytosis
2. B cells and immunoglobulins
B cells – cells of specific (acquired) immunity, after antigen recognition by antigen specific receptors,
they differentiate into plasma cells producing antibodies (immunoglobulins)
Immunoglobulins – glycoproteins composed of two identical heavy (H) and two identical light (L)
polypeptide chains. H chains contain 1 variable and 3 eventually 4 constant domains (depending on
immunoglobulin isotype). L chain possesses 1 variable and 1 constant domain. Variable domains of H and
L chain combine together to form an antigen binding site. The heavy chain type (, µ, , , ) determines
immunoglobulin isotype (class), (IgA, IgM, IgG, IgE, and IgD).
Monoclonal antibodies – produced by one clone of plasma cells, are of identical specifity and isotype,
under physiological conditions present in very low concentration, increased level in individuals with
myeloma (tumor growth of plasma cells). Monoclonal antibodies can be prepared in vitro by hybridoma
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technology (fusion of B cells with myeloma cell culture) and are used in therapy (e.g. tumor
immunotherapy) or as reagents in research.
Fig.2 Basic structure of immunoglobulin
Functions of antibodies

neutralization of bacterial toxins, activity of viruses and other microorganisms (bond to crucial
epitopes)

opsonization (bond to antigenic epitopes) – facilitation of phagocytosis, mediation of antibody
dependent cell-mediated cytotoxicity (ADCC)

protection of mucosa (mucosal IgA, eventually IgM)

activation of the classical complement pathway (binding of C1q complement component to Fc region
of IgG or IgM)

degranulation of mast cells and basophils – protection against multicellular parasites, mediation of
allergic reaction (IgE)

regulation of the immune response (idiotypic network, competition with BCR for antigen)
Antigen specific receptor of B cells (BCR)
•
surface immunoglobulin (antigen recognition)
•
transmembrane proteins CD79 and CD79 associated with cytoplasmic protein-tyrosine kinases of
the Src family (signal transduction into the cells)
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B cell development
B cells develop in the bone marrow, where they undergo rearrangement of genes encoding for variable
immunoglobulin domains. Gene rearrangement (recombination) enables existence of large repertoire of B
cells and subsequently antibodies which differ from each other in antigen specifity. Genes for H and L
immunoglobulin chains are composed of V, D (only in H chain), J, and C segments. V, (D), and J
segments encode together for variable domain of immunoglobulin chain (each type of gene segments
encodes for certain number of aminoacids), C segments encode for all constant domains.
Rearrangement of gene segments for variable immunoglobulin domains
•
D-J (DNA region between any D and J gene segment is enzymatically cut out), V-DJ rearrangement of
gene segments for H chain (DNA region between any V segment and DJ complex is cut out)
•
µ chain formation, pre-B cell receptor expression (µ chain, substitutive L chain, and associated
transmembrane chains with signaling function)
•
V-J rearrangement of gene segments for L chain
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expression of complete BCR with surface IgM
•
alternative splicing (simultaneous formation of surface IgM and IgD in mature naive B cells
Fig.3 Rearrangement of gene segments for immunoglobulins
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B cell activation - takes place in secondary lymphoid organs, in two phases (primary and secondary
response). Primary response – B cells recognize antigen by antigen specific receptors (BCRs). Antigen
presenting cells (e.g. dendritic cells) process this antigen (engulfment and cleavage) and display antigenic
peptides in a complex with MHC class II molecules to precursors of helper T cells (Th) which recognize
these complexes by their antigen specific receptors (TCRs). If IL-4 is present, clone of antigen specific
Th2 cells is formed. Th2 cells provide costimulation signal to B cells in the form of intercellular contact
and cytokine secretion. B cells differentiate into memory B cells or plasma cells producing mainly IgM
antibodies with low binding affinity to the antigen. Secondary response – memory B cells recognize
immunocomplexies (antigen+IgM) on the surface of follicular dendritic cells and cooperate with Th2
cells, which results in another circle of proliferation and differentiation into plasma cells or memory B
cells. Plasma cells produce antibodies of other isotype than IgM (class switch) and with high binding
affinity to the antigen (affinity maturation). Memory B cells are activated during repeated encounter with
the same antigen.
Class (isotype) switch – certain number of C gene segments is enzymatically removed from DNA
encoding for the heavy immunoglobulin chain and therefore other than C gene segment for μ chain is
placed in the position adjacent to VDJ complex. Class switch leads to isotype change but antigen specifity
remains the same.
Affinity maturation – somatic mutations of rearranged V gene segments for the heavy and the light
immunoglobulin chain. B cells which underwent mutation leading to higher binding affinity to the antigen
survive and differentiate into plasma cells producing high affinity antibodies.
3. MHC molecules and antigen presentation
See study materials on www.lf3.cuni.cz (http://www.img.cas.cz/mci/lectures.php - Antigen processing and
presentation.pdf)
4. T cells
Antigen specific receptor of T cells (TCR)
•
transmembrane chains  and , eventually  and  (antigen recognition)
•
CD3 complex associated with cytoplasmic protein-tyrosine kinases of the Src and Syk family (signal
transduction into the cell)
Subpopulations of T cells
1. classification in order of TCR
 T cells – majority of T cells, recognize antigenic peptides in a complex with MHC molecules
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γ T cells – in humans 5 % T cells, able to recognize native antigens (without processing in antigen
presenting cells)
2. classification of  T cells
Cytotoxic T cells – CD8+ T cells, recognize antigenic peptides in a complex with MHC class I molecules,
elimination of virus-infected and tumor cells
Helper T cells (Th) – CD4+ T cells, recognize antigenic peptides in a complex with MHC class II
molecules, cooperation with B cells (Th2), cooperation with macrophages (Th1)
T cell development – begins in the bone marrow, continues in thymus, where T cells undergo
rearrangement of gene segments encoding for variable domains of TCR (similar process as in B cells),
negative and positive selection.
Positive selection – T cells with functional TCR (recognizing MHC molecules in a complex with
antigenic peptides) survive
Negative selection (elimination of autoreactive T cells) – T cells which express TCR with high binding
affinity for self MHC molecules displaying autoantigens are programmed to die by apoptosis.
Approximately 5% T cells survive both positive and negative selection and leave the thymus as mature T
cells able to bind MHC class I or II molecules.
Peripheral tolerance
Immunological ignorance – T cells cannot be activated if the autoantigen is physically separated from
them (hematoencephalic barrier) or if it is present in organism in very low concentration
Anergy (long-termed inhibition of T cell function) – T cells cannot be activated without costimulation
signal from antigen presenting cells
Suppression – activation of autoreactive T cells can be inhibited by regulatory T cells (e.g. secretion of
cytokines IL-10, TGF-)
T cell activation
•
Recognition of antigenic peptides in a complex with MHC molecules on the surface of antigen
presenting cells by means of TCR
•
Receiving of costimulation signal from antigen presenting cells (interaction between molecules CD28
on T cells and CD80 or CD86 on antigen presenting cells)
Cytotoxic mechanisms of T cells (see Apoptosis)
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Fig. 4 Activation of T cells
Department of Immunology
Fig. 5 TCR
5. Complement
Complement – usually soluble component of the innate (nonspecific) immunity, complex of 30-40 serum
and membrane (glyco)proteins synthesized by hepatocytes, eventually by monocytes, macrophages and
epithelial cells
Complement activation
There are three different complement activation pathways: classical, lectin and alternative. Complement
activation presents cascade system till formation of C5b fragment (the previous component activates the
next one by proteolytic cleavage), subsequent lytic phase is common to all types of activation pathways.
Classical pathway is initiated by binding of C1q complement component to Fc region of IgG or IgM,
which recognized antigenic epitopes on the cell surface or within soluble immunocomplexes. The
activated C1 component cleaves enzymatically C4 and C2 component into two fragments. C4bC2a forms
C3-convertase that splits C3 component to give C3b and C3a fragments. Large C3b fragment joins
C4bC2a complex resulting in formation of classical C5-convertase (C4bC2aC3b) that cleaves C5
component into C5a and C5b fragment.
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Fig. 6 Classical complement activation pathway
Lectin pathway is the alternative of the classical pathway. It is activated by linking of serum mannosebinding lectin (MBL) to carbohydrate structures typical for microorganisms. MBL is structurally very
similar to C1q and associates with enzymes (MASP-1, MASP-2) which cleave C4 and C2 component.
Following steps of the cascade are the same as in the classical pathway.
Alternative pathway is triggered by the spontaneous cleavage of C3 component in the presence of
activation surfaces (bond to zymosan of yeast, lipopolysaccharide of G- bacteria, tumor cells, etc.). C3b
fragment joins B factor that undergoes the cleavage by enzymatic factor D to generate alternative C3convertase (C3bBb). C3-convertase is stabilized by properdin. After the addition of other C3b fragment to
C3bBb C5-convertase arises.
Lytic phase of complement activation – nonenzymatic formation of membrane attack complex (MAC,
C5bC6789). Components C6, C7, C8 and 15-20 molecules of component C9 bind to C5b fragment. This
complex inserts into membrane of the target cell creating pores (cell lysis).
Functions of complement and complement receptors CR1-CR4 (binding to complement fragments)

lysis of target cells (MAC) - bacteria, tumor cells, graft cells, incompatible erythrocytes

participation in inflammatory reaction (C5a, C4a, C3a) – chemotactic factors, anaphylatoxins
(increase permeability of blood vessels), degranulation of mast cells and basophils

elimination of circulating immunocomplexes

opsonization – facilitation of the phagocytosis (C3b, iC3b, C4b)

cell adhesion (CR3, CR4)

regulation of antibody response (interaction CR2-C3dg)

protection of self cells against destruction mediated by complement (CR1)
Regulation of complement

synthesis of complement components in the form of inactive precursors
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
unstableness of active complement fragments (short half-life)

specific regulatory proteins - regulation of initial phase (C1-inhibitor), C3- and C5-convertase activity
(DAF, MCP, I and H factors, CR1), MAC formation and insertion into the cell membrane (protectin)
Fig. 7 Alternative complement activation pathway
6.
Membrane molecules of immune cells
1. Fc receptors – bind Fc regions of immunoglobulins, individual types of Fc receptors have different
specifity for antibody isotypes (mainly for IgG - FcRI, II, III and IgE - FcRI, II)
Functions

facilitation of phagocytosis of cells coated with antibodies

mediation of antibody dependent cell-mediated cytotoxicity (Fc receptor with low binding affinity for
IgG =FcRIII)

degranulation of mast cells and basophils (FcRI)

negative regulation of the specific immune response (FcγRIIB = CD32B)

transcytosis of polymeric immunoglobulins (poly-Ig receptor)
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2. Adhesion molecules – enable intercellular communication based on direct contact
Classification in order of the structure:
a. Integrins – bind components of extracellular matrix or membrane molecules belonging to
immunoglobulin superfamily, they consist of transmembrane chains α and β
Group β1 (VLA) – mediate binding of monocytes and eosinophils to VCAM-1 of endothelial cells during
diapedesis
Group β2– mediate binding of neutrophils to ICAM-1,2,3 of endothelial cells during diapedesis
b. Adhesion molecules belonging to immunoglobulin superfamily – contain domains typical for
immunoglobulin molecule (ICAM-1,2,3, VCAM-1, CD4, CD8, CD28, CD80, CD86, TCR, BCR)
c. Lectins – bind carbohydrate structures
Selectins – mediate binding of endothelial cells to leukocyte oligosaccharides during the initial phase of
diapedesis
C- type lectins – e.g. surface NK cell receptors
herbal lectins – act as polyclonal mitogens of T cells, i.e. activate large number of T cells independently
on their antigen specifity (concanavaline A, phytohemaglutinin)
d. Mucins – strongly glycosylated glycoproteins (CD34, GlyCAM-1)
3. CD markers – immune cell surface molecules are members of CD classification system
7. Cytokines
(Glyco)proteins regulating immune response, they are synthesized by leucocytes, eventually by
endothelial, epithelial, dendritic and mast cells
Characteristics - pleiotropic effect (cytokines affect the activity of multiple cell types), redundancy
(individual cytokines can be functionally replaced by others), cascade system (one cytokine induces
secretion of the other one). Cytokines have either autocrine (on the cell producing the cytokine),
paracrine (on the cells located close to the site of cytokine secretion) or endocrine effect (on distant
tissues).
Classification in order of the function

pro-inflammatory cytokines – the amplification of inflammation (IL-1α a β, IL-6, IL-8, TNF)

anti-inflammatory cytokines (IL-4, IL-10, TGF-β)

growth factors of hemopoetic cells (IL-3, IL-7, IL-15, GM-CSF, SCF)

cytokines stimulating the humoral immunity (IL-4, IL-5, IL-6)

cytokines stimulating the cell-mediated immunity (IL-2, IL-12, IFN-γ, TNF-)

Cytokines with antiviral effect (IFN-α a β)

Chemokines (IL-8, MCP-1, MCP-2)
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6. Antigens and apoptosis
Antigens – substances which are recognized by the immune system and provoke the immune response
Classification of antigens
1. in order of their origin
exogenous – e.g. microorganisms and their products
endogenous (autoantigens) – e.g. self old, impaired or tumor-transformed cells
2. in order of the relation between donor and recipient during transplantation
autologous – self antigens
syngenic – antigens of the genetically identical individual (e.g. identical twins)
alogenic - antigens of the genetically different individual belonging to the same biological specimen
xenogenic - antigens of individual belonging to the different biological specimen
3. in order of their solubility
corpuscular (insoluble) – microorganisms, cells and their subcellular structures
soluble
4. according to the way of B cell stimulation
T- dependent – help of TH2 cells is required for B cell activation (direct intercellular contact between B
and TH2 cells, secretion of cytokines)
T- independent - direct activation of B cells (independently on TH2 cells), enabled by the character of
antigen structure
Allergens - especially exogenous antigens which invoke the pathological (allergic) immune response in
sensitive persons with hereditary predisposition (e.g. food components, mites, pollen)
Superantigens - exogenous antigens (mainly microbial toxins) stimulating large number of T or B cells
independently on their antigen specifity.
Adjuvants - substances which are administered simultaneously with the antigen to increase the immune
response (they facilitate ingestion of antigen by APC, stimulate proliferation of lymphocytes, etc.)
Small region of the antigen that is recognized by immune cell receptors is termed epitop. There are 2 types
of epitopes – linear (determined by primary structure of the protein) and conformational epitopes (formed
by aminoacids situated in proximity by means of the protein spatial structure).
Characteristics of antigens
Immunogenicity - ability to induce the immune response
Biochemical composition – proteins, glycoproteins, nucleoproteins and lipopolysaccharides are the
strongest antigens (due to their heterogeneous structure).
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Molecule size – in general all the proteins with Mr > 10 000 are strong antigens. Small molecules which
are not able to induce the immune response itself, become strong antigens after coupling to a carrierprotein (so called haptens - e.g. some pharmaceuticals and metals).
Structural stability – substances with rapid metabolism or with flexible structure are not able to form
stable epitopes and therefore are not strong antigens.
Apoptosis (programmed cell death) – physiological process, removing of excessive, already useless or for
organism potentially dangerous cells without damage of surrounding tissue. As for the immune system
apoptosis plays role in the elimination of autoreactive B and T cells and useless effector cells. NK cells
and cytotoxic T cells can trigger apoptosis of their target cells (activation of caspases mediated by
granzymes, interaction FAS-FASL, TNF-TNFR).
Morphologic manifestations of apoptosis- changes in the cell membrane and cytoskeleton, condensation
and fragmentation of nucleus resulting in break up of the cell into apoptotic bodies. Apoptotic cells are
phagocytosed by macrophages (recognition by means of scavenger receptors).
Obr.8 Induction of apoptosis
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