Introduction to immunology. LESSON 3: ANTIBODIES/BCR/B-CELL RESPONSES Today we will get to know: The antibodies How antibodies are produced, their classes and their maturation processes Antigen recognition by antibodies How B-cells work 1
Adaptive immunity B-cells B-lymphocytes (B-cells) are the only cells of the body which produce antibodies. These are soluble molecules (belonging to different classes) also known as gamma globulins or immunoglobulins. Antibodies are very efficient players of humoral immunity. B-cells have characteristic big nuclei, with large endoplasmic reticulum and numerous ribosomes. These features reflect their active transcriptional state (they produce large amounts of antibodies). Antibodies are produced as both transmembrane and soluble forms. In the transmembrane form, they are the antigen receptors of B-cells (called B-Cell Receptor, BCR). In the soluble form, they act as humoral immunity effector molecules which bind to microbes and toxins. Abbas et al. 2
The adaptive immunity molecular basis of antigen recognition by antibodies Similarly to antigens recognized by T-cells, epitopes recognized by B-cells are millions (but less than that of T-cells). And, they don t have limitations: Can be linear peptides of folded peptides Can also be polysaccharides, lipids, nuclei acids and small chemical molecules Can be on cells or soluble Do not need to be presented by MHC molecules 3
Adaptive immunity The antibodies All the antibodies (also called immunoglobulins, Ig) have a similar structure, but they display an enormous variability in the antigens that they can recognize. Unlike T-cell epitopes, B-cell epitopes (generally called determinants) can be linear or conformational (3D). Abbas et al. 4
Adaptive immunity The antibodies Always present: Two identical heavy chains and two identical light chains, which form a symmetric structure. The variable regions of heavy and light chains (V H and V L ) participate to antigen recognition, forming the antigen-binding site. Each of them has 3 CDR which bind to the antigen. The constant regions of heavy chains are responsible for the functions of the antibodies. I.e., they can activate the complement system, or be recognized by phagocytes. Abbas et al. 5
Adaptive immunity The antibodies The light chains: There are two isoforms of light chains, called k and l. They differ at the C-term but they have identical functions. In humans, c.ca 60% of all antibodies have k chains, while 40% have l chains. The two light chains in each antibody are identical. So, each antibody presents either two k or two l chains, never a mix. The heavy chains: There are five isoforms of light chains, called m, d, e, g and a (some of them also include subclasses). They differ at the C-term and they confer different functions to the antibodies. Antibodies are classified by the heavy chain the have: m chain IgM d chain IgD e chain IgE These classes are better called isotypes g chain IgG a chain IgA The two heavy chains in each antibody are identical 6
Adaptive immunity The antibodies The general structure of antibodies has been identified with proteolytic experiments, where antibodies were digested with different enzymes (papain and pepsin). The products of the proteolytic digestion are still used to identify different functional regions of the antibodies. Fab (fragment, antigen binding) is the product which retains the ability to bind to the antigen. Fc (fragment, crystallizable) is the part which tends to precipitate forming a reticulate. F(ab ) 2 contains both the Fab from the same antibody. Abbas et al. 7
Adaptive immunity The antibodies Abbas et al. 8
Adaptive immunity The antibodies Unlike TCR, antibodies are plastic: they can change their shape to better bind to antigens. The ability of antibodies to change their shape depends on the hinge region. This is a sequence long 10 60 residues (depending on the isotype) located between C H 1 and C H 2 Also, V H domains can rotate. C H domains can not. ANTIBODIES ARE VERY FLEXIBLE Abbas et al. 9
Adaptive immunity The antibodies: affinity, avidity and valence Affinity: the strength of antigen-binding site antigen bond. Generally defined by K d, indicates how easily antibodies bind to the antigens. The lower the better. Antibodies produced during immune reactions have K d between 10-7 M and 10-11 M. It s a physical property of each specific antigen-binding site. Avidity: the total strength of antibody antigen bond. It depends on the sum of the affinity of each antigen-binding site in the antibody. Since each antibody has at least two antigensbinding sites, avidity is always bigger than the affinity. Valence: the total number of antigen-binding sites in the antibody. For monomeric Ig (like IgG) it s equal to two. In pentameric IgM, it s ten (2 antigen-binding sites per antibody, 5 antibodies bound together). 10
Adaptive immunity The B-Cell Receptor (BCR) All immunoglobulins exist as transmembrane forms on the surface of B-cells. In this form, they are the antigen receptor of the B-cells (BCR). Similarly to the TCR, BCR needs co-receptors to activate B-cells. BCR, as TCR, has cytoplasmic tails which are too short to transduce any signal. Hence, just like the TCR needs CD3 and z chains, the BCR needs to form a complex with Iga and Igb chains, which will activate intracellular signaling pathways. Unlike TCR, which needs CD4/CD8 and other costimulatory molecules to activate T-cells, the BCR can co-operate with other molecules, like CD21 (CR2), to recognize antigens bound to the complement. Anyway, this is not mandatory for B-cell activation. Abbas et al. At least two BCR must bind to the same antigen to activate B-cells (receptor cross-linking or clustering). 11
Adaptive immunity The B-Cell Receptor (BCR) There is a fundamental difference in the BCR of naïve and mature B-cells: naïve BCR is always IgM or IgD (also both on the same cell), while mature BCR can be also IgA, IgE and IgG. This depends on the isotype switch phenomenon, which is fundamental during the maturation of B-cells. IgA IgD IgM mature B-cell IgE Naïve B-cell IgG mature B-cell mature B-cell Regardless of the isotype, all Ig on the membrane are ALWAYS MONOMERIC. Conversely, when secreted, IgA and IgM can form multimers. IgE and IgG remain as monomers. IgD is NEVER SECRETED and has only a function as BCR on naïve B-cells. 12
Adaptive immunity somatic rearrangements and mutations in Ig/BCR and TCR genes What makes antibodies/bcr and TCR so variable and able to recognize millions of different epitopes? VDJ recombination Junctional diversification Hypermutation SOMATIC VARIABILITY: Each TCR and Ig/BCR gene in each T- or B-cell is different, while those genes are equal in the germ line of all individuals In other words, we do not inherit our ability to recognize antigens. WE DEVELOP IT! 13
Adaptive immunity V(D)J recombination Heavy (H) and light (L) chains of Ig/BCR, as well as a and b chains of the TCR, have many different genetic segments (exons) in the CDR regions. Those different segments are organized in groups: Variable (V) segments Diversity (D) segments Joining (J) segments V and J segments are present in all Ig/BCR and TCR genes, while D segments are only in the heavy chains of the immunoglobulins/bcr and in the b chain of the TCR. Abbas et al. 14
Adaptive immunity V(D)J recombination V(D)J recombination is a process where one single V segment, one single J segment and one single D segment (when present) are chosen among the many others. This process is somatic, non-homologous recombination proceeding in a very tightly regulated way. Abbas et al. 15
Adaptive immunity V(D)J recombination V(D)J recombination depends on two enzymes, called RAG (Recombination- Activating Gene)-1 and - 2. RAG-1 and RAG-2 assemble in a tetramer also known as V(D)J recombinase. All J segments after the one which underwent recombination are left untouched. They will be eliminated during RNA processing. Abbas et al. 16
Adaptive immunity V(D)J recombination RAG-1 and RAG-2 recognize specific sequences called RSS (Recombination Signal Sequences), which are differentially distributed in V, D and J genes. RSS are eptameric/nonameric sequences spaced by 12 o 23 bp. They are at 3 of each V gene, at the 5 of each J gene and at both ends of the D genes. V(D)J recombination occurs via deletion or inversion. V(D)J recombination occurs in BOTH H and L chains of Ig (as well as in a and b chains of TCR) INDEPENDENTLY. This increases variability and possibilities for antigen recognition. Abbas et al. 17
Adaptive immunity Junctional diversification Two different mechanisms can add de novo nucleotides in the joining sites among recombined V-D-J segments: those nucleotides are called N and P nucleotides. During V(D)J recombination, the two ends of the site to recombine are cut in an asymmetrical way. There must be new nucleotides to fill the gaps. These are the P nucleotides. Also, up to 20 new nucleotides can be added by the TdT enzyme in between newly-formed P nucleotides sites. These are called N nucleotides. Abbas et al. 18
Adaptive immunity other variability mechanisms With V(D)J recombination and junctional diversification, the story ends for TCR. But, in Ig genes two other mechanisms can operate further: hypermutation and isotype switch. In the lymph nodes, under the influence of cytokines released from Th cells, V genes of Naïve B-cells start to accumulate mutations 1000 times more frequently than the rest of their genome (somatic hypermutation). B clones where these mutations increase the affinity of the antibody against the antigen are positively selected and proliferate. This process is defined as affinity maturation. Abbas et al. 19
Adaptive immunity other variability mechanisms In the lymph nodes, under the influence of different cytokines produced by Th cells, B-cells will start to produce antibodies with the same V(D)J structure but with different C (constant) regions. Since each different C region determines the isotype of the antibodies, this mechanism will produce an isotype switch. VDJ segments C m C d C g C e C a VDJ segments VDJ segments C m C d IgM IgD In naïve B-cells, transcription of the C regions starts from the strong promoters which are near m and d segments. This is why naïve B-cells express IgM and IgD. 20
Adaptive immunity other variability mechanisms VDJ segments C m C d C g C e C a VDJ segments C g IgG VDJ segments C e IgE VDJ segments C a IgA In activated B-cells, different cytokines produced by Th cells will trigger AID enzyme, which controls recombination in the genomic regions coding for the C segments. Th1, Th2 and Th17 cytokines will induce the switch towards the C segment (hence, towards the isotype) which works best with ongoing T-cell responses. In example, Th1 responses are against intracellular bacteria and viruses. Th1 cytokines will induce the switch towards IgG, which have a strong ability to opsonize the microbes and to activate the complement, ensuring the maximal response by innate immunity. 21
Adaptive immunity different functions of Ig isotypes Ig isotype Functions Outcome IgD Naïve BCR Activation of B-cells IgM IgA Naïve BCR, opsonization of microbes, complement activation Mucosal immunity and neonatal immunity (opsonization of invading pathogens) Activation of B-cells, stimulation of innate immunity Stimulation of innate immunity IgG IgE Strong opsonization of microbes, strong complement activation, neonatal immunity Opsonization of parasites, degranulation of mast cells Stimulation of innate immunity and adaptive immunity (CTL) Stimulation of innate immunity 22
Adaptive immunity antigens recognized by B-cells Antigens recognized by B-cells include proteins, carbohydrates, lipids, nucleic acids and small molecules, either in linear or 3D configuration. These antigens are better described by their ability to trigger antibody production and to need cooperation of T- and B-cells. Hence, B- cell antigens are divided into: T-independent antigens (TI) Carbohydrates, lipids and nucleic acids. They are polymeric, so they will easily attract two BCR and co-receptors, thus activating antibody production T-dependent antigens (TD) Proteins and peptides. They must bind to BCR, be phagocytized, be conjugated to MHC-II and finally presented to Th cells. These, in turn, will stimulate antibody production 23
Adaptive immunity antigens recognized by B-cells Remember: antigens must crosslink BCRs to activate B-cells! In other words, at least two BCR must bind to the same antigen in order to start signaling. TI antigens are generally LONG and POLYMERIC structures which easily aggregate two BCR. TD antigens are, conversely, too small to aggregate two BCR. The immune system circumvents this problem by exploiting T-cells. In this context, B-cells are used in the first step as APC and, in the second stage, stimulated to produce antibodies. Abbas et al. 24
Adaptive immunity different responses triggered by TI and TD antigens T-independent antigens (TI) Carbohydrates, lipids and nucleic acids. T-dependent antigens (TD) Proteins and peptides. Induce the production of low-affinity IgM Isotype switch almost absent (sometimes some IgG and IgA) Unable to induce affinity maturation Only some TI antigens stimulate the formation of memory B-cells (generally short-term) Induce the production of high-affinity IgG, IgA and IgE Isotype switch Induce affinity maturation Prominent formation of memory B-cells (generally long-term) 25
Adaptive immunity haptens and carriers Small molecules which bind to BCR are a problem for B-cells. Being small, they re not able to induce BCR crosslinking and activation. And, if they are not linear (or, even worse, non-protein) they are unsuitable for T-cells. So, what are they? HAPTEN Small molecules which are recognized by Ig/BCR but that are unable to stimulate antibody production. CARRIER A big molecule which may be antigenic or not. It will bind to the hapten and form a complex. The hapten-carrier complex (hapten-carrier adduct) will induce antibody formation. Generally, antibodies will be against epitopes from both the hapten and the carrier. 26
Y Y Y Y Y H Y Introduction to immunology. Lesson 3 Adaptive immunity haptens and carriers Natural carriers are generally albumin and other globulins in the serum (many exogenous and synthetic ones are used in vaccine science). Activation of antibody production by haptencarrier adducts require T-cells and is MHC-II restricted. Carrier proteolysis, binding of carrier fragments to MHC-II and presentation to Th cells B-cell Carrier binds to the hapten Under the influence of Th cytokines, B-cell proliferate and produce anti-hapten antibodies B-cell BCR binds to the adduct through the hapten and internalizes the adduct B-cell Th-cell TCR is activated by MHC-II carrying the carrier epitope and release cytokines that stimulate B-cell proliferation 27
Adaptive immunity plasma cells Plasma cells are special states of activated B-cells, in which the production of Ig is pushed towards the soluble forms by alternative splicing of the C domains. Activation of B-cells by TI and TD antigens (but mostly by TD, because of the cytokines produced by Th) will push splicing and poly-adenylation of Ig mrna towards the secretory form. Also, the rate of Ig synthesis will be strongly increased (some say around 10000/sec). Those are the characteristics of plasma cells. Abbas et al. 28
Adaptive immunity memory B-cells The mechanisms leading to the formation of memory B-cells are widely unknown. Still, they have some features: They can either be found in lymphoid organs or circulating in the blood They are generally induced by TD antigens They show hypermutated V segments and, thus, produce antibodies with strong affinity As a consequence of isotype switch they generally produce IgG, IgE and IgA They activate faster than naïve B-cells in response to the same antigen 29
Adaptive immunity antibody feedback and B-cell responses switch-off Antibody production is inhibited by the antibodies themselves (antibody feedback). Complexes made of antibody+antigen (immunocomplexes) bind to BCR, with the possibility to activate it. But, if they simultaneously bind to a receptor called FcgRIIB (which recognizes the C domains of Ig), BCR activation will be inhibited. FcgRIIB is also expressed on almost all myeloid cells, and mediates their suppression. IgM, produced in the first stages of B-cells activation, do not bind to FcgRIIB. Conversely, isotypes generated by the isotype switch (those antibodies that are produced later in the response) bind to FcgRIIB. So, the more the B-cells go on producing antibodies the more the chances that they will shut-down. There is also another inhibitory receptor on B-cells, called CD22. However, its natural ligand is unknown and we do not know much about this shut-down mechanism. Treg can switch off B-cells, by either releasing immuno-suppressive cytokines or inducing B-cells apoptosis. 30