Introduction to immunology. LESSON 2: THE ADAPTIVE IMMUNITY Today we will get to know: The adaptive immunity T- and B-cells Antigens and their recognition How T-cells work 1
The adaptive immunity Unlike the innate immunity, that is fast and inborn but non-specific, the adaptive immunity ensures a specific, though slow, response to an enormous amount of pathogens. The main features of adaptive immunity are: Feature Specificity Diversity Memory Clonal expansion Specialization Homeostasis Self tolerance Function Ensures that the response to a microbe (or a non microbial antigen) is specific for that microbe (or antigen) only Makes the immune system able to respond to a great variety of antigens Ensures that the immune system is able to respond to repeated infections by the same microbe Increases the number of lymphocytes specific for that particular microbe/antigen Generates optimal responses against a specific microbe Ensures that the immune system will be able to get back to its normal functions and respond to a new challenge in a short time Prevents damage of the host during infections 2
The adaptive immunity cells The cellular components of the adaptive immunity are the T and B lymphocytes. B lymphocytes (or B-cells) are the only cells of our body able to produce antibodies. They differentiate into either plasma cells (antibody-producing cells) or memory cells (cells which are stored to ensure a fast antibody response against the same pathogen). They are involved in humoral immunity. T lymphocytes (or T-cells) are involved in cellular immunity because they do not produce antibodies. Conversely, they are able to recognize antigens and either attack the cells expressing them or stimulate/suppress the immune response against the antigen. Hence, they show a very wide differentiation ability, including: T helper (Th) cells. Upon antigen recognition, they will produce soluble mediators of immunity (the cytokines) which will regulate the strength and duration of the response. Cytotoxic T-cells (CTLs). Upon antigen recognition, they will kill the cells expressing the antigens. Regulatory T cells (Treg). They will play a role in suppressing the immune response, ensuring that no damage to the host occurs. Their excess/deficit also play a role in many pathologies. Natural killer and natural killer T-cells (NK and NKT). Subpopulations of lymphocytes which directly kill the cells expressing the antigens (similar to CTLs). But, they show a very poor diversity and they are generally considered as innate immunity effectors. 3
The adaptive immunity cells Humoral immunity Wide variety of effects mediated by cytokines Cellular immunity Active regulation of the immune response Cellular immunity 4
The adaptive immunity time-course Note that T- and B- cells which have never seen an antigen are called naïve. They will need time to activate mechanisms which will allow them to respond to the stimulus. T- and B-cells will eventually produce memory cells 5
The adaptive immunity primary and secondary reactions The advantage of memory cells is that they live for a long time and that they are already armed against the same pathogen. So, a second challenge with the same pathogen will lead to faster and stronger specific responses. Conversely, innate immunity cells cannot turn into memory cells and need to reactivate at every challenge. Note that the example drawn here is for B- cells, but the same applies to T-cells. 6
The adaptive immunity lymphoid organs and lymphocytes routes Naïve lymphocytes will need to travel to secondary lymphoid organs to meet the antigens and activate 7
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). They are infrequently found in the blood (where they generally circulate as naïve B cells), as they mainly localize in the bone marrow where they can survive for many years. 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. B-cells are sub-grouped in B-1 and B-2 cells 8
Adaptive immunity overview of the immunoglobulins (Ig) All Ig have a similar structure, but they display an enormous variability in the antigens that they can recognize. 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. 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. 9
Adaptive immunity maturation of B-cells 10
Adaptive immunity maturation of B-cells B-1 cells originate from fetal hematopoietic stem cells localized in the liver. They are a subpopulation of total B-cells which produce natural antibodies. B-2 cells originate from adult hematopoietic stem cells localized in the bone marrow. They are the majority of total B-cells and produce specific antibodies. During their differentiation, they transit through a short intermediate stage, called transitional B-2 cells, and then fully differentiate into either follicular and marginal-zone B-2 cells. Follicular B-2 cells are the majority, circulating in between the blood, the lymphatic organs and the bone marrow. They produce all antibodies isoforms. Marginal-zone B-2 cells are a minority, mostly present in the spleen and lymph nodes. They produce antibodies with poor diversity and also natural antibodies (similarly to B-1 cells) 11
The adaptive immunity T-cell maturation and selection The name T-cells was originally used to identify those lymphocytes that need the thymus to mature. 1 3 4 2 12
The adaptive immunity T-cell maturation and selection In the thymus, many important processes take place. These will ensure that T-cells have highaffinity receptors (able to recognize antigens in a strong way) and that they re not autoreactive (they will discriminate self cells). 1 Immature T-cells coming from the bone marrow (pro-t) need proliferative signals to survive in the thymus. These are provided by specialized cells in the cortical area of the thymus, but only if pro-t are able to bind to them with their receptor. If they don t, they will die by nutrient deprivation (death by neglect). 2 Pro-T-cells which manage to survive in the cortical area will be tested for their ability to discriminate normal body cells (self cells, represented by thymus epithelial cells). If T-cells bind too tightly to those cells (meaning that they are autoreactive clones, which would attack normal body cells), they are induced to die by apoptosis. If they survive this negative selection, they move towards the medullar area and become pre-t-cells. 13
The adaptive immunity T-cell maturation and selection 3 Pre-T which have good receptors and are not autoreactive are induced to proliferate and expand by cells of the thymus. This is the basis of positive selection, which will induce T-cells to become CD4 + (Th) or CD8 + (CTLs) T-cells. 4 Newly-formed CD4 + and CD8 + T-cells will be thoroughly tested again to ensure that they are not autoreactive. If they show autoreactive behavior, they will be suppressed and induced to die by apoptosis by regulatory T-cells of the thymus. 14
Y Introduction to immunology. Lesson 2 The adaptive immunity molecular basis of T-cell recognition Macromolecules able to stimulate an immune response are called antigens. T-cells recognize only small parts of each antigen, called epitopes or determinants. Each macromolecule can have many epitopes. Antigen recognition by T-cells always require presentation by MHC molecules. Antigen Epitope 1 Single epitope Epitope-MHC complex Epitope 2 Epitope 3 H MHC T-cell Diversity Specificity 15
The adaptive immunity molecular basis of T-cell recognition Unlike PAMPs of the innate immunity, epitopes recognized by T-cells are millions. Still, they have some limitations: Must be linear peptides Must be small Must be on a cell, not soluble Must be conjugated to MHC molecules All of these features depend on the ability of the antigen receptor of T-cells (called the T-cell receptor, TCR) to recognize an epitope only if conjugated to a presenter molecule (the MHC molecules). This phenomenon is known as MHC restriction, and so all T-cells are MHCrestricted (apart from strange T-cells like NK, NKT and gd T-cells) 16
The adaptive immunity molecular basis of T-cell recognition T-cell receptor (TCR) only gets activated if it binds to an antigen together with some specific parts of MHC molecules. This is why T-cells are MHC-restricted. The antigen-binding pocket of MHC molecules is small and linear, and this is why T-cells epitopes must be linear and short. MHC molecules are only expressed on the surface of cells. This is why T-cells can not recognize soluble antigens. 17
The adaptive immunity the Major Histocompatibility Complex (MHC) MHC has been discovered in mice and humans during studies to understand the genetic basis for transplant rejection. Today we know that those reactions depend mostly on T-cell activation. George Snell Major Histocompatibility Complex genes were identified at different times in mice and humans, so they have (or had) various names: In mice, they are still called H-2 (histocompatibility-2 genes). During the 60 s/70 s, the genes involved in the regulation of the immune response were called Ir (immune response) genes. Later they were mapped in the H-2 locus and the term Ir was abandoned. In humans, they re either called HLA (human leukocyte antigens) or MHC. Baruj Benacerraf MHC molecules are a set of different molecules grouped into class-i, class-ii and class-iii subtypes. Only class-i and -II are involved in antigen recognition by T-cells, while class-iii includes genes for complement proteins, HSP and cytokines. Jean Dausset 18
The adaptive immunity HLA locus Very big locus (E. Coli genome is c.ca 4500 kb). MHC genes are co-dominantly expressed. This means that each person expresses both paternal and maternal MHC genes at the same time. This maximizes the number of MHC available for antigen binding. MHC are the most polymorphic genes in our genome. At the moment, 3500 possible HLA isoforms have been postulated, but they re likely to be much more. The series of all the HLA alleles in each chromosome is called haplotype. 19
The adaptive immunity MHC-I Each MHC-I molecule: Is made of an a chain (44-47kD) transcribed from HLA class-i genes and the b2-microglobulin (12kD). Has polymorphic regions in a1 and a2 domains. Their variability depends on the class-i allele from which they derive (HLA-A, HLA-B and HLA-C). The antigen-binding pocket can accommodate peptides made of 8-11 residues. Has an invariant a3 domain which is recognized by the TCR. Only CTLs can recognize this region. Binds to intracellular antigens. VIRTUALLY ALL NUCLEATED CELLS OF THE BODY EXPRESS MHC-I MOLECULES ON THEIR SURFACE. 20
The adaptive immunity MHC-II Each MHC-II molecule: Is made of an a chain (32-34kD) and a b chain (29-32kD). Both a and b chains are transcribed from HLA class-ii genes. Has polymorphic regions in a1 and b1 domains. Their variability depends on the class-ii allele from which they derive (HLA-DR, HLA- DQ and HLA-DP). The antigen-binding pocket can accommodate peptides made of 10-30 residues. Has an invariant b2 domain which is recognized by the TCR. Only Th can recognize this region. Binds to extracellular antigens. ONLY MACROPHAGES, DENDRITIC CELLS, B- CELLS AND FEW OTHER EXPRESS MHC-II ON THEIR SURFACE. 21
The adaptive immunity MHC-I and MHC-II pathways 22
The adaptive immunity T-Cell Receptor (TCR) The TCR: Is made of an a and a b chain (all Th and CTLs). A small subpopulation of T-cells has its TCR made by a g and a d chain. Has variable domains (V) in both a and b chains. Those regions are involved in antigen recognition and mutations here provide diversity. Va and Vb have three hyper-variable regions which recognize the antigen- MHC complex. These are called Complementarity-Determining Regions (CDR). Vb also has another region (CDR4) which has a role in pathologies. TCR needs other proteins to activate the T-cell response. 23
The adaptive immunity TCR and accessory molecules TCR complex = TCR+CD3+z chains Accessory molecules 24
The adaptive immunity T-cell diversity Cytotoxic T cells = CD8 + T-cells TCR CD8 MHC-I CD8 + T-cells (CTLs) recognize antigens presented by MHC-I molecules. Since MHC-I is expressed by all nucleated cells and captures intracellular antigens, this ensures that every cell infected by viruses and intracellular bacteria, as well as transformed cells expressing tumor antigens, will be recognized and killed by CTLs. All nucleated cells CTLs will kill the cells that express microbial antigens presented by MHC-I 25
The adaptive immunity T-cell diversity T helper cells = CD4 + T-cells CD4 APC TCR MHC-II Th cells will produce cytokines to control and regulate the immune reaction CD4 + T-cells (Th) recognize antigens presented by MHC-II molecules. Since MHC-II is expressed only by specific cells involved in immune reactions (APC) and captures extracellular antigens, this ensures that microbes in the extracellular space can be ingested by APC and presented to Th. This is a fine example of cooperation in between innate (APCs are mostly DC and macrophages) and adaptive immunity. Th cells will respond by differentiating into different Th subpopulations and producing cytokines which will boost the immune activity of other cells (phagocytes, CTL, B-cells, etc.) 26
The adaptive immunity Antigen-Presenting Cells (APC) 27
The adaptive immunity Antigen-Presenting Cells (APC) Regardless of the different routes that antigens can take, they will always end up being captured by APC, which will present them to CTL and Th cells and ensure an optimal adaptive response. 28
The adaptive immunity The role of co-stimulatory molecules Co-stimulatory molecules (especially the family of B7-1/B7-2) are essential to activate Th cells (and also to strongly boost CTLs activity). In the absence of co-stimulation, T- cells become anergic. Anergic T-cells do not clonally expand after antigen recognition. Rather, they undergo apoptosis and die (clonal anergy). Presenting self antigens to developing T-cells without costimulatory molecules is the strategy used in the thymus to delete autoreactive T-cell clones. 29
The adaptive immunity T-cell expansion and differentiation Clonal expansion ensures the generation of a high number of effector T- cells from a small number of naïve T-cells activated by an antigen. CD8 + T-cells expand approx. 50000 times CD4 + T-cells expand approx. 10000 times 30
The adaptive immunity Th differentiation While CTL are directly part of cellular immunity, CD4 + cells (Th) respond to antigenic stimuli releasing a wide variety of cytokines. These are paracrine mediators of immunity, which stimulate and empower the functions of surrounding immune cells. Upon activation and clonal expansion, CD4 + T-cells undergo a crucial step of differentiation (sometimes called polarization) which will tailor their cytokine production towards three major combinations, which will have different effects. Naïve T-cell Actually, the situation is more complicated, as different subtypes can express a mix of cytokines from different subpopulations. Th subpopulations can sometimes interconvert! Th1-cell Th2-cell Th17-cell 31
The adaptive immunity Th differentiation 32
The adaptive immunity Th1 Th1 differentiation is induced by viruses, intracellular bacteria (L. monocytogenes and Mycobacteria, i.e.), parasites (Leishmania) and some antigens administered with strong adjuvants. It is specific against intracellular microbes. Intracellular microbes infect DC and macrophages Infected APC start producing IL-12, IFN-g and other IL-12 and IFN-g induce Th1 differentiation Th1 cells produce mainly IFN-g, which inhibits the growth of intracellular microbes IFN-g inhibits naïve T-cell differentiation towards Th2 and Th17 33
The adaptive immunity Th2 Th2 differentiation is induced by helminthes parasites, allergens and some weak antigens that do not stimulate Th1 and Th17 differentiation. It is specific against worms and some persistent bacterial infections. Granulocytes and mast cells recognize helminthes Activated granulocytes and mast cells produce IL-4 IL-4 induces Th2 differentiation Th2 cells produce mainly IL-4, IL-5 and IL-13 which coordinate mucosal defenses active against worms IL-4 inhibits naïve T-cell differentiation towards Th1 and Th17 34
The adaptive immunity Th17 Th17 differentiation is induced by inflammatory cytokines produced by APCs in response to bacteria and fungi. It is not as specific as Th1 and Th2, but has a wider range of action and is stronger. APC recognize bacteria and fungi in the extracellular space Activated APC produce IL-1, IL-6, IL-23, TGF-b and other All of these cytokines together induce Th17 differentiation Th17 cells produce mainly IL-17 and IL- 22, which strongly stimulate inflammation in the mucosae IL-23 and TGF-b (mainly) suppress Th1 and Th2 differentiation 35
The adaptive immunity Memory T-cells (T mem ) Memory T-cells (T mem ) arise either from effector T-cells or from naïve T-cells. What drives the differentiation into memory T-cells is largely unknown. Their features are: Their quantity depends on the strength of the antigen stimulus. The stronger the stimulus, the more T mem will be generated. They survive for many years (sometimes for the whole life) after their differentiation. To compare, effector T-cells live for weeks or months. They outnumber the quantity of naïve T-cells for the same antigen. Their number is 10-100 times bigger than that of naïve T-cells for the same antigen. Their responses to the antigen are faster than the responses of naïve T-cells. In mice, naïve T-cells require 5 7 days to respond to an antigen, while T mem activate in 1 3 days. T mem cells constitute a pool which ensures SPECIFIC, RAPID and STRONG responses to antigens (pathogens) already met by the immune system 36
Adaptive immunity switch-off feedback mechanisms and regulatory T-cells (T reg ) Spontaneous termination of T-cell responses occur with pathogens eradication: as soon as the number of microbes decreases, the number of activated APC will decrease too. This means that those cytokines which stimulated Th differentiation will decrease, and that differentiated Th cells will die by apoptosis. Similarly, CTLs die by apoptosis soon after they destroyed target cells. Regulatory T-cells (T reg ) arise spontaneously in the thymus and in peripheral tissues from immature CD4 + naïve T-cells. Their primary function is to dominantly suppress Th1, Th2, Th17, B-cells and innate immunity cells by releasing immunosuppressive cytokines (like IL-10), by cell-to-cell inhibition and also by cytolysis. They are of a crucial importance to ensure non-self reactivity and to avoid damages caused by excessive immune responses in peripheral tissues. 37