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Showing posts with label Immunology. Show all posts
Showing posts with label Immunology. Show all posts

Monday, May 3, 2021

The B‐Cell Surface Receptor For Antigen (BCR)

The B‐Cell Surface Receptor For Antigen (BCR)


The B‐Cell Surface Receptor For Antigen (BCR)
The B‐cell displays a transmembrane immunoglobulin on its surface
In Chapter 2 we discussed the cunning system by which an antigen can be led inexorably to its doom by activating B‐cells that are capable of making antibodies complementary in shape to itself through interacting with a copy of the antibody molecule on the lymphocyte surface. It will be recalled that binding of antigen to membrane antibody can activate the B‐cell and cause it to proliferate, followed by maturation into a clone of plasma cells secreting antibody specific for the inciting antigen (Figure 4.1a).
B‐cells and T‐cells “see” antigen in fundamentally different ways

Figure 4.1 B‐cells and T‐cells “see” antigen in fundamentally different ways. (a) In the case of B‐cells, membrane‐bound immunoglobulin serves as the B‐cell receptor (BCR) for antigen. (b) T‐cells use distinct antigen receptors, which are also expressed at the plasma membrane, but T‐cell receptors (TCRs) cannot recognize free antigen as immunoglobulin can. The majority of T‐cells can only recognize antigen when presented within the peptide‐binding groove of an MHC molecule. Productive stimulation of the BCR or TCR results in activation of the receptor‐bearing lymphocyte, followed by clonal expansion and differentiation to effector cells.

Immunofluorescence staining of live B‐cells with labeled anti‐immunoglobulin (anti‐Ig) (e.g., Figure 2.8c) reveals the earliest membrane Ig to be of the IgM class. Each individual B‐cell is committed to the production of just one antibody specificity and so transcribes its individual rearranged VJCk (or λ) and VDJCμ genes. Ig can be either secreted or displayed on the B‐cell surface through differential splicing of the pre‐mRNA transcript encoding a particular immunoglobulin. The initial nuclear μ chain RNA transcript includes sequences coding for hydrophobic transmembrane regions that enable the IgM to sit in the membrane where it acts as the BCR, but if these are spliced out, the antibody molecules can be secreted in a soluble form (Figure 4.2).
The T‐Cell Surface Receptor For Antigen (TCR)

The T‐Cell Surface Receptor For Antigen (TCR)


The T‐Cell Surface Receptor For Antigen (TCR)
As alluded to earlier, T‐cells interact with antigen in a manner that is quite distinct from the way in which B‐cells do; the receptors that most T‐cells are equipped with cannot directly engage soluble antigens but instead “see” fragments of antigen that are immobilized within a narrow groove on the surface of MHC molecules (Figure 4.1b). 

As we shall discuss in detail in Chapter 5, MHC molecules bind to short 8–20 amino acid long peptide fragments that represent “quality control” samples of the proteins a cell is expressing at any given time, or what it has internalized through phagocytosis, depending on the type of MHC molecule. In this way, T‐cells can effectively inspect what is going on, antigenically speaking, within a cell at any given moment by surveying the range of peptides being presented within MHC molecules. Another major difference between B‐ and T‐cell receptors is that T‐cells cannot secrete their receptor molecules in the way that B‐cells can switch production of Ig from a membrane‐bound form to a secreted form. These differences aside, T‐cell receptors are structurally quite similar to antibody as they are also built from modules that are based upon the immunoglobulin fold.
The Generation Of Diversity For Antigen Recognition

The Generation Of Diversity For Antigen Recognition


The Generation Of Diversity For Antigen Recognition
We know that the immune system has to be capable of recognizing virtually any pathogen that has arisen or might arise. The awesome genetic solution to this problem of anticipating an unpredictable future involves the generation of millions of different specific antigen receptors, probably vastly more than the lifetime needs of the individual. As this greatly exceeds the estimated number of 25 000–30 000 genes in the human body, there are some clever ways to generate all this diversity, particularly as the total number of V, D, J, and C genes in an individual human coding for antibodies and TCRs is only around 400. Let’s revisit the genetics of antibody diversity, and explore the enormous similarities, and occasional differences, seen with the mechanisms employed to generate TCR diversity.


Intrachain amplification of diversity
Random VDJ combination increases diversity geometrically
We saw in Chapter 3 that, just as we can use a relatively small number of different building units in a child’s construction set such as LEGO® to create a rich variety of architectural masterpieces, so the individual receptor gene segments can be viewed as building blocks to fashion a multiplicity of antigen specific receptors for both B‐ and T‐cells. The immunoglobulin light chain variable regions are created from V and J segments, and the heavy chain variable regions from V, D, and J segments. Likewise, for both the αβ and γδ T‐cell receptors the variable region of one of the chains (α or γ) is encoded by a V and a J segment, whereas the variable region of the other chain (β or δ) is additionally encoded by a D segment. As for immunoglobulin genes, the enzymes RAG‐1 and RAG‐2 recognize recombination signal sequences (RSSs) adjacent to the coding sequences of the TCR V, D, and J gene segments. The RSSs again consist of conserved heptamers and nonamers separated by spacers of either 12 or 23 base‐pairs and are found at the 3′ side of each V segment, on both the 5′ and 3′ sides of each D segment, and at the 5′ of each J segment. Incorporation of a D segment is always included in the rearrangement; Vβ cannot join directly to Jβ, nor Vδ directly to Jδ. To see how sequence diversity is generated for TCR, let us take the αβ TCR as an example (Table 4.2). Although the precise number of gene segments varies from one individual to another, there are typically around 75 gene segments and 60 Jα gene segments. If there were entirely random joining of any one V to any one J segment, we would have the possibility of generating 4500 VJ combinations (75 × 60). Regarding the TCR β‐chain, there are approximately 50 Vβ genes that lie upstream of two clusters of DβJβ genes, each of which is associated with a Cβ gene (Figure 4.11). The first cluster, associated with Cβ1, has a single Dβ1 gene and 6 Jβ1 genes, whereas the second cluster associated with Cβ2 again has a single Dβ gene (Dβ2) with 7 Jβ2.

Thursday, April 29, 2021

Invariant Natural Killer T‐Cell Receptors Bridge Innate and Adaptive Immunity

Invariant Natural Killer T‐Cell Receptors Bridge Innate and Adaptive Immunity


Invariant Natural Killer T‐Cell Receptors Bridge Innate and Adaptive Immunity
The highly variable nature of the TCR confers on the conventional T‐cell population the ability to respond to an immense array of different antigens, with individual T‐cells specific for a single antigen. Invariant natural killer T‐cells (iNKT) are a unique subset of T‐cells that display a semi‐variant TCR that equips individual iNKT cells with the ability to detect a broad array of microbial lipid antigens, presented on CD1d antigen‐presenting molecules on antigen‐presenting cells (APCs). Although conventional T‐cells are activated by APCs that have first been activated by microbial antigen (in a process that takes some time), iNKTs can respond directly to PAMPs, secreting cytokines and presenting co‐stimulatory molecules in a manner more reminiscent of innate immune cell PRR activation than T‐cell stimulation.

Natural killer T‐cells

Figure 4.15 Natural killer T‐cells. (a) Schematic representation of type I and type II natural killer T (NKT) cells. These two subsets use different variable (V) region gene segments in the α and β chains of their T‐cell receptors (TCRs), and they recognize different CD1d‐ restricted antigens. (b) The αβTCR is composed of two chains, with the V domains containing the complementarity determining region (CDR) loops. The CDR3 loops are encoded by multiple gene segments and also contain nontemplated (N) regions, which add further diversity to the TCR repertoire. The color coding is the same as that used for the type I NKT TCR in (a). APC, antigen‐presenting cell; C, constant; D, diversity; J, joining. (Reproduced with permis­ sion from the authors Rossjohn et al., (2012) Nature Reviews Immunology 12, 845–857 © Nature Publishing Group.)

Although conventional CD4+ T‐cells provide help to B‐ cells as part of an adaptive immune response, iNKTs are unique in that they can provide help to B‐cells in an innate and adaptive manner, with differing outcomes. iNKTs that are activated by antigen presented on B‐cell C1d can directly license B‐cell activation in a cognate, innate‐like manner, through co‐stimulation with CD40L and the production of various cytokines, such as IFNγ and IL‐21. This leads to a restricted form of B‐ cell activation, with plasmablast expansion, early germinal center development, modest affinity maturation, and primary class‐switched antibody production, but lacking the development of plasma cells and B‐cell memory responses. Alternatively, iNKTs that have been activated by DCs presenting antigen can drive full B‐cell activation in a noncognate, or adaptive fashion, by enlisting the help of CD4+ T‐cells to license B‐cells, driving the generation of mature germinal centers, robust affinity maturation, the development of antibody‐producing plasma cells, and a B‐cell memory response.
NK Receptors

NK Receptors


NK Receptors
Natural killer (NK) cells are a population of leukocytes that, like T‐ and B‐cells, employ receptors that can provoke their activation, the consequences of which are the secretion of cytokines, most notably IFNγ, and the delivery of signals to their target cells via Fas ligand or cytotoxic granules that are capable of kill­ ing the cell that provided the activation signal (Figure 1.40 and Figure 1.41; see also Videoclip 3). 
However, in addition to activating NK receptors, NK cells also possess receptors that can inhibit their function. As we shall see, inhibitory NK cell receptors are critical to the correct functioning of these cells as these receptors are what prevent NK cells from indiscriminately attacking healthy host tissue. Let us dwell on this for a moment because this is quite a different set‐up to the one that prevails with T‐ and B‐cells. A T‐ or B‐lymphocyte has a single type of receptor that either recognizes antigen or it doesn’t. NK cells have two types of receptor: activating receptors that trigger cytotoxic activity upon recognition of ligands that should not be present on the target cell, and inhibitory receptors that restrain NK killing by recognizing ligands that ought to be present. Thus, NK cell killing can be triggered by two different situations: either the appearance of ligands for the activating receptors or the disappearance of ligands for the inhibitory receptors. Of course, both things can happen at once, but one is sufficient.
We have already discussed NK cell‐mediated killing in some detail in Chapter 1, here we will focus on how these cells select their targets as a consequence of alterations to the normal pattern of expression of cell surface molecules, such as classical MHC class I molecules, that can occur during viral infection. NK cells can also attack cells that have normal expression levels of classical MHC class I but have upregulated nonclassical MHC class I‐related molecules because of cell stress or DNA damage.

NK cells express diverse “hard‐wired” receptors
Unlike the antigen receptors of T‐ and B‐lymphocytes, NK receptors are “hard‐wired” and do not undergo V(D)J recombination to generate diversity. As a consequence, NK cell receptor diversity is achieved through gene duplication and divergence and, in this respect, resembles the pattern recognition receptors we discussed in Chapter 1. Thus, NK receptors are a somewhat confusing ragbag of structurally disparate molecules that share the common functional property of being able to survey cells for normal patterns of expression of MHC and MHC‐related molecules. NK cells, unlike αβ T‐cells, are not MHC‐restricted in the sense that they do not see antigen only when presented within the groove of MHC class I or MHC class II molecules. On the contrary, one of the main functions of NK cells is to patrol the body looking for cells that have lost expression of the normally ubiquitous classical MHC class I molecules; a situation that is known as “missing‐self ” recognition (Figure 4.17). Such abnormal cells are usually either malignant or infected with a microorganism that interferes with class I expression.
We saw in Chapter 1 that many pathogens activate PRRs such as Toll‐like receptors that induce transcription of inter­feron‐regulated factors, which subsequently direct the transcription of type I interferons (IFNα and IFNβ). PRRs, such as TLR3, TLR7–9 and the RIG‐like helicases, that reside within intracellular compartments are particularly attuned to inducing the expression of type I interferons (see Figure 1.16). Such PRRs typically detect long single or double‐stranded RNA molecules that are characteristically produced by many viruses. One of the downstream consequences of interferon secretion is the cessation of protein synthesis and consequent downregulation of, among other things, MHC class I molecules. Thus, detection of PAMPs from intracellular viruses or other intracellular pathogens can render such cells vulnerable to NK cell‐mediated attack. Which is exactly the point? Many intracellular pathogens also directly interfere with the expression or surface exposure of MHC class I molecules as a strategy to evade detection by CD8+ T‐cells that survey such molecules for the presence of nonself peptides.
Natural killer (NK) cell‐mediated killing and the “missing‐self” hypothesis

Figure 4.17 Natural killer (NK) cell‐mediated killing and the “missing‐self” hypothesis. (a) Upon encounter with a normal autologous MHC class I‐expressing cell, NK inhibitory receptors are engaged and activating NK receptors remain unoccupied because no activating ligands are expressed on the target cell. The NK cell does not become activated in this situation. (b) Loss of MHC class I expression (“missing‐self”), as well as expression of one or more ligands for activating NK receptors, provokes NK‐mediated attack of the cell via NK cytotoxic granules. (c) Upon encountering a target cell expressing MHC class I, but also expressing one or more ligands for activating NK receptors (“induced‐self”), the outcome will be determined by the relative strength of the inhibitory and activating signals received by the NK cell. (d) In some cases, cells may not express MHC class I molecules or activating ligands and may be ignored by NK cells, possibly owing to expression of alternative ligands for inhibitory NK receptors.

Because of the central role that MHC class I molecules play in presenting peptides derived from intracellular pathogens to the immune system, it is relatively easy to understand why these molecules may attract the unwelcome attentions of viruses or other uninvited guests planning to gatecrash their cellular hosts. It is probably for this reason that NK cells coevolved alongside MHC‐restricted T‐cells to ensure that pathogens, or other conditions that may interfere with MHC class I expression and hence antigen presentation to αβ T‐cells, are given short shrift. Cells that end up in this unfortunate position are likely to soon find themselves looking down the barrel of an activated NK cell. Such an encounter typically results in death of the errant cell as a result of attack by cytotoxic granules containing a battery of proteases and other destructive enzymes released by the activated NK cell.
The Major Histocompatibility Complex (MHC)

The Major Histocompatibility Complex (MHC)


The Major Histocompatibility Complex (MHC)
Molecules within this complex were originally defined by their ability to provoke vigorous rejection of grafts exchanged between different members of a species (Milestone 4.2). We have already referred to the necessity for antigens to be associated with class I or class II MHC molecules in order that they may be recognized by T‐lymphocytes (Figure 4.8). How antigenic peptides are processed and selected for presentation within MHC molecules and how the TCR sees this complex are discussed in detail in Chapter 5, but let us run through the major points briefly here so that reader will appreciate why these molecules are of huge importance within the immune system.
MHC molecules assemble within the cell, where they associate with short peptide fragments derived either from proteins being made by the cell (MHC class I molecules bind to peptides derived from proteins being synthesized within the cell) or proteins that have been internalized by the cell through phagocytosis or pinocytosis (MHC class II molecules bind to peptides derived from proteins made external to the cell). There are some exceptions to these general rules, which we deal with in Chapter 5. We have already made the analogy that this process represents a type of “quality control” checking system where a fraction of proteins present in the cell at any given moment are presented to T‐cells for inspection to ensure that none of these is derived from nonself. Of course, if a cell happens to harbor a nonself peptide, we want the immune system to know about this as quickly as possible, so that the appropri­ ate course of action can be taken. Thus, MHC class I molecules display peptides that are either self, or that are being made by an intracellular virus or bacterium. MHC class II molecules display peptides that are either extracellular self proteins or proteins being made by extracellular microorganisms. The whole point is to enable a T‐cell to inspect what is going on, antigenically speaking, within the cell.
As we shall see, MHC class I molecules serve an important role presenting peptides for inspection by CD8 T‐cells that are mainly preoccupied with finding virally infected or “abnormal” cells to kill. Should a TCR‐bearing CD8 T‐cell recognize a class I MHC–peptide combination that is a good “fit” for its TCR, it will attack and kill that cell. MHC class II molecules, on the other hand, are not expressed on the general cell population but are restricted to cells of the immune system, such as DCs, that have an antigen‐presenting function as we already outlined in Chapter 1. Upon recognition of an appropriate MHC class II–peptide combination by a CD4 T‐cell, this will result in activation of the latter and maturation to an effector T‐cell that can give help to B‐cells to make antibody for example. Although this is an oversimplification, as we will learn in later chapters, please keep in mind the general idea that MHC class I and II molecules present peptides to CD8‐ and CD4‐ restricted T‐cells, respectively, for the purposes of allowing these cells to determine whether they should become “activated” and differentiate to effector cells. Let us now look at these molecules in greater detail.
 
Figure M4.2.1 Main genetic regions of the major histocompatibility complex (MHC).

 


Class I and Class II Molecules Are Membrane Bound Heterodimers
MHC class I
Class I molecules consist of a heavy polypeptide chain of 44 kDa noncovalently linked to a smaller 12 kDa polypep­ tide called β2‐microglobulin. The largest part of the heavy chain is organized into three globular domains (α1, α2, and α3) that protrude from the cell surface, a hydrophobic section anchors the molecule in the membrane, and a short hydrophilic sequence carries the C‐terminus into the cytoplasm (Figure 4.19).

Sunday, April 11, 2021

Pathogen Recognition Receptors Provide The First Line Of Detection For Microbial Antigen

Pathogen Recognition Receptors Provide The First Line Of Detection For Microbial Antigen

Pathogen Recognition Receptors Provide The First Line Of Detection For Microbial Antigen

As we learned in Chapter 1, the innate immune system employs an impressive battery of defense mechanisms that specifically detect the presence of invading microbes, to coordinate a series of rapid responses that deal directly with the invader, while at the same time sowing the seeds for a more specific and long‐lasting adaptive immune response. Over many millennia of co‐evolution, vertebrate immune systems have become impressively adept at accurately identifying the presence of potentially harmful microbes, through the detection of microbial structures that are essential for viability and, therefore, refractive to the pressures of natural selection. These conserved microbial antigens, called pathogen‐associated molecular patterns (PAMPs), are unique to individual classes of microbes, and as such, convey pathogen‐specific information to the innate immune system, to facilitate an appropriate response tailored to the particular threat at hand.
Detection of PAMPs is facilitated by a family of evolutionarily conserved germline‐encoded receptors called pathogen recognition receptors (PRRs), expressed on innate immune cells such as DCs, macrophages, and neutrophils. PAMP detection is often the first indication to the innate immune system of microbial presence and consequently, PAMP‐induced PRR activation rapidly promotes the production of a host of cytokines, chemokines, and type 1 interferons that mobilize innate immune cells to directly confront the invader. Additionally, PRR stimulation acts as a crucial line of communication between the innate and adaptive immune systems by instructing antigen‐presenting cells, such as DCs, to effectively
license a T‐cell‐mediated adaptive immune response against a particular antigen. As will be discussed in later chapters, the particular mode of T‐cell activation is further shaped by PRR‐ induced DC‐derived cytokines, which effectively tailor the T‐ cell‐mediated response to the particular type of microbe. As PRR signaling has also been shown to be important for instructing B‐cells to respond to particular types of microbial antigen, it should be clear that the recognition of microbial PAMP by PRRs plays a crucial role in coordinating both innate and adaptive immune responses to infection.
To date, several different classes of PRRs have been charac­ terized, including Toll‐like receptors (TLRs), NOD‐like receptors (NLRs), RIG‐1‐like receptors (RLRs), DNA receptors, and C‐type lectin‐like receptors, which together sense a wide range of conserved microbial antigen. TLRs are among the best‐characterized PRRs and we will next turn our attention to this important immune receptor family.
TLR family structure, ligand specificity, and signaling mechanism (a) Structures of TLRs bound to ligand and arranged into a phylogenetic tree.
Figure 4.30 TLR family structure, ligand specificity, and signaling mechanism (a) Structures of TLRs bound to ligand and arranged into a phylogenetic tree. The ligands are colored red, and TLRs are blue and green. (b) Overview of LPS recognition by TLR4/MD‐2. LPS binding induces dimerization of the TLR4/MD‐2 complex, which is proposed to enable dimerization of the intracellular TIR domains and recruitment of adapter molecules such as MyD88. Aggregation of the death domains (DD) of MyD88 brings four IRAK4 and four IRAK2 molecules together forming a large tower‐like structure called the “Myddosome.” (Source: Park B.S. et al. (2013) Experimental and Molecular Medicine 45(12), 1–9. Reproduced with permission of Nature Publishing Group.)

Toll‐like receptors detect a wide range of conserved microbial PAMP
Named after a Drosophila protein that was originally discovered as important for embryogenesis and later, as required for anti­fungal immunity, Toll‐like receptors (TLRs) are a key family of mammalian PRRs involved in the detection of a wide variety of PAMPs. To date, 10 TLRs have been described in humans, and 12 have been characterized in mice. TLR1, 2, 4, 5, and 6 are expressed on the cell surface and detect ligands from bacteria, fungi, protozoa, and certain self antigens, whereas expression of TLR3, 7, 8, and 9 are confined to intracellular endocytotic compartments, where they recognize nucleic acids signatures unique to bacteria and viruses (Figure 4.30a).
What antibodies see

What antibodies see


The acquired immune responses mounted by lymphocytes depend upon specific recognition of antigen by the B‐cell receptor (BCR, a transmembrane version of the antibody molecule) or the T‐cell receptor (TCR). Following clonal selection the antigen‐specific lymphocytes undergo proliferation to produce sufficient numbers of effector cells and also to generate memory cells. In the case of B‐cells the main effector cells are the plasma cells that secrete a soluble version of the same antibody that was used as the BCR on the original B‐cell. In the case of T‐cells the effector cells are cytokine‐secreting helper or regulatory cells, or cell‐killing cytotoxic cells.
Introduction
In acquired immunity, specific antigens are recognized by two classes of molecules: (i) antibodies, present either as soluble proteins or as transmembrane molecules on the surface of
B‐cells; and (ii) T‐cell receptors, present as transmembrane molecules on the surface of T‐cells. Antibodies recognize antigens on the outside of pathogens or as soluble material such as toxins, whereas αβ T‐cell receptors recognize peptides in the context of MHC molecules on the surface of host cells. Antibodies can thus be thought of as scanning for foreign material directly whereas T‐cells (particularly cytotoxic T‐cells) are scanning for cells that are infected with pathogens.
 
Complementarity of the antibody combining site and the epitope recognized on the antigen
Figure 5.1 Complementarity of the antibody combining site and the epitope recognized on the antigen. The structure of the complex of the Fab of the antibody pertuzumab and its antigen HER2 is shown. HER2, the human epidermal growth factor receptor, is overexpressed on some breast cancer cells and pertuzumab is an antibody, similar to Herceptin®, with potential as a therapeutic against breast cancer. Below, the two molecules are shown separately with the interaction footprint shown on each. (Source: Robyn Stanfield. Reproduced with permission.)


What antibodies see
Antibodies recognize molecular shapes (epitopes) on antigens. Generally, the better the fit of the epitope (in terms of geometry and chemical character) to the antibody combining site, the more favorable the interactions that will be formed between the antibody and antigen and the higher the affinity of the antibody for antigen. The affinity of the antibody for the antigen is one of the most important factors in determining antibody efficacy in vivo.

Thursday, April 8, 2021

Immunology In The Laboratory

Immunology In The Laboratory


Immunology In The Laboratory
The ability to measure accurately and sensitively different aspects of immunological function is an important part of both experimental and clinical immunology (see Chapter 44). Some of the most commonly used techniques in the immunological laboratory (immunological assays) are shown in the figure. Some techniques, such as the differential blood count, have hardly changed in over a hundred years. Others, such as flow cytometry and the PCR continue to evolve at a rapid rate as new technologies are developed. In all cases, clinical laboratories are making increased use of robotics and sophisticated computational analysis to automate all aspects of the process, to make it faster, cheaper and more reliable. The ability to integrate many different measurements (immunological, haematological, psychological, genetic, etc.) taken from each patient rapidly and reliably is also driving the development of ‘personalized medicine’, where doctors will be increasingly able to tailor each treatment precisely to match the needs of individual patients.

Immunology In The Laboratory

Flow cytometry (Fig. 45.1–45.3) is one of the most powerful techniques in the immunologist’s repertoire. Cells are sucked into a fine jet of liquid so that they pass rapidly across a beam of one or, in more sophisticated machines, several lasers. Cells scatter the incoming beam of light by refraction and reflection. Light scattered through a small angle is called ‘forward scatter’ and is proportional to the size of the cells. Light scattered through a 90° angle is called ‘side scatter’ and depends on the granularity of the cell; e.g. a granulocyte has a much larger side scatter than a lymphocyte (see Fig. 45.1).
Investigating Immunity

Investigating Immunity


Investigating Immunity
As most of the cells and molecules of the immune system spend some or all of their time in the blood, sampling them is usually straightforward, and the standard tests illustrated here account for a substantial part of the routine work of the immunology and haematology laboratories, and often of microbiology and biochemistry too. The assays are mainly automated and a report will usually give the normal values and indicate which results are abnormal. Nevertheless, because of the considerable differences between individuals, and in the same individual with time, interpretation is not always obvious, even to those well versed in other aspects of medicine. The interpretation of tests for autoimmunity is particularly tricky.

Investigating Immunity

Assays of both immunological molecules (e.g. antibody) and cells mainly make use of standard reagents, which are themselves frequently monoclonal antibodies, designed to react with only one molecular feature or cell-surface marker. The use of antibodies, whether monoclonal or not, to detect antigens of any kind is referred to as immunoassay (see Fig. 45). For detection, antibodies may be labelled with a radio-isotope (radioimmunoassay), an enzyme (ELISA) or a fluorescent molecule (immunofluorescence).
Transplant Rejection

Transplant Rejection


Transplant Rejection
The success of organ grafts between identical (‘syngeneic’*) twins, and their rejection in all other cases, reflects the remarkable strength of immunological recognition of cell-surface antigens within a species. This is an unfortunate (and in the evolutionary sense unforeseeable) result of the specialization of T cells for detecting alterations of MHC antigens, upon which all adaptive responses depend (for a reminder of the central role of T-helper cells see Figs 19 and 21), plus the enormous degree of MHC polymorphism (different antigens in different individuals; see Fig. 11). It appears that when confronted with ‘non- self’ MHC molecules, T cells confuse them with ‘self plus antigen’, and in most cases probably ‘self plus virus’; several clear examples of this have already been found in mouse experiments. This may be one of the reasons for MHC polymorphism itself: the more different varieties of ‘self’ a species contains, the less likely is any particular virus to pass undetected and decimate the whole species. Differences in red cell (‘blood group’) antigens also give trouble in blood transfusion (top right) because of antibody; here the rationale for polymorphism is less obvious, but it is much more restricted (e.g. six ABO phenotypes compared with over 1012 for MHC). The ‘minor’ histocompatibility and blood group antigens appear to be both less polymorphic and antigenically weaker.

Transplant Rejection

Graft rejection can be mediated by T and/or B cells, with their usual non-specific effector adjuncts (complement, cytotoxic cells, macrophages, etc.), depending on the target: antibody destroys cells free in the blood, and reacts with vascular endothelium (e.g. of a grafted organ; centre) to initiate type II or III hypersensitivity, while T cells attack solid tissue directly or via macrophages (type IV). Unless the recipient is already sensitized to donor antigens, these processes do not take effect for a week or more, confirming that rejection is due to adaptive, not innate, immunity.
Antimicrobial Immunity: A General Scheme

Antimicrobial Immunity: A General Scheme


Antimicrobial Immunity: A General Scheme
At this point the reader will appreciate that the immune system is highly efficient at recognizing foreign substances by their shape but has no infallible way of distinguishing whether they are dangerous (‘pathogenic’). By and large, this approach works well to control infection, but it does have its unfortunate side, e.g. the violent immune response against foreign but harmless structures such as pollen grains, etc. (see Fig. 35).

Antimicrobial Immunity: A General Scheme

Would-be parasitic microorganisms that penetrate the barriers of skin or mucous membranes (top) have to run the gauntlet of four main recognition systems: complement (top right), phagocytic cells (centre), antibody (right) and cell-mediated immunity (bottom), together with their often interacting effector mechanisms. Unless primed by previous contact with the appropriate antigen, antibody and cell-mediated (adaptive) responses do not come into action for several days, whereas complement and phagocytic cells (innate), being ever present, act within minutes. There are also (top centre) specialized innate elements, such as lysozyme, interferons, etc., which act more or less non-specifically, much as antibiotics do. Innate molecules that have evolved to block virus infection are sometimes called restriction factors.

Friday, November 6, 2020

DYNAMIC INTERACTIONS AT THE BCR SYNAPSE

DYNAMIC INTERACTIONS AT THE BCR SYNAPSE

DYNAMIC INTERACTIONS AT THE BCR SYNAPSE

Just as TCRs form immunological synapses during contact with specific peptide–MHC, Bcell receptors have also been found to exhibit similar behavior, particularly when antigen is presented on a membrane surface. Although Bcells can be stimulated by soluble antigen, it is now widely accepted that the primary form of antigen that triggers Bcell activation in vivo is localized to membrane surfaces. The most likely culprits here are the follicular DCs that are resident within lymph nodes, as well as macrophages and DCs that migrate there bearing gifts of antigen. Antigens can be immobilized on cell surfaces by complement or Fc receptors as immunocomplexes, or through direct binding to various scavenger receptors. An encounter between a Bcell and membraneassociated antigen provides the opportunity for the Bcell membrane to spread along the opposing membrane, gathering sufficient antigen to trigger Bcell activation, as well as providing an opportunity for other contacts to be made, such as those that can be provided by membrane integrins. This spreading response is driven by BCR engagement of antigen at the leading edge of the Bcell and, apart from increasing the number of BCR–antigen contacts that are then available to trigger Bcell activation, the spreading response also increases the amount of antigen that is ultimately concentrated and internalized by the Bcell, leading to more efficient antigen presentation to activated Tcells when the Bcell subsequently goes looking for Tcell help (Figure 7.30).

THE NATURE OF B‐CELL ACTIVATION

THE NATURE OF B‐CELL ACTIVATION

THE NATURE OF B‐CELL ACTIVATION

Similar to Tcells, active or resting Bcells are nondividing and activation through the BCR drives these cells into the cell cycle. As is the case for the TCR, the BCR (surface Ig) does not possess any intrinsic enzymatic activity. Once again, it is the accessory molecules associated with the antigen receptor that propagate activation signals into the Bcell. It was noted in Chapter 4 that the BCR complex is composed of membraneanchored immunoglobulin that is associated with a disulfidelinked Igα and Igβ heterodimer, the cytoplasmic tails of which each contain a single ITAM motif (see Figure 4.4). As we will now discuss in more detail, antigendriven crosslinking of the BCR results in the initiation of a (Bruton’s tyrosine kinase). Active Lyn also phosphorylates residues on CD19, a component of the Bcell coreceptor complex (discussed in detail in the next section) that rein­ forces signals initiated by the BCR (Figure 7.28). Syk fulfills a critical role within the Bcell activation pro­cess; disruption of the gene encoding Syk in the mouse has profound effects on downstream events in Bcell signaling and results in defective Bcell development. In this respect, Syk serves a similar role in Bcells to that served by ZAP70 inTcells. Active Syk phosphorylates and recruits BLNK (Bcell linker; also called SLP65, BASH, and BCA) to the BCR complex. Upon phosphorylation by Syk, BLNK provides binding sites for phospholipase Cγ2 (PLCγ2), Btk, and Vav. Recruitment of Btk in close proximity to PLCγ2 enables Btk to phosphorylate the latter and increase its activity. Just as in the Tcell signaling pathway, activated PLCγ2 initiates a pathway that involves hydrolysis of PIP2 to generate diacylglycerol and inositol trisphosphate and results in increases in intracellular calcium and PKC activation (Figure 7.28). PKC activation, in turn, results in activation of the NFκB and JNK transcription factors and increased intracellular calcium results in NFAT activation, just as it does in Tcells.

Receptor cross‐linking recruits the BCR to lipid rafts

Figure 7.27 Receptor crosslinking recruits the BCR to lipid rafts. Antigeninduced receptor crosslinking recruits the BCR, which is normally excluded from membrane cholesterolrich lipid raft domains, to membrane lipid rafts where signaling proteins such as the protein tyrosine kinase Lyn reside. Stable recruitment of the BCR to rafts facilitates Lynmediated phosphorylation of ITAMs within the cytoplasmic tails of the Igα and Igβ accessory molecules that initiate the BCRdriven signaling cascade.

The Vav family of guanine nucleotide exchange factors consists of at least three isoforms (Vav1, 2, and 3) and is known to play a crucial role in Bcell signaling through activation of Rac1 and regulating cytoskeletal changes after BCR crosslinking; Vav1deficient Bcells are defective in proliferation associated with crosslinking of the BCR (Figure 7.27).

Signaling cascade downstream of antigen‐driven Bcell receptor (BCR) ligation

Figure 7.28 Signaling cascade downstream of antigendriven Bcell receptor (BCR) ligation. Upon interaction with antigen, the BCR is recruited to lipid rafts where ITAMs within the Igα/βheterodimer become phosphorylated by Lyn. This is followed by recruitment and activation of the Syk and Btk kinases. Phosphorylation of the Bcell adaptor protein BLNK creates binding sites for several other proteins, including PLCγ2 that promotes PIP2 hydrolysis and instigates a chain of signaling events culminating in activation of the NFAT and NFkB transcription factors. The CD19 coreceptor molecule is also phosphorylated by Lyn and can suppress the inhibitory effects of GSK3 on NFAT through the PI3K/Akt pathway. BCR stimulation also results in rearrangement of the cell cytoskeleton via activation of Vav that acts as a guanine nucleotide exchange factor for small Gproteins such as Rac and Rho.


The BCR crosslinking model seems appropriate for an understanding of stimulation by type 2 thymusindependent antigens, as their repeating determinants ensure strong binding to, and crosslinking of, multiple Ig receptors on the Bcell sur­ face to form aggregates that persist owing to the long halflife of the antigen and sustain the high intracellular calcium needed for activation. On the other hand, type 1 Tindependent antigens, such as the Tcell polyclonal activators, probably bypass the specific receptor and act directly on downstream molecules such as diacylglycerol and protein kinase C, as Igα and Igβ are not phosphorylated.

 

Bcells require costimulation via the Bcell coreceptor complex for efficient activation

Similar to Tcells, Bcells also require two forms of costimulation to mount efficient effector responses. One form of co stimulation takes place at the point of initial encounter of the BCR with its cognate antigen and is provided by the Bcell coreceptor complex that is capable of engaging with molecules such as complement that may be present in close proximity to the specific antigen recognized by the BCR. The other form of costimulation required by Bcells takes place after the initial encounter with antigen and is provided by Tcells in the form of membraneassociated CD40 ligand that engages with CD40 on the Bcell. This form of costimulation requires that the Bcell has internalized antigen, followed by processing and presentation on MHC class II molecules to an appropriate Tcell. If the Bcell is displaying an MHCpeptide combination recognized by the Tcell, the latter will be stimulated to produce cytokines (such as IL4) as well as provide costimulation to the Bcell in the form of CD40L. We will consider the nature of the costimulatory signals provided by the Bcell co receptor complex here and deal with CD40Lbased costimulation in a separate section below.

The B‐cell co‐receptor complex provides co‐stimulatory signals for B‐cell activation through recruitment of a number of signaling molecules

Figure 7.29 The Bcell coreceptor complex provides costimulatory signals for Bcell activation through recruitment of a number of signaling molecules, including phosphatidylinositol 3kinase and Vav, which can amplify signals initiated through the Bcell receptor. On mature Bcells, CD19 forms a tetrameric complex with three other proteins: CD21 (complement receptor type 2), CD81 (TAPA1), and CD225 (interferoninduced transmembrane protein 1) (LEU13).

The mature Bcell coreceptor complex (Figure 7.29) is composed of four components: CD19, CD21 (complement receptor type 2, CR2), CD81 (TAPA1), and CD225 (LEU13, interferoninduced transmembrane protein 1). CR2 is a receptor for the C3d breakdown product of complement and its presence within the BCR coreceptor complex enables a innate immune response (complement) to synergize with the BCR to productively activate Bcells. Imagine a bacterium that has activated complement and has become coated with the products of complement activation, including C3d. If the same bacterium is subsequently captured by the BCR on a Bcell, there is now an opportunity for CR2 within the BCR coreceptor complex to bind C3d, which effectively means that the Bcell now receives two signals simultaneously. Signal 1 comes via the BCR and signal 2 via the coreceptor complex. So how does simultaneous engagement of the coreceptor complex and the BCR lead to enhanced Bcell activation?

Well, the answer is that we don’t know for sure, but it is clear that CD19 plays an especially important role in this process. CD19 is a Bcellspecific transmembrane protein that is expressed from the proBcell to the plasmacell stage and possesses a relatively long cytoplasmic tail containing nine tyrosine residues. Upon Bcell receptor stimulation, the cytoplasmic tail of CD19 undergoes phosphorylation at several of these tyrosine residues (by kinases associated with the BCR) that creates binding sites on CD19 for several proteins, including the tyrosine kinase Lyn, Vav, and phosphatidylinositol 3kinase (PI3K). CD19 plays a role as a platform for recruitment of several proteins to the BCR complex (Figure 7.28), much in the same way that LAT functions in TCR activation.

Vav is recruited to CD19 upon phosphorylation of the latter by Lyn and, along with PI3K that is also recruited to CD19 as a result of Lynmediated phosphorylation (Figure 7.28), plays a role in the activation of the serine/thre­onine kinase Akt; the latter may also enhance NFAT activation through neutralizing the inhibitory effects of GSK3 (glycogen synthase kinase 3) on NFAT. Because GSK3 can also phosphorylate and destabilize Myc and cyclin D, which are essential for cell cycle entry, Akt activation also has positive effects on proliferation of activated Bcells.

Similar to the role that CD28 plays on Tcells, the Bcell coreceptor amplifies signals transmitted through the BCR approximately 100fold. As we have discussed above, because CD19 and CR2 (CD21) molecules enjoy mutual association, this can be brought about by bridging the Ig and CR2 receptors on the Bcell surface by antigen–C3d complexes bound to the surface of APCs. Thus, antigeninduced clustering of the Bcell coreceptor complex with the BCR lowers the threshold for Bcell activation by bringing kinases that are associated with the BCR into close proximity with the coreceptor complex. The action of these kinases on the coreceptor complex engages signaling pathways that reinforce signals originating from the BCR.

Figure 7.30 CD40–CD40Ldependent Bcell costimulation by a Thelper cell. Independently activated T and Bcells can interact if the Bcell is presenting the correct peptide–MHC complex sufficient for stimulation of the Tcell. Successful antigen presentation by a Bcell to an activated Thelper cell results in CD40Ldependent costimulation of the Bcell as well as the provision of cytokines, such as IL4, by the Tcell that are essential for class switching, clonal expansion and differentiation to effector cells.

Bcells also require costimulation from Thelper cells

Just as Tcells require costimulatory signals from DCs in the form of B7 ligands for productive activation (Figure 7.3), Tdependent Bcells also require costimulation from Thelper cells in order to cross the threshold required for clonal expansion and differentiation to effector cells (Figure 7.24). The sequence of events goes much like this. Upon encountering cognate antigen through direct binding to a microorganism, the BCR undergoes the initial activation events described above. This culminates in the internalization of the BCR, along with captured antigen, which is then processed and presented on MHC class II molecules (Figure 7.30). To continue the process of maturation to either a plasma cell or a memory cell, the Bcell must now encounter a Tcell capable of recognizing one of the antigenic peptides the Bcell is now presenting from the antigen it has internalized. Note that this need not be the same epitope recognized by the Bcell to undergo initial activation. Upon encountering a Tcell with the appropriate TCR, the Bcell provides stimulation to the Tcell in the form of MHC–peptide as well as costimulatory B7 signals (Figure 7.30). In turn, the Tcell upregulates CD40 ligand (CD40L) that can provide essential costimulation to the Bcell, enabling the latter to become fully activated and undergo clonal expansion and class switching. If CD40L help is not forthcoming, Bcells rapidly undergo apoptosis and are eliminated. This help is provided by a special class of Tcell called a follicular helper Tcell, a distinct branch of CD4+ Tcells that express the cell surface receptor CXCR5, which targets them to Bcell follicles in the secondary lymphoid organs. Thus, Bcells and Tcells provide mutual costimulation as a means of reinforcing their initial activation signals (Figure 7.30). In effect, the Blymphocyte is acting as an APC and, as mentioned above, it is very efficient because of its ability to concentrate the antigen by focusing onto its surface Ig. Nonetheless, although a preactivated Thelper can mutually interact with and stimulate a resting Bcell, a resting Tcell can only be triggered by a Bcell that has acquired the B7 costimulator and this is only present on activated, not resting, Bcells.

Presumably the immune complexes on follicular DCs in germinal centers of secondary follicles can be taken up by the Bcells for presentation to Thelpers, but, additionally, the complexes could crosslink the sIg of the Bcell blasts and drive their proliferation in a Tindependent manner. This would be enhanced by the presence of C3 in the complexes as the Bcell complement receptor (CR2) is comitogenic.

Damping down Bcell activation

We have already discussed how Tcell enthusiasm for antigen can be dissipated by engaging CTLA4; similar mechanisms also operate to damp down signals routed through the BCR. Several cell surface receptors, including FcγRIIB, CD22, and PIRB (paired immunoglobulinlike receptor B), have been implicated in antagonizing Bcell activation through recruitment of the protein tyrosine phosphatase SHP1 to ITIMs (immunoreceptor tyrosinebased inhibitory motifs) in their cytoplasmic tails. SHP1 impairs BCR signaling by antagonizing the effects of the Lyn kinase on Syk and Btk; by dephosphorylating both of these proteins SHP1 blocks recruitment of PLCγ2 to the BCR complex. Coligation of the BCR with any of these receptors is therefore likely to block Bcell activation. CD22 appears to be constitutively associated with the BCR in resting Bcells and in this way may raise the threshold for Bcell activation. Successful formation of a Bcell receptor synapse ay physically exclude CD22 from the BCR complex.


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