Immunologie

Differentiatie leukocyten

Monocyten en macrofagen

Macrophages express a variety of receptors, each of which is able to recognize specific components of microbes. Some, like the mannose and glucan receptors and the scavenger receptor, bind cell-wall carbohydrates of bacteria, yeast, and fungi. The Toll-like receptors (TLRs) are an important family of pattern-recognition receptors present on macrophages, dendritic cells, and other immune cells. TLRs recognize different microbial components; for example, a heterodimer of TLR-1 and TLR-2 binds certain lipopeptides from pathogens such as Gram-positive bacteria, while TLR-4 binds both lipopolysaccharides from Gram-negative and lipoteichoic acids from Gram-positive bacteria. Other pattern-recognition receptors are intracellular, such as the nucleotide-binding and oligomerization domain (NOD)-like receptors. NOD1, for example, binds to diaminopimelic acid, a component of the cell wall of most Gramnegative bacteria.

T-helper 1 cellen activeren macrofagen zeer sterk

When an effector TH1 cell specific for a bacterial peptide contacts an infected macrophage, the T cell is induced to secrete the macrophage-activating factor IFN-γ and to express CD40 ligand. Together these newly synthesized TH1 proteins activate the macrophage.
Monocytes circulating in the blood migrate into infected and inflamed tissues. Adhesion moleculeson the endothelial cells of the blood vessel wall capture the monocyte and cause it to adhere to the vascular endothelium. Chemokines bound to the vascular endothelium then signal the monocyteto migrate across the endothelium into the underlying tissue. The monocyte, now differentiating into an inflammatory monocyte, continues to migrate, under the influence of chemokines released duringinflammatory responses, toward the site of infection. Monocytes leaving the blood are also able to differentiate into dendritic cells (not shown), depending on the signals that they receive from their environment.
Important cytokines and chemokines secreted by dendritic cells and macrophages in response to bacterial products include IL-1β, IL-6, CXCL8, IL-12, and TNF-α. TNF-α is an inducer of a local inflammatory response that helps to contain infections. It also has systemic effects, many of which are harmfu. The chemokine CXCL8 is also involved in the local inflammatory response, helping to attract neutrophils to the site of infection. IL-1β, IL-6, and TNF-α have a crucial role in inducing the acute-phase response in the liver and inducing fever, which favors effective host defense in various ways. IL-12 activates natural killer (NK) cells and favors the differentiation of CD4 T cells into the TH1 subset in adaptive immunity.

Dendritische cellen

Antigen-presenterende cellen (APC)

Cytokines en chemokines

T-cel cytokines

Macrophages encountering bacteria or other types of microorganisms in tissues are triggered to release cytokines (left panel) that increase the permeability of blood vessels, allowing fluid and proteins to pass into the tissues (center panel). Macrophages also produce chemokines, which direct the migration of neutrophils to the site of infection. The stickiness of the endothelial cells of the blood vessel wall is also changed, so that circulating cells of the immune system adhere to the wall and are able to crawl through it; first neutrophils and then monocytes are shown entering the tissue from a blood vessel (right panel). The accumulation of fluid and cells at the site of infection causes the redness, swelling, heat, and pain known collectively as inflammation. Neutrophils and macrophages are the principal inflammatory cells. Later in an immune response, activated lymphocytes can also contribute to inflammation.

Chemokine networks

Chemokines are classified into four families on the basis of structural differences: CXCL, CCL, XCL, and CXC3CL. Chemokines can also be classified as pro-inflammatory (red), homeostatic (green), and mixed function (yellow). Chemokines bind to a subfamily of seven-transmembrane G-protein-coupled receptors, which are classified as CXCR, CCR, XCR, and CX3CR on the basis of the class of chemokines they bind. Many, but not all, of the chemokine-chemokine receptor networks that coordinateimmune modules are represented here. The connection between receptors and cell types on which they are expressed is indicated by the ‘circuit’ representation of lines and connecting nodes. To connect chemokines and their receptors to target cells, follow a horizontal line and turn on a vertical one at each node; the rhomboids (diamond shapes) link vertical lines to the cell type. Note that most chemokine receptors can bind multiple chemokines.

B-lymfocyten

B-celactivatie 

Lymfeklier

Blauw: T-cel zoneLichtgeel: B-cel zoneGeel: kiemcentrum

Origine van lymfomen

VDJ recombinatie (genherschikking)

V-region genes are constructed from gene segments. Light-chain V-region genes are constructed from two segments (center panel). A variable (V) and a joining (J) gene segment in the genomic DNA are joined to form a complete light-chain V-region exon. Immunoglobulin chains are extracellular proteins, and the V gene segment is preceded by an exon encoding a leader peptide (L), which directs the protein into the cell’s secretory pathways and is then cleaved. The light-chain C region is encoded in a separate exon and is joined to the V-region exon by splicing of the light-chain RNA to remove the L-to-V and the J-to-C introns. Heavy‑chain V regions are constructed from three gene segments (right panel). First, the diversity (D) and J gene segments join, and then the V gene segment joins to the combined DJ sequence, forming a complete VH exon. A heavy-chain C-region gene is encoded by several exons. The C-region exons, together with theleader sequence, are spliced to the V-domain sequence during processing of the heavy-chain RNA transcript. The leader sequence is removed after translation, and the disulfide bonds that link the polypeptide chains are formed. The hinge region is shown in purple.
The immunoglobulin isotypes are encoded by a cluster of immunoglobulin heavy-chain C-region genes. The general structure of the main immunoglobulin isotypes (above in upper panel) is indicated, with each rectangle denoting an immunoglobulin domain. These isotypes are encoded by separate heavy-chain C-region genes arranged in a cluster in both mouse and human (lower panel). The constant region of the heavy chain for each isotype is indicated by the same color as the C-region gene segment that encodes it. IgM and IgE lack a hinge region but each contains an extra heavy-chain domain.

Rol van cytokines in antibody class switchting

Functie van verschillende immunoglobulines

Constant and variable region

Fab fragmenten

Zware en lichte ketens

Lichte ketens zijn van type kappa (60%) of lambda (40%).

Nanobodies

The single variable domain on a heavy chain (VHH fragments) represents the absolute smallest antibody fragment necessary to specifically bind an antigen with exceptionally high affinity.

T-cellen

CD4+ bindt aan MHC-II, CD8+ bindt aan MHC-I

Drie signalen voor T-cel activatie/proliferatie

Stages of α:β T-cell development in the mouse thymus

The stages of α:β T-cell development in the mouse thymus correlate with the program of gene rearrangement, and the expression of cell-surface proteins, signaling proteins, and transcription factors.
The state of the T-cell receptor genes, the expression of some essential intracellular proteins, and the expression of some cellsurface molecules are shown for successive stages of α:β T-cell development. Note that because the T-cell receptor genes do not undergo further changes during antigendriven development, only the phases during which they are actively undergoing rearrangement in the thymus are indicated. The antigen-dependent phases of CD4 and CD8 cells are depicted separately, and are detailed in Chapter 9.

A summary of the development of human α:β T cells.

Schematic structure of T-cell receptor

The TCRα:β heterodimer is very similar to a Fab fragment of immunoglobulin.

Signaal 1: peptide:MHC complex

Signaal 2: co-stimulatie van T-cellen

Co-inhibitie

Peptide:MHC tetramer

Downstream signaling in T-cellen na co-stimulatie

Signaling via CD28 leidt uiteindelijk tot activatie van transcriptiefactore (NFAT, AP-1, NFKB) en uiteindelijk expressie van o.a. IL-2, wat een essentieel cytokine is voor T-cel proliferatie, survival en differentiatie naar effector T-cellen.

Klonale expansie

Expressie van IL-2 receptor op geactiveerde T-cel

Leden van de TNF-TNFR superfamilie kunnen de canonical NFκB pathway activeren

Signaal 3: CD4 T-cell effector differentiatie door cytokines

Geactiveerde T-cellen brengen andere moleculen op oppervlak tot expressie

Fate-specifying cytokines initiëren differentiatie van CD4+ T-cellen

Differentatie naar subset is afhankelijk van cytokines en intracellulaire pathways

With the exception of TGF-β, which participates in both TH17 and pTreg development, the cytokines that specify the development of distinct effector cells typically activate different members of the STAT family of transcription factors

Effector moleculen geproduceerd door verschillende T-cel subsets

CD8 T cells are predominantly killer T cells that recognize peptide:MHC class I complexes. They releaseperforin (which helps deliver granzymes into the target cell) and granzymes (which are pro-proteases that are activated intracellularly to trigger apoptosis in the target cell), and often also produce thecytokine IFN-γ. The various functional subsets of CD4 T cells recognize peptide:MHC class II complexes. TH1 cells are specialized to activate macrophages that are infected by or have ingested pathogens; they secrete IFN-γ to activate the infected cell, as well as other effector molecules. They can express membrane-bound CD40 ligand and/or Fas ligand.

T-cel cytokines

CD4+ effector T-cel subsets

Verandering van oppervlaktemoleculen na T-cel activatie

Effector T cells change their surface molecules, allowing them to home to sites of infection

Checkpoint inhibitie

PD-1/PD-L1 inhibitie

CTLA-4 inhibitie

Some, but not all, tumors express ligands for PD-1, such as PD-L1 or PD-L2. Left panels: T cells reacting to tumors without PD-1 ligands do not encounter the inhibitory signal from PD-1 and may deliver their cytotoxicaction when recognizing tumor-specific antigens. In contrast, tumors expressing PD-1 ligands can repress T cells and be protected from cytotoxicity. Right panels: For tumors expressing PD-1 ligands, checkpoint blockade can be achieved by antagonisticantibodies against PD-1 or against the ligand, PD-L1 in this case.

Complement

C3 ios Activatie van het complementsysteem via lectine, klassieke of alternatieve route leidt tot:- Chemotaxis van immuuncellen (door productie van anafylatoxines zoals  C3a en C5a)- Opsonizatie (via C3b dat bindt aan pathogenen = 'opsonisatie')- Beschadigen van membranen van pathogenen (via C5b-9 = MAC)

Pattern recognition receptors

The four classes are toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD) leucinerich-repeat (LRR)–containing receptors (NLRs), and retinoic acid–inducible gene I protein (RIG-I) helicase receptors. NLRs, the central components of the inflammasomes, are complex protein platforms that lead to the activation of caspase 1 and interleukin-1β processing. The most extensively studied inflammasomes are as follows: the NOD leucine-rich-repeat and pyrin domain–containing protein 3 (NLRP3) inflammasome, activated by bacterial and fungal pathogen-associated molecular patterns; the NLR family caspase recruitment domain–containing protein (CARD) 4 (NLRC4) inflammasome, activated during intracellular bacterial infections by flagellin; and the absent in melanoma 2 (AIM2) inflammasome, activated by double-stranded (ds) DNA. ASC denotes apoptosis-associated speck-like protein containing a CARD, HIN hematopoietic interferon-inducible nuclear protein, IRF3 interferon regulatory factor 3, LPS lipopolysaccharide, MAL myelin and lymphocyte protein, MDA5 melanoma differentiation-associated protein 5, MR mineralocorticoid receptor, MyD88 myeloid differentiation factor 88, NF-κB nuclear factor-κB, NLRC4 NLR family CARD-domain–containing protein 4 (also known as IPAF), ss single-stranded, SYK spleen tyrosine kinase, TRAM TRIF-related adaptor molecule, and TRIF toll-like–receptor adaptor moleculeBron: NEJM 2011

Toll-like receptors

Locatie van TLR

Stranger and danger models. Dendritic cells (DCs) are present in all tissues, where they gather antigens from the local environment but are not in an immunostimulatory state. In Janeway’s ‘stranger’ model, antigen-presenting cells (later appreciated to be DCs) were endowed with pattern-recognition receptors (PRRs) that recognize the unique features of microbial molecules (pathogen-associated molecular patterns, PAMPs). When PAMPs were present — for example, from an infection or adjuvant — then DCs were stimulated to migrate to lymphoid tissues and present both antigen and co-stimulatory molecules (CD80 and/or CD86) to T cells. In Matzinger’s ‘danger’ model, the crucial event controlling the initiation of an immune response was not infection, but the production of danger signals known as damage-associated molecular patterns (DAMPs) from cells stressed, damaged and/or dying in the local tissue. These were postulated to act on DCs in a manner that also caused them to migrate to lymphoid tissue and present antigens to T cells in an immunostimulatory manner. It has been speculated that DAMPs might be produced in response to PAMPs and therefore that DAMPs might be the final mediator promoting immune responses in all situations, including infection. This might occur; however, it is also possible, and in our view probable, that DAMPs and PAMPs can alert the immune system to a problem independently and possibly even in a synergistic manner. TCR, T-cell receptorBron
Discriminating between viable cells, necrosis and apoptosis. The hidden self model proposes that innate immune cells have receptors that detect certain intracellular molecules, known as damageassociated molecular patterns (DAMPs), that are normally hidden in the interior of cells and that are only revealed after necrosis. This model can explain why live cells, which contain pre-existing danger signals, don’t stimulate the innate immune system. Moreover, it can also explain why necrotic cells always stimulate the innate immune system, whereas apoptotic cells are only stimulatory in some situations and not in others100. This is because necrotic cells always lose membrane integrity and release their intracellular contents. By contrast, apoptotic cells initially maintain membrane integrity. If apoptotic cells are thenrapidly cleared by phagocytes, the dead cells don’t release their intracellular DAMPs and the immune system is not stimulated. However, if apoptotic cells are not rapidly cleared, as might occur in a solid organ, they undergo secondary necrosis and become permeable. The resulting release of DAMPs then stimulates the innate immune system. So, the event that communicates ‘danger’ to the immune system is not how the cell dies per se, but whether or not it eventually loses membrane integrity and releases its intracellular contents into the extracellular milieu. This concept of hidden signals being revealed might also apply to DAMPs of extracellular origin (for example, when revealed through the action of enzymes released after cell damage) and even, as has been proposed, hidden portions of molecules.Bron

NLRs en inflammasoom

Cellular damage activates the NLRP3 inflammasome to produce pro-inflammatory cytokines. The LRR domain of NLRP3 associates with chaperones (HSP90 and SGT1) that prevent NLRP3 activation. Damage to cells caused by bacterial pore-forming toxins or activation of the P2X7 receptor by extracellular ATP allows efflux of K+ ions from the cell; this may dissociate these chaperones from NLRP3 and induce multiple NLRP3  molecules to aggregate through interactions of their NOD domains (also called the NACHT domain). Reactive oxygen intermediates (ROS) and disruption of lysosomes also can activate NLRP3 (see text). The aggregated NLRP3 conformation brings multiple NLRP3 pyrin domains into close proximity, which then interact with the pyrin domains of the adaptor protein ASC (PYCARD). This conformation aggregates the ASC CARD domains,  which in turn aggregate the CARD domains of pro-caspase 1. This aggregation of procaspase 1 induces proteolytic cleavage of itself to form the active caspase 1,  which cleaves the immature forms of pro-inflammatory cytokines, releasing the mature cytokines that are then secreted.
The inflammasome is composed of several filamentous protein polymers created byaggregated CARD and pyrin domains. Schematic interpretation of NLRP3 inflammasome assembly. In this model, CARD regions of ASC and caspase 1 aggregate into a filamentous structure. The adaptor ASC translatesaggregation of NLRP3 into aggregation of pro-caspase 1.

Selectines en integrines

Selectines, ingegrines en VCAM/ICAM

Transmigratie van lymfocyten

Naive T cells are induced to roll along the surface of a high endothelial venule (HEV) by the interactionsof selectins expressed by the T cells with vascular addressins on the endothelial cell membranes. Chemokines present at the HEV surface activate receptors on the T cell, and chemokine receptorsignaling leads to an increase in the affinity of integrins on the T cell for the adhesion molecules expressed on the HEV. This induces strong adhesion. After adhesion, the T cells follow gradients of chemokines to pass through the HEV wall into the paracortical region of the lymph node.
Adhesion molecules involved in leukocyte interactions. Several structural families of adhesion molecules have a role in leukocyte migration, homing, and cell–cell interactions: the selectins, the integrins, and proteins of the immunoglobulin superfamily. The figure shows schematic representations of an example from each family, a list of other family members that participate in leukocyte interactions, their cellular distribution, and their ligand in adhesive interactions. The family members shown here are limited to those that participate in inflammation and other innate immune mechanisms. 

Transmigratie van neutrofielen

Neutrophils leave the blood and migrate to sites of infection in a multi-step process involving adhesive interactions that are regulated by macrophage-derived cytokines and chemokines Top panel: the first step involves the reversible binding of a neutrophil to vascular endothelium through interactions between selectins induced on the endothelium and their carbohydrate ligands on the neutrophil, shown here for E-selectin and its ligand, the sialyl-LewisX moiety (s-Lex). This interaction cannot anchor the cells against the shearing force of the flow of blood, and thus they roll along the endothelium, continually making and breaking contact. Bottom panel: the binding does, however, eventually trigger stronger interactions, which result only when binding of a chemokine such as CXCL8 to its specific receptor on the neutrophil triggers the activation of the integrins LFA-1 and CR3 (Mac-1; not shown). Inflammatory cytokines such as TNF-α are also necessary to induce the expression of adhesion molecules such as ICAM-1 and ICAM-2, the ligands for these integrins, on the vascular endothelium. Tight binding between ICAM-1 and the integrins arrests the rolling and allows the neutrophil to squeeze between the endothelial cells forming the wall of the blood vessel (i.e., to extravasate). The leukocyte integrins LFA-1 and CR3 are required for extravasation and for migration toward chemoattractants.

Laboratoriumtechnieken

Immunisatie

Antibodies can be elicited by small chemical groups called haptens only when the hapten is linked to an immunogenic protein carrier. Following immunization with a hapten–carrier conjugate, three types of antibodies are produced. One set (blue) binds the carrier protein alone and is called carrier-specific. One set (red) binds to the hapten on any carrier or to free hapten in solution and is called hapten-specific. One set (purple) binds only the specific conjugate of hapten and carrier used for immunization, apparently bindingto sites at which the hapten joins the carrier, and is called conjugate-specific. The amount of antibody of each type in this serum is shown schematically in the graphs at the bottom; note that the original antigen binds more antibody than the sum of anti-hapten and anti-carrier antibodies as a result of the additional binding of conjugate-specific antibody.

Affinity chromatography

Affinity chromatography uses antigen–antibody binding to purify antigens or antibodies. To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by alteringthe pH, which can usually disrupt antibody-antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown).

ELISA

To detect antigen A, purified antibody specific for antigen A is linked chemically to an enzyme. The samples to be tested are coated onto the surface of plastic wells, to which they bind nonspecifically; residual sticky sites on the plastic are blocked by adding irrelevant proteins (not shown). The labeled antibody is then added to the wells under conditions that prevent nonspecific binding, so that only binding toantigen A causes the labeled antibody to be retained on the surface. Unbound labeled antibody is removed from all wells by washing, and bound antibody is detected by an enzyme-dependent color-change reaction. This assay allows arrays of wells known as microtiter plates to be read in fiberoptic multichannel spectrometers, greatly speeding the assay. Modifications of this basic assay allow antibody or antigen in unknown samples to be measured.

Mass spectrometry

Following immunoprecipitation of a multiprotein complex using antibodies specific for one component of the complex, the individual proteins are separated by gel electrophoresis. An individual band representing one protein is isolated and digested with a protease such as trypsin. The digested protein sample is injected into the mass spectrometer, which ionizes the peptides, transfers them to the gas phase, and then separates them based on differences in their mass to charge (m/z) ratio by subjecting them under high vacuum to a magnetic field. A detector collects information on the signal intensity for each peptide ion and displays the information as a histogram. This histogram, usually referred to as a spectrum, is compared with a database containing potential cleavage sites for the proteolytic enzyme used in all known protein sequences, allowing for identification (ID) of the protein in the sample.

Multidimensional mass spectrometry (MS/MS).

Multidimensional mass spectrometers (MS/MS) consist of two mass spectrometers linked in tandem but with an interceding middle sector that fragments ions. In the first sector, the first mass spectrometer separates peptide ions, as shown in Fig. A.16. Each peptide ion from this first separation is then fragmented in the middle sector of the apparatus by collision with other molecules (often an inert gas such as N2). Since fragmentation occurs primarily in the peptide backbone, a mixture of fragments is generated in which the fragments each differ by one amino acid residue. The resultant fragments are then separated in the second mass spectrometer, the final sector. The sequence of the peptide can be read directly from the second mass spectrum. The order of amino acid residues in the peptide can be deduced because of the exquisite precision of the measurements ofeach ion together with knowledge of the exact mass of each possible amino acid residue.

Competitive inhibition assay for antigen in unknown samples

A fixed amount of unlabeled antibody is attached to a set of wells, and a standard reference preparation of a labeled antigen is bound to it. Unlabeled standard or test samples are then added in various amounts and the displacement of labeled antigen is measured, generating characteristic inhibition curves. A standard curve is obtained by using known amounts of unlabeled antigen identical to that used as the labeled species, and comparison with this curve allows the amount of antigen in unknown samples to be calculated. The green line on the graph represents a sample lacking any substance that reacts with anti-A antibodies.

Western blotting

Like immunoprecipitation (see Section A-13), immunoblotting is used for identifying the presence of a given protein in a cell lysate, but it avoids the problem of having to label large quantities of cells with radioisotopes. Unlabeled cells are placed in detergent to solubilize all cell proteins, and the lysate is run on SDS-PAGE to separate the proteins (see Section A-13). The sizeseparated proteins are then transferred from the gel to a stable support such as a nitrocellulose membrane. Specific proteins are detected by treatment with antibodies able to react with SDS-solubilized proteins (mainly those that react with denatured sequences); the bound  antibodies are revealed by anti-immunoglobulin antibodies labeled with an enzyme. The term Western blotting as a synonym for immunoblotting arose because the comparable technique for detecting specific DNA sequences is known as Southern blotting, after Edwin Southern, who devised it, which in turn provoked the name ‘Northern’ for blots of size-separated RNA, and ‘Western’ for blots of sizeseparated proteins. Western blots have many applications in basic research and clinical diagnosis. They are often used to test sera for the presence of antibodies against specific proteins, for example, to detect antibodies against different constituents of HIV (Fig. A.15).

PBMC isolation: Fycoll gradient

Diluted anticoagulated blood (left panel) is layered over Ficoll-Hypaque™ and centrifuged. Red blood cells and polymorphonuclear leukocytes or granulocytes are denser and travel through the Ficoll-Hypaque™, while mononuclear cells consisting of lymphocytes together with some monocytes band over it and can be recovered at the interface (right panel).

Flow cytometrie en FACS analyse

Flow cytometers allow individual cells to be identified by their cell-surface antigens and to be sortedCells to be analyzed by flow cytometry are first labeled with fluorescent dyes (top panel). Direct labeling uses dye-coupled antibodies specific for cellsurface antigens (as shown here), while indirect labeling uses a dye-coupled immunoglobulin to detect unlabeled cell-bound antibody. The cells are forced through a nozzle in a singlecell stream that passes through a laser beam (second panel). Photomultiplier tubes (PMTs) detect the scattering of light, which is a sign of cell size and granularity, as well as emissions from the different fluorescent dyes. This information is analyzed by computer (CPU). By examining a large number of cells, the proportion of cells with a specific set of characteristics can be determined and levels of expression of various molecules on these cells can be measured. The lower part of the figure shows how these data can be represented, the example in this case being the expression of two surface immunoglobulins, IgM and IgD, on a sample of B cells from a mouse spleen. The two immunoglobulins have been labeled with different-colored dyes. When the expression of just one type of molecule is to be analyzed (IgM or IgD), the data are usually displayed as a histogram, as in the left-hand panels. Histograms display the distribution of cells expressing a single measured parameter (for example, size, granularity, fluorescence intensity). When two or more parameters are measured for each cell (IgM and IgD), various types of two-dimensional plots can be used to display the data, as shown in the right-hand panel. All four plots represent the same data, and in each case, the horizontal axis represents intensity of IgM fluorescence, and the vertical axis the intensity of IgD fluorescence. Two-color plots provide more information than histograms; they allow recognition, for example, of cells that are ‘bright’ for both colors, ‘dull’ for one and bright for the other, dull for both, negative for both, and so on. For example, the cluster of dots in the extreme lower left portions of the plots represents cells that do not express either immunoglobulin, and are mostly T cells. The standard dot plot (upper left) places a single dot for each cell whose fluorescence is measured. This format works well for identifying cells that lie outside the main groups, but tends to saturate in areas containing a large number of cells of the same type. A second means of presenting these data is the color dot plot (lower left), which uses color density to indicate high-density areas. A contour plot (upper right) draws 5% ‘probability’ contours, with contour lines drawn to indicate each successive 5% of the population; this format provides the best monochrome visualization ofregions of high and low density. The lower right plot is a 5% probability contour map, which also shows outlying cells as dots.

Lymphocyte isolation using antibody-coated magnetic beads.

Although FACS is superb for isolating small numbers of cells in pure form, mechanical means of separating cells are preferable when large numbers of lymphocytes must be prepared quickly. A powerful and efficient way of isolating lymphocyte populations is to couple paramagnetic beads to monoclonal antibodies that recognize distinguishing cell-surface molecules. These antibody-coated beads are mixed with the cells to be separated and are run through a column containing material that  attracts the paramagnetic beads when the column is placed in a strong magnetic field. Cells binding the magnetically labeled antibodies are retained; cells lacking the appropriate surfacemolecule can be washed away (Fig. A.22). The retained cells are recovered by removing the column from the magnetic field. In this case, the bound cells are positively selected for  expression of the particular cell-surface molecule, while the unbound cells are negatively selected for its absence.

ELISPOT

A modification of the ELISA antigen-capture assay (see Section A-4), called the ELISPOT assay, is a powerful tool for measuring the frequency of T-cell responses and also provides information about the cytokines produced. Populations of T cells are stimulated with the antigen of interest, and are then allowed to settle onto a plastic plate coated with antibodies against the cytokine that is to be assayed (Fig. A.25). If an activated T cell is secreting that cytokine, the cytokine is captured by the antibody on the plastic plate. After a period of time the cells are removed, and a second antibody against the cytokine is added to the plate to reveal a circle (‘spot’) of bound cytokine surrounding the position of each activated T cell; it is these circles that give theELISPOT assay its name. By counting each spot and knowing the number of T cells originally added to the plate, one can easily calculate the frequency of T cells secreting that particular cytokine. ELISPOT can also be used to detect specific antibody secretion by B cells, in this case by using antigen-coated surfaces to trap specific antibody and labeled anti-immunoglobulin to detect the bound antibody.

Peptide:MHC tetrameren voor identificatie TCR specificiteit

Peptide:MHC complexes coupled to streptavidin to form tetramers are able to stain antigen-specific T cells. Peptide:MHC tetramers are formed from recombinant refolded peptide:MHC complexes containing a single defined peptide epitope. MHC molecules that contain biotin can be chemically synthesized, but usually the recombinant MHC heavy chain is linked to a bacterial biotinylation sequence, a target forthe Escherichia coli enzyme BirA, which is used to add a single biotin group to the MHC molecule. Streptavidin is a tetramer, each subunit having a single binding site for biotin; hence the streptavidin:peptide:MHC complex creates a tetramer of peptide:MHC complexes (top panel). Although the affinity between the T-cell receptor and its peptide:MHC ligand is too low for a single complex to bind stably to a T cell, the tetramer, by being able to make a more avid interaction with multiple peptide:MHC complexes binding simultaneously, is able to bind to T cells whose receptors are specific for the particular peptide:MHC complex (middle panel). Routinely, the streptavidin molecules are coupled to a fluorochrome, so that the binding to T cells can be monitored by flow cytometry. In the example shown in the bottom panel, T cells have been stained simultaneously with antibodies specific for CD3 and CD8, and with a tetramer of HLA-A2 molecules containing a cytomegalovirus peptide. Only the CD3+ cells are shown, with the staining of CD8 displayed on the vertical axis andthe tetramer staining displayed along the horizontal axis. The CD8– cells (mostly CD4+), on the bottom left of the figure, show no specific tetramer staining, while the bulk of the CD8+ cells, on the top left, likewise show no tetramer staining. However, a discrete population of tetramer positive CD8+ cells, at the top right of the panel, comprising some 5% of the total CD8+ cells, can clearly be seen. 

Identification of T-cell receptor specificity using peptide:MHC tetramers (zie plaatje links)

For many years, the ability to identify antigen-specific T cells directly through their receptor specificity eluded immunologists. Foreign antigen could not be used directly to identify T cells because, unlike B cells, T cells do not recognize antigen alone but rather the complexes of peptide fragments of antigen bound to self MHC molecules. Moreover, the affinity of interaction between the T-cell receptor and the peptide:MHC complex is in practice so low that attempts to label T cells with their specific peptide:MHC complexes routinely failed. The breakthrough in labeling antigen-specific T cells came with the idea of making multimers of the peptide:MHC complex, so as to increase the avidity of the interaction.Peptides can be biotinylated using the bacterial enzyme BirA, which recognizes a specific amino acid sequence. Recombinant MHC molecules containing this target sequence are used to make peptide:MHC complexes, which are then biotinylated. Avidin, or its bacterial counterpart streptavidin, contains four sites that bind biotin with extremely high affinity. Mixing the biotinylated peptide:MHC complex with avidin or streptavidin results in the formation of a peptide:MHC tetramer—four specific peptide:MHC complexes bound to asingle molecule of streptavidin (Fig. A.30). Routinely, the streptavidin moiety is labeled with a fluorochrome to allow detection of those T cells capable of binding the peptide:MHC tetramer. Peptide:MHC tetramers have been used to identify populations of antigenspecificT cells in, for example, patients with acute Epstein–Barr virus infections (infectious mononucleosis), showing that up to 80% of the peripheral T cells in infected individuals can be specific for a single peptide:MHC complex.They have also been used to follow responses over timescales of years in individuals with HIV or, in the example we show, cytomegalovirus infections. These reagents have also been important in identifying the cells responding,for example, to nonclassical MHC class I molecules such as HLA-E or HLA‑G, in both cases showing that these nonclassical molecules are recognized by subsets of NK receptors.