Haptens must be bound to carriers to induce a humoral response. The same hapten-carrier conjugate must be used to elicit a secondary response; to generate a secondary response to a hapten bound to a different carrier, the patient must first be immunized against the new carrier. T cells bind carriers; B cells bind haptens. The response of hapten-primed B cells to a hapten-carrier conjugate requires carrier-primed T_H cells. Remember that CTL activity only occurs with T_H help. One-Way MLR

1. Are any IA alleles present in the stimulator that are not present in the responder?
2. Are any IE alleles present in the stimulator that are not present in the responder?

1. There is disparity at none/one/two of the Class II loci, leading to no/weak/strong proliferative response.

1. Are any K alleles present in the stimulator that are not present in the responder?
2. Are any D alleles present in the stimulator that are not present in the responder?
3. Are any L alleles present in the stimulator that are not present in the responder?

1. There is disparity at none/one/two/three of Class I loci, causing no/weak/strong/strong cytotoxicity.
2. If there is a weak proliferative response, there will be a weak cytotoxic response.
3. If the result above is no proliferation but the cytotoxic response is strong, then in fact there will be very weak proliferative and cytotoxic responses.

If you are asked whether B cells in a bone marrow chimera can respond to an extracellular bacterial infection:

1. Are the donor and recipient IA alleles the same?
2. Are the donor and recipient IE alleles the same?

1. If the answer to both questions is yes: B cells can respond in a thymus-dependent pathway since APCs, T_H cells and B cells all share the same Class II MHC alleles. APCs bind and activate the T_H cells, which then recognize and activate B cells. Also, B cells can respond in a thymus-independent pathway.
2. If the answer to one question is yes: B cells can respond in a thymus-dependent pathway since some APCs, T_H cells and B cells will have one Class II MHC allele in common (although response will be weaker than above). APCs bind and activate the T_H cells, which then recognize and activate B cells. Also, B cells can respond in a thymus-independent pathway.
3. If the answer to neither question is yes: B cells will respond to the bacterial infection only in a thymus-independent manner because no APCs can present antigens to the T cells, which are not positively selected to interact with the B cell Class II MHC.

If you are asked whether a bone marrow chimera can respond to an intracellular bacterial or viral infection:

1. Are the donor and recipient IA alleles the same?
2. Are the donor and recipient IE alleles the same?

1. If the answer to either question is yes: T_H cells will be activated by APCs showing foreign antigens, marking the APC for lysis by CTLs, allowing the T_H cells to them activate B cells and leading to both B and T cell memory. More donor and recipient Class II allele overlap increases T_H help.
2. If the answer to neither question is yes: the CTLs will not be activated by infected cells since there will be no positive selection for the MHC molecules present on APCs.

1. Are the donor and recipient K alleles the same?
2. Are the donor and recipient D alleles the same?
3. Are the donor and recipient L alleles the same?

1. If the answer to three questions is yes: CTLs will be able to recognize and kill infected cells due to the Class I MHC match.
2. If the answer to two or one questions is yes: CTLs will be able to recognize and kill infected cells due to the Class I MHC match, although slightly slower than if all Class I alleles matched.
3. If the answer to none of the questions is yes: no functional CTL response. Bone-marrow derived cells cannot will not be killed, since their Class I MHC haplotype does not match the recipient Class I MHC haplotype at any alleles.

When analyzing proliferation of a bone-marrow chimera responder against a stimulator:

1. Is the stimulator IA (Class II) allele the same as either of the recipient or donor IA alleles?
2. Is the stimulator IE (Class II) allele the same as either of the recipient or donor IE alleles?

1. If the answer to both questions is yes: zero T cell proliferation.
2. If the answer to one of these questions is yes: some T cell proliferation.
3. If the answer to none of these questions is yes: strong T cell proliferation.
4. Reason: chimera T cells are negatively selected in thymus to not respond to donor Class II haplotype (spell it out, IA^?IE^?), and peripheral tolerance prevents reactivity to recipient Class II haplotype (again, spell it out IA^?IE^?). The stimulator is different from none/one/two of the tolerated alleles, leading to none/weak/strong proliferation.

When analyzing cytotoxicity of a bone-marrow chimera responder against a stimulator:

1. Is the stimulator K (Class I) allele the same as either of the recipient or donor K alleles?
2. Is the stimulator D (Class I) allele the same as either of the recipient or donor D alleles?
3. Is the stimulator L (Class I) allele the same as either of the recipient or donor L alleles?

1. If the answer to all of these questions is yes: no cytotoxicity.
2. If the answer to two of these questions is yes: some cytotoxicity.
3. If the answer to one of these questions is yes: more cytotoxicity.
4. If the answer to none of these questions is yes: strong cytotoxicity.
5. Reason: chimera T cells are negatively selected in thymus to not respond to donor Class I haplotype (spell it out, K^?D^?L^?), and peripheral tolerance prevents reactivity to recipient Class I haplotype (again, spell it out K^?D^?L^?). The stimulator is different from none/one/two/three of the tolerated alleles, leading to none/weak/more/strong cytotoxicity (cell lysis).

Antibodies are effective during the extracellular stage, preventing virions from spreading and protecting against reinfection. sIgA is a very important part of mucosal secretions, blocking viral attachment to mucosal epithelial cells. Also, antibodies may directly activate complement-mediated lysis of virion particles with lipid envelopes.

However, once the virus enters a cell it is inaccessible to antibodies and infected cells must be eliminated by CTLS. CD8 T cells recognize viral antigens presented in a Class I MHC context on the surface of an infected cell. CTL activation requires co-stimulation — if the virally infected cell is not an APC, then it must be phagocytosed by a professional APC. The CD8 T cell then recognizes endogenously synthesized (synthesized within the cell) viral proteins presented in a Class I MHC context on the cell surface. Cytokines produced by CD4 T cells (T_H cells) drive CD8 differentiation into effector CTLs that use antigen-specificity to locate and kill infected nucleated cells.

DCs present peptides on both Class I and II MHC. T cell proliferation, indicated by H3 incorporation, indicates presentation of an immunogenic peptide on Class II while lysis indicates presentation on Class I. To mount an effective immune response against a viral infection a strong CD8 T cell response is required to kill infected cells. This requires help from CD4 T cells. Therefore, an effective vaccine requires the priming of both CD4 and CD8 T cells. Peptide 3 is presented on Class I and primes CD8 T cells against the virus while Peptide 2 primes CD4 T cells. Lysis is augmented using these peptides together because of T cell help. LCMV LCMV is a non-cytopathic virus. Mice deficient of T cells become chronic LCMV carriers, while normal mice develop meningitis to due to CTL killing of meningeal cells. It is frequently used for experiments. Parasites There are many animal parasites, ranging from protozoa (unicellular eukaryote) to helminths (large worms). The principal innate response to parasites is phagocytosis, but many parasites are resistant to phagocytosis and can even replicate within macrophages. Phagocytes secrete microbicides to kill organisms too large for phagocytosis, but many helminths have thick teguments that resist neutrophil and macrophage attacks. Also, some helminths activate complement — but many of them are also resistant to lysis via complement.

Helminthic infections are eliminated by secretion of IL-4 and IL-5 by activated T_H2 cells. IL-4 and IL-5 then stimulates production of sIgE that binds the worm. This IgE binds F(c)ε receptors on eosinophils, activating the eosinophils to secrete granule enzymes that destroy the parasite.

Some pathogenic protozoa (such as Leishmania spp.) survive within macrophages by deactivating the macrophage. Adaptive immunity is then necessary, with T_H1 cells secreting large amounts of IFN-γ to re-activate the infected macrophages. Protozoa such as malaria that replicate within and lyse host cells stimulate specific antibody and CTL responses. Another parasite, trypanosomes, change the expression of their surface antigen to evade immune response. ‘C’ Anything If you see a chemical that starts with the letter ‘C’ (but not ‘CD’) then it is likely involved in complement. C3a, C4a and C5a are small-peptide byproducts of complement and are anaphylatoxins, binding mast-cells and basophils to induce degranulation and cause vascular permeability and smooth muscle contraction. Also, anaphylatoxins amplify the inflammatory response by inducing synthesis of pro-inflammatory cytokines. C5a is the most potent anaphylatoxin, and also is a chemoattractant and activator of white blood cells (macrophages, etc). C5a can attract white blood cells to extravasate from local capillaries and migrate into the tissue space where complement has been activated. Phagocytes Phagocytes have receptors which directly recognize bacteria and lead to phagocytosis, activation, microbicidal activity and cytokine secretion. Macrophage Tissue macrophages: trap, engulf and destroy pathogens; produce cytokines (including IL-12); induce co-stimulatory molecules; and present antigens for the adaptive immune response. Macrophages present antigens on their cell surface, they are known as antigen presenting cells (APCs). These antigens are then recognized by effector cells (B cells and T cells). Macrophages also bear CD14 (and LPS receptor), CD11b/CD18 complex (binds C3b and C4b, complement byproducts), scavenger receptor (binds sialic acid), TLR and F(c)R. F(c)R binds antigen-antibody complexes, absorbs them, degrades them in the lysosome and then presents the fragments on the cell surface. Adaptive Immunity Adaptive immunity is triggered when an infection eludes innate defenses and generates a threshold of antigen. Acquired immunity is effective only after several days, the time required for antigen-specific T and B cells to proliferate and differentiate into effector cells. See more at the acquired immunity overview. T Cells T cells do not recognize antigens floating in solution. They only recognize antigens presented by antigen-presenting cells. There are two kinds of T cells: CD4 T cells (aka helper T cells or T_H cells) which respond to Class II MHC; and CD8 T cells (aka cytotoxic T cells or T_C cells) which respond to Class I MHC. CD4 T cells are split into T₁1, T_H2 and the poorly-understood T_H17. CD4 cells play important roles in B cell activation and release lots of cytokines. CD8 T cells do not release as many cytokines, but eliminate virally infected and cancer cells, and are important for autograft rejection. Remember that CTL activity only occurs with T_H help. T₁ vs T_H2 Naïve CD4 T cells activated in presence of IL-12 and IFN-γ are committed to T_H1 lineage. Naïve CD4 T cells activated in presence of IL-4 (and especially if IL-6 is also present) are committed to T_H2 lineage. These cytokines are secreted by the cells which respond appropriately to a given pathogen. T_H1 and T_H2 cells amplify their own populations. T_H1 cells secrete IFN-γ, inhibiting T_H2 proliferation. T_H2 cells secrete IL-10 and TGF-β, inhibiting activation and growth of T_H1 cells.

However, it is unclear from [³H]-thymidine uptake how much each individual population has proliferated. The one-way mixed-lymphocyte reaction (aka the one-way mixed-leukocyte reaction or one-way MLR) resolved this issue. One population — the stimulator — is first inactivated (via mitomycin c or lethal x-irradiation) before being added to the MLR well. These inactivated cells merely provide foreign alloantigens to the responder population. Within 24-48 hours, the responder T cells have begun proliferating and within another 48 hours an expanding population of functional CTLs has been formed. CD4 (T_H) cells, dendritic cells and certain accessory cell types are all critical for the MLR to function.

When activated CTLs are incubated for 1-4 hours with washed target cells (to remove unabsorbed Na₂⁵¹CrO₄) the amount of ⁵¹Cr in the supernatent correlates directly to the extent of target cell lysis by the CTLs. Thus, the specificity of CTLs for allogeneic cells tumor cells, virus-infected cells and artificially modified cells can be assayed. CTLs will typically only lyse cells of the same MHC Class I haplotype.

Antigen-antibody crosslinking leads to phosphorylation of the tyrosines within the Ig-α and Ig-β ITAMs. This phosphorylation is performed by the receptor-associated PTKs Lyn, Blk and Fyn (similar to p56^Lck activity on TCRs). This ITAM phosphorylation creates docking sites for the critical proteins Syk (also a PTK, analogous to the TCR’s ZAP-70) and B cell linker protein (BLNK). These critical proteins provide docking sites for further proteins. Once BLNK has been phosphorylated by Syk, it recruits Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) so Syk can activate Btk, and so that Btk can then phosphorylate PLCγ2. Once PLCγ2 has been phosphorylated, it activates early calcium signaling and the initiation of pathways dependent on protein kinase C (PKC). The pathways activated by the BCR include small G protein pathways (for growth), PKC-dependent pathways and NF-κB production pathways — note the similarities to T cell activation.

In addition to the BCR, there are two membrane-bound components which provide stimulation (the B cell coreceptor) or inhibition (CD22). The B cell coreceptor is a complex of three proteins: CD19, which provides a long cytoplasmic tail with docking sites; CD21 (aka CDR2), which is a receptor for C3d; and CD81. C3d is a byproduct of complement that coats antigens — while the immunoglobulin binds the antigen, CD21 cross-links with C3d. This forms a BCR-antigen-BCcoR complex, allowing CD19′s cytoplasmic tail to interact with Ig-α and Ig-β and undergo phosphorylation. CD19′s phosphorylated cytoplasmic tail then binds signaling molecules, including the protein tyrosine kinase (PTK) Lyn, and hugely amplifies the activating signal. This explains how naïve B cells with low antigen affinity are still able to respond to low concentrations of antigen.

CD22 delivers a negative signal that makes activation of B cells more difficult. Activation of B cells leads to phosphorylation of the immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic tail of CD22. Tyrosine phosphatase then binds the CD22 ITIM, stripping phosphates from the ITAMs of neighboring signaling complexes. Since ITAM phosphorylation is the core of B cell activation, phosphate removal deactivates the cell. CD22 knockout mice develop autoimmune diseases as they age, illuminating the importance of negative regulation.

Two distinct signalling events are needed to push the resting naïve B cell into the cell cycle: signal 1 followed by signal 2. T_H cells are essential for activation of a B cell by thymus-dependent antigens. Binding of thymus-dependent antigens to a B cell’s mIg does not alone induce proliferation and differentiation without additional interaction with T_H membrane molecules as well as appropriate cytokines. The steps are described below:

The two chains are connected at a cysteine residue by a disulfide bond. The transmembrane region is 21 or 22 amino acids and positively charged, anchoring the TCR into the cell membrane and promoting TCR-CD3 interaction. Lastly, the cytoplasmic tail (carboxy-terminal end) is 5 to 12 amino acids. αβ and γδ TCR heterodimers resemble an Fab fragment attached to the cell membrane; due to this structural homology, they are included under the immunoglobulin superfamily.

αβ TCRs react only with peptide antigens. However, γδ TCRs can react to glycolipids and phospholipids (nonpeptides) presented by CD1. Furthermore, some γδ cells react with unprocessed peptide antigens not even presented by an MHC. Also, the most common γδ TCRs within a given species bind the most common pathogens that species encounters. These γδ TCRs directly binds microbes, and the γδ cells might directly kill pathogens.

Both innate and adaptive immunity rely heavily upon binding of the TCR to a peptide-MHC complex. γδ cells are characterized as part of innate immunity due to their rapid responses, low specificity and ability to bind free antigens. αβ cells, conversely, are clearly part of adaptive immunity. In addition to these functional differences, γδ cells and αβ cells are also structurally different. Allison, Garboczi and their coworkers found that a γδ receptor specific for non-presented microbial phospholipids has a deep binding cleft. Also, separate research found that γδ TCRs have a 111° bend between their V and C regions while αβ TCRs have a 147° bend.

In B cells, membrane-bound antibodies associate with the Ig-α/Ig-β heterodimer to form the B cell antigen receptor. In T cells, the T cell receptor associates with its fellow membrane-bound component CD3 to form the TCR-CD3 membrane complex. Please note that CD3 is another component of the cell surface on T cells — TCR and CD3 are together on the T cell surface and interact together to transduce signals into the T cell. CD3 is essential for membrane expression of the TCR and for signal transduction. All CD3 chains contain a negatively charged amino acid (aspartic or glutamic acid) which interacts with one or two positively charged transmembrane TCR residues. Please note that B and T cell accessory molecules only participate in signal transduction after antigen interaction and do not actually influence antigen interaction.

Alone, the T cell receptor has low affinity for the peptide-MHC complex. For this reason, additional accessory molecules known as coreceptors or cell-adhesion molecules strengthen the bond between a T cell and a target or antigen-presenting cell. CD4 and CD8 are coreceptors which also distinguish cytotoxic T cells from helper T cells; CD4 binds Class II MHC molecules and CD8 binds Class I MHC molecules. Once bound, CD4 and CD8 also activate signal transduction. Additional coreceptors include CD2, LFA-1, CD28 and CD45R, all of which bind independently to other ligands on target or antigen-presenting cell. Once these coreceptors bind, the TCR scans for its appropriate peptide-MHC complex. Once TCR binds, membrane expression of cell-adhesion molecules is activated to tighten the intracellular bond. Cell-adhesion molecules play a role in tissue graft acceptance or rejection. If the grafted tissue bears MHC molecules which are similar to the host, then T cells will still respond to the MHC molecules.

Genes encoding α, β, γ and δ chains are expressed only in T cells. Functional TCR genes are formed by rearrangements of V and J segments in α and γ genes, and of V, D and J segments (like IgH) in β and δ genes. The α, β, γ and δ genes also include C segments which do not rearrange. The C region contains a long exon encoding much of the C domain, followed by shorter exons encoding the connecting, transmembrane and cytoplasmic regions. In humans, α and δ gene segments are located on chromosome 14, while β and γ gene segments are located on chromosome 7. In mice, the α, β and γ gene segments are located on chromosomes 14, 6 and 13. In both mice and humans, δ gene segments are located between V and J α segments. The location of the δ gene segments is important: functional rearrangement of the α gene segments deletes C_δ, so that a T cell cannot co-express αβ TCRs with γδ TCRs. The table below describes human and murine TCR genes, and counts both pseudogenes (nonfunctional mutants) and functional genes.

Like the antibody L chain, the α chain is encoded by V, J and C segments. Like the antibody H chain, the β chain is encoded by V, D, J and C gene segments. Rearrangement of the TCR gene segments results in α VJ joining and β VDJ joining. After the rearranged TCR genes are transcribed, the α and β chains are expressed as a disulfide-linked heterodimer on the T cell membrane. Immunoglobulins can be bound or secreted, but TCRs are only membrane-bound. The TCR constant region, as shown in the figure above, includes constant (C), connecting, transmembrane and cytoplasmic sequences. Also, as opposed to the multiple different immunoglobulin C gene segments encoding different isotypes, α DNA has only one C segment and β DNA has duplicated J and C segments.

Pre-T cells expresses the recombination activating genes RAG-1 and RAG-2, as do pre-B cells. The RAG-1/2 recombinase recognizes conserved recombination signal sequences (RSSs) flanking each V, D and J gene segments in non-recombined TCR DNA. RAG-1/2 catalyzes V-J and V-D-J joining via the same deletions and inversions which occur in immunoglobulin genes. First, Rag-1/2 knicks one DNA strand between the coding and signal sequences and excises the resulting DNA loops. SCID mice (which lack B and T cells) are defective for a gene which repairs double-stranded DNA breaks; as a result, in SCID mice, the Ab and TCR D and J gene segments are not rejoined. RAG-1/2 only recombines TCR genes in T cells and Ig genes in B cells. This specificity is due to different recombinase regulatory systems, and uniquely configured chromatin in each lineage that allows recombinase to access only appropriate sites.

δ genes are located between the V and J segments of the &alpha gene. Thus, when the α gene recombines, the δ DNA is excised. In a phenomenon known as allelic exclusion, this mechanism prevents αβ TCRs and γδ TCRs from co-expressing. Another instance of allelic exclusion involves the two duplicate β gene J and C clusters. Having two duplicates means that at least one functional rearrangement is likely to take place. Once a functional rearrangement occurs, then rearrangement of the other cluster is inhibited. However, more than one α allele can undergo functional recombination; while this can lead to different α chains being expressed on the same cell surface, only one allele is MHC restricted and therefore functional. The mechanisms of TCR diversity are described below.

Before birth, the yolk sac, fetal liver and fetal bone marrow are the major sites of B cell maturation; after birth, B cells mature in the bone marrow. Many B cells are produced, but most die after a few weeks unless they encounter their specific antigen or nestle into a supportive lymphoid organ. An immature B cell bearing IgM in its membane leaves the bone marrow and matures to express both membrane-bound IgM and IgD (mIgM and mIgD) with a single antigenic specificity.

Naive B cells (have not encountered antigen) circulate in blood ad lymph and are carried to secondary lymphoid organs (notable the spleen and lymph nodes). If a B cell’s mIgM or mIgD interacts with its antigen, then the cell activates undergoes clonal expansion. This creates a population of genetically identical B cells (which express an identical antibody) that differentiates into memory B cells and plasma B cells. Also, some B cells undergo affinity maturation, whereby the average affinity of the antibodies they produce increases. Also, many B cells undergo class switching, whereby the B cells switch from producing µ isotype antibodies (IgMs) to produce γ, α or ε isotype antibodies.

Naive thymocytes are those which have not yet encountered a peptide-MHC complex. Arrested in G₀, naive thymocytes have condensed chromatin, minimal cytoplasm and little transcriptional activity. They continuously recirculate through blood, lymph an lymph nodes. To activate, they need additional costimulatory signals to those described above. Signal 1 is the initial interaction between TCR-CD3 and peptide-MHC. Signal 2 is is provided by thymocyte CD28 and CD152 interaction with B7 proteins on the antigen-presenting cell. B7 proteins are constitutively expressed on dendritic cells, and in activated macrophages and activated B cells. B7 binds two proteins — CD28 and CD152 — which are both found on thymocyte membranes as disulfide-linked dimers. Binding of CD28 induces the cell to activate, while binding of CD152 represses activation. CD152 has a much higher B7 affinity than CD28, and its expression is activated by binding of CD28. Thus, CD152 is essentially a braking mechanism that maintains homeostasis; CD152 knockout mice have enlarged lymph nodes, enlarged spleen and die 3-4 weeks after birth. In the absence of a costimulatory signal, an unresponsive state called clonal anergy ensues (as opposed to clonal proliferation).

In essence, activation occurs when a dendritic cell simultaneously binds itself to a T_H‘s antigen receptor (primary signal) and to its CD28 receptor (secondary signal). This signals to the dendritic cell that the antigen is foreign (dangerous) and that the next encountered cytotoxic thymocyte must be activated. Other times, dendritic cells are directly activated by an antigen via toll-like receptors and activate cytotoxic thymocytes — this is a critical example of how innate immunity activates adaptive immunity.

T Cell Death

Over 98% of all thymocytes die during positive and negative selection, with the remaining cells entering the circulatory system to differentiate into effector or memory thymocytes. These T cells express two cell-surface proteins, Fas and Fas ligand (FasL), which are both essential for apoptosis via the Fas pathway. Upon activation, thymocytes increase Fas/FasL expression — the result is that over-stimulated cells are killed. This is essential for avoiding over-proliferation of thymocytes, and also for killing any self-reactive cells which avoided thymic selection.

Antibodies make contact with protein antigens, usually 15-22 amino acids on the antigen contact a similar number on the antibody giving a complementary surface of 650-900 Angstroms. The amino acids comprising the epitope are adjacent in 3D space, but not necessarily in linear sequence.

When exposed to an antigen, B cells producing antibodies reactive to that antigen will begin to produce vast amounts of antibody. Each B cell produces a monoclonal population of antibodies, meaning each antibody binds the same epitope (site) on the same antigen; however, the B cells together produce a polyclonal population of antibodies, consisting of different antibodies binding a different epitope (site) on the same antigen.

One problem with the study of antibodies was that they are heterogenous. For example, an electrophoresis pattern of an animal immunized against albumin (a homogenous protein) would show a spike of albumin and then several much smaller spikes of antibodies (meaning the albumin antibodies are polyclonal, or consisting of different subsets binding different sites on the same antigen). In multiple myeloma, tumorous plasma cells all secrete the tame type of immunoglobulin. This leads to a huge monoclonal spike of antibodies, since there will be huge amounts of a single antibody. The experiments below were all performed to determine the structure of antibodies. After all these experiments, the antibody structure shown above was determined.

Type I hypersensitivity (most commonly allergies) stem from overproduction of IgE. Thus, it requires an initial encounter so that isotype switching can occur — in secondary responses, the IgE crosslinks the antigen (and F(c)εRI and causes mast-cell and basophil degranulation. Atopic allergies and asthmatics are becoming more common in economically developed nations due to changes in exposure to childhood infectious disease (atopy is negatively associated with measles, hepatitis A or tuberculosis infections), environmental pollution (diesel exhaust enhances T_H2 responses, leading to higher IgE production), allergen levels (crazy cat ladies) and dietary changes.

IL-4 is pivotal in regulating the IgE response: IgG1 and IgE account ∼2% of all antibodies secreted by splenic B cells incubated with LPS; IgG1 accounts for ∼50% and IgE accounts for ∼20% of all antibodies secreted by B cells incubated with LPS and IL-4. IL-4 knockout mice cannot mount an IgE response to parasites. IL-4 stimulates isotype switching by activating the promoters for Iε, and Iγ1 (the I regions for ε and γ1 isotype genes). T_H1 cells enhance response to IL-3, IL-4 and IL-10. T_H2 cells reduce response to IFN-γ.

Delayed-type hypersensitivity (DTH) is a T_H1-mediated localized inflammatory reaction that effectively clears intracellular pathogens (such as viruses) and contact allergens (such as poison oak and poison ivy). DTH is characterized by large influxes of inflammatory cells (like mφs), tissue damage and delayed onset. DTH reactions functional antibodies and a functional complement system; it cannot occur without B1 cells nor C5. There are two phases related to delayed-type hypersensitivity:

Contact allergens (such as poison oak) are usually small molecules which complex with skin proteins; these complexes are internalized by Langerhans cells (APCs found in skin) and then presented to T_H cells. Also, when DTH is continuously activated, normal tissue is damaged due to a continuous assault of lytic enzymes. This can occur in the lungs of Mycobacterium tuberculosis patients, as well as lesions in Mycobacterium leprae patients.

Thymic selection is a two-step process: positive selection, which induces apoptosis in thymocytes whose TCR cannot bind self MHC molecules; and negative selection, which induces apoptosis in thymocytes which bind self MHC molecules too well or in presence of a self peptide. Positive selection results in MHC restriction, and negative selection results in self-tolerance (meaning the thymocytes will not attack healthy self cells).

There are two proposed models as to how CD4⁺CD8⁺ cells mature into CD4⁺CD8^- or CD4^-CD8⁺ cells: the instructive model and the stochastic model. Neither model has been definitely proven nor disproven. The instructive model postulates that double-positive cells interact with either a Class I or a Class II MHC molecule, and are somehow signaled to differentiate into either CD4 or CD8 cells. The schotastic model postulates that repression of CD4 or CD8 is random and has nothing to do with TCR specificity. Only thymocytes whose TCR and coreceptor bind the same MHC molecule continue to mature.

Part of the immunoglobulin superfamily, the γ, δ and ε chains contain extracellular, transmembrane and cytoplasmic domains. The ζ chain is distinct, with shorter extracellular and longer cytoplasmic domains. All CD3 chains contain a negatively charged amino acid (aspartic or glutamic acid) which interacts with one or two positively charged transmembrane TCR residues.

CD3 chains have an immunoreceptor tyrosine-based activation motif (ITAM) located in their cytoplasmic tail. Also found on the Ig-α/Ig-β heterodimer of the B cell receptor complex and IgE and IgG F(c) receptors, ITAMs interact with tyrosine kinases and are critical for signal transduction. In CD3, the γ, δ and ε chains each contain a single ITAM, while ζ and η chains contain three ITAMs. CD4 CD4 binds to Class II MHC molecules, and is a 55kD monomeric membrane glycoprotein containing: four extracellular immunoglobulin-like domains (D1, D2, D3 and D4); a hydrophobic transmembrane region; and a long cytoplasmic tail containing three serine residues that can be phosphorylated. Like CD8, the extracellular domain of CD4 binds to the conserved regions of MHC molecules on antigen-presenting cells. The membrane-distal domain (furthest from the membrane) of CD4 binds Class II MHC molecules at a hydrophobic pocket formed by the α2 and β2 domains. CD5 CD5 is a marker typically found on T cells, but also present on B-1 cells (not B-2 cells, aka conventional B cells). CD8 CD8 binds to Class I MHC molecules, and is usually a disulfide-linked αβ heterodimer or αα homodimer. The α and β chains are both 30-38kD glycoproteins with: an extracellular immunoglobulin-like domain; a stalk; a hydrophobic transmembrane region; and a cytoplasmic tail of 25 to 27 residues, several of which can be phosphorylated. Like CD4, the extracellular domain of CD8 binds to the conserved regions of MHC molecules on antigen-presenting cells. CD8 binds to Class I α2 and α3 domains, and interacts somewhat with β₂-microglobulin. Upon binding CD8, the Class I α3 domain changes slightly; only a single CD8 can bind a Class I MHC molecule at a time. CD11 See CD18 CD16 An F(c)γ receptor, CD16 stimulates binding and uptake of antigens for antigen presentation. CD18 Integrins are heterodimeric, composed of a CD18 β subunit bound to a CD11a, b or c α subunit. The three integrins are: LFA-1 (CD18/CD11a), essential for adherence of T cells to APCs for T cell activation; macrophage-1 antigen (CD18/CD11b), aka integrin αMβ2, a receptor of complement byproducts on macrophages; and integrin αxβ2 (CD18/CD11c), also a complement receptor. CD19 Part of B cell coreceptor, with a long cytoplasmic tail with docking sites. Other components of the B cell coreceptor are CD21 and CD81. CD21 Aka CR2, CD21 is part of B-cell coreceptor and binds C3d, a byproduct of complement. Since B cells are part of acquired immunity and complement is part of innate immunity, this is an example of different branches of the immune system interacting together. Other components of the B cell coreceptor are CD19 and CD81. CD22 Present on the membrane of resting B cells, CD22 delivers a negative signal that makes activation of B cells more difficult. CD23 CD23, aka F(c)εRII, binds Ige. CD24 A molecule known as heat stable antigen (HSA). CD25 The α chain of the IL-2 receptor, present on pre-B cells. CD28 A co-receptor of the T cell receptor. Important for T cell activation. CD32 An F(c)γ receptor, CD16 stimulates binding and uptake of antigens for antigen presentation. CD34 Present on about 1% of hematopoietic stem cells. Not unique to stem cells, but irradiated mice (completely lacking stem cells) inoculated with an enriched population of CD34⁺ cells can restore hematopoiesis. CD40 A molecule on the surface of B cells which binds CD154 (aka CD40L) on the T_H cell surface. CD40 is involved in the formation of a T-B conjugate. Also, CD40 is a tumor necrosis factor — a family of cell surface proteins and cytokines which regulate cell proliferation and apoptosis. Its ligand, CD40L (CD154) belongs to the TNF receptor (TNFR) family. CD40 binding to T cell CD40L is necessary for immunoglobulin gene rearrangement for a functional antibody. CD40L See CD154 CD43 Leukosialin. Only expressed on pro-B cells. CD45R Aka B220, CD45R is a protein tyrosine phosphatase found on leukocytes. As a marker unique to B cells, B220⁺ cells are usually assumed to be B cells. CD64 An F(c)γ receptor, CD16 stimulates binding and uptake of antigens for antigen presentation. CD80 Also known as B7-1, CD80 is a principal costimulatory molecule present on antigen presenting cells. CD81 Also known as TAPA-1, CD81 is part of the B cell coreceptor. Other components of the B cell coreceptor are CD19 and CD21. CD86 Also known as B7-2, CD86 is a principal costimulatory molecule present on antigen presenting cells. CD152 Also known as CTLA-4. CD154 Expressed on the activated T_H cell membrane, CD154 (aka CD40L) is a tumor necrosis factor receptor (TNFR) protein which binds CD40 (a tumor necrosis factor) on the surface of B cells. Involved in the formation of a T-B conjugate. Toger with AID, induces isotype switching. B cell CD40 binding to T cell CD40L is necessary for immunoglobulin gene rearrangement for a functional antibody. ]]> http://studentreader.com/clusters-of-differentiation/feed/ 0 http://studentreader.com/mouse-models/ http://studentreader.com/mouse-models/#comments Thu, 30 Oct 2008 08:05:08 +0000 Levi Clancy http://studentreader.com/?p=2203 Mus musculus is the common house mouse. Its genome is the same size as that of human, with 20 chromosome pairs and 3×10⁹ base pairs. Its life cycle takes two months, and gestation takes 21 days. The physical map of the genome is more complete than that of the human, making it relatively easy to positionally clone a gene identified by mutation. Because it is a mammal, its developmental processes are most similar to that of human. Techniques are available to “knock out” any gene that has been cloned. The entire genome has been sequenced and is available at http://www.informatics.jax.org.

Mice are excellent research models because they are mammals (their biochemical pathways are similar to humans) and their genome sequence is known (99% of human genes are present). Also, it is very easy to control a mouse’s environment (diet, climate, etc) and also to control its genetics. Genetically, there are hundreds of inbred strains of genetically identical mice available; also, it is possible to alter gene sequences and expression levels within mice via transgenics and gene targeting.

Immunologists use inbred strains of genetically identical mice so that organs and tissues can be transplanted between mice without graft rejection. This is because the genetically identical mice have the same MHC molecules. Mating two mice with homozgyous MHCs (for example, H-2^b/b and H-2^k/b) will lead to H-2^b/k heterozygous offspring. The heterozygous progeny can accept grafts from either parent or with each other; however, the homozygous parents cannot accept grafts from their heterozygous offspring.

Natural killer cells lack TcRs, CD4s and CD8; instead, they have: cell-surface activating receptors, which bind noncovalently to molecules with ITAMs; and on the cytoplasmic side, inhibitory receptors with ITIM(s) which — upon phosphorylation — recruit and activate SHP-1 & -2, which inhibit the activating receptors. The balance between activating signals and inhibitory signals is what determines whether a natural killer cell will destroy or bypass a microbe it encounters. There are many different inhibitory and activating receptors, but two well-studied ones are:

Digested antigen fragments are presented on the macrophage or monocyte cell-surface (in an MHC context) so other cells can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Since macrophages present antigens on their cell surface, they are known as antigen presenting cells (APCs). These antigens are then recognized by effector cells (B cells and T cells). Macrophages also activate cytokines, which stimulate differentiation and reproduction of lymphoid cells.

Macrophages are continuously maturing from circulating monocytes. Please read about the precursor to macrophages, the monocyte.

A thoroughly-studied disease of cytokine receptors is SCID, a genetic disorder which has helped reveal the roles that cytokines play.

In the bone marrow, B cells complete their hematopoietic differentiation from stem cells into IgM⁺,IgD^weak immature virgin B cells. Next, in the medulla, B cells fully activate to become IgM⁺,IgD⁺ mature virgin B cells. Upon exposure to antigens, B cells in the medulla begin producing antibodies that flow through the lymph to the entire body. This helps create the body’s immune memory, with large amounts of antibody loaded in the bone marrow and at germinal centers. B cell activation occurs in the following steps:

1. An antigen flows through an afferent lymphatic vessel and into a node’s cortical sinus.
2. The antigen percolates through the node until getting trapped by reticular cells and dendritic cells.
3. Macrophages within the node trap and present the antigen. If it is a foreign antigen, either or both virgin B cells or memory B cells may react.

The B cells discussed so far are conventional B cells (aka B-2 cells). There is another subset of B cells known as B-1 cells (aka CD5 B cells, since some species’ B-1 cells display CD5). B-1 cells arise from stem cells during fetal life and self-renew via division of existing cells. While conventional B cells usually produce IgG, B-1 cells typically produce IgM and have little or no IgD. CD5 B cells secrete antibodies in response to TI-2 polysaccharides, leading to complement and removal of bacteria. This occurs within 48 hours of antigen exposure, as a bridge until the adaptive T cell response can activate. Unlike the T cell response, the B-1 response does not have memory. The table below (adapted from Immunology, 6^th edition) further compares and contrasts conventional (aka B-2) and B-1 cells.

Granulocytes are a type of white blood cell containing granules. Granulocytes are aka polymorphonuclear neutrophilic leukocytes (PMNs/PMLs) or polys, although PMN can refer specifically to neutrophils since neutrophils are the most common granulocyte. The granules inside granulocytes are secretory vesicle containing a molecule (for example, basophil granules contain histamine). The granules are dormant in the cytoplasm until a cell signal instructs the granules to release their components. This signal is typically shock or distress. Degranulation is when granules release their contents from the cell.

Lymphocytes bind to specific antigens and generate from stem cells in primary lymphoid tissue via hematopoiesis. Mφs, dendritic cells and B cells originate in bone marrow; and T cells originate in the thymus. After maturing, lymphocytes migrate to secondary lymphoid tissues (lymph nodes, blood and other tissues) to fulfill their role.

Macrophages (aka mononuclear phagocytes or Mφs) have two main functions: phagocytosis and antigen presentation. In phagocytosis, the macrophage or monocyte cell-surface F(c) Receptor (FcR) binds to the antibody-antigen complex (an antibody bound to an antigen on the cell-surface of a pathogen). The pathogen, antigen and antibody are engulfed and degradative granules and enzymes break it down in the lysosome. Digested antigen fragments are bound to MHC molecules and presented on the macrophage or monocyte cell-surface so that effector cells (B and T cells) can recognize the antigen. Because macrophages present antigens, they are antigen presenting cells (APCs). Macrophages play a tremendous role in clearing tagged antigens and releasing degradative enzymes to damage tissues and initiate healing.

Macrophages: trap, engulf and destroy pathogens (phagocytosis); present antigens for the adaptive immune response (antigen presentation); produce cytokines (including IL-12); and induce co-stimulatory molecules. Also, macrophages also bear CD14 (and LPS receptor), CD11b/CD18 complex (binds C3b and C4b, complement byproducts), scavenger receptor (binds sialic acid), TLR and F(c)R (described above). Please read about the precursor to macrophages, the monocyte.

Immune system cells can be organized many different ways. The categories above are based primarily on origin. However, other broad classes are frequently used which are important to understand.

Neutrophils have a lobulated nucleus, meaning it is condensed and dead — hence the term polymorphonuclear. Their cytoplasm is filled with granules containing degradative enzymes such as lysozyme, collagenase, elastase and others. During phagocytosis, these granules combine with phagosomes to break down pathogens.

Neutrophils are abundant in blood, constituting 50-60% of circulating white blood cells. However, neutrophils are absent from healthy (uninfected) tissues and only live about six hours. After performing phagocytosis several times, the neutrophil dies and degranulates (thus releasing its degradative enzymes). These enzymes damage and inflame local tissues — making neutrophils important in inflammation — but these corrosive enzymes also initiate healing.

1. Each activated B cell reproduces, to create an expanding population of identical B cell clones. This population is called a clone.
2. Some members of the clone become plasma cells. Plasma cells produce and secrete copies of the antibody displayed on the B cell surface.
3. Other members of the clone are stored as memory cells. Thus, there is a large population of cells to create a strong response when the antigen enters the system again.

Clonal Expansion Theory explains:

1. Specificity, since only antigen-reactive clones are triggered and only antigen-specific antibody is produced. Diversity is not explained but incorporated into this theory.
2. Memory, since clonal expansion explains why subsequent responses to an antigen are exponentially stronger than the initial response.
3. Tolerance, since B and T cells with potential self-reactivity are destroyed or rendered anergic (unable to respond).

Generation of Diversity

An enormous number of specific antibodies and T cells must be generated to respond to the enormous and changing antigenic universe. Immunoglobulin (for B cells) and TCR (for T cells) genes have multiple and similar methods for creating diverse antigenic receptors:

1. Multiple genes encode different protein sequences with different specificities.
2. Multiple segments of genes differentially combine to create increased specificities.
3. Differential junctions following gene segment recombination yield different amino acid sequences.
4. Somatic (after clonal expansion has begun) mutation of receptor genes in various clones causes changes in germline sequences.

B cells were discovered approximately fifteen years after Burnett’s Clonal Selection Theory, proving that antibodies all pre-exist and that the repertoire is independent of the antigenic universe. This validated Burnett’s theory as well as Ehrlich’s similar (although much older) proposal. However, two key facets of Ehrlich’s theory were also disproven: B cells are exclusively dedicated to antibody production; and antibodies are not from food. The Clonal Expansion Theory is the modern explanation for antibodies and their origin.

Adaptive immunity is triggered when an infection eludes innate defenses and generates a threshold of antigen. Acquired immunity is effective only after several days, the time required for antigen-specific T and B cells to proliferate and differentiate into effector cells. Also, adaptive immunity ensures that mammals surviving an initial infection by a pathogen are generally immune to further illness from by that same pathogen. The adaptive immune system is based on T and B cells (aka leukocytes, a kind of white blood cell) that are produced by bone marrow stem cells and mature in the thymus and/or lymph nodes. The fundamental steps of acquired immunity are:

Innate and adaptive immunity are quite intertwined. For example, antigen presentation by dendritic cells (part of the innate immune system) activates thymocytes to proliferate and differentiate (part of the adaptive immune system). In many species, including mammals, the adaptive immune system can be divided into two major sections:

In addition to determining self from non-self, the immune response has B and T cells which identify different forms of non-self. Distinguishing various forms of non-self is crucial. For example, effectively responding to a bacterial infection would have no effect on an intracellular pathogen. B and T cells rely upon subtle differences in the biochemical structures of foreign proteins, carbohydrates, lipids and other building blocks. Known as antigens, these non-self structures are the target of the immune response and cause it to produce antigen-specific antibodies in response.

The immune response can only detect bio-organic antigens, essentially limiting it only to chemicals encoded or controlled by genes. Thus, all antigens are based on carbon and the atoms which bond to carbon (hydrogen, oxygen, nitrogen, phosphorous and sulfur — aka CHONPS). The immune response ignores any chemical not based on the six atoms listed. This means that sand, mercury, minerals and other contaminants are not subject to the immune response.

B and T cells bear receptors to distinguish self from non-self. B cells present immunoglobulin (antibody molecule) and T cells present T cell receptor (TCR). Much like enzyme binds substrate, the function of both molecules is to bind antigen. Binding of antigen by both B and T cells leads to removal of antigen from the system. The configuration of CHONPS is the antigenic determinant.

At this point, please study Clonal Expansion Theory to understand how the body specifically detects countless antigens; next, memorize important cells of the immune system to grasp the team of cells that make up the immune system.

Macrophages process pathogens and present on their cell surface antigens from the pathogen. After antigen presentation, antigen-specific cells under clonal expansion. Antigen-specific cells with high antigen affinity (due to their particular antibody configuration) respond efficiently and preferentially expand over time. This explains why subsequent responses to an antigen are anamnestic (stronger and faster) after initial exposure.

Immunity requires a primary stimulus, meaning nobody is immune to an antigen until they have been exposed to that antigen. However, some people may be resistant to an antigen for genetic reasons or may have strong innate protection. Innate responses and acquired (anamnestic) responses evolved together to confer resistance and protection against microbial invasion and malignant cells. Activation of innate immunity cells (such as via TLRs) promotes antigen-presenting cells to not only present antigens but also activate B and T cells.