Immunology Ch 8 B Cell Maturation

Transitional B cells​

-After 24 hours immature cells leave bone marrow and become transitional B cells which express IgD together with IgM as the BCR​ -First to respond to the cytokine BAFF (B lymphocyte factor)​ -BAFF is made by multiple cells (monocytes, dendritic cells, stromal cells) as a survival signal​

Thymus Independent plasma Cells​

-Antigens that do not require T-cell help ​ -Polysaccharide components of bacterial capsules are one clinically important set of TI antigens​ -Generally rapid and almost exclusively IgM​ -Made by short-lived plasma cells in the marginal zone of the spleen ​ -Can agglutinate antigen and activate the complement system and provides a crucial early protection against bacterial infections ​

Sites of Early B-Cell Differentiation​

-B lymphocytes arise from hematopoietic stem cells​ -B-cell differentiation occurs throughout life and the B-cell repertoire is continuously replenished ​ -Stroma, the nonlymphoid cells in bone marrow, provide critical adhesive interactions ​ -Also produce cytokines (IL-7) that promote the survival and enhance proliferation​

Development of B Lymphocytes: Overview​

-B-cell differentiation is predominantly in the bone marrow and occurs throughout life ​ -Key stages in the B-cell differentiation involve specific immunoglobulin (Ig) gene rearrangements ​ -Stages represent developmental checkpoints​ -Signals received determine specific pathway​ -Lack of a signal may result in the death of the cell​

CD10

-CD10 is expressed by early cells in the lineage—pro-B and pre-B cells—as well as by germinal center B cells​

CD19

-CD19 is expressed on all cells in the B-cell lineage from the pro-B to memory B cell, (not plasma cell)​

CD20

-CD20 is expressed on all cells from the immature B cell to the memory B (not plasma cell)​

CD27

-CD27 is expressed exclusively on memory& plasma cells ​

B-Cell Membrane Proteins:​ Stage-Specific Markers​

-Different cell-surface molecules are expressed at different stages in B-cell development and activation​ -CD10 is expressed by early cells in the lineage—pro-B and pre-B cells—as well as by germinal center B cells​ -CD19 is expressed on all cells in the B-cell lineage from the pro-B to memory B cell, (not plasma cell)​ -CD20 is expressed on all cells from the immature B cell to the memory B (not plasma cell)​ -CD27 is expressed exclusively on memory& plasma cells ​

Plasma Cells: Main Ig secretor

-Do NOT express a membrane form of Ig ​ -Secretes antibody of a single antigenic specificity and of a single isotype: IgM, IgG, IgA, or IgE. ​ -Express CD27 uniquely (lose CD10, CD19, CD20)​ -Plasma cells are generated in the germinal center of lymph nodes and spleen, and in MALT.​ -TD antigens (thymus dependent) plasma cells require helper T cells to activate ​

Pro-B cell: D to J Rearrangement.​

-Earliest cell in the B lineage is the pro-B cell ​ -The first rearrangement is the heavy-chain locus (DH gene segment to JH segment)​ -Pro-B cells express CD19 ​ -CD19 is expressed on all further B-cells except plasma cells so CD19 is a useful marker of B-cells ​ -Pro-B cells also express CD10​

Mature B Cells​

-Expresses higher levels of IgD than does the transitional B cell​ -Referred to as naïve, or immunocompetent ​ -Known also as follicular B cells since found in primary and secondary follicles​ -In the marginal zone of the spleen, are called splenic marginal zone B cells, they do not recirculate through the blood like follicular cells​

The Pre-B Cell Receptor​

-Expression of the pre-BCR is a key early checkpoint ​ -Consists of μ chain & 2 sets of associated proteins: ​ 1.Surrogate light chains CD179a and CD179 which are structurally similar to a λ light chain​ 2. The Igα (CD79a) and Igβ (CD79b) are part of the BCR and is used for signal transduction in pre-BCR and BCR -Signal transduction of Igα/Igβ is due to tyrosine-based activation motifs (ITAMs) in the cytoplasm​ -Signaling via pre-BCR causes further differentiation, proliferation, shuts down surrogate light-chain synthesis, and starts light-chain gene rearrangement​

Pre-B Cell: V-DJ Rearrangement

-Heavy-chain has a second rearrangement ​ -V(D)J recombinase joins heavy-chain VH gene to rearranged DHJH forming a VDJ unit ​ -Positioned close to Cμ, pre-B synthesizes a μ chain ​ -If not productive on the first chromosome, proceeds to the second chromosome​ -If rearrangement is productive, a μ chain is made​

Negative Selection and the Development of Central Tolerance.​

-Nonself-antigen cells leave the bone marrow and develop into mature B cells ​ -Self-antigens recognized by immature B cells are likely expressed by bone marrow stromal cells -Negative selection and central tolerance is the process where self B cells die by apoptosis ​ -Some self-reactive cells change their receptor, via receptor editing by further rearranging light chain variable regions (heavy chain not possible)​​

Stroma

-Stroma, the nonlymphoid cells in bone marrow, provide critical adhesive interactions

How TI antigens work​

-T-independent antigens have repeating epitopes that induce B cell recognition and activation without T cells ​ -A second signal, (interaction of TLRs with PAMPs) is also required for activation of the B cell ​

Immature B Cells​

-The next stage is the immature B cell​ -Light chains pair with μ chains to form monomeric IgM, which are expressed on the cell surface to form the BCR ​ -Immature B cells express CD20, also expressed in the next stages of B-cell differentiation ​ -Thus, CD20 is a marker for the later stages of B-cell development. ​

Synthesis, assembly & secretion of Immunoglobulins

1) Translation and assembly (L+H) in the ER 2) Processing in the Golgi 3) Creation of secretory vesicles that fuse with the cell membrane

Gene expression

A gene has been expressed when its product, a protein, is made and is operating in the cell -RNA translation into protein occurs on ribosomes -Ribosomes are either free in cytoplasm or attached to the rough ER (secreted and membrane proteins) -The first few amino acids of a polypeptide act as a signal peptide which determines if proteins belong in the cell or inserted into membranes -Further processing includes addition of carbohydrates, phosphates, or lipids

What key event occurs in the germinal center of lymph nodes during B cell maturation Somatic hypermutation Selection for high affinity antibody Class switch recombination Development of plasma and memory B cells All of the above

All of the above

Which of the following characterize B cells? They function in antibody mediated immunity against bacteria. They are manufactured and mature in the bone marrow. They directly recognize antigens and undergo clonal selection. All of the choices characterize B cells. None of the choices characterize B cells.

All of the choices characterize B cells.

B cell maturation Summary

Both B and T lymphocyte precursors originate in red bone marrow. B cells mature in the bone marrow and develop​ immunocompetence & self-tolerance.​ Immunocompetent but still naive lymphocytes leave the thymus and bone marrow.​ When a lymphocyte's antigen receptors bind its antigen, that lymphocyte can be activated.​ Activated lymphocytes proliferate and then differentiate & mature​

Outcomes of Ab-Ag Interactions

Cross-linking of antigen molecules with antibody is required to form complexes (agglutination, precipitation) Both antigen and antibody need to be multivalent (2 or more binding sites) in order to form large cross-linked complexes that will become insoluble

The antibodies found in mucus, saliva, and tears are IgG. IgM. IgA. IgD. IgE.

IgA.

The antibodies found almost entirely and only on the surface of B cells (not secreted from them), and which always exist as monomers, are IgG. IgM. IgA. IgD. IgE.

IgD.

The central Dogma of DNA to RNA to Protein

The DNA template strand is used to make a complimentary copy of RNA (mRNA) in an anti-parallel direction. The template is 3' to 5' and mRNA is produced 5' to 3' making a mirror image of the template The mRNA is translated into a specific protein based on codons used

Plasma cells are activated by a(n) antigen. B cell. T cell. memory cell. APC.

antigen.

Which of the following bacterial components would most likely result in B cell stimulation by T-independent antigens? capsule plasmid pili ribosome

capsule

Without CD40L-CD40 interaction, B lineage cells fail to undergo which of the following? development from the pro-B cell. immunoglobulin gene rearrangement. class (isotype) switching. formation of an antigen specific receptor.

class (isotype) switching.

The early development of B cells is conducted under the influence of which of the following? antigen. AID. MHC class II. cytokines.

cytokines.

The cell responsible for producing a rapid antibody response against a foreign protein when it is encountered years after the first encounter is a memory T cell. plasma cell. memory B cell. killer T cell.

memory B cell.

The earliest stages of B-cell differentiation: A. occur in the embryonic thymus B. require the presence of antigen C. involve rearrangement of gamma-chain gene segments D. involve rearrangement of surrogate light-chain gene segments E. occur in the bone marrow

occur in the bone marrow

organization of Light Chains Genes

κ and λ light chains consist of two major domains, a variable region (VL) and a constant region (VC) Two gene segments—variable (V) and joining (J) code for the variable region

Immunofluorescence

•Ab's are labeled (covalently attached) with a fluorescent compound for detection •Fluorescein (FITC) is commonly used and is excited with UV light then emits green light (400 nm) •FITC is coupled to free amine groups on proteins •Direct is when primary Ab is labeled •Indirect is when primary Ab is not labeled, but a labeled anti-Ab is used (secondary Ab)

Assays of lymphocyte function

•B or T cells respond to mitogenic stimuli that activate proliferation •Mitogenic or antigen-driven stimulation that results in antibody production (for B cells) or cytokine production (for T cells) Many T-cell type specific assays are used to evaluate their functional integrity based on the target cell type (B cells, macrophages etc.

Cell culture

•Cell culture systems have facilitated major breakthroughs including B cell hybridoma monoclonal antibody technology •Knowledge of growth factors required which make it possible to grow functional cells in vitro •Recombinant DNA technology combined with cell culture techniques enables production of engineered immune molecules and receptors •Cell culture studies help elucidate the biologic consequences of receptor expression

Agglutination Reactions

•Cross-linking results in the clumping or agglutination of cellular antigens by antibodies •Highest dilution (e.g. 1:1000) of serum that still causes agglutination is termed the antibody titer •Assay is performed by mixing twofold serial dilutions of serum with a fixed [antigen] •Cross-linking does not occur at high concentrations of antibodies (prozone) since not enough antigen to bind to both Fab sites

Experimental Systems to Study Immunology

•Deep dive into In vitro techniques, assays, and experimental systems used in the lab •Many techniques use antibody-based tests •Also novel use of molecular biological methods, genetic engineering, cell culture techniques, developed since Human Genome sequenced (2000); used to discover Immunogenic epitopes expressed by pathogens (novel vaccines)

Passive or Indirect Agglutination

•Direct agglutination is when RBC and antibody combination is a natural constituent •Passive (aka. indirect) agglutination is for detecting natural Ig not bound to RBC (or other inert particle) by attaching serum Ig first to an insoluble particle in a test tube •Agglutination reactions (direct or passive) are widely used clinically 1) erythrocyte (RBC) typing in blood banks, 2)hemolytic diseases 3) rheumatoid (Rh) factor 4) test for syphilis, 5) human chorionic gonadotropin (HCG) for pregnancy

Intro to Ab structure

•Every person can generate billions of different BCR or TCR molecules theoretically up to 1 x 1011 •Nobel prize awarded to Dr. Tonegawa in 1987 for discovering how BCR and TCR genes rearrange in the genome •Recombinant gene rearrangement is the key process that allows about one hundred genes and gene segments (mini-genes) to produce this huge number of unique Ig proteins

genetically engineered molecules and receptors

•Genes encoding membrane receptors expressed on cells have also been cloned and genetically engineered to allow for gene transfer to cells that do not normally express these receptors •Human mAb are being produced using genetic engineering techniques •Humanized antibodies consist of the constant region of human Ab's and variable region of a mouse Ab's

Immunoabsorption & Immunoadsorption

•Immunoabsorbtion involves binding of Ag and Ab in solution •Immunoadsorption involves capture on some insoluble support (plastic etc. like ELISA) •Immunoadsorption can be used to purify either Ag or Ab with immuno-affinity matrix •Purified Ag/Ab can be removed with gentle treatments such as high salt and low/high pH

Immunoelectrophoresis of Serum proteins

•Immunoelectrophoresis involves separating proteins in an electric field followed by detection with antibodies •After separation by electrical charge, a trough is cut to add anti-protein serum •Antigen & Ab diffuse in agar and form precipitation lines

Ab production by B cells

•In the case of B cells, the polyclonal activators LPS or PWM can be used to assess the ability of a population of B cells to produce antibody •ELISAs are the most used quantitative assays for measuring antibody levels

Florescence activated cell sorting

•Instrument forces cells into narrow channel that can only allow one cell to pass in single file •Passed through beam of UV light, and cells are detected by fluorescent emissions •Droplets containing a single cell can be sorted based on intensity of various fluorescent markers

Adoptive Transfer

•Is when protection via cell-mediated immunity by antigen-specific T cells as opposed to antibody-mediated (humoral) immunity (passive) •Adoptive transfer of T cells is usually performed using genetically identical donor and recipients (e.g. inbred strains) matching MHC receptors

Adoptive Transfer

•Is when protection via cell-mediated immunity by antigen-specific T cells as opposed to antibody-mediated (humoral) immunity (passive) •Adoptive transfer of T cells is usually performed using genetically identical donor and recipients (e.g. inbred strains) matching MHC receptors

Analysis of gene expression

•Microarrays (gene chips) are powerful tools for measuring the expression of thousands of genes simultaneously •DNA fragments representing all or subsets of genes are attached to plastic •Labeled cDNAs are washed over the microarray and allowed to hybridize to matching fragments •Unhybridized material is washed away, leaving fluorescence where matching has occurred •Results compared to a gene database to determine increase or decrease in expression

B cell and T cell proliferation assays

•Mitogens are nonspecific stimuli that stimulate growth of B- and/or T-cell populations •A mitogen that selectively activates B cells, is lipopolysaccharide (LPS) •Similarly, several lectins (Con A & PHA), are very effective T-cell mitogens PWM stimulates activation of both B and T cells

How mAb are made

•Monoclonal antibodies (mAb) are identical antibody molecules (clones), derived from a single antibody-producing cell •Malignant plasma cells are fused (hybridized) with freshly harvested B cells •Fusions are selected by requiring production of a specific enzyme (HGPRT) not in the malignant cells •Single cell clones are selected and expanded if express Ab of interest

Solid phase Immunoassays

•Most widely used immunoassay where proteins are adsorbed to solid plastic; usually a microtiter plate •Ab then binds to Ag and is detected via enzymes that are conjugated to the Ab, and the products the produce •Allows for quantification of Ag and/or Ab by detection of the enzyme product (color, light etc.) •Called ELISA for enzyme-linked immunosorbent assay

Primary Interactions Antibody:Antigen

•No covalent bonds are involved in the interaction between antibody and an antigen •Binding forces are relatively weak van der Waals forces, electrostatic and hydrophobic forces •Interaction requires a close fit between an epitope and the antibody, (lock and a key, or shape) to allow many weak interactions •Can be readily dissociated by low or high pH, by high salt concentrations etc.

mRNA have introns that need to be removed

•Pre-mRNA is composed of exons and introns -The exons will be expressed -The introns, occur in between the exons -The exons are "expressed" and introns are "in the way" •Intron removal is done using spliceosomes -A spliceosome uses a ribozyme (enzyme made of RNA rather than just protein) to cut and remove the introns -Remaining exons are spliced back together -The result is a mature mRNA transcript

Precipitation Reactions

•Precipitation reaction is between Ab's and soluble antigen •Antibody cross-links multivalent antigens to form a lattice •Reaches a certain size, Ag-Ab complex become insoluble and precipitates out of solution •The amount of light scatter caused by antigen-antibody complexes in solution can quantify complexes

Diffusion Tests

•Precipitation reactions can also take place in a solid matrix such as agar gels •Reactants diffuse in gel and form gradients •Visible lines of precipitation form based on the rate of diffusion and concentration of each reactant in the gel •Ab is distributed equally in radial diffusion test, but wells contain different concentrations of antigen (Double diffusion test and Radial diffusion tests)

Primary cell cultures and cloned lymphoid cell lines

•Primary lymphoid cells consisting of heterogeneous populations of T and/or B cells can be cultured on plates etc. •Used to study the molecular mechanisms in B and T cells development (e.g. gene rearrangement) •Transformation of B and T cells cloned from a specific cell can generate immortal cell lines •Used to develop many useful diagnostic and therapeutic reagents (e.g. monoclonal antibodies)

Direct Binding Immunoassays

•RIA = radioimmunoassays not generally used due to easier non-radioactive assays today, but are very sensitive for very low amounts •Known amount of radioligand is mixed with limiting Ab, most ligand is bound but some is free •Next a known amount of unlabeled Ag is titrated •Competition occurs causing more labeled Ag to become free Ag; based on dose & binding affinity •A standard concentration curve of Ag is then used to determine the [Ag] in an unknown sample

Antigen-Antibody Interactions

•Strong binding between antigen and antibodies serves as the basis for many immune assays •Widely used for diagnostic purposes, detection and identification of either antigen or antibody •Outcomes= precipitation (soluble antigen), agglutination (particulate, cellular), complement activation if crosslinking occurs

Effector Cell assays for T cells and NK cells

•T-cell assays are varied since functionally diverse T-cell subsets exist •Functional assays for T helper cells focus on activation of B cells, macrophages etc. •Cytotoxic activity of CD8+ T cells are measured by the ability to kill radiolabeled target cells •Functional property of NK cells is known as antibody-dependent cell-mediated cytotoxicity (ADCC)

Association/Dissociation Constant

•The binding of a monovalent antigen (Ag) with each antibody (Ab) binding site (Fab) can be represented by the equation for a 1:1 molecular interaction: KA = k1/k-1 = [AbAg]/[Ab][Ag] •where k1 represents the forward (association) rate constant and k−1 the reverse (dissociation) rate constant. •Calculated by determining the ratio of bound [AbAg] complexes to unbound (free) [antigen] and [antibody] •Association constant (KA) is a measure of the affinity (strength) of the antibody for the antigen and is the inverse of the Dissociation constant KD = k-1/k1

Affinity and Avidity

•The intrinsic binding strength for antibody binding to a single epitope (or hapten) is termed affinity, often very strong, KD < pM (10-12 M) •Avidity involves the sum of the affinities of all the epitopes involved in multivalent interactions •Avidity denotes the overall binding strength between antibodies and a multivalent antigen. •IgM antibodies have higher avidity than IgG antibodies, due to larger number of Fab's

Genetic Events in the synthesis of Ig chains

•The organization of genes coding for Ig Heavy and Light chains show similarities with typical membrane proteins, but also important differences •Genes coding for antibodies (as well as genes coding for the TCR) have the unique property of rearranging during different stages of development •Like mRNA splicing, but occurs in the genomic DNA of B and T cells, not mRNA

A review of Normal gene expression

•Transcription occurs when RNA polymerase binds along with transcription factors to a gene promoter region (green light) •Mature messenger RNA (mRNA) has noncoding introns removed and exons spliced together •Newly synthesized heavy (H) and light (L) polypeptide chains are processed inside the ER and Golgi before they reach the cell surface

COOMBs test for anti-RBC Ab

•Uses anti-immunoglobulins (e.g. rabbit anti-human Ig) that bind to anti-RBC IgG for detection •Facilitates agglutination of erythrocytes by IgG antibodies by overcoming zeta potential; (negative charge on RBC surface IgM stance)

Flow Cytometry

•Uses fluorescent Ab's specific for cell surface Ag •Used for surface marker analysis as well as cell sorting to purify different types (e.g. B vs T cells) •Commonly uses CD surface markers (clusters of differentiation) of which about 100 are known

Western Blots (Immunoblots)

•Western blots first separate proteins in acrylamide gels (usually by size) which are then transferred to paper •Paper is then soaked in anti-serum •Proteins are detected using labeled antibodies (radioactive, fluorescent or enzyme linked)

K- Chain synthesis

•κ locus (located on chromosome in 2) exists in every cell of the body (only B cells use it) •There are 40 different Vκ gene segments separated by introns •There are 5 different Jκ segments •There is a single Cκ gene downstream of the Jκ genes