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How do the variable portions of antibody genes look in cells which don't produce antibodies?

How do the variable portions of antibody genes look in cells which don't produce antibodies?



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There are several families of antibodies found in mammals. They may have two or more antibody domains which contain heavy and light chains. The variable regions of the light and heavy chains genes in the chromosome are spliced in antibody producing cells so that each cell produces a different antibody, with a unique sequence of amino acids in the variable regions.

Here's a cartoon:

The variable regions are disproportionately large here, but it gives some idea…

What I'm having trouble finding is how the DNA regions of these genes look in cells which don't produce antibodies. Are they the same as that in the germline? Do they undergo recombination but are not expressed?

Any help would be appreciated.


Perhaps you want to ask how VDJ recombination is regulated in non lymphoid cells…

Well even in immature lymphoid cells VDJ recombination is regulated. And also Ig genes are suppressed in T-cell and TCR genes are suppressed in B-cells… Also, the recombination of Ig is suppressed in T-cell and vice-versa.

RAG-1,2(Recombination activating gene) downregulation partly does the job, but not fully.

Earlier reports say that transcription in the locus is essential and epigenetic regulation of transcription in turn regulates recombination. But what it exactly means is that accessibility to the RSS (Recombination signal sequences) is limited in a repressed chromatin and recent reports say that nucleosomes are appropriately positioned over the RSS to control recombination.

Have a look at these:

http://www.cell.com/abstract/S0092-8674%2802%2900675-X

http://www.sciencedirect.com/science/article/pii/S0952791506000057

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC204470/


13.1D: Generation of Antibody Diversity

  • Contributed by Gary Kaiser
  • Professor (Microbiology) at Community College of Baltimore Country (Cantonsville)
  1. Define gene translocation and relate it to each B-lymphocyte being able to produce an antibody with a unique shaped Fab.
  2. Define the following:
    1. combinatorial diversity
    2. junctional diversity
    3. affinity maturation

    In this section we will look at generation of antibody diversity through gene translocation. As mentioned earlier, the immune system of the body has no idea as to what antigens it may eventually encounter. Therefore, it has evolved a system that possesses the capability of responding to any conceivable antigen. The immune system can do this because both B-lymphocytes and T-lymphocytes have evolved a unique system of gene-splicing called gene translocation, a type of gene-shuffling process where various different genes along a chromosome are cut out of one location and joined with other genes along the chromosome.

    To demonstrate this gene translocation process, we will look at how each B-lymphocyte becomes genetically programmed to produce an antibody functioning as a B-cell receptor(BCR) having a unique shaped Fab. As mentioned above, the Fab portion of an antibody is composed of 2 protein chains: a heavy and a light (see Figure (PageIndex<1>)).

    The variable heavy chain portion of the Fab is coded for by a combination of 3 genes, called VH (variable heavy), DH (diversity heavy), and JH (joining heavy). The variable light chain portion of the Fab consists of either a kappa chain or a lambda chain coded for by a combination of 2 genes, VL (variable light) and JL (joining light). In the DNA of each B-lymphocyte there are multiple forms of each one of these variable determinant genes. Although the exact number of each gene isn't known and varies from person, there are approximately 38-46 VH genes 23 DH genes 6 JH genes 34-38 kappa VL genes 5 kappa JL genes 29-33 lambda VL genes and 4-5 lambda JL genes.

    While a person inherits alleles for the various V(D)J genes from each parent, an individual B-lymphocyte will only express an inherited allele set from one parent. This increases a greater diversity of antibodies in that individual.

    Through random gene translocation, any combination of the multiple forms of each gene can join together (see Figure (PageIndex<2>)) resulting in thousands of possible gene combinations. This is known as combinatorial diversity.

    Gene translocation of the V(D)J genes is initiated when an enzyme called V(D)J recombinase recognizes recombination signal sequences located at the 3' end of V genes, the 5' end of J genes, and both ends of D genes. As a result, the chromosome forms a loop allowing different genes from different regions along the chromosome to align (see Figure (PageIndex<3>)). In the heavy chain any J-heavy gene and any D-heavy gene align and bind together as the genes are cut from one location and pasted into another. Subsequently, any one of the V-heavy genes is attached to this DJ segment. In the light chain, chromosomal looping enables any V-light gene to attach to any J-light gene.

    During gene translocation, specialized enzymes in the B-lymphocyte cause splicing inaccuracies wherein additional nucleotides are added or deleted at the various gene junctions. This change in the nucleotide base sequence generates even greater diversity in Fab shape. This is called junctional diversity.

    Furthermore, as B-lymphocytes proliferate, they undergo affinity maturation, a process that "fine tunes" the shape of the Fab epitope binding site. This is because the immunoglobulin V genes of B-lymphocytes have a mutation rate between 1000 to 10,000 times greater than other human genes in the body. This somatic hypermutation creates a great opportunity for selection of variant B-lymphocytes with even better fitting antigen-binding sites that fit the epitope more precisely. The longer and more tightly the antigen binds to the B-cell receptor, the greater the chance that B-lymphocyte has of surviving and replicating. In other words, the "fit" of the antibody can be improved over time. Affinity maturation occurs in the germinal centers of the lymph nodes.

    Most likely humans produce at least 10 11 different shaped BCRs. Keep in mind that the 3-dimensional shape of a protein is ultimately determined by the sequence of its amino acids and the sequence of amino acids is determined by the order of nitrogenous bases in the genes coding for that protein. Between combinatorial diversity, junctional diversity, and affinity maturation, there are probably billions of possible gene combinations and rearrangements that can code for the Fab portions of an antibody. Chances are, then, each B-lymphocyte will carry out a unique series of gene translocations and be able to produce an antibody with a unique shaped epitope-binding site.

    Because gene translocation is a random process, some immature B-lymphocytes do wind up making B-cell receptors that fit the body's own antigens. Immature B-lymphocytes with self-reactive B-cell receptors may be stimulated to undergo a new gene rearrangement to make a new receptor that is no longer self-reactive. Recognition of self antigen can reactivate genes that allow the B-lymphocyte to carry out new light chain V-J recombinations and enabling that cell to express a new B-cell receptor. This process is called receptor editing.

    Alternately, self-reactive B-lymphocytes can also undergo negative selection. Since the bone marrow, where the B-lymphocytes are produced and mature, is normally free of foreign substances, any B-lymphocytes that bind substances there must be recognizing "self" and are eliminated by apoptosis, a programmed cell suicide. Apoptosis results in the activation of proteases within the target cell which then degrade the cell's structural proteins and DNA.


    Contents

    Human antibody molecules (including B cell receptors) are composed of heavy and light chains, each of which contains both constant (C) and variable (V) regions, genetically encoded on three loci:

    • The immunoglobulin heavy locus ([email protected]) on chromosome 14, containing the gene segments for the immunoglobulin heavy chain.
    • The immunoglobulin kappa (κ) locus ([email protected]) on chromosome 2, containing the gene segments for part of the immunoglobulin light chain.
    • The immunoglobulin lambda (λ) locus ([email protected]) on chromosome 22, containing the gene segments for the remainder of the immunoglobulin light chain.

    Each heavy chain or light chain gene contains multiple copies of three different types of gene segments for the variable regions of the antibody proteins. For example, the human immunoglobulin heavy chain region contains 2 Constant (Cμ and Cδ) gene segments and 44 Variable (V) gene segments, plus 27 Diversity (D) gene segments and 6 Joining (J) gene segments. [2] The light chain genes possess either a single (Cκ) or four (Cλ) Constant gene segments with numerous V and J gene segments but do not have D gene segments. [3] DNA rearrangement causes one copy of each type of gene segment to go in any given lymphocyte, generating an enormous antibody repertoire roughly 3×10 11 combinations are possible, although some are removed due to self reactivity.

    Most T-cell receptors are composed of a variable alpha chain and a beta chain. The T cell receptor genes are similar to immunoglobulin genes in that they too contain multiple V, D, and J gene segments in their beta chains (and V and J gene segments in their alpha chains) that are rearranged during the development of the lymphocyte to provide that cell with a unique antigen receptor. The T cell receptor in this sense is the topological equivalent to an antigen-binding fragment of the antibody, both being part of the immunoglobulin superfamily.

    An autoimmune response is prevented by eliminating cells that self-react. This occurs in the thymus by testing the cell against an array of self antigens expressed through the function of the autoimmune regulator (AIRE). The immunoglobulin lambda light chain locus contains protein-coding genes that can be lost with its rearrangement. This is based on a physiological mechanism and is not pathogenetic for leukemias or lymphomas. A cell persists if it creates a successful product that does not self-react, otherwise it is pruned via apoptosis.

    Heavy chain Edit

    In the developing B cell, the first recombination event to occur is between one D and one J gene segment of the heavy chain locus. Any DNA between these two gene segments is deleted. This D-J recombination is followed by the joining of one V gene segment, from a region upstream of the newly formed DJ complex, forming a rearranged VDJ gene segment. All other gene segments between V and D segments are now deleted from the cell's genome. Primary transcript (unspliced RNA) is generated containing the VDJ region of the heavy chain and both the constant mu and delta chains (Cμ and Cδ). (i.e. the primary transcript contains the segments: V-D-J-Cμ-Cδ). The primary RNA is processed to add a polyadenylated (poly-A) tail after the Cμ chain and to remove sequence between the VDJ segment and this constant gene segment. Translation of this mRNA leads to the production of the IgM heavy chain protein.

    Light chain Edit

    The kappa (κ) and lambda (λ) chains of the immunoglobulin light chain loci rearrange in a very similar way, except that the light chains lack a D segment. In other words, the first step of recombination for the light chains involves the joining of the V and J chains to give a VJ complex before the addition of the constant chain gene during primary transcription. Translation of the spliced mRNA for either the kappa or lambda chains results in formation of the Ig κ or Ig λ light chain protein.

    Assembly of the Ig μ heavy chain and one of the light chains results in the formation of membrane bound form of the immunoglobulin IgM that is expressed on the surface of the immature B cell.

    During thymocyte development, the T cell receptor (TCR) chains undergo essentially the same sequence of ordered recombination events as that described for immunoglobulins. D-to-J recombination occurs first in the β-chain of the TCR. This process can involve either the joining of the Dβ1 gene segment to one of six Jβ1 segments or the joining of the Dβ2 gene segment to one of six Jβ2 segments. [3] DJ recombination is followed (as above) with Vβ-to-DβJβ rearrangements. All gene segments between the Vβ-Dβ-Jβ gene segments in the newly formed complex are deleted and the primary transcript is synthesized that incorporates the constant domain gene (Vβ-Dβ-Jβ-Cβ). mRNA transcription splices out any intervening sequence and allows translation of the full length protein for the TCR β-chain.

    The rearrangement of the alpha (α) chain of the TCR follows β chain rearrangement, and resembles V-to-J rearrangement described for Ig light chains (see above). The assembly of the β- and α- chains results in formation of the αβ-TCR that is expressed on a majority of T cells.

    Key enzymes and components Edit

    The process of V(D)J recombination is mediated by VDJ recombinase, which is a diverse collection of enzymes. The key enzymes involved are recombination activating genes 1 and 2 (RAG), terminal deoxynucleotidyl transferase (TdT), and Artemis nuclease, a member of the ubiquitous non-homologous end joining (NHEJ) pathway for DNA repair. [4] Several other enzymes are known to be involved in the process and include DNA-dependent protein kinase (DNA-PK), X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1 also known as Cernunnos or XRCC4-like factor [XLF]), the recently discovered Paralog of XRCC4 and XLF (PAXX), and DNA polymerases λ and μ. [5] Some enzymes involved are specific to lymphocytes (e.g., RAG, TdT), while others are found in other cell types and even ubiquitously (e.g., NHEJ components).

    To maintain the specificity of recombination, V(D)J recombinase recognizes and binds to recombination signal sequences (RSSs) flanking the variable (V), diversity (D), and joining (J) genes segments. RSSs are composed of three elements: a heptamer of seven conserved nucleotides, a spacer region of 12 or 23 basepairs in length, and a nonamer of nine conserved nucleotides. While the majority of RSSs vary in sequence, the consensus heptamer and nonamer sequences are CACAGTG and ACAAAAACC, respectively and although the sequence of the spacer region is poorly conserved, the length is highly conserved. [6] [7] The length of the spacer region corresponds to approximately one (12 basepairs) or two turns (23 basepairs) of the DNA helix. Following what is known as the 12/23 Rule, gene segments to be recombined are usually adjacent to RSSs of different spacer lengths (i.e., one has a "12RSS" and one has a "23RSS"). [8] This is an important feature in the regulation of V(D)J recombination. [9]

    Process Edit

    V(D)J recombination begins when V(D)J recombinase (through the activity of RAG1) binds a RSS flanking a coding gene segment (V, D, or J) and creates a single-strand nick in the DNA between the first base of the RSS (just before the heptamer) and the coding segment. This is essentially energetically neutral (no need for ATP hydrolysis) and results in the formation of a free 3' hydroxyl group and a 5' phosphate group on the same strand. The reactive hydroxyl group is positioned by the recombinase to attack the phosphodiester bond of opposite strand, forming two DNA ends: a hairpin (stem-loop) on the coding segment and a blunt end on the signal segment. [10] The current model is that DNA nicking and hairpin formation occurs on both strands simultaneously (or nearly so) in a complex known as a recombination center. [11] [12] [13] [14]

    The blunt signal ends are flush ligated together to form a circular piece of DNA containing all of the intervening sequences between the coding segments known as a signal joint (although circular in nature, this is not to be confused with a plasmid). While originally thought to be lost during successive cell divisions, there is evidence that signal joints may re-enter the genome and lead to pathologies by activating oncogenes or interrupting tumor suppressor gene function(s)[Ref].

    The coding ends are processed further prior to their ligation by several events that ultimately lead to junctional diversity. [15] Processing begins when DNA-PK binds to each broken DNA end and recruits several other proteins including Artemis, XRCC4, DNA ligase IV, Cernunnos, and several DNA polymerases. [16] DNA-PK forms a complex that leads to its autophosphorylation, resulting in activation of Artemis. The coding end hairpins are opened by the activity of Artemis. [17] If they are opened at the center, a blunt DNA end will result however in many cases, the opening is "off-center" and results in extra bases remaining on one strand (an overhang). These are known as palindromic (P) nucleotides due to the palindromic nature of the sequence produced when DNA repair enzymes resolve the overhang. [18] The process of hairpin opening by Artemis is a crucial step of V(D)J recombination and is defective in the severe combined immunodeficiency (scid) mouse model.

    Next, XRCC4, Cernunnos, and DNA-PK align the DNA ends and recruit terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that adds non-templated (N) nucleotides to the coding end. The addition is mostly random, but TdT does exhibit a preference for G/C nucleotides. [19] As with all known DNA polymerases, the TdT adds nucleotides to one strand in a 5' to 3' direction. [20]

    Lastly, exonucleases can remove bases from the coding ends (including any P or N nucleotides that may have formed). DNA polymerases λ and μ then insert additional nucleotides as needed to make the two ends compatible for joining. This is a stochastic process, therefore any combination of the addition of P and N nucleotides and exonucleolytic removal can occur (or none at all). Finally, the processed coding ends are ligated together by DNA ligase IV. [21]


    How COVID-19 Vaccines Work

    COVID-19 vaccines help our bodies develop immunity to the virus that causes COVID-19 without us having to get the illness.

    Different types of vaccines work in different ways to offer protection. But with all types of vaccines, the body is left with a supply of &ldquomemory&rdquo T-lymphocytes as well as B-lymphocytes that will remember how to fight that virus in the future.

    It typically takes a few weeks after vaccination for the body to produce T-lymphocytes and B-lymphocytes. Therefore, it is possible that a person could be infected with the virus that causes COVID-19 just before or just after vaccination and then get sick because the vaccine did not have enough time to provide protection.

    Sometimes after vaccination, the process of building immunity can cause symptoms, such as fever. These symptoms are normal and are signs that the body is building immunity.


    Genes Explain Race Disparity in Response to a Heart Drug (5/29)

    1. DNA rearrangement occurs during development -- therefore each cell has a unique set of genes for antigen binding protein.

    2. DNA rearrangement occurs before exposure to Ag.

    3. One protein with unique binding site made per cell. (Either one antibody/BCR or one TCR. Note this means only one of two alleles of gene are expressed in this case).

    4. Protein (BCR or TCR) has a variable part (specific for antigen) and a constant part (not dependent on Ag).

    5. Mechanism of clonal selection

    a. Ag binding protein acts as trap/receptor for A g. Antigen binding to surface Ab (BCR) or TCR serves to select cells of appropriate specificity. (Cells making the 'right' Ab or TCR.)

    b. Clonal expansion (or suppression) occurs in response to Ag binding

    (1). Destruction. If Ag is perceived as "self" → cell (T or B) destroyed or suppressed ( → tolerance). See Sadava fig. 18.7 (8th ed. only)

    (2). Activation. If Ag is perceived as foreign → cell divides → clonal expansion, further differentiation into effector or memory cells. (See below for details.)

    (3). Whether antigen is perceived as "self" or "foreign" depends on time of exposure to the antigen (embryonic vs adult) and additional factors. (This turns out to be very complicated, so we are ignoring the "additional factors.")

    B. Features that are unique to T cells (See top of handout 25A for B vs T, and TC vs TH )

    1. Protein made by T cell is T cell receptor, not Ab . (See Sadava fig. 18.12 (18.13)). Each T cell makes a unique TCR (also called T cell antigen receptor) due to DNA rearrangements of TCR genes.

    2. T cell receptor always remains on cell surface never secreted

    3. Antigen must be on eukaryotic cell surface:

    a. Antibody will bind to free antigen in solution (or to part of a whole bacterium). TCR will not.

    b. TCR only binds to Ag on surface of another (euk.) cell. .

    c. TC vs TH

    (1). TC

    (a). Target cell: TC binds to ordinary (euk.) cell with abnormal epitopes on surface for example, an infected cell.

    (b). Result of binding to target cell: TC destroys target.

    (c). Surface marker: TC is CD8 + -- has protein called CD8 on surface

    (a). Target cells: TH binds to immune cell with abnormal epitopes on surface, usually called an antigen presenting cell (APC).

    (b). Result of binding to target cell: Binding activates the TH cell &/or APC. (Promotes the immune response -- details vary depending on type of APC.)

    (c). Surface marker: TH is CD4 + -- has protein called CD4 on surface


    II. Activation of B and T cells -- what triggers clonal expansion? See Sadava fig. 18.15 (18.17) for overall picture.

    A. What is required? To activate a B or T cell, cell must get a juxtacrine signal and a paracrine signal. See Sadava fig. 18.14 (18.16)

    1. Paracrine = a cytokine = secreted protein that affects development of the immune system & some related functions.

    a. Terminology: Cytokines made by leucocytes often called interleukins, abbreviated IL-1, IL-2, etc.

    b. Example of action: Activation of both B and TC cells requires paracrines from TH cells. See texts if you are interested in names and functions of various cytokines. (No details of paracrines will be covered in class some details are included here and in problem book FYI only.)

    2. Juxtacrine -- Involves contact between surface proteins on two cells -- a T cell & its partner (infected target cell or APC). See handout 25A and Sadava fig. 18.14 (18.16). At least two juxtacrine interactions are required:

    a. T cell must have: TCR & CD4 or CD8: CD4 (if it's a TH) or CD8 (if it's a TC)

    b. Partner must have epitope attached to MHC (MHC = cell surface protein details below).

    (1). TCR binds to epitope

    (2). CD4 or CD8 binds to MHC

    c. Other proteins are involved too we are ignoring them. Consult advanced texts if you are interested.

    1. Clonal Expansion: An activated B or T cells divides and specializes, forming an expanded clone containing both memory cells and effector cells. See Sadava figs. 18.6 & 18.15 (18.7 & 18.17).

    2. What do the effector cells do?

    a. Effector B cells secrete antibody

    b. Effector TC cells kill targets

    c. Effector TH cells provide juxtacrine and paracrine signals that promote the functioning of other immune cells.

    C. More on MHC -- How helper T's and cytotoxic T's distinguish their respective targets. (For pictures of MHC molecules see picture from Alberts two types of MHC)

    1. What is it? MHC = very variable surface protein. (MHC is an acronym for major histocompatibility complex.)

    a. Types: There are 2 main types, and many versions of each type.

    b. Genes: Each individual has several different genes for each of the two main types of MHC. Each of these genes has 20-40 or even more variants (alleles).

    c. Person to person variation: Since there are several genes per person and many different alleles of each gene in the population, there is a lot of variation in the actual MHC proteins (and DNA) from person to person.

    d. No cell to cell variation: These genes, unlike genes for antibodies and TCR's, do not rearrange during development. All cells in person have the same MHC DNA. So there is variation from person to person, but all cells in a single person have the same MHC genes and make the same MHC proteins (multiple types made per cell).

    2. Two basic types of MHC

    a. MHC I. All nucleated cells have MHC I on their surface.

    b. MHC II. Cells of immune system (phagocytes & B cells) have MHC II on their surface. (Not all T cells have MHC II at all times, & we will assume T cells do not have MHC II.)

    3. How is MHC involved in T & B cell activation? See Sadava fig. 18.15 (18.17).

    a. What does MHC do? MHC and small pieces of antigen (epitopes or antigenic determinants) form a complex. Complex is on cell surface, so epitopes are 'displayed' on the cell surface, stuck to the MHC molecules.

    b. How does T cell bind to euk. cell?

    (1). Euk cell must have MHC + Antigen (epitope) on its surface

    (2). TCR binds to variable part of MHC-Ag complex = binds to epitope itself

    (3). CD4 or CD8 binds to part of corresponding MHC (II or I respectively).

    c. Two types of T's bind to different MHC's (w/ Ag) -- this is how T cells tell immune cells (that have captured Ag) and infected (ordinary) cells apart.

    (1). Cytotoxic T's (CD8 + ) bind to target cells with Ag + MHC I on surface.

    (a). TC are said to be "MHC I restricted" -- note target must have MHC I and Ag.

    (b). Target cells for cytoxic T's are usually ordinary cells making abnormal proteins -- infected cells, for example.

    (c). Binding to target (abnormal) cell → activation of TC and killing of target cell.

    (2). Helper T's (CD4 + ) bind to target cells with Ag + MHC II on surface.

    (a). TH are said to be "MHC II restricted" -- note target must have MHC II and Ag.

    (b). Target cells for helper T's are usually cells of the immune system, especially B cells and phagocytic cells that have internalized foreign antigens. These are called 'antigen presenting cells' or APC's.

    (c). Binding to target cell (APC) → activation of TH and activation of target cell (if it is a B cell).

    d. Activation of B cells -- usually requires a TH, but can be one or two step (See Sadava fig. 18.15 (18.17))

    (1). One step: B and TH bind to and activate each other. B acts as APC to activate TH TH in turn activates B.

    (2). Two step:

    (a). Phagocytic APC (not a B cell) binds to and activates a TH (See Sadava fig. 18.13 (18.15)).

    (b). Activated TH detaches from APC binds to and activates a B cell.

    4. How does epitope get on MHC? How are antigens 'presented' or 'displayed' on the cell surface?

    a. How the pieces get to attached to MHC -- depends on type of cell and where protein comes from.

    (1). Infected cells -- proteins made inside the cell are digested in proteosomes protein fragments (epitopes) enter the ER using a special transporter, and bind to MHC I.

    (2). Cells of immune system -- proteins made outside the cell are engulfed (by phagocytic cells) or endocytosed (after binding to antibody on surface of B cells). Protein fragments bind MHC II in endosomes.

    b. How MHC + epitope reaches cell surface -- MHC's are transmembrane proteins in subcellular membranes (of endomembrane system) MHC's bind epitope and complex reaches cell surface by exocytosis.

    c. Many epitopes displayed per cell -- every APC (antigen presenting cell) 'presents' many different pieces of whatever antigen(s) it engulfed, endocytosed or made.

    Try Problems 13-5 & 13-9. For a review of the information so far, try 13-6 , 13-11 (skip C) & 13-12.

    III. How do T cells get activated? Summary Table. See also Sadava, fig. 18.14 (18.16).

    Notes:
    (1). Activation of lymphocytes also requires appropriate cytokines. TH cells need IL-1 from the APC's TC cells need IL-2 from TH and B cells need various IL's class of Ab made by B cell depends on type of IL it gets.

    (2) In a TH -- B cell combo, each can activate the other. Alternatively, a helper T can be activated first, and then activate a B cell.


    IV. Ab Structure -- See handout 25B, picture of an immunoglobulin (from Alberts), or Sadava fig. 18.9 (18.10). What is the molecular structure of antibody molecules?

    A. V vs C -- types of Immunoglobulin (Ig).

    1. There are 5 main classes of Antibody -- IgM, IgD, IgG, IgE, and IgA. See table on handout 25B & Sadava Table 18.3.

    2. V & C: Each Ab or Ig is made up of a V section ("variable" region or Vee) & a C section ("constant" region or Cee).

    3. Variable region

    a. V is specific for Ag (or epitope). Determines what Ag will be bound = grabbers.

    b. V is variable due to differences in sequence, not just differences in folding around Ag.

    c. Every Ab or Immunogloblin (Ig) has (at least) 2 grabbers.

    d. All grabbers in one Ab are the same.

    e. All the antibodies made by one Ab-producing cell have the same V. All the antibodies made by descendents of that cell have very similar V's. (Minor differences are due to somatic mutation see advanced texts if you are interested. We will ignore somatic mutation for the rest of this discussion, and assume all the antibodies made by the descendents of one cell have the same variable region.)

    a. C determines biological effects -- localization of Ab, and what will happen as consequence of binding Ag. (Whether complement will be activated, whether Ab will be found primarily in blood or secretions, etc.)

    b. 5 main types of C regions, therefore 5 main classes of antibody. (For properties of the dif. classes see handout 25B or Sadava Table 18.3 )

    c. The same V's can go with different C's. (Called "Class Switching")

    (1). All the antibodies made by one Ab-producing cell do not necessarily have the same C.

    (2).The antibodies made by descendentsof a single cell may have different C's. The same variable region can go with different constant regions as B cell clone expands. How is this possible? Need a closer look at Ig structure

    B. H vs L. See handout 25B or Sadava fig. 18.9 (18.10)

    1. Every Ig has 2 kinds of chains , L ("light") and H ("heavy"). Light and heavy refer to relative differences in mol. wt.

    2. Basic unit is 2 of each for a total of 4 chains. (For number of basic four-chain units per Ig, see table.)

    3. Variable region (grabber) made of parts of each.

    4. Each chain has a constant region

    a. 2 kinds for L (kappa or lambda)

    b. 5 basic kinds for H (mu, delta, gamma, epsilon or alpha)

    c. Hc (constant part of H) determines class (IgM, IgD, IgG, IgE, IgA)

    d. Class (determined by Hc) determines location & other aspects of function (see "special properties" in table)

    d. Class switching involves the H chains only, not the L chains.

    5. Myelomas & Hybridomas: Ig structure was figured out by studying proteins made by myeloma cells (cancers derived from Ab-producing cells) or hybridomas (hybrids of Ab-producing normal cells and cancer cells). Only way to get large numbers of cells all making the same Ab/Ig. See texts for significance of hybridomas and monoclonal antibodies. (Sadava 18.11 (18.12))

    C. Classes of Ab and class switching during development of immune response

    1. Order of events (see handout 25A, bottom) during immune response as B cell matures

    a. First make M, then M + D -- all on surface.

    b. Meet Ag → primary response: secrete M.

    c. Meet Ag a second time → secondary response secretes usually G but can be E or A.

    d. All these Ig's combine with same Ag

    2. Implications of structure and switching

    a. Can make different variable regions -- zillions of them, one for each dif. epitope. So the IgG, for example, in a person is a mixture -- all IgG molecules have the same constant regions but have different variable regions.

    b. During different stages of immune response, can make Ab with same variable region but different constant region (for H). Can switch class and/or secreted vs. surface. How is this possible?

    3. What we already know: How switch from membrane bound to secreted works. Variable part stays same Hc changes from hydrophobic to hydrophilic by alt. splicing/poly A addition.

    4. What we don't know so far: How do you make so many dif. variable regions AND What changes when you switch classes (from IgM to IgG? M to M + D)? Must be rearrangement of DNA or alternate splicing of RNA. Different solutions at different steps.

    Try Problems 13-1 to 13-3.

    V. Structure of the DNA coding for Antibodies -- Basis of Generation of Diversity (G.O.D) and Class Switching

    A. Basic idea: genes for H and L are mosaic -- Each "Gene" has several parts. See texts or handout 25B or Sadava 18.16 (18.18.)

    B. How "gene" is divided -- region coding for each chain (H or L) has parts coding for each type of constant region and several parts coding for the variable region .

    C. How DNA is used to make different antibodies (With different V's) -- DNA is rearranged -- See Handout 25C.

    1. Pre Ag

    a. Rearrange V/D/J region of DNA to make one coding region for variable part of H chain per naive/virgin B.

    b. A similar process of DNA rearrangement occurs in DNA coding for variable part of L chain.

    c. Net: Only one H chain allele and one L chain allele are rearranged and used. Therefore each cell makes only one type of variable region.

    2. Post Ag -- Somatic Mutation → minor changes in region of DNA coding for V regions of H & L chains. (No change in DNA coding for C regions ). In the secondary response, there is a second round of clonal selection for B cell variants making 'better Ab' -- Ab that binds Ag better (higher affinity Ab). This is why Ab made in secondary response is better at binding Ag than primary Ab.

    3. Reminder: Switching at DNA level is unique to immune system.

    Note: We are going to ignore the effects of somatic mutation, but it is included here for reference.

    D. Summary of G.O.D (generator of diversity) -- how get so many V's?

    1. H & L mix and match -- any H chain can go with any L chain

    2. Mosaic V genes -- V parts (V, D, J) of DNA coding for each chain mix and match

    3. Joins are inexact -- bases can be added when you rearrange the DNA -- when join V to D etc.

    4. Somatic mutation -- post Ag

    E. TCR genes are similar, except no somatic mutation. Genes are mosaic, and are rearranged. The proteins that are made have more than one chain each TCR has constant and variable regions. See TCR picture from Alberts.

    F. Class Switching -- How DNA is used to make different versions of the same antibody (with different C regions) See Handout 25C.

    1. Definition of Class switching -- cell makes antibody with same variable region and different constant region.

    2. Mechanism -- Switching occurs at DNA and RNA levels.

    3. Pre Ag -- M vs D

    Alternate splicing allows cell to produce M and D antibodies with same V/D/J (but different constant region of H chain). For details see Sadava fig. 18.17 (18.19).

    3. Post Ag -- Alt splice of RNA and/or further rearrangement of DNA → new mRNA → new version of antibody with same variable region. Can have either of the following:

    a. Rearrangement (usually deletion) of DNA → gene for H chain with original variable region and a new "constant" region. Make new class of antibody. (See Sadava fig. 18.18 (18.20))

    b. Alternative splicing → mRNA for secreted version of cell surface antibody -- Same H chain except it's missing part that anchors the protein in the plasma membrane. Go from making "BCR" to making secreted antibody.

    Try recitation problem 14-3 & problem 13-13.

    VI. Evolutionary Aspects (FYI)

    A. Clonal vs. Natural Selection . Note how clonal selection and natural selection compare. In both cases, need to have many variants (diff. antibodies or dif. organisms) to be able to respond to unpredictable environmental challenges. How is this done? In both cases, make many variants and conditions select (promote propagation of) cells making the few suitable Ab (or carrying out a rare, useful function) the rest are wasted. Random generation of variants seems wasteful, but is the biological solution to preparing for change without conscious planning ahead.

    B. The Major Proteins of the Immune System are Related

    1. The immune system uses 3 types of proteins that have a common evolutionary origin. These are antibodies, TCR and MHC. For additional pictures see Sadava fig. 18.9 (18.10) for antibodies & Sadava fig. 18.12 (18.13) for TCR. Here are links to parallel pictures (from Alberts) of the two types of MHC, a TCR, and an immunoglobulin (showing the domains).

    2. All 3 types of proteins have a "constant" part and a "variable part."

    a. Constant part determines where protein is (cell surface? What kind of cell? etc.) and its general function.

    b. All 3 proteins bind epitopes -- Variable part determines what antigen/epitope will bind to the protein.

    3. All 3 proteins include one or more copies of the immunoglobulin domain -- a section of the protein that is similar in structure and function. this is a common theme -- the same domains are found over and over in different proteins. (Examples are SH2 domains DNA binding domains, etc.)

    4. Variable part of antibodies and TCR's are generated by rearranging the DNA the variable part of MHC's is encoded in the germ line -- the DNA inherited in the zygote is the DNA used to code for the MHC's. The DNA from MHC is NOT rearranged. However the genes for MHC's are polymorphic (have many different common alleles).


    VII. Summary of Major Players in the Immune System:

    Cells B cells, TC cells, TH cells, phagocytic cells, APC's
    Secreted Proteins Antibodies (Ab or immunoglobulins 5 classes), Perforin*, Cytokines*
    Cell Surface Proteins MHC, BCR, TCR, CD4, CD8

    The chart above summarizes the major players in immunology. You should be able to describe what each item is, its significance, and how it is related to all the others. "Secreted proteins" refers to those made by B and T cells. Proteins involved in the immune response (such as complement*) that are not made by lymphocytes are not listed. See ans. to problem 13-6, table above, and the table in lecture 24 (IV-C).

    *Terms with a star have not been discussed in detail, but you should be aware of their general roles.


    What is a secondary antibody?

    A secondary antibody is an antibody designed to target a primary antibody. Secondary antibodies are often used in combination with primary antibodies to detect target proteins in various immunoassays, like western blots, ELISA, and immunofluorescence. Many secondary antibodies are conjugated to other molecules, like Alexa Fluor dyes or horseradish peroxidase (HRP), which enable detection of the secondary antibody.

    It is possible to use conjugated primary antibodies, but secondary antibodies provide many advantages. Using a secondary antibody makes it easy to choose a different conjugate no matter the primary antibody. For example, the same primary can be used with a secondary antibody conjugated to HRP in a western blot and with a different secondary antibody conjugated to Alexa Fluor 488 dye in immunofluorescence.

    Also, secondary antibodies enhance detection by localizing more conjugate at the antigen than is possible with a labeled primary antibody. By using secondary antibodies, one avoids needing to chemically label (conjugate) primary antibodies, which are more specialized and costly to obtain. Conjugation (which is amino acid-specific) can interfere with the primary antibody’s recognition of the antigen.


    What are Antigens?

    An antigen is a foreign or &ldquonon-self&rdquo macromolecule (typically a protein) that reacts with cells of the immune system. However, not all antigens will provoke a response . For example, each of us produce a large number of self-antigens. Each of us has a unique set of self-antigens that do not trigger an immune response within ourselves. The absence of this immune response very important and highly regulated, it prevents scenarios where the immune cells begin to attack host cells. In the presence of foreign atnigens, proteins called antibodies attach to the antigens on the plasma membrane of the cell containing the antigen.

    Antigens and ABO Blood Types

    Like other cells, our red blood cells may or may not have self-antigens present on their cell membrane. The ABO blood typing is a naming scheme that states the presence or absence of just two antigens: antigen A and antigen B. The antigens that are present on the surface of our red blood cells determine our blood type. If we looking at the table below, we&rsquoll see that:

        • &rarr Blood type A has A-antigens
        • &rarr Blood type B has B-antigens
        • &rarr Blood type AB has both A-antigens and B antigens
        • &rarr Blood type O has neither antigen.


        Mechanism of Action

        Antibodies and antibiotics also differ in their mechanism of action: the way they kill pathogens and fight off infection. Antibodies produced in B cells bind to specific factors, called antigens, found on the pathogen. Once an antibody binds an antigen, the antibody triggers an activation of the immune system. The antibody signals for immune system cells to engulf and digest the infectious invader, helping to neutralize the infection.

        Antibiotics, on the other hand, typically work by inhibiting essential cellular functions the infectious bacteria requires to live and divide. Penicillin, the first discovered antibiotic, works by preventing synthesis of the cell wall, an essential step in bacterial cell division, according to Elmhurst College 2. Without proper cell wall formation, water can rush into the bacteria and cause the cell to burst, thereby treating the infection.


        Immune training underway

        To see if the usual plasmablast wave was followed by a germinal center response, the team extracted cells from participants’ armpit lymph nodes at multiple timepoints and used flow cytometry to quantify the proportion of germinal center–resident B cells there, which carry specific cell markers. Fluorescently labeled probes were used to isolate cells that target SARS-CoV-2’s spike protein, which is produced by the mRNA vaccine.

        Sure enough, the researchers observed spike-binding germinal center B cells in all participants three weeks after the first jab. The proportion of these cells increased after the second shot and stayed at high levels in most participants for at least seven weeks—a good sign that memory B cells and plasma cells are being generated. The longer germinal center reactions go on, the more rigorous training antibody-producing cells are getting. “You get higher affinity B cells, but you get also more B cells differentiating into the memory B cells and [a] long-lived plasma cell pool,” he says. “This is what the vaccine is meant to do.”

        The fact that the mRNA vaccine elicits a strong germinal center response “is not a huge surprise,” Ellebedy says a good plasmablast response is usually a good indication that the germinal center reactions will follow. What was surprising is that the response appeared to be stronger than they’d previously observed in the influenza study, when only three of eight participants’ lymph nodes harbored hemagglutinin-binding germinal center B cells. And in those three, “the magnitude of the response was modest,” Ellebedy recalls.

        With the mRNA vaccine, “in every node we looked at, we found very nice, beautiful germinal center responses specific to the spike,” Ellebedy says. He suspects that the difference may have to do with the fact that the seasonal influenza vaccine they investigated doesn’t contain efficacy-boosting compounds known as adjuvants. Instead, the vaccine essentially only consists of a piece of protein, selected for inducing a strong immune response, and relies heavily on the fact that many people already have pre-existing immunity to influenza in order to produce protection. The SARS-CoV-2 mRNA vaccine, in contrast, contains adjuvants, and mRNA itself can elicit immune reactions and is potentially translated into larger quantities of protein than those contained in the flu vaccine, Ellebedy hypothesizes.

        The results are in line with recently published mouse data that suggest a stronger germinal center response after a single shot of a SARS-CoV-2 mRNA vaccine than to a recombinant protein vaccine with an adjuvant. Although the germinal center responses to non-mRNA vaccines haven’t yet been tested in people, Ellebedy’s data underscore the potency of mRNA vaccines, he says.


        Decoding the Immune System

        A remarkable range of insights and new drugs might result from new T cell technologies, including medicines to fight cancer and immune diseases.

        T cells spend their lives talking the language of disease, and we should listen to what they have to say.

        Almost every cell in the body chews up a small fraction of its proteins and presents them as antigens on its surface to enable immune system monitoring. T cells surveil these antigens and represent the critical first arm of the adaptive immune system. This component of the overall immune system kills diseased cells, coordinates antibody production by B cells, and provides the essential memory of past disease. For every antigen that a T cell probes with its highly variable T cell receptor (TCR), the cell must integrate the resulting signaling with a variety of its past and current experiences. These include the core functional training the T cell received in the thymus, memories of previous antigen exposure, all previous and concurrent immune interactions, and the microenvironment and functional state of the target cell it is probing. In choosing among many potential paths, each T cell, and the T cell compartment as a whole, must find a balance between killing cancer and aberrantly attacking healthy tissue, thereby causing autoimmune disease. T cells must walk that fine line every day, and in doing so they collectively demonstrate a fundamental and severely underappreciated ability to continuously monitor every cell in our body.

        The enormous potential and limitations of current T cell therapeutics are best exemplified by two of today’s most vaunted avenues of therapeutic research: checkpoint inhibitors that disinhibit T cells to enable antigen-specific tumor killing, and CAR T cell therapies that redirect a patient’s own T cells to known antigen targets on cancer cells. Both therapeutics leverage T cell potency to kill cancer cells, but they do it with almost no knowledge of the native antigen specificity of the cells, often resulting in toxicity—particularly with checkpoint inhibitors, when the activated T cells turn against healthy tissue to cause autoimmunity.

        What we can learn from B cell development

        The current state of T cell antigen discovery can be understood by considering its sibling B cells. With B cells, revolutions in antibody screening and protein engineering have allowed antibodies and their derivatives to transform medicine and dominate the biologics market. As with T cell therapeutics today, the first B cell therapeutics were poorly defined mixtures of B cell–produced antibodies targeted to specific antigens. The result was antiserum therapeutics that were initially used as antibacterials (and predated antibiotics by over 40 years) and are still used to great effect today as antivenoms. The turning point came in the 1970s with technologies that allowed high-throughput antibody interrogation. Hybridoma technology and, later, surface display technologies provided high-throughput screening methods to identify individual B cell antibody clones that bind a specific target antigen. This ability to produce, screen, and optimize antibodies resulted in five of the top six biologics on the market today, whose mechanisms include the core targeting moieties of both the checkpoint inhibitors and CAR T therapeutics.

        T cells should be our teachers

        T cells should follow a similar path to antigen discovery and therapeutic value, but there are critical differences in T cell biology that will increase both the difficulty of antigen discovery and the potential value of the antigens and therapeutic modalities that result. T cells function through direct cell-cell interactions in which they use their highly diverse T cell receptors (TCRs) to inspect antigens loaded on a target cell’s surface. Upon interaction with their target antigen, activated T cells often stay at the target site, rapidly expand, and kill the target cell. This makes T cells a potent weapon against infections and diseases such as cancer. But it also makes them ideal immune teachers, because their presence can be used not only to isolate the TCRs that bind to diseased cells but also to identify antigens that protect against infectious disease, cancer, and autoimmune disease. Perhaps most important, these antigens are derived from proteins in all cellular compartments, including the cytoplasm and organelles, providing a much larger range of disease-specific targets than are possible for B cell antibodies, which target only surface and secreted molecules.

        What we need is a method to decipher the functional state of T cells, the TCRs they carry, and the immunogenic antigens that they see.

        3D rendered illustration of white blood cells attacking a cancer cell. Photo via AdobeStock

        T cells see color, not black and white

        Many of the antigens that T cells see in these diseases are not the ones that immunologists typically look for. Antigens generated by pathogens and cancer mutations have never been produced by the body or encountered by the body’s T cell repertoire, so they are typically described as foreign, or non-self. Cells that display these non-self antigens are recognized by T cells and killed. Then again, a core tenet of T cell biology is that all T cells that react to self are killed or converted to regulatory T cells self-reactive cells that escape this clearance and central and peripheral tolerance cause autoimmunity. And yet time and again self-reactive T cells are found in tumors. Early examples were identified from testes and other immune-privileged sites where cells and their presented antigens are shielded from T cell surveillance. Proteins from immune-privileged sites that are aberrantly expressed in cancer cells can be immunogenic, and these cancer testes antigens, such as NY-ESO-1 and MAGE-A1, are the foundation of many of today’s targeted T cell therapy trials.

        Yet this vision of immune-privileged proteins as sources of cancer antigens may vastly underestimate the ability of T cells to recognize disease. The entire human genome can theoretically produce immunogenic antigens if the antigens are transcribed, translated, and presented on the surface of the cell for T cell recognition. Around 1.5 percent of the genome codes for conventional proteins that may be subject to T cell deletion and tolerance. But some fraction of those genes, such as many endogenous retrovirus genes, are transcriptionally repressed, and they are never expressed in normal tissue or thymus, leading to immune recognition when they are activated in disease. No doubt a large fraction of the remaining 98.5 percent of the genome is non-coding and does not produce protein. But a significant portion does in fact encode short or frameshift proteins that can be highly immunogenic when aberrantly expressed in cancer.

        Recent examples of these germline cancer antigens are almost certainly the first of many potential therapeutic targets that could be far more broadly useful and cost effective than current personalized neoantigen mutations that are rarely shared between patients.

        Clearance of cancer and infection may be the least interesting thing T cells do

        Even this broadened view of T cell surveillance dramatically underestimates their role in the body. The body selects a significant subset of T cells, known as regulatory T cells (Tregs), to specifically recognize self and actively protect cells and tissues expressing these antigens from immune attack. Thus, Tregs sit at the interface of self and non-self to convert the conventionally stark black-and-white image of self and non-self into a spectrum of grays. They are most often studied in the context of barrier homeostasis in the intestine and lung, but their emerging role in adipose and cardiovascular tissue strongly suggests that they play essential homeostatic roles in all tissue. Importantly, the antigens that these cells see in tissue are almost entirely unknown.

        The size of the puzzle

        Why don’t we already know what T cells see? T cells can produce at least 10^13 different TCRs. They can recognize a theoretical diversity of over 10^11 antigens loaded on surface receptors known as major histocompatibility complex (MHC) proteins. These proteins are the most polymorphic in the human population, with thousands of variants. The problem becomes one of massive potential diversity on both sides. When targeted to any one person, the practical diversities of the TCRs and antigens are restricted to the person’s defined set of MHCs and the roughly 10-100 million T cell clones that exist in any person at any one time—but the numbers remain nonetheless daunting. Tetramer technology, developed in seminal work over 20 year ago, enabled visualization and isolation of T cells that recognize a single antigen. But inherent limitations in fluorescence and isotope-based isolation have severely restricted the number of antigens that can be effectively screened. Cell-based screening methods enable high-throughput antigen discovery, but they are limited to the interrogation of relatively small numbers of T cell clones. On the other hand, recent advances in single-cell-sequencing technology now allow high-throughput characterization of T cells and the TCRs they encode. Single-cell sequencing has already begun to revolutionize all areas of immunology, but it will be essential to couple high-throughput antigen detection with single-cell sequencing to break the T cell field wide open.

        T cell therapeutics

        A remarkable range of insights and drugs might result from the new T cell technologies. Every nucleated cell in our body presents MHC-bound antigens on its surface, and those antigens provide a distinct signature of the cell’s identity and functional state. Novel cancer-specific antigens will dramatically improve autologous T cell therapy, in which a patient’s own T cells are specifically activated and expanded to increase anti-cancer activity. Furthermore, the TCRs that bind these antigens will provide an entirely new reservoir for recombinant TCRs that can be used to redirect patient cells to cancer, much like CAR T technology today.

        Beyond their immediate utility in cancer treatment, TCRs and their antigen targets will almost certainly be useful in fighting all immune diseases, particularly autoimmunity and transplantation, in which T cell activity must also be redirected to ameliorate disease. Therapeutic modalities that provide antigen-specific immune tolerance are in early development, and they are in desperate need of better antigen targets. But most broadly and perhaps most impactfully, these antigens and TCRs can target any cell in the body, either healthy or diseased, delivering therapeutic cargo wherever it is needed.

        Finally, high-throughput interrogation of antigen–T cell interactions would provide an enormous data set that could be used to predict the antigen specificity of potentially every TCR. This would dramatically improve the accuracy and safety of TCR therapeutics. It would also provide an easy and ubiquitous method to diagnose disease at potentially a very early stage. Periodic blood draws and T cell sequencing would provide the functional state and TCR sequence of circulating T cells. For example, activated T cells with specificity to pancreatic cancer antigens could catch early-stage pancreatic cancer that is treatable but largely asymptomatic. A drop in vaccine-induced memory T cells to measles antigens could be followed by a simple booster vaccine. T cells with reactivity to specific cardiomyocyte antigens could predict underlying heart disease or subclinical atherosclerosis. All of these benefits may be achieved with a blood draw, standard sequencing, and an AI platform trained on a massive antigen-TCR interaction map.

        If we ask the right questions with the right technology, the answers will come quickly. And if we listen to T cells carefully, they will show us where to look and what we will find. At Flagship Pioneering, we have built a foundational platform within Repertoire Immune Medicines to decode T cell–antigen interactions at unprecedented scale, and we have begun to apply it across cancer, infection, and autoimmune disease. Our ears are attuned.