Immunogenetic

Tonegawa’s demonstration 1976—used restriction enzymes and DNA probes to show that germ cell DNA contained several smaller DNA segments compared to DNA taken from developed lymphocytes (myeloma cells)

Bone marrow stromal cells drive Pro-B cell proliferation and maturation. Important molecules and interactions: SCF-cKit IL-7-IL-7R

Antibody Diversity How do we acquire so many different types of antibodies? Somatic recombination Junctional diversity hypermutations

Summary of Splices Light chain Heavy chain D-J joining by DNA splice V-JC joining by DNA splice (lambda) V-J joining by DNA splice (kappa) VJ-C intron removal (RNA splice) Heavy chain D-J joining by DNA splice V-DJ joining by DNA splice VDJ-C intron removal by RNA splice IgM membrane bound 3rd C domain to soluble 3rd C domain by RNA splice Class switching by DNA splice

Genes for immunoglobulin proteins are found on different chromosomes

Heavy chain rearrangement

Kappa light chain rearrangement

Lymphocyte-specific and ubiquitous enzymes are required RAG-1 and RAG-2 are lymphocyte-specific Fibroblasts transfected with RAG-1 + RAG-2 undergo somatic recombination of Ig genes RAG-KO mice have no B or T cells RAG is active in G0 and G1 mitotic phase and is off in the proliferative status. RAG digest DNA in un-precise pattern.

Recombination occurs at specific sites Recombination signal sequences (RSS) occur adjacent to coding sequences in V, D, and J segments Heptamer-spacer-nonamer 12/23 rule

12/23 rule for gene recombination

See gene recombination animation on CD Marker of cells that have undergone V(D)J recombination

√ √ Recombination signal sequences (RSS) Vb Db Jb Vb 7 23 9 Db 12 Jb 7 HEPTAMER - Always contiguous with coding sequence NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer Vb 7 23 9 Db 12 Jb √ √ Vb 7 23 9 Db 12 Jb 12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

V1 D J 9 7 Molecular explanation of the 12-23 rule V2 V3 V4 V8 V7 V6 Loop of intervening DNA is excised V8 V7 V6 V5 Heptamers and nonamers align back-to-back The shape generated by the RSS’s acts as a target for recombinases 7 9 23-mer 12-mer V9 D J An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the 12-23 rule)

D D Junctional diversity V J V J 7 12 9 23 7 12 9 23 Mini-circle of DNA is permanently lost from the genome Signal joint Coding joint V D J V D J Imprecise and random events that occur when the DNA breaks and rejoins allows new nucleotides to be inserted or lost from the sequence at and around the coding joint.

Linkage of C gene to VDJ at the m-RNA level.

Allelic Exclusion These diversity mechanisms often generate non-functional Ig genes: genes that contain stop codons or don't stay in the proper reading frame. The developing B cells use a mechanism called "allelic exclusion", in which each B cell makes only 1 active L chain and 1 active H chain. The cell tries each copy of the L genes and each copy of the H genes in turn: If an active chain is made, no further DNA splicing occurs. However, if a non-functional Ig is made, the cell then tries the next L or H gene. This process continues until an active product is made from both H and L, or until all genes have been tried (in which case the cell dies).

Multiple gene segments increase Ig diversity Combinatorial diversity: Heavy chains 40 x 25 x 6 = 6000 Light chains 40 x 5 = 200 k 30 x 4 = 120 l Total possible: 320 x 6000 = 1.9x106

Junctional diversity Nucleotide deletion can also occur Occurs in HV3 (CDR3) region What problem could these events cause??

N nucleotide addition at joining segments: the addition of random bases

Randomness in joining process helps generate diversity by creating hypervariable of antigen binding site

U U V D J V D J V D J Generation of the palindromic sequence TC AG U D J AT TA Regions to be joined are juxtaposed Endonuclease cleaves single strand at random sites in V and D segment V TC AG U D J AT TA The nicked strand ‘flips’ out V TC~GA AG D J AT TA~TA The nucleotides that flip out, become part of the complementary DNA strand In terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic. The nucleotides GA and TA were not in the genomic sequence and introduce diversity of sequence at the V to D join.

Junctional Diversity – N nucleotide additions Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA V TC~GA AG D J AT TA~TA CACTCCTTA TTCTTGCAA V TC~GA AG D J AT TA~TA CACACCTTA TTCTTGCAA Complementary bases anneal D J TA~TA Exonucleases nibble back free ends V TC~GA CACACCTTA TTCTTGCAA V D J DNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands TC~GA AG AT TA~TA CACACCTTA TTCTTGCAA V TC D TA GTT AT AT AG C

Imprecise joining generates diversity

V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity Germline-encoded nucleotides Palindromic (P) nucleotides - not in the germline Non-template (N) encoded nucleotides - not in the germline Creates an essentially random sequence between the V region, D region and J region in beta chains and the V region and J region in alpha chains.

Additional Diversity Mechanisms In addition to the DNA splicing, other variants in the antibody molecules are generated by two mechanisms: First, the DNA splices do not occur at a precise point: they can vary by several bases, which can lead to the addition or deletion of 1 or 2 amino acids at each splice point. These variations can lead to different specificities of the antibodies. The enzymes that do the DNA splicing (RAG1 and RAG2) produce double stranded breaks in the DNA, which is repaired imprecisely.

P and N region nucleotide alteration adds to diversity of V region During recombination some nucleotide bases are cut from or add to the coding regions (p nucleotides) Up to 15 or so randomly inserted nucleotide bases are added at the cut sites of the V, D and J regions (n nucleotides). TdT (terminal deoxynucleotidyl transferase) a unique enzyme found only in lymphocytes (20 N added) Since these bases are random, the amino acid sequence generated by these bases will also be random

B lymphocyte development

IgM and IgD are coexpressed in mature naïve B cells

Alternative RNA processing generates transmembrane or secreted Ig

Synthesis, assembly and secretion of immunoglobulins

Combination of heavy and light chains adds final diversity of variable region 8262 possible heavy chain combinations 320 light chain combinations Over 2 million combinations P and N nucleotide additions and subtractions multiply this by 104 Possible combinations over 1010

IL-7 has critical role in the proliferative activity of B and T lymphocytes precursor.

Somatic hypermutation adds even more variability B cell multiplication introduces additional opportunities for alterations to rearranged VJ or VDJ segments These regions are extremely susceptible to mutation compared to “regular” DNA, about one base in 600 is altered per two generations of dividing (expanding) lymphocyte population

Further Ig diversity arises through affinity maturation

Affinity maturation is due to somatic hypermutation Silent Neutral Deleterious Positive

Somatic Hypermutation Second, there is a “somatic hypermutation” mechanism by which random base change mutations occur in the V regions in B cells. This mechanism doesn't work in other cells and doesn't affect other genes: only a region of about 1.5 kb is affected. It only occurs as the B cell is maturing: after it has been stimulated to divide by an antigen, somatic hypermutation occurs to modify the antigen binding region. Those cells that bind the antigen most tightly survive and divide more than the others. This process is called “affinity maturation”. It is triggered by the enzyme “activation-induced cytidine deaminase” (AID), which deaminates cytidine to uracil. This base mismatch can be incorrectly repaired by several different mechanisms to generate mutations.

Isotype switching Irreversible Only occurs after a given B cell has encountered antigen Mechanism not fully understood Requires AID Requires DNA repair enzymes Requires external signals (helper T cells)

Isotype switching occurs in activated B cells

Class Switching Heavy chains fall into 5 classes, based on their C regions. Each H gene has C regions for all 5 classes arranged on the chromosome, with the IgM C region nearest to the V regions. There are several different C regions for some of the classes. IgM is the initial Ab made by each B cell. However, after a while the B cell switches to a different class. This is done using a third DNA splice, in which the DNA between the VDJ and the constant region for the new class is spliced out.

Class switching among constant regions: generation of IgG, IgA and IgE with same antigenic determinants—idiotypes

7 means of generating antibody diversity

Location of variability occurs within CDR regions of V domains (antigen binding sites)

Mature B cells undergo Positive and negative selection in the bone marrow and then migrate to the peripheral blood. Negative selection carried out with membrane bound self antigen for immature expressing IgM+ B cells and resulted in apoptosis. In some cases receptor editing (mainly for k gene) resulted in changes in the specificity of B cells.

Stages in B-cell maturation in the bone marrow

From Pro- to Pre-B cell transition events

B cell development

T cell development

T Cells These are some of the General characteristics of the T Cell receptor for antigen recognition.

T cell development occurs in the thymus The Thymus has a Capsule with Trabeculae. The trabeculae divide the thymus into Lobules. Per lobule you have an outer cortex and an inner medulla. The cortex is formed of dense lymphoid tissue which lacks nodules. Since the stroma of the medulla is less heavily infiltrated with lymphocytes than the cortex, the medulla stains more lightly than the cortex. Immature lymphoid cells enter the cortex proliferate, mature and pass on to the medulla. From the medulla mature T lymphocytes enter the circulation. Arteries supplying the thymus follow the connective tissue septa and give off branches that enter the lobular cortex and break up into capillaries, which supply the cortex. Epithelial reticular cells sequester developing lymphocytes and form a sheath covering capillaries and lymphatic vessels. The sheathing forms what is called the blood-thymus barrier, preventing antigen contamination of developing and programmed T lymphocytes. The blood-thymus barrier is not found in the medulla, which appears to have a richer blood supply than the cortex. The capillaries terminate in thin-walled veins located in the connective tissue septa along with arteries. Lymphatic vessels arise within the thymic lobule and join to form larger vessels, which accompany the arteries and veins in the septa. In contrast to lymph nodes, the thymus contains no lymph sinuses or afferent lymphatic vessels. The following cell types are present: lymphoid cells, epithelial cells, macrophages, other supporting cells Thymic epithelial cells have different appearances in different locations within the gland. They form a continuous sub-capsular layer and a network in the cortex and medulla. Deep in the medulla they are also aggregated into Hassall's corpuscles. Morphologically, the thymus differs from the lymph node in its lack of nodules and afferent lymphatic vessels, and by the presence of Hassall's corpuscles. Efferent lymphatic vessels take origin close to the trabeculae and leave through them. Blood vessels also enter and leave through the trabeculae. Capillaries but no venules can be seen in the cortex. In the medulla, venules are present. Blood vessels in the thymic parenchyma have a sheath of reticulo-epithelial cells external to the basal lamina of their endothelium; this establishes the blood-thymic barrier. Most developping T-cell dies within the thymus. T cell precursor arriving in the thymus from the BM spend up to a week differentiating there before they enter a phase of intense proliferation. Approximately 98% of the lymphocyte will fail their maturation and die whithin the thymus. They die by apoptosis and their residual bodies are seen inside the macrophages throughout the thymic cortex. This apparent waste is a crucial part of T-cell development as its reflects the intensive screeming that each new thymocyte undergoes fopr the ability to recognize self-MHC and for self tolerance. Lack of thymus in mice and human resulted to nude mice and digeorge syndrome in human.

Progression through development correlates with rearrangement TCRbeta rearrangement begins during DN stages.

TCR b locus has two D-J clusters Allows a 2nd rearrangement if 1st is nonproductive

Diversity in the TCR gene locus

How do you test for successful TCRbeta chain rearrangements if you have not rearranged TCRalpha? Pre-Talpha Analagous to the surrogate light chain for immunoglobulin Pre-T cell receptor

The TCR and BCR gene is most variable in the CDR3 region

Allelic exclusion occurred in B-TCR but not in a-TCR, So 30% of Ln T has TWO types TCR and co-expressed both allele of a-TCR.

Thymocytes at different developmental stages are found in distinct parts of the thymus Maturation

Development of T cells in a mouse. RAG  rearrangement

Tolerance to self antigens encountered in the thymus is achieved by eliminating T cells that are reactive to these antigens.

Major Thymocyte Subsets: (cont.) CD4-CD8- (Double Negative, DN) cells: 3-5% of total thymocytes Contain least mature cells, considerable cell division 2/3rds are triple negative (TN) based on TCR expression Can be further divided based on CD44 and CD25 (discussed later) TCR b,g and d rearrangements occur at this stage 1/3rd are TCR gd+ CD4+CD8+ (Double Positive, DP) cells: 80-85% of total thymocytes TCR a rearrangement occurs at this stage Most have rearranged TCR ab genes and express low levels (1/10th mature level) of TCR Small subset has high levels of TCR (most mature, positively selected cells) Small subset is actively dividing (earliest DPs) Most apoptosis occurs here, very sensitive to apoptosis inducing agents, especially sensitive to glucocorticoids CD4+CD8- and CD4-CD8+ (Single positive, SP) cells: 10-15% of total thymocytes Most are mature cells with high levels of CD3 and TCR ab CD4:CD8 approx 2:1 ratio Most SP cells are functionally mature and are destined to leave the thymus Small subset of SP are immature (ISP) (CD8 in mouse, CD4 in human) and have low CD3 and no TCR ab - transitional cells that are on the way from DN -> DP

How do DP Cells Become SP? Instructive Model Fitting Stochastic Model Random

Mechanism of Selection Avidity Differences in signal strength is dictated by TcR:MHC avidity “Quantitative” Model Differential Signaling Selection outcomes are dictated by different signals Qualitative

TCR a locus has 50 Ja segments

T Cell Receptors The TCR protein has 2 subunits and one antigen binding site. The alpha subunit has V and J segments (similar to Ig light chains) The beta subunit has V, D and J regions, like the Ig heavy chain. Both segments undergo DNA splicing rearrangements like the Ig genes. The joining is not precise and short additions or deletions of bases can occur, as in the Ig genes. However, affinity maturation and somatic hypermutation do not occur. The TCR protein is membrane bound. It is only found on T cells.

Sequentially making and expressing a pre-TCR and TCR

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

Summary of Thymic Development

Lineage Commitment Models DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Newer Lineage Commitment Models DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Positive Selection Results in MHC restriction Mechanism: Immature thymocytes cluster with MHC molecules on the cortical cells of the thymus If TCR interacts with MHC  protective signal results that prevents apoptosis. If TCR does not interact with MHC  no protective signal and apoptosis occurs. Result? Only reactive thymocytes survive.

Negative Selection Ensures self-tolerance Weeds out High affinity thymocytes Mechanism: APC’s bearing MHC’s interact with thymocytes If avidity is too strong  thymocyte undergoes apoptosis. Details unknown… Result? Only self-tolerant thymocytes survive.

Generation of diversity in the TcR COMBINATORIAL DIVERSITY Multiple germline segments In the human TcR Variable (V) segments: ~70, 52 Diversity (D) segments: 0, 2 Joining (J) segments: 61, 13 The need to pair  and  chains to form a binding site doubles the potential for diversity JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR

Estimate of the number of human TcR and Ig Excluding somatic hypermutation Immunoglobulin  TcR Element H    Variable segments 40 59 52 ~70 Diversity segments 27 2 D segments in all 3 frames - - Yes Yes Joining segments 6 9 13 61 Joints with N & P nucleotides 2 (1)* 2 1 2360 3640 No. of V gene pairs Junctional diversity ~1013 ~1013 Total diversity ~1016** ~1016 * Only half of human k chains have N & P regions **No of distinct receptors increased further by somatic hypermutation

Summary: Ig vs TCR

Summary: mechanisms that generate diversity in lymphocyte receptors

Thanks