Lymphocytes_Mandl Flashcards
What are hallmarks of pyroptosis?
- Secretion of contents
- Inflammasome assembly
- Activation of caspase-1
- IL1-b secretion
*NOT DNA fragmentation (this is apoptosis)
What is clonal selection theory?
- Cells of the adaptive immune system, B and T lymphocytes, generate a RARE and UNIQUE antigen receptor from incomplete gene segments
- Each lymphocyte expresses only ONE antigen receptor specificity = expression is clonal
- All progeny from that single clone will inherit the same receptor specificity (clonal selection + expansion)
- only the lymphocytes which encounter an antigen to which their receptor binds will be activated to proliferate and become effector cells = clonal expansion
What is the general sturcture of immunoglobulin?
2x identical light chains + 2x identical heavy chains
Chains are connected by disulfide bonds
There is a hinge region at the y separation that confers flexibility to the immunoglobulin
- Membrane bound form of Ig = BCR
- Secreted form of Ig = Antibody
*They have the same antigen specificity and come from the same B cell
What is the result of a papain digestion?
Gives 3 subparts → 2x Fab region (Ag binding) + 1x complete Fc region (parts of both heavy chains still connected)
What is the purpose of the Fc region?
non-Ag binding portion, can:
- Bind to Fc receptors on phagocytic cells
- Bind to C1q complement
- Regulate secretion
What is the result of a pepsin digestion?
get linked Fab = F(ab)2 + Fc fragments
*Both light chains are still connected through part of the heavy chains
How are antigens bound by immunoglobulins?
Vh (variable heavy) and Vl domain pairs from the antigen-binding site at the tip of each arm of Ig
- Formed by 3 hypervariable loops in each of the polypeptides = complementarity-determining regions (CDR1, 2 and 3)
- Ig can bind to pockets, grooves, extended surfaces of knobs within the antigen binding site
- Can recognize linear and conformational epitopes
- In addition to proteins can bind: polysaccharides, lipids, small molecules, etc
How is the structure recognized by an Ig called?
Antigenic determinant = epitope
What are the 5 major classes of heavy chains? And 2 classes of light chians of antibodies?
Light chains → lambda (l) and kappa (k)
Heavy chains → gamma, μ (mu), alpha, epsilon, delta, corresponding to the 5 immunoglobulin classes: IgG, IgM, IgA, IgE, IgD = isotypes
What differentiates IgG, IgM, IgA, IgE and IgD?
IgM and IgE → 5 C domains in their heavy chains
IgG, IgD, IgA → 4 C domains in their heavy chains
Also differentiated by their assemblies (monomers, dimers, pentamers), which dictates where they are found in the body.
- IgM in serum is always a pentamer
- IgA can be monomers, dimers, trimers
- IgG can be further subdivided into IgG1, IgG2a, IgG2b, IgG3
Ex: IgA and IgM are good at being transported across epithelial layers
What is class-switching?
When an Ig becomes a different isotype
ex IgG → IgA
*Different isotypes are better at different functions (ex: IgGs are best at neutralizing)
How do neutralizing Abs function?
Reminder: Abs are the secreted immunoglobulins of B and T cells (antigen specific)
They bind to their receptors to: block the action of toxins, prevent pathogens from infecting cells (prevent them from binding their receptor by binding the receptor)
What is opsonization?
Coating with antibody facilitates phagocytosis via FcR binding
*non-neutralizing effect of Abs
What are 3 possible ways antibodies mediate responses to extracellular forms of pathogens and their toxic products?
- neutralizing Abs → Ingestion by macrophages
- Opsonization → Ingestion by macrophages
- Complement activation → Lysis and ingestion
What is the structure of the BCR?
BCR = membrane-bound for of Ig → identical structure to antibodies
- Modified c-terminus of heavy chain to anchor BCR into plasma membrane
- Hase 2x light chains + 2x heavy chains
- Before activation B cells express IgM and IgD
- BCR requires invariant signaling proteins, Iga (CD79A) and Igb (CD79B), to transmit the signal (as they have to short of an intracellular portion)
ITAM = immunoreceptor tyrosine-based activation motifs (on the intracellular portion of Igalpha and Igbeta) → gets phosphorylated of the BCR binds to its antigen
What are the invariant signaling proteins/coreceptors of the BCR?
Ig alpha (CD79A)
Ig beta (CD79B)
*They dimerize and have an ITAM (immunoreceptor tyrosine-based activation motifs) which gets phosphorylated when BCR binds its antigen
What is involved/required for B cell activation?
- Multiple BCRs on a B cell bind their specific antigens
- Receptor cross-linking and clustering leads to BCR signaling and B cell activation
- BCR signaling with input from B cell coreceptor: CD19:CD81:CD21 complex
CD21 (complement receptor 2) binds C3dg complement fragment (on pathogen) → amplifies the signal
Members of the Src family of tyrosine kinases are recruited to the BCR → phosphorylate ITAM + coreceptors
How does the B cell phenotype/state change with B cell activation/BCR signaling?
Before → quiescent cells has very little cytoplasm
After → Large cell ready to divide, protein manufacture producing Abs
*Within 48hrs
BCR signal leads to changes in cell:
- metabolism
- cytoskeleton
- transcription factor expression
- clonal expansion
What is the antibody repertoire?
The total number of antibody specificities within an individual
- Each B lymphocyte has a unique antigen receptor
- Humans have ~1011 = 100 billion B cells!
What is combinatorial diversity?
Each Ig chain rearrangement uses a random selection of V(D)J
Light chains → kappa or lambda → can have different V, J and C regions
Heavy chains can have different V, D, J, C regions
What is the different betweent the lambda and the kappa light chain loci?
What is the difference with the heavy chain locus as well?
Lambda:
- V1–…– V29/32 —- J1 - C1 - J2 - C2 - J3 - C3 - J4 - C4 -
Kappa:
- V1 –…– V31/35 ——- J1 –…– J5 – C
*Only 1x C
*Each V segment has an L segment just before it
WHat is the structure of the heavy chain locus?
What is expressed in immature B cells?
Heavy:
- has a series of C gene regions, each of which corresponds to a different Ig isotype
- initially B cells express μ and d, and thus express IgM and IgD
- after activation B cells can undergo class switching to express other isotypes
- L1-V1–…– L40-V40 ———— D1-23 — J1-6 - C1 - C9
Wha are recombination signal sequences (RSS)?
What VDJ rules are associated with them?
DNA rearrangements are guided by conserved non-coding DNA sequences called recombination signal sequences (RSS) found adjacent to gene segments
RSS made up of: block of 7 nucleotides (heptamer) followed by a spacer (12 or 23 bp) and then a block of 9 nucleotides (nonamer)
V(D)J rules:
1. Recombination happens between gene segments on one chromosome
2. Gene segment flanked by RSS with 12bp spacer can be joined only to one flanked by a 23bp spacer
What is the role of RAG1/2?
*recombinase activating genes: RAG1 and RAG2
(lymphocyte specific genes)
- Recognize and bind RSS, enforces 12/23 rule, aligns the two RSS that will undergo rearrangement (juxtaposition and looping out of DNA in between)
- Make a DNA break at end of each RSS (DNA excision circles are lost with cell division)
- VJ form a coding joint (not RAGs)
What are the precise steps of V(D)J recombination and which proteins are involved?
- Juxtaposition of RSS by RAG1/2 + Generation of DNA hairpin ends
- Ku70:Ku80 binds DNA ends (DNA repair protein) + Artemis:DNA-PK opens hairpins
- TdT adds non-template nucleotides (in the middle between both segments → for end-joining)
- Increases diversity (~1-3 Nt) - 2x DNA ends are ligated DNA ligase IV and XRCC4 (also involved in DNA repair mechanisms)
- Gaps are filled to make the coding joints → addition of P (palindromic) nucleotides
Result 2/3 times = addition of a stop codon/frameshit causing a stop codon → non-functional chain
What part of the V(D)J recombination forms the CDR3?
What is the CDR3?
complementarity-determining regions → in the hypervariable loops of the antigen-specific region
contains the PNP nucleotides added during recombination of the D and J segments
What are the 5 main ways diversity is generated in the B cell repertoire?
- multiple gene segments exist at each Ig loci
- combinatorial diversity – H chain with L chain
- P-nt additions (not always there?)
- exonuclease trimming (random nt loss)
- non-templated N-additions by TdT (90% of diversity!)
Loss of function in which enzymes would result in a complete lack of B (and T) cells?
- Artemis
- Ku70
- TdT (can still male B and T cells, bu loss of a lot of diversity)
- DNA-PK
- Rag1
Causes severe combined immunodeficiency (SCID) due to lack of B and T cells
- no adaptative immune system, can’t build memory
What is the concept of allelic exclusion?
Only one single unique H and one L chain are expressed , but we have 2 copies of every Ig gene from paternal and maternal
- Need to ensure B cells make Ig of only a single specificity
From progenitor B cell:
1. Test parent 1 recombination of the heavy chain
2. If works, great! If not, try the other parent’s gene
3. If works, move on, if not, cell death
*When we do recombination, we can’t go back bc we cut out pieces of DNA
- Do the same for kappa light chain
- If both parents kappa light chain rearrangement does not work → move onto lambda chains
- If both parents lambda chains don’t work → cell death
- If you find a combination that works (no stop codons) → test combination of heavy and light chain for self-reactivity
Where does development, Ig rearrangement and testing for self-reactivity take place?
In the BONE MARROW for B cells
- Stages of development are defined by cell surface markers, TF expression and Ig gene rearrangement
What happens as the heavy chain rearrangement is expressed, but can’t be properly tested as it also needs a a light chain to be expressed at the cell surface?
Rearranged heavy chain associates with a surrogate light chain → pre-B cell receptor
- Then if works, light chain rearrangement is induced
Surrogate light chain components = VpreB + lambda5
*Signal through the surrogate is not antigen specific (cross-linked)
What are the different B cell developmental stages?
Stem cell → Germline, not Ig
Early pro-B cell → D-J heavy chain rearrangement
Late pro-B cell → V-DJ heavy chain rearrangement
Large pre-B cell → VDJ heavy chain rearranged + test heavy chain with surrogate light chain
- mu chain transiently at surface as part of heavy chain test
Small pre-B cell → V-J light chain rearrangement
- intracellular mu chain
Immature B cell → VDJ heavy chain + VJ light chain rearranges + IgM expressed on the surface
When during development are RAG-1 and RAG-2 expressed?
*RAG = lymphoid specific recombinase
During heavy chain rearrangement → pro-B cell
During light chains rearrangement →pre B cell
What mechanisms are involved in central tolerance of B cells?
- Location
- 2 mechanisms
What surface receptor needs to be expressed?
Antigen receptor sIgM is tested for reactivity to self-antigens
Central tolerance arises in central lymphoid organ (BM)
- Receptor editing → deleting self-reactive light chain and replacing it with another sequence
- can be repeated until no additional light chain V and J gene segment available - Clonal deletion → immature B cell undergoes apoptosis
- Only non-autoreactive B cells leave the BM
- Requires expression of sphingosine-1 phosphate receptor 1 (S1PR1) which binds to S1P (high blood concentration) and promotes migration into the vasculatures
What does peripheral tolerance imply for B cells?
B cells not functionally mature which encounter cross- linking antigen will undergo apoptosis
What stage of development are B cells at when they emerge from the bone marrow?
When immature B cells emerge from the bone marrow into periphery, they are transitional B cells: sIgM (no IgD)
2 transitional B cells stages:
T1 (sIgM+ sIgD- CD21- CD23-) short-lived → they enter the spleen → T2 (sIgM+ sIgDlo- CD21+ CD23+)
In the spleen, most of them become follicular B cells, but some also migrate splenic marginal zone
What is the difference between Follicular B cells and B cells in the marginal zone?
(In the spleen)
Both are considered mature B cells
Follicular B cells → sIgMlo sIgD++ CD21+ CD23+
MZ B cells → sIgM++ sIgDlo CD21+ CD23- CD1+
- Specific properties to be activated quickly by cytokine signals (fast responders)
- Around the B follicle
What are 3 ways by which antibodies contribute to immunity?
- Neutralization
- prevent pathogen entry into cells
- prevent action of toxins - Complement activation
- trigger complement by activating C1 and directly kill bacterial cells through membrane attack complex - Opsonization
- Fc region can bind FcRs helping phagocytes to uptake and ingest pathogen
**Antibody isotype (constant region different) impacts function
What are 2 type of B cell activations?
T-dependent response
T-independent response
What is the T-dependent response leading to B cell activation?
T-dependent response requires two distinct signals:
- B cell receptor cross-linking
- Interaction with an activated CD4 T cell
- B cell internalizes the Antigen it has recognized
- Digests it and presents it on MHC-II to CD4+ helper T cells
- TCR recognize the peptide for which it is specific + costimulation through CD40/CD40L
*CD40 on the B cell and ligand on the T cell
*BOTH B and T cells (bone marrow and thymus cells) are required for specific antigen immune response
A T-dependent response leading to B cell activation is a process where a B cell, after encountering an antigen, presents it to a helper T cell (CD4+) which then becomes activated and releases cytokines, signaling the B cell to proliferate and differentiate into antibody-producing plasma cells; essentially, the B cell needs the “help” of a T cell to fully activate and generate a strong immune response against an antigen.
- Primarily in the spleen or lymph nodes where B and T cells encounter
In what location does the T cell-dependent activation of B cell occur?
B cell activation is a highly dynamic process in space, guided by chemokines
Day 0:
1. B cells encounter antigen in B cell follicle
2. T cells are activated in the T cell zone
Day 1:
B and TH cells come together at the boundary of T cell zone and B cells follicle (interfollicular region)
*Chemokine driven process
Day 3-5:
- Some fully activated B cells downregulate CCR7 and express EBI2 (ligand: oxysterol)
- Move towards subcapsular sinus (lymph node) or bridning channels (spleen)
- Differentiate into cluster of activated B cells → Primary Foci
- Become plasmablasts that proliferate extensively and secrete large quantities of antibodies (IgM)
→ first phase of humoral response
What chemokines ensure localization of T cells in the T cell zone and of naive B cell in the follicle before encounter?
What expression leads to encounter of B and T cells for B cell activation?
T cell localization in T cell zone → CCR7 interaction with CCL21/CCL19
Naive B cell localization in B cell follicle → CXCR5 interaction with CXCL13
For activation:
- B cell antigen encounter leads to CCR7 expression which enables repositioning to interfollicular region
- TH cell activation can lead to CXCR5 expression and thus migration to interfollicular region
Migration to the interfollicular zone allows for encounter of 2 very rare antigen-specific cells
- If the B cell doesn’t get help from the T cell, it becomes anergic
What is tested to know if someone if in the early days of a humoral response?
Levels of IgM specific to that antigen
What are 2 options or the fully activated B cells that have encountered the T helper cell?
OPTION 1:
Some downregulates CCR7 and express EBI2 (ligand: oxysterol) → move toward the subcapsular sinus (lymph node) or bridging channels (spleen)
They differentiate into clusters of activated B cells: primary foci
→ become PLASMABLASTS that proliferate extensively and secrete large quantities of antibodies (IgM)
*1st phase of humoral response
OPTION 2:
Other fully activated B cells move back into the B cell follicle (downregulation of EBI2) where they expand to form the germinal center (GC)
*2nd phase of humoral immune response
- They generate higher affinity antibodies
- Produce class-swtiched Abs (class switch recombination)
- Leads to differentiation of memory B cells
What happens to T-dependent activated B cells that downregulate EBI2?
Move back into the B cell follicle (downregulation of EBI2) where they expand to form the germinal center (GC)
*2nd phase of humoral immune response
- They generate higher affinity antibodies
- Produce class-swtiched Abs (class switch recombination)
- Leads to differentiation of memory B cells
What is a germinal center? (structure)
B cell follicles in which there is a germinal center: secondary lymphoid follicle
- mainly made up of proliferating B cells + 10% TFH cells
Mantel zone = resting B cells pushed to the edges (not specific for that antigen)
Dark zone = CXCL12+; centroblasts that express both CXCR4 and CXCR5
- The dark zone expresses CXCL12 which is a ligand for CXCR4
Light zone = CXCL13+; less densely packed with centrocytes that have downregulated CXCR4 and express CXCR5 only
What are the receptor signatures of B cells in the light vs dark zones of the germinal center?
GC B cells are high in Bcl6 expression: master transcriptional regulator
light zone B cell (centrocytes): CXCR4lo CD83hi CD86hi
dark zone B cell (centroblasts): CXCR4hi CD83lo CD86lo
*B cells move between light and dark zones
What allows for iterative round of somatic hypermutations and selection?
The movement between the dark and light zones
What are somatic hypermutations?
Movement of B cells between the light and dark zones allows iterative rounds of somatic hypermutation and selection
- V regions (particularly CDRs) accumulate mutations in every division that are passed down to daughter cells
- Most mutations lead to worse Ag binding or frameshifts → B cell apoptosis (mopped up by macrophages in GC)
- Some mutations improve Ag binding leading to affinity maturation
*That is why B cells from the germinal center (downregulated EBI2) eventually form higher affinity Ab, but slower process
What is the concept of affinity maturation?
*Occurs after T-dependent B cell maturation, with cells that downregulate EBI2 and form germinal centers
Back and forth movement between dark and light zones:
Dark Zone → somatic hypermutations + division of B cells
Light Zone → B cells obtain Ag from FDCs → presents to TFH cells to obtain help → If obtain help are ‘selected’ and survive → go back into DZ
*If can’t bind Ag or obtain help, they die
Cyclic reentry model → continual refinement of B cell affinities for antigen during the germinal center response
*Stops when the antigen is cleared as it requires the antigen
At the DNA level, how does affinity maturation occur?
Somatic Hypermutations are carried out by the enzyme AID: activation-induced cytidine deaminase
→ Deaminates cytidine in single-stranded DNA (partially open DNA) → Initiates mismatch repair and/or base excision repair with an error-prone DNA polymerase which inserts mutations
There are mutational hot spots in the V regions: directed by DNA sequence motifs that AID favours
What is the effects of loss-of-function mutations in AID?
Lack both somatic hypermutation and class switching: can only make lower affinity IgM antibodies
*No other classes than IgM
At the DNA level, how does class switch recombination occur?
Switching from IgM/D to other antibody isotypes only occurs following B cell activation with the help of AID which mediates class switch recombination
- AID deaminates cytidines in the S regions → can generate a dsDNA break in 2 switch regions (repetitive DNA sequences)
- These are joined, looping out DNA in between
Transcription is required → DNA has to be open
Ex: Cμ to Ce switch
What directs AID to prefer some class-switches over others?
Different cytokines direct B cells to undergo CSR to different heavy chain classes → define which regions are accessible to AID
IL-4 → IgG1, IgE
IFN-y → IgG3, IgG2a
TGF-b → IgG2b, IgA
IL-21 → IgG3, IgG1, IgA
IL-5 → IgG1, IgA
What part of the germinal center can we find Helper T cells?
Light zone
What are 2 outcomes of the germinal center?
Plasmablasts → lots will stay in the lymph nodes, Ab producing machines
- 10-20% will migrate to the BM and survive for long
Memory B cells precursor cells
- quiescent cells with higher affinity
What are Long-lived plasma B cells?
Plasmablasts that migrate to the bone marrow and stay for long term immunity against specific pathogen
*Not memory B cells
Was found because plasma serum Ab levels stay elevated, but Abs have a very short half-life
- Even after irradiation which depletes memory B cells
What is the T-independent B cell activation?
T-INdependent responses lead to predominantly low-affinity antibody responses and only IgM production
- No memory B cells
When encounter antigens with repetitive structures that can directly cross-link multiple B cell receptors on the B cell surface, triggering activation signals without the need for antigen presentation to a T cell
- Usually involves Ag that has polyvalent, repeating determinants and is found across many microbes
- Cross-linkage (binds to multiple BCRs) of BCRs by repetitive nonprotein antigen epitopes
2 types:
T1 → B cell mitogens (ex: LPS)
T2 → repetitive structures (repetitive polysaccharides)
*more similar to an innate response
What is the different between both types of T-independent B cell activations?
T1 → B cell mitogens:
- can induce B cell division regardless of Ag specificity = polyclonal activation
Ex: bacterial DNA (CpG) and LPS
- Activate TLRs on B cells
T2 → repetitive structures:
- can only activate mature B cells: splenic
MZ B cells & CD5+ B1-B cells
Ex: bacterial capsular polysaccharides, polymerized flagellin
