Lymphocyte Receptor Signaling, B cell Development

transmembrane receptor signaling

transmembrane receptors convert extracellular signals into intracellular biochemical events

different sorts of RTKs - some have a kinase domain as an intrinsic part of the receptor molecule while others have a closely noncovalently associated but still separate kinase

intracellular signal propagation is mediated by large multiprotein signaling complexes

• adaptor proteins and kinases - classical signal transducers, engage in conformational changes that allow for differential interactions

• scaffolds hold related proteins together to facilitate signal handoff

antigen signal → receptor → transducers → changes in cell activity → response

T and B activation

naive B/T cells have a large nucleus, very condensed DNA, not much gene expression, not much cytoplasm

full activation of naive T and B cells requires 2 signals:

1. antigen receptor (BCR/TVR) and co-receptor (CD4/CD8)

2. co-stimulatory receptor (CD28 on T cells and CD40 on B cells)

TCR receptor complex

the TCR us a multi-subunit signaling complex

• a and B chains bind antigen presented on MHC

• epsilon, delta, and zeta invariant chains transduce the signal

  • epsilon and delta chains in pairs (sometimes also with gamma chain) on either side of the aB TCR

  • pair of thin zeta chains connecting to the transmembrane domain of the aB TCR

  • called invariant chains because they are the same across T cells

• epsilon, delta, and zeta chains have ITAMs on their intracellular regions - immunoreceptor tyrosine-based activation domains → phospho-tyrosines which activate protein scaffolding

• once a signal has been received, the ITAMs become phosphorylated, allowing them to recruit scaffolding proteins like ZAP-70

  • zeta chain associated protien

  • activated by ITAM phosphorylation

  • zap-70 is a key kinase that propagates T cell activation cascades

  • deficiency of zap-70 does not prevent signal reception but prevents signal transduction - lack of T cell activation, severe immunodeficiency - also prevent T cell development since signaling is key there too

co-receptors

co-receptors assist in signal transduction

examples are CD8 and CD4

stabilize TCR-MHC interaction and enhance ITAM phos

enhance phosphorylation of ITAMs

Lck (lick, to remember) - another key kinase, associates with co-receptors

• associated with CD4 and 8

• phosphorylates TCR ITAMS to then activate zap-70

multiple pathway activation

upon activation, zap-70 phosphorylates LAT and SLP-76, initiating four downstream signaling modules

simultaneous initiation of multiple pathways to control multiple aspects of cell behavior

• metabolic - increased

• gene expression - TF activation, lots of genes

• cytoskeleton - actin reorganization, rearranged to become motile

• migration - integrin conformational change (activate, so cells can grab on to receptors in vessels at infection sites and make it to the infected tissue

immune synapse

intimate junction to narrow activation to target cell

TCR-MHC interaction initiates an actin-dependent immunological synapse between T cell and APC/target cell

the immune synapse is the interface formed between T cells and other antigen-presenting cells (APCs) or target cells

allows for direct communication and sustained signaling

e.g. killer T and tumor cell synapse

MHC-TCR+CD interactions as well as the release of secretory contents from vesicles within the main synapse, integrin binding (LFA-1, ICAM-1) at the edge of the synapse (check from previous knowt) - stabilizing interactions

BCR complex

main receptor associated with invariant chains that carry out the signaling function of the receptor

membrane bound BCR is also a multisubunit signaling complex

• heavy and light Ig chains bind antigen

• Ig a and B invariant chains transduce the signal - connect to base of the F region on the bound antibody

antibody transmembrane domain removed via alternative splicing

receptors involved here are the IgM or D ones

BCR signal transduction

binding of antigen to BVR induces intracellular signaling

overall process very similar to TCR but with some different proteins

• Syk instead of ZAP

• don’t need to remember signaling details

• some components are B cell specific, others are shared between BCR and TCR pathways

• Btk - Bruton’s tyrosine kinase - drug target to disable pathogenic B cells in autoimmune disease

ITAMs get phosphorylated and recruit downstream kinases and scaffolds

signal propagates

similar outcomes as in T cells - gene expression, metabolism, migration

signals and stimulation

for naive B and T cells, TCR/BCR signaling alone is insufficient for proper activation

• need secondary signal (co-stimulation) via CD28 (T cells) or CD40 (B cells)

• also exist inhibitory receptors on lymphocytes to prevent activation

remember, co-receptors are not co-stimulators

• co-receptor - recognized MHC-TCR

• co-stimulator - verifies foreign antigens

CD28 for naive T cell activation

B7.1 and 2 are CD28 ligands expressed on specialized antigen presenting cells (APC)

referred to as CD80 and 86, expressed by activated APCs

greatly enhances T cell activation

w/o the costimulator, the initial TCR signal does down before the T cell can properly mature

costim ligands only produced by antigen cells

costim gives T cell additional context, informing it that it is binding something foreign

DCs and their central role

key antigen presenting cells that are especially good at activating naive T cells

• express high levels of MHC I and II

• become more efficient at activation in the presence of danger signals (PRR activation) - upregulate CD80 and 86 presentation

• PRR signaling also lets them know that they are infected

CD40 and B cell activation

activation of naive B cells via CD40 done using CD40L (ligand) on CD4 T cells (helper Ts)

activates NFkB pathway

inhibition by interfering with co-stimulation

inhibitory receptors like CTLA-4 block co-stimulation via CD28 by competing for the CD28 ligands

other types of inhibitors send negative signals to the T cell to prevent activation

theraputics - antibodies blocking CTLA-4 are used in cancer immunotherapy to boost weak anti-tumor T cell responses - immune checkpoint blockade

inhibitory receptors have ITIMs - immunoreceptor tyrosine-based inhibitory motifs

they recruit phosphatases that reverse the kinase cascades from ITAMs, preventing activation

PD-1 is an example of an inhibitory signal

general B cell development stages

early (central) development in the bone marrow (T cells go from bone marrow to the thymus)

• VDJ recombination and expression of BCRs

• deletion of self-reactive cells

peripheral development in spleen and lymph nodes

• lymohocytes interact with other cells

• deletion of self-reactive cells

• B cell follicles and survival of foreign antigen-specific B cells

positive and negative selection

positive selection - no functional BCR, cells do not survive

negative selection - BCRs recognize self, cells are killed or inactivated

antigen receptor assembly

antigen receptor genes are assembled by somatic gene rearrangements of incomplete receptor gene segments

deletion/inactivation of self-reactive lymphocytes

lymphocytes activated by antigens give rise to clones of antigen-specific effector cells that mediate adaptive immunity

central and peripheral selection

central - bone marrow or thymus

• initial receptors may be edited to alter (and enhance) specificity

• self antigen receptors are clonally deleted

• other initial antigens proceed

peripheral - lymph nodes and spleen

• costimulatory and foreign antigen signal received - activation and clonal expansion of effector/memory cells

• self-antigen receptor found faces clonal deletion or clonal inactivation

immune tolerance - non-reaction against self antigens, have to cull self-reactive cells

B cell development

central

• hematopoetic stem cell comits to being a B cell

• B-cell precursor rearranges immunoglobin genes (VDJ)

• express functional B cell receptor (IgD or M)

• immature B cell bound to self antigens is removed from repertoire

• leave bone marrow and populate B-cell follicles in spleen, etc.

peripheral

• mature B cell bound to foreign antigen and activated

• activated B cells give rise to plasma and memory cells

• absence of B cell stimulation will eventually result in apoptosis

have to have functional B cell receptor during development as the signals are key to proliferation, coordination, maturation

derivation of lymphocytes

T and B lymphocytes come from hematopoietic stem cells in bone marrow

then mature:

• B in Bone (the B does not stand for bone)

• T in Thymus

both will eventually migrate to populate peripheral lymphoid organs - spleen and lymph nodes

B cell derivation

signals provided by bone marrow stromal cells promote induction of specific TFs that coordinate early B cell development

• stroma - supporting cells and tissue, type of epithelial cell, promote a subset of hematopoetic stem cells and promote maturation and commitment to B cell lineage

beginning of B cell development

protein activity - growth factor receptors, recombinase and tdt nucleotide addition with light chain surrogate, signal transduction at mature stage for activity

• make pre-B receptor after heavy chain recombination but before light chain - standin

• tdt only really being expressed during heavy chain recombination, seems to stop early into light chain recombination

• 2 bursts of RAG 1/2, 1 for the heavy chain and 1 for the light chain

• IL-7R like growth factor for early B cells

• Btk - downstream kinases from B cell receptor, successful B cell signaling stims B cell to finish developing because the signaling is an indication that the cell is properly functioning and this clear to move on.

• Iga and B - ITAMs, constant accesory chains, mediate signal transduction and recruit intracellular machinery

• surrigate light chain (lamda5 and VpreB) - stuff to stand in for the light chain in pre-B receptor - stabilizes not-very-stable pre-receptor

• RAG 1/2, tdt, Btk, and Iga and B are all expressed in a coordinated fashion (expression overlap)

VDJ recombination

begins with VDJ, specifically with the heavy chain (happens several steps ahead of the light chain)

• early stage - activation of just heavy chain recombination

• middle stage- tdt, surrogate light chain, pre-B receptor with Iga and B, can signal at this stage to verify functionality so B cell development can proceed

• late stage - light chain recombination, some tdt but nearly as much as with the heavy chain, fully functional B cell arises

• IgM first expressed then IgD d

more specifics

• first J and D segments join

• V segment added to J and D, then paired with surrogate light chain to create a pre-B receptor - used to test heavy chain signaling

• a productively rearranged immunoglobin gene is immediately expressed as a protein by the developing B cell

light chain V and J then joined

if heavy chain recomb fails to yield a proper chain, can abort the rearrangement of that allele and then it will rearrange the second allele instead

• if both fail, cell engages in apoptosis

• example of a fail would be somethig like tdt adding nucleotides in a way that causes frameshifts

pre-B receptor

tests for successful production of a complete heavy chain and signals for the transition from the pro-B cell to the pre-B cell stage

autosignaling through the pre-B cell receptor confirms the successful heavy chain rearrangement so development can move forward

• don’t need antigen to activate the pre-B receptors - VpreB and lamda5 termini can interact between 2 receptors to stimulate them and initiate next phase of B cell development: light chain rearrangement

• further heavy chain rearrangement is stopped

rescue of light chain rearrangements

B cell randomly picks either kappa or lamda light chain gene while the other gets innactivated

nonproductive light chain rearrangements can be rescued by further rearrangement

many opportunities for sequential rearrangements in the light chain

can also rearrange the lambda gene chain if the kappa fails and vice versa

eventually exclusively kappa or lambda will be expressed

testing immature Bs for autoreactivity (central tolerance)

immature B cells tested before they leave the bone marrow - central tolerance

self-reactive B cells are eliminated

• strong B cell signaling early on is often a bad sign - self-signaling with high affinity

• B cells undergo apoptosis or become anergic (very unresponsive, eventually die if not stimulated)

• in central development, will only be able to recognize and flag cells that are self-reactive to tissues and cells in the bone marrow - need another screening when they reach the periphery

self-reactive B cells can also be rescued by receptor editing (additional VDJ recomb of light chain genes)

self-reactive B cells also have a second chance by banking on light chain allele rearrangements (checking for reactivity is done after heavy chain rearrangement but before light chain)

receptor editing

RAG1/2 still expressed - autoreactive B cells can rearrange light chain to change the BCR

peripheral tolerance

lymphocytes that encounter sufficient quantities of self-antigens for the first time in the periphery are eliminated or inactivated

no receptor editing option at this stage - no second chance

result in mature B cells ready for antigen reception

some self-recognizing B cells do still escape detection but they can be regulated by lack of activation of costimulatory receptors

• need PRRs to upreg costimulatory receptors to verify original signal, will not kill unsuported prey

• T helper also selectively activates when it receives pathogens it knows are foreign - self-reactive Bs and Ts should not be able to activate due to lack of secondary signal

immature cells in the spleen

immature B cells arriving in the spleen turn over rapidly and require cytokines and positive signals through the B cell receptor for maturation and long-term survival

specialized zones for B and T cells in lymphoid organs - lymph nodes

• B cell follicles for B cells

• germinal centers within follicles - areas with a lot of B cell proliferation - causes node swelling

B cell maturation

B cells complete maturation in the follicles in the spleen or lymph nodes

• undergo further selection

• majority will die unless they get BCR stimulation and survival factors

follicular dendritic cells (FDC) are specialized cells that capture and retain antigens for very long periods (months or years)

• not actually related to DCs

• unlike other DCs, they present these antigens to B cells

• FDCs provide foreign antigen and survival factors (BAFF, etc) - capture antigens as complexes and do NOT process them because they aren’t going to be talking to T cells

• follicular B cells are B cells that survive selection and stay - make up the majority of mature be cells

• since they can hang onto antigens for years, they can help stimulate and activate B cells for years - selection for useful Bs and kick out Bs that aren’t reactive to anything they bring

follicular B cells are classic B cells, but there are other subsets

other B cells

will mostly be talking about follicular Bs but there are others:

• innate B cells - can make a lot of receptors really fast, stick on but they don’t have as high affinity as slower but better follicular Bs

• as we age, immune memory becomes more biased