17-18. B cell activation and antibody production

where are B cells located in the secondary lymphoid organs?

• lymph node: outer cortex → arranged in follicles

• spleen: follicles in white pulp

what is clonal selection of B cells?

selection and activation of specific B cells based on antigen recognition

what types of antigens can B cells bind?

• native protein antigen

• polysaccharides

• lipids

• nucleic acids

• chemicals

(much broader recognition than T cells)

what were nude (athymic) mice used to study?

• defective thymus → lack T cells

• used to determine role of T cells

what are thymus-dependent antigens? what type of antigen is thymus-dependent?

1. antigens dependent upon helper T cells to induce antibody production

   • without T cells, B cells are not activated

2. proteins

what are thymus-independent antigens? what types of antigens are thymus-independent?

1. antigens that do NOT need helper T cells to induce antibody production

2. polysaccharides, lipids (non-protein antigens)

what do follicular B cells respond to? what do they produce?

1. recognize T-dependent antigens

   • protein antigen + helper T cell

2. produce isotype-switched, high-affinity antibodies and long-lived plasma cells

• broad antigen receptor diversity

where are follicular B cells found?

spleen, other lymphoid organs (ex. lymph nodes)

what types of B cells recognize T-independent antigens (polysaccharides, lipids)?

• marginal zone B cells → important for bloodborne antigens

• B-1 B cells

• limited antigen receptor diversity

where are marginal zone B cells found?

spleen (major) and other lymphoid organs

where are B-1 B cells found?

mucosal tissues, peritoneal cavity

what do T-independent antigen-recognizing B cells produce?

• mainly IgM and short-lived plasma cells

• not producing long-lived plasma cells or immunological memory

• not switch Ab isotype

how does a secondary antibody response differ from a primary antibody response?

• 2° is a stronger, more accelerated (quicker) response

• presence of memory cells → higher starting levels of antibody

• switch Ab production from IgM to IgG (higher affinity for antigen)

where are memory B cells found? where are long-lived plasma cells found?

1. usually found in secondary lymphoid tissues

2. most long-lived plasma cells are found in bone marrow

what is required for B cell activation?

• 2 signal-model of activation → simultaneous engagement of:

  1. B cell receptor complex

  2. co-receptor complex (or TLR)

• need inflammation/danger signal with innate immune activation to activate naive B cells

what are the components of the B cell receptor complex?

• B cell receptor (IgM) → interacts with antigens

• Igα & Igβ → associated proteins that provide signaling

what are the components of the co-receptor complex?

• CR2

• CD19

• CD81

how does complement activation contribute to B cell activation?

C3b (produced by complement) bound to a microbe binds to CR2 (part of co-receptor complex)

what provides the second signal if complement is not activated?

TLR recognizing PAMPs (still need danger signal from innate immunity)

what are the results of B cell activation?

1. biochemical signal transduction

2. endocytosis/internalization of antigen → antigen-specific APC

   • only endocytose antigen

effects of B cell receptor-mediated signal transduction?

• increased survival, proliferation

• interaction with helper T cells (present antigens via MHC II)

• increased responsiveness to cytokines

• migration from follicle to T cell zone (increased expression of CCR7)

• antibody secretion

what is CCR7?

• receptor that is receptive to chemokines in T cell zone — CCL19/21

• expression increased in activated B cells

• expression downregulated in activated T cells

linked recognition

• T-dependent antigen must contain both B and T cell epitopes

• epitopes must be on the same antigen, though they are not necessarily the same epitope

• very important for vaccine development

how do B cells present antigens to helper T cells?

1. B cell recognition of native protein antigen

2. receptor-mediated endocytosis of antigen

3. antigen processing and presentation (via MHC II pathway)

4. (CD4+) T cell recognition of antigen

how do dendritic cells contribute to T-B cell interaction?

DC activates naive helper T cells → activated T cell downregulates CCR7 → starts to migrate out of T cell zone, toward B cell zone at the same time B cell is moving toward T zone

helper T-cell dependent activation of B lymphocytes

• effector T cells produce cytokines that further amplify B cell response and direct isotype switching

• TCR-MHC II signaling → T cell produces CD40 ligand & B cell expresses CD40 molecule

  • CD40 signaling is critical → modifies B cell transcription

what are consequences of CD40 or CD40L deficiency?

• no secondary response

• hyper-IgM syndrome (cannot switch isotype)

• immunocompromised

  • no macrophages activation (also needs CD40 signaling)

  • poor antibody response

what happens to B cells after interacting with T cells?

• some differentiate into short-lived plasma cells → produce Abs quickly to control infection

• some head back to follicles and start germinal center reaction

how do extrafollicular helper T cells become follicular helper cells (T_fh)?

after interacting with B cells, some helper T cells start expressing CXCR5, which is attracted to CXCL13 in the B cell zone → go to follicles with B cells and form germinal centers

what 3 processes are involved in the germinal center reaction?

• affinity maturation

  • somatic hypermutation of Ig genes

• isotype switching (also occurs out of germinal centers)

• generation of memory B cells & long lived plasma cells

what process(es) occur in the dark zone of the germinal center?

high levels of B cell proliferation

what process(es) occur in the light zone of the germinal center?

• somatic hypermutation

• affinity maturation

• isotype switching

affinity maturation

process that leads to increased affinity of antibodies for a particular antigen

• result of somatic hypermutation of Ig genes, followed by selective survival of B cells producing antibodies with the highest affinity for the antigen

how is activation-induced deaminase (AID) induced? what process(es) is it involved in?

1. induced by CD40 signals

2. key role in somatic hypermutation (and isotype switching)

during somatic hypermutation, in what part of the antibody do the mutations accumulate?

in or near the CDRs (variable region)

how can we experimentally show that the mutations increase affinity?

• measure Kd (dissociation constant) → how well Ag & Ab stick together (how easy it is to separate once bound)

  • ↓ Kd = ↑ affinity

• measure Kd of antibodies during primary and subsequent immune responses → decreased Kd as mutations accumulate → increasing affinity

what is the function of activation-induced deaminase (AID)? what is the significance of this reaction for somatic hypermutation?

1. converts dC (deoxycytidine) → dU (deoxyuridine)

2. dU is an unnatural nucleotide in DNA, so it is removed and replaced with dT (deoxythymidine)

C → T change in DNA sequence is an error prone mechanism, leading to even more mutations

what two survival signals do B cells need in order to not undergo apoptosis after somatic hypermutation?

1. antigen recognition

2. CD40 signaling from helper T cells (CD40/CD40L)

where is antigen found in the germinal center?

localized on follicular dendritic cells (FDCs)

• Fc receptors → Ag-Ab complexes

• C3b/C3d (complement) receptors → C’-Ag-Ab complexes

is antigen freely available to all B cells in the germinal center?

• limited amount of antigen in germinal center

  • competition for antigen becomes more intense as immune response goes on → selecting higher and higher affinities

• compete with other antibodies (including Ab on FDCs) to obtain antigens from FDCs

what component(s) of the antibody contribute(s) to isotype switching?

heavy chain only! (light chain does not contribute to isotype)

how is isotype switching induced?

cytokines produced by CD4+ helper T cells (or mucosal tissues)

IgG switching is induced by what cytokine?

INF-γ (produced by T_H1)

IgE switching is induced by what cytokine?

IL-4 (produced by T_H2)

IgA switching is induced by what cytokine? where is this cytokine produced?

1. TGF-β (transforming growth factor beta) (& others not emphasized)

2. produced in the mucosal tissues (MALT), which are constantly exposed to antigens → need to suppress immune response

what is a switch region?

region of DNA located at the 5’ end of each constant region gene segment

• become transcriptionally active depend on cytokines present

what is the mechanism of switch recombination?

• switch region at a particular heavy chain locus becomes transcriptionally active (cytokines) → targeted by AID

• AID converts dC → dU, which is then replaced with dT; creates ds DNA breaks

• intervening DNA is deleted; new heavy chain is next to VDJ region

does switch recombination affect antigen specificity?

• NO → VDJ region unchanged during this process

• switch recombination specializes the effector functions (F_c region)

what is a polyvalent antigen? what types of antigens tend to be polyvalent?

1. multiple copies of the same epitope on the same antigen

2. thymus-independent antigens

how do polyvalent antigens interact with B cells?

induce maximal crosslinking of membrane Ig on B cells, without a need for T cell help

how does the overall antibody response differ for T-independent antigens?

• low level switching to IgG

• little to no affinity maturation

• produces short-lived plasma cells

• no memory B cells produced

**no immunological memory produced

how can we induce a very strong antibody response to polysaccharides?

• conjugate a t-independent antigen to a t-dependent antigen → stimulate isotype-switched, high affinity antibody responses to the t-independent antigen

• B cells can then present processed antigen to CD4+ T cells via MHCII to do T cell dependent processes

(create a T-dependent response from a T-independent antigen)