Lecture 24: Humoral Immunity I: Activation of B Cells

• Humoral Immunity I involves the activation of B cells.

Concepts of B Cell Activation

• B cell activation leads to antibody production, starting with BCR crosslinking and B cell co-receptor ligation
• Activation of Src kinases (Lyn, Fyn, Blk) and Syk result in clonal expansion
• There is increased Ag presentation molecule and cytokine expression
• There are altered chemokine responsiveness

Follicular Dendritic Cells (FDCs)

• Follicular DC (FDC) have extensive dendrite surface area and are non-phagocytic
• CR1 and CR2 are used to tether Ags for naive B cells to screen and centrocytes to undergo affinity maturation
• FDCs produce IL-6, IL-15, and BAFF, promoting centroblast proliferation.

Activated B Cells

• Activated B cells initially expand in the medulla and then migrate to the follicle
• They undergo differentiation from centroblasts to centrocytes to GC B cells to plasma cells (SLPC, LLPC, and memory cells)
• CD40-CD40L and cytokine signaling (IL-2, IL-4, IL-5, IL-6, IL-10, IL-21) are involved

Learning Objectives

• Igα and Igβ play a role in BCR signaling
• Activated Syk and the B cell co-receptor components have defined actions
• B cell co-receptor proteins CR2, CD19, and CD81 enhance B cell sensitivity to Ag
• Lacking functional expression of B cell co-receptor proteins affects patient outcomes

FDC Features

• FDCs capture, preserve, and display Ag to naive B cells using specific features

Cell Types

• Centroblasts, centrocytes, germinal center (GC), GC B cells, plasma cells (PC), and somatic hypermutation are various cell types

FDC Mechanism

• FDC selects high affinity BCR among centrocytes for affinity maturation

Roles of TFH and FDC

• TFH cells and FDC contribute to B cell differentiation into Ig-producing PC, including cell-cell contact and cytokines
• AID is stimulated for somatic hypermutation and affinity maturation
• Cytokines contribute to B cell activation for memory B cell formation
• SLPC, LLPC, and memory cells have recognized differences

Overview of B-Lymphocyte Activation

• Mature, naive B lymphocytes, markers, migration involved
• B lymphocyte activation stages: First and second signals, cellular responses, differentiation
• Terminal effector cells and molecules are produced

Humoral Immune Response

• The production of immunoglobulins or antibodies is the sole function of this response
• Antibodies present in blood, lymph and extracellular fluids
• Antibodies bind to protein or carbohydrate epitopes on the surface of a microbe or infected host cell
• Five classes of antibodies: IgM, IgD, IgA, IgE, IgG
• Differences in antibodies enable antibody-coated pathogens to interact with specific types of effector cells

FDC Ag Capture

• FDCs capture, concentrate, and slowly release Ags
• Antigen arrives from tissues via afferent lymphatics
• Small antigens delivered to follicles via conduits
• Larger antigens are taken up by macrophages in subcapsular sinus and by dendritic cells in medulla

Signal 1-BCR Signaling

• Initial BCR contact with an Ag epitope induces movement of the BCR to the site of this contact to increase Ag-BCR encounter
• A cluster of BCRs engages Ag, and multimeric Ags containing repeats of the epitope results in BCR crosslink
• Signaling molecules are Igα and Igβ, which contain cytoplasmic tails with ITAMs
• Activation of the BCR triggers phosphorylation of ITAMs by Src kinases (Lyn, Fyn, Blk)
• Syk (spleen tyrosine kinase) binds to the P-ITAMs and acts similar to ZAP-70 in T cells
• PLC and Ras/Rac pathways are activated
• Transcription factors bind to accessible promoters in the B cell DNA and induce an activation signal

Thymus Independent (TI) and Thymus Dependent (TD)

• B cell activation pathways include TI-1 antigen, TD antigen, CD40/CD40L
• Mitogen and Contact Dependent are two different modes

Signal 2-B Cell Activation

• B cell co-receptor signaling is required for B cell activation
• B cell co-receptor components:
  • CR2 (CD21) recognizes iC3b and C3d derivatives of C3b fragments
  • CD19 is the signaling chain of the co-receptor
  • CD81 binds CD19 and aggregates the co-receptor and BCR
• Src kinases like Lyn phosphorylate the cytoplasmic tail of CD19
• P-CD19 enhances signaling pathways associated with BCR crosslinking
• Co-receptor activation can increase BCR signaling events up to 10,000 fold
• Patients lacking functional B cell co-receptor components CD19 or CD81, have low levels of serum antibodies and limited isotype switching

FDC Store

• FDC have an extensive dendrite surface area to display large quantities of Ags and lack phagocytic activity
• Antigens captured by FDC are preserved along the cell surface for months/years
• During complement activation, C3b (CR1 ligand) attaches to pathogen/Ag and may degrade to C3d (CR2 ligand)
• FDC expresses both CR1 and CR2 to capture Ags coming into the LN or delivered by DC
• FDC tethers the Ags on CR1 and CR2 to be screened by naive B cells
• Sinus macrophages aid FDC by similarly displaying Ags on CR2

Clonal Expansion

• Activation of B lymphocytes causes proliferation and increased anti-apoptotic factors

Ag Presentation

• Activation of B lymphocytes causes increased MHC I + MHC II and B7 (CD80/CD86)

Cytokine Receptors

• Activation of B lymphocytes leads to increased IL-2R, IL-4R, IL-5R, IL-21R

Chemokine Receptors

• Activation of B lymphocytes leads to increased CCR7+ and decreased CXCR5

Interfollicular Zone

• Pre-TFH cells reside here.
• These cells can be either CCR7+ naive Th cells or CXCR5+ naive Th cells (pre-TFH)

DC Engagement

• DC engagement activates Pre-TFH cells
• Proliferation, anti-apoptotic factors, CD40 ligand, IL-2, IL-4, IL-5 (Th2-like) are induced
• Other cytokines are dictated by the signal 3 cytokine

Pre-TFH Activation

• Activated Pre-TFH cells engage activated B cells
• Interfollicular Zone and B cell zone are distinct areas

Differentiated TFH

• Pre-TFH differentiate into TFH cells
• CD40 ligand and CXCR5 expression are stabilized
• Production of IL-10, IL-21 and the IL-21 receptor (IL-21R) are induced

AID and B Cell Proliferation

• TFH cells induce B cell proliferation and AID
• Defective CD40L expression = X-linked hyper-IgM syndrome

Clonal Expansion Focus

• Primary focus of B cell clonal expansion occurs in the Medulla
• Activated B cells and TFH cells migrate to the medulla and divide
• Division is promoted by IL-2, IL-4, IL-5
• B cells differentiate into Plasma cells (PC) secreting the initial IgM detected in the blood
• TFH produced IL-5, IL-6, and IL-10, promoting differentiation of B lymphoblasts into PC

Secondary Focus

• Secondary focus of B cell clonal expansion occurs in primary and secondary Follicle
• Some of the B lymphoblasts leave the medulla still attached to their TFH cells and traffic back to the follicle
• B lymphoblasts begin to rapidly proliferate creating a secondary follicle

Germinal Center Focus

• Secondary focus of B cell clonal expansion occurs in the Germinal Center
• Rapidly dividing B cells are known as centroblasts
• TFH cells also undergo some proliferation but not to the extent of the centroblasts
• Rapidly proliferating centroblasts push out naïve B cells and many TFH cells leading to formation of a germinal center
• Germinal centers represent B cell responses to a specific Ag

Centroblasts

• TFH cytokines (IL-2, IL-4, IL-5) initiate proliferation, and FDC cytokines [IL-6, IL-15, BAFF (B cell activating factor)] sustain proliferation
• Surface Ig (ie. BCR), or slg, is lost

Somatic Hypermutations

• Interactions of centroblasts with TFH cells in the follicle/germinal center induce AID
• Mutations occur in the Variable (V) region of both Heavy and Light Ig chains
• There is a high rate of mutation (hypermutation)
• AID action results in Igs with varying affinities for Ag
• Centrocytes are generated, indicating completed somatic hypermutation and expression of a BCR (slg)
• AID = activation-induced cytosine deaminase, acts by deaminating cytosines in DNA, converting them into uracils, DNA repair replaces the erroneous bases and generates mutation.

Centroblasts + BCR

• Centroblasts + BCR = Centrocytes
• Centrocytes express a BCR
• They engage FDC for affinity maturation

Affinity Maturation

• Mediated by FDC
• CD21 molecule serves as a receptor for cleavage fragments of the C3 component of complement-iC3b, C3dg, and C3d
• Mice deficient in CD21 also have impaired responses to T cell-dependent and T cell-independent antigens and a defect in germinal center formation

Centrocytes with High Affinity

• Centrocytes require BCR and CR signaling to survive and remain attached to FDC

Surviving Centrocytes

• Surviving centrocytes (GC B cells) engage TFH cells and differentiate into plasma cells
• IL-10 and IL-21 signaling promotes proliferation and differentiation of GC B cells into plasma cells
• GC B cells then undergo isotype switching
• Centrocytes undergo apoptosis if they fail to interact with TFH and FDC, and are phagocytosed by tingible body macrophages
• Tingible body macrophage = so named because these macrophages contain many phagocytized apoptotic cells (body) in various stages of degradation

Short-Lived Plasma Cells

• Short-lived plasma cells are generated first and produce IgM
• IgM response is released in the lymphoid medulla several days after Ag encounter
• If IgM detected in serum within two days, source is marginal zone B cells
• Short-lived plasma undergo apoptosis while long-lived are generated

Long-Lived Plasma Cells

• Long-lived plasma cells are found in inflamed/infected tissues.
  • They secrete high levels of Ig at Ag source site and undergo apoptosis as Ag decreases
• Long-lived plasma cells are found at the Bone marrow
  • They reside associated with bone marrow stromal cell up to the lifetime of the person
  • They don't migrate. They are memory plasma cells and constantly secrete low levels of Ig (Ag exposure maintains levels)

Memory B Cells

• Memory B cells remain in blood circulation or reside transiently in lymphoid organs
• Memory B cells do NOT produce Ig but express a BCR
• Memory B cells serve as a reservoir for memory B cell responses

TFH Cytokine

• A switch of TFH cytokine induces memory B cell formation.