Immunology Unit 3 - Chapter 11 B-Cell Activation, Differentiation, and Memory Generation

Finally, there are temporal differences in the memory response of the two subsets of memory B cells.

. (a) In the presence of high concentrations of serum antibody, high affinity memory cells bearing class switched Ig receptors (swIg+ memory) can respond, but lower affinity IgM+ memory cells are unable to compete for antigen. High affinity class-switched memory cells form plasma cells and new memory cells, but do not re-enter the germinal centers. (b) As serum antibody levels decline, low affinity IgM+ memory cells are able to bind antigen and are stimulated to form IgM+ and swIg+ plasma cells, as well as new IgM+ and swIg+ memory cells. In addition, these IgM+ memory cells are able to enter the germinal center, where they can undergo somatic hypermutation, creating high affinity B cells. This decreases the tendency of the memory responses to be exhausted by frequent encounters with the same, or cross-reacting antigens. It further allows the lower affinity, IgM-bearing memory cells to mutate in response to variants of the original antigen Why waste time making memory cells from the original IgM? So we have a B cell, it gets activated, we have first responders (the IgM), but we know these cells are not the antibodies with the best affinity (which is why we go through the germinal centers to create a better antibody, preform class switching, etc) SO why don't we just keep those cells that we know have a better affinity? why do we make the IgM's into memory cells? Well later in the memory response when all of the swIg+ plasma cell antibodies have done their job and you have less antigen left, the IgM+ memory cells are what binds what left of the antigens. Next, these IgM+ memory cells can then re-enter germinal centers to reperform somatic hypermutation and class switching You see......class-switched memory cells cannot reenter germinal centers and cannot reperform somatic hypermutation and hence is stuck producing that one antibody. BUT IgM+ memory antibodies can rebind antigens and re-enter germinal centers producing a new class-switch higher affinity antibody SO why is this important? For two reasons 1. it's possible we can exhaust our immune response. So we have memory cells that are expressing class-switched antibodies but we only have a limited number of these antibodies. Therefore upon a second challenge by an antigen, these limited numbers of antibodies might not survive the contraction. Therefore the IgM is acting as a fail-safe mechanism. 2. These IgM help increase the antigens we can bind to considering they have a lower binding affinity and hence provide a faster immune response

How does the enzyme AID work?

AID deaminates a deoxycytidine residue, creating a uridine-guanosine (U-G) mismatch. Resolution of this mismatch may be mediated by any one of several pathways: (1)The DNA mismatch may be repaired with high fidelity by proteins of the base excision repair (BER) or mismatch repair (MMR) pathway. (2)The deoxyuridine residue may be interpreted by the DNA replication machinery as if it were a deoxythymidine, resulting in the creation of an A-T pair in place of the original G-C pair in one of the daughter cells. (3)The mismatched uridine may be excised by uridine DNA glycosylase, leaving an abasic site that is filled in by any of the four bases, in a reaction known as short-patch BER, catalyzed by one of a number of error-prone polymerases. (4)Enzymes of the MMR pathway, such as MSH2-MSH6, may recognize the gap and ExoI excises a long stretch of the DNA surrounding the U-G couple. Error-prone polymerases are then recruited to the hypermutable site, and these polymerases can introduce a number of mutations around the original mismatch. Thus, depending on the repair mechanism, a mutation may occur only in the originally altered base or in one or more bases surrounding it. so basically it creates a mismatch and we get a mutation or it is fixed and we do get anything.

So how did we figure out that B cells needed T cell help to generate an antibody response?

Adoptive transfer experiments demonstrated the need for these two cell populations during the generation of antibodies to T-dependent antigens. Early adoptive transfer experiments reconstituted irradiated mice with syngeneic bone marrow cells, thymus-derived cells, or a mixture of bone marrow- and thymus-derived cells. These mice were then challenged with a T-dependent antigen. Only recipient mice reconstituted with both bone marrow- and thymus-derived cells were able to mount an antibody response.

what is long-term humoral immunity?

Although not technically defined as memory cells, a second type of B-cell progeny also provides long-term humoral immunity. Once established in a long-term niche, often the bone marrow, long-lived plasma cells produce antigen-specific antibodies for a very long period after antigen stimulation, apparently without the need for further antigenic stimulation. Around 10 days into an ongoing response, interaction occurs between TFH PD-1 receptor and its ligands on B cells. This initiates the B cell differentiation into plasma cells. Then, changes in chemokine expression allow the cells to home to different body sites and in particular the bone marrow. Those homing to the bone marrow can be very long-lived (CXCL12 and APRIL cytokines help them to persist). As you might expect, the niches occupied by fully differentiated plasma cells differ from those inhabited by developing B cells. So there are two ways to have memory as far as b cells are concerned. 1. b cells become activated --> diff. into plasma cells and create memory. 2. an activated b cell diff. directly into a memory cell. But with regards to plasma cells the can provide long term humeral immunity. these cells are traveling from germinal centers as plasmablast into the bone marrow.

As explained on the previous slide, BCR-mediated endocytosis transports antigen into vesicles, where it is broken down into peptides. These peptides are subsequently loaded onto MHC class II molecules and returned to the B-cell surface.

Antigen engagement results in up-regulation of the expression of CD40, CD80, and CD86 on the B-cell surface. These molecules bind with coreceptors on T cells, further facilitating productive B-cell interactions with cognate T cells. SO now that we have internalized an antigen and went through the exogenous pathway, created peptides which can bind to MHC class II molecules on the surface of APCs there is still an issue of how we find the right T cell. we make this process more likely to occur by placing the two in the same areas and hope for the best. But how does this occur? Next notecard.

How are B cells activated?

As is the case for T cells, antigen binding to the B-cell receptor leads to activation of a signal transduction cascade. Antigen engagement by the BCR induces phosphorylation of tyrosine residues in the ITAMs of Igα and Igβ by Src family kinases. This phosphorylation initiates a response leading to the formation of a cytoplasmic signal-transducing complex called a signalsome. Multiple outcomes are possible following BCR antigen binding, which depend on the strength and duration of antigen binding and are further regulated by interactions between the BCR and other cell receptors, including CD21, CD40, IL-4R, IL-21R, and BAFF-R. In this example, CD21 binds to complement fragment C3d enhancing antigen signaling. SO the BCRs aggregate which activates lyn and syk kinase --> this triggers a lipid phosp. and signaling --> leading to Ca+2 release (so you have tyrosine phos. lipid phos then Ca+2 signaling just as in the case for T cells) BUT the bottom line is the activation of these particular signaling pathways leads to the activation of certain transcription factors (CREB, JUN, EIK-1, Egr-1, and NFAT). BUT ALSO the survival of the b cell - because it has now been activated, proliferation - so it can make an antibody, and differentiation - because now the b cell has to transition from being a b cell with a receptor on its surface to a cell thats expressing a secreted form of the BCR (or antibody). This also leads to the formation of memory cells and germinal centers.

As we saw previously, activated B cells do not just differentiate into plasma cells at primary foci, they also initiate a _______ _________ _________. What are there one or two zones in the germinal center? If more then one what are the functions of each?

As we saw previously, activated B cells do not just differentiate into plasma cells at primary foci, they also initiate a germinal center response. Formation of a germinal center requires interactions between CD40 & CD40L between B/T cells as well as cytokines from follicular dendritic cells (FDCs) and TFH cells to B cell stimulate proliferation. Depending on the nature of the antigen, the size of the GC peaks around 7 to 12 days after antigen stimulation, and GCs normally resolve within 3 to 4 weeks. Anatomically, we can distinguish two zones: •Dark zone — densely packed with proliferating B cells •Light zone — B cells interspersed with a network of FDCs SO germinal centers form inside follicles in the lymph node. They contain 2 zones: a light zones which contains FDCs that are binding to b cells and a dark zone where b cells are proliferating

At the start of a T-dependent B-cell response, the B cell binds antigen via?

At the start of a T-dependent B-cell response, the B cell binds antigen via its Ig receptors (signal 1). Some of the bound antigen is internalized into specialized vesicles, where it is processed and re-expressed in the form of peptides in the antigen-binding groove of MHC class II molecules. In other words, the B cell is acting as a professional antigen presenting cell. Signal 2 is provided by an activated T cell, which binds to the B cell both through its antigen receptor and via a separate interaction between CD40 on the B cell and CD40L (CD154) on the activated TH cell. On binding to the B cell, the T cell releases its activating cytokines (signal 3) directly into the T-cell/B-cell interface. The nature of the response is also affected by cytokines released by other cells in the vicinity of the antigen encounter, as described later in this section. Signal 2 and 3 originate from the helper t cell (that have already been activated)

But how can a B cell, with a receptor that is normally expressed at extremely low frequency within the receptor repertoire, can possibly find and bind to a T cell specific for the same antigen, which is also present at low frequency among all the available T cells?

B cells are highly motile cells, programmed to respond to chemoattractant signals such as the ones listed in the table below. The take home message is that chemokine interactions with their receptors on B cells direct B-cell migration through the lymph node in the absence as well as in the presence of antigen. Furthermore, chemokine receptor expression is temporally modulated during B-cell activation. so via the expression of different chemokine receptors which will make the b cell move around the follicle (b cell zone) in the lymph node the stage of progression varies with the expression of certain chemokine's as well.

So what happens in the germinal centers? what is somatic hypermutation and what zone does is occur in?

B cells in dark zones divide rapidly and undergo somatic hypermutation, a period of extremely high rates of mutations. In light zones, B cells interact with TFH and follicular dendritic cells, and B cells bearing high-affinity, mutated receptors are selected. SO we have our t cell zone. the naive B cell finds its antigen and t helper cells and becomes activated. What happens next? 1. we have the formation of an IgM+ memory cell 2. some of the activated B cells can either becomes plasma cells which are found in the primary focus 3. or they can migrate back into the germinal centers. The cells that end back up in the germinal center are going to undergo a larger number of mutation in their antigen binding region to select for a better antibody (antibodies that are binding with even stronger affinity then the original receptor that was expressed on the cell surface) This process is called somatic hypermutation and at the end of this process we get an activated b cell which expresses an antibody (IgM, IgG, IgE, etc) with a different heavy chain region and which binds to the antigen with higher specificity and higher affinity giving a more robust response.

What type of B cells are t independent? what types of B cells are t dependent?

B-1B cells and marginal B cells are t independent while B-2B cells are T dependent and need a helper t cell for activation

B-cell memory provides a rapid & strong response to secondary infection. But why is that?

B-cell memory provides a rapid & strong response to secondary infection. But why is that? •There are signaling differences (Ca2+-based signaling) between naïve and memory cells that help to account for the ability of memory B cells to enter the cell division cycle more rapidly than naïve cells •Memory B cells also constitutively express higher levels of molecules that mediate signaling interactions with T cells, such as CD40, CD80, and CD86 •Some memory B cells undergo proliferation in response to innate stimuli such as CpG, alone, whereas naïve B cells require concomitant stimulation through the BCR and TLR •Memory and naïve B-cell populations also differ in their requirements for help from cell types other than T cells. Some virus-specific memory B cells have been shown to entirely lose the requirement for T-cell help on restimulation (though this is exceptional) basically the threshold for activation is lower

What is the clonal selection hypothesis?

Before we start looking in more details at the events surrounding the activation of B cells, let's review some fundamental principles and in particular the clonal selection hypothesis: •Immature B lymphocytes bear immunoglobulin (Ig) receptors on their cell surfaces. All receptors on a single B cell have identical specificity for antigen. •On antigen stimulation, the B cell will mature and migrate to the lymphoid organs, where it will replicate. Its clonal descendants will bear the same receptor as the parental B cell and secrete antibodies with an identical specificity for antigen. •At the close of the immune response, more B cells bearing receptors for the stimulating antigen will remain in the host than were present before the antigenic challenge. These memory B cells will then be capable of mounting an enhanced secondary response. •B cells with receptors for self-antigens are deleted during embryonic development.

Let's focus on the formation of germinal centers and their maintenance.

Beginning in the center of B-cell follicles as just a few rapidly dividing B cells, a germinal center can grow to include as many as 10,000 cells in a matter of a few days. GC B cells are highly susceptible to apoptosis, and some of the anti-apoptotic signals that they require to survive are provided by follicular helper T (TFH) cells. B cells also interact closely with FDCs and must do so in order to survive. FDCs hold antigen on their surfaces for long periods of time. This antigen can be found in the form of antigen-antibody complexes, or as antigen covalently bound to complement fragments secured to the FDC surface by FDC complement receptors. B cells interacting with FDC-bound antigen receive survival signals from the cells that help to maintain the integrity of the germinal center structure. So how do we select for an antibody that binds to an antigen with higher affinity? We use a couple of different players. FDCs which can bind to antigens either directly or through Fc receptors (which are antibody receptors) which can bind to the antibody which can then bind to the antigen. We also have player number 2 helper t cells or in other words the Tfh cells which express a t cell receptor and are located in the germinal centers and are specific for the peptide processed for the antigen. and player 3 is the b cell itself. it turns out B cells are prone to apoptosis and they have to constantly receive signals from cytokines to survive. so if they do not receive the signals they die (march 31st 53 min)

what is the role of follicular dendritic cells (FDCs)?

Finally, let's consider of the role of the follicular dendritic cells (FDCs). The dendrites of FDCs are studded with antigen-antibody complexes, retained on the surface of the FDC through interaction either with Fc or with complement receptors. Because of the high surface density of antigen on FDCs, it is postulated that FDCs serve as antigen concentration site for future selection and differentiation (for example during germinal center differentiation). The picture shows you how FDCs kind of jam themselves in there so they can immobilize antigens as they travel through the lymph nodes.

The activation of a B cell by a cognate antigen is followed by a series of events inside the lymph node that will lead to the expression of antibodies that bind the antigen with higher affinity (characteristic of the late primary response) as well as the generation of memory cells. What is affinity maturation?

Following antigen encounter, the first B cells to produce antibodies are the antibody-forming cells (AFCs) in the extrafollicular primary foci of lymph nodes and spleen. In general, foci of AFCs arise around 3 days after immunization. The AFCs initially produce only IgM antibodies, but IgG antibodies can be detected by 5 to 6 days after immunization. These cells provide high concentrations of specific antibodies that can neutralize or opsonize antigen by the first 5 to 6 days of an immune response and thus are the major source of protective humoral immunity early after antigen contact. These antibodies will also help drive affinity maturation. We are first going to look at plasma cells. SO a naive B cell expressing cell surface IgM becomes activated. it can then diff. into plasma cells and start expressing soluble IgMs. these cells are also called AFCs Affinity maturation is the process of selecting for better antibodies and how we switch from producing IgM to IgG

How does affinity testing occur? what happens in the dark zone?

How does affinity testing occur? On entering the dark zone, B cells lose expression of MHC class II molecules, and hence much of the antigen they had acquired originally and processed for presentation to TFH cells is lost. However, on cycling to the light zone, they re-express MHC class II and therefore they must now seek out fresh antigen within the light zone for presentation to TFH cells. Those B cells with higher affinity receptors will have a competitive advantage over their sibling B cells and be able to gather and express higher levels of antigen on their cell surfaces. A light zone B cell with a receptor that can no longer bind antigen will be unable to attract T-cell help and will die by apoptosis. What happens in the dark zone? once a b cell have been selected for survival and goes back into the dark zone it will proliferate. we have somatic hypermutation and then we get a better antibody. BUT upon the b cell coming back into the dark zone an important event happens. It loses the ability to express MHC class II molecules which means there will be no antigen found on the cell surface so when it goes back into the light zone it will have to redo the process of finding an antigen, processing, etc.

Where do B cell become activated? Where do naive B cells encounter antigens?

Let us first study how naïve B cells encounter antigen in the lymph nodes and spleen. Here are the fundamentals: B cells migrate to the lymphoid follicles, where one of two things can occur. The B cell can interact with antigen and become activated. Or, in the absence of immediate antigen stimulation, the B cell recirculates through the blood and lymphatic systems and back to the lymphoid follicles. Recirculating mature B cells have a half-life of approximately 4.5 months. Antigen from tissue spaces drained by the lymphatic system is filtered by regional lymph nodes. In the nodes, the mechanism of B-cell antigen acquisition varies according to the size of the antigen. Some low-molecular-weight antigens (less than 70 kDa or 4 nm hydrodynamic radius) enter the lymph nodes via a leaky network of conduits that are sampled by the follicular B cells. Higher molecular weight antigens are taken up first by Fc or complement receptors on subcapsular sinus macrophages or by similar receptors on B cells, dendritic cells, and circulating macrophages, and subsequently passed on to the B cells. Examples include viruses and bacteria tagged by complement or antigens already bound by antibodies. SCSM: subcapsular sinus macrophages FRCC: fibroblastic reticular cell conduits SO b cells become activated in the lymph nodes. the follicles are where the B cells reside. Antigen size plays into which b cell is activated and what response is given. Small antigens can travel more freely through scs but larger antigens cannot interact directly with the BCR. So instead they might be taken up my macrophages that reside on the scs and be displayed to the BCR instead. The antigen can also be bound by follicular dendritic cells and displayed to B cells.

are all antigens processed the same way?

NO they are processed in two different ways.

What are the two different ways antigens are processed?

Now let's see how bound is processed and re-expressed in the form of peptides in the antigen-binding groove of MHC class II molecules. There are two methods: When a B cell forms a synapse with an antigen-presenting cell, lysosomes move toward the immunological synapse. On reaching the plasma membrane, the lysosomes spill their contents into the synaptic junction, acidifying the junction and allowing their proteolytic enzymes to cleave the bonds between the antigen and the antigen-presenting cell. The B cell internalizes the proteolytically generated antigen in complex with the BCR into the endosomal pathway and antigen presentation occurs via the traditional exogenous pathway. Alternatively, actomyosin fibers of B cell exert a pulling force on BCR that with a sufficiently high affinity pulls antigen from the antigen-presenting cell. From there, BCR-antigen complexes enter the endosomal system and are processed for antigen presentation. Before we go into what happens to the B cell lets remember we need signal 2. In signal 2 some part of the antigen thats recognized by the b cell receptor has to be internalized by the B cell and degraded (processed) in order to create peptides which will then be bound by the MHC class II molecule with the hope of activating a coordinate helper T cell. BUT before we go into finding the correct helper t cell let's look at how antigens are processed. This can happen in two different ways. 1. vesicles that contain lysosomal enzymes can fuss with the immune synapse that forms between the b cell and the APC and cut off some of the antigens creating smaller molecules which can be internalize by the b cell which can then activate the endogenous pathway which can produce peptides that can bind to MHC class II at the cell surface and be presented 2. after the B cell binds to an antigen you have actin/myosin fibers that change the b cell membrane in a way that it begins to pull the b cell membrane away from the APC and while doing so we internalize the BCR and some of the APCs membrane, which can then go through the exogenous pathway for processing. (look into exogenous and endogenous pathway stuff) March 31st 26:30

How does the cell make the choice as to which S regions will be broken and rejoined? or in other words, how does the cell decide with antibody class it wants to be?

Once again, we have to consider the roles of cytokines. In the example below, the cell is synthesizing IgG1. Cytokine signals lead to the transcription of germline DNA over the targeted switch regions. Transcription is initiated at promoters upstream of a noncoding "I" exon, located 5´ of each of the switch regions. Transcription continues through the associated S region and terminates downstream of the relevant CH exons. Different cytokines secreted by T cells or other immune cells, can stimulate transcription from different I region promoters. Finally, B cells must also receive costimulatory signals from CD40 or B-cell Toll-like receptors in order to engage in CSR So once again this is where cytokines comes in again. It has to do with which cytokine the helper t cell is expressing. Different cytokines are going to lead to the expression of different transcription factors which are targeted towards different promoter regions

What is the path followed by activated B cells within the germinal center?

Once the germinal center is fully established, up to 50% of the B cells transition from the dark zone to the light zone every 4 to 6 hours, which suggests that very little time elapses between the occurrence of a mutation and when it is tested for increased antigen binding.

Following stimulation of primary B cells at the T-cell/B-cell border within the lymph node, some B cells differentiate quickly into what two things? What determines the B cells fate?

Plasma cells or germinal centers. Following stimulation of primary B cells at the T-cell/B-cell border within the lymph node, some B cells differentiate quickly into plasma cells that form primary foci and secrete an initial wave of IgM antibodies while some form germinal centers. A regulatory network of transcription factors controls the germinal center B cell/plasma cell decision point. •Differentiation into plasma cells requires the up-regulation of the plasma cell transcription factors IRF-4 and BLIMP-1. •Migration to the primary follicles to form germinal centers requires the transcription factor Bcl-6 and low levels of IRF-4. NOW we are looking at the differentiation into germinal centers vs. plasma cells. This diff. depends on the expression of specific transcription regulators/factors This process is similar to cross regulation of TH1 vs. TH2. So for ex. if the plasma cell expresses IRF-4 it will inhibit the TF SHM which is important for the fact of germinal centers. So overall the expression of a specific TR in a plasma cell not only drives diff into a plasma cells BUT ALSO prevents the diff into a germinal center and visa versa.

what are B-1B cells? what are marginal zone B cells?

T-independent responses generated by B-1 and marginal zone (MZ) B cells give rise to relatively low-affinity, primarily IgM antibodies: B-1 B cells - CD5+, 5% of B-cell population in humans/mice, primarily produce IgM - Produce "natural" antibodies independent of T-cell help that bind a broad spectrum of antigens with low affinity Marginal zone B cells - Must receive low-level signals through BCR for survival - Can renew themselves in the periphery - Differentiate from T2 B cells in the spleen - Specialized to respond to blood-borne antigen entering the immune system through the spleen

Certain subsets of B cells have evolved mechanisms to secrete antibodies to particular classes of antigens, without T-cell help. Antigens capable of eliciting T-independent antibody responses tend to have polyvalent, repeating determinants that are shared among many microbial species, thus echoing characteristics of many PAMPs. These antigens are:

TI-1 antigen — typically bacterial cell-wall components, bind to innate immunity PRRs on B cells TI-2 antigen — polymeric protein antigen and capsular polysaccharides, crosslink many mIgM BCRs SO now we are looking a T independent b cell responses - so b cells that can be activated without a helper t cell. The antigens that activate these cells are different than the ones that activate t dependent b cell responses. TI-1 ex. a lipopolysaccharide activating the BCR or a TLR giving signal I and 2 triggering cell activation In the case of TI-2 antigens the binding is so strong, signal 1 alone is enough to activate the cell

Do b cells move between the dark and light zones of the germinal centers? What are cells in the dark zone called? what cells in the light zone called?

The distinct B cell populations respond to distinct cytokines that guide their movement through the distinct locales in the follicles. B cells in the dark zone are referred to as centroblasts; those in the light zone are referred to as centrocytes. Movement between the two zones is orchestrated by modifications in cell-surface chemokine receptors. SO just as you would image you have movement between the b cell zone and t cell zones but also the light zone and dark zone. the expression of different chemokine receptors helps guide movement.

What are the function of B cells?

The function of a B cell is to give rise to plasma cells that secrete antibodies capable of binding to an organism or molecule that poses a threat to the host. The secreted antibodies have antigen-binding sites identical to those of the receptor molecules on the B-cell surface. Antibodies, once secreted, can protect the host against the pathogenic effects of invading viruses, bacteria, and parasites in a variety of ways. But how are B cells activated and what happens after activation? This is what we are going to investigate in this lecture. The activation of B cells is a little different than T cells considering B cells NEED T helper cells to be activated.

What is class switching recombination CSR?

The molecular machinery that allows the cell to switch from expressing μ to expressing any heavy-chain class other than μ or δ operates at the level of DNA recombination, and the process by which it occurs is referred to as class switch recombination (CSR). Recombination occurs between donor and acceptor switch (S) regions. These are tandem repeats of short, G-rich sequences 20-80 bp long that contain targeting sites for AID. AID-induced cytidine-to-uridine lesions are created on both strands within a switch region, and these lesions are then converted into double-stranded breaks by members of the base excision repair or mismatch repair pathway, as described earlier. Ligation between two broken S regions is catalyzed by proteins of the nonhomologous end-joining pathway. So now we have moved on to class switching bc remember we are still making IgMs but we also need to make IgG, etc. So class switching occurs in the primary focus and germinal centers (whereas somatic hypermutation only occurs in the germinal centers) So how does class switching work? well it turns out AID can also introduce lesions in areas known as s regions. These lesions lead to breaks in sequence which can eventually lead to the removal of Cμ and the addition or the Cγ fragment which in turn switches the antibody class from IgM to IgG

There are two major types of B-cell responses, which are elicited by structurally distinct types of antigens. What are the two different types of B cell responses?

There are two major types of B-cell responses, which are elicited by structurally distinct types of antigens. The first type of response is generated following recognition of protein antigens and requires the participation of CD4+ helper T cells. This class of B-cell response is therefore known as a T-dependent (TD) response. It is mediated by B-2 B cells binding to TD antigens. The second type of response is directed toward multivalent or highly polymerized antigens and does not require T-cell help. This type of response is referred to as a T-independent response, and the antigens that elicit such responses are T-independent (TI) antigens. TI-1 antigens bind to innate receptors on B cells and (at high antigen concentrations) elicit a polyclonal, antibody-secreting response. In contrast, TI-2 antigens are highly multivalent and bind only to Ig receptors. SO there are T cell dependent signals and T cell independent signals and it really depends on the type of antigen bound (size of the antigen etc.)

What is somatic hypermutation?

This process produces individual point mutations in Ig heavy- and light-chain rearrangements. The mutations increase over time and with repeated exposures and are followed by affinity selection to produce antibodies with increased affinity for the antigen over time. Somatic hypermutation is initiated by a cytidine deamination reaction catalyzed by the enzyme activation-induced deaminase (AID). DNA repair mechanisms then cause alterations of the sequence of the variable region of immunoglobulin genes. basically its just random mutations of the individual heavy and light chain variable regions. The mutations are induced by the enzyme AID which transforms a deoxycytidine into a deoxyuridine.

So with regards to chemokine receptors, what happens when the B cell enters the lymph node?

When a B cell enters the lymph node, the B-cell chemokine receptor, CXCR5, responds to CXCL13 signaling and migrates to the follicle. B cells move randomly within the follicle, but it all changes if a B cell meets an antigen capable of binding to its BCR (its "cognate" antigen). B cells being expressing CCR7 after antigen encounter. Its ligands (CCL19/CCL21) are secreted by stromal cells in T-cell-rich areas of lymphoid organs. So, the expression of CCR7 directs B-cell migration to B- and T-cell boundary. B cells move in the T-cell-rich zone until they encounter their counterpart antigen-specific T cell. B cells then engage their cognate T-cell partners. After that, expression of CCR7 is downregulated and B cells leave T-cell areas to eventually enter follicles to form germinal centers. Meanwhile, other activated B cells form primary foci of proliferating B cells that will eventually differentiate into plasma cells. These cells secrete the unmutated, predominantly IgM antibodies of the early immune response. SO when the B cell arrives in the lymph node it starts expressing chemokine receptors that help guide it towards the follicle where they are more likely to find an antigen from here it will more to a zone where it is more likely to find a Tfh helper cell.

What happens when the BCR recognizes its cognate antigen?

When the BCR recognizes its cognate antigen, the receptors on the B-cell membrane briefly spread over the surface of the antigen-presenting cell membrane, and then contract, resulting in B-cell receptor clustering. This is achieved in a matter of minutes and represents the earliest phase of B-cell activation. By the end of the contraction phase of the membrane response, microclusters of BCRs collapse into a single central cluster of receptors. As part of the contraction we get a large cluster of b cell receptors bound to antigen Receptor clustering leads to the formation of an immunological synapse between the B cell and its antigen. On the B-cell surface, an inner ring of BCRs forms the central part of the supramolecular activating cluster, or cSMAC, which is surrounded by a ring of adhesion molecules, referred to as the pSMAC, encircled by a distal ring of polymerized actin, the dSMAC. so on the cell surface of the b cell you have multiple IgM membrane bound receptors that form clusters (these clusters are what allow for a robust response if you recall)

How do we ensure we are not introducing mutations in areas that do not encode for antigen binding?

well it turns out there are mutational hot spots which are usually found in the variable regions of antibodies The mutational apparatus targets so-called mutational hot spots: sequence motifs far more likely to be targeted by AID. These include the following sequence motifs: Comparison of the sequences in the antigen-binding, complementarity-determining regions (CDRs) with the rest of the amino-terminal variable regions of antibodies indeed showed a higher concentration of mutational target sequences in CDRs than in the framework sequences. Consequently, the rate of hypermutation is orders of magnitude higher in Ig variable region DNA than in other genes in germinal center B cells. These spots are more likely to be targets by AID than other locations and are mainly found in regions that code for antibody binding

Shutting down BCR signaling may be necessary to stop proliferation when it is no longer required. but how do we regulate B cells?

•Negative signaling through CD22 shuts down unnecessary BCR signaling. This receptor bears immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of these motifs recruits SHP-1 tyrosine phosphatase. SHP-1 then removes phosphates from tyrosine residues of neighboring signaling complexes. •Negative signaling through FcγRIIb (CD32) receptor inhibits B-cell activation. This receptor possesses ITIMs similar to those in CD22. Circulating IgG can bind this receptor and shut down B-cell activation. •CD5 acts as a negative regulator of B-cell signaling. CD5 induced on B-2 B cells following BCR-CD40 engagement. Many CD5+ B cells secrete IL-10. •B-10 B cells act as negative regulators by secreting IL-10 upon antigenic stimulation. Negative signaling involves dephosphorylation (because if you think about it B cells signaling starts with tyrosine phosp. therefore negative regulation involves dephosp)