Ativação e diferenciação de linfócitos B

Question 1

Fases de uma resposta imunológica humoral

Answer 1

Naive B lymphocytes (IgM+, IgD+) recognize antigens, and under the influence of helper T cells and other stimuli (not shown), the B cells are activated to proliferate, giving rise to clonal expansion, and to differentiate into antibody-secreting plasma cells.

Some of the activated B cells undergo heavy-chain isotype switching (–> IgG) and affinity maturation (–>High affinity IgG), and some become long-lived memory cells.

Question 2

Ativação de linfócitos B (esquema)

Answer 2

T-dependent and T- independent antibody responses.

Antibody responses to protein antigens require T cell help!!! and the antibodies produced typically show isotype switching and are of high affinity!! Most T-dependent responses are made by follicular B cells.

Non-protein (e.g., polysaccharide) antigens are able to activate B cells without T cell help!!
Marginal zone B cells and B-1 cells play greater roles in T-independent responses.
+other signals (eg. complement protein)

Question 3

Ativação de linfócitos B

Answer 3

• 2 tipos de respostas: dependentes de linfócitos T (Thymus (T)-dependent
responses) ou independentes de linfócitos T (T independent responses)

=> Dependentes de linfócitos T (TD): Requer antigénios proteícos e linfócitos
T CD4+ auxiliares

1) Ag multivalente liga-se ao BCR e promove crosslinking de recetores
2) Linfócito T CD4+ ativado interage com o linfócito B via TCR e interação CD40L (T)/ CD40 (B).

=> Independentes de linfócitos T (TI): Ag multivalentes, sem necessidade de linfócitos T auxiliares

• TI-1: Envolve sinalização via BCR e PRRs, e.g. LPS. Reconhecido por PRRs
  expressos à superfície de LB.
• TI-2: Envolve sinalização via BCR e CD21, e.g. polissacáridos da cápsula
  bacteriana. Ag muito repetitivos. Não mitogénicos mas capazes de ativar
  muitos BCRs (crosslinking de vários recetores). Muitos destes Ag estão associados ao fragmento C3d.

Question 4

Ativação de LB por antigénio dependente de células T

Answer 4

-Sinalização via BCR (sinal 1)
-Sinalização via CD40 (sinal 2)
-Sinalização via citocinas (sinal 3)
(sobrevivência, proliferação e diferenciação)

T-dependent (TD) antigens bind to the Ig receptor of B cells. Some of the antigen is processed and presented
to helper T cells. T cells bind to the MHC-peptide antigen, and deliver further activating signals to the B cell via
interaction between CD40L (on the T cells) and CD40 (on the B cells).
In addition, T cells secrete activating cytokines, such as IL-4, IL-5 and IL-21, which are recognized by receptors on the B-cell surface. Cytokines deliver differentiation, proliferation, and survival signals to the B cells.

Question 5

Ativação de LB por antigénio independente de células T (TI-1 e TI-2)

Answer 5

-Dão origem a Ab de baixa
afinidade, primariamente IgM

-Ativação de células B
independente de LT não gera memória imunológica

• CD21 co-receptor das células B (faz parte do complexo BCR) é também o recetor da molécula
  de complemento C3d

-TI-1 Ag:
Sinalização via BCR e TLRs
Funcionam como ativadores
policlonais ou mitogénios B

T-independent-type 1 (TI-1) antigens bind to B cells through both Ig and innate immune receptors. For
example, LPS from gram-negative organisms binds to B cells via both membrane-bound immunoglobulin (mIg)
and TLR4, resulting in signaling from both receptors.

-TI-2 Ag:
Sinalização via BCR (crosslinking de BCR e CD21)
Epitopos repetitivos que induzem cross-linking

T-independent-type 2 (TI-2) antigens are frequently bound by C3d complement components and cross-link
both mIg and CD21 receptors on B cells. Cross-linking of between 12 and 16 Ig receptors by TI-2 antigens has been shown to be sufficient to deliver an activating signal

Question 6

Complexo BCR

Answer 6

• O complexo BCR inclui co-recetores como o CD21 (CR2), que aumentam a capacidade da célula de ser ativada por
Ag complexado com componentes do complemento.

O complexo BCR também
inclui as proteínas Iga e Igb, que possuem ITAMs que promovem a transdução de sinal a partir do BCR para o interior da célula, assim como as moléculas CD19 e CD81.

• Aumenta grandemente a sinalização via BCR e diminui o threshold de ativação da célula B
• A molécula de CD19 é precisa

Immunoreceptor tyrosine-based activation motif

CR2/CD19/CD81 co-recetor da célula B

Question 7

Papel da imunidade inata na ativação de linfócitos B

Answer 7

Signals generated during innate immune responses to microbes and some antigens cooperate with recognition of antigen by antigen receptors to initiate B cell responses.

A, Activation of complement by microbes leads to the binding of a complement breakdown product, C3d, to the microbes.
The B cell simultaneously recognizes a microbial antigen (by the immunoglobulin receptor) and bound C3d (by the CR2 receptor).
CR2 is attached to a complex of proteins (CD19, CD81) that are involved in delivering activating signals to the B cell.

B, Molecules derived from microbes (PAMPs) may activate Toll-like receptors (TLRs) of B cells at the same time as microbial antigens are being recognized by the antigen receptor.

Question 8

Sinalização via BCR

Answer 8

Crosslinking do BCR por Ag desencadeia fosforilação dos ITAMs
pelas tirosina cinases Src (Lyn, Fyn, Blk)

–> Docking da tirosina cinase Syk

• -> Propagação da sinalização (fosforilação de outras proteínas):
• ativação de PLCgamat –> IP3, aumento Ca2–> ativação de e enzimas dependentes de Ca2+ e –> DAG –> PKC
• troca GTP/GDP em Ras, Rac –> Ras-GTP e Rac-GTP –> ativação enzimas ERK e JNK

–> Ativação de fatores de transcrição (Myc, NFAT, NFKB e AP-1)

Mutações em BTK causam agamaglobulemia associada ao cromossoma x ou doença de Burton. Deficiência em todos os isotipos de Ig!!

Cross-linking of antigen receptors on B cells by antigen triggers biochemical signals that are transduced by the Igassociated proteins Igα and Igβ. These signals induce early tyrosine phosphorylation events, activation of various
biochemical intermediates and enzymes, and activation of transcription factors. Similar signaling events are seen in
T cells after antigen recognition. Note that maximal signaling requires cross-linking of at least two Ig receptors by
antigens, but only a single receptor is shown for simplicity.

Question 9

Resposta humoral primária e secundária

Answer 9

In a primary immune response, naive B
cells are stimulated by antigen, become
activated, and differentiate into antibody-secreting cells that produce
antibodies specific for the eliciting antigen. Ag T dependent and independent!!

A secondary immune response is elicited when the same antigen stimulates memory B cells, leading to production of greater quantities of
specific antibody than are produced in the primary response. Ag T dependent!!

Question 10

Consequências funcionais
da ativação de linfócitos B
(reconhecimento de Ag
via BCR)

Answer 10

The activation of B cells by antigen in lymphoid organs initiates the process of B cell proliferation and IgM secretion and prepares the B cell for interaction with helper T cells.

Changes in activated B cells + Functional consequences:

• Expression of proteins that promote survival and cell cycling –> increased survival, proliferation
• Antigen presentation, increased B7 expression –> interaction with helper T cells
• Increased expression of cytokine receptors –> responsiveness to cytokines
• Increased expression of CCR7 –> Migration from follicle to T cell zone
• Generation of plasma cells –> Antibody secretion

Question 11

Sequência de eventos na ativação de linfócitos B por Ag dependentes de células T

Answer 11

Células T auxiliares ativadas por DCs migram na direção da zona B do
gânglios linfáticos e interagem com linfócitos B ativados por Ag na borda dos folículos linfoides!

A, Interação T-B inicial: Immune responses are initiated by the recognition of antigens by B cells and CD4+ T cells. The activated lymphocytes migrate toward one another and interact at the interface of T and B cell zones.

B, Ativação de LB: The initial T-dependent B cell proliferation and differentiation results in the formation of an extrafollicular focus (FOCO EXTRAFOLICULAR), in which B cells proliferate, can undergo isotype switching, and differentiate into plasma cells (mostly short-lived).

Some of the T cells that are activated in the extrafolicular focus develop into follicular helper T cells and migrate back into the follicles, together with some activated B cells, to form a germinal center!

The late events in B cell responses occur in germinal centers and include somatic mutation and the selection of high-affinity cells (affinity maturation), additional isotype switching, memory B cell generation, and the generation of longlived plasma cells.

Question 12

Migração de linfócitos B e células T CD4+ auxiliares e interação B-T

Answer 12

Antigen-activated helper T cells and B cells move toward one another in response to chemokine signals and make contact adjacent to the edge of primary follicles.

Lymph node:
Antigen presentation by DCs –> T cell activation
=> CCR7 baixa, CXCR5 sobe and migration of activated T cells to edge of follicle

Antigen uptake and prcessing; B cell activation in B cell zone (primary follicle),
=> CCR7 sobe, and migratio of activated B cells to edge of follicle

==> B cells present antigen to activated helper T cells

Question 13

Apresentação de Ag a LT auxiliares por células B

Answer 13

LB que reconhecem o Ag via BCR, endocitam o mesmo, processam-no em
vesículas endossomais e apresentam os
péptidos resultantes em moléculas de
MHC II para reconhecimento por LT CD4+

“Linked recognition” é o processo pelo qual
um linfócito B é optimamente ativado por
uma célula T ativada que responde ao mesmo Ag ou a um Ag fisicamente associado

B cells specific for a protein antigen bind and internalize that antigen, process it, and present peptides attached to class II major histocompatibility complex (MHC) molecules to helper T cells. The B cells and helper T cells are specific for the same antigen, but the B cells recognize native (conformational) epitopes, and the helper T cells recognize peptide fragments of the antigen bound to class II MHC molecules. B cells also express costimulators (e.g., B7 molecules) that may play a role in T cell activation (not shown).

Question 14

Mecanismos de ativação de linfócitos B por células T CD4+ auxiliares

Answer 14

Helper T cells recognize peptide antigens presented by B cells (and costimulators, e.g., B7 molecules, not shown) on the B cells. The helper T cells are activated to express CD40 ligand (CD40L) and secrete cytokines, both of which bind to their receptors on the same B cells and activate the B cells.

Mutações LOF no gene CD40L resultam em Síndrome de hiper IgM tipo 1 (ou ligada ao
cromossoma X), que é caracterizado por deficiência na produção de Ab, nomeadamente na maturação da afinidade e mudança de classe, assim como imunidade celular deficiente.

Question 15

Apresentação de Ag a células B foliculares nos gânglios linfáticos

Answer 15

CXCR5- quimiocina CXCL13

Follicular dendritic cells (FDCs) are cells of stromal origin and are located in the central region of primary follicles and in the light zone of germinal centers. They provide a reservoir of Ag to B cells to bind as they undergo mutation, selection and differentiation during germinal center
differentiation.

• Small Ag directly captured by B cells
• Larger Ag captured by macrophages (via C and FC receptors) in the subcapsular sinus as well as by FDC and are presented to B cells

Antigen presentation to follicular B cells in the lymph node. Lymphatic fluid containing antigens (red) and cytokines and chemokines (blue) reaches the lymph node through the afferent lymph vessel and enters the subcapsular sinus (SCS) region (SEIO SUBCAPSULAR). The SCS region is lined with a porous border of SCS macrophages (SCSMs) that lie on or just under the lymphatic endothelial border and that prevent the free diffusion of the lymph fluid into the lymph node. Larger antigens are bound by surface receptors, such as complement and Fc receptors, on the SCSMs and then presented directly to B cells. Smaller antigens, and chemokines less than approximately 70 kDa in molecular weight, access B cells in the follicles either by diffusion or by passage through conduits emanating from the sinus. Ag also binds to FDCs via complement and other receptors and can bypassed from these cells to B cells.

Question 16

Comparação Respostas células B Extrafolicular e Germinal Center!!

Answer 16

Extrafollicular response - GC response

Localization:
medullary cords of lymph nodes and at junction between T cell zone and red pulp of slpean - Germinal centers of secondary follicles

CD40 signals:
required - required

Specialized T cell help:
extrafollicular helper T cells - Tfh cells in GC

AID expression:
Yes - Yes

Isotype switching:
Yes - Yes, extensive

Somatic hypermutation:
Low rate - High rate

Affinity maturation of antibody:
Low - High

Terminally differentiated B cells:
Short-lived plasma cells (life span of ~3 days) - Long-lived plasma cells, which migrate to bone marrow, and memory cells

Transcription factors activated in B cells:
Blimp-1 - Bcl-6

Plasmócitos do foco extrafolicular produzem grandes quantidades de Ab do isotipo IgM e IgG não-mutados que providenciam uma primeira resposta humoral protetora.

Question 17

Papel de fatores de transcrição na determinação da diferenciação de LB ativados em plasmócitos ou entrada na reação de centros germinativos

Answer 17

A regulatory network of transcription factors controls the germinal center B cell/plasma cell decision point. The transcription factors that control germinal center B-cell versus plasma-cell states of differentiation are related to one another through a mutually regulatory network.

Pax-5 and Bcl-6, along with low levels of IRF-4, favor the generation of proliferating, germinal center cells. (GC fate)

Conversely, the expression of BLIMP-1 and of high levels of IRF-4 support the generation of antibody-secreting cells. (plasma cell fate)

Question 18

Reação centro germinativo

Answer 18

Activated B cells migrate into the follicle and proliferate, forming the dark zone of the germinal center (zona escura, centroblastos).

These B cells undergo somatic hypermutation of Ig V genes and migrate into the light zone (zona clara, centrólitos), where they encounter follicular dendritic cells displaying antigen and Tfh cells.

B cells with the highest affinity Ig receptors are selected to survive, and they differentiate into antibody-secreting cells and memory B cells. The antibody-secreting cells leave and reside in the bone marrow as long-lived plasma cells, and the memory B cells enter the recirculating lymphocyte pool.

Question 19

Seleção de células B nos centros germinativos

Answer 19

B cell activation by protein antigen and helper T cells

–> induction of AID and migration into GC

–> B cells with somatically mutated Ig V genes and Igs with varying affinities for antigen

–> Only B cells with high-affinity antigen receptors recognize antigen on follicular DCs and present it to Tfh cell

–>Only B cells with high affinity antigen receptors are selected to survive

Somatic mutation of V genes in germinal center B cells generates antibodies with different affinities for antigen. Binding of the B cells to antigen displayed on follicular dendritic cells is necessary to rescue the B cells from programmed cell death. B cells may also present antigen to germinal center Tfh cells, which promote B cell survival. The B cells with the highest affinity for antigen thus have a selective advantage for survival as the amount of available antigen decreases during an immune response. This leads to an average increase in the affinity of antibodies for antigen as the humoral immune response progresses.

Question 20

Eventos moleculares na geração de células T auxiliares foliculares (TFh)

Answer 20

The generation of Tfh cells requires sequential activation of T cells, first by DCs and then by activated B cells. DC-Naive CD4+ T cell –> CXCR5 –> Tfh cell (Bcl-6+++) ligado a activated B cell por ICOS-ICOSL.
The differentiated Tfh cells migrate into germinal centers, where they activate B cells.

Within 4 to 7 days after antigen exposure, activated antigen-specific B cells induce
some previously activated T cells to differentiate into Tfh cells, which express high levels of the chemokine receptor CXCR5, are drawn into lymphoid follicles by
CXCL13, the ligand for CXCR5, and play critical roles in germinal center formation
and function.

Question 21

Papel da IL-21 na reação do centro germinativo

Answer 21

In GCs, B cells that have undergone somatic hypermutation pass through checkpoints where they get tested for their ability to bind Ag presented in the form of immune complexes on the surface of follicular DCs. If B cells recognize an Ag via BCR, they can process and present it to Tfh cells and elicit their help via CD40Lmediated and IL-21-mediated signals. This results in survival, proliferation, CSR, and differentiation of B cells to IgM-, IgG-, or IgA-secreting PCs.

Question 22

Hipermutação somática ou

maturação de afinidade

Answer 22

Left panel: the primary antibody repertoire is initially composed of IgM containing
variable regions (red) produced by V(D)J recombination and constant
regions (blue) from the μ gene segment.

Right panel: somatic hypermutation results in mutations (shown as black lines) being introduced into the heavychain and light-chain V regions (red), altering the affinity of the antibody for its antigen.

!!Mutações acumuladas tendem a localizar-se nas regiões hipervariáveis ou determinantes de complementaridade!!

Mutações na região variável aumentam com o tempo e com imunizações repetidas Afinidade do Ab para o Ag tende também a aumentar

Somatic mutations in Ig V genes. Hybridomas were
produced from spleen cells of mice immunized 7 or 14 days previously with a hapten, oxazolone, coupled to a protein and from spleen cells obtained after secondary and tertiary immunizations with the same
antigen. Hybridomas producing oxazolone-specific monoclonal antibodies were produced, and the nucleotide sequences of the V genes encoding the Ig
heavy and light chains were determined. Mutations in V genes increase with time after immunization and with repeated immunizations and are clustered in the complementarity-determining regions (CDRs). The location of CDR3 in the heavy chains is approximate. The affinities of the antibodies produced also tend to increase with more mutations, as indicated by the
lower dissociation constants (Kd) for hapten binding.

!!AID (activation-induced cytidine deaminase) é necessária!!

!AID induz mutações no DNA por desaminação da citosina em uracilo (que é reconhecido como timina). Ou seja, induz a mudança de C:G em U:G!

!!Regioes hipervariáveis: Vh e Vl!!

Question 23

Mudança de classe (isotipo)

Answer 23

B cells activated by helper T cell signals (CD40L, cytokines) undergo switching to different Ig isotypes, which mediate distinct effector functions. Selected examples of switched isotypes are shown. All isotypes are capable of neutralizing microbes and toxins.

Helper T cell with CD40L - CD40 of activated B cell
–> IgM+ B cell

=> isotype switching:

• IgM: complement activation
• IgG subclasses (IgG1, IgG3): Opsonization and phagocytosis, complement activation, neonatal immunity (placental transfer)
• IgE, IgG4: Immunity against helminths, mast cell degranulation (immediate hypersensivity)
• IgA: Mucosal immunity (transport of IgA through epithelia)

Question 24

Recombinação switch

Answer 24

In class switch recombination, the initial μ heavy chain C regions (blue) are replaced by heavy-chain regions of another isotype (shown as yellow), modifying the effector activity of the antibody but not its antigen specificity

Switch de IgM (μ) para IgE (e)

Organização de um locus da cadeia pesada após rearranjo VDJ e antes de class switching

Envolve recombinação entre regiões de switch específicas (sequências de DNA repetitivas localizadas a montante de cada gene C)

Processo iniciado pela AID (activation induced cytidine
deaminase). UNG, APE1 e maquinaria DSBR (e.g. DNA-PK e Ku) também estão envolvidas.

Na recombinação switch ocorre mudança de classe nos anticorpos produzidos, mantendo-se a região variável antes rearranjada

Top panel shows the organization of a rearranged immunoglobulin heavy-chain locus before class switching. Switch regions (S) are repetitive DNA sequences that guide class switching and are found upstream of each of the immunoglobulin C-region
genes, with the exception of the δ gene. 

Switching is guided by the initiation of transcription by RNA polymerase through
these regions from promoters (shown as arrows) located upstream of each S. Due to the repetitive sequences, RNA
polymerase can stall within the S regions, allowing these regions to serve as substrates for AID, and subsequently for UNG and APE1. These enzymes introduce a high density of single-strand nicks into the non-template DNA strand and the template strand. Staggered nicks are converted to double-strand breaks by a mechanism that is not yet understood. These breaks are then recognized by the cell’s double-strand break repair machinery, which involves DNA-PKcs, Ku proteins, and other repair proteins.

Bottom two panels: the two switch regions, in this case Sμ and Sε, are brought together by the repair proteins, and
class switching is completed by excision of the intervening region of DNA (including Cμ and Cδ) and ligation of the Sμ and Sε regions.

Question 25

AID: Crítica para SHR e CSR

Answer 25

• Ratinhos KO para AID não são capazes de efetuar hipermutação somática nem mudança de classe das Igs (Muramatsu et al. 2000 Cell)
• Pacientes com mutações em AID não efetuam mudança de classe e exibem uma redução dramática na capacidade de hipermutação somática (Revy et al. 2000 Cell)
• Ratinhos KO para AID e doentes com mutações em AID produzem elevados títulos de Ab IgM contra proteínas

Síndrome de hiper IgM tipo 2

Síndrome de hiper IgM tipo 1 resulta de mutações em CD40L (X-linked)

Question 26

A diferenciação de um LB em plasmócito requere alteração das cadeias pesadas da forma membranar para a secretada

Answer 26

Substituição da região hidrofóbica C- terminal que permite ancoragem na membrana plasmática por uma sequência de aminoácidos hidrofílica

When a B cell is stimulated, it secretes a soluble form of its receptor that differs in sequence from the membranebound BCR at the C terminus but has the same antigen binding site as the membrane receptor. The hydrophobic region that anchors the membrane-bound form onto the surface of the B cell is replaced, by differential mRNA splicing, with a soluble (hydrophilic) amino acid sequence in the secreted antibody.

The biochemical difference between the membrane-bound receptor and the secreted form of the antibody lies at the carboxyl terminus of the heavy chains. Secreted antibodies have a hydrophilic
amino acid sequence of various lengths at the carboxyl terminus. In membrane-bound
immunoglobulin receptors, this hydrophilic region is replaced by three sequentially arranged regions.

Question 27

A forma membranar (BCR) ou secretada (Ab) das Ig deriva da
mesma sequência da cadeia pesada e é determinada
por splicing alternativo

Answer 27

Exões M1 e M2 codificam para a região transmembranar;
Sequência SC (secretion coding) codifica para o C-terminal da forma secretada

At the end of the heavy-chain C gene, there are two exons (M1 and M2, yellow) that together encode the transmembrane region
and cytoplasmic tail of the transmembrane form. Within the last C-domain exon, a secretion-coding (SC) sequence (orange) encodes the carboxy terminus of the secreted form. In the case of IgD, the SC sequence is in a separate exon (not shown), but for the other isotypes, including IgM as shown here, the SC sequence is contiguous with the last C-domain exon. The events that dictate whether a heavy-chain RNA will result in a secreted or a transmembrane immunoglobulin occur during processing of the pre-mRNA transcript. Each heavy-chain C gene has two potential polyadenylation sites (shown as pAs and pAm).

Left panel: the transcript is cleaved and polyadenylated (AAA) at the second site (pAm). Splicing occurs from a site located within the last Cμ4
exon just upstream of the SC sequence (orange), to a second site at the 5ʹ end of the M1 exons (yellow). This results in removal of the SC sequence and joining of the Cμ4 exon to the exons M1 and M2 and generates the transmembrane form of the heavy chain.

Right panel: polyadenylation occurs at the first poly(A) addition site (pAs), and transcription terminates before the exons M1 and M2, preventing the generation of the transmembrane form of the heavy chain, and producing the secreted form.

Question 28

Regulação das respostas humorais: Feedback por anticorpos

Answer 28

Secreted IgG antibodies form immune complexes (antigenantibody
complexes) with residual antigen (shown here as a
virus but more commonly is a soluble antigen).

The complexes interact with B cells specific for the antigen, with the membrane immunoglobulin (Ig) antigen receptors recognizing epitopes of the antigen and a certain type of Fc receptor (FcγRIIB) recognizing the bound antibody.

The Fc receptors block activating signals from the antigen receptor, terminating B cell activation.

The cytoplasmic domain of B cell FcγRIIB contains an
ITIM that binds enzymes that inhibit antigen receptor–mediated B cell activation.

ITAM, Immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif.