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1

Desenvolvimento de Linfócitos B

• Em muitos vertebrados (ratinho e humanos), após o nascimento os linfócitos B (LB) desenvolvem-se na medula óssea.

Fases de desenvolvimento B:

• Rearranjo da cadeia pesada e da cadeia leve das imunoglobulinas (geração de diversidade).

• Tolerância central: eliminação de células autoreactivas.

• LB imaturos que expressam IgM deixam a medula --> Migração para o baço --> Processo de maturação (T1 --> T2 --> LB maturo).
No estadio de desenvolvimento T1, linfócitos são submetidos a mecanismos de tolerância periférica.

• LB maturo deixa o baço (expressão de IgM e IgD) --> migração para os folículos linfóides.

Encontro com Ag nos centros germinativos --> Proliferação, hipermutação e mudança de classe (isotipo).

2

Diferenciação de células B

• Na medula óssea, a partir de precursores imaturos

Precursor linfoide

--> Pro-B, comprometido para linhagem B, rearranjo cadeia pesada (early/late)

--> Pré-B, Pre-BCR, rearranjo cadeia leve (large/small)

--> B imatura, IgM, tolerância central

--> Tolerência periférica T1 --> T2, Baço; B matura

3

Estadios de desenvolvimento na medula e maturação B no baço

Estadios de desenvolvimento B na medula

HSC (Hematopoietic stem cell)
--> MPP (Multipotent progenitor)
--> LMPP (Lymphoid-primed multipotent progenitor cell)
--> ELP (Early lymphoid progenitor cell)
--> CLP (common lymphoid precursor
--> Pre-Pro B
--> Early Pro-B (DJ H cain recombination; Start of V-DJ H chain recombination)
--> Late Pro-B (V-DJ H chain recombination)
--> Pre-B (H chain expressed as pre-BCR, several rounds of cell division, VJ L chain recombination)
--> Immature (MIgM expression, negative selection: deletion, receptorediting)

--> immature B cell in blood vessel --> baço

Transitional-1 --> transitional-2 --> Mature B

B-cell development begins with a hematopoietic stem cell (HSC) and passes through progressively more committed
lymphoid progenitor cell stages until it reaches the pro-B-cell stage. At this stage, the precursor cell is irreversibly
committed to the B cell lineage, and the recombination of the immunoglobulin genes begins. Once the completed
IgM is expressed on the cell surface, the immature B cell leaves the bone marrow to complete its maturation
through the transitional T1 and T2 stages in the spleen.

4

Interação de HSC e progenitores B com populações celulares

-estabelecem contacto com várias populações celulares da medula (nichos) à medida que progridem no desenvolvimento!!

CXCL12 (Stromal cell-derived factor 1) :
• Essencial para os estadios iniciais do desenvolvimento B
• Possivelmente importante para a retenção de progenitores B na medula óssea


HSCs begin their developmental program close to the osteoblasts (top).

An HSC is also shown entering from the blood (lefthand
side), illustrating the fact that HSCs are capable of recirculation in the adult animal.

Progenitor cells then move to gain contact with CXCL12-expressing stromal cells, where they mature into pre-pro-B cells.

By the time differentiation has
progressed to the pro-B-cell stage, the developing cell has moved to receive signals from IL-7–producing stromal cells.

After leaving the IL-7–expressing stromal cell, the pre-B cell completes its differentiation and leaves the bone marrow as an
immature B cell.

Immature B cells express the S1P receptor, which recognizes the lipid chemoattractant sphingosine 1-phosphate (S1P) in the blood.

CXCL12 is shown in purple; IL-7 in blue; S1P in red.

5

Interação de progenitores com células estromais da medula é fundamental para o desenvolvimento B!!

• Estroma providencia citocinas (solúveis e “membrane bound”) e quimiocinas que controlam a diferenciação e proliferação de linfócitos

• IL-7: sobrevivência e proliferação de linfócitos B durante o seu processo de desenvolvimento

• SCF (stem-cell factor) promove proliferação dos progenitores de células B


Progenitor cells bind to the adhesion molecule VCAM-1 on stromal cells through the integrin VLA-4 and also
interact through other cell-adhesion molecules (CAMs).

The adhesive interactions promote the binding of the
receptor tyrosine kinase Kit (CD117) on the surface of the pro-B cell to stem-cell factor (SCF) on the stromal cell,
which activates the kinase and induces the proliferation of B-cell progenitors.

6

Fatores de transcrição na diferenciação de células B

E2A e EBF: Expressão de proteínas críticas para recombinação V(D)J, incluindo Rag1 e Rag2

Pax5: Expressão de CD19, Iga e BLNK

Pax5-/-: Bloqueio no estadio Pro-B (necessário para o comprometimento de células pro-B para a linhagem B)


Pluripotent HSC --> CLP (B, T, NK cells) -EFB, E2A, Pax5-> ProB --> FOB/MZB/B-1B

ou Pluripotent HSC --> CLP (B, T, NK cells) --> T, NK cells -Notch1, GATA3-> Pro-T --> alfabetaT

ou Pluripotent HSC --> CLP (B, T, NK cells) --> T, NK cells --> NK

7

Recetor da célula Pré-B

composition of the pre-BCR: two μ heavy chains (blue) associate with two surrogate light chains, each made up of γ5 (red) and VpreB (yellow), with extra peptide tails at the C terminus of VpreB and at
the N terminus of γ5.

self-aggregation/cross-linking induced signaling (-+IL-7R signals-> 1 e --> 3)

1. survival, proliferation
2. baixa RAG1/2, baixa VH-DJH gene rearrangement (HC allelic exclusion)
3. baixa Pre-BCR and IL-7 signals, baixa proliferation
4. sobre RAG1/2
5. sobe Light chain VLJL gene rearrangement

8

Exclusão alélica em LB

Sinalização via Pré-BCR promove exclusão alélica da
cadeia pesada:

• Reduz a expressão de Rag1 e Rag2
• Promove degradação dos enzimas Rag1 e Rag2

• Reduz o acesso da maquinaria de recombinação VDJ ao locus da cadeia pesada

Garante que apenas uma especificidade é gerada em cada LB



In rabbits, all of the B cells in an individual homozygous for the a allele of the immunoglobulin heavy-chain locus (Igha/a) will express immunoglobulin of allotype a, whereas in an individual homozygous for the b allele (Ighb/b) all the B cells make immunoglobulin of allotype b. In a heterozygous animal (Igha/b), which carries the a allele at one of the Igh loci and the b allele at the other, individual B cells can be shown to express surface immunoglobulin of either the a-allotype or the b-allotype, but not both (bottom panel). This allelic exclusion reflects the productive rearrangement of only one of the two Igh alleles in the B cell, because the production of a successfully rearranged immunoglobulin heavy chain forms a pre-B cell receptor, which signals the cessation of further heavy-chain gene rearrangement.

9

Pré-BCR vs BCR

Pré-BCR (na Pré-B):

"surrogate light-chain", Vpre-B, lambda5
Iga/Igb


BCR (na B imatura):

K ou lambda, miu,
Iga/Igb

10

Tolerância central na medula óssea

A ligação do BCR a componentes (Ag) do próprio na medula óssea promove deleção clonal ou inativação de células B imaturas



First panels (no self reaction): immature B cells that do not encounter antigen mature normally; they migrate from the bone marrow to the peripheral lymphoid tissues, where they may become mature recirculating B cells bearing both IgM and IgD on their surface.

Second panels (multivalent self molecule): when developing B cells express receptors that recognize multivalent ligands, for example, ubiquitous cell-surface self molecules such as those of the MHC, these
receptors are deleted from the repertoire. The B cells either undergo receptor editing,
thereby eliminating the self-reactive receptor, or the cells themselves undergo programmed cell death (apoptosis), resulting in clonal
deletion.

Third panels (soluble self molecule): immature B cells that bind soluble self antigens able to cross-link the B-cell receptor are rendered unresponsive
to the antigen (anergic) and bear little surface IgM. They migrate to the periphery, where
they express IgD but remain anergic; if in competition with other B cells in the periphery,
anergic B cells fail to receive survival signals and die.

Fourth panels (low-affinity non-crosslinking self molecule): immature B cells whose antigen is inaccessible to them, or which bind monovalent or soluble self antigens with low affinity, do not receive any signal and mature normally. Such cells are potentially
self-reactive, however, and are said to be clonally ignorant because their ligand is
present but is unable to activate them.

11

Tolerância central na medula óssea (cont.)

Seleção negativa (deleção clonal) e edição de receptor (“receptor editing”) ocorre em LB imaturos autoreactivos na medula óssea!!!


Evidência experimental:

The presence or absence of mature peripheral B cells expressing a transgene-encoded IgM BCR that reacts
with the H2 MHC class I molecule Kk was determined in
H2d/d mice (a) and in H2d/k mice (b and c).

(a) In the H2d/d mice transgenic for the Kk -specific BCR,
there was no self antigen for the immature B cells to bind to and consequently they went on to mature, so that splenic B cells expressing the transgene-encoded anti-Kk as membrane Ig were found in the periphery.

(b) In the H2d/k heterozygous mice transgenic for the Kk
-specific BCR, there were no mature B cells in the periphery expressing the BCR specific for the self antigen Kk protein.

(c) More detailed analysis revealed two fates of the
transgenic B cells in the H2d/K mice. Recognition of self-
Kk induced many to undergo apoptosis. There were also
some peripheral B cells that expressed the transgene-encoded μ chain but a different light chain, resulting from light-chain editing in some of the immature B cells. With the new light chains these B cells no longer bound the Kk molecule and consequently escaped negative selection.

12

Edição do recetor (“receptor editing”) - passos geral

A substituição da cadeia leve por este processo pode “salvar” algumas células B autoreativas da deleção clonal, por alteração da sua especificidade (nova cadeia leve associa-se com a cadeia pesada prévia e se a célula deixar de reconhecer Ag multivalentes sobrevive)


When a developing B cell expresses antigen receptors that are strongly cross-linked by multivalent self antigens such as MHC molecules on cell surfaces (top panel), its development is arrested. The cell decreases
surface expression of IgM and does not turn off the RAG genes (second panel).

Continued synthesis of RAG proteins allows the cell to continue light-chain gene rearrangement. This usually leads to a new productive
rearrangement and the expression of a new light chain, which combines with the previous heavy chain to form a new receptor; the process is
called receptor editing (third panel).

If this new receptor is not selfreactive, the cell is ‘rescued’ and continues normal development, much like a cell that had never reacted with self antigen (bottom right panel).

If the cell remains self-reactive, it may be rescued by another cycle of rearrangement; however, if it continues to react strongly with self antigen, it will undergo apoptosis, resulting in clonal deletion from the repertoire of B cells (bottom left panel).

13

Edição do recetor - como?

Edição do recetor utiliza um rearranjo secundário com segmentos Vk e JK a montante e a juzante, respetivamente, do rearranjo original

In the event of a productive light-chain rearrangement that leads to the formation of an autoreactive antibody, additional rounds of rearrangement may occur between upstream V and downstream J gene segments. Here, the primary rearrangement between V3 and J3 is edited out with a secondary rearrangement between V2 and J4.

14

Maturação dos linfócitos B

• LB imaturos deixam a medula óssea, via corrente sanguínea, e migram para o baço:

-> 2 subpopulações de “transitional B cells” (LB ainda em processo de maturação, transição)

Baço: T1 -> T2 -> LB maturo

Se auto-reactivos, LB T1 não se diferenciam em T2 (submetidos a mecanismos de Tolerância periférica)

• “Apenas” os LB T2 conseguem entrar nos folículos

Immature B cells leave the bone marrow via the blood. As T1 transitional immature B cells they enter the splenic marginal sinuses, percolating into the T-cell zones. There they differentiate into T2 transitional B cells, which gain the ability to enter the B-cell follicles, where they complete their differentiation into mature
follicular B cells and recirculate in the blood. Marginal zone cells have also been shown to derive from T2 B cells.

Algumas B T2 --> Zona marginal do baço (Células B da zona marginal)

imagem: arteríola central e bainha periarteriolar linfoide...

15

Processo de seleção positiva e negativa no baço

Nos folículos (T2 B):
Sinalização tónica via BCR

=> sobe BAFFR
Sinalização via BCR e BAFFR
=> Sobrevivência e diferenciação em células B maturas

T1 transitional B cells that recognize antigen, including self antigen, with high affinity in the spleen are eliminated by negative selection and never reach the splenic follicles. Those T1 B cells that escape negative selection differentiate into T2 B cells and enter the follicles. In the follicles their
BCRs tonically deliver a stimulatory survival signal, either without binding a ligand or from interactions with an unknown molecule(s).

T2 B cells that have received this survival signal upregulate their BAFF receptors and bind BAFF, also contributing to survival. Survival due to these two sets of signals constitutes positive selection. Those T2 B cells that fail to receive these stimulatory signals die in the spleen.

16

Tolerância periférica

Estadio T1 (no baço)

Transitional B cells that recognize self antigens undergo peripheral tolerance. After emigrating from the bone
marrow and entering the circulation, immature B cells are known as transitional B cells.
Not yet fully mature, these
cells are still subject to tolerance in the spleen following engagement of their sIgM receptor by a self antigen.

Transitional B cells that encounter a multivalent self antigen receive a strong B-cell receptor signal and undergo
cell death.

Transitional B cells with sIgM that binds to a soluble self molecule are rendered anergic, and ultimately die within a few days due to being excluded from the B-cell follicles in the spleen.

Transitional B cells that bind with low affinity to a soluble self molecule remain clonally ignorant of the self antigen and continue their maturation.
Transitional B cells with no self reaction also continue their maturation into mature B cells.

The final stages of Bcell maturation lead to upregulation of sIgD, and take place in the B-cell follicles in the spleen

17

Principais populações de LB na periferia

• LB foliculares: subset LB mais abundante; Ab alta afinidade e células memória. Expressam MHC II, CD19, CD23 e recetores Fc.

• LB da zona marginal: Mais abundantes no baço.

• Linfócitos B1: Residem nas mucosas e cavidade peritoneal.

18

LB foliculares

Major sites
secondary lymphoid organs

Progenitors first appear in mice
HSC: on day E10.5

Source of new B cells in adults
From HSC in bone marrow

Dependence on IL-7 and BAFF
Yes

V-region diversity
HIghly diverse

Somatic hypermutation
Extensive

Requirements for T-cell help
Yes

Isotypes produced
HIgh levels of IgG

Response to carbohydrate antigens
Possibly

Response to protein antigens
Yes

Memory
Yes

19

LB da zona marginal

Major sites
Peritoneal an pleural cavities

Progenitors first appear in mice
Progenitor: on day E9.5

Source of new B cells in adults
Self-renewing (division of existing B-1 cells)

Dependence on IL-7 and BAFF
No

V-region diversity
Restricted diversity: limited VH and VL usage and N nucleotide addition

Somatic hypermutation
Some

Requirements for T-cell help
No

Isotypes produced
Primarily IgM, some IgG

Response to carbohydrate antigens
Yes

Response to protein antigens
Possibly

Memory
Some

20

Linfócitos B1

Major sites
Marginal zones of spleen

Progenitors first appear in mice
From HSCs, aso earlier progenitor?

Source of new B cells in adults
From HSCs in bone marrow, long-lived

Dependence on IL-7 and BAFF
Yes

V-region diversity
Somewhat restricted

Somatic hypermutation
Unclear

Requirements for T-cell help
Varible

Isotypes produced
Primarily IgM; some IgG

Response to carbohydrate antigens
Yes

Response to protein antigens
Yes

Memory
Unknown

21

Mutações em proteínas que intervêm no processo de
recombinação V(D)J

Causadoras de imunodeficiências severas, que se refletem na população B (e T)

Rag1-/-
Rag2-/-
Rag1-/- Rag2-/-

Artemis-/-

DNA-ligase IV-/-

DNA-PK p350

22

Imunodeficiências primárias que afetam o desenvolvimento precoce de células da linhagem B

Btk- agamaglobulinemia ligada ao cromosoma X (XLA)

IGHM-/-
Iga (CD79A) -/-
Igβ (CD79B) -/-
λ5 (IGLL1) -/-

BLNK-/-

23

Mutações que causam deficiências em células B

A) The pre-BcR and mature BcR complexes consist of an immunoglobulin dimer associated with the Ig-a and Ig-β
subunits that generate intracellular signals.
The pre-BcR differs from the mature complex owing to the
presence of a surrogate light chain (indicated by a dotted line) in the pre-BcR dimer.
Further associated with the BcR are CD19, CD21, CD81 (TAPA-1), and CD225 (Leu-13, not shown), which act as coreceptors to modulate the threshold of signaling.

Mutations in the receptor complexes that have been linked to B-cell immunodeficiencies are
indicated with a red X. (Iga, CD19)

B). B-cell differentiation entails a progression from a progenitor stem cell (|- ADA deficiency) to a pro-B cell (|- RAG1, RAG2, Artemis mutation) to a pre-B cell (|- BTK; BLNK, Iga, miu, lambda5 mutations) to an immature B cell and, finally, to a mature B cell (|- CD19 mutations). The pre-BcR provides signals for pre-B-cell differentiation.

ADA denotes adenosine deaminase, RAG recombination-activating gene, BTK Bruton's tyrosine kinase, and BLNK mutated B-cell linked protein.

24

Conceitos chave

• Beginning around the time of birth and extending through adult life, hematopoiesis, including B-cell development, occurs in the bone marrow
and is influenced by various niches established by stromal cells.

• Stages of B-cell development are defined by the presence of sets of cellsurface markers (which include cytokine and chemokine receptors, and
adhesion molecules e.g. VLA-4 integrin binding to VCAM-1 on stromal cells), expression of specific transcription regulators, and the rearrangement status of immunoglobulin genes.

• The stages of B-cell development are controlled by networks of transcription factors and by epigenetic changes that influence the expression of key genes. The cells become increasingly committed to
becoming B lymphocytes.

• Stages of B-cell development can also be defined by the status of immunoglobulin gene rearrangements:
=> The heavy-chain V genes rearrange first in pre-pro- and pro-B cells, with D-to-J recombination occurring initially, followed by V-to-DJ recombination. The heavy chain is then expressed on the cell surface in combination with the surrogate light chain, which is made up of VpreB and λ5. Together they form the pre-B-cell receptor, which is expressed on the cell surface along with the Iga/Igβ signaling complex.

• Signals from the pre-B-cell receptor and the IL-7 receptor in mice, stop V gene rearrangement (ensuring heavy-chain allelic exclusion), confer a
survival signal, and activate several rounds of cell division, followed by light-chain gene rearrangement.

• The cell divisions allow multiple B cells to use the same successfully rearranged heavy chain in combination with many different light chains.
Expression of the pre-BCR and initiation of these events constitutes the pre-B-cell (first) checkpoint in B-cell development.

• After light-chain rearrangement at the small pre-B-cell stage, expression
of the completed mIgM B-cell receptor on the cell surface of immature B cells shuts down further light-chain gene rearrangement and confers a
survival signal, constituting the immature B-cell (second) checkpoint in the formation of mature B cells.

• Immature B cells are very sensitive to the induction of self-tolerance through the elimination of self-reactive cells. Immature B cells whose
BCRs are strongly activated by self antigens present in the bone marrow are induced to undergo receptor editing to change the specificity of their
BCRs. Self-reactive B cells that have not undergone receptor editing are deleted by apoptosis. These processes are part of the central tolerance
mechanisms for B cells.

• Immature B cells that migrate from the bone marrow to the spleen are called transitional 1 (T1) B cells. Interaction with self antigens in the spleen can induce these cells to undergo apoptosis. T1 cells are then
submitted at this stage to peripheral tolerance mechanisms.

• In T1 B cells that escape peripheral tolerance mechanisms, the level of
IgD expression increases, and they differentiate into T2 B cell that enter the follicles. Signals from the cytokine BAFF binding to the BAFF receptor are necessary for the survival of transitional and mature B cells.

• In addition to the predominant population of conventional B cells that arises in the bone marrow, called B-2 or follicular B cells, other subsets of B cells (B-1 and marginal zone [MZ] B cells) arise through other developmental programs, occupy distinct anatomical locations, and have
distinct functions.

• B-1 B cells (B-1a and B-1b) and MZ B cells differ from B-2 cells in surface markers, and in having a more limited repertoire of antibody specificities, which include cross-reactions between self and microbial antigens. They spontaneously generate natural antibodies and rapidly generate antibody responses to microbial infection without T-cell help.

• Recent studies have provided strong evidence that B-1a B cells are derived from early embryonic precursors, distinct from and preceding HSCs, that seed out into the pleural and peritoneal cavities and are
capable of self-renewal, generating B-1a cells throughout life.

• In contrast, B-1b and MZ B cells seem to be derived mostly from HSCs and B-2 lymphoid progenitors. MZ B cells inhabiting the splenic marginal
zone arise from T2 B cells