Desenvolvimento de Linfócitos B Flashcards
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).
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
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.
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.
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.
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
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)
- survival, proliferation
- baixa RAG1/2, baixa VH-DJH gene rearrangement (HC allelic exclusion)
- baixa Pre-BCR and IL-7 signals, baixa proliferation
- sobre RAG1/2
- sobe Light chain VLJL gene rearrangement
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.
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
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.
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.
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).
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.
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…
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.
