Ativação e diferenciação de Linfócitos T na periferia Flashcards Preview

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1

Papel dos linfócitos T na erradicação de infecções

A, CD4+ T cells (Th1, Th17) recognize antigens of phagocytosed and extracellular microbes and produce cytokines that recruit
and activate the phagocytes to kill the microbes. CD8+ T cells can also secrete some cytokines and participate in
similar reactions.


B, CD8+ cytotoxic T lymphocytes (CTLs) recognize antigens of microbes residing in the cytoplasm of infected cells and kill the cells.

2

Sequência de eventos para o estabelecimento da resposta imunológica mediada por linfócitos T CD4+

CD4+ T cells recognize peptides that are derived from protein antigens and presented by dendritic cells in peripheral lymphoid organs.

The T lymphocytes are stimulated to proliferate and differentiate into effector (and memory) cells, which enter the circulation and
migrate to sites of infection in peripheral tissues.

In the tissues, effector T cells
recognize the antigen and respond by secreting cytokines that recruit more
leukocytes and activate phagocytes to eradicate the infection.

3

Reconhecimento de antigénio e ativação de linfócitos T - Iniciação de uma resposta imunológica adquirida

Interação entre a célula T naïve e a APC:

• Sinal 1: Fornecido pela sinalização via TCR (interação do TCR com complexo MHC/péptido), co-recetor CD4 ou CD8 com MHC, e moléculas de adesão. Relevante para a iniciação da ativação de uma célula T naïve e ativação de células T efetoras.

• Sinal 2: Fornecido pela interações do recetor CD28 na célula T com moléculas coestimuladoras expressas pela APC (B7.1/B7.2). Aumenta a sobrevivência e a proliferação de células que receberam o sinal 1.

ICOS: recetor na célula T e
ICOSL: expresso por APCs =Sinal 2

• Sinal 3: Citocinas. Sinal que dita qual o tipo de diferenciação será adotado pelas células T ativadas (e.g. Th1, Th2, Th17).


The TCR/MHC-peptide interaction, along with CD4
and CD8 coreceptors and adhesion molecules, provide Signal 1. Costimulation by a separate set of molecules, including CD28 (or ICOS, not shown) provides Signal 2. Together, Signal 1 and Signal 2 initiate a signal transduction cascade that results in
activation of transcription factors and cytokines
(Signal 3) that direct T-cell proliferation (IL-2) and
differentiation (polarizing cytokines). Cytokines can act in an autocrine manner, by stimulating the same cells that produce them, or in a paracrine manner, by stimulating neighbouring cells.

4

Interações responsáveis pela ativação de células T

A schematic of the interactions between a CD4 T cell (left) or CD8 T cell (right) and its activating dendritic cell.

Dendritic cells process antigens and present peptides associated with MHC class II to CD4 T cells and present peptides associated with MHC class I to CD8 T cells.

Binding of TCR to MHC-peptide is enhanced by the binding of coreceptors CD4 and CD8 to MHC class II and class I, respectively. CD4 and CD8 also associate with Lck, which helps initiate signaling.

CD28 interactions with CD80/86 provide the required
costimulatory signals.

Adhesion molecule interactions, two of which (LFA-1/ICAM-1, CD2/LFA-3) are depicted, markedly strengthen the connection between the T cell and APC or target cell so that signals can be sustained.


(a) A successful T-cell/DC interaction results in the organization of signaling molecules into an immune
synapse. A scanning electron micrograph (left) shows the binding of a T cell (artificially colored yellow) and dendritic cell (artificially colored blue). A fluorescence micrograph (right) shows a cross-section of the immune synapse, where the TCR is stained with fluorescein (green) and adhesion molecules (specifically LFA-1) are
stained with phycoerythrin (red). Other molecules that can be found in the central part of the synapse (central supramolecular activation complex [cSMAC]) and the peripheral part of the synapse (pSMAC) are listed:

cSMAC:
TCR/CD3
Coreceptors CD4 or CD8
Costimulatory receptors
(CD28)

pSMAC:
Adhesion molecules
LFA-1/ICAM-1
LFA-3/CD2

5

Ativação de Linfócitos T

--> Iniciada pela interação do complexo TCR-CD3 com péptidos apresentados
por moléculas de MHC:
• CD8+ - MHC classe I
• CD4+ - MHC classe II
+ co-estimulação
+citocinas

--> Inicia uma cascata de eventos bioquímicos que induz uma célula a entrar
em fase ativa do ciclo celular, proliferar e diferenciar-se em célula efetoras e de memória

reconhecimento do Ag + ativação --> expansão clonal + diferenciação --> contração --> memória

6

Sinalização via TCR

Cinases LcK & ZAP-70 --> Fosforilação de ITAMs no
complexo CD3 e adaptadores (LAT e SLP-76)

Antigen recognition by T cells induces early signaling events, which include tyrosine phosphorylation of molecules of the T cell receptor (TCR) complex and the recruitment of adaptor proteins to the site of T cell antigen recognition.

These early events lead to the activation of several biochemical intermediates, which in turn activate transcription factors that stimulate transcription of genes whose products mediate the responses of the T cells.:

PLCgama1 activation --> increased cyt Ca2+ --> calcineurin --> NFAT
AND Diacylglycerol --> PKC --> NFkB, AP-1

GTP/GDP exchange on Ras, Ras --> Ras/Rac-GTP --> ERK, JNK --> NFkB, AP-1

Activation of Pl3 kinase --> PIP3 --> Akt, mTOR --> NFkB, AP-1; +protein synthesis


The possible effects of costimulation on these
signaling pathways are not shown. These signaling pathways are illustrated as independent of one another, for simplicity, but may be interconnected in more complex networks.

7

Co-estimulação é necessária para ativação de linfócitos T?

1. Co-estimulação é necessária para ativação de linfócitos T!!:
Experimental setup used anti-CD3 monoclonal antibodies or a mitogen, PMA, to provide Signal 1, and anti-CD28 antibodies to provide Signal 2.

(b) [3H]uridine incorporation an indicator of RNA synthesis was measured, in response to various treatments. Addition of stimulating anti-CD28 antibody increased RNA synthesis in response to activation by PMA or anti-CD3.

8

Papel da co-estimulação na ativação de linfócitos T

Resting antigen-presenting cells (APCs), which have not been exposed to microbes or adjuvants, may present peptide antigens, but they do not express costimulators and are unable to activate naive T cells. T cells that recognize antigen without costimulation may become unresponsive (tolerant) to subsequent exposure to antigen.

Microbes, as well as cytokines produced during innate immune responses to microbes, induce the expression of costimulators, such as B7 molecules, on the APCs. The B7 costimulators are recognized by the CD28 receptor on naive T cells, providing signal 2. In conjunction with antigen recognition (signal 1), this recognition initiates T cell
responses. Activated APCs also produce cytokines that stimulate the differentiation of naive T cells into effector
cells. IL, Interleukin.

9

Recetores de coestimulação e inibição

Recetores de co-estimulação:
• CD28
• ICOS

Recetores de inibição:
• CTLA4
• PD1

Ligands on APCs that are homologous to B7 bind to receptors on T cells that are homologous to CD28. Different ligand-receptor pairs serve distinct roles in immune responses. CD28 and ICOS are stimulatory receptors on T
cells, and CTLA-4 and PD-1 are inhibitory receptors.

10

Exemplo expressão/função CTL4?

• Células T naïve expressam CD28 que, por interação com moléculas coestimuladoras
presentes em APCs (B7.1
e B7.2) promove a sobrevivência e expansão das células T.

[cross-linking of CD28 delivers the co-stimulatory signal during activation of naive T cells and induces the expression of CTLA-4 (CD152)]

• Células T ativadas expressam níveis aumentados de CTLA4 (CD152) que possue uma maior afinidade para moléculas de B7.1 e B7.2, fornecendo um sinal inibitório à célula T ativada.

• CTLA4 serve assim como um regulador da fase proliferativa das células T

[CTLA-4 binds B7 (CD80 or CD86) more avidly than does CD28 and delivers inhibitory signals to activated T cells]

11

Células T efectoras podem ser ativadas na ausência de sinal 2!!

Sinal 1 (via TCR) é sempre
necessário para ativação

naive T cell stimulated by virus-infected DC --> T cell recognizes same antigen on infected epithelial cell --> activated T cell kills infected epithelial cells

12

Papel dos Recetores de “inibição”

• Papel importante na manutenção da tolerância na periferia

• Ajudam a reduzir a inflamação no curso natural de uma infeção e durante
infeções crónicas

ver a tabela?? específica para CTLA-4 e PD-1


Naïve T cells are activated when they engage both TCR and CD28 costimulatory receptors on antigen-presenting cells. Activated T cells up-regulate the coinhibitory molecule CTLA-4, which binds CD28 ligands even more strongly. This interaction inactivates the T cell. However, when the CTLA-4 engagement is blocked with the antibody ipilimumab, the T cell is reactivated.

costimulation via CD28: tcell activation

CTLA- 4 blocks costimulation: no tcell activation

ipilimumab blocks CTLA-4: t-cell activation

13

Sinais que induzem anergia

Resting T cells that engage antigen and costimulatory ligands CD80/86 are activated and expand.

(b) T cells that engage antigen on the surface of a non-antigen-presenting cell (such as a pancreatic islet beta cell) will not receive costimulatory signals and, instead, are inactivated or anergized.

(c) Anergy can also be induced when a T cell engages antigen and receives inhibitory rather than costimulatory signals through coinhibitory receptors such as CTLA-4 and PD-1.

14

Células apresentadoras de antigénio (APCs) profissionais

3 major classes of professional APCs.


- Dendritic cells are the best activators of naïve T cells. This may be due, in part, to their relatively high levels of expression of MHC and costimulatory molecules
when they are mature and activated.

-Activated B cells interact most efficiently with differentiated T cells that are specific for the same antigen that activated them.

-Macrophages play several different roles, processing and distributing antigen in
secondary lymphoid tissues as well as interacting with effector cells in the periphery.

!It is important to recognize that the distinctions shown are rules of thumb only. Functions among the APC classes overlap, and investigators now recognize
many different subsets within each major group of APCs, each of which may act independently on different T-cell subsets.

This diversity may be a consequence of activation by different innate immune receptors or may reflect the existence of independent cell lineages. Note that activation of effector and memory T cells is not as dependent on costimulatory interactions.

15

Key features - DCs and macrophages

-phagocytic
-express receptors for apoptotic cells, DAMPs, and PAMPs
-localize to tissues
-localize to T-cell zone of lymph nodes following activation (DCS)
-constitutively express high levels of MHC class II molecules and antigen-processing machinery
-express costimulatory molecules following activation
-variety of subtypes, some migrating, some resident, some better at cross-presenting
-specific subtypes may specialize in activating different T cells

16

Key features - B cells

-internalize antigens via BCRs
-constitutively express MHC class II molecules and Ag-processing machinery
-express costimulatory molecules following activation

17

Expressão de moléculas após ativação de células T

CTLA4 induzido 1 a 2 dias após ativação

The approximate kinetics of expression of selected molecules during activation of T cells by antigens and
costimulators are shown.

The illustrative examples include a transcription factor (c-Fos), a cytokine (IL-2), and
surface proteins. These proteins are typically expressed at low levels in naive T cells and are induced by activating signals.

CTLA-4 is induced 1 to 2 days after initial activation.

The kinetics are estimates and will vary with the nature of the antigen, its dose and persistence, and the type of adjuvant.

B, The major functions of
selected surface molecules are shown: Naive T cell + TCR --> CD69 Retention in lymph node --> IL-2R (CD25) Proliferation --> CD40L Amplification of effector functions --> CTLA-4 Control of Response

18

Papel do CD40 na ativação dos linfócitos T

T cells recognize antigen (with or without B7 costimulators)

=> CD40L is expressed on T cells and binds to CD40 on DC; leads to DC activation

=> DC expresses B7 and secretes cytokines, which enhance T cell activation

=> Enhanced T cell proliferation and differentiation

Pico expressão ~1 dia após ativação

Antigen recognition by T cells together with costimulation (not shown) induces the expression of CD40 ligand (CD40L) on the activated T cells. CD40L engages CD40 on APCs and may stimulate the expression of more B7 molecules and the secretion of cytokines that activate T cells. Thus, CD40L on the T cells makes the APCs better at promoting and amplifying T cell activation

Licensing of APC
Amplificação da função efetora

19

Papel do CD40L e citocinas na função de células T CD4 auxiliares

CD4+ T cells that have differentiated into effector cells express CD40L and secrete cytokines.

CD40L binds to CD40 on
macrophages or B lymphocytes, and cytokines bind to their receptors on the same cells.

The combination of signals delivered by CD40 and cytokine receptors activates macrophages in cell-mediated
immunity (A --> killing of phagocytosed microbes) and activates B cells to produce high-affinity isotype-switched antibodies in humoral immune responses (B --> secretion of antibodies with enhanced abilities to neutralize and eliminate antigens).

20

Regulação da expressão do recetor da IL-2

Resting (naive) T lymphocytes express the IL-2Rβγc complex, which has a moderate affinity for IL-2.

Activation of the T cells by antigen, costimulators, and IL-2 itself (secretion) leads to expression of the IL-2Ra chain (also called CD25) and increased levels of the high-affinity IL-2Rαβγc complex!!

--> IL-2-induced T cell proliferation

21

Atividade biológica da IL-2

IL-2 stimulates the survival, proliferation, and differentiation of T lymphocytes, acting as an autocrine growth factor, leading to the generation of effector and memory cells.

IL-2 also promotes the survival of regulatory T cells and maintains their functional capability, and thus controls immune responses (e.g., against self antigens).

22

Expansão e declínio da resposta imunológica
mediada por células T

• LT ativados por Ag na presença de co-estimulação começam a proliferar em 1 ou 2 dias, resultando na expansão de clones específicos para Ag
microbianos

• CD8- D0 1:10^5-10^6 específicos; 1 semana após infeção 10 a 20% de CD8 específicos para o vírus.
Expansão de CD4 100x a 1000x menor

• Algumas células T específicas para Ag microbianos diferenciam-se em células efetoras, cuja função é irradicar a infeção. Uma outra fração destas células diferenciam-se em
células de memória.


The numbers of CD4+ and CD8+ T cells specific for various antigens, and the clonal expansion and contraction during immune responses, are illustrated. The numbers are approximations based on studies of model microbial and other antigens in inbred mice; in humans, the numbers of lymphocytes are about 1000-fold greater.

23

Desenvolvimento de células T de memória

In response to antigen and costimulation, naive T cells differentiate into effector and memory cells.

According to the linear model of memory T cell differentiation, most effector cells die and some survivors develop into the memory population.

According to the branched differentiation model, effector and memory cells are alternative fates of activated T cells.

24

Propriedades das células de memória

=> As células de memória são capazes de sobreviver num estadio quiescente após eliminação do Ag e de montar respostas mais rápidas e de maior magnitude que as células naive.

• As células de memória expressam níveis mais elevados de proteínas anti-apoptóticas, que podem ser responsáveis pelo aumento da sua sobrevivência.
-> expressão de Bcl2 e Bcl-XL aumenta


• Estas respondem mais rapidamente a estimulação antigénica que células
naive específicas para o mesmo Ag.
-> Diferenciação de células naive ou células de memória em células efectoras: 5 a
7 dias vs 1 a 3 dias (modificações epigenéticas)

• O número de células de memória específicas para um Ag é consideravelmente maior que o numero de células naives específicas
para o mesmo Ag.
-> 10-100x > frequência

• Células de memória têm a capacidade de migrar para tecidos periféricos e responder a Ag nesses locais.
-> expressão de moléculas de adesão e recetores de quimiocinas diferentes; menor dependência de co-estimulação que células T naive

• As células de memória exibem proliferação lenta, e esta capacidade de auto-renovação (self-renewal) pode contribuir para a sua sobrevivência aumentada.

• A manutenção das células de memória é dependente de citocinas e independente de Ag.
-> IL-7 importante para a manutenção de células CD4 e CD8 de memória
-> As células de memória CD8 dependem também de IL-15

25

Subsets de células de memória

There are 3 major subsets of TM cells: TCM (central), TEM (effector) and TRM (resident).

These subsets are identified on the basis of differential expressions in several chemokine receptors and adhesion molecules.

While TCM cells express high levels of CCR7 and CD62L (L-selectin) (CCR7hiCD62Lhi),

TEM cells express low levels of CCR7 and CD62L (CCR7loCD62Llo).

These phenotypes indicate that
TCM cells circulate to SLOs, whereas TEM cells circulate to peripheral organs.


TRM cells express low levels of CCR7 and high levels of CD69 and CD103 (CCR7loCD69hiCD103hi) to remain in peripheral organs

26

Declínio da resposta imunológica mediada por células T

• A eliminação do Ag leva à contração das resposta T, e este declínio é responsável pela manutenção da homeostasia no sistema imunológico.

-> Ag desce --> co-estimulação e IL-2 desce --> expressão de moléculas anti-apoptóticas Bcl-2 e Bcl-XL desce

-> deprivação de fatores de crescimento --> ativação de sensors de stress celular -->
indução de apoptose de células ativadas à contração da pool de linfócitos T ativados específicos

-> Mecanismos reguladores, como expressão de recetores de inibição e.g. CTLA-4 e
PD-1 assim como a atividade de células T reguladoras, pensam-se estar envolvidos
na contração das respostas imunológicas