Respuesta inmune adaptativa

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Ángel Torregrosa Rojas
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1 CURSO DE FORMACIÓN CBMSO-UAM 2012 Respuesta inmune adaptativa Balbino Alarcón CBMSO 19 de enero de 2012

7 La respuesta inmune adaptativa tiene dos características distintivas: -Existencia de un repertorio inmenso de receptores específicos para prácticamente cada antígeno. -Memoria.

8 La recombinación de segmentos génicos es uno de los orígenes del repertorio

9 El resultado es un conjunto de linfocitos B (y T) que tienen receptores preparados para reconocer de forma específica su antígeno y sólo su antígeno. Receptor para antígeno de los linfocitos B (BCR)

10 Desarrollo y origen de la memoria en la respuesta humoral

11 Los centros germinales son esenciales para la respuesta de memoria humoral

12 Germinal centers are sites of intense B cell proliferation, somatic hypermutation and class switch

13 Características de la memoria en linfocitos B: -Expansión clonal (mayor número de linfocitos capaces de responder). -Cambio de isotipo (inmunoglobulinas G, A, E en lugar de M). -Rapidez de respuesta. -Maduración de afinidad.

14 THE T-CELL ANTIGEN RECEPTOR (TCR-CD3) COMPLEX V V ITAM- Immunoreceptor Tyrosine-based Activation Motif

15 T cell developtment Progenitors * Negative selection DN1 CD8+ CD8+ DN2 DP Positive selection gd DN3 b-selection DN4 CD4+ CD4+ Cortex Medula Thymus

16 Los receptores de los linfocitos T siempre reconocen antígenos unidos a receptores de las células que los presentan

17 El antígeno reconocido por los linfocitos T CD8+ (citotóxicos) es un péptido en una cavidad de la cadena pesada de MHC de clase I

18 El antígeno reconocido por los linfocitos T CD4+ (cooperadores) es un péptido en una cavidad de la cadena pesada de MHC de clase II

19 no. thymocytes Affinity model for positive and negative selection death by neglect positive selection negative selection Avidity

20 Los linfocitos T necesitan para activarse integrar señales de distintos receptores

21 Dependiendo de las citoquinas presentes, las señales del TCR son moduladas para conducir a la diferenciación de los linfocitos T CD4+ a diferentes tipos de células efectoras

22 Type Priming cytokines Transcription factors Effector cytokines Functions Immunopathology Th1 IL-12 T-bet, STAT-1 STAT-4 IFN-γ Intracellular pathogens Autoimmunity, cell-mediated response Th2 IL-4 GATA-3, STAT-6 IL-4 Extracellular pathogens Asthma, allergy, atopy Th17 TGF-β + IL-6 ROR-γt, ROR-α, STAT-3 IL-17, IL-22 Extracellular bacteria Autoimmune inflammation Th1 differentiation is a complex process at a molecular level initiated by activation of CD4 + T cells with antigen in the presence of IL-12, a heterodimeric cytokines composed of p35 and p40 subunits. The production of IFN-γ from Th1 cells further enhances Th1 development by STAT-1 signaling pathway, which in turn up-regulates T-bet, a master transcription factor required for Th1 differentiation and development [7]. Activation of T-bet further increases the responsiveness of Th1 cells to IL-12 by up-regulating IL-12 receptor β2, which in turn stabilizes Th1 phenotype [8].

24 PROXIMAL SIGNALLING COMPLEXES AND DOWNSTREAM RESPONSES INDUCED BY T- CELL RECEPTOR LIGATION Src family domains Syk family domains

25 TCR signaling pathways T cell ACTIN CYTOSKELETON INTEGRIN AVIDITY GENE EXPRESSION

26 -T cells are activated by as few as apropriate antigen/mhc complexes in a sea of hundred thousands of MHC molecules with self-peptides. T CELLS SHOW HIGH SENSITIVITY -The difference between an agonist peptide and a null agonist is frequently of only one amino acid. T CELLS SHOW HIGH SPECIFICITY -The difference between the first concentration of antigen that causes some activation effect and the concentration at which the response becomes saturated is often of 10E5-to-10E7 fold. T CELLS HAVE A WIDE DYNAMIC RANGE -T cells recognize antigen using a receptor, the TCR, that bears poor thermodynamic and kinetic properties.

27 TCR signaling models -Kinetic proofreading koff kon -Crosslinking -Conformational change -Seggregation phosphatases -Dissociation in composing subunits -Serial triggering internalization

28 TCR crosslinking as the molecular basis for initiation of TCR signaling SIGNAL TRANSDUCTION -Boniface, JJ,...Davis, MM. (1998). Immunity 9: Cochran, JR,... Stern, LJ. (2000). Immunity 12:

29 APC with a diameter of 20 mm loaded with 100 agonist peptide molecules 2 mm 10 nm - - plasma membrane

30 The induced binding of the TCR to the N-terminal SH3 domain of Nck shows the existence of a conformational change in the TCR MHC/antigen a b a b g e z z d e Nck P P Nck N Nck SH3.1 SH3.2 SH3.3 SH2 C Cbl CD3-e Pak Sos Cbl WIP WASp SLP76 EGFR

31 Vibration of the de dimer in solution CD3e CD3d

32 UCHT1 d e STALK

Amino Some acid amino residues acids of the at the CD3e bottom stalk sequence of CD3e undergo may a stabilize major shift the after active anti-cd3 conformation binding em eh A B C C E F G STALK

33 Amino Some acid amino residues acids of the at the CD3e bottom stalk sequence of CD3e undergo may a stabilize major shift the after active anti-cd3 conformation binding em eh A B C C E F G STALK DDAEN I------EYKVSISGTSVELTCPLDSDENLKWEKNGQEL DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFS P-QKHDK HLVLQDFSEVEDSGYYVCYTPA S---NKNTYLYL KARVCEYCVEVD ELEQSGYYVCYPRGSKPEDANFYLYL RARVCENCMEMD TM C80- -C80 active (stiffed) inactive (loose) Martínez-Martín, & Alarcón, (2009). Cooperativity between T cell receptor complexes revealed by CD3e conformational mutants. Science Signaling 11;2(83):ra43

34 Expression of conformational mutants of CD3e exert a dominant-negative effect on the transmission of the conformational change d e anti-cd3 2B4 hybridoma GST-Nck pull-down

35 A conformational change contributes to initiation of TCR signaling OLIGOMERIZATION CONFORMATIONAL CHANGE SIGNAL TRANSDUCTION

36 Wang. & Kappler. (2009). A conserved CXXC motif in CD3e is critical for T cell development and TCR signaling. PLOS Biol. 7(12): e

37 Expression of conformational mutants of CD3e exert a dominant-negative effect on the transmission of the conformational change d e anti-cd3 2B4 hybridoma GST-Nck pull-down

38 2B4 hybridoma Doses of mutant as low as 3% of total CD3e exert a dominant negative effect

39 5x 100x The TCR is organized on the surface of pre-activated T cells as oligomers of different length

40 Phosphorylation of the largest TCR oligomers at low doses of antigen

41 The Journal of Experimental Medicine Schamel et al, 2005 Aug 15;202(4): Epub 2005 Aug 8.

42 Cooperativity between TCR complexes

43 The presence of multivalent TCR complexes could be necessary for the conformational change, could increase the avidity of antigen/mhc, and could constitute a platform for signal amplification signal spreading MHC/antigen conformational change

44 Models of allostery for membrane receptors Monod-Wyman-Changeux Monod-Wyman-Changeux agonist peptide self peptide Koshland-Néméthy-Filmer Bray-Duke conformational spread Martínez-Martín, & Alarcón, (2009). Cooperativity between T cell receptor complexes revealed by CD3e conformational mutants. Science Signaling 11;2(83):ra43

45 Both proportion and size of multivalent TCR complexes increase in antigen-experienced T cells naïve Ag-experienced

46 % of total number of gold particles Both proportion and size of multivalent TCR complexes increase in antigen-experienced T cells naïve * ** *** Ag-experienced > >20 Cluster size (gold particles per cluster) OT-I TCR transgenic

47 Antigen sensitivity of memory vs. naïve CD8+ T cells IFNg expression vs. Tetramer binding IFNg (MFI) naive IFNg naïve MFI IFN-g memoria IFNg memory MFI IFN-g Tetramer naive unión naïve Tetramer memoria memory unión tetramer binding (MFI) 0 0, ,001 0, MHC tetramer (nm) 0 OT-I transgenics LN

48 Una mutación en el dominio transmembrana de CD3z impide la formación de oligómeros del TCR L C Y L LD G IL F I Y G V I I T A L Y L L CY L LD G IAF I Y G V I I T A L Y L L CY L LD G IL F I Y G V I I T A AY L m

49 CD3z podría ser el pegamento entre unidades del TCR, permitiendo la formación de oligómeros

50 Avidity maturation in T cells?

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Respuesta inmune adaptativa

CURSO DE FORMACIÓN CBMSO-UAM 2010 Respuesta inmune adaptativa Balbino Alarcón CBMSO 5 de julio de 2010 La respuesta inmune adaptativa tiene dos características distintivas: -Existencia de un repertorio