The Ras/MAPK Pathway Is Required for Generation of iNKT Cells

iNKT cells derive from CD4+CD8+ DP thymocytes, and are selected by thymocyte-thymocyte interactions through signals from their invariant Vα14-Jα18 TCR and from the costimulatory molecules SLAMF1 and SLAMF6. Genetic studies have demonstrated the contribution of different signaling pathways to this process. Surprisingly, current models imply that the Ras/MAPK pathway, one of the critical mediators of conventional αβ T cell positive selection, is not necessary for iNKT cell development. Using mice defective at different levels of this pathway our results refute this paradigm, and demonstrate that Ras, and its downstream effectors Egr-1 and Egr-2 are required for positive selection of iNKT cells. Interestingly our results also show that there are differences in the contributions of several of these molecules to the development of iNKT and conventional αβ T cells

Estudio de las asociaciones moleculares y la función del receptor linfocitario CD6

[spa] Los linfocitos T (LT) son activados cuando reconocen a través de su TCR el péptido antigénico presentado por el MHC. Esta es la primera señal y ha de ser amplificada para una correcta activación de los LT. Esta amplificación es realizada por parejas ligando-receptor con función co-estimuladora, como B7-CD28, que dan la llamada segunda señal. La zona de contacto entre el LT y la célula presentadota (APC) se denominada sinapsis inmunitaria (SI), y las moléculas accesorias y de señalización se van reorganizando formando una estructura denominada Complejo Supramolecular de Asociación o SMAC. CD6 pertenece a la superfamilia de receptores con dominios ricos en cisteínas tipo Scavenger (SRCR, Scavenger Receptors Cistein Rich). Tradicionalmente ha sido descrito como un receptor con función co-estimuladora pero el mecanismo por el que realiza esta función no es conocido. Este trabajo de tesis doctoral pretende ampliar los escasos conocimientos existentes sobre esta proteína. 1. Estudio de posibles asociaciones de CD6 con otros receptores linfocitarios: Mediante estudios bioquímicos (co-inmunoprecipitación) y de biología celular (FRET y modulación) hemos detectado la asociación física de CD6 a CD5 y al complejo CD3/TCR y proponemos una posible asociación a otras dos moléculas con función de adhesión como son CD44 e ICAM-3. 2. Implicación de CD6 y CD5 en los procesos de activación linfocitaria: Los receptores CD5 y CD6, así como el ligando de CD6 (ALCAM), son reclutados a la zona central de la SI. También describimos el reclutamiento de CD6 a los rafts. La presencia de la proteína recombinante soluble (rs) CD6 (rsCD6), a diferencia de rsCD5, produjo una inhibición de la proporción de SI madura. En estudios de proliferación de PBLs con diferentes estímulos también tanto rsCD6 como rsCD5 demostraron capacidad inhibitoria de forma dosis dependiente de ciertos estímulos. Por tanto la interacción de CD5 y CD6 a sus respectivos ligandos es importante para la correcta activación de los LT, demostrando estas proteínas tener capacidad inmunomoduladora. 3. Posibles asociaciones realizadas por la región citoplasmática de CD6: Mediante ensayos de doble híbrido detectamos la asociación de la región más C-terminal de CD6 a la proteína adaptadora syntenin-1. Syntenin-1 contiene una zona N-terminal seguida de dos dominios PDZ en tándem y fue descrita por primera vez por su interacción con syndecan. Nuestros resultados demostraron que en la interacción CD6-syntenin-1 participan los dos dominios PDZ de syntenin-1 así como los aa más C-terminales de CD6. También esta interacción la demostramos en PBLs humanas así como el acúmulo de syntenin-1 en la zona central de la SI. CONCLUSIONES 1- CD6 está físicamente asociado a CD5, lo cual sugiere que estos dos receptores pueden formar una unidad funcional compartiendo señales similares o complementarias. 2- CD6 está físicamente asociado al complejo CD3/TCR de forma independiente a CD5. Esta asociación convierte a CD6 en un co-receptor de este complejo y lo capacita para modular las señales mediadas por él. 3- CD6 y su ligando se acumulan en la zona central de la SI co-localizando con CD5 y el complejo CD3/TCR. Esta localización está de acuerdo con su capacidad potencial de modular la activación linfocitaria. 4- La región intracelular de CD6 interacciona con syntenin-1, una proteína con dominios PDZ. La demostración de la presencia de syntenin-1 en la zona central de la SI sugiere que esta proteína funciona como una proteína adaptadora permitiendo la unión de CD6 al citoesqueleto o a proteínas señalizadoras durante la formación y/o maduración de la SI. 5- La forma recombinante soluble CD6 (rsCD6) ha demostrado tener capacidad inmunomoduladora. Esto demuestra la relevancia de las interacciones mediadas por este receptor en los procesos de activación linfocitaria y plantea la su posible utilidad terapéutica.[eng] T cell activation needs the interaction of co-stimulatory molecules with their ligands presented by the APC. CD6 is a co-stimulatory molecule which exact function is not well known. For all that is the object of this doctoral thesis. 1- Study of the interactions of CD6 with other lymphocitary receptors: Biochemical (co-immunoprecipitation) and cell biological studies (FRET and modulation) showed physical association of CD6 to CD5 and to the CD3/TCR complex and we proposed a possible association to others molecules with adhesion function like CD44 and ICAM-3. 2. CD6 and CD5 implication on the lymphocitary activation process: Our results demonstrated that the CD5 and CD6 receptors, and the CD6 ligand (ALCAM), accumulated at the central zone of the immunological synapse (IS). Soluble forms of these proteins showed immunomodulatory capacity. 3- Study of interactions thought the CD6 cytoplasmic region: By two-hybrid assays we detected the association of the most C-terminal region of CD6 with the adaptor protein syntenin-1 with a tandem of PDZ domains. Our results showed that in the interaction CD6-Syntenin-1 were implicated both syntenin-1´s PDZ domains and the four amino acids most C-terminals of CD6. Syntenin-1 also accumulated at the central zone of the IS. CONCLUSIONS: 1- CD6 is physically associated to CD5. This suggest that both receptors can form a functional unit shearing similar or complementary signals. 2- CD6 is physically associated to the CD3/TCR complex, independently of CD5. This association converts CD6 in a CD3/TCR co-receptor with capacity of modulate its signals. 3- CD6 and its ligand (ALCAM) accumulate at the central zone od the IS, co-localizing with CD5 and the CD3/TCR complex. This association agrees with its potential capacity of modulate the lymphocitary activity. 4- The intracellular region of CD6 interacts with syntenin-1, a protein with PDZ domains. The demonstration of the presence of syntenin-1 at the central zone of the IS suggests that this protein functions as an adaptor protein allowing the union of CD6 to the citoskelleton or to signalling proteins during the formation and/or maturation of the IS. 5- The soluble form of CD6 has immunomodulatory capacity. This demonstrate the relevance of the CD6 mediated interactions during the lymphocitary activation process and propose its possible therapeutic utility

dnRas mice lack iNKT cells.

<p>(<b>a</b>) Thymic profile of WT and dnRas mice (<b>b</b>) Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of normal littermate controls (WT) and dnRas mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. (<b>c</b>) Percentages and absolute numbers of iNKT cell subpopulations in gated TCR^hiPBS57-CD1dtet⁺ thymocytes from WT and dnRas mice. Results representative of nine independent dnRas and WT pairs analyzed in five experiments<b>,</b> except for the CD44/NK1.1 histograms (n = 2). The bar graphs show the average and SEM of all the experiments. Significance as assessed using a two-tailed unpaired t-test. ***<0.001, **<0.01. (<b>d</b>) Gating strategy used to sort the different populations. (<b>e</b>) Expression of Egr-1, Egr-2 and Id3 in sorted Tet⁺ HSA^hi and Tet⁺ HSA^lo from normal littermate control (WT) and dnRas mice. Bar graphs show relative expression of dnRas compared to WT for three independent experiments. Each experiment was an independent sort of a WT and dnRas pair. Expression in each experiment was normalized to the expression levelis in Tet⁺ HSA^hi WT cells. Significance as assessed using a two-tailed unpaired t-test. **<0.01.</p

Defects in Slamf1, Slamf6 and CD1d expression in dnRas, but not Egr-1,2 double knockout mice.

<p>Slamf1, Slamf3, Slamf6, Slamf5 and CD1d expression levels in DP thymocytes mice were assessed by flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations. In <b>(A)</b> WT vs. dnRas (n = 5). In <b>(B)</b> WT vs. Egr1^-/-;Egr2^f/f-<i>lck</i>-Cre (Egr-DKO) (n = 3). <b>(C)</b> SAP and Bcl_xL expression levels in DP thymocytes from WT or dnRas mice were assessed by intracellular flow cytometry. Shown are representative histograms, and the mean and SEM of the normalized MFI of DP populations (n = 5). To normalize the MFI, we averaged the MFI for the WT mice in each experiment and considered that value 1. The bar graphs show the average and SEM of all the experiments. Significance was assessed using the unpaired t-test ***<0.001, **<0.01 *<0.05<b>.</b></p

Egr1^-/-; Egr2^f/f<i>lck</i>cre mice have a complete block in iNKT development.

<p>(<b>a</b>) Thymic profile of littermate wild-type (WT), or Egr1^-/-;Egr2^f/f<i>lck</i>cre (DKO) mice <b>(b)</b> Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and EgrDKO mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. <b>(c)</b> Percentages and absolute numbers of iNKT cell subpopulations in gated thymic TCR-βhiPBS57-CD1dtet⁺ of WT and EgrDKO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance as assessed using the unpaired t-test. ***<0.001, **<0.01</p

Egr1 and Egr2 contribute in a quantitatively different manner to iNKT cell development.

<p>(<b>a</b>) Contribution of WT and Egr1^-/- (Egr1KO) cells to the iNKT compartment in thymus (T), spleen (S) and liver (L) of mixed bone marrow chimeras generated by injecting Egr1^-/- (CD45.2) and F1(C57BL/6xB6-LY5.2/Cr) (CD45.1;CD45.2) bone marrow cells into lethally irradiated B6-LY5.2/Cr recipient mice (CD45.1). Mean and SEM is shown on the right (n = 5). Significance was assessed using a paired t-test. **<0.01, *<0.05. This is one out of two independent experiments. <b>(b)</b> Percentages and absolute numbers of iNKT cells in the thymus (T), spleen (S) and liver mononuclear cells (L) of WT and Egr-2^f/f-<i>lck</i>-Cre (EGR2KO) mice stained with CD4, CD8, PBS57-loaded CD1d tetramer and TCRβ. <b>(c)</b> Percentages and absolute numbers of iNKT cell subpopulations in gated TCR-β^hiPBS57-CD1dtet⁺ thymocytes from WT and EGR2KO mice. Results representative of five independent pairs in three independent experiments. The bar graphs show the average and SEM of all the experiments. Significance was assessed using an unpaired t-test. ***<0.001, **<0.01.</p