Characterization of the in vivo immunomodulatory properties of CD5 and CD6

Abstract

Our goal in this doctoral thesis was to study the immunomodulatory effects of CD5 and CD6, two proteins expressed on the lymphocytes membrane. These two proteins belong to the Scavenger Receptors Cystein- Rich superfamily, characterized by the presence of one or more cysteine rich domains. The interaction between CD6 and ALCAM, its principal ligand, is well established but recently more ligands of CD6 have been described. On the other hand, to date there is no consensus about the CD5 ligand/s, although several candidates have been proposed. Previous work carried out with CD5 and CD6-deficient mice revealed the role as negative modulators of the T/B cell receptor signaling. It was observed how its blockade/absence led to a hyper-activation post T cell activation by increment of cell proliferation and calcium mobilization.

We decided to generate a transgenic mouse that would expresses high levels of the extracellular region of human CD5 constitutively, in order to block the interactions mediated by membrane-bound CD5 with its ligand/s. This “functional knockout" would resemble what would happen in a clinical application, by injecting the soluble recombinant protein to humans. These mice presented indeed soluble human CD5 in serum, which resulted in immunophenotypical changes that were reproduced in wild-type mice after repeated administration of exogenous rshCD5 protein. The transgenic mice did not reveal any gross alterations in major lymphocyte subpopulations. However, a decrease in T and B cell subsets with regulatory function, as well as an increment in NKT cells were observed. They also presented more severe disease outcome to two different induced experimental autoimmune disease models and a better anti- tumor response against murine melanoma cells (B16-F0) and a genetically modified line of thymoma tumor cells (EG7-OVA). We have observed that this effect is due to the increase in number and activity of the effector cells of the immune system as well as the lowering of the regulatory T cells in the tumor draining lymph nodes. On the other hand, wild-type mice challenged with melanoma showed an improved anti-tumor response as well as changes in the tumor draining lymph node after therapeutic administration of the protein. In addition, in this model, a decrease in IL-6 expression levels was observed, and the loss of efficacy obtained with CD5 administration by depleting NK cells.

We also generated another murine model expressing elevated levels of human soluble CD6 in a constitutive way, with the objective of blocking the heterophillic and homophillic interactions of CD6 and its ligands. These mice had a decrease in the spleen and lymph nodes total cell number. Their B cells have reduced proliferative capacity and the regulatory T cell less suppressive activity aside to a reduction of their number. We observed an increment of the anti-tumor response to different tumor cell lines, but their autoimmune response was not exacerbated, as it was expected. These results were reproduced when recombinant soluble human CD6 was injected repeatedly into wild-type mice.

The results obtained demonstrate that counteracting the function of membrane-bound CD5/CD6 with sCD5/CD6 may enhance current immunotherapies for cancer, as well as be used in other possible applications such as in acute or chronic infections (CD5 recognizes fungal and viral components, and CD6 bacterial components) and vaccines.

El objetivo de esta tesis doctoral ha sido el estudio de los efectos inmunomoduladores de CD5 y CD6, dos proteínas expresadas en la membrana de los linfocitos. Estas dos proteínas pertenecen a la superfamilia de receptores "Scavenger Receptor Cystein-Rich", caracterizada por la presencia de uno o varios dominios ricos en cisteínas. En cuanto a CD6, están descritos varios ligandos, sin embargo, a día de hoy no existe un consenso acerca del ligando/s de CD5.Trabajos previos llevados a cabo con ratones deficientes para CD5/CD6, pusieron de manifiesto sus papeles como moduladores negativos de la señal del receptor de células T/B.

Decidimos generar un ratón que expresara niveles elevados de la región extracelular de CD5 humano constitutivamente, con el objetivo de bloquear las interacciones mediadas por el CD5 de membrana con su ligando/s. Observamos que estos ratones presentaron una respuesta autoinmune exacerbada pero mejor respuesta anti-tumoral frente a células murinas de melanoma y linfoma. Este efecto se debió al aumento de número y actividad de las células efectoras del sistema inmune, así como a la bajada de las células T reguladoras en los ganglios drenantes del tumor. Por otro lado, ratones silvestres a los que se les indujo melanoma mostraron una respuesta anti-tumoral mejorada, así como cambios en el ganglio drenante, tras la administración de la proteína de forma terapéutica. Además, la eficacia obtenida con la administración de CD5 se perdió al deplecionar las células NK.

Además, se generó otro modelo murino que expresara niveles elevados de CD6 soluble humano de manera constitutiva, con el mismo objetivo. Estos ratones presentaron una bajada en cuanto al número de células en bazo y nódulos linfáticos debido a la capacidad proliferativa reducida de los linfocitos B. Además de una disminución en número, las células T reguladoras presentaron una menor actividad supresora. Así, observamos un aumento de la respuesta anti-tumoral frente a diferentes líneas tumorales, pero su respuesta autoinmune no se encontraba exacerbada. Estos resultados se reprodujeron cuando se inyectó CD6 soluble a ratones silvestres.

Los resultados obtenidos denotan la importancia de CD5 y CD6 en la respuesta inmune, y su posible aplicación en combinación con las actuales terapias inmunomoduladoras.