CD6 modulates thymocyte selection and peripheral T cell homeostasis

The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6(-/-) thymi showed a reduction in both CD4(+) and CD8(+) single-positive subsets, and double-positive thymocytes exhibited increased Ca(2+) mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell-autonomous selective disadvantage of CD6(-/-) T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6(-/-) mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4(+)TEM and CD8(+)TCM) and regulatory (T reg) T cells. The suppressive activity of CD6(-/-) T reg cells was diminished, and CD6(-/-) mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells

Implicacions funcionals i terapèutiques de les interaccions moleculars mitjançades per CD5 i CD6 en les respostes immunitàries

[cat] Els receptors de superfície CD5 i CD6 són receptors altament homòlegs amb dominis tipus scavenger, expressats principalment als timòcits i cèl·lules T madures. Els dos es troben associats al receptor de cèl·lules T (TCR) actuant com a moduladors de les senyals transmeses a nivell intracel·lular. Pel que fa a CD5, s’ha pogut determinar en diferents estudis la seva funció com a modulador negatiu de la senyal del TCR en timòcits i cèl·lules T madures a través del seu domini citoplasmàtic. En canvi, la manca de models animals ha fet que el rol biològic de CD6 encara sigui una incògnita, tot i que sí s’ha demostrat que esta implicat en la formació i estabilització de la sinapsi immunològica a través de la unió al seu lligand CD166/ALCAM. Estudis del nostre laboratori van demostrar que curiosament les regions extracel·lular de CD5 i CD6 son capaces d’unir-se a fongs i bactèries, respectivament, tot i que la rellevància biològica d’aquesta interacció encara esta per determinar. Per tal d’aprofundir en el paper que juguen aquests dos receptors limfocitaris, el nostre laboratori ha adquirit recentment dos ratolins transgènics knock-out per CD5 i CD6 (CD5-/- i CD6-/-) amb la finalitat de caracteritzar fenotípica i funcionalment la nova línia de ratolins deficients per a CD6-/- tant en condicions normals como d’agressió biològica, i establir analogies fenotípiques i funcionals entre els nous ratolins CD6-/- i els prèviament descrits CD5-/-. L’anàlisi de les poblacions limfocitàries va revelar que els ratolins CD6-/- presentaven una disminució al timus de les poblacions CD4+ i CD8+SP (single positive), i que les cèl·lules doble positives CD4+CD8+ (DP) presentaven augmentada l’alliberació de calci intracel·lular en resposta a l’estimulació via TCR. Es van realitzar experiments amb quimeres mixtes de medul·la òssia que van demostrar una desavantatge selectiva de les cèl·lules T CD6-/- durant el desenvolupament, fet confirmat al observar que ratolins transgènics pel TCR (OT-I i Marilyn) presentaven una selecció de cèl·lules T anormal en absència de CD6. L’estudi de les cèl·lules T perifèriques CD6-/- va revelar que aquestes proliferaven menys in vivo però més in vitro davant l’estimulació del TCR amb anticossos anti-CD3, així com que els ratolins CD6-/- tenien més cèl·lules de tipus memòria/efectores i T reguladores. De manera rellevant, es va veure que els ratolins CD6-/- eren més sensibles a patir fenòmens d’autoimmunitat, degut a una artritis induïda per col·lagen més severa en aquests animals relacionada amb una patró de citocines pro-inflamatòries més elevat. Pel que fa a la resposta davant un xoc sèptic en la seva funció com a receptors de patòngens, els ratolins CD6-/- van presentar una major sensibilitat a la mort per peritonitis polimicrobiana, però no en canvi els ratolins CD5-/- a una peritonitis induïda per derivats fúngics. Un estudi més detallat va revelar que la soca de ratolins C57BL/6 era deficient en la secreció d’IFN-γ comparat amb la soca CD1, i que l’administració d’IFN-γ recombinant juntament amb la proteïna CD5 soluble augmentava la supervivència dels animals de manera estadísticament significativa. En conclusió, en aquesta tesi doctoral s’ha demostrat que CD6 regula el llindar d’activació dels timòcits i de les cèl·lules T perifèriques, i que l’expressió d’aquest receptor dóna resistència a fenòmens autoimmunitaris i a la mort per peritonitis polimicrobiana. Per altra banda, també s’ha vist que l’administració d’IFN-γ recombinant soluble juntament amb la proteïna CD5 protegeix de la mort induïda per un xoc sèptic induït per derivats fúngics.[eng] The CD6 glycoprotein, as its related receptor CD5, is a lymphocyte surface receptor putatively involved in T-cell development and activation. CD6 facilitates adhesion between T-cells and APC through its interaction with CD166/ALCAM, and physically associates with the TCR at the centre of the immunological synapse. However, its precise role during thymocyte development and peripheral T-cell immune responses remains to be defined. Interestingly, a report published by our group showed that both CD6 and CD5 can bind bacteria and fungi, respectively, although so far the biologic relevance of this interaction has not been defined. Here, we analyze the in vivo consequences of CD6-deficiency. CD6-/- thymi showed a reduction in both CD4+ and CD8+ single-positive subsets, and double-positive thymocytes exhibited increased Ca2+ mobilization to TCR-crosslinking in vitro. Bone marrow chimera experiments revealed a T-cell autonomous selective disadvantage of CD6-/- T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T-cell selection events occur in the absence of CD6. CD6-/- mice displayed increased frequencies of antigen-experienced peripheral T-cells generated under certain level of TCR signal strength and/or co-stimulation such as effector/memory (CD4+TEM, CD8+TCM) and regulatory (Treg) T-cells. Furthermore, CD6-/- mice presented an exacerbated autoimmune response to collagen as well as higher mortality in a polimicrobian induced septic shock. In contrast, CD5-/- mice responded equally to WT in a fungi induced septic shock. However, a detailed analysis revealed that C57BL/6 mice were deficient in the secretion of IFN-γ, and that the administration of soluble IFN-γ in combination with soluble CD5 protects mice from the septic shock like syndrome. Taken together these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T-cell subpopulations including Treg cells

Multifaceted effects of soluble human CD6 in experimental cancer models

© Author(s) (or their employer(s)) 2020.[Background]: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. [Methods]: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEμTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. [Results]: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. [Conclusions]: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.This work was supported by the Worldwide Cancer Research (14-1275), Fundació La Marató TV3 (201319-30), and Ministerio de Economía y Competitividad (SAF-2016-80535-R) co-financed by European Development Regional Fund 'A way to achieve Europe' ERDF to FL; SAF2016-75195-R to JM, SAF2017-82905-R to RM, and SAF2015-70028-R to GG-A. ITS, MO-M, MV-dA, CC, SC-L and FA are recipients of fellowships from Fundação para a Ciência e a Tecnologia (SFRH/ BD/75738/2011), Ministerio de Economía y Competitividad (BES-2011-048415; BES-2014-069237; BES-2017-082107), Ministerio de Educación Cultura y Deporte (FPU15/02897), and Instituto de Salud Carlos III (Sara Borrell Programme, CD15/00016), respectively

Multifaceted Effects of Soluble Human CD6 in Experimental Cancer Models

Background: CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. Methods: High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckEμTg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. Results: Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. Conclusions: Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer

CD6 modulates thymocyte selection and peripheral T cell homeostasis

The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6(-/-) thymi showed a reduction in both CD4(+) and CD8(+) single-positive subsets, and double-positive thymocytes exhibited increased Ca(2+) mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell-autonomous selective disadvantage of CD6(-/-) T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6(-/-) mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4(+)TEM and CD8(+)TCM) and regulatory (T reg) T cells. The suppressive activity of CD6(-/-) T reg cells was diminished, and CD6(-/-) mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells