RESUMEN
Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.
Asunto(s)
Antineoplásicos/metabolismo , Sustancias de Crecimiento/metabolismo , Proteínas de la Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Células HT29/efectos de los fármacos , Humanos , Ligandos , Prueba de Cultivo Mixto de Linfocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Unión Proteica , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Homología de Secuencia de Aminoácido , Linfocitos T/efectos de los fármacos , Distribución Tisular , Factor de Transcripción AP-1/metabolismo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
TR2 (TNFR-related 2) is a recently identified member of the TNFR family with homology to TNFRII. We have demonstrated previously that TR2 mRNA is expressed in resting and activated human T cells and that TR2-Ig partially inhibits an allogeneic mixed leukocyte proliferation response. We now characterize TR2 further by the use of specific mAbs. Flow-cytometry analysis using TR2 mAbs confirmed that resting PBL express high levels of cell surface TR2, and that TR2 is widely expressed on all freshly isolated lymphocyte subpopulations. However, stimulation of purified T cells with either PHA or PHA plus PMA resulted in decreased surface expression within 48 h of activation before returning to resting levels at 72 h. TR2 mAbs inhibited CD4+ T cell proliferation in response to stimulation by immobilized CD3 or CD3 plus CD28 mAbs. Assay of culture supernatants by ELISA showed inhibition of TNF-alpha, IFN-gamma, IL-2, and IL-4 production, which, for IL-2 and TNF-alpha was also confirmed by intracellular cytokine staining. Furthermore, expression of activation markers on CD4+ T cells, including CD25, CD30, CD69, CD71, and OX40 (CD134), was inhibited. TR2 mAbs inhibited proliferation in a three-way MLR, and a response to soluble recall Ag, tetanus toxoid. In conclusion, these results suggest that TR2 is involved in the activation cascade of T cell responses and TR2 mAbs prevent optimal T cell proliferation, cytokine production, and expression of activation markers.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Citocinas/antagonistas & inhibidores , Activación de Linfocitos/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Virales/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Citocinas/biosíntesis , Epítopos de Linfocito T/inmunología , Inhibidores de Crecimiento/farmacología , Humanos , Inmunosupresores/farmacología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Simplexvirus/inmunología , Solubilidad , Linfocitos T/citología , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Lymphocyte proliferation assays were used to determine the ability of human and BALB/c T-lymphocytes to recognise and respond to in vitro challenge with purified preparations of four respiratory syncytial (RS) virus proteins. Human peripheral blood lymphocytes (PBLs) collected from adult donors as well as primed BALB/c mouse splenocytes each responded specifically to challenge with intact RS virus and preparations of the fusion (F), attachment (G), 23 kilodalton (23K), and 34K phospho- (P) proteins of the virus. F protein was recognised most frequently by human PBLs, and elicited higher levels of response than equivalent concentrations of the other protein preparations examined. The human PBL proliferative responses elicited by in vitro challenge with intact virus antigen as well as with each of the four protein preparations were found to be confined to the CD4+ T-helper (Th) sub-population of lymphocytes. However, proliferative responses to intact virus and F protein were found to be accompanied by only modest and inconsistent production of Interleukin-2 (IL-2). Finally, no evidence was obtained to indicate that any of the challenge antigens employed in this study were intrinsically mitogenic, as neither naive human cord blood lymphocytes, nor un-primed BALB/c mouse splenocytes proliferated when challenged with intact RS virus or with F, G, 23K, or P protein preparations.