HLA-DR Alpha 2 Mediates Negative Signalling via Binding to Tirc7 Leading to Anti-Inflammatory and Apoptotic Effects in Lymphocytes In Vitro and In Vivo

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRα2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-ζ chain & ZAP70, and inhibition of IFN-γ and FasL expression. HLA-DRα2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRα2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway

Identifizierung und Charakterisierung eines neuen Membranproteins, TIRC7, (T cell immune response cDNA7), als Target für Immunmodulation

HabilschriftThe membrane protein, TIRC7 (T cell immune response cDNA7) was identified as a molecule transiently expressed in T cells following activation by mitogens or allo-antigens via DDRT-PCR analysis. Results of several in vitro and in vivo studies, including those with TIRC7 null mice, indicate that TIRC7 is a key negative regulatory T cell molecule upstream of certain inhibitory pathways. Signals downstream to TIRC7 induce suppression in both T and B cell immune responses. TIRC7 molecule has been shown to serve as potential immunoregulatory target for the development of novel therapies for various immunological disorders.Durch DDRT-PCR-Analyse wurde das Membranmolekül TIRC7 (T cell immune response cDNA 7) identifiziert und gezeigt, dass es nach Aktivierung durch Mitogene oder Allo-Antigene vorübergehend auf T-Zellen exprimiert ist. Ergebnisse verschiedener in-vitro und in-vivo Studien (darunter auch solcher mit Knockout-Mäusen) weisen darauf hin, dass TIRC7 eine negativ-regulatorische Funktion ausübt und dabei die Unterdrückung der durch T-Zellen und B-Zellen vermittelten Immunantwort bewirkt. Es wurde gezeigt, dass TIRC7 als immun- modulatorisches therapeutisches Target zur Entwicklung neuartiger Therapien für die Behandlung verschiedener immunologischer Erkrankungen in Frage kommt

The Transmembrane Receptor TIRC7 Identifies a Distinct Subset of Immune Cells with Prognostic Implications in Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a heterogeneous malignancy with a dismal prognosis. Therapeutic options are largely limited to surgery and conventional chemotherapy offers limited benefit. As immunotherapy has proven highly effective in various cancer types, we have undertaken a quantitative immunohistopathological assessment of immune cells expressing the immunoinhibitory T cell immune response cDNA 7 receptor (TIRC7), an emerging immunoinhibitory receptor, in a cohort of 135 CCA patients. TIRC7+ immune cells were present in both the tumor epithelia and stroma in the majority of CCA cases with the highest levels found in intrahepatic CCA. While intraepithelial density of TIRC7+ immune cells was decreased compared to matched non-neoplastic bile ducts, stromal quantity was higher in the tumor samples. Tumors exhibiting signet ring cell or adenosquamous morphology were exclusively associated with an intraepithelial TIRC7+ phenotype. Survival analysis showed intraepithelial TIRC7+ immune cell density to be a highly significant favorable prognosticator in intrahepatic but not proximal or distal CCA. Furthermore, intraepithelial TIRC7+ immune cell density correlated with the number of intraepithelial CD8+ immune cells and with the total number of CD4+ immune cells. Our results suggest the presence and prognostic relevance of TIRC7+ immune cells in CCA and warrant further functional studies on its pharmacological modulation

Soluble HLA-DR alpha 2 domain inhibits IFN-γ cytokine expression and phosphorylation of STAT4, but not STAT6.

<p>A. Human PBL were activated with PHA for 48 h and co-cultured with either sHLA-DRα2 or control protein. Supernatants were subjected to quantitative sandwich ELISA. sHLA-DRα2 at a concentration of 50 µg/ml significantly inhibited the IFN-γ expression of stimulated PBL, whereas control protein exhibited no significant effect. B. No inhibition of IL-10 expression was observed. The results shown represent the means of five independent experiments, respectively. C. Human PBL were allo-activated for 4 h in the presence of anti-TIRC7 mAb and subjected to Western blot analysis. Equal volume of cells were fractionated by SDS-PAGE, and the phosphorylation status of STAT4 and STAT6 was determined by Western blot analysis. STAT4 phosphorylation was decreased in the presence of sHLA-DRα2 whereas pSTAT6 remained unchanged. No changes were observed using STAT4, and anti-Tubulin mAb. Shown is one representative experiment out of three independent experiments. D. Human PBL were isolated using standard Ficoll gradient centrifugation protocol. Cells were activated with PHA, anti-CD3/CD28 mAb, and MLR, respectively, and co-cultured with either sHLA-DRα2 or control protein in varying concentrations (50, 100, and 150 µg/ml). Cells were subjected to CFSE proliferation assays. A significant inhibition of proliferation was observed using sHLA-DRα2 in all proliferation assays whereas the control protein did not show any inhibition. The results shown represent the means of four independent experiments, respectively. E. The inhibition of proliferation of 48 h anti-CD3/CD28 stimulated human PBL by sHLA-DRα2 (100 µg/ml) was prevented by co-incubation of the anti-TIRC7 mAb 136 (100 µg/ml). F. Cell lysates were prepared from 1 h allo-activated PBL and immunoprecipitated (IP) with anti-TIRC7 mAb and subjected to western blot analysis. The immunoblot (IB) with anti-TIRC7 and anti-SHP-1 mAb showed the co-precipitation of SHP-1 and TIRC7. G. The phosphorylation of TCR-ζ chain and ZAP70 induced by IL-2 is inhibited by sHLA-DRα2. Human PBL were isolated and activated using anti-CD3/CD28 mAb for 18 h in the presence and absence of sHLA-DRα2. In the presence of sHLA-DRα2 (50 µg/ml) the phosphorylation of TCR-ζ chain and ZAP70 in stimulated cells (right lane) was reduced to a level similar to that of non-stimulated cells (left lane) while in stimulated cells without sHLA-DRα2 (middle lane) substantial phosphorylation of both proteins was observed.</p

sHLA-DR α2 binds to TIRC7 expressed in CD4 and CD8 T cells.

<p>CD4+ and CD8+ T cells were separated by magnetic beads, stimulated with anti-CD3/CD28 mAb for 48 h, and incubated with either sHLA-DR α2 or control protein for 30 min prior to confocal microscopic analysis. Using anti-human Fc-Cy3 mAb as secondary antibody, binding of sHLA-DRα2 or control protein to TIRC7 protein was analyzed. The results show a binding in CD4+ and CD8+ human T cells co-incubated with sHLA-DRα2 (upper panel) whereas no binding was observed in various control experiments using either control protein (lower panel) or anti-Fc-Cy3 conjugated secondary mAb only. Shown is one experiment out of three.</p