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

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

HLA-DR alpha 2 domain interacts with TIRC7 protein.

<p>A. Strain AH109 carrying GAL4 activation domain, fused to cDNA containing HLA-DR, was tested for interaction with indicated domains of TIRC7 (N-terminus aa 1-173, large extracellular domain aa 438-512 or C-terminus aa 586-614) as bait. The growth of combined clones on Histidine-negative agar plates indicate a specific interaction between the HLA-DR alpha 2 and TIRC7 extracellular domain whereas no interaction between HLA-DR alpha 2 to the C – terminal or N- terminal domain of TIRC7 was observed. The growth of colonies on Histidine positive agar plates which represents a positive control, is shown in the left panel. B. Lysates were prepared from 1 h allo-activated PBL and Jurkat cells. Lysates were immunoprecipitated (IP) with anti-TIRC7 mAb and immunoblotted (IB) with specific antibody against HLA-DR protein or TIRC7 in denaturing conditions. Co-precipitation of TIRC7 and HLA-DR is observed in PBL whereas only TIRC7 was precipitated in HLA-DR negative Jurkat cells. C. COS7 cells were transiently transfected with a TIRC7-<i>myc</i> fusion protein vector construct, incubated with sHLA-DRα2 and stained with secondary anti-human Fc protein-Cy3 conjugated mAb. Flow cytometry analysis revealed that sHLA-DRα2 solely binds to TIRC7 transfected COS7 cells and fail to bind to non-transfected COS7 control cells. Shown is one experiment out of four. D. COS7 cells transfected with TIRC7-<i>myc</i> fusion protein showed concentration-dependent binding of sHLA-DRα2 (0, 25, 50, 150 µg/ml) using direct immunofluorescence method. No binding of control protein was observed in transfected COS7 cells. Shown is one experiment out of three. E and F. TIRC7 deficient and wild-type mouse splenocytes were isolated and either stimulated with PHA for 48 h or remained unstimulated. Cells were incubated with either human HLA-DR alpha 2 or human control protein prior to flow cytometry (E) or confocal microscopic analysis (F) using anti-human Fc-specific Cy3 as secondary antibody. The results show that HLA-DR alpha 2 solely binds to stimulated WT cells in flow cytometric and microscopic analyses. Shown is one experiment out of three, respectively.</p

Scheme of proposed model of regulation of immune activation via TIRC7-HLA-DR alpha 2 binding.

<p>TIRC7 serves as ligand for HLA-DR alpha 2 upon TCR activation in the early phase of immune activation. After positive signals were received and immune cells are activated, TIRC7 is expressed on the cell surface (A) and its binding to HLA-DR alpha 2 transduces negative signals to lymphocytes (B).</p

TIRC7 signals triggering prevents T cell - APC interaction reflected in inhibition of several cytokines.

<p>A. Human PBL were isolated using standard Ficoll gradient centrifugation protocol and culture 14 days to induce monocyte differentiation. Either sHLA-DRα2 (50 µg/ml) or control (50 µg/ml) were co-incubated and subjected to cytokine release assays. Cytokine levels were measured by quantitative real time PCR which revealed a profound inhibition of MCP-1, IL-12, IFN-γ, TNF-α and Rantes expression compared with the control. B. Human sHLA-DRα2 showed specific cross-reactivity on balb/c mice splenocytes (left side). For the in vivo functional analysis balb/c splenocytes were isolated and further subjected to microscopic analysis for cross-reactivity of human sHLA-DRα2 to mouse TIRC7 which resulted in significant binding on activated splenocytes. C. In balb/c mice (n = 14) LPS was administered followed by a single dose (100 µg/mice, i.p.) of human sHLA-DRα2 (200 µg/day, i.p.). Human sHLA-DRα2 resulted in a significant down-regulation of IFN-γ, TNF-α and Rantes after 24 h treatment. sHLA-DRα2 or control protein treated splenocytes were subjected to immunobloting using mAb against caspase 7. Activation of caspase 7 is indicated by the appearance of cleaved fragments with a size of 20 kDa in sHLA-DRα2 treated animals (a1 and a2) whereas no activation of caspase 7 was observed in controls. Shown are two examples out of five.</p

Targeting of TIRC7 in lymphocytes induces apoptosis via the mitochondrial intrinsic pathway.

<p>A. PBL were co-incubated 50 µg/ml sHLA-DRα2 or control protein for 5 h, and immunobloting using mAb against caspase 9, caspase 8, caspase 7 and caspase 3 was performed. Activation of caspase 9 and caspase 7 is indicated by the appearance of cleaved fragments with a size of 37 and 20 kDa, respectively. No activation of caspase 8 and 3 was observed. B. Human PBL were isolated, activated with anti-CD3/CD28 antibodies (a) cultured in the presence of 50 µg/ml sHLA-DRα2 (d), and subjected to flow cytometric analysis. In comparison to non-treated controls (b,c) stimulated human T cells incubated with 50 µg/ml sHLA-DRα2 (d) demonstrated a remarkably reduced expression of FasL as detected by FITC labeled anti-FasL mAb. C. Human PBL were recall antigen stimulated for 6 days and incubated with FITC labeled anti-TIRC7 mAb. A monocyte cell line, THP-1, was incubated with anti-HLA-DR mAb and secondary stained with Cy3-conjugate. To increase cell interaction, PBL and THP-1 cells were mixed and incubated at 37°C for 20 min and subjected to confocal microscopic analysis. The data show that HLA-DR (red color) and TIRC7 (green color) co-localize (merge, yellow color) at the physical site of T cell/APC interaction. Shown is one experiment out of three.</p

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