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Prevention of Acute Allograft Rejection by Antibody Targeting of TIRC7, a Novel T Cell Membrane Protein

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

TIRC7 Deficiency Causes In Vitro and In Vivo Augmentation of T and B Cell Activation and Cytokine Response

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7−/− mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7−/− mice exhibited significantly increased proliferation and expression of IL-2, IFN-γ, and IL-4 in response to different stimuli. Resting T cells from TIRC7−/− mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7−/− mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.

TIRC7 Inhibits T Cell Proliferation by Modulation of CTLA-4 Expression

Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.

Antibody targeting of TIRC7 results in significant therapeutic effects on collagen-induced arthritis in mice

TIRC7 is a cell surface molecule which is expressed in T and B lymphocytes and negatively regulates their function. Anti‐TIRC7 specific monoclonal antibody (mAb) inhibited T cell memory response to recall antigens. Up‐regulation of TIRC7 on lymphocytes from joint tissue of patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggested TIRC7 as a novel target to promote anti‐inflammatory reaction. Anti‐TIRC7 mAb administration significantly inhibited the induction and progression of CIA and the anti‐collagen IgG1 and IgG2a antibody response. Combination therapy of anti‐TIRC7 mAb and soluble TNF‐α receptor demonstrated an increased inhibitory effect over the single compounds on CIA. The results demonstrate the therapeutic potential of TIRC7 targeting with mAb in diseases associated with exaggerated T and B cell responses.

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.

THE HUMAN HOMOLOG OF DROSOPHILA CORNICHON PROTEIN IS DIFFERENTIALLY EXPRESSED IN ALLOACTIVATED T-CELLS

To identify novel genes induced in the early stage of T-cell activation, mRNA expression in alloactivated human lymphocytes was examined. Differential display-reverse transcription PCR analysis revealed a 207-bp cDNA fragment which was upregulated 24 h after allostimulation of a human T-cell line. The corresponding complete 1396 bp cDNA, named TGAM77, encodes a predicted 134 amino acid protein which shares 63% homology with the cornichon (cni) protein of Drosophila melanogaster. Upregulation of TGAM77 mRNA in the early phase of T-cell activation was confirmed by Northern blot and RT-PCR analysis of activated human lymphocytes. TGAM77 mRNA is expressed in a variety of human tissues with various expression levels. In analogy to cni which is involved in an epidermal growth factor-like signaling pathway inducing cellular asymmetry in Drosophila oogenesis, TGAM77 might function in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation.

Tissue-infiltrating plasma cells are an important source of carboxylesterase 2 contributing to the therapeutic efficacy of prodrugs.

Carboxylesterase 2 (CES-2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. CES-2 expression was analyzed by immunohistochemistry in colorectal cancer (CRC) compared to colonic inflammation as well as in liver and peripheral blood. In CRC, tumor grades showed no correlation with levels of CES-2 expression, which was heterogeneous within these tumors. Cellular infiltrates in the immediate tumor vicinity expressed high levels of CES-2. Thus, tissue adjacent to the tumor was a substantial source of CES-2 with high expression in plasma cells. CES-2(high) plasma cells were abundantly found in the colon of patients with inflammatory bowel disease. CES-2 expression is strong in hepatocytes of normal livers, while CES-2 expression in peripheral blood mononuclear cells of healthy donors was overall low at protein and mRNA levels. In summary, the conversion of ester-containing prodrugs by CES-2 is mainly to occur in the periphery, during liver passage and in the colon after enterohepatic recirculation. We here demonstrated plasma cells as strong producers of CES-2. Further studies should elucidate the role of CES-2(+) plasma cells in intestinal inflammation and cancer.

TIRC7 and HLA-DR axis contributes to inflammation in multiple sclerosis

Background and objective: Interactions between TIRC7 (a novel seven-transmembrane receptor on activated lymphocytes) and its ligand HLA-DR might be involved in the inflammatory process in multiple sclerosis (MS). Methods: Methods comprised immunohistochemistry and microscopy on archival MS autopsies, proliferation-, cytokine-, and surface-staining assays using peripheral blood lymphocytes (PBLs) from MS patients and an in vitro model. Results: TIRC7 was expressed in brain-infiltrating lymphocytes and strongly correlated with disease activity in MS. TIRC7 expression was reduced in T cells and induced in B cells in PBLs obtained from MS patients. After ex vivo activation, T cell expression of TIRC7 was restored in patients with active MS disease. The interaction of TIRC7+ T lymphocytes with cells expressing HLA-DR on their surface led to T cell proliferation and activation whereas an anti-TIRC7 mAb preventing interactions with its ligand inhibited proliferation and Th1 and Th17 cytokine expression in T cells obtained from MS patients and in myelin basic protein-specific T cell clone. Conclusion: Our findings suggest that TIRC7 is involved in inflammation in MS and anti-TIRC7 mAb can prevent immune activation via selective inhibition of Th1- and Th17-associated cytokine expression. This targeting approach may become a novel treatment option for MS.

Anti-TIRC7 Antibody Treatment Prevents Rejection of Mice Cardiac Allografts while Immune Competency to Viral and Bacterial Antigens Remain Normal

Introduction T cell immune response c-DNA (TIRC7) is expressed in activated lymphocytes binding to its ligand HLA-class II protein in response to alloantigens. Notably, TIRC7 is mainly induced in activated lymphocytes at the site of the inflammation. Thus, unlike other immunosuppressive agents, treatment with an anti-TIRC7 does not interfere with an overall immune competency. Here, anti-TIRC7 antibody (mAb) treatment was compared with Cyclosporin A (CyA) treatment in a mouse cardiac transplant model. As currenty used therapeutics to prevent rejection act unspecifically with severe side effects including infections, we tested the overall immue competency in the presence of anti-TIRC7 mAb treatment and challanged mice with infectious antigens. Method Fully vascularized heterotopic allogeneic heart transplants were performed across a full-mismatch barrier (C57BL/10 into CBA). Recipients mice received anti-TIRC7 mAb (40mg/kg, day 0-7) and compared to treatments with CyA (10mg/kg for either 7 or 14 days or an untreated control n=5-7/group. In an additional experiment, two experimental model systems for viral infections - non-cytopathic lymphocytic choriomeningitis virus (LCMV) and acutely cytopathic vesicular stomatitis virus (VSV) in addition to Listeria monocytogenes (LM) infections were used to analyse fundamentally different types of antiviral and bacterial antibody responses in the presence or absence of anti-TIRC7 mAb. LCMV responses were examined after injecting 200pfu LCMV into the footpad of B6 mice and response was assessed by CTL assays. VSV triggers the production of IFN-I in mice leading to augmentation of specific B cell responses. IgG and IgM titer against VSV was analyzed up to 20d post infection. Listeria monocytogenes (LM) provides a means for introducing antigens into the class I pathway of antigen presentation and induces CD4 response. 1000cfu LM was injected i.v and LM-titers were assessed in spleen and liver (n=4/experiment). Results Mean cardiac allograft survival in anti-TIRC7 mAb treated mice was 52 days with some grafts working > 140 days, in sharp contrast to CyA treated mice and controls with a mean graft function of 8days. Morphology of allografts showed diminished lymphocyte infiltration in anti-TIRC7 mAb treated allografts. Mice cleared the LCMV infection within 15 d. Anti-VSV IgG and IgM antibody titers were highest at 15 d post infection. Mice liver and spleen infected with LM were protected in anti-TIRC7 mAb treated mice as assessed at day 4 post infection. Conclusion Ligation of TIRC7 regulates immune activation at the site of inflammation while the function of peripheral blood lymphocytes remains normal. Mice treated with anti-TIRC7 mAb mounted normal immune responses to infections. Thus, targeting TIRC7 may provide a novel therapeutic approach to specifically modulate alloimmune responses.