Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors.

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

The phenotype of circulating follicular-helper T cells in patients with rheumatoid arthritis defines CD200 as a potential therapeutic target.

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily affecting synovial joints in which the development of autoantibodies represents a failure of normal tolerance mechanisms, suggesting a role for follicular helper T cells (T(FH)) in the genesis of autoimmunity. To determine whether quantitative or qualitative abnormalities in the circulating T(FH) cell population exist, we analysed by flow cytometry the number and profile of these cells in 35 patients with RA and 15 matched controls. Results were correlated with patient characteristics, including the presence of autoantibodies, disease activity, and treatment with biologic agents. Circulating T(FH) cells from patients with RA show significantly increased expression of the immunoglobulin superfamily receptor CD200, with highest levels seen in seropositive patients (P = 0.0045) and patients treated with anti-TNFα agents (P = 0.0008). This occurs in the absence of any change in T(FH) numbers or overt bias towards Th1, Th2, or Th17 phenotypes. CD200 levels did not correlate with DAS28 scores (P = 0.887). Although the number of circulating T(FH) cells is not altered in the blood of patients with RA, the T(FH) cells have a distinct phenotype. These differences associate T(FH) cells with the pathogenesis of RA and support the relevance of the CD200/CD200R signalling pathway as a potential therapeutic target.

An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation.

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.

The tachykinin NK1 receptor is crucial for the development of non-atopic airway inflammation and hyperresponsiveness.

Mast cell activation, bronchoconstriction, inflammation and airway hyperreactivity are prominent features of non-atopic hypersensitivity reactions in mouse airways. We studied the role of tachykinin receptors in mice that were skin-sensitized with dinitrofluorobenzene (or vehicle) and challenged intranasally with dinitrobenzene sulfonic acid. Tachykinin NK1 receptor blockade, by treatment with the antagonist RP67580, or absence of the tachykinin NK1 receptor resulted in a strong reduction in the accumulation of neutrophils in the bronchoalveolar lavage fluid, and in the development of tracheal hyperreactivity in mice 48 h after challenge. In contrast, treatment with the tachykinin NK2 receptor antagonist SR48968 did not affect the dinitrofluorobenzene-induced hypersensitivity reaction. We have previously shown that mast cells play a crucial role in the development of non-atopic asthma. However, we did not observe an inhibitory effect of the tachykinin receptor antagonists or the genetic absence of tachykinin NK1 receptors on mast cell protease release. In conclusion, distal from mast cell activation, the tachykinin NK1 receptor is crucial for the infiltration of pulmonary neutrophils and the development of tracheal hyperreactivity in non-atopic asthma.

Renal transplant immunosuppression impairs natural killer cell function in vitro and in vivo.

BACKGROUND: Despite an increasing awareness of the importance of innate immunity, the roles of natural killer (NK) cells in transplant rejection and antiviral and cancer immunity during immunosuppression have not been clearly defined. METHODS: To address this issue we have developed a quantitative assay of NK cell function that can be used on clinical samples and have studied the influence of immunosuppression on NK cell function. NK cell degranulation and intracellular interferon (IFN)-γ production were determined by flow cytometry of peripheral blood samples. RESULTS: Overnight ex vivo treatment of peripheral blood cells from healthy controls with ciclosporin or tacrolimus inhibited NK cell degranulation and IFN-γ production in a dose-dependent manner. A similar impairment of function was seen in NK cells from patients treated in vivo with calcineurin inhibitors. In the early post-transplant period, there was a variable reduction of NK cell counts after treatment with alemtuzumab and basiliximab. CONCLUSIONS: The functional inhibition of NK cells in early transplant patients coincides with the period of maximum susceptibility to viral infections. The ability to assay NK cell function in clinical samples allows assessment of the impact of immunosuppression on these effector cells. This information may be helpful in guiding the titration of immunosuppression in the clinical setting.

Genetic deficiency in the chemokine receptor CCR1 protects against acute Clostridium difficile toxin A enteritis in mice.

BACKGROUND & AIMS: The role of the CC chemokine receptor (CCR) 1 in acute enteritis was investigated by subjecting CCR1 knockout mice to Clostridium difficile toxin A treatment. METHODS: Toxin A or vehicle was injected into ileal loops in anesthetized wild-type, CCR1-/- and macrophage inhibitory protein (MIP)-1alpha-/- mice. After 1-4 hours, fluid accumulation was calculated, and the loops were processed for histology, myeloperoxidase activity, regulated on activation, normal T cell expressed and secreted (RANTES) production, and messenger RNA measurements. RESULTS: Toxin A induced in all mice a significant (P < 0.05) increase in ileal fluid accumulation, epithelial damage, and neutrophil infiltration, with all parameters being significantly (P < 0.01) lower in CCR1-/- and MIP-1alpha-/- mice. Ileal messenger RNA expression of the CCR1 ligands MIP-1alpha and RANTES and RANTES synthesis were increased in toxin A-treated wild-type mice. The RANTES antagonist Met-RANTES significantly (P < 0.01) reduced the toxin A-induced increases in ileal fluid accumulation and myeloperoxidase activity in wild-type mice. CONCLUSIONS: C. difficile toxin A-induced murine enteritis involves CCR1 and its ligands MIP-1alpha and RANTES, which may be important mediators of the neutrophil recruitment characterizing acute, enterotoxin-mediated enteritis.

Elevated vasoactive intestinal peptide concentrations in patients with irritable bowel syndrome.

The aim was to assess the roles of gut hormones and immune dysfunction in irritable bowel. In Study I, rectal mucosal samples examined blindly showed no histological evidence of inflammation in 16 irritable bowel patients compared to 17 healthy controls. The proinflammatory mediators interleukin-1beta and prostaglandin E2 also failed to show evidence of inflammation. Vasoactive intestinal peptide was elevated in irritable bowel (P = 0.01), but substance P, calcitonin gene-related peptide, and somatostatin levels were similar to control values. In Study II, 30 irritable bowel patients had elevated (P = 0.002) plasma concentrations of vasoactive intestinal peptide compared to 30 controls, and peptide levels were unrelated to whether the patient's predominant bowel habit was constipation, diarrhea, or both in alternation. In conclusion, no evidence of inflammation was detected in irritable bowel patients, but elevated vasoactive intestinal peptide concentrations were observed in both studies and might represent a potential diagnostic tool for irritable bowel syndrome.

C5L2, a nonsignaling C5A binding protein.

C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.