No abstract

Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P ‫؍‬ 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R ‫؍‬ ؊0.36; P ‫؍‬ 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human Fc␥RIIIA and mouse Fc␥RIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common

Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (T) and T follicular helper (T) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4 ICOS-expressing T cells with a T-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS T-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)-SSc mice and contributed to dermal fibrosis via an interleukin-21- and matrix metalloproteinase 12-dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS T-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS T-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.

Summary Human cluster of differentiation (CD) antigen 19 is a B cell‐specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI‐551 is an affinity‐optimized and afucosylated CD19 mAb with enhanced antibody‐dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI‐551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI‐551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI‐551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI‐551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC‐enhanced CD19 mAb with an anti‐CD20 mAb could be a novel approach for the treatment of B cell lymphomas.