Integrins: Integrating the Biology and Therapy of Cell-cell Interactions.

Purpose: Although the role of integrins has been described in a variety of diseases, these roles seem to be distinct. To date, no study has attempted to provide links to the various pathways by which such integrins can be involved in these diverse disease settings. The purpose of this review was to address this gap in our knowledge with the hypothesis that there is, in fact, a common pathway by which integrins may function.

Heat shock protein-90 inhibitors enhance antigen expression on melanomas and increase T cell recognition of tumor cells.

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines.

A screening assay to identify agents that enhance T-cell recognition of human melanomas.

Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity.

Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases.

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal.

Strategies to overcome obstacles to successful immunotherapy of melanoma.

The immunogenicity of malignant melanomas has been recognized by the observed recruitment of tumor-specific cytotoxic T-cells (CTL), leading to the identification of several melanoma associated antigen (MAA). However, numerous strategies to treat melanoma with immunotherapy have resulted in only partial success. In this editorial, we discuss recent data related to the ability of tumors to elude immune responses.

Enhancement of human melanoma antigen expression by IFN-beta.

Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines.

Role of the mitogen-activated protein kinase signaling pathway in the regulation of human melanocytic antigen expression.

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase.

Induction of "antigen silencing" in melanomas by oncostatin M: down-modulation of melanocyte antigen expression.

We previously reported that antigen expression in melanoma cell lines is down-regulated by proteins secreted by antigen-negative melanoma cells. Here we report the purification and characterization of one of these down-regulatory factors, the cytokine, oncostatin M (OSM), which transmits its signal via the gp130 cell surface receptor, resulting in the selective down-modulation of the melanocyte lineage antigens: Melan-A/MART-1, gp100, tyrosinase, tyrosinase-related proteins 1 and 2, and the M isoform of microphthalmia transcription factor. Furthermore, we have found that some melanoma cell lines produce as yet uncharacterized factors distinct from OSM which also down-modulate antigen expression via signaling pathways different from that employed by OSM.

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein.

Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.

Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs.

Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5.

Prior studies indicate that 3 bacterial outer-membrane proteins (OMPs) are released into serum associated with lipopolysaccharide (LPS) and are bound by IgG in antiserum to Escherichia coli J5 (anti-J5 IgG). The present studies analyzed the interaction of the OMPs with anti-J5 IgG and evaluated their release in an infected burn model of gram-negative sepsis. Affinity purification studies were performed on filtrates of bacteria incubated in human serum and plasma from rats with sepsis by use of O chain-specific anti-LPS IgG and anti-J5 IgG.

Melanoma antigen recognition by tumour-infiltrating T lymphocytes (TIL): effect of differential expression of melan-A/MART-1.

We have isolated, from an individual patient with metastatic melanoma, a series of eight TIL clones capable of lysing autologous melanoma cell targets. Six of the eight clones expressed TCRAV2S1 and lysed targets expressing HLA-A2 and the Melan-A/MART-1 peptide: AAGIGILTV. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis showed that the Melan-A/MART-1-specific clones were predominant in the bulk culture prior to cloning.

Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes.

In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma.

Oligoclonality of Vdelta1 and Vdelta2 cells in human peripheral blood mononuclear cells: TCR selection is not altered by stimulation with gram-negative bacteria.

Despite the enormous potential repertoire of gammadelta T cells, there are several observations which suggest that the expressed gammadelta repertoire in the periphery of normal individuals is often quite restricted. To assess selective expansions among gammadelta T cells from both adult and newborn blood samples, PBMC from 12 normal adults and cord blood from 15 normal newborns were analyzed for TCRDV1 and TCRDV2 junctional diversity by CDR3 size spectratyping and single-strand conformational polymorphism. Although TCRBV usage showed extensive heterogeneity in adults and newborns, both populations often showed CDR3 region restriction for TCRDV1 and TCRDV2.

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria.

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting.

T cell receptor usage by HLA-DR3-specific T cell clones isolated from a renal allograft.

In order to evaluate the T cell receptor (TCR) usage by clones of human allograft infiltrating lymphocytes, this study utilized polymerase chain reaction (PCR) amplification of TCR transcripts from five clones which were previously shown to react with a human leucocyte antigen (HLA)-DR3 mismatch between a living related kidney donor and recipient. The five CD4+ (CD8-) clones, which were selected for TCR analysis, proliferated in response to HLA-DR3 and three of the clones were also cytotoxic against the same target cells. After identification of the TCRAV and TCRBV usage of the clones, the sequence of the TCR alpha and beta were determined by direct sequencing of the PCR product.

In vivo accumulation of the same anti-melanoma T cell clone in two different metastatic sites.

In a patient with progressing metastatic melanoma, we showed that the same autologous tumor-cytolytic CD8+ tumor infiltrating lymphocyte (TIL) clone accumulated in two separate metastatic sites. This clone, which represented three of eight independently derived clones from a tumor deposit on the skin of the abdomen, also represented two of eight clones derived from a skin lesion on the shoulder. This clone could be identified by its use of a unique TCRBV2-nD1n-J1S6 sequence, and could also be detected by single-stranded conformational polymorphism (SSCP) as the dominant TCRBV2-expressing clone among CD8+ TILs propagated from both shoulder and abdominal lesions.

Escherichia coli and Pseudomonas aeruginosa induce expansion of V delta 2 cells in adult peripheral blood, but of V delta 1 cells in cord blood.

Human peripheral blood T cells proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We observed that during the first few days after stimulation a large percentage of the responding PBMC were gamma delta T cells. In our study we characterized the early T cell responses of freshly isolated adult and newborn PBMC to soluble preparations of heat-killed E.

Responses of human T cells to dominant discrete protein antigens of Escherichia coli and Pseudomonas aeruginosa.

Normal human beings have circulating T lymphocytes that proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We performed the present study to characterize the nature of the responding T cells and to determine whether distinct or shared conventional antigens, superantigens or polyclonal activators account for T cell proliferation. Long term antigen-specific T cell lines were generated by repeated stimulation of PBMC from four donors with soluble antigen preparations of E.

Longitudinal analysis of T cell receptor (TCR) gene usage by human immunodeficiency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals a limited TCR repertoire.

Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope.

T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients.

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the 'dominant' rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TIL-5). These IL-2-propagated TIL cell lines had a CD8+ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells.

T lymphocyte responses to antigens of gram-negative bacteria in pyelonephritis.

We showed previously that large numbers of T lymphocytes accumulate within a few days in the kidneys of rats with ascending pyelonephritis induced with Escherichia coli or Pseudomonas aeruginosa. CD4+ T cells propagated from the lesions exhibited MHC-restricted proliferative responses to formalin-fixed bacteria of the species used to induce infection. In the present study we investigated further the nature of the antigens responsible for the T cell proliferation and studied the ability of different bacterial strains and species to produce proliferative responses.