James T. Kurnick

1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor.

The monocyte factor, interleukin 1, or other factors homologous with interleukin 1, modulates functions of a variety of cells, including T and B lymphocytes, synovial cells, and chondrocytes. We have reported that a human monocyte cell line, U937, produces interleukin 1 when incubated with a soluble factor from lectin-stimulated T lymphocytes. We have also shown that U937 cells have a specific cytosolic receptor for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3). We now report that 1 alpha,25(OH)2D3(10(-11)-10(-10) M) induces maturational changes in the U937 cells similar to those produced by conditioned medium from lectin-stimulated T lymphocytes (increase in Fc receptors and OKM1 binding and decrease in proliferation), but does not induce monokine production as measured by mononuclear cell factor activity. 1 alpha,25(OH)2D3 is 200-300-fold more effective than 25-hydroxyvitamin D3, which is consistent with the known biological potency of these vitamin D3 metabolites. 1 alpha,25(OH)2D3 and the lymphokine together markedly augment maturational effects and, in addition, augment monokine production. The specificity of the interaction is further demonstrated by the lack of augmentation of monokine production with 1 beta,25-dihydroxyvitamin D3 in the presence of lymphokine. These interactions of a classical hormone and the hormonelike product(s) of the immune system with U937 cells serve as a model for human monocyte/macrophage differentiation and suggest a role for these interactions in some aspects of inflammation.

Tumor-derived interleukin-2-dependent lymphocytes in adoptive immunotherapy of lung cancer

SummaryA trial of adoptive immunotherapy was performed in which long-term cultured, interleukin-2 (IL2)-dependent T-lymphocytes were administered to patients with metastatic adenocarcinoma of the lung. Lymphocytes were isolated from explants of cancer tissues that were cultured in medium with recombinant IL-2. These T-cells expressed surface markers of activation, and killed a broad panel of tumor targets. Intravenously injected 111indium-labeled T-cell blasts distributed primarily to lungs, liver, and spleen. Despite a paucity of infused lymphocytes detected by external imaging at sites of tumor, five of seven patients showed reduction of their cancers. However, in no case was greater than 50% reduction of total tumor burden achieved. Evidence of increased delayed cutaneous hypersensitivity to protein antigens was observed in three patients following therapy. We conclude that long-term cultured tumor-derived T-cells can be transferred safely into humans and that these cells may be capable of enhancing immune responses and mediating tumor reduction in vivo.

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. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1–specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy. (Mol Cancer Res 2006;4(10):779–92)

Functional characterization of T lymphocytes propagated from human lung carcinomas.

Tissue fragments from biopsies of six patients with malignant tumors of the lung were cultured in interleukin 2 (IL-2). Cultures of proliferating lymphocytes were isolated from all cases. Tumor cell lines (small cell carcinoma and adenocarcinoma) were established in parallel cultures from two of these patients. Lymphocytes that proliferated in vitro were virtually all mature T lymphocytes (greater than 95% T3+, T11+). The T8+ subset accounted for an average of 70% while T4+ cells averaged 20% of the cells in culture. HNK-1 antigen was presented on 23% of cells. Seventy-four percent of cells expressed Ia (HLA-DR) antigens. B cells did not proliferate under these conditions. In all cases the cells lysed K562 targets and were active in lectin-mediated cytolysis against human lymphoblasts. All cultures produced lymphokines (IL-2 and IFN-gamma) when stimulated with PHA. Lymphocytes grown from a tissue specimen with adenocarcinoma were capable of killing autologous tumor cells in vitro. Specific cytotoxicity has been maintained by these cultured lymphocytes for greater than 6 months. IL-2 activated peripheral blood cells in this case showed little specific cytotoxicity for autologous tumor cells. Lymphocytes from another specimen of adenocarcinoma also lysed this tumor, but cells from the other four specimens did not. Lymphocytes propagated from the specimen of small cell undifferentiated cancer did not lyse autologous tumor cells. These data show that primary lung tumors contain activated T cells which will respond to IL-2 in vitro. These tumor-infiltrating lymphocytes have demonstrable function, which can include cytolytic activity against autologous lung tumor.

Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released by Escherichia coli Bacteria into Serum

ABSTRACT 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 coliJ5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

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. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.

Release of Gram-Negative Outer-Membrane Proteins into Human Serum and Septic Rat Blood and Their Interactions with Iminunoglobulin in Antiserum to Eschevichia 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. All 3 OMPs were captured from septic rat blood by anti-LPS IgG. Release of OMPs into serum was highest for immature bacterial cultures and was increased by antibiotics in vitro and in vivo. Anti-J5 IgG selectively captured an 18-kDa OMP released into serum and into plasma from septic rats. The results raise the possibility that anti-J5 IgG may, in part, protect via anti-OMP antibodies.

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. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.

Dominant rearrangements among human tumor-infiltrating lymphocytes. Analysis of T-cells derived from 32 patients with melanoma, lung, and renal cell carcinoma

Dominant rearrangements of T‐cell receptor (TCR) β‐chain genes are reported among tumor‐infiltrating lymphocytes (TIL). After interleukin‐2 expansion of TIL from renal and lung carcinoma and melanoma biopsy tissues, rearrangements of TCR βchain genes were analyzed by Southern blotting. Nongermline restriction fragments, indicating dominant rearrangements, were detected among the TIL from all 6 patients with renal cell carcinoma, 17 of 20 patients with melanoma, and 3 of 6 patients with lung tumors. The restriction‐fragment sizes of these dominant rearrangements were heterogeneous among the various patients. Rearrangements into Cβ1 were more common than Cβ2 rearrangements. Phenotypic analyses indicated that dominant rearrangements occurred in both CD4 and CD8 predominant TIL populations. The TIL populations that were extracted were expanded to derive large cell numbers suitable for in vivo transfer in an interleukin‐2 and TIL immunotherapy program. The data indicated that the cells delivered to these patients usually were characterized by dominant populations of T‐cells with selective TCR gene rearrangements. The significance of selective TCR use requires evaluation of the function and specificity of the TIL comprising these dominant populations both in their native in vivo setting and in the context of therapeutic transfer.