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.

4Complexes 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. 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 MLPdeficient 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.Bacterial cell wall components released into the bloodstream are believed to be important in the pathogenesis of gram-negative sepsis. Although prior investigators have reported that bacteria release lipopolysaccharide (LPS) into serum (62, 63) and into the circulation (4,18,56,66), the full composition of released bacterial products has not been established. Very little is known about release of non-LPS gramnegative outer membrane components such as outer membrane proteins (OMPs) in sepsis. Fragments containing LPS, OmpA, and another faintly staining protein, of 17 kDa, were affinity purified from filtrates of human serum incubated with Salmonella enterica serovar Abortus equi bacteria using Ochain-specific anti-LPS immunoglobulin G (IgG) (20). Similarly, we have affinity purified complexes containing LPS and at least three OMPs, with estimated molecular masses of 35, 18, and 5 to 9 kDa, from filtrates of normal human serum incubated with Escherichia coli bacteria, using O-chain-specific anti-LPS IgG (29,30).Previous studies indicated that passive and active immunity directed to rough mutant bacteria such as S. enterica serovar Minnesota Re595 and E. coli J5 protect in experimental and clinical gram-negative sepsis (1,5,11,42,43,68). Protection has been attributed to antibodies directed to conserved core components of LPS (lipid A and core oligosaccharide). However, it has been difficult to prove that antisera to rough strains of bacteria contain cross-reactive anti-lipid A or anti-core oligosaccharide IgGs (15, 57), and the exact mechanism of protection remains unclear and controversial.We have demonstrated that IgG in antiserum raised to heatkilled E. coli J5 (J5 antiserum) binds to the same three gramnegative bacterial OMPs that are released into serum in the OMP-LPS complexes described above (30). OMP-LPS complexes are also released into the bloodstream of burned rats with E. coli O18K ϩ sepsis (29). In addition, at least one OMP, with an estimated molecular mass of 18 kDa, is released from bacteria separately from the OMP-LPS comple...

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.

No abstract

In a patient with progressing metastatic melanoma, we showed that the same autologous tumorcytolytic 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. Using SSCP analysis, we also demonstrated that this clone was dominant in the fresh tumor tissue and in all TILs in which CD8 ؉ were strongly represented, including several separate but parallel cultures. The SSCP pattern for this clone was not apparent among CD4 ؉ TILs or CD8؉ peripheral blood mononuclear cells. The SSCP analysis of the tumor tissue prior to in vitro culture is an indication that the selection for this anti-tumor cytotoxic T cell clone was a ref lection of its in vivo accumulation. Thus, we provide evidence that melanomas are immunogenic and able to select for cytotoxic antitumor-specific TIL clones that are expanded in vivo and can circulate to accumulate in different tumor sites. However, because these clones were isolated from progressing tumor metastases, the accumulation of these specific cytotoxic T cells was not sufficient to contain tumor growth.It has been shown that the immune response to human melanoma includes cytotoxic T lymphocytes capable of lysing autologous tumor cells in vitro (1-15). Some of the melanoma tumor antigens recognized have been identified and cloned (16)(17)(18)(19)(20)(21)(22). Despite the presence of these cytotoxic lymphocytes, it is evident that the immune response fails to eradicate clinically apparent tumors. Several mechanisms have been proposed to explain tumor escape from immune surveillance, including the loss of HLA alleles needed for T cell recognition (23, 24), as well as other cell surface molecules necessary for induction of T cell activation and proliferation (25).Several laboratories have shown that the tumor infiltrating lymphocytes (TILs) propagated from human melanoma often show restricted T cell antigen receptor (TCR) usage (1,4,12,(26)(27)(28)(29)(30)(31)(32)(33)(34). In some cases it has also been possible to demonstrate that the dominant T cells among the TILs are able to lyse autologous tumors (3,12,35). Still, an unresolved concern has been the lack of unequivocal evidence that the dominant clones isolated in vitro were representative of the in vivo accumulation of these cells within the tumor, rather than a reflection of an in vitro selection. By comparing the TCR usage among the freshly isolated TILs with the TCR which could be sequenced from anti-tumor cytotoxic clones, we addressed the question of whether significant accumulations of melanoma-specific cytotoxic T cells...

Antibodies to the clonally unique variableregion determinants (idiotype) of the antigen-specific ac heterodimeric receptor of a clone of cytotoxic T cells (CTLs) were shown previously to render diverse cells, regardless of their own surface antigens, susceptible to lysis by that clone of CTLs. To extend these findings, we have sought to develop a general means for targeting cells for destruction by any CTL, without regard to its afi idiotype and specificity for antigen.We explored the use of heteroantibody duplexes formed by joining covalently an antibody to the T3 complex (anti-T3), which is associated with the 43 receptors on all human mature T cells, and a second antibody, specific for an antigen on the intended target cell. The second antibody selected in this study was specific for the idiotype (Id) of the surface immunoglobulin of a human B-lymphoma (anti-Ig Id). In the presence of the anti-T3/anti-Ig Id heteroantibody duplex the B-lymphoma cells were lysed by a clone of human T8' CTLs (of unrelated specificity) but not by a noncytotoxic clone of human T4' helper T cells, and lysis by the CTLs was specifically blocked by the uncoupled anti-T3 or the uncoupled anti-Ig Id antibodies. The extent of the heteroantibody-dependent cytolysis depended both on the heteroantibody concentration and on whether the intended target cells or the CTL effectors were initially preincubated with the heteroantibody. Under (2). In the present study, we sought to generalize these results and to develop the means by which any CTL, regardless of its own antigen-specificity, could be made to lyse any particular cell chosen to serve as a surrogate target. For this purpose, it seemed appropriate to use antibodies that are specific for the constant domains, rather than the variable domains (2), of the aj3 receptors, since such antibodies might be able to activate all CTLs. However, antibodies to constant domains of the aid receptor were not available, and so we turned to antibodies that are specific for the T3 molecular complex.T3 differs from the other clonally invariant surface proteins on T cells in its intimate association with the af3 receptor on all human mature T lymphocytes (3). It seemed likely, therefore, that anti-T3 antibodies might function like antibodies to the constant domains of the ap3 receptors. Indeed, although free anti-T3 antibody inhibits CTL lysis of natural targets (4), we found in preliminary experiments that the covalent attachment of anti-T3 antibodies to surrogate target cells rendered them susceptible to lysis by human CTLs having an unrelated specificity (data not shown). To explore the anti-T3 antibody further, we chose not to attach it covalently to an intended target cell but rather to use a bifunctional heteroantibody duplex in which anti-T3 antibody was covalently linked to a second antibody that was specific for a surface antigen on the intended target cell. For the second antibody, we selected a monoclonal antibody (anti-Ig Id) specific for the idiotype of the surface immunoglobulin o...

The role of cell adhesion molecules (CAM) LFA1, ICAM-1, LFA3, VLA1, VLA4, CD29, CD44, and 0 5 6 in tumorinfiltrating lymphocyte (TIL) and natural killer cell (NK)-mediated killing of target cells was studied. Melanoma cell lines and autologous TIL were derived from seven patients with metastatic melanoma, and cytotoxicity assays were done in the presence and absence of monoclonal antibodies (MoAb) to CAM expressed on melanoma cells or TIL. The melanoma cell lines analyzed were all positive for CD29 and LFA3 expression, negative for LFAl expression, but showed variable expression of ICAM-1, VLA1, VLA4, CD44, and CD56. The effects of anti-CAM antibodies on TIL-mediated melanoma killing fell into three categories: (1) consistent inhibition of TILmediated killing was observed when melanoma cells were pretreated with anti-ICAM1 and anti-LFA-3 MoAb or when TIL were pretreated with anti-LFAl; (2) no effect was observed when melanoma cells were pretreated with anti-CDS@ or (3) a discreet, but significant, inhibition was observed when target cells were pretreated with anti-CD29, anti-VLAI, anti-VLA4, and anti-CD44. Cytotoxicity was significantly enhanced by pretreatment of

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.