SummaryHuman immunodeficiency virus 1 (HW-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using fleshly isolated peripheral blood mononudear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-l-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. In the present study, a longitudinal analysis of T cell receptor (TCK) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-l-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using Vol and Vfl family-specific primers was performed on each done, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized Vo114 and VB4 genes. Sequence analysis of the TCK revealed the first nine clones isolated to also be identical at the nudeotide level. The TCK-o~junctional region sequence of the tenth done was identical to the junctional region sequences of the other nine, but this clone utilized distinct DB and JB gene segments. This study provides evidence that the observed high degree of HIV-l-specific CTL activity may be due to monodonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.

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.

Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) 13-chain genes in 13 of 14 cultures showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture.These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders.

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.

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.

A 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.

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...

Subclasses of lymphocytes can be separated on gradients of non-toxic polyvinylpyrrolidone-coated colloidal silica (Percoll) by virtue of differential densities. Such gradients can yield functionally active lymphocyte populations after brief centrifugation. Gradients can be generated in a discontinuous step fashion and centrifuged in standard table-top laboratory centrifuges or as self-generating gradients during ultracentrifugation. The density medium has low viscosity and can be made isotonic for virtually any use. Gradients have proved useful in both human and experimental animal studies, and high percentage yields allow for separations from small cell numbers. Methods are described for separation of whole blood and lymphoid subpopuctions. The cytoxic capability of various density fractions was evaluated for mixed lymphocyte culture-induced allogeneic killing and spontaneous, so-called "natural" killer cell activity. The lower density associated with blast transformation allows for significant enrichments of stimulated cells from in vitro cultures. Higher thymidine incorporation, restimualtion in mixed lymphocyte reactions, and greater cytotoxic capacity are associated with these "blast" fractions.