Lenora A. Boyle

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.

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.

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.

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.

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. However, the tumour progressed in vivo even in the presence of these tumour cell‐lytic clones. Using the anti‐Melan‐A/MART‐1 MoAb (A‐103), we noted that Melan‐A/MART‐1 expression on three melanoma cell lines varied considerably during in vitro culture, in the absence of T cell immunoselection, relative to cell density. Tumour cells which spontaneously decreased Melan‐A/MART‐1 expression were less susceptible to specific TIL lysis. Melan‐A/MART‐1 expression and susceptibility to lysis increased in cells cultured at lower density. These data suggest that modulation of tumour antigen may account for tumour progression in the presence of tumour cell‐lytic T lymphocytes. The observations suggest a possible explanation for the common finding of Melan‐A/MART‐1‐specific lytic TIL in clinically progressing melanomas, as well as a possible pathway for therapeutic intervention.

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 TlL‐5). These IL‐2‐propagaled 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. We derived 40 clones from TIL‐1 and 23 from TIL‐5. All tested clones were CD3+, CD4−, CD8+ and expressed the α/β TCR. From TIL‐1.27 of 40 clones, and 13/19 of the TIL‐5 clones lysed autologous tumour cells. In contrast to the NK, ‐negative bulk cultures, K‐562 killing was detected in 21 of the TIL‐1 clones and 17 of the TIL‐5 clones. TIL‐1 contained eight clones and TiL‐5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectimediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti‐CD3 or anti‐HLA class I MoAbs, Of the 34 TlL‐1 clones analysed, 15 shared a rearranged TCRβ EcoR1 restriction fragment of approximately 9 5 kb with the bulk culture. Clones sharing the EcoR1 10 5‐kb dominant band present in TIL‐5 bulk culture were also isolated. When the pattern of TCRβ rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response lo autologous tumour cells were also detected among those contributing to the dominant rearrangement; (ii) some clones that share an apparently identical rearranged band different from the “dominant” rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti‐tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour. Further study of the TCR gene rearrangements should help to clarify how selection at this level can benefit future immunotherapeutic approaches.

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. The results indicate that several different TCRAV and TCRBV gene segments are used among the different clones, but the two clones that were both cytotoxic and proliferative in response to HLA-DR3 shared identical TCRAV27-J42-C and TCRBV13-D1-J1S2-C1 transcripts. The additional three clones showed various TCRAV and TCRBV transcripts, but evaluation of the CDR3 region of the TCR beta chain, corresponding to the peptide antigen binding sites, demonstrated shared amino acid motifs which resulted both from germline sequences and combinations of n-region and germline-derived codons. These results suggest that the repertoire for anti-HLA-DR3-reactive clones can include a diverse expression of TCR, but there may be selection for some clones, as well as conserved motifs in the CDR3 region of anti-DR3 specific clones.

T-CELL REcEPTOR UsAGE AMoNG GRAFT INFILTRATING T LYMPHOCYTES

Recognition of peptide-containing MHC molecules by the α/β T-cell receptor (TCR) has been shown to be the central controlling interaction which initiates specific cell-mediated immunity.1,2 Although some stimuli, such as PHA and other polyclonal activators, can induce proliferation in virtually all T lymphocytes,3 and “super antigens” can activate whole subsets of TCR-V region-selected T-cells4, recognition of non-self MHC molecules (allo-antigens) remains the strongest truly antigen-specific stimulus yet described.5–7 Thus, the rapid deployment of specifically-reactive T-cells to grafted allogeneic tissue and cells is a consistent feature of the immune response to allografts.