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Background/Methods: In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. Results: The results showed that: (1) Transfection of the class I negative Fen cell line with normal β-microglobulin (β2-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the β2-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700–3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 × 106 of positive Fen cells contained about 200 µg total protein of which about 0.10 µg was class I and about 2 ng was class I-associated peptides. Conclusions: These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.

Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.

A modified tetrazolium reduction assay (MTT) was used to assess the relation between HLA class I antigen expression on tumour cells and their susceptibility as a target for non-MHC restricted LAK/NK cytotoxicity using interleukin-2 activated peripheral blood mononuclear cells (MNC) from normal individuals. At 20/1 effector/target ratio this ranged from no killing to 77%. The efficiency of killing was dependent on duration of effector cell culture with IL-2, peaking at day 10 and declining thereafter. This killing could be enhanced by addition of other cytokines including interferons alpha, beta and gamma. Study of a panel of 15 tumour cell lines using a single effector showed that there was no statistically significant inverse correlation (using Spearman rank test) between the degree of tumour class I expression and LAK/NK killing at 20/1 (r = 0.23 P = 0.39) and 10/1 (r = 0.30, P = 0.27) and at 5/1 E/T ratio r = 0.47, P = 0.08) respectively. Lack of inverse correlation between these two parameters came from study of one bladder tumour line (FEN), whose absent class I antigens had been corrected by transfection with beta 2 microglobulin gene. At high E/T ratio (20/1) there was an increase in the susceptibility of target cells to lysis (36% parent cell, 45% transfected cell), whilst at lower E/T ratios (1/1) there was significantly more killing of the non-transfected cells (10% vs 31%). The addition of anti-class I antibody W6/32 increased killing by 18% but this was non-specific as the same increase occurred with a class II antibody. These data suggest that overall there was not an inverse correlation between class I expression and LAK/NK killing at high E/T ratios, whilst at low (5/1 or lower) E/T ratios this correlation nearly reached statistical significance suggesting that the conflicting literature reports may be due to a threshold levels of effector cells above which the masking effects of MHC antigens disappears.

Tissue sections from 22 seminoma (Se) and 10 teratoma (Te) patients were investigated for correlation between the presence of tumour infiltrating T-lymphocytes (TIL) and the expression of major histocompatibility complex (MHC) antigens using an immunoperoxidase staining technique. Complete absence of both class I and II antigens was observed in all Te and 20 out of 22 Se. The two positive Se showed only weak expression on 2% of tumour cells. Despite the absence of human leucocyte antigens (HLA) there were a large number of TIL scattered throughout the tissues in the case of Se with no predominance of CD4 or CD8 subpopulations in either group. T gamma positive cells were less than 5% of total CD3 positive cells in both Se and Te. The majority of the TIL were found to express activation markers, i.e. HLA class II antigens. Culture of tumour cell suspension with IL-2 produced passageable IL-2-dependent T cells from 10 out of 15 tumours. Studies with testis cell lines showed the complete absence of class I antigens in 2 out of 5 cases and the inability of interferon gamma (IFN gamma) to induce expression. IFN gamma also failed to induce class II antigens in three out of five of these lines. The immunological paradox of the presence of a large number of activated T-cells in testicular tumours despite the complete absence of MHC antigens remains unexplained and needs further investigation. Possible hypotheses are reviewed.

Using radiobinding, transfection and colorimetric assays, the biological significance of epidermal growth factor (EGF) and its receptor on established human tumour cell lines was investigated. The intensity of class I major histocompatibility antigen (MHC) and EGF receptor (EGFR) expression on 20 tumour cell lines was investigated and showed no direct correlation (coefficient of correlation r = 0.43 and P = 0.06). furthermore, transfection of the beta 2-microglobulin gene into a class I negative bladder tumour cell line, resulting in the re-expression of fully assembled cell surface class I antigens, did not result in alteration of EGFR expression. However, there was an inverse correlation between the intensity of EGFR expression and the stimulatory response of cells to exogenously added EGF. The per cent inhibitions of cell proliferation by EGF at 100 ng/ml for A431 (highest EGFR expressor) and Scaber (lowest EGFR expressor) were 37 and -7%, respectively. The results also showed that cell lines isolated from testis tumours positive for epithelial markers (using pan keratin antibody LP34 as an epithelial marker), expressed significantly lower EGFR levels than cell lines from bladder tumours. The expression of EGFR receptor was not modulated by interferons (IFN-alpha and -gamma and only a minor effect with IFN-beta) or active supernatant containing a mixture of cytokines. Whilst the pretreatment of tumour cells with IFNs resulted in a significant increase in the susceptibility of tumour cells to interleukin-2-activated peripheral blood mononuclear cells, EGF treatment resulted in their protection. Thus, the per cent killing at an effector:target ratio of 20:1 for untreated cells and EGF (100 ng/ml), IFN-alpha (1000 U/ml), -beta (2000 U/ml) and -gamma (100 U/ml) were 53%, 33% (P = 0.004), 64% (P = 0.004), 69% (P = 0.001) and 66% (P = 0.001), respectively. These results indicate the complex interactions between EGF and EGFR and their relevance in modifying tumour cell behaviour. The hypothesis that the resistance to cytolysis of tumour cells induced by EGF stimulation may be a factor in the accelerated tumour growth seen in patients after traumatic tissue damage is discussed.

The induction of major histocompatibility complex antigens by interferons (IFN) on 17 established tumour cell lines was investigated by radio binding. One bladder (Fen) and two testis lines (Tera I and Ha) lacked class I antigens and IFN-gamma failed to induce their expression. However, IFN-gamma upregulated these antigens on lines expressing low class I antigens (Tera II and EP2102) with little or no significant effect on high class I expressing lines (T24 and RT112). In one bladder line (Wil) IFN-gamma, whilst failing to alter monomorphic class I, upregulated polymorphic HLA-A2 and A3 antigens. None of the 17 lines expressed class II antigens, but could all be induced by IFN-gamma except T24, TccSup, Tera II and Lan lines. This defect was not due to the absence of IFN-gamma receptor, since under the same conditions intracellular adhesion molecule 1 was upregulated. IFN-alpha, whilst failing to have any effect on class II, induced class I antigens. IFN-beta showed no activity on either class I or II antigens when used alone. However, in combination, it inhibited IFN-gamma induced class II antigens. Thus, it may be possible to study cells from fresh tumours to preselect the minority of patients who might benefit from cytokine therapy.

Analysis of tissue sections from transurethrally resected bladder tumours using anti-CD3 antibody showed the presence of T lymphocytes in intra-epithelial layers in eight of 12 cases investigated. In a larger group of patients, Tumour Infiltrating Lymphocyte (TIL) growth was established from six of 19 cases using Interleukin-2 (IL-2) and conditioned medium (CM) and resulted in the expansion of TILs up to 100-fold. TILs from these individuals were phenotyped with W6/32 (anti-HLA-A,B,C), HB55 (anti-DR) and anti-CD3 antibodies using FAC sorter. The mean +/- s.d. frequency of positive staining with these antibodies were 96.7 +/- 4.0%, 87.5 +/- 10.0% and 82.5 +/- 7.8% respectively, indicating the activated nature of these T cells. The cytotoxic activity of these TILs against Daudi (ie, LAK activity) cell line at 25/1 E/T ratios varied from 26.3 +/- 3.2 to 62.8 +/- 5.2%. In one case where TILs and autologous tumour cell line were established, cytotoxicity studies showed low level of cytotoxicity against the autologous tumour cells (15.8 +/- 1.6%) compared with 62.8 +/- 5.2% against Daudi. Staining of tumour sections from these 19 individuals with W6/32 and BBM.1 revealed positive staining in six of six that developed TILs but only six of 13 (46%) cases, whose tumour failed to grow TILs (P less than 0.02, Fisher exact test). These results are indicative of the presence of IL-2 passageable T cells in bladder cancer biopsy and demonstrate that the successful expansion of these cells correlates with the normal expression of class I antigens on the tumour cells. Images Figure 5