Christina Hising

Mechanisms of escape from CD8+ T‐cell clones specific for the HER‐2/NEU proto‐oncogene expressed in ovarian carcinomas: Related and unrelated to decreased MHC class 1 expression

We have developed an in vitro model to study mechanisms by which ovarian tumor cells that over‐express the HER‐2/neu proto‐oncogene escape recognition by TCD8+. Nine tumor‐specific, HLA A2‐restricted TCD8+ clones were isolated from 2 ovarian tumor‐specific TCD8+ lines derived from tumor‐infiltrating or ‐associated lymphocytes. Of these, 2 clones recognized the previously defined HER‐2/neu epitope E75 (a.a. 369–377) and one recognized the C85 epitope (a.a. 971–979), whereas the specificity of the remaining 6 clones was unknown. Three different tumor escape variants (EVC8, EVC22 and EVC36) were produced by co‐culturing an ovarian tumor line over‐expressing HER‐2/neu with these autologous TCD8+ clones. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER‐2/neu, ICAM‐1 or LFA‐3 molecules was found. There was a correlation between the level of tumor‐specific recognition and HLA A2 expression among the tumor clones isolated from 2 of the escape variants (EVC8 and EVC36). In contrast, high HLA A2‐expressing tumor clones isolated from the EVC22 variant, or EVC22 which had regained high HLA A2 expression through IFN‐γ treatment, were not recognized by the HER‐2/neu‐specific TCD8+ clone C‐22. No mutations were found in the cDNA or the genomic DNA derived from the PCR product corresponding to a 496 bp fragment including the region coding for the E75 epitope of the HER2/neu gene in the EVC22 variant. Collectively, this in vitro model underlines the importance of decreased expression of the HLA restriction element for escape from tumor‐specific TCD8+ but also demonstrates that additional mechanisms exist.

Human ex vivo carcinoma cells produce transforming growth factor β and thereby can inhibit lymphocyte functions in vitro

Abstract We tested 20 human carcinoma samples for the production of transforming growth factor β (TGFβ) in vitro. Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells. The proliferation of these cells is inhibited by TGFβ. According to this assay, the supernatants contained both active and latent TGFβ. In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations. A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFβ. In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFβ-specific mAb. It is important to note that, when TGFβ-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often. These results thus substantiate the assumption that production of TGFβ may help the survival of potentially immunogenic tumour cells in immunocompetent patients.

Correlation between HLA-A2 gene frequency, latitude, ovarian and prostate cancer mortality rates

Molecular-target therapies are novel approaches to the treatment of prostate and ovarian cancer, but to ensure the best response, a very careful selection of patients, based on immunological characteristics, must be performed. We screened for HLA type, 24 patients with advanced ovarian cancer and 26 patients with hormone-refractory prostate cancer, in order to be recruited to vaccine protocols. HLA typing was performed with PCR in ovarian cancer patients and with serological assay in prostate cancer patients. The results were then extended to a population level, comparing the HLA genotype frequencies in Europe with ovarian and prostate cancer mortality rates. An overrepresentation of HLA-A2 phenotype was observed in both patient groups compared to the normal Swedish population (p=0.01). As it is already known, the higher phenotype frequency of this allele found in Scandinavian countries decreases significantly as one moves further south in Europe. Ovarian and prostate cancer mortality rates decrease as well as the demographic changes in HLA-A2. These observations have to be confirmed by more extended investigations in order to elucidate if HLA-A2 higher frequency is already present at the diagnosis (risk factor) or is selected during the course of the disease (prognostic factor). Moreover, this fact would suggest different strategies for specific immunotherapy in addition to first line conventional treatments.

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon γ and tumour necrosis factor α treatment of tumour cells potentiates their interaction with autologous blood lymphocytes

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon γ and tumour necrosis factor α elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.

Diverse effect of cytokine treatment of tumor cells on specific versus non-specific cytotoxicity

Abstract(First submitted 15 Nov 1991;accepted 11 December 1991) The effect of IFN-γ and TNF-α treatment of an ovarian carcinoma line on the sensitivity to lysis by specific CTL clones and non-specific Tumor Associated Lymphocytes (TAL), isolated from the ascites fluid, was analyzed. Thein vitro established TAL line displayed a non-specific lytic activity against the autologous tumor as well as against several allogeneic tumor lines. Pretreatment with IFN-γ alone, or in combination with TNF-α, rendered the carcinoma line less susceptible to lysis by the autologous TAL line. Conversely, susceptibility to lysis by tumor specific T cell clones, isolated from the TAL line, was increased as a result of cytokine pretreatment. Several TCR-α/β+, CD8+ T-cell clones showing a more specific pattern of lysis against the autologous tumor were isolated. Lysis of the autologous tumor by these clones involved the TCR-α/β via a MHC-class I restricted mechanism dependent on the adhesion molecules ICAM-1 and LFA-3, as inferred from antibody blocking studies. The enhanced sensitivity to specific CTL clones seen after cytokine treatment may be related to theenhanced expression of ICAM-1 molecules on the ovarian carcinoma. These results have implications for cytokine based immunotherapy, where IFN-γ may enhance the effects of tumor associated specific CTL while decreasing that of non-specific effector cells.

Cytotoxic susceptibility and defective MHC class I expression do not correlate with mutation of p53 in human carcinomas

Twenty-nine samples of ex vivo ovarian and lung carcinomas were investigated for the relationship between the presence of mutated protein 53 (mp53) and cytotoxic susceptibility. Unaltered expression of MHC class I alleles was required for the cytotoxic susceptibility of tumour cells to the autologous ex vivo blood lymphocytes, i.e. all 4 sensitive tumours belonged to the group of 11 tumours without defect in MHC class I expression. In contrast, the susceptibility did not correlate with the presence of mp53, i.e. cases with mp53 were randomly distributed between the sensitive and resistant tumours (2/4 and 10/17 respectively). There was no correlation either between the p53 mutation and down-regulation of MHC class I alleles. The results suggest that in these tumours the mutated p53 is not the source of immunogenic peptides and that the lack of recognition of the tumours with mp53 is not caused by a defect in the expression of MHC class I molecules.

Oligoclonal T Cells Expressing the TCR V-ß 6 Gene Product Following IL-2 Culture of Ovarian Carcinoma Derived Lymphocytes

T-cell populations obtained from tumor tissues (Tumor infiltrating lymphocytes, TIL) have been proposed to be enriched in lymphocytes specific for the autologous tumor. Experimental models have shown that TIL expanded in recombinant interleukin 2 (IL-2) are 50-100 times more effective in their therapeutic potency than IL-2-expanded LAK-cells derived from peripheral blood1. In clinical trials, therapies based on TIL combined with IL-2 have however so far yielded disappointing results, with response rates not better than those reported for LAK cells in combination with IL-22,3,4. Also, it has generally been difficult to demonstrate a specific effector function of TIL against the autologous tumor, and IL-2 activated TIL from most types of tumors showed non-specific cytotoxic activity similar to that of LAK cells5,6,7. Moreover, there is a considerable practical problem in obtaining a sufficient number of TIL to be used for therapy.