Role of T-lymphocytes in production of a cancer-associated protein factor in serum from lung cancer patients.

Oligoclonal T-cells have been generated by sensitization of peripheral blood mononuclear cells from lung cancer patients to a lung cancer tumor-associated antigen (TAA). A factor similar to the antigen-specific glycoprotein factor in the serum of these cancer patients was found in the supernatant of the oligoclonal T-cells. The factor from the T-cell supernatant had specificity for lung cancer TAA and induced stimulation of normal lymphocytes of the CD8 phenotype when mixed with lung cancer TAA. Furthermore, the factor blocked the ability of lymphocytes from lung cancer patients to recognize lung cancer TAA. Both the factor from lung cancer serum and from the oligoclonal T-cells were absorbed on a lung cancer-associated antigen-coupled immunosorbent column. On FPLC-gel filtration the desorbed fractions from the immunosorbent column from both sources showed activity in the same molecular weight range, 70-90 kD. Heteroantisera raised against the factor from serum and against the factor from the oligoclonal T-cell supernatant bound about the same portion of lymphocytes from lung cancer patients as measured by immunofluorescence, while only a minor fraction of cells from patients with unrelated cancers and from healthy persons were labelled on incubation with the antisera. These results support the hypothesis that an antigen-specific factor found in serum of cancer patients is produced by antigen-stimulated T-cells, possibly of the CD8 phenotype. This putative antigen-specific suppressor factor and the tumor antigen-reactive lymphocytes of the patient seem to share similar idiotopes.

Humoral leukocyte adherence inhibition response in patients with oral cancer.

Antitumor immunity was assessed in a group of 10 patients suffering from squamous cell carcinoma of the oral cavity using the humoral leukocyte adherence inhibition assay (H-LAI). A tumor extract from a person with cancer of the tongue was used as antigen. Sera from five patients with carcinoma of the tongue were all positive in the test. Of the other five patients, three had cancer of the lips while in two patients the type was unspecified. All patients except one with cancer of the lips gave a positive response in the test. Thus, of the 10 patients with carcinoma of the oral cavity, nine responded. Among nine controls, one person gave a positive response.

Cell-populations involved in the humoral leukocyte adherence inhibition reaction. The T8-suppressor/cytotoxic cells are both effectors and responders.

In an attempt to identify the cell subpopulation involved in the humoral leukocyte adherence inhibition (H-LAI) reaction, mononuclear leukocytes were fractionated and the response with the different cell populations was measured in the H-LAI assay using sera from lung cancer patients and a lung cancer associated antigen. The monocytes were found to exhibit a suppressive effect on the H-LAI response. The B-lymphocytes seemed not to take an active part in the reaction. Depletion of the T-lymphocytes abolished the reaction. Subsequent studies on the T-lymphocyte subpopulations revealed that removal of the T4-cells had little effect while removal of the T8-cells resulted in complete abrogation of the H-LAI response. Direct blocking by monoclonal antibodies against the T4 and T8 surface determinants gave additional support for the T8-cell dependency of the H-LAI assay. The cells that specifically were induced to loose adherence in the H-LAI assay upon incubation with serum from a lung cancer patient and lung cancer associated antigen were also analyzed. Compared to the total mononuclear leukocyte population, the relative content of monocytes and B-cells were lower in the non-adherent population. An increase was found in the relative number of T-cells. While the relative number of T4-cells decreased, the content of T8-cells was significantly increased. Furthermore, a direct correlation was found between the loss of adherence of the T8-subpopulation and the index of the H-LAI response. The present study indicates the T8-subpopulation of the T-lymphocytes to be involved both in the effector and responder side of the H-LAI reaction and gives implications for an immunological reaction behind the response of the H-LAI assay.

Detection of ovarian cancer by the humoral leukocyte adherence inhibition test using a purified tumor-associated antigen.

The defined, purified ovarian cancer-associated antigen, NB/70K, was used as tumor antigen in the humoral leukocyte adherence inhibition (H-LAI) assay. Bell-shaped dose-response relationships were obtained with increasing concentrations of NB/70K and constant serum concentration from ovarian cancer patients. The concentrations of NB/70K used in this study were 100-250 times less than what had been used in previous H-LAI studies with crude antigen extracts. The response rate of ovarian cancer patients was greatly influenced by the stage of the disease. Thus 75% (6/8) of the patients with disease restricted to the pelvis (stages I and II) reacted while only 20% (5/24) of the patients with more advanced stages (stages III and IV) gave response. Of patients with other gynecological cancers, 17% (1/6) were positive and only 8% (1/13) of patients with other malignant diseases reacted. One of the 11 controls (9%) was found to give positive response. The importance of the present investigation was the finding that a defined, purified, cancer-associated antigen elicited reactivity in the H-LAI assay and that a high sensitivity for detection of early stages of ovarian cancer was obtained with this antigen. The results suggest that TAAs involved in immune recognition of cancer can be identified by the H-LAI assay.

Purification of a cancer-associated protein factor in serum of lung cancer patients.

The serum factor responsible for the humoral leukocyte adherence inhibition (H-LAI) reaction in lung cancer patients has been purified. It is precipitable by ammonium sulfate between 30-70% saturation. On DEAE ion exchange chromatography activity is eluted in the 0.12-0.2 M acetate fraction. The serum factor has affinity for Con A. This gives evidence for a glycoprotein nature of the factor. Electrophoresis indicates an apparent mol. wt of 71,000 dalton. The data suggest that the protein can be separated into subunits of 43,000 and 28,000 dalton under reducing conditions. Isoelectric focusing gives a mean pI of 4.5. The applied fractionation procedure gave a 1120-fold purification relative to lung cancer serum. Antiserum against the purified factor has been produced in rabbits. The factor can be demonstrated in high concentrations in serum of lung cancer patients, but is also found in smaller quantities in sera of other types of cancer. Minor quantities are present in normal serum. The results suggest that quantitation of the H-LAI factor can be used in cancer diagnosis.

Early detection of breast cancer by leukocyte adherence inhibition assay.

The hematocytometer leukocyte adherence inhibition technique was used to study cell-mediated immune activity against breast carcinoma. In a group of 83 patients with untreated breast cancer in stage I, 74% showed a positive response, among 47 patients in stage II, 64% responded, while only 51% of the 37 patients in stages III and IV responded. Of 86 control persons, only two women showed a positive response. In a group of 296 women with benign breast disease, 21% showed a positive reaction against breast carcinoma antigen. The percentage of positive responses was higher than the average among women with risk factors such as: mother or sister with breast cancer, previous removal of benign breast lumps, microcalcifications, and increased intraductal epithelial proliferation found in the biopsies. Women with benign breast disease having two or more of the above risk factors were assigned to the high risk group. Of 49 women in this group, 47% had reactive leukocytes. Ninety-two women had only one risk factor, and 27% of those showed a positive reaction. Of the 155 women with none of the risk factors, only 10% had a positive reaction. The results suggest that leukocyte adherence inhibition test may be used to identify groups of women with an increased possibility of developing breast cancer.

A serum immune factor in detection of an occupational group with increased risk for lung and nose cancer.

Epidemiological studies have demonstrated that workers in the nickel refinery industry have an increased risk for respiratory tract carcinoma. In the present study, serum from 51 workers at a Norwegian nickel refinery have been tested against lung, nasal and breast carcinoma antigens in the humoral leukocyte adherence inhibition test. The breast cancer antigen was used as a non-specific antigen. The frequency of positive response against the lung carcinoma antigen was significantly higher among the refinery workers (21/51) than in the controls (3/17) (P = 0.07). Moreover, among workers employed for 10 years or more, the response was higher than found for workers with shorter employment. Of the nickel workers with nasal dysplasia, 56% (15/27) gave a positive reaction against the lung carcinoma antigen compared to 25% (6/24) of the workers without dysplasia (P = 0.03). The same trends were also found for the nasal carcinoma antigen. The study gives further support for the usefulness of the humoral leukocyte adherence inhibition test in identification of individuals with an increased risk for developing cancer.

Leukocyte adherence inhibition assay in human pulmonary neoplasia.

The hemocytometer leukocyte adherence inhibition technique was used to study cell-mediated immuno-activity of patients with lung cancer. KCl extracts (3.5 M) from the lung cancer cell line Calu-1 and the breast cancer cell line MCF-7 were used as antigens. Of 138 patients with lung cancer, 85% showed a positive response against the Calu-1 antigen. The response was independent of the histological type of the tumor and was the same among untreated patients, patients undergoing different types of treatment and patients who died within 3 months after blood collection. Twenty-five percent of the untreated lung cancer patients also reacted against the breast cancer antigen. Among lung cancer patients undergoing different types of treatment, 36% reacted while 50% of the patients who died within 3 months after blood collection reacted against the breast cancer antigen.

Humoral antitumor immune responses in patients with breast cancer measured with the leukocyte adherence inhibition technique.

A modification of the hemacytometer leukocyte adherence inhibition (LAI) test was described. In this modification, 0.25% serum from patients with breast cancer was added with the relevant antigen to the assay system with the use of trypsinized leukocytes from control persons as indicator cells. The modified assay measured a humoral immune response. In studies of patients with untreated breast cancer (stages I and II) with the use of a KCl extract from a breast carcinoma or from MCF-7 cells as antigens, the modified LAI test was found to be at least as sensitive as was the ordinary test. In a blind study on sera collected from patients with breast cancer 0.5-2 years before the LAI measurements and stored at -20 degrees C, 15 of 18 (83%) patients had a positive response. Whereas the ordinary LAI test is limited to the use of fresh blood, the present test can be performed with small amounts of serum that can be frozen and stored.

Role of circulating antibodies in the humoral leukocyte adherence inhibition response of lung and breast cancer patients.

A modified leukocyte adherence inhibition (H-LAI) assay has recently been developed in which 0.25% serum from the patient is added to the assay system in combination with the relevant antigen. Trypsinized leukocytes from control persons are used as indicator cells. In the present work, the nature of the humoral factor in serum from breast and lung cancer patients is studied. 3.5 M KCl extracts from the cell lines MCF-7 and Calu-1 were used as breast and lung cancer antigen, respectively. It was found that the humoral factor involved in the H-LAI response was precipitated from the sera by addition of ammonium sulphate to 50% saturation. This factor could be removed by passage through an affinity column with the relevant antigen bound to the matrix. Stable complexes were formed between the humoral factor and the relevant antigen, and could be precipitated by polyethylene glycol. When different anti-immunoglobulins were added to the sera, the humoral factor was specifically removed by addition of anti-IgG antibodies. The data presented indicate that the humoral factor in sera from patients with breast and lung cancer is antitumor antibodies of IgG nature.

Quantitation of proteins in sputum from patients with chronic obstructive lung disease. II. Determination of albumin, transferrin, alpha1-acid glycoprotein, IgG, IgM, lysozyme and C3-complement factor.

Albumin, transferrin, alpha1-acid glycoprotein, IgA, IgG, IgM, lysozyme and C3-complement factor have been immunologically determined in sputum and serum samples from 16 patients with chronic bronchitis. The sputa were effectively solubilized prior to the analysis. This is necessary for correct determination of the compositions of sputum. IgA (approx. 3 g/l) and lysozyme (approx. 1 g/l) were present in the highest concentrations. Lactoferrin was qualitatively shown to be present in all the sputa. The concentration of IgG, albumin and transferrin were much higher in the sera than in the sputa, their presence in sputum probably being a result of a passive "leakage" from serum. The ratios for IgA, IgM and lysozyme indicated that these macromolecules are locally synthesized in the respiratory tract. The concentrations of IgA and lysozyme were closely correlated, indicating that the biosynthesis of secretory IgA and bronchial lysozyme may be coupled or controlled by the same mechanism. Except for a weak correlation between the concentration of IgA in sputum and viscosity, no such correlations were obtained for the other proteins determined.

Immune responses in lung cancer patients measured by a modified leukocyte adherence inhibition test using serum.

A modified leukocyte adherence inhibition (H-LAI) assay was used to study immunological factors in serum from lung cancer patients. In this test, 0.25% serum was added to the assay system, together with tumor antigen and trypsinized leukocytes from control persons. Extracts from a human lung cancer cell line (Calu-1) and a human breast cancer cell line (MCF-7) were used as antigens. The results obtained were compared with data found with the original hemocytometer (C-LAI) assay. Of 21 lung cancer patients studied, 20 (95%) gave a positive response in both the H-LAI and the C-LAI assay systems against Calu-1 antigen. Only 1 of the patients gave a positive response in the H-LAI system against MCF-7 antigen, while 3 patients (14%) responded in the C-LAI assay. None of the 14 control persons tested gave a positive response. While the C-LAI assay was limited to the use of fresh blood, the H-LAI system was performed on small amounts of serum. The serum could be stored in the frozen state for a long time period. The results indicate that the H-LAI assay possesses at least the same sensitivity and specificity as the original C-LAI test.