Specific Immune Response in Human Skin Carcinoma

Eight out of nine patients with squamous cell carcinoma of skin have shown immunological reactivity against their own tumour cells by one or more tests with their sera or peripheral blood lymphocytes. The tests included membrane and cytoplasmic immunofluorescence, and, with cultured tumour, complement-dependent serum cytotoxicity and lymphocyte attack. One case examined in depth had an unusually conspicuous lymphocyte and plasma cell reaction on histological examination, and was positive by all four tests; a time-lapse cinephoto-micrographic record over seven days was obtained of the attack on the carcinoma cells in culture by the patient''s lymphocytes.     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Colonic carcinoma: clinicopathological correlation with immunoreactivity.

The relation between tumour spread, histological differentiation, and in-vitro antitumour immunoreactivity was studied in 132 cases of carcinoma of the large bowel. Positive correlations were found between blood lymphocyte antitumour cytotoxicity and both tumour differentiation and absence of recurrence or metastatic spread.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. I. Demonstration of widespread occurrence using reference typing sera.

M0use alloantisera produced against different specificities of the K, I, and D regions of the H-2 gene complex reacted as immunogenetically anticipated with normal lymphoid target cells of different haplotypes in cytotoxicity and indirect immunofluorescence tests. These same alloantisera, however, produced anomalous positive reactions when tested on cultured MCA-induced sarcoma cells from B10 background H-2 congenic mice. Absorption experiments demonstrated that the anomalous activity in these sera was directed against a tumor membrane antigen(s) which was distinct from H-2 region specificities against which the reference alloantisera were produced, and which was shared in common by multiple cultured sarcoma lines. Similar anti-tumor antibody activity could be demonstrated in the serum of older (greater than 12 weeks) but not younger normal unimmunized mice of the strains used as recipients for alloantiserum production. It is suggested that the observed anamalous anti-tumor activity in these alloantisera may be due to the presence of antibodies reactive with envelope antigens of murine leukemia virus which are expressed on sarcoma cells maintained in culture.     

Immunization with Irradiated Tumour Cells and Specific Lymphocyte Cytotoxicity in Malignant Melanoma

A microculture technique has been adapted to assay the cytotoxic properties of the lymphocytes from patients with malignant melanoma when cultured with their autologous tumour cells. In a series of patients with established melanoma specific autologous cytotoxicity was extremely uncommon, being detectable in only 3 out of the 22 cases studied. This cytotoxicity did not correlate with clinical staging of the disease but may well have been related to tumour volume. By autoimmunization of patients with an irradiated suspension of their own tumour cells the appearance of cytotoxic lymphocytes could be provoked in 5 out of the 12 patients studied. This cytotoxicity was detectable at the end of the first week after the autograft and disappeared by the third week. Cytotoxic lymphocytes did not correlate in any obvious way with the appearance of specific antitumour antibodies detected by immunofluorescence. So far there has been no evidence of a serum factor capable of blocking the lymphocyte cytotoxicity in these patients.The presence and possible significance of cytotoxic lymphocytes in patients with malignant disease is discussed.     

Tumour-specific Antibodies in Human Malignant Melanoma and their Relationship to the Extent of the Disease

Biopsy specimens and sera were obtained from 103 melanoma patients. Autoantibodies were demonstrated by (1) complement-dependent cytotoxicity of autologous melanoma cells in short-term culture; (2) complement-dependent inhibition of ribonucleic acid synthesis; (3) immunofluorescent staining of the cytoplasm of killed melanoma cells and of the surface membrane of viable melanoma cells. Over one-third of the sera studied had antibodies to autologous melanoma cells. Although for technical reasons all three tests could not be performed with the cells from every melanoma, whenever multiple testing was possible there was complete concordance. The autoantibodies were virtually confined to patients in whom the disease was not widely disseminated, and over 80% of such patients had positive sera. In a limited number of patients who have been followed autoantibodies disappeared as the disease progressed to become widely disseminated. Two patients with generalized disease developed autoantibodies following inoculation by their own irradiated tumour cells.Two types of autoantibodies were recognized: one, active against antigen(s) in the cell surface membrane, was specific for each tumour—that is, only the autologous serum reacted—and was concerned in the cytotoxic activity; the other reacted with cytoplasmic antigens which appeared to be present in most or all melanoma cells.     

Demonstration of serum alpha1-macroglobulin on leucocyte surfaces in non-pregnant and pregnant rats

alpha1-macroglobulin (alpha1-M) was demonstrated by indirect immunofluorescence staining on the surface membrane of leucocytes obtained from rat blood and lymphoid tissue. The incidence of alpha1-M positive cells correlated with serum levels of the protein. Compared with blood, a significantly higher percentage of staining cells was demonstrated in spleen and lumbar lymph nodes. During pregnancy, when serum levels of alpha1-M were significantly elevated, the proportion of alpha1-M staining leucocytes increased in blood, Thymus and lymph nodes but the incidence of alpha1-M positive spleen cells was unaffected. The comparatively low incidence of alpha1-M staining thymocytes, together with the presence of alpha1-M on glass-adherent cells and on leucocytes from congenitally athymic rats, suggest that this protein is associated predominantly with B cells and monocytes.     

The Kurloff cell in estrogenized guinea pigs as a CT7+ 8BE6- CT6- MR-1- CT10- IgM- lymphocyte with natural killer activity.

The relationship of the Kurloff cell (KC), guinea pig blood mononuclear cell with natural killer (NK) activity, to a known cell lineage was established. Using indirect immunoperoxidase staining and flow cytometric analysis, numerous monoclonal antibodies directed against guinea pig macrophage antigen, Ia antigen or different T lymphocyte markers and a polyclonal anti-IgM serum were tested in unimmunized estrogenized animals. We excluded any relationship between KC and the monocytic macrophage lineage (MR-1-) and between KC and the B lymphocyte lineage (CT10- and IgM-). The KC immunophenotype was pan T CT7 positive but 8BE6 (mature thymocyte) and CT6 (cytotoxic suppressor T lymphocyte) negative. Since KC displays an NK activity, this cell may be classified among the NK effector cells exhibiting some T lymphocyte markers.     

Immunocytochemical Staining For the C-Erbb-2 Gene Product In Breast Aspirates: A Preliminary Report

The results of the determination of c-erbB-2 gene expression by immunocytochemical staining of cytological aspirates, prepared by cytocentrifugation, have been compared with paraffin-embedded tissue sections from the same tumour. Our results show equivalent staining in 20/22 cases, with six cases being both scored positive and fourteen cases being both negative. Two samples gave conflicting results. One case was scored as being positive on the cytological aspirate, whereas in the tissue sections taken from the same tumour positive staining was only seen in areas of non-invasive intraduct carcinoma. This sample was scored as being negative. In another case, cytoplasmic staining with less than 50% of the cells showing any positivity was observed in the cytospin sample, with negative staining in the corresponding tissue section. We conclude that expression of c-erbB-2 immunostaining is detectable on cytological preparations prepared by cytocentrifugation but must be interpreted with caution in tumours which may have a large intraduct component or which give predominant cytoplasmic staining.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.     

Immunofluorescent staining of metastatic carcinoma cells in serous fluid with carcinoembryonic antibody, epithelial membrane antibody, AUA-1 and BER-EP4

Using an indirect immunofluorescence technique, we assessed the accuracy and clinical usefulness of a panel of monoclonal and polyclonal antibodies. the panel consisted of carcinoembryonic antibody (CEA) and epithelial membrane antibody (EMA), AUA-1, and Ber-EP4 conjugated with fluorescein isothiocyanate. Twenty-six specimens from pleural, peritoneal or pericardial effusions known to contain carcinoma cells (adenocarcinoma or large cell anaplastic carcinoma) and 16 specimens without carcinoma were first examined. the sensitivity and specificity for each of the antibodies were as follows: CEA, 71% and 75%; EMA, 96% and 81%; AUA-1, 80% and 100%; and Ber-EP4, 85% and 100%, respectively. the panel of antibodies was then applied to a group of 14 'problematic' fluids. These had been identified as causing dilemmas in interpretation, either because the cells in the fluids were of equivocal appearance on light microscopy, or the cytological diagnosis was different from that expected in the light of the clinical condition of the patient. Insufficient cellular material was present in one specimen. In five (39%) of the cases the immunochemical staining supported the light microscopic diagnosis. In four (30%) cases, however, the results indicated that the original light microscopic report was incorrect. Two of these were examples of large cell carcinoma of the lung, in which false negative reports had been issued on pleural fluids. the other two were cases of benign ovarian tumours in which a false positive report had been issued. the immunostaining also clarified the final diagnosis in the three patients (23%) on whom 'suspicious' cytological reports had previously been issued. the remaining case, fluid from a patient with a high grade mixed Mullerian tumour of the ovary, was unresolved. We conclude that immunofluorescent staining by AUA-1, EMA and Ber-EP4 is an aid in the cytological interpretation of serous fluids. CEA is much less helpful.     

Kinetic study of antitumor lymphocyte cytotoxicity in rats bearing progressive or regressive syngeneic colonic tumors

In order to perform a kinetic study of the antitumor lymphocyte-mediated cytotoxicity in syngeneic BDIX rats bearing progressive (TRb) or regressive (TSb) colonic tumors, the 51Cr release assay was adapted to this experimental model. Two groups of 72 rats were inoculated with TRb or TSb cells. Results failed to show any clear and univocal evolution of lymphocyte cytotoxicities against TRb or TSb cells. On the other hand these cytotoxicities were roughly at the same level than natural cytotoxicity (normal rats), and were always higher against TSb than TRb cells.     

Antitumor activity of equinatoxin.

Equinatoxin, a highly basic protein extracted from Actinia equina, causes an increase in the survival time of mice bearing the ascitic form of Ehrlich carcinoma, whereas it has no effect on L1210 leukaemia. When tested for in vitro cytotoxicity by the dye exclusion test, it shows a potent activity on both tumour cell lines, with ED50 of a few ng/ml. Higher concentrations produce an extensive lysis of the cells. The cytotoxic effects of Equinatoxin are inhibited by phospholipids, thus suggesting that its mechanism of action may be related to interactions with lipids or other charged components of cell membrane. The observed lack of in vivo activity against L1210 leukaemia presumably is due to poor systemic absorption of the protein and/or neutralization by serum factors.     

Neoantigen of the complement membrane attack complex of cytotoxic human peripheral blood lymphocytes.

Neoantigenic determinants (neoAg) specific for the assembling membrane attack complex (MAC) of complement were detected by immunofluorescence microscopy on the surface of cytotoxic lymphocytes during the antibody-dependent cellular cytotoxicity (ADCC) reaction. This study employed antibody-sensitized chicken erythrocytes as target cells, human peripheral blood lymphocytes as effector cells, and RITC-conjugated rabbit F(ab')2-anti-neoAg. NeoAg was present on 60% of ADCC plaque-forming lymphocytes (PFL). Eight out of 182 neoAg-positive PFL were observed in direct contact with their target cells. In these cases MAC-specific neoAg was visualized at the zone of contact between the cells. Anti-neoAg Ig was found to inhibit ADCC plaque assays up to 62%; and 51Cr-release assays up to 79%. Stimulation of lymphocytes by PHA or mixed lymphocyte culture increased the expression of neoAg. In the case of PHA, increased neoAg expression was correlated with an increased incorporation of 14C-leucine into C5, C6, C7, and C8 antigens, which was detected by immunodiffusion and autoradiography.     

Cytotoxicity from cultured cells: analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity.

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr ⁵¹Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.     

Chlamydia trachomatis infections in asymptomatic women. Results of a study employing different staining techniques

A total of 300 cervical smears randomly collected from asymptomatic women in a mass-screening program for the detection of cervical carcinoma was investigated for Chlamydia trachomatis infection by the use of Papanicolaou and immunofluorescence staining. Features of chlamydial infection detected in 18 cases by Papanicolaou-stained smears were confirmed in 11 cases with immunofluorescence; not a single case that was negative in the Papanicolaou-stained smears was positive by immunofluorescence. The presence of Chlamydia in the Papanicolaou-stained smears in ten cases, including two cases that were negative by immunofluorescence, was also proven by either immunoperoxidase staining or in situ hybridization. On the other hand, either immunoperoxidase or in situ hybridization gave false-negative results in two of the ten cases. Therefore, the combined use of different techniques demonstrated that false-negative results occurred with all techniques, except with Papanicolaou-stained smears, whose sensitivity is apparently the highest.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Immunohistochemical studies of colorectal carcinoma with a monoclonal antibody against carcinoembryonic antigen

Immunoperoxidase localization of carcinoembryonic antigen (CEA) was performed on tissue sections of colorectal carcinoma using a monoclonal antibody (MAb) against CEA. CEA has been demonstrated in 20 out of 22 rectum carcinomas (90.9%), in all of 23 colonic carcinomas, in none of 4 hyperplastic polyps and in 2 out of 6 adenomatous polyps (33.3%). CEA was found more often, and the intensity of the staining was stronger in well-differentiated carcinomas than in moderately and poorly differentiated carcinomas. No correlation was found between the presence of CEA in colorectal carcinoma and the stages of the disease. The mean values of serum CEA in patients with colorectal carcinoma and polyps with negative, weakly and strongly positive staining were 5.4 +/- 3.9 ng/ml, 28.3 +/- 23.8 ng/ml and 99.8 +/- 145.3 ng/ml respectively. Elevation of serum CEA occurred in 30 out of 39 (78.9%) cases with strongly positive CEA staining, in 4 out of 6 (66.7%) with weakly positive and in 1 out 9 (11.1%) with negative staining. A significant difference was found in serum CEA activity between the group with negative CEA staining and positive CEA staining (P less than 0.01). Our results suggest that the monoclonal antibody (MAb C27) can be used for the localization of CEA in conventionally prepared tissues of colorectal carcinomas by immunoperoxidase techniques for routine immunopathological diagnosis.     

Specific Immunodiagnosis of Hepatocellular Carcinoma by Leucocyte Adherence Inhibition

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient''s blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.