Biomedical subjects
Publications and source records attributed to J H Dean.
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Cell-mediated immunity (CMI), as detected by the agarose microdroplet macrophage migration inhibition (MMI) assay, was investigated using peritoneal exudate cells (PEC) of BALB/c mice and several crude membrane (CM) and solubilized preparations of murine plasmacytomas. The MMI assay was quite sensitive and detected inhibition of macrophage migration as low as picogram quantities of CM, NP40 detergent- and papainsolubilized preparations (CS) of ADJ-PC5 and LPC-1 plasmacytomas. The data were highly reproducible from one experiment to the next with the same or different lots of the CM or solubilized extracts. Specificity studies demonstrated that ADJ-PC5 and LPC-1 plasmacytomas expressed cross-reactive tumor-associated antigens (TAA) as detected by MMI and confirmed by tumor challenge and Winn neutralization experiments. No cross-reactivity was observed with similar extracts prepared from an unrelated syngeneic simian virus 40 (SV40)-induced sarcoma. The inhibition of macrophage migration observed was mediated by culture supernatants generated from the mixture of plasmacytoma-immune spleen cells with antigens and then assayed in an indirect MMI assay on normal PEC. The agarose microdroplet MMI assay appeared to be a rapid and sensitive method to measure TAA recognition and to monitor TAA isolation and solubilization with minimum numbers of immune cells.
Lymphocyte proliferation (LP) assays were performed in microculture using the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A); the T + B cell mitogens, pokeweed mitogen (PWM) and staphylococcal phage lysate (SPL); and a pool of allogeneic stimulating leukocytes in one-way mixed leukocyte cultures (MLC) in lung and breast cancer patients and normal individuals. The resultant data were expressed in three different ways: (1) as mean counts per minute (CPM) of tritiated thymidine incorporation; (2) as a stimulation index (SI) and (3) as a relative proliferation index (RPI). The RPI is defined as the ratio of net CPM (nCPM) in experimental cultures with stimulant (E) minus medium control cultures (C) of a test individual to the mean nCPM of three or more normal individuals examined in the same assay on the same day. These expressions were then compared for their ability to discriminate between LP responses in cancer patients and normal individuals. The RPI value and selected cut-off values gave the most sensitive measure for the determination of depressed proliferative responses. These analyses demonstrated that lung carcinoma patients were depressed to PHA (50%), MLC (47%), PWM (43%) and Con A (40%). To a lesser degree, breast carcinoma patients were also depressed to MLC (36%), PHA (31%), PWM (27%) and Con A (19%). Our data indicate that the use of the RPI in the analysis of LP response represents an improved method for detecting impaired response of lymphocytes to general mitogens and alloantigens which can consistently reveal immunosuppression in many cancer patients and may be useful for serial monitoring of individual patients.
Cytogenic and immunologic studies were performed on 20 members of a family who had an increased susceptibility to carcinomas of the colon, uterus and of lymphoproliferative malignancy. Chromosomal abnormalities such as small G and/or long submetacentric marker chromosome and other aberrations were observed in members who had cancer as well as some asymptomatic siblings. In addition, impairment of T cell function was noted in many of the members suggesting that defective cell-mediated immunity, which also may be genetically determined, played an important role in the expression of this disease.
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Direct and indirect agarose microdroplet migration inhibition assays were performed with log dilutions (50--5 X 10(-8) microgram/ml) of soluble purified protein derivative (PPD) of tuberculin and leukocytes (4 X 10(5)/droplet) from PPD skin test positive or negative individuals. Some tests were run in parallel with the capillary tube method. Inhibition of migration of leukocytes from 9/11 PPD skin test positive donors studied was observed in direct tests when employing PPD doses ranging from 1--50 microgram/ml PPD. Inhibition of migration of leukocytes from some PPD skin reactive donors was obtained with as little as 5 X 10(-3)--5 X 10(-7) microgram/ml (i.e., 5 nanograms to 0.5 picograms). Some inhibition of leukocyte migration was seen with skin test negative donors (presumably toxicity) with the higher doses of PPD (50 microgram/ml), but generally little inhibition was observed with these donors at lower doses. Comparative experimetns of the agarose micromethod and the capillary tube technique indicated that the agarose assay was more sensitive in that it could detect reactivity with 2--4 logs lower antigen concentration. Indirect assays using supernatants of cultures of PPD triggered mononuclear cells and indicator granulocytes in agarose droplets demonstrating that a lymphokine (presumable leukocyte inhibitory factor) was being produced and suggested that cell-mediate immune (CMI) reactions were operative. The results indicate the usefulness of this technique in the sensitive-detection of CMI against such antigens as soluble PPD. The assay should prove useful in quantitative assessment of cell-mediated reactivity by using a wide range of antigen concentrations and the leukocytes from as little as 2--5 ml of blood.
Lymphocyte proliferation assays with autologous tumor material in mixed leukocyte-tumor interactions (MLTI) were employed to monitor tumor-associated cell-mediated immune responses of peripheral blood lymphocytes from patients with carcinoma of the breast. In addition, leukocyte migration inhibition (LMI) assays were employed to compare reactivity to autologous breast-tumor extracts versus allogeneic breast-tumor extracts. Positive lymphoproliferative responses to tumor-associated antigens (TAA) were observed in the MLTI assay with the use of either intact autologous tumor cells or crude extracts (in mug and ng quantities) in 12 of 34 (35%) breast cancer patients studied. Positive reactivity to tumor, but not to normal tissue of reactive patients, was observed in repeated assays. Finally, patients demonstrating positive MLTI responses to autologous tumor extracts likewise responded in LMI assays to these same autologus extracts as well as to allogeneic breast-tumor extracts, but not to non-breast-tumor extracts. Thus breast tumors appeared to possess common TAA among both male and female patients.
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Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.
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One hundred and seven patients with carcinoma of the lung underwent immunologic testing, and 62 of these patients were randomized to an immunotherapy protocol comparing the effects of Pasteur strain BCG, either alone or combined with allogeneic tumor cells, to the effects of no immunotherapy. Patients with residual disease left at the time of surgery or with metastatic disease at the time of diagnosis showed no increase in survival as a result of this form of immunotherapy. An insufficient number of patients with less advanced disease, in whom we would expect the most beneficial effect, have been entered in this study. In general, we were unable to document substantial effects of immunotherapy on the immunologic parameters tested. Only in recall antigen skin testing was there a statistically significant increase in reactivity in the immunotherapy groups. Tests of general immune status appeared to have a predictive value in monitoring lung cancer patients. Anergic patients had a poorer prognosis than did patients who demonstrated skin test reactivity. Patients with normal percentages of lymphocytes (T cells) forming rosettes with sheep erythrocytes at 29 degrees C were generally normal in other tests of immune competence. In serial studies of rosette formation, all patients who developed recurrent disease had a pattern of depressed or falling rosette values, and these abnormalities occurred an average of 3.1 months prior to clinical detection of recurrence. Patients with large-cell anaplastic carcinoma were found to have a significantly higher incidence of depressed rosette levels than the other histologic types. Both large and small-cell anaplastic patients had significantly depressed lymphocyte proliferation by mitogens and allogeneic cells. Although lung cancer patients have been described as immunologically depressed, they are capable of recognizing tumor-associated antigens. When tested in leukocyte migration inhibition assays with tumor-associated antigens, the majority of the patients in our study were found to be reactive. The use of a 3 M KCl extract of pleural effusion cells from a patient with pulmonary adenocarcinoma has given good reactivity and specificity in lung cancer patients of all histologic types. In addition, these patients have been shown to respond in a mixed lymphocyte/tumor interaction to tumor-associated antigens (Dean, 1976b).
Methods are described by which cryopreserved cells can be utilized in a number of in vitro assays. On a per cell basis, nearly total recovery of function can be demonstrated for lymphocyte transformation (mitogens, antigens and MLC) and rosette-forming cells. Excellent recovery of mononuclear cell production of leukocyte migration inhibitory factor was also observed. Highly reproducible activity was recovered on a per lymphocyte basis in lymphocyte cytotoxicity but with a definite decrement in the percentage recovery. Both for longitudinal studies of immune function and for standardization of these assays in one or more laboratories such cryopreserved cells are ofimmense value and should be widely utilized.
The cell-mediated immune status of B10.D2 (H-2d) mice immunized with spleen cells from a congenic strain, B10.A (H-2a), differing at the H-2 locus and of BALB/c mice immunized with a syngeneic simian virus 40 (SV40)-induced sarcoma (mKSA-TU5) was evaluated by an agarose microassay for migration inhibition factor. The inducing antigens in this experiment were papain-solubilized and partially purified chromatographic preparations of spleen cells from A/J mice (H-2a) and a papain-solubilized antigen extract prepared from a tissue culture-adapted cell line (TU-5), derived from the SV40-induced mKSA tumor. The assay used microliters of normal or immune peritoneal exudate cells (PEC) resuspended in a 2-mul droplet of agarose and cultured in the presence or absence of antigen. Specific migration inhibition of PEC from immunized mice was observed with concentrations of solubilized antigen preparations as low as 2.0 mug/ml (3.67 mug/chamber).
Cell-mediated immunity (CMI) and tumor rejection were studied in the Friend virus leukemia system of C57Bl/6 mice. Mice were immunized with Friend leukemia virus (FLV) or X-irradiated FBL-3 leukemic cells and studied temporally for the development of CMI reactivity by assays of 51Cr release lymphocyte cytotoxicity, lymphocyte transformation, migration inhibition, Winn tumor cell neutralization and transplantation rejection. High levels of specific lymphocyte cytotoxicity were observed by day 7 f0llowing FLV infection; this reactivity reached a peak between 17 and 21 days, and returned to background levels by day 36. Further, positive Winn assays were obtained with spleen cells from mice immunized with FLV at times when the mice resisted live FBL-3 tumor challenge. Positive lymphocyte transformation was obtained with spleen cells from mice immunized with FLV or FBL-3, but not with cells from normal mice or mice immune to a syngeneic methycholanthrene-induced tumor, when cultured with papain-soluble FBL-3 or RBL-5 tumor-cell extracts or mitomycin-C (MMC)-treated FBL-3 or RBL-5 cells. Positive reactivity in the lymphocyte transformation assay occurred after reactivity had peaked in the lymphocyte cytotoxicity test. Similar positive macrophage migration inhibition patterns were also obtained with peritoneal exudate cells (PEC) from FLV-immunized mice using papain-solubilized tumor-associated antigen (TAA) from FBL-3 cells. These data suggest that sequential development and modulation of CMI reactivity occurs as observed in different assays following immunization in this system.
BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.
Four assays of tumor-specific cell-mediated immunity were compared during growth of a syngeneic Simian-virus-40-induced tumor, mKSA. Major differences were found in immunity detected by the tumor neutralization test (the Winn test), direct tumor challenge, the microcytotoxicity assay and the -51chromium-release lymphocytotoxicity assay. Progressive growth of the neoplasm followed by loss of immunity (the eclipse phenomenon) was documented with the Winn test. It was established that this eclipse phenomenon represented a lesion in the T-cell system of tumor-bearing hosts. This lesion was found to be specific and unrelated to anatomic tumor location. The ability to produce graft-versus-host reactions, and the ability to respond to mitogens, were found to be generally intact in tumor-bearing animals. Cells capable of recognizing mKSA tumor antigens and reconstituting an immune response following surgical removal of tumor or upon transfer to normal mice were found in the spleens of mice bearing advanced tumors. No suppressor cells that might account for the eclipse phenomenon could be demonstrated. Tumor-bearer serum did not block neutralizing activity of immune spleen cells in the Winn test, and immune cells were capable of neutralizing tumor even in tumor-bearing hosts. The possibility that the lesion is intrinsic to T-cell precursors of effector cells is discussed.