Radioisotopic 51Cr-leukocyte adherence inhibition (LAI) assay. I. Demonstration of anti-tumor immunity in patients with breast carcinoma.

A simplified radioisotopic leukocyte adherence inhibition assay (51Cr-LAI assay) was used to determine tumor-directed immune responses in patients with cancer of the breast. Essential steps in development of this assay are the standardization of conditions for optimal 51Cr uptake by peripheral blood lymphocytes (PBL) and the inclusion of autologous or normal AB serum in the incubation media. A dextrose salt mixture (GNK) was found to enhance intracellular uptake of 51Cr significantly (8-fold) without affecting viability of the cells or without causing selective loss of lymphocyte subpopulations. The presence of 10% autologous or normal AB serum prevented non-specific LAI responses to unrelated tumor antigens. In a study of 46 preoperative patients with suspected breast cancer, clear and accurate prediction of the presence of cancer was achieved with this new assay. All patients with localized breast cancer showed significant adherence inhibition in response to allogeneic breast tumor extracts whereas normal control women and patients with benign diseases did not respond. Neither patients with cancer nor those with benign breast diseases reacted to extracts of benign breast tissue antigens. LAI reactivities appeared to be directed selectively against tumor-associated antigens (TAA) and reflect specific antitumor immunity. This short term (4 h) 51Cr-LAI assay provides reproducible and specific results analogous to those using tube-LAI assay. The test has the advantages of being accurate, sensitive and free from technical bias.

Multiple dysfunctions in developmental and activational stages of T lymphocytes, B lymphocytes and monocytes in ARC and AIDS patients.

Peripheral blood leukocytes from ARC and AIDS patients were examined before and after phytohemagglutinin (PHA) stimulation by dual color flow cytometry and monoclonal antibodies which identify developmental and activational stages of T lymphocytes, B cells and monocytes. There was a persistent elevation in the total number of circulating Ia+ lymphocytes with progressive selection for B1+ Ia+ lymphocytes and T suppressor cells and a concurrent reduction in the antigen-presenting monocytes. Following PHA stimulation there was a marked decrease in all subsets of Ia+ lymphocytes and monocytes. These results indicate (a) multicellular dysfunctions in the immunosurveillance mechanisms in AIDS, and (b) that many functional subsets of circulating lymphocytes and monocytes were already activated and therefore poorly responsive to additional antigenic or mitogenic stimuli.

Effector lymphocyte response to homologous tumor antigens in various stages of malignant disease as monitored by leukocyte adherence inhibition--cell mediated immunity (LAI-CMI).

Leukocyte adherence inhibition-cell mediated immunity (LAI-CMI) studies were performed on leukocytes obtained from patients with various stages of breast cancer, colon carcinoma and lung cancer in order to monitor cell mediated immunity during tumor progression. In the presence of autologous serum, all patients with localized tumors showed positive LAI-CMI indexes (greater than 20%), while significant reduction of homologous tumor antigen recognition as measured by the LAI-CMI responses was observed in nearly all patients with Stage IV breast cancer, Duke C colon cancer and Stage III lung cancer. On substituting autologous serum with normal AB serum, leukocytes from patients with large tumor burdens responded to homologous tumor antigens. These results indicate the existence of organ-specific serum factor(s) which may mask the receptor sites on effector cells for tumor recognition. Patients with such serum blocking factor(s) showed significant increase of IgG immune complexes IgM, IgA and alpha-2-macroglobulins. Application of a protein A affinity column purification resulted in a major reduction of IgG and other immune globulins but not of alpha-2-macroglobulin. The blocking effects of autologous serum, however, were not completely abrogated by filtration on the protein A column, thus suggesting that SBF may be heterogeneous in nature and may occur in other serum protein fractions beside the immune globulins.

Inverse relationship between HIV-1 p24 antigenemia, anti-p24 antibody and neutralizing antibody response in all stages of HIV-1 infection.

A double blind cohort study was conducted on 149 homosexual males and 36 patients with AIDS to investigate the relationship between HIV-1 antigenemia, the presence of neutralizing antibody (NA) activity and specific anti-viral core protein (p24) antibody (Ab) in the sera of HIV infected individuals during their progression to AIDS. All AIDS patients and 68% (101/149) of the homosexual males were HIV seropositive upon entering the study. Of those 48 (32%) homosexuals who were HIV negative at the onset, three seroconverted during the two year observation period. Retrospective studies of the HIV(-) subjects' sequentially stored serum samples demonstrated an early transient appearance of gag encoded p24 antigen (Ag) which preceded their production of NA and specific anti-p24 Ab. Following their seroconversion, no more circulating p24 Ag could be detected. Among the 101 HIV positive homosexuals, 16% rapidly progressed to AIDS and seven of these 16 (44%) subjects eventually died during the two year observation period. In this group of individuals with poor prognosis, presence of NA and anti-p24 Ab commenced at the onset reaching peak levels just prior to developing AIDS and began to decline as the clinical course worsened. Their circulating level of p24 Ag remained undetectable as long as there was quantifiable NA and anti-p24 Ab in their sera. Reappearance of circulatory p24 Ag, on the other hand, was associated with high risk for progression to AIDS.2+hus, while only 11

Isoprinosine-induced modulation of T-helper-cell subsets and antigen-presenting monocytes (Leu M3 + Ia +) resulted in improvement of T- and B-lymphocyte functions, in vitro in ARC and AIDS patients.

Peripheral blood leukocytes from ARC and AIDS patients were analyzed following phytohemagglutinin- and pokeweed mitogen-induced lymphocyte transformation by dual-color flow cytometry using monoclonal antibodies that identify developmental (HLA-DR) and functional (Leu8) subsets of T cells and monocytes. Significant decreases in both the suppressor regulating helper T subset (Leu3+ Leu8+) and the reciprocal inducer helper T subset (Leu3+ Leu8-) responsible for inducing differentiation of B cells were observed. Simultaneously, the percentages of the effector suppressor T cells and the precursor suppressor T cells were increased, both of which were required for generation of suppression of cell-mediated immunity. There was also a progressive selection of Ia+ cells bearing the Leu2 (Ts) markers and a concurrent reduction of the percentage of antigen-presenting monocytes and activated helper T cells. These results suggest that the functional deficiencies in AIDS may be caused by defects in T-cell activation as well as antigen presentation by monocytes. Isoprinosine induced an increase in both regulator Th (Leu3+ Leu8+) and inducer Th (Leu3+ Leu8+) subsets of helper T cells while potentiating the expression of Ia antigen on helper T cells and monocytes during mitogen-driven DNA synthesis. These events initiated a cascade of cellular interactions leading to partial restoration of cell-mediated immune responses. These interferences with the defective helper/suppressor regulatory pathways may have important therapeutic implications.

Phenotypic and functional correlations in circulating lymphocytes of prodromal homosexuals and patients with AIDS.

Findings of selected phenotypic and functional parameters demonstrated significant aberrations in both prodromal homosexual males and patients with AIDS. An impaired blastogenic response to T-cell dependent B-cell mitogen was associated with a significant decrease in percentage of helper T cells but appeared unrelated to the percentage of suppressor T cells. The functional subsets of the latter were further defined by a panel of monoclonal antibodies (Leu8, Leu15, and HLA-DR) applying dual color flow cytometric techniques. In both homosexuals at risk and in AIDS patients, activated suppressor cells with the phenotype Leu2+Leu15+ and Leu2+HLA-DR+ were elevated while the reciprocal cytotoxic Ts cells (Leu2+Leu15- and Leu2+HLA-DR-) were depressed. Both subsets of suppressor precursor cells and effector cells (Leu2+Leu8+ and Leu2+Leu8-) were elevated particularly in high risk homosexual males. These results suggest a defective feedback loop that regulate both helper and suppressor T cells as well as B-lymphocyte functions and may explain the clinical manifestation of AIDS.

Specificity of tumor antigen recognition by T-lymphocytes in the leukocyte adherence inhibition (LAI) assay in patients with breast cancer.

In an attempt to enhance the specificity with LAI assay in the diagnosis of cancer, we explored the possibility of using highly purified, 51Cr-prelabeled-PBL-subpopulations in performance of the assay. Leukocyte separation was achieved in 2 stages which involved EAET rosetting for the isolation of T-lymphocytes followed by Percoll gradient for separation of monocytes. These procedures provided excellent cellular yield, viability and cellular enrichment. By comparing the reactivity of various isolated subpopulations from breast cancer patients we established the critical role of T-lymphocytes in the recognition of tumor antigen in the LAI reaction, i.e. T-lymphocytes exhibited a breast cancer extract-specific response, reflected in a significant reduction of leukocyte adherence, while B-lymphocytes did not respond. In contrast, monocytes of breast cancer patients showed a non-specific LAI response to several unrelated tumor extracts. Similarly, T-lymphocytes from all patients with benign breast diseases as well as from healthy subjects showed no LAI response while false positive responses were detected in the unseparated PBL and monocytes of these individuals. It is concluded, therefore, that a differential diagnosis of cancer can only be made if isolated T-lymphocytes from PBL are used in the LAI assay procedure.

Impairments in functional subsets of T-suppressor (CD8) lymphocytes, monocytes, and natural killer cells among asbestos-exposed workers.

Peripheral blood leukocytes from asbestos-exposed workers were analyzed by dual color flow cytometry using monoclonal antibodies that identify developmental (HLA-DR) and functional (Leu 8) subsets of T helper, suppressor lymphocytes, and monocytes. An increase in the number of T suppressor cells was closely associated with a decrease in T lymphocyte functions while numerical defects in activated monocytes (Leu M3+Ia+) and natural killer cells (Leu 7+) were correlated with a depressed Th/Ts ratio. Furthermore, among asbestos-exposed workers with depressed T cell functions we have demonstrated a significantly higher number of the effector Ts (Leu 2+ Leu 8-) subset which regulates both the Th/Ts lymphocyte system as well as B cells and NK cell activities. These findings identified changes in the T suppressor feedback regulatory loop as being responsible for the immunoregulatory imbalance among long-term asbestos workers. In double blind analyses of demographic and radiographic data these phenotypic changes were not correlated with age, smoking history, or duration of exposure but were associated with radiographic evidence of asbestos-associated effects. This correlation established a direct link between asbestos exposure and the subsequent development of immune dysfunction.

Immunorestorative properties of isoprinosine in the treatment of patients at high risk of developing ARC or AIDS.

We have evaluated the immunomodulatory effects of isoprinosine in a double blind randomized clinical study on 63 immunosuppressed male homosexuals with persistent generalized lymphadenopathy (PGL) or ARC. The subjects received either placebo or isoprinosine at 1 or 3 g/day for 28 days. All subjects were monitored for performance for a one year period. In the 3 g/day treatment group clinical improvement was reported by 52% of the patients in contrast to 15% in the placebo group. Patients receiving 3 g/day isoprinosine showed significant increase in NK cells, a major subset of which bears the Leu 7 surface antigen, and in NK cell function as early as at the termination of treatment. This normalized NK cell property was still evident 5 months after cessation of therapy. Total T lymphocytes and T helper cells also increased in this group and a concomitant reduction was observed in activated T lymphocytes (HLA-DR+). As a direct result of the therapy an increase was found in the Th regulatory (Leu 3+ Leu 8+) cell population resulting in normalization of Th inducer/Th regulatory cell ratio. A concomitant reduction to normal range occurred in Ts effector (Leu 2+ Leu 8-) and functionally activated Ts (Leu 2+ HLA-DR+) cell populations. The kinetics of these effects suggest that isoprinosine stimulates the production of precursor lymphocytes and initiates a process of cell differentiation capable of producing long-term restoration of host immunity.

Modulation of T- and B-lymphocyte functions by isoprinosine in homosexual subjects with prodromata and in patients with acquired immune deficiency syndrome (AIDS).

Male prodromal homosexuals and patients with acquired immune deficiency syndrome (AIDS) exhibited similar immunological abnormalities but by different degrees. A reduction in the number of circulating T lymphocytes bearing the T-4 surface marker led to an altered ratio of Th to Ts subpopulations in both groups of subjects. Total numbers of suppressor cells (Ts) remained virtually similar in both study groups to that of the control subjects. Proliferative responses to T-cell mitogen (PHA) and T cell-dependent B-cell mitogen (PWM) were severely impaired in prodromal subjects and more so in the AIDS group. The response to PWM was unrelated to the total number of suppressor T cells but was associated with a significant decrease in helper T-cell number. The impaired lymphocyte functions of immunosuppressed subjects were potentiated by coincubation with isoprinosine in a selective fashion. While the percentage of upward modulation among homosexuals with normal lymphocyte functions was comparable to that obtained in control subjects, a higher degree of augmentation was achieved in AIDS patients and in prodromal subjects with impaired blastogenic responses. In none of the AIDS patients with severe immunodeficiencies, however, was the lymphocyte functions restored to the normal range established in the heterosexual controls. These results suggest the feasibility of eventual prophylactic utilization of isoprinosine in male homosexuals at high risk of developing AIDS.

Restoration of impaired B- and T-lymphocyte subsets and functions in vitro by isoprinosine in prodromal homosexuals and AIDS patients.

Functional and phenotypical parameters demonstrated significant aberrations in both prodromal males and patients with the acquired immune deficiency syndrome (AIDS). Impaired B-cell functions as quantitated by Staphylococcus aureas Cowan Strain I (SAC) and pokeweed mitogen (PWM)-induced blastogenesis, intracytoplasmic immunoglobulins and spontaneous immunoglobulin were associated with a significant decrease in Leu3+ cells but unrelated to Leu2+ lymphocytes. The functional subsets of the latter were further defined by monoclonal antibodies (Leu8 and HLA-DR) applying dual color flow cytometry. Activated and effector-suppressor subsets with the phenotypes Leu2+ HLA-DR+ and Leu2+ Leu8- were elevated while both subsets of helper and suppressor-inducing helper lymphocytes, Leu3+ Leu8- and Leu3+ Leu8+, were depressed. These data demonstrated a broad spectrum of dysfunction involving all 3 stages of B-cell development in AIDS as well as possible defects in the feedback suppressor loop which regulates both the helper and suppressor T-lymphocyte system and B-cell functions. While in vitro incubation with isoprinosine had no modulative effect on SAC-induced blastogenesis (resting B-cell activities), it did modulate both PHA, PWM-induced transformation and the spontaneous secretion of immunoglobulins (partially and fully activated B-cell activities). In co-incubation with PWM, isoprinosine augmented the expression of inducer cells for helper functions while enhancing to normal level the number of suppressor-inducing helper cells. In addition, it reduced activated and effector-suppressor cells to near normal range in the PBL of high risk homosexuals. Only marginal modulation, however, was observed in suppressor subsets of AIDS subjects. This interference with the defective feedback loop may account for the selective clinical and immunoregulatory actions of this drug.

Neutralizing antibodies as a prognostic indicator in the progression of acquired immune deficiency syndrome (AIDS)-related disorders: a double-blind study.

A double-blind longitudinal study for the presence of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies (NAb) in the sera of 36 patients with acquired immune deficiency syndrome (AIDS), 149 prodromal homosexual subjects, and 33 heterosexual subjects has been carried out. All AIDS patients and 68% of prodromal homosexual subjects (101/149) were found to be HIV-1 antibody positive by Western blot assay. All heterosexual subjects were HIV-1 antibody negative. Neutralizing antibody(s) was determined by testing the protective activity of sera against HIV-1 infection of human T-cell line H9. Study subjects were divided into NAb(+) (antibody titer, greater than 1:40) and NAb(-) (antibody titer, less than 1:40) groups. During the 24-month observation period 2 of 80 (3%) HIV-1(+) NAb(+) individuals progressed to AIDS and died, as compared to 5 of 21 (24%) of HIV-1(+) NAb(-) subjects who progressed to AIDS. Similarly, among the NAb(+) AIDS patients 8 of 23 (35%) died, while 10 of 13 (77%) of the NAb(-) patients died during the course of the study. In addition, the absence or reduction of HIV-1 p17 and p24 antibodies directed against HIV-1 antigens as well as the low titer or absence of NAb appears to be closely related to the clinical progression of the disease. These studies suggest that a decrease in the virus neutralization capacity of the sera and a decrease or complete loss of HIV-1 p17 and p24 antibodies may be useful as prognostic indicators for the progression of disease in HIV-1-seropositive patients.