Chemiluminescence in peripheral blood mononuclear cells of solid tumor cancer patients

Summary Levels of chemiluminescence were measured in peripheral blood mononuclear cells (PBMC) from normal subjects and from solid tumor cancer patients. Patients with advanced malignant disease were found to have significantly elevated baseline chemiluminescence activity in their resting PBMC as compared to normal subjects or to cancer patients with, at most, minimum residual disease. Patients with either advanced disease or minimum residual disease, however, were found to exhibit significantly elevated activation of chemiluminescence by treatment of cells with phorbol myristic acetate (PMA). Treatment of surgically resected stage I lung cancer patients with Freund's complete adjuvant alone or emulsified with extracted lung cancer antigens was found to elevate chemiluminescence levels in patient PBMC. Serum from those vaccinated patients was found to elevate chemiluminescence levels of resting PBMC from normal subjects. That serum activity did net correlate with levels of immune complexes measurable in the Clq or Raji cell assay.     

Serial immune testing in surgically resected lung cancer patients

Summary Immunoregulation of phytohemagglutinin (PHA) responsiveness by glass-adherent cells and prostaglandin-synthesizing cells was serially monitored in the peripheral blood mononuclear cells (PBMC) of surgically resected stage I and stage II lung cancer patients entered on a trial of adjuvant immunotherapy. The relationship between immunoregulatory cell function, immunocompetence, and disease relapse was determined. Immunoregulatory activity was measured in PHA-stimulated cultures in the presence and absence of 2 g/ml indomethacin and in the presence and absence of glass-adherent cells. In each instance of disease relapse seen, an increase in immunoregulatory cell function to a level significantly different from normal was observed 3 months prior to or coincident with clinical confirmation of disease recurrence. This was usually associated with a decline in PHA responsiveness. In the patients who did not relapse, the levels of PHA responsiveness and immunoregulatory function persisted within normal limits throughout the course of study. Percentages and numbers of leukocytes and leukocyte subsets and delayed cutaneous hypersensitivity were also monitored in this study, but could not be consistently correlated with early changes in clinical disease status. These data suggest that the development of indomethacin-sensitive and glass-adherent suppressor cells may precede and predict for tumor recurrence in surgically resected lung cancer patients.     

Suppressor cell regulation of encephalitogenic T cell lines: generation of suppressor macrophages with cyclosporin A and myelin basic protein.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) can be adoptively transferred using myelin basic protein (BP)-specific helper T cell lines, and suppressor cells may be important in recovery from EAE. In order to generate suppressor cells, spleen cells obtained from BP-complete Freund's adjuvant (CFA) inoculated SJL/J mice and from normal mice were cultured for 7 days with medium, with cyclosporin A (CsA), or with CsA and antigen (BP or purified protein derivative of mycobacterium (PPD)). Cultured spleen cells were assayed for suppressor activity in vitro by coculture with BP-specific and PPD-specific helper T cell lines derived from SJL/J mice. Immunized donor spleen cells cultured with cyclosporin A (CsA) and BP were potent inhibitors of T cell line proliferation, and suppressor activity was increased 17-fold compared with control splenocytes. The number of suppressor cells required to suppress PPD-specific line proliferation by 50% (I50) was significantly higher than the number required to suppress BP-specific line proliferation, suggesting an antigen-specific component to the suppression. The major effector cell required for suppression was a large granular Mac-1+ cell with the functional characteristics of a macrophage. Suppressor activity persisted after depletion of Thy 1.2+ cells, but suppression was no longer antigen-specific, suggesting that culture of spleen cells with CsA and BP may generate suppressor macrophages which are antigen-nonspecific and Thy 1.2+ suppressor cells which are antigen-specific. These suppressor cells may be important in the regulation of CR-EAE and the techniques described for their generation may prove useful for treatment and prevention of disease.     

The interleukin 2 secretion defect in vitro in systemic lupus erythematosus is reversible in rested cultured T cells

Interleukin 2 (IL 2) secretion in response to mitogenic stimulation in vitro is strongly reduced in circulating T lymphocytes from patients with SLE. It is still not clear how this abnormality relates to the B cell hyperactivity in the disease. Some investigators proposed that an intrinsic T helper cell defect could lead to suppressor cell dysfunction and autoimmunity. Others have found that in fact increased suppressor cell activity can cause IL 2 hyposecretion. In the present study we report that the IL 2 secretion in response to PHA plus PMA by T cells from patients with SLE, which initially was decreased by a factor of 10 as compared with the IL 2 secretion in blood donor T cells, was restored when the T cells were rested for 2 to 3 days in culture before stimulation. IL 2 hyposecretion in SLE T cells and the kinetics of normalization in culture were not changed by the addition of normal adherent cells during the stimulation with PHA/PMA, occurred in the absence of significant cell death or proliferation or change of the T4:T8 cell ratio during the resting culture, were not due to a maturation of immature T6-positive cells (less than 1.5% T6 cells in SLE T cells), and also occurred in T8-depleted T4 cells alone. Furthermore, a normalization of IL 2 secretion took place in the presence of either SLE serum or normal serum, and the addition of fresh autologous T cells to 3-day-cultured SLE T cells did not cause suppression of IL 2 secretion. These data show that some rapidly reversible defect occurs in circulating T helper cells in SLE. That this could reflect an exhaustion of T helper cells that have been activated in vivo is discussed.     

Immune response in experimentally induced uremia. II. Suppression of PHA response in uremia is mediated by an adherent, Ia-negative and indomethacin-insensitive suppressor cell

The characteristics of the adherent suppressor cell in uremic rats were examined. We found that: 1) adherent spleen cells from uremic rats display a more potent suppressor activity than do control adherent cells; 2) the suppression that is mediated by uremic adherent spleen cells cannot be eliminated by pretreatment with indomethacin, whereas the suppression that is mediated by naturally present adherent cells in the control rat is reversed by pretreatment with indomethacin; 3) the uremic adherent suppressor cell does not have Ia antigens that can be detected by the monoclonal antibodies OXL, whereas control adherent cells have Ia antigens on their surface; 4) both the control and uremic adherent suppressor cells are insensitive to mitomycin C and do not have any detectable levels of Thy 1 antigens on their surface. It appears that immune suppression in uremic rats is mediated by an adherent cell that differs from adherent cells present in control animals. The suppression in uremic rats is either not mediated by prostaglandins or may be mediated by preformed prostaglandin synthetase products.     

Selection of 3LL tumor subline resistant to natural effector cells concomitantly selected for increased metastatic potency

Summary The numbers of strongly adherent monocytes in the peripheral blood of normal subjects and cancer patients were determined. The method used was to place peripheral blood mononuclear cells in microwells and culture them for 1 week. At the end of that period, adherent macrophages were counted in the Coulter counter after release. Adherent cells per milliliter of blood, per total cells, and per mononuclear cells or monocytes plated were markedly diminished in the peripheral blood mononuclear cells of 44 melanoma, 23 breast cancer, 18 lung cancer, nine colon cancer, and 27 leukemia patients. Median values were 14.8×10⁴ adherent cells per ml peripheral blood for 86 normal subjects, as against 2.5×10⁴ per ml in the peripheral blood of the 125 patients (P<0.001). There was a poor correlation between the adherent cell numbers and the peripheral blood leukocyte counts, but an excellent correlation of the different adherent cell counts with each other. The number of adherent cells in the peripheral blood varied inversely with age in the cancer patients, but not in the normal subjects (r=0.29, P<0.005). When patients under age 50 were compared to the controls, the deficiency of adherent cells was slightly more severe in patients with stage IV lung cancer than in those with stage III lung cancer. In contrast, there was no difference in the degree of deficiency between patients with stage III melanoma and no evident disease and patients with stage IV disseminated metastatic disease. The implications of these results are discussed.     

Absence of suppression in natural and induced tolerance to F antigen

The role of suppression in natural and induced tolerance to F antigen was investigated in two sets of experiments. In the first, CBA mice were submitted to pretreatments which decrease suppression and the antibody response to self- or allo-F type was investigated. The second set of experiments involved the transfer of spleen cells from tolerized or from naturally tolerant mice into normal mice which were then primed with allo-F, as well as the co-transfer of tolerant and primed lymphocytes into normal mice, to test whether tolerant lymphocytes present suppressor cells. The results indicate that the immune response against allo-F antigen is normally kept in a low level by a suppressive mechanism, and that F-specific suppressor T cells are absent from tolerant mice.Abbreviations used in this paper ATx adult thymectomy - BSS buffered salt solution - CFA Freund's complete adjuvant - CY cyclophosphamide - F.1 type-1 F antigen - F.2 type-2 F antigen - PBS phosphate-buffered saline - RIA radioimmunoassay - Th T helper cell - Ts T suppressor cell     

Functional properties of lymphocyte subpopulations in hepatitis B virus infection. I. Suppressor cell control of T lymphocyte responsiveness

Hepatocellular injury in hepatitis B virus infection may be produced by an autoaggressive hepatocytotoxic immune response. To test the hypothesis that acquired suppressor cell defects may participate in such a response, we assessed the functional integrity of 2 suppressor cell populations in patients with type B viral hepatitis. Spontaneous suppression of the 1-way mixed lymphocyte response by radiation-resistant, adherent peripheral blood mononuclear cells decreases during the acute phase of disease, returns towards normal with clinical recovery, but remains depressed in patients with chronic hepatitis. The degree of spontaneous suppressor cell dysfunction correlates inversely with at least 1 biochemical parameter of hepatocellular injury (SGPT). The functional integrity of this suppressor cell fluctuates during chronic hepatitis and may reflect currently undefined biologic variables in this disease. Mitogen-induced suppression on lymphocyte activation by radiation resistant, nonadherent suppressor cells is also depressed in acute and chronic hepatitis, but it does not correlate with biochemical evidence of hepatocellular injury on an individual-patient basis. Documentation of these generalized defects of nonspecific suppressor cell function establishes a basis for the possible existence of specific anomalies of immuno-regulation that may permit the expression of normally suppressed auoaggressive hepatocytotoxic immune mechanisms in viral hepatitis.     

Impaired NK Response of Cancer Patients to IFN-α but Not to IL-2: Correlation with Serum Immunosuppressive Acidic Protein (IAP) and Role of Suppressor Macrophage

In vitro NK responses of cancer patients (N=21) to rIFN-αA and rIL-2 were examined. The serum concentration of IAP (immunosuppressive acidic protein) was determined in parallel. Five out of seven patients whose serum IAP contents were within the normal range (270 μg/ml to 470 μg/ml), had their NK activities significantly augmented by rIFN-αA and rIL-2. On the other hand, NK cells from ten out of fifteen patients whose serum IAP concentrations were 650 μg/ml or more, were not activated by rIFN-αA. NK cells of these fifteen patients yet were capable of responding to rIL-2. NK cells from cancer patients, however, became responsive to rIFN-αA by either removal of adherent cells or treatment with indomethacin. Therefore, macrophages in PBMC of cancer patients with high serum IAP levels seem to selectively suppress NK response to rIFN-αA by an indomethacin-sensitive mechanisms. It was further shown that PGE₂ was not the mediator of this suppression.     

Suppressor cells in the effector phase of autologous cytotoxic reactions in cancer patients

Summary Cytotoxicity was induced in lymphocytes (CL) from 10 out of 15 patients by autologous mixed lymphocyte tumor cell culture and further cultivation with recombinant interleukin-2. In cells from 3 of the 10 patients, cytotoxicity was suppressed by more than 50% when autologous peripheral blood mononuclear cells (PBMC) from the patients with large tumors were added to the autologous killing system. The cells responsible for suppressing the cytotoxicity in the effector phase were adherent or nonadherent to plastic depending on the patient examined. The T cell fraction from 1 patient significantly suppressed the cytotoxic activity, and this suppression was seen only in the autologous system. On the other hand, plastic adherent cells but not T cells from PBMC of 2 subjects suppressed the cytotoxic activity of CL. The reason why the main cell population suppressing the CL activity differed among the patients is unclear. However, the findings that the suppression was mostly abrogated following resection of the tumor mass suggested that suppressor cells, either of macrophage lineage or T cells, are induced in patients with a large tumor mass. This speculation is supported by the finding that the PBMC from a patient with tumor recurrence regained the suppressive activity.     

T and B cells induce macrophages which suppress proliferation but not lymphokine secretion

In vitro culture of mouse spleen cells for 2 days or more leads to the production of adherent, phagocytic, Thy-1-, Ia+, Lyt-2- cells ("suppressor macrophages") which strongly inhibit the proliferative response of T and B lymphocytes to a variety of stimuli: mitogens, specific antigens, and antigen-nonspecific growth factors. Suppressive activity fails to develop, however, in cultured spleen cells from which nonadherent cells have been removed before the initial 48-hr incubation, and only partial suppression is obtained from cell suspensions from which T cells have been depleted before culture. We find that the requirement for nonadherent cells can be replaced by graded doses of lymphocytes. Lyt-2- and Lyt-2+ T cells are about equally potent in inducing suppressive activity in nonadherent cells. Surprisingly, B cells (containing fewer than 0.1% contaminating T cells) are also able to induce suppression in this system. The suppression induced includes both indomethacin-sensitive and indomethacin-resistant components. Interestingly, not all stages of mitogen-induced T-cell activation are blocked by these adherent cells: proliferation is inhibited, but production of interleukin 2 (IL-2) and interleukin 3 (IL-3) is unaffected.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1-3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.     

Mitogen induced cellular cytotoxicity (MICC) in multiple myeloma.

Mitogen induced cellular cytotoxicity (MICC) was noted to be markedly increased in patients with multiple myeloma as compared to normal controls and to patients with chronic lymphocytic leukemia (CLL). Enhanced MICC was present at various effector-to-target cell ratios and at several mitogen concentrations. Removal of adherent, phagocytic cells by carbonyl iron, glass wool, or rayon columns abolished the MICC response from the peripheral blood of both multiple myeloma patients and normal controls. Thus, the effector cell mediating MICC may be monocytic in origin and closely resembles the suppressor cell for immunoglobulin synthesis described in patients with multiple myeloma. Our data suggest that the MICC assay with chicken red blood cells as targets may provide a convenient method for identifying pathologic conditions where this cytotoxic effector cell population plays an active role.     

Suppression of mitogen-induced proliferation of normal spleen cells by macrophages from chickens inoculated with Marek's disease virus

Spleen cells from chickens 7 days after inoculation with Marek's disease virus (MDV) responded poorly to stimulation by phytohemagglutinin (PHA). Addition of these cells to syngeneic normal spleen cells caused of marked suppression of the PHA response of the normal cells. The MDV spleen cells also inhibited the DNA synthesis of MSB-1 lymphoblastoid cells in vitro. The suppressive activity is attributed to the presence in MDV spleen cells of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by treatment with anti-T or anti-B serum with C, but it was reversible by treatment with carrageenan or carbonyl iron/magnet, by passage through glass wool column, and by adherence to plastic Petri dishes. The adherent MDV spleen cells also showed strong suppressor cell activity against syngeneic normal spleen cells.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. I. Induction and suppression of delayed-type hypersensitivity and in vitro proliferative responses

Pretreatment of Lewis rats with a single i.p. injection of ABA-N-acetyl-tyrosine in incomplete Freund's adjuvant induced an unresponsiveness for delayed-type hypersensitivity to subsequent immunization with the same antigen in complete Freund's adjuvant. Complete suppression of in vitro antigen-induced proliferative responses required repeated pretreatment. Passive transfer of lymphoid cells from spleen and lymph nodes but not sera from suppressed rats induced unresponsiveness of hapten-specific T cell functions. Nylon wool-nonadherent cells and cells panned on F(ab')2 of rabbit anti-Lewis rat Ig plates suppressed the induction of DTH and in vitro antigen-stimulated proliferation. Adult thymectomy increased DTH and failed to abolish the induction of suppression.     

Immunoregulation in experimental interstitial nephritis: immunization with renal tubular antigen in incomplete Freund's adjuvant induces major histocompatibility complex-restricted, OX8+ suppressor T cells which are antigen-specific and inhibit the expression of disease

We utilized a model of experimental interstitial nephritis induced by renal tubular antigen in complete Freund's adjuvant to examine a mechanism of immunologic tolerance produced by priming immunization with tubular antigen in incomplete Freund's adjuvant. Brown Norway rats primed with tubular antigen in incomplete adjuvant do not develop significant nephritis after challenge with antigen in complete adjuvant, and this tolerance can be transferred to naive recipients with donor T cells. These T cells also specifically suppress a delayed-type hypersensitivity response to soluble tubular antigen in recipients immunized to produce disease. This suppression is MHC-restricted and is mediated by OX8+ T cells which bind antigen and bear idiotypes cross-reactive with those on antibodies eluted from the tubular basement membrane. Despite the suppression of histologic disease, tolerized animals were able to produce significant titers of antibodies to tubular basement membrane. Our findings demonstrate an additional strategy for altering the natural history of immune-mediated renal disease, and further refine the characterization of the suppressive effect produced by incomplete Freund's adjuvant.     

Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells

Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.     

Depressed lectin-dependent and enhanced antibody-dependent cell-mediated cytotoxicity in patients with stage I cancer of the larynx

Lectin-dependent cell-mediated cytotoxicity (LDCC) of peripheral blood mononuclear cells (PBMC) from patients with stage I cancer of the larynx (LC) was evaluated using human adherent 3H-TdR-prelabeled HEp-2 carcinoma cells as targets at 50:1 effector-target ratio with 25 micrograms/ml concanavalin A (Con A) in a 24-hour assay. Under these conditions, but without Con A, no considerable natural cell-mediated cytotoxicity (NCMC) was performed by PBMC either from control or from LC donors. Depressed levels of LDCC, but augmented ADCC to chicken red blood cells were detected in LC patients. Natural killer activity to K562 targets was not different from that of control subjects. In parallel studies, normal Con A-induced blastogenesis and B cell counts, low T, and active T cell counts, as well as high Leu-11a+ cell counts were detected in patients with LC. The relationship between depressed LDCC and low T, and active T cell counts, and enhanced ADCC and high Leu-11a+ cell counts is suggested in stage I LC patients.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.