Natural killer cells in carcinomatous pleural effusions

Summary Effector cells in carcinomatous pleural effusions of patients with primary or secondary lung cancer were examined for natural killer (NK) activity against K562 cells in a 4-h chromium release assay, and for mitogenic responses and lymphocyte subpopulation constitutions. NK activity of lymphocyte-rich mononuclear cells isolated from carcinomatous pleural effusions by centrifugation on a discontinuous gradient of Ficoll-Hypaque was markedly low in seven of 40 patients studied, and absent in the other 33 cases. NK activity of peripheral blood mononuclear cells from the patients was lower than that of cells from normal donors, but always higher than that of effusion cells from the same patients. NK cells in the peripheral blood and in pleural effusions had some characteristics in common, in that they lacked a capacity to bind sheep erythrocytes, were nonadherent to Sephadex G-10 beads and nylon wool, and belonged to large granular lymphocytes. On the other hand, nonmalignant effusions of patients with congestive heart failure had significant NK activity. The effector cells in the effusions included a higher frequency of T cells than those in the peripheral blood of the same patients. Proliferative responses to phytohemagglutinin and concanavalin A of effusion cells were comparable to those of normal blood cells and were higher than those of blood cells from the same patients. The reason for low NK activity and high mitogenic response in carcinomatous pleural effusions is as yet undefined.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

Suppression of a pokeweed mitogen-stimulated plaque-forming cell response by a human B lymphocyte-derived aggregated IgG-stimulated suppressor factor: suppressive B cell factor (SBF)

The mechanisms whereby formed immune complexes (IC) or immunoglobulin aggregates can suppress further antibody production were explored by culturing normal human peripheral blood mononuclear leukocytes (PBL) with heat-aggregated IgG (HAIgG) and collecting the culture supernatants at 24 hr. These supernatants were found to suppress a pokeweed mitogen (PWM)-induced rheumatoid factor plaque-forming cell (RF-PFC) response in normal individuals. PWM-induced anti-trinitrophenylated sheep red blood cell (TNP-SRBC) PFC were also inhibited by suppressor supernatants from HAIgG-stimulated PBL, suggesting that the polyclonal PFC response was inhibited by a suppressor factor. The suppressor factor inhibited PWM stimulated RF-PFC throughout the culture period, but suppression was maximal at the peak of the RF-PFC response. Suppressor factor was only effective at the initiation of cultures, suggesting that it inhibited early events in the PWM-stimulated RF-PFC response. Molecular weight determination of the suppressor factor by differential membrane fractionation suggested a m.w. range of 30,000 to 50,000, and chromatography on Sephadex G-100 showed a peak activity at an approximate m.w. of 32,000. Studies suggested the factor was not an interferon. Depletion of T lymphocytes by E rosetting and macrophages/monocytes by G-10 adherence did not affect the generation of suppressor factor. Depletion of T lymphocytes (OKT4, OKT8) and NK cells (Leu-11b) by antibody-dependent, complement-mediated cytotoxicity also did not affect the generation of suppressor factor. Depletion of B lymphocytes with OKB7 resulted in the generation of significantly less suppressor factor. Suppression produced by unstimulated purified B lymphocytes was approximately one-half that seen when B lymphocytes were stimulated with HAIgG. Differential membrane fractionation studies suggested that only HAIgG-stimulated B cell cultures contained peak activity in the 30,000 to 50,000 m.w. fraction. Supernatants from unstimulated purified T cells also generated suppression, which was approximately one-half of that seen with HAIgG-stimulated B cells, but no increase in suppressor activity was seen in T cell cultures after incubation with HAIgG. These studies demonstrate that HAIgG is capable of stimulating B lymphocytes to produce a lymphokine, suppressive B cell factor (SBF), which is capable of suppressing a polyclonal PFC response. SBF may be important in feedback control of human immunoglobulin production.     

Carrageenan-induced decline of natural killer activity: II. Inhibition of cytolysis by adherent non-T Ia-negative suppressor cells activated in vivo

The intravenous injection of 1 to 2 mg of ι-carrageenan (CAR) into (C57BL/6 × C3H)F₁ or BALB/c mice causes a prompt and substantial decline of splenic natural killer (NK) activity against YAC-1 lymphoma targets lasting approximately 1 week in F₁ mice. During this time, NK activity can be enhanced by administration of the interferon inducer polyinosinic-polycytidilic acid. The in vivo effect of CAR requires neither an intact thymus nor unimpaired proliferative capacity of lymphomyeloid cells, according to experiments in congenitally athymic BALB/c.nu/nu mice and in preirradiated (700 rad of γ-rays) F₁ hybrids. The splenic cytotoxic activity lowered in vivo by CAR can be restored in vitro by removing subpopulations of cells that adhere to glass wool or carbonyl iron particles, but not to Sephadex G-10. Thus, the lytic function of mature NK cells is reversibly inhibited in the spleens of CAR-treated animals; differentiation and maturation of NK precursors are not inhibited, as judged by the enhancing effect on NK activity of the interferon inducer. Splenocytes of CAR-treated donors suppress cytotoxic effectors of untreated mice in cell mixing experiments. Athymic and preirradiated animals given CAR are fully competent donors of suppressor cells. Suppressor function is insensitive to irradiation (2000 rad of γ-rays in vitro) and to anti Thy-1 or anti-Ia antibody plus complement. Inhibition of NK cytolysis is not restricted by the major histocompatibility complex and can also be mediated by cell-free supernatants in which suppressor cells were incubated. This model of reversible inhibition of NK activity suggests that activation of thymus-independent suppressor cells is one of the regulatory mechanisms of natural cytotoxic activity in vivo.     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.     

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.     

Strong suppression by monocytes of T cell mitogenesis in chicken peripheral blood leukocytes

Peripheral blood leukocytes (PBL) isolated from chicken blood by flotation on Ficoll-Hypaque (FH) have much lower proliferative responses to Con A and PHA than PBL isolated by slow-speed (SS) centrifuging at 60 X G. FH preparations contain all categories of blood cells except erythrocytes, whereas SS are almost devoid of nonlymphoid cells. FH responses approach SS levels after filtration through Sephadex G-10, which removes almost all monocytes detectable by neutral red and nonspecific esterase methods, while only partially depleting granulocytes and thrombocytes. Thus, the low responses of FH are probably associated with suppressor activity of monocytes in these preparations, numbering 6 to 8% of mononuclear cells. G-10 filtration decreases responses of SS preparations, indicating that the few (less than 0.1%) monocytes in SS function mainly as helper cells. In co-cultures, irradiated FH cells (FHX) but not SSX produce as much as 90% suppression of SS or FH responses. Suppression by FHX is totally inhibited by heat killing (56 degrees C for 45 min) or monocyte inactivation by preincubation with Trypan blue, and is removed by G-10 filtration, although not completely. Adherent cells isolated from unirradiated FH on Cytodex beads (ADC-CY) produce up to 99% suppression in co-culture with SS or nonadherent cells derived from FH. Soluble suppressor factors can be detected in conditioned media from supernatants of Con A-stimulated cocultures containing suppressor monocytes, but their suppressor activity is partially opposed by stimulatory factors, possibly interleukin 2, also present in supernatants. It is concluded that in FH preparations and therefore in blood, but not in SS, monocytes that have not been activated exert strong suppressor activity on mitogen-induced T cell proliferation. Occasional chickens of one inbred line, RPRL 72, had exceptionally high suppressor activity of monocytes, as shown by very low FH responses and very high suppressor effects of FHX well outside the normal range of variation found in this line or in line RPRL 63. This abnormal suppressor activity may have resulted from activation of suppressor monocytes as a response to subclinical infection.     

Autologous tumor killing and natural cytotoxic activity of tumor-associated macrophages in cancer patients

Summary Tumor-associated macrophages (TAM) isolated from pleural effusions and ascites fluids of cancer patients were tested for cytotoxicity against freshly isolated autologous tumor cells and K562 in a 4-h⁵¹ Cr-release assay, and in vitro effects of OK432 (a streptococcal preparation) and partially purified human leukocyte interferon (IFN) on their cytotoxicities were examined. Positive cytotoxicities against K562 were recorded for TAM samples from 2 of 23 pleural effusions and 3 of 10 ascites specimens. Tumor-associated macrophages were not cytotoxic to autologous tumor cells, while low but significant lysis was observed with tumor-associated lymphocytes (TAL) samples from 2 of 13 pleural effusions and 1 of 6 ascites specimens. In vitro treatment with OK432 resulted in an enhancement of natural cytotoxicity in 4 of 13 TAM and 10 of 15 TAL samples. An induction or augmentation of autologous tumor killing activity by OK432 was observed in 2 of 10 TAM and 8 of 11 TAL samples. In contrast, IFN failed to induce autologous tumor killing activity, although IFN-enhanced lysis of K562 was detected in 1 of 7 TAM and 2 of 9 TAL samples. These results indicated that autologous tumor killing and natural cytotoxic activities were defective in macrophages and lymphocytes at the site of the tumor growth, and both activities were strongly enhanced by OK432 rather than IFN.     

Cell origins and diagnostic accuracy of interleukin 27 in pleural effusions

The objective of the present study was to investigate the presence of interleukin (IL)-27 in pleural effusions and to evaluate the diagnostic significance of pleural IL-27. The concentrations of IL-27 were determined in pleural fluids and sera from 68 patients with tuberculous pleural effusion, 63 malignant pleural effusion, 22 infectious pleural effusion, and 21 transudative pleural effusion. Flow cytometry was used to identify which pleural cell types expressed IL-27. It was found that the concentrations of pleural IL-27 in tuberculous group were significantly higher than those in malignant, infectious, and transudative groups, respectively. Pleural CD4(+) T cells, CD8(+) T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells might be the cell sources for IL-27. IL-27 levels could be used for diagnostic purpose for tuberculous pleural effusion, with the cut off value of 1,007 ng/L, IL-27 had a sensitivity of 92.7% and specificity of 99.1% for differential diagnosing tuberculous pleural effusion from non-tuberculous pleural effusions. Therefore, compared to non-tuberculous pleural effusions, IL-27 appeared to be increased in tuberculous pleural effusion. IL-27 in pleural fluid is a sensitive and specific biomarker for the differential diagnosing tuberculous pleural effusion from pleural effusions with the other causes.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.     

Physiochemical characterization of human histamine-induced suppressor factor

A product of histamine-stimulated human lymphocytes, histamine-induced suppressor factor or HSF, was characterized by enzyme treatment, sensitivity to reduction and alkylation, by molecular sieve chromatography, and by polyacrylamide gel electrophoresis. HSF was found to have a wide pH stability (pH 3-10), sensitivity to temperatures greater than 80 degrees C, and to have the properties of a glycoprotein by virtue of its sensitivity to chymotrypsin, trypsin, sodium periodate, and neuraminidase. HSF did not appear to have a serine group(s) in its "active" site since its biologic activity remained intact following treatment with an irreversible serine esterase inhibitor (phenylmethylsulfonyl fluoride). Further, HSF did not appear to have inter- or intra-molecular disulfide linkages because treatment with denaturing and/or reducing agents, followed by alkylation, did not significantly alter its activity. Molecular sieve chromatography employing Sephadex G-100 revealed an apparent molecular weight for HSF of 25-40,000. Electrophoresis of HSF in polyacrylamide gels at pH 8.7 under nonreducing conditions revealed two regions of activity, one region migrating with albumin and the other region anodal to albumin. In addition to suppressing lymphocyte proliferation, the 25-40,000 Mr Sephadex G-100 fractions also inhibited the production of leukocyte inhibitory factor. Of particular interest, gel filtration of supernatants generated by stimulating mononuclear cells with either histamine, dimaprit (but not 2-pyridylethylamine), concanavalin A, or candida albicans resulted in similar elution profiles with regard to inhibition of lymphocyte proliferation. That is, 25-40,000 Mr fractions of supernatants generated by each stimulant suppressed lymphocyte proliferation to a similar degree. The latter findings provide indirect evidence that T lymphocytes, triggered in response to antigen-specific and nonspecific stimuli, elaborate suppressor molecules capable of modulating T-cell function that share certain similarities.     

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12-14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10(-5) M), an inhibitor of prostaglandin synthesis, or anti PGE2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE2-mediated suppression. High levels of PGE2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE2 (10(-7)-10(-6) M) but not PGF2 alpha (10(-6) M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE2 produced by decidual cells and decidual macrophages.     

The role of monocytes/macrophages in TCR-zeta chain downregulation and apoptosis of T lymphocytes in malignant pleural effusions

The aim of this study was to assess alterations of zeta chain expression and their relation to apoptosis of T lymphocytes. T lymphocytes were obtained from malignant pleural effusions (MPE) of 15 patients. The work focused on TCR-zeta chain expression, apoptosis of T lymphocytes, and on the content of monocyte/macrophages in effusions. Analysis was performed using three color flow cytometry combining CD3, TCR-zeta and TUNEL reaction. The content of tumor and monocyte/macrophage cells has been determined using CD3, CD14, and cytokeratins, as markers of distinct cell subpopulations. Our findings strongly indicate that decreased zeta chain expression in T cells depends on the content of monocyte/macrophage cells, and that the range of the decrease is inversely proportional to the number of monocytes/macrophages in the effusion. Those having low zeta chain expression were the main subpopulation of T cells undergoing apoptosis. These data suggest that decreased zeta chain expression in T cells in MPE may be related to the abundance of monocyte/macrophages in effusions.     

Reversal of macrophage-augmented MLR reactivity by tumor-induced splenic suppressor T cells and their soluble factor(s)

Augmenting concentrations of macrophages or their supernatants failed to reverse T-cell hyporeactivity in tumor-bearing mice (TBM). Serial passaging over nylon wool columns depleted TBM spleen cells of a mildly adherent tumor-induced suppressor cell and restored mixed lymphocyte reaction (MLR) reactivity to the purified TBM T-cell population. The tumor-induced suppressor cell was extensively plated to remove macrophages and characterized as a T cell by its anti-Thy 1 serum sensitivity. This suppressor T cell, when added to normal T cells, abrogated all enhancing effects caused by addition of macrophages. Suppressor T-cell inhibition was non-contact dependent, since suppressor T-cell supernatants inhibited MLR activity in T cells treated with enhancing concentrations of macrophage supernatants. Thus it appears that tumor-induced T-cell debilitation is a reversible phenomenon, mediated not by macrophages but by soluble factor(s) from a nonphagocytic, mildly adherent, suppressor T cell.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.