Interleukin 2 and stimulator lymphoblastoid cells will induce human thymocytes to bind and kill K562 targets.

Human thymocytes cultured in the presence of IL-2 and an irradiated B cell line became cytotoxic to K562 target cells. Thymocytes cultured alone or with only IL-2 exhibited almost no killing, but thymocytes cultured in the presence of stimulator cells alone exhibited low levels of cytotoxic activity. Removal of Fc gamma receptor-bearing cells from the activated thymocyte population almost completely abolished the binding and lytic activity. Separation of thymocytes into Fc microns+ and Fc microns-cells before culturing with IL-2 and stimulator cells revealed that only the Fc microns+ subpopulation developed into K562 killer cells. These findings indicate that modulation of Fc microns to Fc gamma receptors on the thymocyte cell surface is part of the maturation process of this particular subset of cytotoxic cells. Morphologically, most of the activated Fc gamma+ K562-binding cells were large, granulated lymphocytes. Only very few of the round, nongranulated small thymocytes were bound to K562 target cells.

Systemic administration of human leukocyte interferon to melanoma patients. II. Cellular events associated with changes in natural killer cytotoxicity.

Treatment of patients with malignant melanoma with daily doses of human leukocyte interferon (IFN) resulted in natural killer (NK) cytotoxicity that increased during the 1st week of treatment and subsequently declined to pretreatment levels despite continued IFN administration; events involved in these changes were examined. The increased cytotoxicity was associated with an increased number of cells able to bind K562 targets and an increased proportion of cells able to mediate cytolysis of K562 targets. The period of increasing NK activity was also associated with an increased ability to generate in vitro cytotoxic effectors against K562 targets by stimulation with allogeneic lymphoblastoid cells. The responding cells in this system were depleted of NK activity by adherence to immune complexes. These results suggested that increased NK activity during week 1 of in vivo treatment was associated with augmentation of the development of NK cells from noncytotoxic precursors in addition to direct effects on NK cells. In contrast, the decline in NK activity appeared to be primarily a direct effect on NK cells, because these cells could no longer be augmented in vitro by additional IFN. The refractory state to IFN augmentation of cytotoxicity was limited to the period of IFN treatment in vivo, and responsiveness to IFN was promptly regained upon cessation of treatment. No evidence for the development of suppressor cells of NK effector function could be obtained.

Lymphoid cells infiltrating human pulmonary tumors: effect of intralesional BCG injection.

Tumor-infiltrating lymphocytes (TIL) and regional lymph node lymphocytes (LNL) were isolated by mechanical disaggregation and density gradient centrifugation from 30 untreated human lung tumors and 12 BCG-injected human lung tumors. Lymphocyte populations were characterized by their ability to form erythrocyte (E)-rosettes, erythrocyte-antibody-complement, and erythrocyte-antibody gamma-rosettes, by their proportion of esterase-staining cells, and by their responses in mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML). and natural killer (NK) assays. TIL from untreated tumors had low proportions of E-rosetting cells (mean, 27.3%), relatively high proportions of "null" cells, and poor responses in MLC-CML and NK assays. There were no significant differences between primary lung tumors and lung metastases in rosettes, MLC-CML responses, or NK activity. In contrast, TIL from tumors injected with BCG 14 days before resection had higher proportions of E-rosetting cells (47.8%) and vigorous MLC-CML and NK responses. LNL from 11 patients with untreated tumors had higher proportions of E-rosetting cells (40.5%) than LNL from 9 patients with BCG-injected tumors (35.0%) and LNL from patients with untreated tumors had higher responses than LNL from treated patients in MLC-CML assays. These results suggest that the BCG injection induced an infiltration of functionally reactive NK and T-cells at the tumor site without an associated increased activity of T-cells from regional lymph nodes.

Systemic administration of human leukocyte interferon to melanoma patients. I. Effects on natural killer function and cell population.

Natural killer (NK) cytotoxicity was assessed against K562 targets in 14 melanoma patients who received daily im doses of human leukocyte interferon (IFN) for 42 consecutive days. The most common pattern of NK activity was a decline from pretreatment levels 1 day after initiation of treatment, followed by increasing cytotoxicity with peak activity at day 7 and a subsequent gradual decline to pretreatment levels during the remaining weeks of treatment. This pattern was particularly apparent in patients who received 3 x 10(6) or 9 x 10(6) U IFN/day, while patients who received 1 X 10(6) U IFN/day tended to lack the decline at day 1 and maintained the elevated NK activity past day 7. Changes in NK activity could not be related to changes in absolute lymphocyte counts; to proportions of cells bearing membrane receptors for erythrocyte-antibody-complement, of cells bearing FC gamma receptor; or to clinical response to IFN.

Comparison of histocompatibility antigens on cultured human tumor cells and fibroblasts by quantitative antibody absorption and sensitivity to cell-mediated cytotoxicity.

Human tumor and febroblast tissue culture cells were compared to determine the suitability of fibroblasts as control cells in experiments on human tumor serology and cellular immunology. Fibroblasts expressed the same HL-A antigen profile as did melanoma cells. Furthermore, the quantitative expression of the determinants was similar on both cell types. In four of five pairs tested, the fibroblasts displayed similar sensitivity to effector cells generated by mixed lymphocyte culture as did the tumor cells from the same donor, but there were some differences in the effects of specific alloimmune effector cells at high and low effector-to-target ratios on the two types of target cells. Results indicated that fibroblasts are legitimate control target cells for studies in human tumor immunology, if screening assays are done to verify their antigenicity and sensitivity to cell-mediated cytolysis.

Inhibition of lymphokine-activated killer cell function by human alveolar macrophages.

Tissue- and organ-specific factors may be important in the regulation of cytotoxic lymphocytes. We therefore examined the ability of human alveolar macrophages (AMs) to alter the tumoricidal function of lymphokine-activated killer cells (LAK cells). AMs, obtained by bronchoalveolar lavage from healthy volunteers, or peripheral blood monocytes were added to a standard 4-h chromium release LAK assay at varying concentrations. AMs severely inhibited the killing of both NK-sensitive (K562) and NK-resistant (M14) tumor cells [42 +/- 2.6% (SEM) inhibition of M14 killing at the 0.125:1 AM:LAK ratio and 83 +/- 2.3% inhibition at the 1:1 ratio, n = 9]. Peripheral blood monocytes, in contrast, were only one-eighth as inhibitory as AMs. A positive smoking history was associated with a 3- to 7-fold increase in the number of AMs recovered by bronchoalveolar lavage but had no effect on the inhibition produced per AM cell. The mechanism of inhibition was investigated. Formalin fixation produced an 8-fold reduction in the inhibitory capacity of AMs, suggesting the need for active metabolism or an intact cell membrane. No soluble mediator could be detected with a two-chamber Transwell system, in 24-h AM culture supernatants, or following blocking experiments with indomethacin, catalase, or superoxide dismutase. Binding studies demonstrated selective binding between LAK cells and AMs, yet AMs were not susceptible to LAK-mediated lysis under the usual assay conditions. In summary, AMs are potent inhibitors of in vitro LAK function. Inhibition requires direct cell contact and is independent of soluble reactive oxygen species, prostaglandins, or activation by tobacco smoking. Inhibition is not due to lysis of the AM as a competitive cold target. These results suggest that AMs may actively limit antitumor cytotoxic responses in the lung.

Fetal calf serum-induced blastogenic and cytotoxic responses of human lymphocytes.

Incubation of normal human peripheral blood lymphoid cells in fetal calf serum-supplemented media initiates blastogenic and nonspecific cytotoxic responses. A panel of lymphocytes from 13 normal donors exhibited increased [3H]thymidine incorporation from 2 to 40 times that of control lymphocytes incubated in pooled human AB-supplemented media. Fetal calf serum-sensitized human lymphoid cells were active in cytotoxicity assays against a wide variety of cultured human tumor and normal target cells. Cytotoxicity continued to increase from 3 to 9 days of incubation in fetal calf serum-supplemented media, while blastogenic response peaked at 6 days of incubation. Cytotoxic and blastogenic responses increased with higher concentrations of fetal calf serum. Once stimulated with fetal calf serum, the lymphocytes did not require the continued presence of fetal calf serum to mediate a cytotoxic reaction.

Depression of natural killer cytotoxic activity in lymphocytes infiltrating human pulmonary tumors.

We assessed natural killer activity of lymphocytes present at the site of human tumors to determine the local effects of the tumor on immune function. Lymphocytes were extracted from human pulmonary tumors of varying histological types. In addition to tumor-infiltrating lymphocytes (TIL), peripheral blood lymphocytes from the same patients were prepared. Using a Michaelis-Menten kinetic model and titration of K562 targets in a 51Cr release assay, TIL exhibited a marked depression of maximal lytic capacity (Vmax) when compared to autologous peripheral blood lymphocytes. In a single cell lysis and binding assay to assess the proportion of target-binding lymphocytes and target-lysing binders, both TIL and peripheral blood lymphocytes had equivalent numbers of lymphocytes binding target cells and an equivalent number of target-binding cells that could mediate cytolysis. Analysis of lymphocyte subsets was performed using mouse monoclonal antibodies. The TIL population expressed markers found on natural killer cells, including HNK-1 and B73.1. Thus, natural killer cells are present at the tumor site, show lytic capability, but appear to be unable to recycle for multiple lytic events.

Effects of systemic recombinant interleukin-2 on natural killer and lymphokine activated killer activity of human tumor infiltrating lymphocytes.

The purpose of these studies was to compare local and systemic human lymphokine activated killer (LAK) and natural killer (NK) cytotoxicity and to determine its modulation by the systemic administration of recombinant interleukin-2 (rIL-2). After preoperative systemic rIL-2, we extracted tumor infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from patients with pulmonary tumors and compared pre- and posttreatment spontaneous NK activity and their response to in vitro rIL-2. Spontaneous TIL NK activity was increased in patients receiving 15,000 units/kg rIL-2 preoperatively [6.6 lytic units (LU)] compared to those receiving 1,000-10,000 units/kg (0.8 LU) or no rIL-2 (1.4 LU). After 3 days incubation with 1,000 units/ml rIL-2, TIL NK cytotoxic activity was increased in patients receiving 15,000 units/kg rIL-2 (65.4 LU) compared to those receiving 1,000-10,000 units/kg (6.0 LU) or no treatment (24.9 LU). Spontaneous TIL LAK activity was low overall (1.1 LU) with the exception of two patients receiving 15,000 units/kg who had 3.1 and 3.7 LU spontaneously. TIL LAK precursor activity was only slightly increased in patients receiving 1,000-10,000 units/kg rIL-2, whereas those receiving 15,000 units/kg rIL-2 had an average of 22.8 LU. Systemic rIL-2 also increased spontaneous PBL NK activity. Reincubation of PBL obtained at time of surgery or 3 days after discontinuing systemic rIL-2 resulted in significant increases in cytotoxic response to in vitro rIL-2 compared to pre-IL-2 in vitro responses. Systemic rIL-2 had no effect on spontaneous PBL LAK activity. Thus, the immunosuppressive tumor environment can be partially reversed with 15,000 units/kg systemic rIL-2. Higher doses of systemic rIL-2 also increased spontaneous PBL NK activity at time of surgery and 3 days after discontinuing rIL-2. Both TIL and PBL inducible cytotoxicity were boosted in vitro following higher doses of systemic rIL-2.

Human pulmonary macrophages utilize prostaglandins and transforming growth factor beta 1 to suppress lymphocyte activation.

The ability to activate peripheral blood lymphocytes (PBLs) in vitro with interleukin-2 (IL-2) is suppressed by the presence of autologous human pulmonary alveolar macrophages (AMs). AMs suppress both IL-2-induced proliferation and the induction of lymphokine-activated killer cell (LAK) activity in a dose-dependent manner (79 +/- 6% suppression of LAK activity at a 0.25:1 AM/PBL ratio). Increasing the IL-2 concentration increased baseline LAK activity but did not prevent AM-mediated suppression. At least two different mechanisms of suppression were observed, one diffusible in nature and the other contact dependent. Indomethacin prevented the component of inhibition that diffused across porous polycarbonate membranes, indicating prostaglandins as the diffusible inhibitor. In contrast, indomethacin had no effect when added alone into conventional AM-PBL cocultures, but a combination of indomethacin and anti-transforming growth factor beta 1 (TGF-beta 1) antibody did prevent inhibition. This result suggests that TGF-beta 1 acts as an additional contact-dependent inhibitor. PBLs that were rendered unresponsive to IL-2 completely recovered their responsiveness within 4 days after removing AMs from the coculture. These features suggest that pulmonary macrophages have multiple mechanisms for locally suppressing IL-2 responses and lymphocyte activation.

Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin.

Natural killer (NK) and lymphokine-activated killer (LAK) cell cytotoxic functions can be strongly augmented by the iron-carrier protein lactoferrin (LF). LF significantly enhances NK and LAK activities when added at the beginning of NK or LAK cytotoxicity assays. LF is effective in augmenting cytotoxic activities at concentrations as low as 0.75 microgram/ml, and higher concentrations of LF induce greater augmentation of NK and LAK. Iron does not appear to be essential for LF to increase NK and LAK, as depleting iron from LF with the chelator deferoxamine does not affect the capacity of LF to increase cytotoxicity. LF is known to have RNase enzymatic activity, and LF enhancement of NK and LAK can be blocked by RNA. However, LFs from two different sources with over 100-fold difference in RNase activity are equally effective in enhancing NK and LAK. Furthermore, purified non-LF RNase does not modulate NK or LAK activity and DNA is as effective as RNA in blocking LF augmentation of NK or LAK cytotoxicity. Therefore, the RNase activity is unlikely to be responsible for LF enhancement of the cytotoxicities. Newborn infants are known to have low NK activity and NK and LAK cells have been implicated in host defense against microbial infections. Thus, maternal milk-derived LF may have a role in boosting antimicrobial immunity in the early stages of life. In adults, LF released from neutrophils may enhance NK and LAK functions in the inflammatory process induced by microbial infections.

From cytoprotection to tumor suppression: the multifactorial role of peroxiredoxins.

In the past decade, a new family of highly conserved antioxidant enzymes, Peroxiredoxins (Prxs), have been discovered and defined. There are two major Prx subfamilies: one subfamily uses two conserved cysteines (2-Cys) and the other uses 1-Cys to scavenge reactive oxygen species (ROS). This review focuses on the four mammalian 2-Cys members (Prx I-IV) that utilize thioredoxin as the electron donor for antioxidation. The array of biological activities of these proteins suggests that they may be evolutionarily important for cell function. For example, Prxs are capable of protecting cells from ROS insult and regulating the signal transduction pathways that utilize c-Abl, caspases, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) to influence cell growth and apoptosis. Prxs are also essential for red blood cell (RBC) differentiation and are capable of inhibiting human immunodeficiency virus (HIV) infection and organ transplant rejection. Distribution patterns indicate that Prxs are highly expressed in the tissues and cells at risk for diseases related to ROS toxicity, such as Alzheimer's and Parkinson's diseases and atherosclerosis. This interesting correlation suggests that Prxs are protective against ROS toxicity, yet overwhelmed by oxidative stress in some cells. Prxs tend to form large aggregates at high concentrations, a feature that may interfere with their normal protective function or may even render them cytotoxic. Imbalance in the expression of subtypes can also potentially increase their susceptibility to oxidative stress. Understanding the function and biological role of Prxs may lead to important discoveries about the cellular dysfunction of ROS-related diseases ranging from atherosclerosis to cancer to neurodegenerative diseases.

Universal rosetting reagent for the detection of human cell surface markers.

The extensive use of monoclonal antibodies for the detection of human cell surface markers has necessitated the development of a simple, reproducible reagent which can be applied universally to any mouse anti-human antibody. We have developed a rosetting reagent which is composed of ox red blood cells, rabbit anti-ox RBC antibody, protein A, and rabbit anti-mouse Ig antibody. This reagent binds specifically to human lymphocytes coated with mouse monoclonal antibody, is as sensitive as immunofluorescence and gives comparable results with fresh and frozen peripheral blood lymphocytes. Sensitivity, reliability, and ease of quantitation make this an extremely practical reagent, and its versatility makes it especially useful for large scale studies of human T cell markers.

Lymphocyte surface markers and cytotoxicity following cryopreservation.

The effects of cryopreservation (CP) on lymphocyte subpopulation distribution and functional activity in blastogenic and cytotoxicity assays were tested. Peripheral blood lymphocytes (PBL) from 12 healthy human donors were obtained by Ficoll-Hypaque separation. Half of each sample was tested fresh, while the other half was cryopreserved and then thawed and tested the same day. Each sample of CP-PBL was compared to fresh PBL from the same donor in simultaneous assays. Following CP there was a significant reduction in the percentage of E, EA gamma, and EA mu rosette-forming cells with a reciprocal increase in EAC rosette-forming cells. The blastogenic response to alloantigens was stable following CP while blastogenesis in unstimulated control cultures was significantly reduced. Mixed lymphocyte culture (MLC)-induced cell-mediated lympholysis (CML) was consistently and significantly diminished by CP. Cytotoxicity in 4 h chromium release NK (K562), ADCC, and LDCC assays was also significantly diminished by CP. In contrast, cytotoxicity was unaffected in an 18 h cytotoxicity assay against adherent cultured melanoma target cells.

The use of viable frozen lymphocytes for studies in human tumor immunology.

Viable frozen lymphocytes displayed activity in blastogenesis assays that was indistinguishable from freshly prepared lymphoid cells. Similarly, cytotoxic activity of lymphocytes against melanoma target cells from melanoma patients was only slightly affected by the freezing procedure. Frozen lymphocytes provided a highly reproducible source of cells in these assays. The use of viable frozen peripheral blood lymphoid cells for the retrospective analysis of a cancer patient's immune response is described.

The adenoviral transcription factor, E1A 13S, trans-activates the human tumor necrosis factor-alpha promoter.

The 1311 bp TNF-alpha promoter region fused to a luciferase reporter vector was used in a transient transfection system to study the regulation of TNF-alpha promoter activity by E1A 13S in the U937 macrophage cell line and the MLA 144 T cell line. Co-transfections of the TNF-alpha promoter with an E1A expression vector resulted in a strong trans-activation of the promoter in both cell lines. Sequential truncation of the promoter mapped the E1A responsive region to sequences contained between -120 bp and the transcription start site. Truncation to -95 bp caused a dramatic 87% reduction of E1A activation in MLA 144 cells and further truncation to -36 bp caused a complete loss of E1A activation. In U937 cells, each truncation lowered E1A responsiveness but activity was never completely abolished. Site-directed mutagenesis of putative cis-acting sequences in the TNF-alpha promoter identified the AP-1 site as important for E1A trans-activation in the U937 cell line; the AP-2 and CRE sites also appeared to contribute to a lesser degree. In contrast, only the CRE mutation caused a reduction in E1A induced activity in the MLA 144 cell line. Co-transfection of the E1A expression vector with expression vectors for the cellular transcription factors AP-1, AP-2 and CREB indicated that none of these transcription factors showed any co-operativity with E1A. Thus, cis-acting sequences which contribute to E1A trans-activation of the TNF-alpha promoter have been delineated.

Immunologic defects in lung cancer patients.

Ninety-three patients with lung cancer were evaluated by delayed cutaneous hypersensitivity testing. Twenty-eight of these patients were evaluated by in vitro lymphocyte function. Thirty-three patients were evaluated prior to surgery and defects in the immune response were closely associated with unresectable disease. In vitro and in vivo studies indicate an antigen recognition defect in the cellular immune mechanisms of these patients. Preliminary studies suggest that the immunopotentiating agent Levamisole may be able to augment this defective cellular immunity in patients with lung cancer.

Demonstration of lymphocyte blastogenesis--inhibiting factors in sera of melanoma patients.

The proliferative response of lymphocytes obtained from a healthy volunteer to the mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) was evaluated in the presence of sera from 101 patients with malignant melanoma and 75 normal controls. A significant decrease in blastogenic response was observed in the presence of sera from melanoma patients. Sera from patients with advanced disease were particularly inhibitory, suggesting that the circulating immunosuppressive factors were either tumor derived or tumor induced. Our observation that sera from patients who failed to display a delayed cutaneous hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) are more inhibitory to mitogen stimulation than sera from patients who react to this contact allergen suggests that DNCB skin testing may identify an immunological defect mediated by circulating humoral factors.

Immunologic aspects of unknown primary melanoma.

Immune responses of 36 patients with stage II unknown primary melanoma were compared to those of 83 stage II patients with known primary melanoma. The two groups of patients were similar in age, sex, and extent of disease. Delayed cutaneous hypersensitivity to dinitrochlorobenzene (DNCB) was nearly identical in both groups. Patients with unknown primary melanoma had a mean level of reactivity of 4.7 +/- 0.37 and the patients with known primary had a mean level of 5.0 +/- 0.17. Of unknown primary patients 4.2% were unreactive to DNCB and 4.8% of known primary patients were unreactive. Antibodies to cultured melanoma cells were slightly more prevalent in serum from the unknown primary patients (67%) than from known primary patients (45%), but mean titer of positive sera did not differ significantly. Lymphoproliferative assays did not demonstrate differences in lymphocyte reactivity among the two groups when tested with Concanavalin A, mixed leukocyte culture, or unstimulated lymphocyte blastogenesis. No differences could be detected in these assays of humoral and cellular immune responses when patients with unknown primary melanoma were compared to those with known primary melanoma and similar clinical characteristics.

Effects of operation on immune response in cancer patients: sequential evaluation of in vitro lymphocyte function.

The effect of operation on in vitro lymphocyte function in 35 cancer patients was studied. Lymphocyte proliferative responses to phytohemagglutinin (PHA), poke-weed mitogen (PWM), and concanavalin A (Con A) were measured by 3H-thymidine incorporation. Sheep red blood cell (SRBC) rosette formation also was quantitated. These tests were performed preoperatively and at 24 hours, one week, and 4 weeks postoperatively. Intra-abdominal and intrathoracic procedures, transfusions, and longer operating times depressed the lymphocyte proliferative response. However, an increased lymphocyte proliferative response was noted in sarcoma patients 24 hours postoperatively, possibly as a result of lowered tumor burden. Several of these changes still were evident 4 weeks after operation. Rosette formation also decreased significantly 24 hours postoperatively in patients who had intrathoracic or intra-abdominal procedures, but returned to preoperative levels after one week. In general, operation appears to cause transient depression of lymphocyte function in some cancer patients. However, lymphocyte function returns to normal by the fourth postoperative week in most patients.