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ABSTRACTIn 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 IIV) 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-B (NF-B) 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.

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.

Summary We administered preoperative low-dose interleukin-2 (IL-2) to 10 patients undergoing thoracotomy for pulmonary tumors. The in vivo effect of IL-2 on tumor-associated lymphocyte activity was assessed in the resected specimens by immunohistochemistry and compared with observations in 45 patients who did not receive IL-2. H & E evaluation revealed an increase in intra- and peritumoral lymphocyte infiltration in the IL-2-treated patients. Immunopathological evaluation with monoclonal antibodies revealed that this lymphocyte infiltration was predominantly CD5-positive T cells. The amount of intra-and peritumoral lymphocyte activity correlated with the dose of IL-2 administered (6000–90 000 international units/kg every 8 h for 48 h. IL-2-treated patients showed increases in T-cell-associated activation markers (IL-2 a-receptor, transferrin receptor and HLA-DR) on peritumoral lymphocytes, but not on intratumoral lymphocytes. We previously reported that low-dose IL-2 increases the intrinsic natural killer cell cytotoxicity of intratumoral lymphocytes and suggest that this lymphocyte infiltration is further evidence that low-dose IL-2 can augment in vivo lymphocyte activity at the tumor site.

T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.

Regulation of TNF-α promoter activity by IL-2, IFN-γ, GM-CSF, and IL-4 was examined in the U937 macrophage cell line and the MLA 144 T cell line. Using a transient transfection system, the full-length TNF-α promoter was examined for response to these cytokine signals. Only GM-CSF was able to consistently induce a twofold activation of the TNF-α promoter in the U937 cell line. GM-CSF activation of the promoter region was further analyzed using a series of 5' truncations and site mutations of the AP-1, AP-2, and CRE sites of the promoter. The GM-CSF activation mapped to the region contained within the 95 base pairs upstream from the transcription start site (TSS) with the AP-2 site as a putative cis-acting sequence. IL-4 profoundly inhibited both basal and phorbol ester-induced TNF-α promoter activity as well as protein production. Promoter inhibition by IL-4 required the 95-bp basal promoter sequence.

We have previously shown that RBC significantly augment NK cell-mediated cytotoxicity when added at the initiation of cytotoxicity assays. We now report that RBC cytosol contains a soluble factor that mediates a similar enhancement of NK activity when the factor is precoated on a plastic surface. We have purified this NKEF by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration HPLC. While gel filtration HPLC shows that NKEF has an apparent molecular mass of between 300 and 400 kDa, SDS-PAGE analysis indicates an apparent molecular weight of 48 and 24 kDa under nonreducing and reducing conditions, respectively. Immune serum obtained from a rabbit immunized with purified NKEF blocks NKEF augmentation of NK activity. In contrast, preimmune serum fails to inhibit NKEF activity. Anti-NKEF antibody reacts with the cytosol of NK-sensitive erythroleukemic target cell K562. Western blot analysis indicates that this antibody recognizes a 24-kDa protein both in RBC and in K562. We have generated tryptic peptides from NKEF and obtained partial amino acid sequences from three of the peptides. Two of the sequences show no significant homology with any known sequences. The third sequence shows partial identity with a deduced sequence from a murine erythroleukemia-related gene. Thus, NKEF is a novel protein with unique amino acid sequence. Our results of NKEF in RBC suggest that RBC could play an important role in regulating NK function. Copyright 1993, 1999 Academic Press

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 lympho‐ kine‐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 β1(TGF‐β1)antibody did prevent inhibition. This result suggests that TGF‐ß1acts as an additional contact‐dependent inhibitor. PBLs that were rendered unresponsive to IL‐2 completely recovered their responsiveness within 4days after removing AMs from the coculture. These features suggest that pulmonary macrophages have multiple mechanisms for locally suppressing IL‐2 responses and lymphocyte activation.

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 μg/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.

• Tumor-infiltrating lymphocytes (TILs) show depressed natural killer (NK) activity compared with peripheral blood lymphocytes (PBLs). To determine if TIL NK function can be reactivated in vivo, 11 patients with tumors metastatic to the lung were treated with systemic recombinant interleukin 2 (rlL-2). Spontaneous TIL NK activity and NK activity after three days' incubation with 100 U/mL of rlL-2 were increased in patients receiving 15000 U/kg of rlL-2 preoperatively compared with those receiving between 1000 and 10000 U/kg. Histologically, higher doses of rlL-2 increased the number of intratumoral lymphocytes, the level of peritumoral lymphocytic transferrin, and the expression of HLA-DR. Spontaneous PBL NK activity in patients receiving between 10000 and 15000 U/kg of rlL-2 was also increased and was further increased by in vitro culture with rlL-2. Thus, PBL NK activity and TIL NK function in vivo can be augmented with 15000 U/kg of systemic rlL-2. Both TIL- and PBL-inducible cytotoxicities were further enhanced by in vitro culture with rlL-2.(Arch Surg 1987;122:1446-1450)

Summary The generation of lymphokine-activated killer (LAK) activity and the proliferative response to human recombinant interleukin-2 (IL-2) were significantly reduced by the presence of human recombinant leukocyte interferon (IFNa) in cultures of human peripheral blood mononuclear cells (PBMC). Mature natural killer (NK) cells can be depleted from PBMC with the toxic lysosomotropic agentl-leucine methyl ester. The generation of cytotoxic cells from lymphocytes depleted in leucine methyl ester was also inhibited by indicating that the IFN-a effect is not limited to mature cytotoxic NK cells. Depletion of adherent cells from PBMC did not affect the suppression of LAK induction by IFN-a. Surface marker analyses of Tac antigen and transferrin receptor (TfR) showed that the presence of IFNa throughout the culture period significantly suppressed the typical increase in IL-2-induced Tac- and TfR-positive cells. In contrast, IFNa treatment before and after IL-2 culture enhanced LAK cytotoxic activity. Therefore, combinations of these biological response modifiers for clinical use should take into account the dual effect of IFNa on key features of the IL-2 response.

High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.

Lymphokine-activated killer (LAK) cell activity may be largely the result of activation and/or expansion of peripheral blood natural killer cells by culture with interleukin-2 (IL-2). We have examined the role of proliferation in LAK cell development by either inhibiting or enhancing the proliferative potential of peripheral blood lymphocytes. Inhibition of proliferation was accomplished using irradiation, mitomycin C, or the iron chelator deferoxamine. For each of these agents, a dose-dependent inhibition of proliferation was observed. At doses of inhibitor which nearly completely blocked thymidine uptake, the development of LAK activity was only partially impaired. The mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A) augmented the proliferative response of peripheral blood lymphocytes to IL-2. However, augmentation by PHA, but not Con A, consistently resulted in a decrease in LAK activity. This inhibition of LAK activity by PHA did not appear to be due to inhibition of the effector cell, nor to preferential expansion of irrelevant cells. These data suggests that not all LAK activity is dependent on proliferation, and that high levels of proliferation in the presence of IL-2 do not necessarily lead to LAK activity.

The purpose of these studies was to compare local and systemic human lymphokine activated killer (LAK) and natural killer (NK) cytotoxic activity and to determine its modulation by biologic agents. Local immunity may be an important component in limiting local tumor growth. Therefore, as a model for studying immune function in the local compartment, we assessed NK activity of lymphocytes present at the site of human tumors and in peripheral blood (PBL). We extracted tumor infiltrating lymphocytes (TIL) and PBL from patients with pulmonary tumors and compared NK activity and response to the biological modifiers gamma interferon (IFN-?), indomethacin (INDO), and interleukin 2 (IL-2). We also studied TIL and PBL LAK activity using the NK-resistant M14 target cells and determined the TIL response to IL-2, plus IFN-?. Titration of K562 targets in a 51Cr release assay revealed that untreated TIL have low cytotoxicity (4.32%) compared to untreated PBL (34.3%, P=<0.001). This low level of TIL NK activity was not affected by IFN-?, INDO, or IL-2 at 1 h. However, at 3 days of culture, IL-2 with or without exogenous IFN-? significantly increased TIL NK ctotoxicity (20.5%, P=0.02 without IFN-? and 32.52 lytic units (LU), P=<0.02 with IFN-?). Untreated TIL and PBL both had low cytotoxicity against M14 targets (1.08 LU and 1.26 LU), respectively. After 3 days culture with IL-2 plus IFN-?, both TIL and PBL LAK cytotoxicity were increased (14.34 LU and 40.63 LU). We conclude that local NK and LAK activity is intrinsically low. However, this activity can be modulated by biologic agents, thus giving hope for the development of local antitumor effectors capable of in vivo tumor control.

SENSITISATION of lymphocytes in vitro facilitates the study of cellular immune mechanisms under conditions in which the exact input of reactive cells and antigen is controlled. Such systems have been used to generate cytotoxic effector cells against alloantigens1–3, and more recently against tumour-associated antigens (TAA) in animal models4,5. Less work has been done on TAA induction of immune responses using human antigens and effector cells. Golub et al.6demonstrated in vitro sensitisation against antigens present on autochthonous Burkitt lymphoma cells. Since such sensitised cells mediate tumour or allograft rejection reactions in vivo7,8, the in vitro ‘education’ of lymphocytes against TAA is an appealing potential modality for immunotherapy of human cancer. Several preliminary clinical trials have been reported in which ‘educated’ lymphoid cells were administered to cancer patients9,10, although the clinical benefits of this procedure remain uncertain. We describe here culture conditions that increase cytotoxic activity of melanoma patients' lymphocytes against melanoma-derived target cell lines.

A new method was developed for detecting the phenotype of target binding cells (TBC) in a single-cell assay system. This methodology was evaluated during a clinical trial of recombinant interferon alfa-2a (rIFN alfa-2a) for the treatment of 10 metastatic renal cell carcinoma patients. Total TBC with K562 targets, HNK-1+ TBC, and HLA-DR+ TBC were quantitated during rIFN alfa-2a therapy. A significantly increased proportion of lymphocytes bound to target cells on day 9 of therapy bore the HNK-1 marker. This proportion subsequently declined to pretreatment levels. Total TBC paralleled the rise and fall in HNK-1+ TBC. HLA-DR+ TBC binding to targets remained constant and low throughout therapy. These findings suggest that rIFN alfa-2a early in therapy (day 9) caused the recruitment of additional HNK-1+ cells into binders. However, with continued therapy, this proportion reverts to pretreatment levels. The results of this clinical trial served to illustrate the ability of the modified single-cell assay system to detect TBC phenotype.