Lytic Units Research Articles

Natural killer cell activity in patients with Graves' disease and Hashimoto's thyroiditis.

Natural killer (NK) cell activity of peripheral blood lymphocytes (PBL) against k562 human tumor cell targets was studied in patients with Graves' disease and Hashimoto's thyroiditis. NK activity was measured in a standard 4-hour 51chromium (Cr) release assay. Cytotoxicity was expressed as lytic units (LU)/10(6) PBL. Significantly decreased NK cell activity was demonstrated in both groups of patients, with mean (+/- SE) lytic units of 10.3 (+/- 9.1) and 13.3 (+/- 10.3) for patients with Graves' disease and Hashimoto's thyroiditis, respectively, compared with 36.0 (+/- 26.3) for age- and sex-matched normal subjects. When patients with Graves' disease were analyzed according to their thyroid status; NK activity was significantly depressed in (1) hyperthyroid patients before treatment; (2) hyperthyroid patients receiving antithyroid therapy; and (3) euthyroid patients receiving antithyroid therapy, compared with normal subjects. Graves' disease patients who were hypothyroid after radioactive iodine therapy or thyroidectomy had normal NK activity. No significant differences between hyperthyroid and euthyroid patients or between hypothyroid patients and normal subjects were demonstrated. NK activity in patients with Graves' disease did not correlate with serum levels of thyroxine, the presence or severity of ophthalmopathy, or titers of serum thyroid antibodies. In patients with Hashimoto's thyroiditis there was no correlation between NK activity and goiter size, titers of antithyroid antibodies, or thyroid status. These findings suggest that depression of NK activity in both disorders is secondary to abnormalities of thyroid hormone secretion, although an effect of the underlying autoimmune reactions has not been excluded.

In Vitro Effects of IL-12 on HIV-1-Specific CTL Lines from HIV-1-Infected Children

We studied the in vitro effects of IL-12 on HIV-1-specific CTL lines derived from PBMC of HIV-1-infected children. HIV-1-specific CTL lines were derived by limiting dilution following Ag-specific stimulation of PBMC from HIV-1-infected children and were maintained with repeated anti-CD3 stimulation. Following incubation with IL-12 for 5 to 7 days, HIV-1-specific cytotoxicity was augmented in a dose-dependent fashion (mean increase, 94 +/- 83 lytic units; p = 0.0006). Experiments performed with CD3-blocking Abs and MHC-mismatched targets demonstrated that the IL-12-enhanced activity was MHC restricted and dependent on cells bearing CD3. The effect of IL-12 on proliferation of the CTL lines as tested by [3H]TdR uptake was minimal, with stimulation indexes ranging from 1.25 to 4.9. The effects of IL-12 on cytotoxicity were not significantly altered by addition of Ab to the IL-2R (anti-Tac) in quantities sufficient to block exogenous IL-2 (p = 0.15), demonstrating that endogenous IL-2 activity is not required for IL-12-enhanced cytolytic activity. Likewise, addition of neutralizing Ab specific for IFN-gamma did not change IL-12-enhanced cytotoxicity (p = 0.61). The in vivo role of IL-12 in the generation and the stimulation of CTL remains to be determined; however, its ability to augment HIV-1-specific CTL in vitro adds additional support for IL-12 as a candidate for immune-based therapy of HIV-1.

CYTOTOXICITY OF LAK CELLS AND EBV-SPECIFIC T CELLS AGAINST LYMPHOBLASTOID EBV-POSITIVE B CELLS: A MODEL FOR CELLULAR THERAPY OF PTLD

696 Posttransplant lymphoproliferative disorder (PTLD) is a significant cause of morbidity and mortality in transplant patients. The presence of the Epstein-Barr virus (EBV) in many of these lymphomas has prompted the use of cellular immunotherapy using either EBV-directed T cells or autologous lymphokine-activated killer (LAK) cells. EBV-specific T cells recognize viral epitopes in association with HLA Class I molecules, whereas LAK cell cytotoxicity is enhanced by their depletion. The purpose of this study was to examine the relative levels of in vitro cytotoxicity of these effector cells against either untreated or HLA Class I-depleted EBV-immortalized B lymphocytes. LAK cells, EBV-specific T cell lines and EBV-immortalized lymphoblastoid cell lines were prepared from healthy donors. The degree of cell-mediated lympholysis (CML) was determined by using a standard 4 hour 51Cr release and the results were expressed as lytic units (LU-number of effectors required for 20% lysis of 5×103 targets). In some cases, HLA Class I expression in the target cells was depleted by a brief exposure to pH 3.3 buffer. Cell viability and Class I depletion were confirmed by trypan blue exclusion and flow cytometry, respectively prior to execution of CML assays. LAK cell-mediated cytotoxicity against autologous EBV target B cells was 14.4 LU which increased to 69.6 LU when target cells were HLA-depleted(p<0.05, Wilcoxon test). On the contrary, EBV-specific T cell-mediated cytotoxicity against untreated B cell targets was 765 LU, and this decreased to 316 LU when HLA-Class I depleted cells were used as targets (p<0.05). Additionally, as compared to that of LAK cells, T cell-mediated cytotoxicity against untreated targets was significantly greater (Friedman's test p=0.03, Tukey multiple comparison p<0.05). We conclude that EBV-specific T cells show superior in vitro cytotoxicity against autologous EBV-positive B cell targets, regardless of the level of HLA Class I expression. The results may explain in part the uniform response of PTLD to T cell therapy, and the less frequent responses to that of LAK cell. The level of cytotoxicity for both effector cell types is influenced by the degree of expression of HLA Class I in this model system. This approach may be useful in examining other factors which may enhance or decrease the cytotoxic response against EBV-positive B cells.

Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities.

Culture medium induced morphological changes of melanoma cells associated with change in sensitivity to lysis by lymphokine-activated killer cells.

Three sublines (Clones 1, 2 and 7) of the human melanoma CaCL 73-36 cell line with different cellular morphology, growth patterns, melanin content and surface antigenic profile were maintained in RPMI-1640 medium plus 10% fetal bovine serum (abbreviated as RPMI). Each subline was divided into two groups: one grown in RPMI and the other in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (abbreviated as DMEM) for 96 h. Phenotypically, Clone 2 expressed Class I and II MHC and ICAM-1 on the surface and in the cytoplasm, while Clones 1 and 7 failed to express these antigens in both the cytoplasm and on the cell surface. Melanotic Clones 1 and 7 cells became even more pigmented, had slower growth rates, and exhibited lower saturation densities when incubated in DMEM than when they were incubated in RPMI. On the other hand, Clone 2 cells maintained in RPMI were grossly amelanotic, contained defective-like melanosomes detected ultrastructurally, and had distinct clusters of microvilli polarly located in most of the cells. Such specialized ultrastructures were not affected by medium conditions. Analysis of sensitivity of the clonal sublines to cytolysis by allogeneic effector cells revealed that in spite of low levels of natural killer (NK) cytotoxicity noted, DMEM produced a 2- to 14-fold increase in sensitivity to NK cells, irrespective of which medium was used. Different levels of lymphokine-activated killer (LAK) cytolytic activity were clearly observed in sublines maintained in RPMI, with Clone 2 being the most sensitive and both Clones 1 and 7 being less sensitive. Cells grown in DMEM exhibited significantly higher levels of sensitivity to LAK cytolysis than cells grown in RPMI as revealed by their differences in lytic units (p < 0.05). This was likely due to the high levels of surface ICAM-1 expression in cells incubated in DMEM vs little expression of this adhesion molecule by cells grown in RPMI. Taken together, these results demonstrate the presence of heterogeneous subpopulations within the CaCL 73-36 melanoma cell line regarding their pigmentary status, antigenic profile, growth pattern and responsiveness to NK/LAK cytolysis. The results also call attention to the importance of utilizing a same medium in short- and long-term cultures of melanomas for biological studies and response evaluations of therapeutic agents such as LAK cells, when multiple cell targets from different patients or multi-metastatic cell lines from individual patients are to be compared. Finally, these melanoma sublines may be valuable for further elucidation of the relationship between MHC expression, and increased sensitivity to LAK cytolysis, and the role of the components of DMEM in the mechanism for the observed induction of cell differentiation and enhanced LAK cytolysis.

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice.

This study was designed to investigate the effects of moderate-intensity endurance training on basal natural killer (NK) cell cytolytic activity in murine splenocytes that were enriched for 1) NK1.1+ cells or 2) macrophages and NK1.1+ cells. Mice were assigned to sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn) groups. Splenocyte number, the percentages of NK1.1+, large granular lymphocytes (NK1.1+, LGL-1+), and other subpopulations did not change in Trn mice. Approximately 70% of cells enriched for NK1.1+ expressed this surface antigen. Lytic units (LU) expressed per LGL-1+ cell were significantly lower in Trn [83.9 +/- 3.2 (SE)] compared with Sed (109.5 +/- 7.5) and TM (101.3 +/- 6.4) groups. When macrophages remained in the in vitro assay, LU per LGL-1(+) cell did not differ across groups. The results indicate that highly enriched NK1.1+ cells from Trn mice had lower NK cell activity compared with Sed mice. No differences in NK cell activity were observed when cells were enriched for NK1.1+ cells and macrophages. These findings support the hypothesis that macrophage modulation of NK cells may be one mechanism contributing to augmented basal NK cell activity in endurance-trained individuals.

Natural Killer Cells in the Late Decades of Human Life

We examined the lymphocyte subsets and indices of natural killer (NK) cell activity (lytic unit (LU), index of absolute NK cell activityin vivo(ALU), and NK cell activity on a per-cell basis (PCNK)) in 82 people (age, 30–99 years) who were immunologically normal. Although the number of NK cells was maintained throughout the examined age range, the ALU and PCNK values correlated negatively with age. We then examined whether any of the various immunologic parameters, including the function and cell counts of NK cells, T cells, and neutrophils, related to past infectious episodes and death in the follow-up period in 44 elderly subjects (age, 63–98 years). Only low ALU and PCNK values correlated with a past history of severe infection, while low LU, ALU, and PCNK values were the only parameters which correlated with death due to infection during the follow-up period. We propose that human NK cells do not escape the aging process and that a low NK cell function relates to the development of severe infections, which may be fatal, in elderly subjects.

Splenectomy impairs lymphocytosis during maximal exercise.

To evaluate the role of the spleen for the exercise-induced lymphocytosis, six splenectomized subjects and six matched control subjects cycled for 12 min at two submaximal work rates corresponding to 50 and 75% of their maximal work capacity, followed by a supramaximal intensity maintained until exhaustion (16 +/- 1 min; mean +/- SE). Venous blood samples were taken before, during, and 2 h after the maximal load. In both groups, the concentration of lymphocytes became elevated during exercise, but the increase from the level at rest was impaired in the splenectomized subjects compared with that of the controls (118 +/- 34 vs. 238 +/- 38%; P < 0.05). This was reflected in several lymphocyte subsets: cluster designation (CD) 3+ cells (pan T lymphocytes), 69 +/- 19 vs. 204 +/- 37%; CD8+ cells (T lymphocyte subset), 164 +/- 41 vs. 467 +/- 68%; CD16+ cells [natural killer (NK) cells], 291 +/- 88 vs. 870 +/- 177%; CD56+ cells (NK cells), 301 +/- 108 vs. 753 +/- 187%. Also, the specific NK cell lysis of target cells (NK cell activity) during exercise was lower for the splenectomized subjects (30 +/- 7%) than that of the control subjects (52 +/- 10%), but evaluation of lytic units indicates that this was due to a reduced number of NK cells in the assay rather than insufficient cell lysis. Plasma catecholamines reached the same level in the splenectomized subjects and control subjects, which was taken to reflect that the activity of the sympathetic nervous system was similar in the two groups of subjects. Thus the major finding of this study is that the spleen is important for lymphocytosis during exercise, accounting for two-thirds of the increase in T lymphocytes and NK cells.

Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma

Natural killer (NK) and lymphokine-activated killer (LAK) cell activities were measured in peripheral blood obtained from healthy women to compare a standard 51Cr release assay with a nonradioactive europium (Eu3+) release assay based on time-resolved fluorescence. The two types of cytotoxicity assays were first compared in paired determinations performed on 28 samples of peripheral blood mononuclear cells obtained from healthy women who had normal pap smears or no biopsy evidence of cervical squamous intraepithelial lesions (SIL). Target cells (NK-sensitive K562 and NK-resistant Raji cell lines) were labeled with Eu3+ only, 51Cr only, or both labels and compared in cytotoxicity assays using fresh or interleukin 2 (IL-2)-activated effector cells. Spontaneous release in the Eu3+ release assay was comparable to that observed in the 51Cr release assay, but maximum Eu3+ release always exceeded that of 51Cr. In 4-h assays, specific release of Eu3+ from target cells was more rapid than that of 51Cr, consistently resulting in 30 to 40% higher levels of activity. However, a significant linear correlation (P < 0.001) was observed between cytotoxicity levels based on measurements of Eu3+ and 51Cr release in 4-h assays. The Eu3+ release assay was then used to measure NK and LAK activities in the peripheral blood of women with cervical SIL or cervical squamous cell carcinoma (SCC). Mean NK activity of women with advanced SIL (121 lytic units [LU]) or SCC (93 LU) was found to be similar to that of controls (101 LU) or patients with normal cervical biopsies (90 LU), as was the ability to generate IL-2-stimulated NK activity. However, LAK activity during 18 h of incubation in the presence of IL-2 was reduced in patients with cervical SCC (P < 0.05) compared with that in normal controls. Results of 51Cr assays performed in parallel with patient samples gave comparable results. Advantages of EU3+ release assays for routine evaluation of cytotoxicity are discussed.

Analysis of Antibody-Dependent Cell-Mediated Cytotoxicity in Autoimmune Thyroid Disease

There is no consensus on the role of antibody-dependent cell-mediated cytotoxicity (ADCC) in autoimmune thyroid disease; recent reports have suggested that antibodies mediating ADCC are found particularly in patients with primary myxoedema, occur less frequently in Hashimoto's thyroiditis and are absent in Graves' disease. Using an ADCC assay with a single source of effector and target cells, and expressing results as lytic units, we have found antibodies capable of mediating ADCC in 9 of 17 patients with primary myxoedema, 9 of 22 patients with Hashimoto's thyroiditis and 6 of 22 patients with Graves' disease. There was no significant difference between the groups in this distribution. Mean levels of ADCC activity were not significantly different comparing primary myxoedema and Hashimoto's thyroiditis patients, although levels were lower in Graves' disease patients compared to those with Hashimoto's thyroiditis (P < 0.05). There was no correlation between TPO antibodies (total IgG or IgG subclasses) measured by ELSA and ADCC activity. These results suggest that thyroid antigens besides TPO are involved in ADCC and that antibodies mediating ADCC are not restricted to subgroups of patients with autoimmune thyroid disease.

Natural cytotoxicity and gnrh agonist administration in advanced endometriosis: positive modulation on natural killer activity

To investigate the effects of pharmacologic suppression of ovarian function on the immune system, with respect to the clinical outcome of endometriosis and the possibility of an immunoendocrine combined treatment. After informed consent, 25 of 37 patients with revised American Fertility Society stage III and IV endometriosis who underwent postoperative medical treatment were selected and enrolled for this immunoendocrine longitudinal study. Medical treatment consisted of tryptorelinum depot injection, 3.75 mg/month for 24 weeks. Blood samples were collected before the first injection in the early follicular phase, day 2-3 of the cycle, and during medical treatment (every 4 weeks) and follow-up (every 6 months). At the end of the study, we had ten blood samples per patient to evaluate the cytotoxic activity, the number of natural killer cells, and the serum levels of estradiol. Natural killer activity was determined against the K562 cell line by target cell retention of the fluorescent dye carboxyfluorescein diacetate. A positive immunomodulating effect was observed during GnRH agonist administration. In particular, a significant progressive increase in natural killer cell activity was defined within the first 12 weeks of medical treatment; after three injections, we observed the highest values of cytotoxicity, with a median of 7.1 lytic units (range 0.3-14.0; P = .02). Natural cytotoxicity then decreased toward a plateau, which persisted during therapy completation and follow-up, with slight fluctuations. In patients who had recurrence, the values of natural killer cell activity were constantly lower than those in patients with disease-free follow-up, particularly within the first 12 weeks of medical treatment.

Measurements of Natural Killer (NK) Activity and NK-Cell Quantification

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphology and the ability to kill certain target cells via one or more cytolytic mechanisms. Measurements ofin vitroNK activity from heparinized whole blood, or lymphocytes isolated from human peripheral blood or tissue, may be performed by a variety of techniques. Procedures using standard51Cr-release assays as well as nonradioactive labeling materials are described in detail in this article, and advantages and disadvantages of these methods are discussed. Quantification of assay results, as percentage of specific lysis, lytic units, and cytolytic units, is explained with attention given to the limitations of each of these calculations. Quality control (QC) procedures that are needed to assure reproducible, reliable NK-cell assays for serial monitoring are described, and suggestions are provided for developing a QC program for these assays. Since NK cells can perform multiple functions, and the mechanism of NK-cell-mediated cytotoxicity is complex, the rationale for selecting a particular assay to measure NK activity is discussed.

P36 A comparison of statistical methodologies for the calculation of lytic units derived from the modeling of natural killer cells

The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available.In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice.Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs.Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose.Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.

Natural Immune Reactivity-Associated Therapeutic Response in Patients with Metastatic Renal Cell Carcinoma Receiving Tumor-Infiltrating Lymphocytes and Interleukin-2-Based Therapy

Combination therapy with systemically administered interleukin-2 (IL-2) and tumor infiltrating lymphocytes (TIL) demonstrates significant clinical activity in some patients with metastatic renal cell carcinoma (RCC). The objective of this study was to identify predictors of therapeutic response in patients with IL-2- and TIL-based immunotherapy. We characterized and compared immunologic properties of tumors, TILs, peripheral blood lymphocytes (PBLs) and sera of responding (R, n = 8) with nonresponding patients (NR, n = 9). Before undergoing nephrectomy, responding patients exhibited a higher percentage of circulating natural killer (NK) cells (CD56+ CD3-) (43 +/- 20%) as compared with nonresponders (18 +/- 16%) (p < 0.01). After nephrectomy, the CD56+ CD3-/CD56- CD3+ ratio in responding patients (pre: 2.60 +/- 2.24; post: 0.28 +/- 0.19; p < 0.05) significantly decreased and was similar to that of patients not responding to therapy (0.42 +/- 0.36). Sera from patients responding to immunotherapy, obtained before and after completion of therapy, contained natural killer (NK)-enhancing factor(s) that significantly enhanced the proliferation (3.2 x 10(3) +/- 25%/ 3.6 x 10(3) +/- 13% counts/min) and cytotoxicity [17.6 +/- 4.0/18.0 +/- 1.9 lytic units (LU)] of fresh PBLs as compared with normal serum (1.8 x 10(3) +/- 8% counts/min; 13.4 +/- 2.5 LU) or sera from nonresponders (1.6 x 10(3) +/- 25%/1.5 x 10(3) +/- 20% counts/min; 8.3 +/- 5.9/6.8 +/- 4.8 LU). In contrast to noncultured tumor suspension, IL-2 cultivation induced TIL growth, cytotoxicity, and multicytokine synthesis, and a complete clearance of tumor cells. No significant differences were observed between responders and nonresponders in the in vitro characteristics of tumor/TIL, which include the degree of intratumoral lymphocytic infiltrate, TIL expansion, specific lysis of autologous tumor, phenotype, expansion time, quantity of TIL infused, cytokine release, and degree of tumor aggressiveness. We conclude that clinical response to TIL and IL-2-based immunotherapy is associated with patients' baseline natural immune status. The percentage of circulating NK cells and the presence of serum NK-cell-enhancing factors may serve as potential predictors of response in patients with advanced RCC. The in vitro study of RCC-TIL suggests that activated TIL may provide a synergistic effect to that of administered IL-2 on activation of cellular immune response in situ, rendering a tumor eradication, while the clinical outcome is largely dependent on the pretreatment immune status of patient.

Immunoregulatory properties of ISG15, an interferon-induced cytokine.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

Cytotoxic Activity of CD4+ T Cells against Autologous Tumor Cells

The 51Cr‐release assay is mostly applied to detecting the cytotoxic activity of CD8+ T cells, and little is known about the activity of CD4+ T cells. Therefore, the correlation between the cytotoxic activity of CD4+ or CD8+ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the 51Cr‐release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4+ and CD8+ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin‐2 and immobilized anti‐CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4+ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4+ T cells were increased 67‐fold in comparison with the activities in 4 h assay (range: 5–197). In the case of CD8+ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4+ and CD8+ T cells was calculated as 1.5 in the 20 h assay (range: 0.2‐>7.2) versus 0.4 in the 4 h assay (range: < 0.1–1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h 51Cr‐release assay in all eight cases. These results indicate that CD4+ T cells have cytotoxic activity as strong as that of CD8+ T cells towards autologous tumor cells.

Immune function in marathon runners versus sedentary controls.

Marathon runners (N = 22) who had completed at least seven marathons (X +/- SEM = 23.6 +/- 5.7) and had been training for marathon race events for at least 4 yr (12.3 +/- 1.3) were compared with sedentary controls (N = 18). Although the two groups were of similar age (38.7 +/- 1.5 and 43.9 +/- 2.2 yr, respectively) and height, the marathon runners were significantly leaner and possessed a VO2max 60% higher than that of the controls. Neutrophil counts tended to be lower in the group of marathoners, while other leukocyte and lymphocyte subsets were similar to controls. Mitogen-induced lymphocyte proliferation did not differ between groups. Natural killer cell cytotoxic activity (NKCA) was significantly higher in the marathoners versus controls (373 +/- 38 vs 237 +/- 41 total lytic units, respectively, a 57% difference, P = 0.02). For all subjects combined (N = 40) and within the group of marathon runners (N = 22), percent body fat was negatively correlated with NKCA (r = -0.48, P = 0.002; r = -0.49, P = 0.019, respectively), and age was negatively correlated with Con A-induced lymphocyte proliferation (r = -0.41, P = 0.009; r = -0.53, P = 0.011, respectively). These data indicate that NKCA but not mitogen-induced lymphocyte proliferation is higher in marathon runners relative to sedentary controls.

MIB 1 Immunostaining in Stage I Squamous Cervical Carcinoma: Relationship with Natural Killer Cell Activity

The aim of the study was to investigate the prognostic significance of estimating tumor cell proliferation in stage I cervical squamous carcinoma by analyzing MIB 1 immunostaining with respect to the lesion size, lymphatic spread, and clinical outcome. A possible relationship between MIB 1 index and natural killer activity was also discussed. The medical records of 34 patients with stage I squamous cervical carcinoma who had undergone primary radical surgery at the Institute of Gynecologic and Obstetrics, Ancona University, between 1988 and 1993, were recruited from our series of 57 consecutive cases and reviewed. Thirty-one patients were considered eligible for the study and evaluated for age, demographic characteristics, tumor histologic grade, tumor size, lymphatic spread, and adjuvant radiotherapy. The expression of primary tumor proliferation related to Ki67 antigen was immunohistochemically evaluated by monoclonal MIB 1 antibody (Immunotech, Marseille Cedex, France) on microwave oven-processed formalin-fixed paraffin-embedded tissue. The basal natural killer cell activity of peripheral blood lymphocytes was evaluated against K562 cell line and expressed in lytic units for each patient. The MIB 1 immunostaining was significantly related with tumor size ( P = 0.001) and lymphatic spread ( P = 0.009); in contrast, there was no relationship between grade of histologic differentiation and MIB 1 immunostaining. The Cox proportional hazards analysis showed a significant independent relationship between MIB 1 immunostaining and disease-free survival ( P = 0.004). The analysis of natural cytotoxicity defined a significant inverse relationship between peripheral blood lymphocyte's natural killer activity and tumor MIB 1 immunostaining ( r = -0.07, with P = 0.03). Our data defined the prognostic significance of tumor cell proliferation immunostaining, an interesting parameter correlated with the disease-free survival in locally advanced cervical carcinoma. The relationship between MIB 1 index and natural killer activity is interesting; natural cytotoxicity seems to he altered in the host with respect to the cervical carcinoma characteristics.

Natural killer (NK) cells as effectors of antibody‐dependent cytotoxicity with chimeric antibodies reactive with human squamous‐cell carcinomas of the head and neck

In patients with cancer, antibody-dependent cellular cytotoxicity (ADCC) may be used as a laboratory test or for enhancing immunotherapy with murine monoclonal or chimeric mouse/human anti-tumor antibodies (mMAbs or cMAbs, respectively). We have established an ADCC assay with IgG1 cMAb SF-25, using human squamous-cell carcinoma of the head and neck (SCCHN) cell lines as targets. By flow cytometry, all SCCHN cell lines tested expressed the antigen recognized by cMAb SF-25. Trypsinization of the cell monolayers facilitated binding of cMAb SF-25 to the antigen on the cell surface of SCCHN targets. Using the PCI-50 SCCHN cell line as a target coated with this cMAb at the optimal concentration of 1.0 micrograms/ml, normal human peripheral blood mononuclear cells (PBMC, n = 28) were found to mediate ADCC at a mean level of 283 +/- 42 (SEM) lytic units (LU20/10(7) effector cells). Non-adherent monocyte-depleted PBMC and natural killer (NK) cells purified by negative selection mediated significantly higher levels of ADCC than unseparated PBMC against SCCHN targets. NK cells, defined as CD3-CD56+ cells, could be effectively armed by cMAb SF-25, as confirmed by flow cytometry and ADCC assays. IL2-activated armed NK cells mediated higher levels of ADCC than non-armed NK cells. Binding of cMAb SF-25 to NK cells and their ADCC were enhanced by pre-incubation with polyethylene glycol. Arming of NK cells with chimeric antibodies should be considered in developing novel strategies for treatment of human SCCHN, especially in the adjuvant setting.

Effect of essential fatty acids on natural cytotoxicity in patients with colorectal cancer

Essential fatty acids (EFAs), have been shown to modulate lymphocyte reactivity and destroy various tumour cells in vitro. Natural cytotoxicity, mediated by NK and LAK cells, is believed to play an important anti-cancer role in vivo. The effect of EFAs, given orally as dietary supplementation, on NK and LAK cell cytotoxic activity in patients with localized (n = 10) and advanced (n = 20) colorectal cancer has been studied, using in-vitro 51Cr release cytotoxicity assays with K562 (NK) and DAUDI (LAK) cells. The activity of effector cells was expressed as lytic units. NK cell activity showed no significant change following 15 days ingestion of EFAs in the group with localized cancer, but was significantly reduced in the group with advanced disease and continued to decline, reaching minimal levels following 6 months supplementation (P = 0.017). LAK cell activity showed no overall alteration after 15 days ingestion of EFAs in patients with localized cancer, but in the group with advanced disease, the reduction in the activity occurred at day 15 and steadily declined on prolonged intake, reaching significant minimal levels after 6 months of supplementation (P = 0.05). Cell surface marker analysis (FACS-CD MABs) revealed reduced absolute numbers of CD16+, CD56+ and CD57+ lymphocytes (P < 0.05) in the patients with advanced colorectal cancer. More importantly, the cytotoxicity of NK and LAK cells returned to the pre-supplementation values, 3 months after cessation of EFA intake. Furthermore, there was no alteration in the cytotoxic activity of NK and LAK cells in the control group (advanced colorectal cancer without EFA supplementation) during the 6 months period of evaluation. These results suggest that prolonged EFA supplementation, in the doses used in this study, may have detrimental effects on natural anti-cancer cytotoxic mechanisms in patients with malignant disease.