Inhibition of postbinding target cell lysis and of lymphokine-induced enhancement of human natural killer cell activity by in vitro exposure to ultraviolet B radiation

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to ultraviolet B (uvB) radiation has been shown to inhibit natural killer (NK) cell-mediated cytotoxicity in a dose-dependent fashion. The purpose of this study was to examine the manner by which uvB produced these deleterious effects. Inhibition of NK activity was not due to lethal injury to NK cells since the viability of cell populations enriched for NK activity was greater than 90% with the uvB doses employed. uvB appeared to directly affect NK cells since procedures which removed suppressor mechanisms, such as removal of monocytes and pharmacologic inhibition of the cyclooxygenase pathway, failed to reverse the response. Furthermore, no suppression of activity of unirradiated NK cells could be produced by coincubation of unirradiated NK cells with uv-irradiated NK cells. When the single cell assay for binding and killing was employed to determine at which stage in the lytic sequence inhibition occurred, it was found that binding was normal but lysis of bound targets and the recycling capacity of active NK cells were markedly reduced. At uvB doses above 50 J/m2, both interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were ineffective in augmenting NK cell-mediated cytotoxic reactions after cells had been irradiated with uvB. Furthermore, incubation of NK cells with IFN-alpha prior to irradiation failed to protect against the inhibitory effects. These studies provide evidence that in vitro exposure of NK cells to uvB radiation inhibits their function by a direct nonlethal effect and that this inhibition occurs selectively at the postbinding stage of target cell lysis.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

Morphologic and phenotypic analysis of canine natural killer cells: evidence for T-cell lineage

Canine natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity were studied utilizing a canine thyroid adenocarcinoma cell line and a lymphoblastoid cell line (CT-45S), respectively, as cell targets. Fractionation of peripheral blood mononuclear cells by Percoll discontinuous-gradient centrifugation resulted in a six- to sevenfold enrichment in large granular lymphocytes (LGL) in parallel with a twofold increase in NK activity (%specific lysis) in low-density fractions. Further enrichment in LGL (78 +/- 6%) and NK activity (threefold increase) was obtained by lytic treatment of low-density fractions 2 and 3 with monoclonal antibody WIG4. By means of cytolytic treatment with additional monoclonal antibodies the phenotype of canine NK cells was determined as Dly-1+, Dly-6+, 1A1+, E-11+, DT-2-, WIG4-. Some NK cells were also Ia+. NK activity was relatively radioresistant with 40% specific lysis even after irradiation with 40 Gy. Among the populations examined, the highest NK activity was found in peripheral blood mononuclear cells, followed by splenic mononuclear cells and bone marrow mononuclear cells. These results indicate that canine NK cells have the morphology of LGL, are relatively radioresistant, and express cell surface antigens suggesting a T-cell lineage.     

Regulation of the natural killer activity of lymphocytes from healthy donors and patients with chronic lympholeukemia through the interaction of T- and non-T cells

The role of T- and B-cell cooperative interaction in the regulation of natural killer (NK) activity in human peripheral blood lymphocytes was studied. It has been shown that preincubation of normal donor mononuclear cells (MNC) for 48 h is followed by the loss of NK activity, while the incubation of the isolated T- and non-T-cell subsets does not lead to an analogous fall in the killer lymphocyte function. NK activity of MNC and isolated lymphocyte subsets in normal donors is shown to exceed that of CLL patients. The absence of preincubation effect on NK activity level of MNC, T- and non-T-cells in CLL patients has been also found. The findings obtained suggest that as a result of T- and B-cell interaction during preincubation differentiation of young T lymphocytes with NK cellular properties takes place. It is followed by the loss in NK activity. B-cell defect in CLL patients might cause the absence of preincubation effect on NK activity of T cells.     

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

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

CD8+ natural killer cells have a potential of a sensitive and reliable biodosimetric marker in vitro

The aim of our work was to evaluate peripheral blood lymphocyte subsets as in vitro indicators of the received dose of ionizing radiation (biodosimetric markers) in the range of 3-20 Gy and to determine the appropriate time interval, during which a dose-dependent induction of apoptosis occurs upon gamma irradiation. In lymphocyte subsets characterized by double color surface immunophenotyping, four-color flow cytometry was used for visualizing cell death-associated increase in superficial phosphatidylserine exposure and cytoplasmic membrane permeability by fluorinated Annexin V and propidium iodide, respectively. No differences between sham-treated and lethal dose (7 Gy)-irradiated samples were observed upon 6 h cultivation in vitro. Ten and 18 h later, about 50 % of lymphocytes were apoptotic, but only the minority of them was in the late apoptotic phase. The only difference in radioresistance of the CD4(+)CD8(-) and CD4(-)CD8(+) lymphocyte subsets was seen upon 2-day cultivation when huge depletion of intact cells and prevalence of the late apoptotic population became obvious. A dose-dependence study in 16 and 48 h cultures confirmed the effectiveness of major T cell subsets as biodosimetric indicators. On the other hand, the minor CD8(+) subset of natural killer (NK) cells has been identified as a radiosensitive lymphocyte population the disappearance of which correlated with the received dose. We demonstrated that the CD3(-)CD8(+)NK subset can be used as a lethal/sublethal dose discriminator to 16 h cultivation. In addition, our data indicate that two-day cultivation followed by CD3/CD8 expression analysis in an intact lymphocyte population may provide a clue for low dosage biodosimetry.     

Mechanism of inhibition of human natural killer activity by ultraviolet radiation

Ultraviolet radiation (UVR) has been shown to inhibit various immune functions in vivo and in vitro. We have confirmed that UVR inhibits human natural killer (NK) activity in vitro and have shown that UVR inhibits human ADCC. In this report, the mechanism by which UVR inhibits NK function was investigated by analyzing the stage at which the inhibiting activity occurs and the ability of the NK cells to release cytotoxic factors previously shown to be involved in CMC. Single cell assays in Agarose revealed that inhibition of NK activity was localized at the postbinding lethal hit stage rather than the initial recognition or binding stage of lysis. We then examined whether UV-treated cells were able to release cytotoxic factors after stimulation with target cells. As expected, stimulated cells released cytotoxic factors, yet, surprisingly, these factors were also released in the absence of stimulator cells. The spontaneous release was detectable in the supernatants as early as 30 min after UV irradiation. The lytic material examined in 48- to 72-hr viability assays was not NK specific, because lysis was obtained with a wide range of NK sensitive and resistant target cells. These results demonstrate that UVR does not alter the capacity of the cells to secrete cytotoxic material, but in fact enhances its release. Several possible mechanisms are proposed to explain the UVR-induced NK inhibition.     

Kupffer cells modulate natural killer cell activity in vitro by producing prostaglandins

The effect of Kupffer cells on natural killer (NK) cell-mediated cytotoxicity was examined. Kupffer cells prepared from rat liver suppressed NK activity against K562 cells and other tumor cell lines through a soluble factor secreted into the culture supernatant. When human peripheral blood mononuclear cells were incubated with the Kupffer cell-culture supernatant, a significant reduction of the cytotoxic activity was observed in the 6-hr chromium-release assay. This activity was dose dependent and was evident at various effector/target cell ratios. Lipopolysaccharide stimulated generation of the suppressive factor released from Kupffer cells in a dose-dependent manner. Suppression of the NK activity was observed when the Kupffer cell-culture supernatant was present in the assay system, whereas pretreatment of effector/target cells with the supernatant had minimal inhibitory effects. Autologous monocytes in human peripheral mononuclear cells were not related to this suppression. The suppressive factor in the fraction had a molecular weight below 10,000. Indomethacin, an inhibitor of prostaglandin synthesis, ameliorated the suppressive effects. These results suggest that Kupffer cells may modulate NK activity by producing PGs (E1, E2, and F2 alpha).     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Natural killer cell-mediated antitumor reactivity of rhesus monkeys

We have analyzed natural killer (NK) cell-mediated antitumor activity or peripheral blood mononuclear cells of rhesus monkeys. All monkeys displayed significant NK cell cytolytic activity against the human tumor cell lines K-562, Daudi and CEM in a short-term (3 h) 51Cr-release assay. Similar to NK cells described in other species, the cytotoxic cells of monkeys were relatively nonadherent to nylon wool columns, exhibited low density after separation on discontinuous Percoll density gradients, and displayed large granular lymphocyte (LGL) morphology. Analysis of the mechanism of NK cell cytotoxicity of rhesus monkeys demonstrated that on the average, 7.1% (range: 3.1-13.2%) of lymphocytes bound to K-562 tumor, and that approximately 14.8% (range: 7.9-26.3%) of these tumor-binding cells (TBC) were cytolytically active. Examination of TBC on cytocentrifuge slides indicated that the majority of binders displayed LGL morphology. The cytotoxic reaction mediated by monkey NK cells exhibited Michaelis-Menten kinetics pattern; the maximum rate of lysis (Vmax) of K-562 was found to be 1-2 X 10(4) following 3 h of incubation. Using similar culture conditions, the recycling capacity of NK cells of this species was estimated at 2-6 times. Finally, it was observed that the NK cell activity of most monkeys could be potentiated following in vitro exposure to the biological response modifier, interleukin-2.     

Inhibition of human natural killer cell activity by platelet-derived growth factor

The present report demonstrates that the naturally occurring biologic substance, platelet-derived growth factor (PDGF), substantially inhibits human natural killer (NK) cell activity. More precisely, pretreatment of peripheral blood mononuclear cells for 2 h with nanogram amounts of either partially purified PDGF or highly purified PDGF significantly inhibited peripheral blood NK cell activity (cytotoxicity) in a dose-dependent manner as measured against the NK-sensitive target, K-562. Furthermore, pretreatment of purified NK cells for 2 h with nanogram amounts of purified PDGF also resulted in a significant, dose-dependent inhibition of human NK cell activity (cytotoxicity), as mediated by positively selected, B73.1+ human NK cells sorted on a fluorescence-activated cell sorter. In addition to the inhibition of NK-mediated cytotoxicity, nanogram amounts of purified PDGF also significantly inhibited the single-cell binding of B73.1+ human NK cells to the NK-sensitive target K-562, as determined by routine single-cell-binding assays (i.e. conjugate formation). The implications of these findings are discussed.     

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.     

Suppressor cells for natural killer activity in carcinomatous pleural effusions of cancer patients

Summary Carcinomatous pleural effusions of 25 of 32 patients with lung cancer, which had markedly low or no natural killer (NK) activity against K562 cells in a 4 h chromium release assay, contained cells capable of suppressing the lytic function of blood NK cells from normal donors and cancer patients. Suppressor cells were found to be Sephadex G-10- and serum coated plastic dish-adherent monocyte/macrophages in 21 of 25 patients and nylon wool-nonadherent lymphocytes in the other four cases. Nonmalignant pleural effusions did not contain any type of suppressor cells. Twenty-four-hour preincubation of suppressor cells with effector cells was required for mediation of the suppressor function. Neither culture supernatants of effusion cells and NK cells nor effusion supernatants suppressed NK activity. The presence of indomethacin during the preincubation and cytotoxicity assay did not abrogate suppressor function. Suppressor cells did not reduce the number of lymphocyte/K562 conjugates. Contaminating tumor cells were not responsible for the suppression of cytotoxic activity. NK cells precultured with suppressor cells were not able to show cytotoxic function even after removal of the suppressor cells. When effusion mononuclear cells were passed through a Sephadex G-10 column and then preincubated for 24 h, these cells showed a significant increase in NK activity. The results suggest that carcinomatous pleural effusions contain at least two classes of suppressor cells for NK activity, monocyte/macrophages, and nylon wool-nonadherent lymphocytes, which could be one of the causes of impaired NK activity in carcinomatous pleural effusions.     

Impaired natural killer cell function in hemophiliacs with or without continuous substitution

In the present study, 28 hemophiliacs substituted continuously and 5 hemophiliacs who had received almost no blood products were investigated. Cells of OKT 3+, OKT 4+, and OKT 8+ subsets were counted. Percoll separated fractions of peripheral blood mononuclear cells were examined by morphological criteria and were tested for NK cell activity. We found that the NK cell activity of both groups of hemophiliacs was decreased on testing Ficoll separated cells or low density Percoll separated cells. Normal NK cell activity was found in medium density cells of hemophiliacs. Two possible explanations are discussed: first, the NK cell activity may be suppressed in hemophiliacs and secondly, there may be a block in maturation of NK cell activity. It is unlikely that chronic substitution by blood products counts for these alterations. The possible role of chronic infections is discussed.     

Inhibition of natural killer cell activity by IgA

The in vitro effect of IgA on natural killer (NK) activities of human peripheral blood mononuclear cells was investigated. Purified myeloma polymeric IgA2 (pIgA2) and secretory IgA (S-IgA) from human colostrum inhibited NK activity, while myeloma polymeric IgA1 (pIgA1), monomeric IgA1 (mIgA1), IgG, and IgM were ineffective. Inhibition was proportional to the concentration of pIgA2 (0.125-1 mg/ml) and was observed after as little as 1 hr of incubation at various effector to K562 target cell ratios. pIgA2 and S-IgA also inhibited NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells, but did not interfere with effector-target cell binding. The inhibitory effect was slightly diminished after 24 hr culture in pIgA2-free medium. Inhibition of cytotoxicity was not due to direct toxicity on lymphoid cells by IgA because PBL treated with pokeweed mitogen in the presence of pIgA2 or S-IgA differentiated into immunoglobulin-producing cells. Viability after 24 hr of preculture with pIgA2 and S-IgA was comparable to that of untreated control cells. Morphological examination of effector cells cultured with pIgA2 or S-IgA showed a decrease in the number of granules, and the formation of cytoplasmic vacuoles. These morphological changes appeared to coincide with the depressed cytotoxicity of NK cells. The results demonstrate that purified pIgA2 and S-IgA have significant immunomodulatory effects on human NK activity.     

Human killer cells and natural killer cells: distinct subpopulations of Fc receptor-bearing lymphocytes

Unstimulated human peripheral blood lymphocytes were depleted of K cells, which mediate antibody-dependent cellular cytotoxicity (ADCC) without removing NK cells, which mediate natural killing (NK). K cell depletion was achieved by buoyant centrifugation removal of lymphocytes that bound to glutaraldehyde-treated P815-AB cells at high lymphocyte-to-target ratios. Likewise, NK cells were removed with glutaraldehyde-treated K562 cells without removing K cells. Furthermore, both cytotoxic cell populations were observed directly in one agarose single-cell cytotoxic assay (ASCA) using P815-AB and K562 cells simultaneously as target cells. Moreover, the percentage of total cytotoxic cells was equal to the sum of the percentage of K and NK cells observed in separate ASCA. Collectively, these results indicate that K cells and NK cells are distinct subsets of FcR-bearing lymphocytes. One subset, K cells, has more avid Fc receptors (fcR) than NK cells and are 'activated' via thier FcR to kill antibody-coated target cells. The second subset, NK cells, have less avid FcR and are not 'activated' through their FcR to kill antibody-coated target cells.     

Kinetics of interleukin-2 induced changes in rigidity of human natural killer cells

The success of adoptive immunotherapy is dependent in part on the successful delivery of effector cells to the tumor and the expression of cellular activities, such as adhesion, extravasation, and cytotoxic activity of the effector cells in the tumor. The structural rigidity of the effector cell is an important determinant of these functions. The present study was designed to quantify the changes in cellular rigidity and cytotoxic activity of human natural killer (NK) cells in the presence or absence of interleukin-2 (IL-2). Micropipet aspiration was used to measure the resistance of NK cells to an imposed external deformation. Homogeneous suspensions of NK cells were activated with 1000 U/mL of recombinant IL-2 in vitro and tested for cellular rigidity from 0 to 96 h post stimulation. The IL-2 activated cells increased their rigidity within 24 h and maintained it at this level for 96 h. Prolonged incubation of cells in IL-2 (14 d) resulted in a consistently high rigidity, which was further increased on starvation of the cells from IL-2. The increased rigidity of these cells was maintained throughout 96 h of IL-2 deprivation, although significant relaxation of rigidity was observed between 48 and 96 h. The relaxation of rigidity was associated with an increase in the number of nonviable cells. Reintroduction of IL-2 for 24 h to a culture of NK cells depleted of IL-2 for 48 h did not restore the cells to the predepletion level of rigidity. Cytotoxic activity of the activated NK cells following removal of IL-2 decreased to about 60% of the control activity within 24 h and continued through 72 h postdeprivation. These findings suggest that the initial activation of human NK cells by IL-2 will produce a relatively rapid increase in rigidity that may cause entrapment of these cells in small capillaries in vivo and that removal of IL-2 will produce an additional increase in rigidity, which is associated with decreased functional activity.     

Effects of in vivo ultrasound hyperthermia on natural killer cell cytotoxicity in the hamster

The effects of in vivo ultrasound irradiation of the spleen on immunological functions were assessed with an in vitro natural killer (NK) cell cytotoxic assay. Anesthetized hamsters were exposed to 1 MHz ultrasound at intensity levels currently being used clinically for therapeutic diathermy and hyperthermia (1-5 W/cm2, for 500 sec with constant beam scanning). Hyperthermic levels in the spleen ranged from 38-43 degrees C. Significant depression of natural killer (NK) cell activity was seen 4 h after spleen irradiation as compared to sham irradiated and normal animals. A return towards normal levels was observed in experimental groups at 24 h after exposure. Sham and normal animals were not significantly different in NK activity, indicating no significant stress-related immunosuppressive effects due to handling. Differential leukocyte counts taken for each exposure condition showed significant lymphopenia at 4, 8, and 16 h after exposure, near normal levels at 24 h, and complete recovery by 48 h. The number of circulating mononuclear cells at 4 h showed a dose-related suppression as the exposure intensities were increased.     

Kinetics of interleukin-2 induced changes in rigidity of human natural killer cells.

The success of adoptive immunotherapy is dependent in part on the successful delivery of effector cells to the tumor and the expression of cellular activities, such as adhesion, extravasation, and cytotoxic activity of the effector cells in the tumor. The structural rigidity of the effector cell is an important determinant of these functions. The present study was designed to quantify the changes in cellular rigidity and cytotoxic activity of human natural killer (NK) cells in the presence or absence of interleukin-2 (IL-2). Micropipet aspiration was used to measure the resistance of NK cells to an imposed external deformation. Homogeneous suspensions of NK cells were activated with 1000 U/mL of recombinant IL-2 in vitro and tested for cellular rigidity from 0 to 96 h post stimulation. The IL-2 activated cells increased their rigidity within 24 h and maintained it at this level for 96 h. Prolonged incubation of cells in IL-2 (14 d) resulted in a consistently high rigidity, which was further increased on starvation of the cells from IL-2. The increased rigidity of these cells was maintained throughout 96 h of IL-2 deprivation, although significant relaxation of rigidity was observed between 48 and 96 h. The relaxation of rigidity was associated with an increase in the number of nonviable cells. Reintroduction of IL-2 for 24 h to a culture of NK cells depleted of IL-2 for 48 h did not restore the cells to the pre-depletion level of rigidity. Cytotoxic activity of the activated NK cells following removal of IL-2 decreased to about 60% of the control activity within 24 h and continued through 72 h post-deprivation. These findings suggest that the initial activation of human NK cells by IL-2 will produce a relatively rapid increase in rigidity that may cause entrapment of these cells in small capillaries in vivo and that removal of IL-2 will produce an additional increase in rigidity, which is associated with decreased functional activity.     

Phenotypic studies of natural killer cell subsets in human transporter associated with antigen processing deficiency

Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56(bright) cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, (51)Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56(bright) NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56(dim)CD16(-) NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56(bright) NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.