In vitro and in vivo activity of murine lymphokine-activated killer cells after cryopreservation

The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B-cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.     

Differential effect of cryopreservation on natural killer cell and lymphokine-activated killer cell activities

The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.     

The role of natural killer cells and lymphokine activated killer cells in the pathogenesis of hepatic injury in hepatitis A [corrected]

In order to clarify the mechanism of liver damage in hepatitis A, we studied the role of Natural Killer (NK) cells and Lymphokine Activated Killer (LAK) cells in non-specific immunological reactions using hepatitis A (HAV) infected cells (JTC-12.P3 cell) using the 51Cr release assay. No significant difference in specific cytotoxicity was observed between uninfected cells (JTC) and HAV-infected cells (JTC-HAV) to fresh and Poly I:Cor rIL-2 pretreated peripheral blood mononuclear cells (PBMC) from healthy donors or patients with acute hepatitis A. But in an experiment employing fresh and rIL-2 pretreated PBMC from cynomolgus monkey as effectors, a significant difference in NK and LAK sensitivity between JTC and JTC-HAV was noticed. Cytotoxicity assay was then carried out using as effectors fresh or rIL-2 pretreated cells of B, NK and T cell fractions obtained after separation of PBMC from monkey by the E-rosette formation method. Both fresh and rIL-2 pretreated B/NK cells showed significantly higher cytotoxicity for JTC-HAV than JTC, but neither fresh or rIL-2 pretreated T cells showed cytotoxicity for JTC-HAV or JTC. These results imply that both NK and LAK cells may play a part in the mechanism of hepatocellular injury.     

Lymphokine-activated killer cells. Analysis of progenitors and effectors

IL-2 has been examined for its ability to regulate lymphokine-activated killer (LAK) activity. IL-2 is a potent activator of cytolytic activity against a wide array of tumor cells, including those from fresh autologous and allogeneic tumors. Using subpopulations of lymphoid cells that were separated on Percoll density gradients, and subsequently purified by immunoadsorbance, studies were performed to examine the phenotypes of progenitor and effector cells of human LAK cells and to compare them with the phenotype of activated NK cells. From these studies, it was evident that several lymphoid subsets, including CD3+, CDw16- and CD3-, CDw16+ cells could mediate LAK lysis of fresh tumor cells. Our examination of the kinetics of activation revealed that CDw16+, NKH1+ (NK-active) cells were maximally activated by 1-2 d. In contrast, CD3+ cells appeared not to achieve maximal cytolytic activity against fresh and cultured tumor cells until days 2-3. Using limiting-dilution frequency analysis, we showed that a large percentage of cytolytically active progenitors was present among the CDw16+, NKH1+ cells. The progenitor and effector cell frequencies appear to be 10-50 times higher in these populations compared to CD3+ cells. In addition, the selective blockage by mAb to the CD3 determinant of the T cell receptor complex indicated that these two effector cell phenotypes relied on different receptors to mediate their cytotoxic activity against tumor cells. Therefore, the accumulated data suggest that there is not a single unique progenitor of LAK activity, but rather that multiple subsets of lymphocytes become cytotoxic in response to IL-2. However, the NK cell population forms the largest single component of LAK cell activity in human peripheral blood.     

A novel 120-kD surface antigen expressed by a subset of human lymphocytes. Evidence that lymphokine-activated killer cells express this molecule and use it in their effector function

A human cell clone (SF-16) displaying strong cytolytic activity against fresh tumor target cells was used for production of murine mAbs against surface antigens expressed by lymphokine-activated killer (LAK) cells and their peripheral blood precursors. The preliminary screening of hybridoma supernatants was performed according to the ability to bind SF-16 cells. Selected mAbs were further analyzed for their reactivity with several T and B cell lines and with peripheral blood T and non-T cell populations. A selected mAb, termed anti-LAK-1, only reacted with some T cell lines and with 15-30% of PBMC. Approximately 10-15% E- rosetting (T) cells and 40-50% E-rosette-negative cells were LAK-1+, as determined by cytofluorometric analysis. As the fluorescence distribution of LAK-1 antigen was clearly bimodal, LAK-1+ and LAK-1- cells could be separated by FACS. Positive cells were composed of large granular lymphocytes (LGL), whereas negative cells were mostly small lymphocytes and monocytes without LGL. After culture in rIL-2, purified LAK-1+ (but not LAK-1-) cells acquired the ability to lyse NK-resistant fresh melanoma target cells. In addition, only the LAK-1+ fraction of PBMC cultured for 5 d in rIL-2 lysed fresh tumor targets, thus indicating that the LAK-1 antigen is expressed also on LAK effector cells. Unlike some other LGL/NK cell markers, LAK-1 antigen is characterized by a stable expression: thus, LAK-1+ cell populations cultured for up to 20 d in rIL-2 maintained the LAK-1 antigen expression, whereas HNK-1 and, partially, CD16 were lost. Finally the cytolytic activity of LAK effector cells generated from PBMC cultured for 3 d in rIL-2 was susceptible to inhibition by the anti-LAK-1 mAb.     

Dissection of the lymphokine-activated killer phenomenon. Relative contribution of peripheral blood natural killer cells and T lymphocytes to cytolysis

In vitro culture of human peripheral blood lymphocytes in IL-2 results in the generation of cytotoxic cells that can lyse fresh and cultured solid tumor cells, as well as hematopoietic tumor cell lines, without deliberate immunization or MHC restriction. This has been referred to as the lymphokine activated killer (LAK) phenomenon. Here, we show that the majority of this activity is mediated by NK cells that express the Leu-19 (NKH-1) antigen, but do not express CD3. The precursor of this effector population also expressed the phenotype CD3-, Leu-19+. Peripheral blood CD3+ T lymphocytes contributed little to the LAK phenomenon, although low levels of non-MHC restricted cytotoxicity against hematopoietic tumor cell targets were mediated by a subset of CD3+ T lymphocytes that coexpressed the Leu-19 antigen. These studies clearly indicated that the LAK phenomenon is not mediated by a unique LAK cell, but is mediated mainly by IL-2-activated peripheral blood NK cells.     

Lymphokine-activated killer cells in rats. III. A simple method for the purification of large granular lymphocytes and their rapid expansion and conversion into lymphokine-activated killer cells

A simple method for the purification and rapid expansion of large granular lymphocytes into cells with efficient broad antitumor cytotoxicity after stimulation by human rIL-2 is described. Nylon-wool nonadherent splenic mononuclear leukocytes from Fischer 344 rats were cultured in medium containing 1,000 U/ml rIL-2. The initial response of a small subpopulation of cells (less than 2%) to rIL-2 was their adherence to the plastic surface. This response was noted as soon as 2 h after addition of rIL-2. 2-h rIL-2-activated plastic adherent lymphocytes were 90-98% LGL, expressed surface markers characteristic of rat NK cells (OX8 [CD8]+, asialo GM1, laminin+, OX19 [CD5]-, R1-3B3 [CD5]-, W3/25 [CD4]-, OX39 [CD25]-, Ia-, and Ig-), and expressed very high levels of cytotoxicity against YAC-1 target cells. In addition to the above markers, plastic-adherent LGLs obtained at 24, 48, or 72 h progressively expressed Ia surface antigens, but were not phagocytic and contained less than 1% monocytes/macrophages by morphology. When 24- or 48-h plastic-adherent LGL/NK cells were cultured over 3-4 d in rIL-2, the cells expanded between 30- and 100-fold, reaching densities between 2-3 X 10(6) cells/ml. These rapidly expanding LGL/NK cells also generated very high levels of LAK activity (including lysis of fresh NK-resistant solid tumor cells), expressed a phenotype characteristic of activated rat NK/LAK cells, and incorporated [3H]TdR into DNA. This technique not only provides a novel method for the purification of LGL/NK cells for in vitro studies but also provides a means for the rapid expansion of highly purified cells with high levels of broad antitumor (LAK) cytotoxicity.     

Defective natural immunity: an early manifestation of human immunodeficiency virus infection

Cytotoxicity mediated by natural killer (NK) and lymphokine-activated killer (LAK) cells may be of significance in host defense against viral infections. This study included 347 patients infected with human immunodeficiency syndrome virus (HIV) type 1 and 110 controls. The NK cell activity, either unstimulated or stimulated with interferon-alpha (IFN-alpha) or interleukin-2 (IL-2), and the LAK cell activity were suppressed in patients, but the NK/LAK cell activity did not differ between patients with AIDS and patients without AIDS. However, the IFN- alpha-stimulated NK cell activity and LAK cell activity were reduced in patients with symptoms of HIV disease (CDCIV) when compared with asymptomatic patients (CDCII+III). When the data were analyzed by multiple linear regression, the percentage of CD4+ cells had a positive effect on these two parameters in patients without AIDS, whereas the percentage of CD4+ cells had no significant effect on unstimulated and IL-2-stimulated NK cell activity in these patients. In controls and AIDS patients, the percentage of CD4+ cells had no effect on NK/LAK cell activity in multiple linear models. The total number of CD16+ cells was low in patients compared to controls, whereas the percentages of CD16+, CD56+, and CD16+CD56+ were either normal or elevated. Therefore, the decrease in NK cell subpopulations did not contribute to the observed depression in NK/LAK cell activity in vitro. It is concluded that natural immunity is suppressed in HIV-seropositive patients primarily because of a qualitative defect of the NK/LAK cells. This qualitative defect includes a reduced responsiveness to IFN-alpha, which is progressive until the onset of symptoms, and possibly related to the loss of CD4+ cells.     

Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes

Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13 allogeneic fresh tumors. Culture of PBL in IL-2 for 2-3 d was required for the lymphokine activated killers (LAK) to be expressed, and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets developed in parallel. Autologous IL-2 was functional in LAK activation, as well as interferon-depleted IL-2 preparations. Irradiation of responder PBL before culture in IL-2 prevented LAK development. Precursors of LAK were present in PBL depleted of adherent cells and in NK-void thoracic duct lymphocytes, suggesting that the precursor is neither a monocyte nor an NK cell. LAK effectors expressed the serologically defined T cell markers of OKT.3, Leu-1, and 4F2, but did not express the monocyte/NK marker OKM-1. Lysis of autologous fresh solid tumors by LAK from cancer patients' PBL was demonstrated in 85% of the patient-fresh tumor combinations. Our data present evidence that the LAK system is a phenomenon distinct from either NK or CTL systems that probably accounts for a large number of reported nonclassical cytotoxicities. The biological role of LAK cells is not yet known, although it is suggested that these cells may be functional in immune surveillance against human solid tumors.     

Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells

To study the cellular structures involved in NK and lymphokine-activated killer (LAK) cell function, we have produced a panel of mAbs that modulate the cytolytic function of a population of cells with LAK activity that derive from large granular lymphocyte (LGL)/NK cells (adherent LAK [A-LAK] cells). In this report, we describe an mAb (3.2.3; IgG1k) that recognizes a triggering structure that is expressed on rat LGL/NK cells and A-LAK cells. This epitope is also expressed on polymorphonuclear leukocytes (PMN). The expression of the epitope identified by mAb 3.2.3 increased progressively on A-LAK cells after culture in the presence of rIL-2. mAb 3.2.3 enhanced the cytolytic activity of NK and A-LAK cells against FcR+ target cells, but not FcR- target cells. However, this effect was not induced by F(ab')2 fragments of 3.2.3. This antibody also induced the release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzy esteresterase by A-LAK cells. These data suggest that the epitope identified by mAb 3.2.3 is on a triggering structure expressed on rat NK cells and A-LAK cells. The expression of the epitope recognized by mAb 3.2.3 on LGL/NK cells and PMN suggests that this structure may be analogous to that identified by the anti-CD16 (-FcR) mAbs. However, the molecule immunoprecipitated by mAb 3.2.3 was a 60-kD dimer composed of two 30-kD chains. These data suggest that mAb 3.2.3 recognizes a unique triggering structure. As mAb 3.2.3 is the first antibody recognizing a determinant with functional significance, selectively expressed on both rat NK cells and A-LAK cells, it will be a useful tool for the study of NK cell ontogeny and function, and the development of cells with LAK activity from the NK cell compartment.     

Response of resting human peripheral blood natural killer cells to interleukin 2

The present study shows that recombinant interleukin 2 (IL-2) purified to homogeneity induces a rapid and potent enhancement of spontaneous cytotoxicity of human peripheral blood lymphocytes. The cells mediating cytotoxicity after 18-h treatment with IL-2 have surface markers of natural killer (NK) cells and are generated from the peripheral blood subset containing spontaneous cytotoxic cells. A parallel production of gamma interferon (IFN-gamma) is induced by recombinant IL-2 (rIL-2), and NK cells appear to be the major producer cells, whereas T cells are unable to produce IFN-gamma under these experimental conditions. However, the kinetics of the enhancement of cytotoxicity are faster than those of IFN-gamma production, and monoclonal anti-IFN-gamma antibodies do not suppress this effect, making it unlikely that the IFN- gamma produced is responsible for the enhancement. The enhancement of NK cell activity induced by rIL-2 precedes any proliferative response of the lymphocytes, which is instead observed in longer-term cultures of both NK and T cells.     

Enhancement of natural killer function through activation of the T11 E rosette receptor.

Natural killer (NK) cells, which represent a small fraction of normal peripheral blood mononuclear cells, were purified by immunofluorescent cell sorting of NKH1+ cells. cytotoxicity of NKH1+ cells could be enhanced through activation by monoclonal antibodies (anti-T11(2) and anti-T11(3)) specific for epitopes of the sheep erythrocyte receptor or by recombinant interleukin-2 (rIL-2). After 18 h, incubation with both anti-T11(2/3) and rIL-2 resulted in similar levels of enhanced cytotoxicity against NK-resistant as well as NK-sensitive targets. Before and after induction, cytotoxicity was found predominantly within the NKH1+ population. These results suggest that several distinct mechanisms may be capable of enhancing NK activity and that the cells responsible for lymphokine-activated killing are likely to be the same population capable of spontaneous or natural killing before activation in vitro.     

Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor

TIL from metastatic melanoma proliferated by greater than 1,000-fold (840-3,675, mean 1,543) after 6 wk in culture of mixtures of TIL and tumor cells with rIL-2 alone. Cytolysis was restricted to autologous tumor cells. CD8+ T cells were the predominant population of TIL before and after expansion, and were primarily responsible for autologous tumor-specific CTL activity. No other rIL-2-activated lymphocytes from peripheral blood, lymph nodes with melanoma metastasis, or TIL from sarcoma or renal cell carcinoma had autologous tumor-specific CTL activity. There were few or no CD16+ NK cells in TIL from metastatic melanoma before or after incubation with rIL-2, respectively. However, TIL from sarcoma or renal cell carcinoma contained a substantial proportion of CD3-CD16+ NK cells, which increased in number in culture with rIL-2. Purified CD16+ NK cells as well as CD3+CD16- T cells from rIL-2-activated TIL of renal cell carcinoma displayed MHC-nonrestricted cytotoxicity. At the clonal level as determined by limiting dilution, 8 of 10 clones from melanoma TIL displayed cytotoxicity restricted to autologous tumor cells, while all 13 clones from renal cancer TIL equally lysed autologous and allogeneic tumor cells. Anti-T cell receptor (TCR)-alpha/beta(WT31) mAb as well as anti-CD3 mAb inhibited autologous melanoma cell-specific CTL activity mediated by rIL-2-activated TIL at the effector phase. These two mAbs also inhibited rIL-2-dependent proliferation of these TIL when added to the culture. Pretreatment of fresh melanoma cells with mAb to MHC antigens followed by washing inhibited specific CTL activity. These results suggest that both TCR-alpha/beta on effector TIL and MHC antigens on fresh tumor cells are involved in the specific immune-recognition. After reaching maximum propagation, TIL from metastatic melanoma responded poorly to rIL-2 alone. However, stimulation with fresh autologous melanoma cells restored both CTL activity and proliferation in response to rIL-2. The latter is associated with IL-2 receptor (Tac antigen) expression on the surface. These results indicate that TIL from metastatic melanomas may have unique characteristics different from lymphocytes obtained from the other sources, and may contain precursor CTL sensitized in vivo to autologous tumor cells, and thus can be propagated in larger numbers with rIL-2 alone while retaining autologous tumor-specific CTL activity.     

Studies of lymphokine-activated killer (LAK) cells. I. Evidence using novel monoclonal antibodies that most human LAK precursor cells share a common surface marker

Separation of LAK precursor (LAKp) cells (as defined by LAK effector generation after incubation with IL-2 for 7 d) from cells with NK activity/LGL morphology was achieved on Percoll gradients using a longer, slower centrifugation than that used for optimal NK enrichment. mAb were generated using the various Percoll fractions as the immunizing cells and used for separation and depletion studies. Two mAbs DM-1 (IgM,k) and DM-2 (IgM,k) recognizing 2-15% and 15-30% of PBL, respectively, abrogated a large proportion of LAK generative potential after complement depletion, but had little effect on NK or LAK effector activity. Cell sorting experiments indicated that the majority of LAKp cells are found within the DM-1+ population and that DM-1+ cells are not simply an accessory cell required for LAKp generation. Further, these two mAbs do not recognize cells that are responsible for generating cytotoxicity during MLC or co-culture with the PR-1 EBV lymphoblastoid cell line. Western blot analysis indicated that DM-1 and DM-2 recognize a 38,000 and 44,000 dalton moiety, respectively. The frequency of cells bearing these antigens and the intensity of cell surface staining decreased during the 7-d culture period, suggesting that these antibodies recognize determinants found only at the precursor level. These findings indicate that cells other than NK effectors or mature T cells are capable of generating a LAK cell response. These LAK precursor cells share a common differentiation surface antigen and are different from AK or antigen-specific CTL precursors. The possibility exists that these cells are identical to, or include, the NK precursor cell.     

Antitumor activity of killer cells stimulated with both interleukin-2 and interleukin-12 on mouse glioma cells.

Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor or cytotoxic lymphocyte maturation factor, has potential for use as an immunomodulator in cancer therapy because it significantly retards the growth of some murine tumors. In this study, we analyzed the antitumor effects of lymphocytes stimulated in vitro with both recombinant IL-2 (rIL-2) and rIL-12. When IL-12 was added to mouse splenocytes (SPCs) or human peripheral blood monocytes (PBMCs) incubated with IL-2 for > 4 days, IL-2-induced cytotoxicity against glioma cells was augmented. In contrast, IL-12 inhibited IL-2-induced lymphokine-activated killer (LAK) cell activity when added concurrently to cultures. The concentration of IL-10 induced by IL-12 increased in the supernatant of human PBMCs costimulated with IL-2 and IL-12. Endogenous IL-10 augmented the cytotoxicity of SPCs stimulated with IL-2 or IL-12 or both. However, tumor-bearing mice treated with PBMCs stimulated with both IL-2 and IL-12 did not survive longer than those treated with PBMCs stimulated with IL-2 alone (LAK cells).     

On the heterogeneity of murine natural killer cells

The heterogeneity of cells capable exerting spontaneous cytotoxicity in vitro was explored using antisera to several genetically determined surface markers on mouse lymphocytes. Four phenotypes of cells derived either from fresh or cultured murine lymphoid tissue were found to exert natural killer (NK) activity in vitro. One affector cell subset, termed NKI cells, had the serological phenotype of Thy-1-, Lyt-2-, Qa5+, and lysed measles virus persistently infected target cells (HeLa- Ms) but not P815 mastocytoma cells. It corresponds with the NK cells described in most systems in which lymphoma targets are commonly used. A second subset, with the same target cell specificity, termed NKT is a thymus-independent cell with the phenotype Thy-1+, Lyt-2-, Qa-5+, Ly- 5+. A third subset of NK cells, termed T killer (TK) cells deriving from cultures of conventional but not nude mouse spleens, mediated spontaneous cytotoxicity of P815 mastocytoma cells, but not of virus- infected targets. It has a phenotype of Thy-1+, Lyt-2+, Qa-5-, Ly-5+, apparently identical with that of conventional, antigen-specific cytotoxic T lymphocytes. The fourth phenotype of NK cells, termed NKM, derived primarily from cultures of bone marrow, is cytotoxic for HeLa- measles but not P815, and expresses only Ly-5+ among the various markers tested. Beige mice possess normal TK and NKM activities, but had normal NKI, NKT as well as NKM activity. All NK cell subsets express the Ly-5 surface marker. The existence of four phenotypically distinct NK effector cells was strengthened by studies on selective regulation of their activity by two different biological factors. Interferon (IFN) augmented NK activity of primarily one of the subsets examined, the NKI cell; the activity of IFN on NKT cells could not be directly tested, but IFN was without positive effect on TK or NKM cells. In contrast, partially purified IFN-free interleuken 2 (IL-2) augmented the activities of both the TK and NKT subsets, but not of NKI or NKM cell. IL-2 was active in augmenting NK activity in spleen cells obtained from both conventional and nu/nu mice, but was without effect on spleens of nu/nu mice depleted of Thy-1+ cells. These and other data suggest that IL-2 acts primarily, if not exclusively, on THy-1+ cells. These results strengthen the view that natural cytotoxicity in vitro can be mediated by several distinct cell populations under different genetic and regulatory control and indicate the importance of defining and delineating the cell lineages of each and the role of the independent subsets in resistance to virus infections and tumors in vivo.     

The generation of natural killer (NK) cells from NK precursor cells in rat long-term bone marrow cultures

In this report, we describe a novel long-term bone marrow culture (LTBMC) system to study the origin and generation of natural killer (NK) cells from NK precursors. Rat bone marrow was cultured for 4 wk in RPMI 1640 with 5% fetal calf serum and 2-mercaptoethanol to allow the formation of an adherent stromal cell layer containing NK precursor cells. After addition of interleukin 2 (IL-2), the LTBMC generated high numbers (up to 100-fold expansion in 7 d) of pure 3.2.3+ large granular lymphocytes with lytic activity against NK-sensitive and -resistant tumor targets, as well as antibody-dependent cellular cytotoxicity. NK activity in LTBMC could be detected 3 d after addition of as little as 1 U/ml rIL-2, whereas lymphokine-activated killer activity was found 5 d after addition of at least 10 U/ml rIL-2. In vivo depletion and in vitro complement lysis studies showed that the NK precursor cells in LTBMC did not express the NK-associated surface markers asialo GM1 or 3.2.3. We also found that LTBMC cells did not exhibit colony growth in granulocyte/macrophage or spleen colony-forming unit assays. The generation of NK cells from NK precursors required, in addition to IL-2, a second growth/maturation factor(s), which was present in the conditioned medium of the LTBMC. This LTBMC system provides a unique in vitro model to study the development of NK cells from precursor cells, the role of the bone marrow stromal microenvironment in this development, and the lineage relationship of NK cells to other hematopoietic cells.     

Human herpesvirus-6 enhances natural killer cell cytotoxicity via IL-15.

The marked tropism of human herpesvirus-6 (HHV-6) for natural killer (NK) cells and T lymphocytes has led us to investigate the effect of HHV-6 on cellular cytotoxicity. We describe here how HHV-6 infection of peripheral blood mononuclear cells (PBMC) leads to upregulation of their NK cell cytotoxicity. The induction of NK cell activity by HHV-6 was abrogated by monoclonal antibodies (mAbs) to IL-15 but not by mAbs to other cytokines (IFN-alpha, IFN-gamma, TNF-alpha, TNF-beta, IL-2, IL-12) suggesting that IL-15 secreted in response to viral infection was responsible for the observed effect. Furthermore, NK activation by HHV-6 was blocked with mAb to CD122, as well as by human anti-HHV-6 neutralizing antibodies. Using RT-PCR, we were able to detect IL-15 mRNA upregulation in purified monocyte and NK cell preparations. IL-15 protein synthesis was increased in response to HHV-6. Finally, addition of IL-15 to PBMC cultures was found to severely curtail HHV-6 expression. Taken together, our data suggest that enhanced NK activity in response to viral infection represent a natural anti-viral defense mechanism aimed at rapidly eliminating virus-infected cells.     

Down regulation of human natural killer cell-mediated cytolysis induced by blood transfusion: role of transforming growth factor-β(1), soluble Fas ligand, and soluble Class I human leukocyte antigen

BACKGROUND: Human natural killer (NK) cells are thought to play a role in antiviral response and tumor immune surveillance. The molecular mechanisms of down regulation of NK‐cell activity observed after red blood cell (RBC) transfusion is still undefined. STUDY DESIGN AND METHODS: Both effects of blood transfusion (ex vivo) and supernatants (SNs) derived from RBC units unstored (RBC‐0) or stored for 5 or 30 days (RBC‐5 or ‐30, respectively) in vitro were analyzed on NK cell–mediated cytolytic activity. RESULTS: We have found that NK cells isolated from transfused patients on Day 3 lysed the NK‐sensitive target cells K562 to a lesser extent than before transfusion. This down regulation of NK‐cell activation was evident also for NK‐cell killing mediated through the engagement of NK cell–activating receptors as NKG2D, NKp30, NKp46, and CD16. Transfused patients reacquired NK cell–mediated cytolytic activity from Day 5 to Day 7 after transfusion. SN from RBC‐30, but not from RBC‐0 or RBC‐5, strongly inhibited the generation of lymphokine‐activated killer (LAK) cells and lysis of the NK‐resistant target cell Jurkat in a dose‐dependent manner. Transforming growth factor‐β1 (TGF‐β1) blocking antibodies partially restored the generation of LAK activity. In addition, the depletion of both soluble Class I human leukocyte antigens (sHLA‐I) and soluble Fas ligand (sFasL) from SN of RBC‐30 completely restored the generation of LAK activity. CONCLUSIONS: Altogether, these findings would support the idea that blood transfusion–mediated down regulation of NK‐cell activity is mediated by sHLA‐I, sFasL, and TGF‐β1.     

Interleukin 5 enhances interleukin 2-mediated lymphokine-activated killer activity

IL-5 expresses various biologic effects on several types of lymphocytes, including B cells, eosinophils, and T cells. We demonstrated that the incubation of resting splenocytes from C57BL/6 mice in murine rIL-5 enhances IL-2-mediated lymphokine-activated killer (LAK) activity against various tumor cells. IL-5 alone, however, does not induce killer activity. IL-2-mediated LAK activity increases in proportion to the dose of IL-5. During the late phase of the culture period, IL-5 seems to have some effect on the induction of IL-2-mediated LAK activity. We expect that IL-5 will prove useful for adoptive immunotherapy.