Adherent lymphokine-activated natural killer cells in normal and severe combined immunodeficiency mice: large granular lymphocytes with natural killer cell phenotype and high cytolytic activity

Plastic-adherent lymphokine-activated natural killer (LANK) cells were generated from nylon wool-nonadherent murine splenocytes cultured in recombinant interleukin-2 (IL-2). Under such conditions, adherent lymphokine-activated killer cells capable of killing natural killer (NK)-resistant targets were not generated. Adherent LANK cells proliferated rapidly and closely resembled NK cells in their morphology, cytotoxic reactivity, and surface marker expression. Mice with severe combined immunodeficiency (scid) were used to generate adherent LANK cells to define the role of T cells in LANK cell development. Scid lymphocytes responded to IL-2 by becoming adherent LANK cells with potent NK-like activity, suggesting that soluble lymphokines other than IL-2 that may have been produced by T cells were not required for the generation of LANK cell activity in mice.     

Analysis of mechanisms by which NK cells acquire increased cytotoxicity against class I MHC-eliminated targets

Acid treatment, where cells are exposed to 0.2 M citric acid buffer at pH 3 for 2 min, was described in a previous paper to be a method which specifically eliminates class I MHC antigens from the membrane of viable cells. We applied this method to characterize functional roles of class I MHC antigens on the target cells in NK cell cytotoxicity. When NK target cells, U937, Molt-4, and Raji, were subjected to acid treatment, the treated cells lost their surface class I MHC antigens and became more sensitive to NK cell killing. On the other hand, the susceptibility of K562 cells which initially lacked class I MHC antigens did not significantly change after such treatment. We then examined the mechanism which enables NK cells to become more cytotoxic against class I MHC antigen-eliminated target cells. Single cell binding assay and cold target inhibition assay demonstrated that class I MHC antigen-eliminated target cells did not acquire high binding affinity to NK cells. However, the interaction between NK cells and class I MHC antigen-eliminated targets resulted in a greater increase in production of NKCF-like factor than did the interaction between NK cells and untreated targets. Class I MHC antigen-eliminated targets did not acquire high killer susceptibility to NKCF-like factor. The present study utilizing the acid treatment method confirmed that surface class I MHC antigens on the targets are important immunoregulatory molecules not only for cytotoxic T lymphocytes but also for NK cells and elucidated some of the underlying mechanisms.     

Role of human circulating and tumor-infiltrating lymphocytes in cancer defense and treatment

We have analyzed the anticancer efficacy of various subsets of human circulating and tumor-infiltrating lymphocytes (TIL). These studies showed that circulating natural killer (NK) cells mediate the most potent oncolytic activity against a variety of tumor targets, after enrichment or stimulation with interleukin-2 (IL-2). Interestingly, NK cell oncolytic activity was directed also against tumor targets frequently designated as 'NK-resistant'. This indicates that NK cells display a broader spectrum of killing than is commonly recognized. TIL did not display any tumoricidal activity when unstimulated, but acquired cytotoxic potential after activation with IL-2. Comparative studies of TIL and circulating lymphocytes from patients with ovarian cancer demonstrated that these two groups of lymphocytes manifested similar levels of cytotoxicity and the same spectrum of target cell killing. No specificity in autologous tumor cell killing was displayed by TIL; instead, TIL were effective against autologous as well as allogeneic tumor targets. The lack of TIL tumor specificity was not detected only in ovarian tumors, but was manifested also in renal- and squamous-cell cancers. Characterization studies demonstrated that the primary oncolytic cells in the periphery and among TIL are NK cells. T lymphocytes displayed some, but rather negligible cytotoxic activity. In contrast, when IL-2-activated NK and T cells were analyzed for lytic activity against normal hematopoietic cells, T cells displayed high levels of bone marrow killing. The anti-bone marrow lytic activity of IL-2-activated T lymphocytes may be harmful after therapy with conventionally prepared lymphokine-activated killers. In light of these observations, new directions to adoptive immunotherapy are discussed.     

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.     

Interferon-gamma upregulates the susceptibility of human neuroblastoma cells to interleukin-2-activated natural killer cells

The influence of interferon-gamma on the susceptibility of neuroblastoma cells in cell-mediated killing was investigated. Neuroblastoma cells were only weakly susceptible targets for peripheral mononuclear cells. However, enrichment of natural killer (NK) cells or activation of NK cells with interleukin-2 resulted in a considerable increase of neuroblastoma cell lysis. Pretreatment of neuroblastoma targets with interferon-gamma additionally increased the susceptibility to enriched NK cells as well as to interleukin-2-activated NK cells. The conjugate formation between enriched NK cells and the neuroblastoma targets was not affected by the pretreatment of the targets with interferon-gamma. Concomitantly, treatment of the neuroblastoma targets with interferon-gamma resulted in a strong induction of otherwise poorly expressed major histocompatibility complex (MHC) class I antigen expression. These results suggest that the increased expression of MHC class I antigens on target cells is not always correlated with decreased sensitivity for NK cells but can also be followed by an increased susceptibility for NK cells.     

Lysis of human T cell leukemia virus infected T and B lymphoid cells by interleukin 2-activated killer cells

The human T cell leukemia (HTLV-1) retrovirus is the etiologic agent for adult T cell leukemia. Interleukin 2 (IL-2) activated killer (AK) cells have been shown to lyse freshly explanted tumor cells in vitro and have been used as a form of adoptive immunotherapy for the treatment of cancer. In this report, the ability of AK cells to lyse HTLV-1-infected targets was examined. Normal lymphocytes, when cultured in recombinant IL-2 for periods of 3 to 7 days, killed infected T and B cell lines. The precursor for these AK cells resided in the CD-16 antigen-positive subset (i.e., natural killer (NK) cells). Resting T cells, NK cells, or unfractionated lymphocytes did not lyse the infected targets. However, when isolated NK cells were incubated for 24 hr in IL-2, suboptimal cytolysis was induced whereas activation of NK cells with a four pulse of IL-2 was insufficient to generate effector cells. The results of performing cold target inhibition studies with Epstein-Barr virus-infected B cell lines and HTLV-1-infected T and B cell lines suggest that there are discrete subsets (i.e., clonotypic) in the AK population that preferentially lyse a given virally infected cell line. Thus to consider AK cells as true polyspecific killer cells may be inaccurate. Alternately AK cells may express a number of different receptors with variable affinities for the Epstein-Barr virus- and HTLV-1-infected cell lines. In addition, it was shown that HTLV-1-infected B cells are relatively resistant to AK cell-mediated lysis. These results clearly indicate that AK cells but not resting NK cells kill HTLV-1-infected cells.     

Inhibition of human NK cell and LAK cell cytotoxicity and differentiation by PGE2

Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression.     

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.     

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.     

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

Exercise stress and murine natural killer cell function

Male C3He mice were trained to run on a treadmill (final speed, slope, and duration of 30 m/min, 8 degrees, 30 min/day, 5 days/week, respectively) for 10 weeks or they remained sedentary. At the end of the training program, half of the mice were sacrificed and half were given a single bout of exercise to exhaustion (50% stepwise increases in final running speed for 2-min intervals). Splenic catecholamine concentrations, splenic natural killer cell cytolytic activity against YAC-1 tumor targets, and frequency of asialo GM1 (a murine natural killer cell surface glycolipid)-positive splenocytes were assessed. Exhaustive exercise in both trained and untrained mice reduced the in vitro killing of tumor targets by splenic natural killer cells relative to killing by splenocytes from mice which did not undergo the acute exercise bout (P less than 0.05). The frequency of asialo GM1-positive splenocytes was also reduced in the exhaustively exercised animals (P less than 0.05). Training alone, without the additional stress of exhaustive exercise, reduced the frequency of asialo GM1-positive splenocytes relative to a sedentary condition (P less than 0.05), but did not compromise natural killer cell cytolytic activity against the tumor targets. Splenic epinephrine concentrations in the exhaustively exercised animals were elevated 3- to 5-fold above the concentrations observed in trained and sedentary mice. These results suggest that a single, acute exercise bout reduces the capacity of splenic natural killer cells to kill tumor targets in vitro and that training enhances splenic natural killer cell cytolytic activity, on a per cell basis, against tumor targets.     

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1-5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.     

Effects of cyclosporin on C57BL/6 splenocytes before and after culture with high-dose recombinant interleukin-2: Implications for immunosuppression with cyclosporin

Summary Lymphokine-activated killer cells appear to arise from precursor cells bearing natural killer (NK) cell antigens. Cyclosporin (CsA) is a well-known immunosuppressive agent that can down-regulate NK cell cytotoxicity. Studies were initiated to evaluate the effects of CsA on splenocytes before and after exposure to recombinant interleukin-2 (rIL-2). Normal C57BL/6 mice receiving CsA at a dose of 100 mg/kg demonstrated a decrease in NK cell lysis against the YAC-1 lymphoma target in a 4-h chromium-release assay. When splenocytes obtained from CsA-treated mice were cultured for 3 days in complete medium containing 1000 U rIL-2/ml, they demonstrated a return of NK cell lysis to normal (mean cytotoxicity = 65 LU versus 60 LU for control and CsA-exposed splenocytes respectively;P, NS, five consecutive experiments) but revealed a decrease in the lysis of a NK-resistant target: the MCA-102 sarcoma (mean cytotoxicity = 20 LU vs 12 LU for control and CsA-exposed splenocytes respectively;P <0.02, five consecutive experiments). Fresh splenocytes cultured in media containing rIL-2 and CsA demonstrated a decrease in proliferation, cell-cycle S-phase fraction and cell yields compared to splenocytes cultured in media containing rIL-2 alone. In addition, a decrease in tumor cell lysis for NK-cell sensitive (mean percentage lysis = 98% vs 60%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) and resistant targets (mean percentage lysis = 68% vs 28%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) was also seen. CsA had no effects on the phenotypic antigenic expression of splenocytes cultured with high-dose rIL-2 although activated T cell antigens were down-regulated when fresh splenocytes were evaluated after in vivo exposure to CsA. These studies support the down-regulating effects of CsA on NK cell lysis and suggest that the rIL-2-activated cell population is heterogeneous as demonstrated by the differential down-regulation and recovery of NK-resistant cell lysis versus NK-sensitive cell lysis.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid)

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.