Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Association of decreased natural and antibody-dependent cellular cytotoxicity and production of natural killer cytotoxic factor and interferon in neonates

Cord blood lymphocytes (CBL) were compared with adult peripheral blood lymphocytes (a-PBL) for their: (i) natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, (ii) target-binding capacity, (iii) ability to induce soluble natural killer cytotoxic factor (NKCF), (iv) interferon (IFN)-, interleukin 2 (IL-2)-, and lectin-induced augmentation of NK activity, and (v) ability to produce IFN against tumor targets in vitro. CBL depleted of adherent cells and Percoll-separated, NK-enriched subpopulations demonstrated significantly lower NK, ADCC, and target-binding activities compared to a-PBL. CBL produced significantly lower levels of NKCF directed against K562 tumor targets in comparison with a-PBL. Although the NK activity of CBL was not stimulated by either IFN or IL-2 to the same levels shown by a-PBL, the percentage enhancement of cytotoxicity of CBL by IFN and IL-2 was greater than that of a-PBL. Lectin-induced enhancement of cytotoxicity was significantly greater for CBL in comparison with a-PBL. Further, the ability of CBL lymphocytes to produce IFN-gamma in vitro against K562 target cells was significantly lower than that of adult PBL. These studies suggest an association between decreased NK, ADCC, and target-binding activities, induction of NKCF and IFN production by CBL, and increased susceptibility of neonates to infection.     

Role of interferon in human natural killer activity against target cells infected with HSV-1

Human natural killer (NK) cells show high cytotoxic activity against target cells infected with herpes simplex virus type 1 (HSV-1). Substantial amounts of interferon (IFN) were generated in co-cultures of NK effector cells and infected target cells; however, the cytotoxic activity seen against a specific infected cell target did not correlate with the amount of IFN induced. The production of IFN increased steadily from 4 to 18 hr of co-culture, as did NK activity; however, IFN production peaked 4 hr later than NK activity. Pretreatment of NK effector cells with exogenous IFN increased cytotoxic activity against all targets tested, but the differential pattern of reactivity against cells infected with wild type and mutant viruses was unaltered. When effector cells were treated with the RNA synthesis inhibitor actinomycin D before co-culture with virus-infected targets, IFN production was markedly reduced, without a concomitant reduction in cytotoxicity. Similarly, the addition of anti-IFN antiserum to co-cultures greatly decreased the available IFN present, but had no effect on NK activity. We conclude that the induction of cytotoxic activity in co-cultures of NK effector cells and HSV-1-infected target cells is independent of the induction of IFN.     

Autologous enhancement by interferon of natural killer activity of human peripheral blood lymphocytes

The in vitro action of interferon (IFN)-alpha and IFN-gamma from six healthy donors and ten patients with multiple sclerosis (MS) on natural killer (INK) activity of peripheral blood lymphocytes (PBL) was studied in an autologous system. The NK activity of PBL was detected by a cytotoxic test using (3)H-uridine human erythromyeloblast K562 cells. Autologous IFN-alpha and IFN-gamma did not augment NK activity of PBL from healthy donors in vitro, whereas in samples from MS patients the IFNs strongly stimulated NK cell cytotoxic function. This stimulation suggests the existence of an inhibitor of regulatory IFN action, that is produced in healthy donors simultaneously with IFN in response to IFN induction, but which is lacking in commercial IFN preparations. The factor-containing supernatants from healthy donors reduced the stimulatory action of autologous IFNs in patients with MS almost until complete blockade. Because this inhibitor was absent in patients with MS, deficiency of an inhibitor of IFN regulatory action in MS could open the way to treatment of this compartment of the immune system.     

Leu-11+ lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rIL 2)-induced activated killer (AK) cells

Precursors of activated killer (AK) cells cytotoxic for human noncultured metastatic melanoma and colon carcinoma were characterized. These cells required 3 days incubation with recombinant interleukin 2 (rIL 2) and DNA synthesis for the induction of AK activity. Both negative and positive cell purification methods were used to identify the subpopulation of cells containing AK precursors. By complement-mediated cell depletion studies, AK precursors were largely present in the Leu-11+ fraction, and to a much lesser extent in the Leu-7+ and Leu-2a+ fractions; they were absent in Leu-3a+ and Leu-4+ cells. Lymphocyte subpopulations were then purified with a cell sorter to positively select for the subset containing AK precursors. Leu-11+ cells had the highest level of AK activity and proliferative response when cultured for 3 days with rIL 2 as well as the highest level of NK activity before culture. Leu-7+ cells had neither AK activity nor a proliferative response when cultured with rIL 2, although they still possessed high NK activity. The same levels of AK and NK activity were found in Leu-2a+ and Leu-2a- fractions, but both activities were absent among Leu-4+ and Leu-3a+ cells. Further fractionation with a two-step sorting technique showed that the highest AK activity resided in the Leu-7-Leu-11+ cell fraction. Morphologically, this subfraction was granular lymphocytes. Titration experiments or rIL 2-responsive cells showed that the number of cells required to achieve a comparable level of rIL 2 proliferative response were as follows: 35 X 10(3) cells from unseparated PBL, 10 X 10(3) cells from Leu-11+ cells, 3.3 X 10(3) from Leu-7-Leu-11+ cells, and 640 X 10(3) cells from Leu-7+ cells. These results indicate that the lymphocyte subpopulation that proliferates in the presence of rIL 2 and then develops AK activity was a subpopulation of Leu-11+ granular lymphocytes, which also possessed the highest NK activity. These Leu-11+ cells lacked the antigens defined by the Leu-7, Leu-3a, or Leu-4 antibodies. Although Leu-7+ cells did not respond to rIL 2 by themselves, they may play a role in the induction of AK activity.     

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge-132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat- or acid-treatment, suggesting that the induced IFN is of gamma-nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge-132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge-132, neither induction of IFN nor augmentation of NK activity occurred, and X-ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge-132 are mediated by the induced IFN.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

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.     

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retarets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8⁺ and CD4⁺ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V11, in particular CD8⁺ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.     

Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.     

Common precursor pool marker for allospecific (CTL) and nonspecific (NK and activated) cytotoxic cells in the bone marrow

A recently described monoclonal mouse IgG1 antibody, NK-9, reacts with practically all peripheral blood large granular lymphocytes (LGL). It also detects a population of non-LGL lymphoid cells that harbors precursor cells for both nonspecific activated killer (AK) and allospecific killer cells. In the bone marrow, the NK-9-positive population represented 9% of all nucleated cells, which was 40% of all lymphoid cells. This population was initially noncytotoxic, but when appropriately stimulated the NK-9-positive cells gave rise to AK and allospecific cytotoxic cells, whereas no such activity could be generated from the NK-9-negative cells. When the NK-9-positive cells were cultured with high concentrations of T cell growth factor, the results were cultures consisting of over 80% cells with LGL morphology and exhibiting effective cytotoxicity against K562 targets. It is concluded that the precursor cells for various modes of nonspecific and antigen-specific cytotoxicity are related and appear to be harbored in the NK-9-positive pool in the bone marrow.     

Acquisition of suppressive activity and natural killer-like cytotoxicity by human alloproliferative "helper" T cell clones

To investigate potential functional changes in alloantigen-specific proliferative CD3+, CD4+, CD8-, Leu-8-, interleukin 2 (IL 2)-secreting noncytotoxic in vitro primed human helper T cells, a set of 12 clones was studied sequentially throughout their finite life spans. Clones surviving to greater than 30 population doublings (PD) retained their requirements for exogenous IL 2 and filler cells for continued growth, but lost the ability to proliferate and to secrete IL 2 when specifically restimulated. This was not accompanied by changed surface marker phenotypes or acquisition of abnormal karyotypes, but was accompanied by the acquisition of MHC-unrestricted potent radioresistant suppressive activity for lympho-proliferative responses. Suppression was not caused by absorption of IL 2, secretion of interferons, de novo mycoplasma contamination, or cytotoxic activity. At least two suppressive mechanisms were demonstrated: 1) the induction of suppressor effectors in naive lymphocyte populations, which required cell to cell contact and could be inhibited by certain monoclonal antibodies against MHC class II determinants; and 2) a direct effect on responding lymphocytes, shown by suppressive activity on cloned PLT-active reagents. Moreover, the majority (75%) of originally allo-proliferative clones also acquired a previously absent cytotoxicity against natural killer (NK) cell-susceptible, but not NK-resistant, target cell lines. This modulation of function from specific alloproliferative, IL 2-secreting nonsuppressive status to strong nonspecific suppressive and NK-like cytotoxic status represents a novel functional activity of human T helper lymphocytes under conditions of clonal propagation.     

The role of adherent cells in supporting natural cytotoxicity reactions

The cytotoxic activity of natural killer (NK) cells and interferon-alpha (IFN-alpha) production by NK cells were studied. It was shown that in healthy donors adherent cells (AC) play an important role in reactions of natural cytotoxicity: they provide the main IFN production in the NK system. AC suppresses the cytotoxic activity of NK cells in patients with scleroderma and AC has a high cytotoxic activity in patients with multiple sclerosis. The role of AC in appearance and development of NK immunodeficiency in these pathologies was discussed.     

Homeostatic defects in interleukin 18-deficient mice contribute to protection against the lethal effects of endotoxin

Toll-like receptor-4-lipopolysaccharide (LPS)-mediated inflammation is used to delineate signals involved in cross-talk between antigen-presenting cells (APCs) and lymphocytes such as natural killer (NK) cells. Following APC stimulation and cytokine release, NK cells produce interferon (IFN)-γ. High levels of LPS induce endotoxicosis, a systemic inflammatory disease in which IFN-γ causes significant morbidity and mortality. Several studies have highlighted the role of interleukin (IL)-18, IL-1β, IL-17A and IFN-γ in the development of endotoxicosis, but whether these cytokines interact with each other is yet to be determined. Our data demonstrate that IL-18 and IL-17A have important roles in NK cell IFN-γ production during endotoxicosis. Importantly, we provide the first evidence that IL-18 also has a role in IL-17A production by T-cell receptor (TCR)-δ cells. Furthermore, we demonstrate that IL-18-deficient mice have a defect in γδ T-cell homeostasis and IL-1β production, both of which can contribute to the development of disease through induction of IL-17A. These results reveal novel requirements for IL-18 in innate immune cell homeostasis and activation, demonstrating that the role of IL-18 in innate immunity occurs at a level other than activation.     

Involvement of the 2'-5' A pathway in the augmentation of natural killer activity

Pretreatment of human large granular lymphocytes (LGL) or unseparated peripheral blood mononuclear cells with interferon (IFN) resulted in a significant augmentation of natural killer (NK) activity. This increase was paralleled by an increase in the 2'-5'A synthetase activity. In order to investigate the possibility that IFN might be inducing augmentation of NK cells via the 2'-5'A pathway, we tested the effects of nonphosphorylated core material [(A2'p)2A] and of the triphosphorylated form of the 2'-5'A [ppp(A2'p)2A]. The core material had no detectable effect on NK activity. In contrast, when experiments were performed with the triphosphorylated form of 2'-5'A, NK activity was stimulated. In order to achieve activation, permeabilization of LGL with calcium chloride was necessary and, under these conditions, a dose-dependent augmentation of NK activity was seen. However, the calcium treatment had considerable toxic effects on basal levels of NK activity. Collectively, these results suggest that IFN may be inducing augmentation of NK activity via the 2'-5'A pathway. Further studies will be necessary to determine the effects of IFN and/or 2'-5'A on subsequent activation steps in the process leading to cytotoxicity by NK cells.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Regulation of human natural cytotoxicity by IgG. III. Interaction between negative regulation by monomeric IgG and positive regulation by interferons of different types

Our prior reported results have demonstrated the dose-dependent inhibition of human natural killer (NK) cell activity upon treatment of peripheral blood mononuclear cells (PBMC) with monomeric IgG (mIgG) prior to the cytotoxic assay. In the present study, the combined effects on NK activity of human interferon (IFN) of each of the three types and mIgG, respectively, were determined. NK cells incubated with IFN alpha or IFN beta had augmented cytotoxicity against K562 target cells but remained responsive to negative regulation by mIgG. PBMC treated with human recombinant IFN gamma had unchanged cytotoxic activity but became partially resistant to suppression by mIgG. This ability of IFN gamma to interfere with the negative regulation of NK activity by cytophilic mIgG was seen when the cytokine was preincubated with effector cells prior to, simultaneously with, or after their exposure to inhibitor protein. These data provide some clues regarding the possible biological significance of the mIgG-induced down-regulation of NK cells which, when required for host protection, might be appreciably reversed or blocked by IFN gamma produced by NK cells or T cells in response to various agents.     

Activation Mechanisms of Natural Killer Cells during Influenza Virus Infection

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.