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.     

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.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.     

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization

Summary We have previously demonstrated that cancer patients' peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h ⁵¹Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-AK cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-AK effector cell was OKT3+, OKT8+, OKT4–, OKM1– and could be generated by just 3 days of allosensitization. The precursors for A-AK cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4–. These allocatived PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of non-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

Evidence that lymphokine-activated killer cells and natural killer cells are distinct based on an analysis of congenitally immunodeficient mice

The in vitro incubation of lymphoid cells in RIL 2 results in the generation of LAK cells that are broadly lytic to autologous, syngeneic, and allogeneic fresh tumor cells, but which do not lyse fresh, normal cells. Strains of mice with congenital immunodeficiencies were tested both for the presence of NK cells and for their capacity to generate LAK cells after in vitro incubation with IL 2. Splenocytes obtained from two immunodeficient mouse strains (NIH-Beige-Nude and NIH-Beige-Nude-XID) failed to generate LAK cells, but displayed significant activity. Splenocytes from another immunodeficient mouse strain (NIH-Beige-XID) generated LAK cells but did not display NK cell activity. This dissociation of activation of LAK cells from NK cells among the immunodeficient strains indicates that the LAK and NK cell lytic systems are distinct.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.     

Clonal and frequency analyses of tumor-infiltrating T lymphocytes from human solid tumors

A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19+ large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.     

Inhibition of normal lymphocyte responses by cell membranes

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Spontaneous in vitro evolution of lytic specificity of cytotoxic T lymphocyte clones isolated from murine intestinal epithelium

Three long-term clonally derived cytotoxic lines have been established from isolates of murine intraepithelial lymphocytes (IEL). All three lines were selected for with antigen and represent two allospecific cytotoxic T lymphocyte (CTL) clones and a major histocompatibility complex (MHC)-restricted clone specific for a murine minor histocompatibility antigen. On long-term in vitro culture, IEL clones gradually lost antigen-specific lytic activity and simultaneously acquired the capacity to lyse natural killer (NK)-sensitive target cells which, in some cases, required high-level lymphokine activation. Of interest was the finding that, despite changes in lytic specificity, IEL clones remained strictly antigen-dependent for proliferation. A murine CTL clone of splenic origin, which was propagated under culture conditions identical to those used for IEL, did not exhibit changes in lytic specificity, suggesting that acquired changes in IEL function cannot be attributed solely to the influence of in vitro culture. Phenotypic analyses of IEL clones with altered lytic specificity revealed that all lines remained Thy-1+, Lyt-2+, L3T4-, with or without lytic activation by lymphokines. The expression of CT-1, a murine CTL activation antigen, and asialo GM1, a murine NK cell marker, were variable on IEL clones, and their presence did not correlate with the changes in lytic behavior. Collectively, these findings provide evidence, at the clonal level, that at least some NK activity present in isolates of murine IEL may originate from antigen-specific CTL. The data also indicate that, on binding antigen, different signals are conveyed to T cells, resulting in proliferation or target cell lysis.     

The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes

Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies.     

Monocyte- and natural killer cell-mediated spontaneous cytotoxicity against human noncultured solid tumor cells

Unstimulated human peripheral blood mononuclear cells from healthy donors exhibited spontaneous cytotoxicity against noncultured solid tumor targets in a 12- to 24-hr 51Cr release or 111In release assay. Both purified monocytes (greater than 99% monocytes) and natural killer (NK)-enriched lymphocytes exhibited comparable levels of spontaneous cytotoxicity against fresh melanoma tumor targets. This cytotoxicity was observed under endotoxin-free conditions. NK-depleted lymphocytes did not lyse the melanoma targets. Culture supernatants of monocytes incubated with the melanoma tumor cells did not exhibit cytotoxic activity against these targets. Purified monocytes lacked NK activity against the K562 targets in a 4-hr 51Cr release assay. Treatment of the monocytes with anti-Leu 1 1b and anti-Leu7 monoclonal antibodies plus complement did not reduce monocyte-mediated lysis of the melanoma targets, demonstrating that contaminating NK cells, if any, were not responsible for the lysis of noncultured melanoma targets by monocytes. In contrast, Leu 1 1b+ NK cells were responsible for the lysis of the melanoma targets by NK-enriched lymphocytes. The addition of recombinant interferon-gamma (rIFN-gamma), but not lipopolysaccharide, into the 51Cr release assay or pretreatment of monocytes with rIFN-gamma significantly increased their cytotoxicity against noncultured solid tumor cells. Monocytes cultured for 3 days with medium alone lost their cytotoxic activity. The addition of rIFN-gamma from the beginning of these cultures prevented the loss of the cytotoxic activity of monocytes. In summary, both unstimulated monocytes and NK-enriched lymphocytes exhibit comparable levels of spontaneous cytotoxicity against fresh solid tumor targets.     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Modulation of natural cytotoxicity by alloantibodies. V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab)₂ fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR⁺ target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.     

Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p15E

It has been shown previously that the retroviral envelope protein p15E suppresses certain monocyte and lymphocyte functions. In this paper, we describe the effects on natural killer (NK) activity of a synthetic peptide (CKS-17) with homology to a region of p15E conserved among numerous retroviruses. Enriched human NK cells were assayed against K562 tumor target cells in a 51Cr-release cytotoxicity assay. Pretreatment of NK cells with CKS-17 at concentrations as low as 1.5 microM, but not with equivalent concentrations of control materials, markedly and reproducibly suppressed NK lytic activity. Prior exposure of NK cells to interferon-alpha (IFN-alpha) at 1000 U/ml did not alter their sensitivity to CKS-17-induced inhibition. Pretreating NK cells with CKS-17 almost entirely diminished their responsiveness to IFN-alpha and IFN-gamma, but not to interleukin 2 (IL 2). Kinetics experiments demonstrated that CKS-17-mediated suppression of both endogenous and activated NK cells was reversible after 18 hr at 37 degrees C. Experiments designed to examine the CKS-17 mechanism of action revealed that the peptide bound to all Leu-11+ lymphocytes, as shown by two-color flow cytometry. CKS-17 did not, however, inhibit effector cell/target cell conjugate formation. These data suggest a new mechanism for immune suppression mediated by retroviruses; inhibition of NK function. They moreover imply that the CKS-17 peptide interferes with the lytic phase of NK cytolysis.     

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.