Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.     

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

Summary Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [³H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.     

Resting human peripheral blood lymphocytes can be activated to cytolytic function by antibodies to CD3 in the absence of exogenous interleukin-2

We investigated the ability of anti-CD3 antibodies to activate resting human peripheral blood lymphocytes (PBL) to a cytolytic function. We found that two anti-CD3 antibodies, but not an anti-CD4, anti-CD8, or anti-CD2 antibody, could activate resting unseparated PBL to become killer cells in the absence of exogenous interleukin-2 (IL-2), although exogenous recombinant IL-2 (rIL-2) synergized with anti-CD3. We also found that these anti-CD3 antibodies were active in the absence of rIL-2 only when linked to a solid surface such as a Sepharose bead or a plastic tissue culture plate. Cytolytic activity was measured in several ways: (i) by the ability of activated PBL to lyse the NK-sensitive line K562, and (ii) by the ability of these cells to lyse a CD10+ (CALLA+), NK-resistant target in the presence of either concanavalin A (lectin-dependent lysis) or an anti-CD10-anti-CD3 heterodimer. At least two different types of cytolytic cells were activated by anti-CD3 antibodies, an NK-like cell, which was CD2+CD3-CD4-CD8-CD16+-NKH1a+, and a CTL-like cell, which was CD2+CD3+CD4-CD8+CD16-NKH1a-. The former cell lysed the K562 line and the latter cell lysed Namalwa in the presence of the anti-CD10-anti-CD3 heterodimer or concanavalin A. The NK-like cell was probably activated by endogenous IL-2 produced by the anti-CD3-activated CD3+ cells and both the NK and CTL-like cells required the presence of adherent cells for maximal activity. The dose response and the kinetics of anti-CD3 activation of PBL to cytolytic activity were also studied. The use of the anti-CD3-activated cytolytic cells as effectors in anti-CD3 heterodimer-mediated lysis of tumor cells may be a novel approach to the therapy of cancer, and a comparison with the well-studied rIL-2/lymphokine-activated killer (LAK) system is discussed.     

Selective lysis of target cells by interleukin-2-expanded peripheral blood mononuclear leukocyte clones

The presence of distinct cytolytic subsets within interleukin-2-expanded peripheral blood leukocytes (IEL) cultures was demonstrated by clonal analysis. Thirty-seven IEL clones were isolated from two healthy blood donors; 21 destroyed both Daudi and K562 cell lines. Of those 21 clones, 1 clone could destroy autologous PBM, 7 clones could destroy fresh allogeneic ovarian carcinoma (OVA-CA) cells, and 6 clones could destroy normal autologous PBM and fresh OVA-CA cells. Twelve of the 37 clones destroyed only one of the four targets tested: 8 clones destroyed K562, 2 clones destroyed Daudi, and 1 clone each was selective for autologous PBM or OVA-CA. Of the remaining 4 clones, 1 destroyed OVA-CA and Daudi cells, 1 destroyed PBM and K562, 1 destroyed PBM and Daudi cells, and 1 destroyed PBM, Daudi, and OVA-CA. These results suggest that these functionally heterogeneous cytolytic clones may use different cell recognition or cytolytic mechanisms to enable these distinct and, at times, reciprocal patterns of target cell selectivity.     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

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.     

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.     

Separation of lymphokine-activated killer cell precursors and T-cells: differential growth characteristics and apparent lack of a T-cell suppressor of LAK-cell induction

Lymphokine activated killer (LAK) cell clinical effectiveness may be limited by the total cell dose and cytotoxic activity. We have, therefore, examined methods to expand the number of LAK-cells by serial passage of unfractionated and fractionated peripheral blood lymphocytes. Human purified lymphocytes were obtained by Ficoll Hypaque gradients followed by exposure of resultant mononuclear cells to phenylalanine methyl ester to remove monocytes. Lymphocytes were then fractionated on a six-step Percoll gradient (50%, 47.5%, 45%, 42.5%, 40%, and 37.5% Percoll). Unfractionated cells and fractions were cultured in standard media (RPMI-1640, 10% human sera, antibiotics and 10 mM HEPES) containing 10 nM of recombinant Interleukin-2 (rIL-2). Lymphocytes were cultured at 1 X 10(6)/ml and recultured every 3 to 4 days in fresh standard media and rIL-2. Utilizing unfractionated and fractionated lymphocytes from seven donors we made the following observations: (1) Continued passage of unfractionated lymphocytes resulted in a loss of LAK-cell activity by greater than or equal to 14 days (e.g., percent lysis of Raji at 10:1 effector:target ratio on days 0, 4, 7, and 21 was 0.38 +/- 11, 41 +/- 17, and 8 +/- 1, n = 4, respectively). (2) LAK-cell functional precursors were predominantly confined to the lymphocytes in the upper (0-4) Percoll fractions (e.g., on day 4, the percent lysis by pooled fractions 1-4 was 63 +/- 5 vs. pooled fraction 5 plus pellet, 18 +/- 7%). (3) As expected, the upper fractions (0-4) were enriched for Leu 19 positive cells (approximately 40%) and large granular lymphocytes (LGL) by morphology (approximately 30%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Spontaneous proliferation of HTLV-II-infected peripheral blood lymphocytes: HLA-DR-driven, IL-2-dependent response.

Peripheral blood lymphocytes obtained from HTLV-II-infected persons (n = 13) and cultured in the absence of exogenous stimulator demonstrated augmented spontaneous proliferation (17,672 +/- 5,498 cpm) when compared with cells from healthy donors (1,921 +/- 1,306 cpm). Removal of non-T population did not abrogate the proliferative response of patients' PBMC, suggesting that the proliferation is not related to the autologous mixed lymphocyte reaction. Addition of recombinant interleukin-2 (rIL-2; 0.1 U/ml) to spontaneously proliferating cultures from HTLV-II-infected persons resulted in a 3- to 4-fold increase in proliferation (61,985 +/- 16,003); in contrast, PBMC from controls demonstrated 38- to 42-fold increase in their proliferative capacity in response to rIL-2 (77,256 +/- 13,044). Antibodies to both IL-2 receptor and HLA-DR were able to inhibit the spontaneous proliferation of PBMC from HTLV-II-infected persons in a dose-dependent manner. Furthermore, addition of cyclosporin A, which preferentially blocks accumulation of IL-2 mRNA, also inhibited spontaneous proliferation in a dose-dependent manner. These observations suggest that the spontaneous proliferation of HTLV-II-infected PBMC is at least in part an HLA-DR-driven, IL-2-dependent event, which is not analogous to the AMLR.     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Heterogeneity in retinoic acid signaling in neuroblastomas: Role of matrix metalloproteinases in retinoic acid-induced differentiation

Causes of retinoid resistance often observed in neuroblastomas are unknown. We studied all trans-retinoic acid (RA) signaling in neuroblastoma cells differing in N-myc levels in terms of neurite formation, expression of tissue transglutaminase, neuronal marker proteins, matrix metalloproteinases (MMPs), and activation of Rac1 and Cdc42. Poor invasiveness observed in SH-SY5Y, LA-N-5, and SMS-KCNR cells was associated with RA-induced neurite formation, Cdc42 activation and N-myc down regulation; expression of constitutively active Cdc42 down regulated N-myc expression and reduced invasion in RA-resistant SK-N-BE(2) and IMR32 cells. RA treatment for 24 h transiently increased invasion and expression of MMP9 in SH-SY5Y, LA-N-5 and MMP2 in SMS-KCNR cells. MMP inhibition prevented RA-induced neurite formation indicating a role in differentiation. Variation in RA signaling thus follows a defined pattern and relates to invasive potential. A defective RA signaling might result in retinoid resistance and unpredictable clinical outcome observed in some neuroblastomas.     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.