Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.     

Induction of autoreactive cells by the preculture of human peripheral blood mononuclear cells with the autologous fresh plasma.

An AMLR in which precultured cells proliferated in response to fresh non-T cells is described. In our system, the responder is human peripheral blood mononuclear cells precultured in the autologous fresh plasma for up to 16 days, and the stimulator is fresh autologous non-T cells. Results suggested that there were two subpopulations of autoreactive cells obtained from the preculture; the high and low density small lymphocytes, both having ERF activity. The autoreactivity of low density cells was augmented when either macrophages or N-ERF-cells were depleted from PBM and thereafter precultures wre performed. A survey of the functional characteristics of the responding cells showed that the responding cells had NK activity against Molt-4 cells but had no significant ADCC activity against target CRBC. Mechanisms for the induction of autoreactive cells by the preculture in the presence of autologous fresh plasma are discussed.     

Phorbol ester-induced lymphocyte adherence: selective action on NK cells

Treatment of human peripheral blood lymphocytes (PBL) with phorbol dibutyrate (PDBU) for 20 to 45 min at 37 degrees C induces adherence of 5 to 30% of the cells to plastic. The adherent cells (pAd) were highly enriched in NK cells on the basis of the following findings: 1) they exhibit high NK and ADCC activity but do not lyse the NK-resistant cell line, Daudi; 2) cytotoxic activity is enhanced by pretreatment with interferon-alpha (IFN-alpha); 3) the surface markers of these cells, as determined with monoclonal antibodies, are consistent with NK cells; and 4) they are enriched with cells morphologically similar to large granular lymphocytes. Conversely, the PDBU-nonadherent cells were substantially depleted of NK cells. The fact that the pAd cells do not lyse the Daudi line and that their NK activity can be further augmented by IFN-alpha would suggest that the pAd are enriched for NK cells rather than changed in their characteristics as a result of the separation procedure. Moreover, no consistent and appreciable modulation of NK activity induced by PDBU was observed. This report therefore demonstrates that PDBU selectively induces NK adherence of NK cells, which may have practical as well as biological implications.     

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.     

Phenotypic characterization of lymphokine-activated killer cells from human lymph node lymphocytes

We previously reported that lymphokine-activated killer (LAK) activity can be generated in human lymph node lymphocytes (LNL) at the same level as that in peripheral blood lymphocytes (PBL), despite the absence of active natural killer (NK) cells. In the present study, we investigated the surface phenotype of LNL-LAK cells by fractionation of lymphocytes, using a panning method. LNL isolated from lung cancer patients were cultured in the presence of recombinant interleukin 2 for 8 days and separated into T cells and non-T cells according to the expression of CD3 antigen. LAK effectors were enriched in the CD3- non-T cells. However, the CD3+ cells also mediated a low but substantial level of LAK activity, which was attributed to a CD8+ T-cell subset. Further investigation of the CD3- cells revealed that most of the CD3- effector cells expressed neither B-cell (CD20) nor NK-cell (CD16) markers. Precursors of this CD3-CD20-CD16- (null) population appeared to be also CD3-, CD20-, and CD16-. From these results, we would stress the significant contribution of CD3-CD20-CD16- null cells to the LAK phenomenon, which has not been focused on in PBL.     

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.     

Phorbolester-treated human lymphocytes are susceptible to natural killer cell-mediated cytolysis

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was found in this study to render normal human lymphocytes susceptible to allogeneic and autologous natural killer (NK) cell lysis. Both T and non-T cells became susceptible after culture for 3 days in the presence of 1 ng/ml TPA. Effector cells were nonadherent mononuclear cells of low density, enriched for large granular lymphocytes and HNK-1+ cells. Activation of effector cells with interferon increased lysis of TPA-treated lymphocytes. The present system may provide a new model to study NK cell target structures.     

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.     

Natural killer cell number and function in the spontaneously diabetic BB/W rat

The BB/W rat provides a good model of spontaneous autoimmune diabetes. Diabetes-prone (DP) rats have a virtual lack of OX 8+ OX 19+ T cytotoxic/suppressor cells in peripheral blood lymphocytes (PBL) and spleen, suggesting that the OX 8+ OX 9- natural killer (NK) cells are the predominant cytotoxic cell in this animal. In this study, we have shown that rat NK cells belong to the OX 8+ OX 19- asialo GM1 bright population, and that rat NK cell function may be depleted in vivo by administration of OX 8 antibody. Furthermore, evidence is provided to indicate that NK cell number and activity are enhanced on a per cell basis in DP rats as compared to the diabetes-resistant W line rat. DP rats had about threefold more NK cells than did W-line rats. The cytotoxic activity mediated by spleen and PBL against the YAC-1 target generally correlated with the relative number of cells having the OX 8+ OX 19- phenotype. DP lymphocytes mediated low levels of cytolytic activity against the relatively resistant NK target cell K562. To more directly compare the activity of W-line and DP NK cells, spleen NK cells were isolated by flow sorting of the OX 8+ OX 19- population. At a 5:1 E:T ratio, DP OX 8+ OX 19- cells elicited 21% +/- 3 specific lysis and W-line cells elicited 7% +/- 2 specific lysis. To determine whether the elevated levels of NK cells and NK cell activity in DP rats were a consequence of NK cell proliferation, spleen cells were size-separated by centrifugal elutriation. The NK cell activity was predominantly mediated by small to medium-size lymphocytes and not blast-size enriched populations. Moreover, when the DNA content of splenic OX 8+ cells was measured, 98% of the cells were in the G0-G1 phase of the cell cycle. These data indicate that NK cell number and activity are elevated in DP rats, and support a role for NK cells in the pathogenesis of BB/W diabetes.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

Depletion of NK cells with the lysosomotropic agent L-leucine methyl ester and the in vitro generation of NK activity from NK precursor cells

Human natural killer (NK) cell activity in peripheral blood lymphocytes (PBL) is totally inhibited by pretreatment of the effector cells with a lysosomotropic agent, L-leucine methyl ester (LeuOMe). This treatment specifically eliminates cells expressing the NK cell markers HNK-1, OKM1, B73.1, or Leu-11b, but has minimal effect on viability of cells with T cell markers Leu-1, OKT3, Leu-2a, or Leu-3a. LeuOMe also drastically decreased the proportion of K562 target-binding lymphocytes among PBL. PBL pretreated with LeuOMe respond normally in thymidine uptake to stimulation by phytohemagglutinin or allogeneic lymphocytes as long as irradiated autologous accessory cells are provided, indicating that the treatment is not toxic to T cells. NK activity can be regenerated in the NK cell-depleted PBL population by incubation with IL 2 or by mixed lymphocyte cultures, but not by alpha-interferon. Cells responsible for regeneration of such NK activity are probably large agranular lymphocytes, because they are resistant to LeuOMe treatment but have the same low buoyant density as NK cells in Percoll density gradient separation. The in vitro-generated NK is still sensitive to LeuOMe inhibition, but a higher concentration of the reagent is required to achieve total inhibition of the activity.     

Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells

A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.     

Activated B lymphocytes: stimulators of an augmented autologous mixed leukocyte reaction

The characteristics of the non-T cell(s) which stimulate T-lymphocyte proliferation in the autologous mixed leukocyte reaction (AMLR) have been at issue since this in vitro reaction was first described. Dendritic cells have been shown to be the most potent stimulator cells, but B cells, null cells, and macrophages have also been demonstrated to have the capacity to stimulate autologous T-cell proliferation. A cell preparation obtained from human peripheral blood was highly enriched for surface immunoglobulin-positive B cells. These cells were activated by brief culture with various B-cell mitogens and then compared to untreated B cells with regard to stimulatory activity in the AMLR. Mitogen-activated B cells were markedly augmented in their capacity to stimulate autologous T-cell proliferation when compared with untreated B cells. Fractionation of the B-cell preparation into high- and low-density subpopulations demonstrated that the high-density cells, enriched in resting B cells, had minimal stimulatory activity but could be activated to have increased AMLR-stimulatory capacity. Proliferation of the activated B lymphocytes was not required for the generation of the augmented AMLR. Response to both untreated and mitogen-activated B cells was a property of T4-positive T lymphocytes. The increase in stimulatory capacity was associated with a decrease in cell surface immunoglobulin, but no significant alteration in the percentage or fluorescence intensity of anti-Ia staining cells was detected. Activated B cells which are generated in vivo may acquire the capacity to generate T effector cells or factors important in the regulation of B-cell function.     

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.     

Active suppression of host-versus-graft reaction in pregnant mice. VIII. The uterine decidua-associated suppressor cell is distinct from decidual NK cells

The fetus resulting from allogeneic mating expresses a variety of antigens that may serve as targets for rejection by the maternal immune system. Accumulation of non-T suppressor cells into the uterine decidua of allopregnant mice may serve to prevent such rejection. It has been previously shown that the suppressor activity in decidua during the second half of murine pregnancy is predominantly associated with a population of small lymphocytes with cytoplasmic granules that lack T-cell markers and inhibit the generation of cytotoxic lymphocytes (CTL) against paternal alloantigens both in vitro and in vivo. Since natural killer cells (NK) also possess cytoplasmic granules and may regulate the murine immune response, we examined the hypothesis that the decidua-associated non-T suppressor cell may represent a regulatory type of NK cell. Similar to NK cells, the decidua-associated suppressor cell expressed FcR for IgG. Unlike NK cells, the decidua-associated suppressor cell proved resistant to treatment with anti-asialo GM1 + C'. Sedimentation velocity examination demonstrated that decidua-associated NK activity was associated with cell population with a modal sedimentation of 4 mm/hr that was larger than the decidua-associated suppressor population. Potent suppressor cell activity was also recovered from the decidua of NK deficient allopregnant bg/bg mice. Therefore, decidua-associated NK cells and suppressor cells represent two distinct populations.     

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.     

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.     

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12-14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10(-5) M), an inhibitor of prostaglandin synthesis, or anti PGE2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE2-mediated suppression. High levels of PGE2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE2 (10(-7)-10(-6) M) but not PGF2 alpha (10(-6) M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE2 produced by decidual cells and decidual macrophages.