Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Differential effects of recombinant human interferon-gamma and interleukin 2 on natural killer cell activity of peripheral blood in early human development

Ontogenic development and the lymphokine responsiveness of human NK cell activity against K562 target cells in peripheral blood lymphocytes were evaluated in fetuses, premature infants, and term neonates by using a 4-hr 51Cr-release assay. Basal NK activity and NK boosting by lymphokines were comparatively assayed after an 18-hr incubation with medium alone, recombinant human IFN-gamma (1000 U/ml), and recombinant human IL 2 (25 U/ml), respectively. Lymphocytes from 20-wk-old fetuses lacked NK cell activity even after the pretreatment with IFN-gamma. Low, but significant levels of NK activity and NK boosting by IFN-gamma were observed in premature infants after 27 wk of gestation, with a progressive intrauterine maturation of these activities. Both basal NK activity and NK boosting by IFN-gamma in term neonates were still lower than those of adult controls. The grade of NK boosting by IFN-gamma appeared to depend on the development of basal NK activity. Contrary to IFN-gamma, IL 2 could induce marked NK activity even in 20-wk-old fetuses who lacked both basal and IFN-gamma inducible NK activities. NK boosting by IL 2 was much more efficient than that by IFN-gamma at any period of human life. The facts that IL 2-induced NK boosting could occur without any appreciable production of IFN-gamma in neonatal lymphocytes, and that ample neutralizing doses of anti-IFN-gamma antibody hardly suppressed IL 2-mediated NK boosting even in adult lymphocytes, indicated that the effect of IL 2 on NK boosting might be independent of IFN-gamma production. On the basis of the ontogenic differences in the development of the lymphokine responsiveness of NK cell activity and on the different NK boosting mechanisms of these lymphokines it was suggested that so-called human "pre-NK cells" might be divided into IFN-gamma sensitive and IL 2-sensitive cells. Whether these cell populations belong to different cell lineages or different maturation stages of the same cell line, however, remains unsettled.     

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

Recombinant interleukin 2 enhanced natural killer cell-mediated cytotoxicity in human lymphocyte subpopulations expressing the Leu 7 and Leu 11 antigens

Highly purified recombinant human interleukin 2 (rIL 2) markedly augments the natural killer (NK) cell-mediated cytotoxicity of peripheral blood lymphocytes. In this study, we examined the cellular and metabolic basis of rIL 2-mediated activation of human lymphocyte subpopulations expressing the NK cell-associated surface antigens Leu 7 and Leu 11. All rIL 2-responsive cytotoxic NK cells were found within the subset of lymphocytes expressing the Leu 11 marker, an antigen associated with the Fc-IgG receptor on human NK cells. Cells lacking the Leu 11 antigen, including cells expressing another NK cell-associated marker, Leu 7, did not express NK cell-mediated cytotoxicity either before or after rIL 2 treatment. By contrast, rIL 2 augmented the NK activity of both Leu7-,11+ and Leu 7+,11+ subpopulations. Activation of Leu 11+ NK cells resulted from a direct effect of rIL 2 on these cells and neither required nor was amplified by the presence of T lymphocytes. Enhanced NK cell-mediated cytotoxicity occurred within 4 hr after exposure to rIL 2, and was blocked by the protein synthesis inhibitor cyclohexamide, but not by the DNA synthesis inhibitor mitomycin C or 1500 rad of x-irradiation. Neither Tac antigen, a high-affinity receptor for IL 2, nor other activation markers, such as transferrin receptor or HLA-DR antigen, were detectable on a significant proportion of Leu 11+ cells, either before or after incubation with rIL 2 for 48 hr. In addition, saturating concentrations of antibodies to each of these markers had no effect on the enhancement of NK activity by rIL 2. Finally, preliminary experiments with neutralizing antibodies to gamma- and alpha-interferons also failed to prevent rIL 2 enhancement of NK cell-mediated cytotoxicity, suggesting that rIL 2 does not mediate its effect via release of these cytokines.     

Mode of in vitro augmentation of natural killer cell activity by recombinant human interleukin 2: a comparative study of Leu-11+ and Leu-11- cell populations in cord blood and adult peripheral blood

Human newborn natural killer (NK) cell activity against K562 target cells was observed to be low compared with adult controls. Although Leu-7 (HNK-1)+ cells were negligible in cord blood, the proportions of Leu-11+ cells were equal to those of adult peripheral blood. Leu-11+ cells sorted from cord blood lymphocytes, as well as from adult lymphocytes exhibited the morphology of granular lymphocytes. In this study, we have investigated the phenotypic characterization of recombinant interleukin 2 (rIL 2)-induced cytotoxic lymphocytes against K562 cells by using anti-Leu-11 monoclonal antibody. Spontaneous cytotoxicity of lymphocytes was restricted to Leu-11+ cells in cord blood, as well as in adult blood, but this activity was low in cord blood Leu-11+ cells as compared with that of adult ones. NK cell activity of adult Leu-11+ cells could not be additionally enhanced after an 18-hr incubation with rIL 2(25 U/ml), whereas rIL 2 could potentiate the cytotoxicity of cord blood Leu-11+ cells approximately to the adult levels. It should be noted that cytotoxic activity of both Leu-11- cells from cord blood and adult blood that had no basal NK cells activity could be significantly potentiated by rIL 2. On the other hand, lymphokine-activated killer cells cytotoxic for HL-60 cell line could not be generated, and no proliferation of the lymphocytes was detected after an 18-hr incubation with rIL 2. It was shown that rIL 2 could not enhance the ability to bind to target cells in Leu-11+ and Leu-11- cells by means of a single cell conjugate assay, but the rate of target lysis of Leu-11+ cells from cord blood was significantly enhanced by rIL 2. These results suggested that rIL 2-induced cytotoxic effector cells were heterogeneous, and rIL 2 might potentiate the cytotoxicity of functionally immature NK cells or NK precursor cells.     

rIL 2-induced proliferation of human circulating NK cells and T lymphocytes: synergistic effects of IL 1 and IL 2

Cells participating in the rIL 2-induced proliferation of resting PBMC were identified by using different methods of cell purification. NK cells recovered in the light density fraction of Percoll gradients responded, as already known, directly to rIL 2 by strong proliferation. In contrast, large T lymphocytes co-purifying with NK cells, and small T cells sedimenting in the high density area of the Percoll gradients, were virtually unresponsive when cultivated in the sole presence of rIL 2. However, the addition of either irradiated autologous monocytes or highly purified IL 1 allowed both kinds of T cells to undergo cell division. Stringent elimination of possibly contaminating NK cells (NKH-1+) and/or activated T cells (TNKTAR, Tac+, HLA-DR+) from the high density T cells by complement lysis did not impair rIL 2-induced cell proliferation, indicating entire responsiveness of these cells to the synergistic action of IL 1 plus IL 2. Both high density CD4+ and CD8+ participated in this phenomenon, with an apparent advantage for CD4+ cells. All Tac+ cells emerging in a 6-day culture of these cells expressed the WT31 antigen, which indicates that T cells involved in rIL 2-induced proliferation are conventional mature T cells. The relative precursor frequencies of NK cells, large T lymphocytes, and small T lymphocytes that proliferated in response to rIL 2 were analyzed by limiting dilution analysis. The frequencies of clonal growth of NK cells and low density T lymphocytes were approximately the same (1/103 vs 1/185), whereas that of high density T cells was four times lower (1/458). Thus, we clearly demonstrate that resting T cells, defined as such by morphological, density, and phenotypic criteria, are able to proliferate in response to IL 2 in the presence of IL 1 without antigenic or mitogenic triggering.     

Tumor necrosis factor-alpha enhances cytolytic activity of human natural killer cells

The effect of recombinant tumor necrosis factor-alpha (rTNF alpha) on human natural killer (NK) function was examined. Lysis of both the NK-sensitive K562 erythroleukemia line and the relatively insensitive renal carcinoma line Cur by nonadherent peripheral blood lymphocytes was significantly enhanced as a result of an 18-hr preincubation with either rTNF alpha or recombinant interleukin 2 (rIL 2). When cells were preincubated with rTNF alpha and low doses of rIL 2 (1 to 10 U/ml), marked additional augmentation of lysis of both targets was noted which was greater than that caused by either cytokine alone. Similar results were observed when responses of CD16+ large granular lymphocytes selected with the fluorescence-activated cell sorter after staining with the NK-specific monoclonal antibody Leu-11 were examined, indicating that the action of the cytokines was directly on the cytotoxic cells. Augmentation of tumor cell lysis could not be ascribed to a cytolytic activity of rTNF alpha on the targets, because no combination of rIL 2, rTNF alpha, or interferon-gamma caused lysis of K562 or Cur. By flow cytometric analysis, it was found that expression of IL 2 receptors was induced on purified CD16+ large granular lymphocytes by rTNF alpha alone and to an even greater degree by the combination of rTNF alpha and rIL 2. Additional analysis of the expression of surface antigens and blocking studies with monoclonal antibodies showed that enhanced tumor cell lysis was not caused by the augmentation of leukocyte function-associated antigen-1-mediated effector/target interactions. These data indicate that rTNF alpha alone, or in combination with rIL 2, directly augments NK cytotoxic activity.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

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.     

Induction of proliferation in vitro of resting human natural killer cells: IL 2 induces into cell cycle most peripheral blood NK cells, but only a minor subset of low density T cells

We report that resting human peripheral blood natural killer (NK) cells proliferate in response to recombinant interleukin 2 (rIL 2), and addition of irradiated lymphoblastoid B cells significantly increase their proliferative response. Interaction of IL 2 with the Tac IL 2 receptor expressed on activated NK cells is necessary to maintain continued growth of these cells. Experiments in which NK cell mitosis is prevented by colchicine show that the majority of peripheral blood NK cells are induced into the first cell cycle over a 6-day culture period in the presence of rIL 2. The addition of the irradiated lymphoblastoid B cell line, Daudi, to colchicine blocked cultures does not increase the proportion of cells entering cell cycle in response to rIL 2 alone. In limiting dilution analysis, only 1/1700 B73.1+ cells grow clonally in response to rIL 2. The frequency of clonal growth of NK cells in response to irradiated Daudi cells alone is minimal, whereas the addition of irradiated Daudi cells to rIL 2 stimulated cultures resulted in a 10-fold increase in clonal frequency compared with the cultures in rIL 2 alone. Therefore, Daudi cells may act by maintaining continuous proliferation of the NK cells originally responsive to IL 2. Unlike NK cells, only a minimal proportion of peripheral blood T cells proliferate in response to IL 2. These IL 2 responsive T cells are characterized by a lower bouyant density than the majority of peripheral blood T cells. These results indicate physiologic differences between peripheral blood resting NK and T cells in their ability to be induced to cycle. IL 2 is a growth factor for both cell types, but although the presence of the growth factor is sufficient for quiescent NK cells to be induced into cycle, T cells require antigenic or other mitogenic stimuli to respond to IL 2. The small proportion of light density IL 2 responsive T cells might represent in vivo activated T cells.     

In vivo recombinant interleukin 2 administration enhances survival against a lethal challenge with Toxoplasma gondii

Administration of recombinant interleukin 2 (rIL 2) resulted in a significant (p less than 0.01) decrease in mortality in mice infected with a dose of Toxoplasma gondii that killed 100% of untreated mice. Mice treated with rIL 2 had a significantly (less than 0.005) lower numbers of cysts in the brains. The protection afforded by rIL 2 could not be correlated with increased antibody synthesis or be explained by increased macrophage killing in the treated mice. Mice treated with rIL 2 after Toxoplasma infection demonstrated increased natural killer (NK) cell activity compared with either Toxoplasma-infected or rIL 2-treated mice. rIL 2 failed to reverse the suppressed proliferative response of lymphocytes to concanavalin A and lipopolysaccharide in mice acutely infected with a virulent strain of T. gondii. These results reveal that rIL 2 may have a remarkably protective effect against intracellular parasites.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.     

Generation of cytotoxic cells from murine bone marrow by human recombinant IL 2

Murine bone marrow (BM) cells were cultured in recombinant IL 2 (rIL 2) and interferon-alpha, -beta, and -gamma, and cytotoxic activity against YAC cells was determined in a 4-hr 51Cr-release assay. rIL 2 at 20 U/ml was the only lymphokine that consistently induced significant cytotoxic activity within 3 days of culture, peaking around 5 to 7 days. The cytotoxic cells generated are heterogeneous, consisting of at least two populations of cells: a) NK-1+, Qa-5+, AsGm-1+ Thy-1+/-, Lyt-2- cells, similar to natural killer (NK) cells, and b) NK-1-, Qa-5+, AsGm-1+ Thy-1+, Lyt-2+ cells, similar to cytotoxic T lymphocytes. The precursor/accessory cells of these BM cytolytic cells maintained in 20 U/ml of rIL 2 were Qa-2+, Qa-5+, Thy-1+/-, AsGM-1+/-, and NK-1+/- but Lyt-2-. They also lysed NK-resistant targets, P815 and BW5147, and the antigenic phenotypes of these cells were similar to those that lysed YAC cells. These studies indicate that IL 2 alone is adequate to generate cytotoxic activity from BM and that these cytotoxic cells were similar to splenic NK cells.     

Role of CD2 and IL 1 beta in T cell responsiveness to IL 2

We have evaluated the effects of an anti-CD2 monoclonal antibody (moAb), Leu-5b, on inhibition of lymphocyte responsiveness. Given previous reports that anti-CD2 moAb prevent production of interleukin 2 (IL 2), as well as up-regulation of IL 2 receptor, we chose a test system in which lymphocytes were activated by exogenously added recombinant IL 2 (rIL 2). Our studies thus allowed us to evaluate other possible mechanisms by which anti-CD2 moAb could act. We found that Leu-5b not only blocked the proliferative response of human peripheral blood leukocytes (PBL) to IL 2, but also the development of cytotoxicity as assayed in lectin-dependent cytotoxicity (LDCC). Addition of recombinant interleukin 1 beta (rIL 1 beta) reconstituted the proliferative response in the presence of Leu-5b but did not allow development of cytotoxicity. Our findings are consistent with the concept that Leu-5b delivers a signal to IL 2-responsive cells that prevents their subsequent proliferation and development of cytotoxicity, but that only the former event, i.e., proliferation, is reconstituted by the addition of IL 1 beta.     

Differences in murine gut-associated lymphoid tissues in generating broadly nonspecific cytotoxic cells in response to interferon alpha A/D and interleukin 2

We examined the response of cells of murine gut-associated lymphoid tissues to agents that augment the activity of natural killer (NK) cells. Specifically, we studied the effect of polyinosinic: polycytidylic acid (Poly I:C) in vivo, and recombinant interferon alpha A/D (rIFN alpha A/D) and recombinant interleukin 2 (rIL2) in vitro on lymphoid cells of mesenteric lymph nodes (MLN) and Peyer's patches (PP) in generating cytotoxicity against NK-sensitive (YAC-1) and NK-insensitive (B16BL6) tumor targets. The effect of these agents on spleen cells was examined for comparison with their effect on MLN and PP cells and as a positive control. MLN and PP cells lacked spontaneous NK activity: however, NK activity could be augmented to different levels by the three agents. The treatment of mice in vivo with Poly I:C induced considerable cytotoxicity in the spleen and MLN but only a weak cytotoxic response in PP. The in vitro enhancement of NK activity by rIFN alpha A/D was strong in the spleen, intermediate in MLN, and consistently poor in PP. The weak NK augmentation by rIFN alpha A/D in PP was not restricted to a single mouse strain. PP cells from five strains of mice responded poorly to rIFN alpha A/D. Furthermore, NK augmentation by rIFN alpha A/D in PP cells did not improve after passing the responder cells through nylon wool, indicating that the lack of augmentation of NK activity was not the result of a preponderance of B cells or the masking of NK cells by adherent lymphoid populations in PP. In contrast to weak augmentation of NK activity by rIFN alpha A/D, considerable IL2-induced lymphocyte-activated killer (LAK) activity against NK-insensitive B16BL6 tumor cells was induced in PP. Limiting-dilution analysis showed that the frequency of LAK precursors in the MLN and PP was not markedly different from that of the spleen. The differences among spleen, MLN, and PP lymphoid populations in generating the broadly nonspecific cytotoxic effector cells in response to rIFN alpha A/D or rIL2 may result from differences in the pools of different pre-NK cells or to differential sensitivity of the same pool of pre-NK cells to rIFN alpha A/D and rIL2 in different anatomical locations.     

Precursor phenotype of lymphokine-activated killer cells in the mouse

Lymphokine-activated killer (LAK) activity has been proposed to functionally differ from natural killer (NK) activity largely on the basis of a broader target cell spectrum and different kinetics of response to interleukin 2 (IL 2). Similarly, it has been proposed that the precursor cells for LAK activity are phenotypically distinct from NK cells. In most precursor studies, phenotype comparisons have been made between fresh NK cells and LAK cells which have been generated by 3 to 5 days of culture in IL 2. In the present study, we utilized positive selection with monoclonal antibodies to characterize the surface phenotype of precursor cells which give rise to rIL 2-augmented NK activity within 24 hr and to classically generated LAK activity which appears after 3 to 5 days of culture in rIL 2. The results demonstrated that highly purified (93 to 95%) Lyt-2+ or L3T4+ T lymphocytes were unable to generate appreciable amounts of either augmented NK activity or LAK activity when cultured with rIL 2, whereas the highly purified (98%) Lyt-2-, L3T4-, asialo GM1+ lymphocyte subset gave rise to both augmented NK and LAK activities. These findings demonstrate that both augmented NK and LAK activities can arise from precursors expressing the same phenotype. Overall, the results suggest that NK cells in mouse spleen constitute a major precursor component for the generation of LAK activity from that organ.     

Effects of natural and recombinant IL 2 on regulation of IFN gamma production and natural killer activity: lack of involvement of the Tac antigen for these immunoregulatory effects

Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro synthesis of human IgE by neonatal lymphocytes: enhancing effect of interferon-gamma

The present study demonstrates that neonatal human lymphocytes that are incapable of producing IgG and IgA antibodies may differentiate into IgE-secreting cells under the influence of IL4. Indeed, the addition of recombinant IL4 to cultures of unfractionated umbilical cord blood mononuclear cells (CBMC) induces a dose-dependent synthesis of IgE but not of the other classes of Ig. Moreover, IgE-secreting B lymphoblastoid cell lines can be derived from neonatal lymphocytes costimulated with EBV and IL4. Comparison of the mechanisms regulating the in vitro IgE synthesis by adult and neonatal lymphocytes indicates that in most cases IFN-gamma markedly potentiates the IgE synthesis in CBMC cultures whereas it has a reverse effect on adult lymphocytes. These reciprocal effects of IFN-gamma are specifically blocked by a neutralizing mAb to IFN-gamma; they are dose-dependent and they are observed when IFN-gamma is added at the initiation of the culture or shortly thereafter. Moreover, in a small number of cases IFN-gamma may also potentiate IgE synthesis by adult lymphocytes. The potentiation or the suppression of IgE synthesis by IFN-gamma is not explained by a differential effect of IFN-gamma on the production of soluble CD23 (sCD23); indeed in both cases IFN-gamma slightly increases the IL4-induced production of sCD23. Moreover, the spontaneous and the IL4-induced production of sCD23 by CBMC is comparable to that of adult lymphocytes. The IgE response is dependent upon the expression of Fc epsilon RII (CD23) inasmuch as it is specifically blocked by anti-CD23 mAb.     

Characteristics of mouse lymphoid cells proliferating in vitro by recombinant human interleukin 2 (TGP-3): comparison between normal and nude mice

Various lymphoid cells obtained from BALB/c and BALB/c nu/nu mice were cultured in vitro with recombinant human interleukin 2 (rIL 2), and the characteristics of responder cells to rIL 2 were analyzed. Spleen cells, lymph node cells, and thymocytes except for bone marrow cells obtained from BALB/c mice remarkably proliferated in response to rIL 2. On the other hand, among lymphoid cells obtained from BALB/c nu/nu mice, only lymph node cells showed significant proliferation by rIL 2. Flow cytometric analysis revealed that mainly two types of lymphoid cells were proliferating in response to rIL 2 in BALB/c mice, i.e., Thy 1+, Lyt 1-, Lyt 2- and Thy 1+, Lyt 1-, Lyt 2+ cells. On the other hand, most of the proliferating cells were Thy 1+, Lyt 1-, Lyt 2- cells in BALB/c nu/nu mice. Treatment with various antibodies plus complement revealed that the majority of IL 2-responsive cells in BALB/c mice were Thy 1+, Lyt 1+, and Lyt 2+, although a minor part of them were Thy 1-, Lyt 1-, and Lyt 2-. On the other hand, a predominant type of the IL 2-responsive cells in BALB/c nu/nu mice were Thy 1-, Lyt 1-, and Lyt 2-, though some were Thy 1+. Nonspecific killer activity against tumor cells increased to variable extents in all of the lymphoid cells of both strains after culture with rIL 2. Our results indicate that mouse responder cells to rIL 2 have the following characteristics. First, the responder cells exist abundantly among spleen, lymph nodes, and thymus in normal mice, though their cell lineages are heterogeneous; one is of T cell lineage and the other of natural killer (NK) cell lineage. Second, nude mice are defective in the responder cells of T cell lineage but not of NK cell lineage. Moreover, the responder cells in nude mice predominantly accumulate in the lymph nodes but not other lymphoid organs.