Interleukin-21 enhances NK cell activation in response to antibody-coated targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.     

NK cells eliminate Cryptococcus neoformans by potentiating the fungicidal activity of macrophages rather than by directly killing them upon stimulation with IL-12 and IL-18

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.     

Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells

NK cells protect hosts against viral pathogens and transformed cells, and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma, despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast, IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition, blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally, presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming.     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.     

Differentiation of murine NK cells into distinct subsets based on variable expression of the IL-12R beta 2 subunit

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rbeta1 and IL-12Rbeta2. The expression of IL-12Rbeta2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-gamma; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8-12 days of culture. Once established, IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rbeta2(high) population. Both IL-12Rbeta2(low) and IL-12Rbeta2(high) NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-gamma and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rbeta2(high) and IL-12Rbeta2(low) murine NK subsets expressed high levels of IFN-gamma, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.     

NK dendritic cells expanded in IL-15 exhibit antitumor responses in vivo

NK dendritic cells (NKDC) are a novel subtype of DC with NK cell properties. IL-15 is a pleiotropic cytokine that plays an obligate role in the proliferation and survival of NK cells. We hypothesized that IL-15 is also essential for NKDC development. NKDC were nearly absent in IL-15(-/-) mice, but restored by administration of exogenous IL-15. Treatment of wild-type mice with IL-15 caused a 2- to 3-fold expansion of both NK cells and NKDC. After 7 days of culture with IL-15, sorted splenic NKDC expanded 10-fold while NK cells increased 5-fold. NKDC expanded in IL-15 retained their cytolytic capacity but lost the ability to stimulate naive T cells. Meanwhile, NKDC expanded in IL-15 produced 10 times more IFN-gamma as fresh NKDC and conferred protection in a tumor prevention model. Thus, IL-15 is essential to the proliferation and survival of NKDC and IL-15 expanded NKDC possess antitumor properties.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

A hybrid protein between IFN-gamma and IL-2

The complementary DNAs encoding human interferon-gamma (IFN-gamma) and human interleukin-2 (IL-2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E. coli to investigate the interactions of these two proteins at the molecular level. Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN-gamma and IL-2. The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN-gamma and the ability to support the growth of natural killer (NK) cells derived from IL-2. Comparing the enhancement of NK cell activity of this hybrid protein with IFN-gamma and IL-2, this hybrid protein appears to conserve each activity almost completely without diminishing the other.     

Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3-), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3-) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-gamma, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-gamma, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-gamma and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-gammaR knockout mice demonstrated that IFN-gamma was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.     

Immunobiology of infection with recombinant vaccinia virus encoding murine IL-2. Mechanisms of rapid viral clearance in immunocompetent mice.

CD8+ cytotoxic T (Tc) lymphocytes mediate recovery from vaccinia virus (VV) infection. In mice, anti-VV Tc cells are detectable on or after day 3 after infection, and cytolytic activity peaks between days 5 and 6. A rVV encoding murine IL-2, VV-hemagglutinin (HA)-IL-2, was cleared more rapidly, compared with a control rVV, VV-HA-thymidine kinase (TK), from tissues of infected euthymic normal mice. The mechanism of VV-HA-IL-2 clearance was operative early in infection and correlated with an elevated NK cell response, before the induction of anti-VV Tc cell response. We have investigated the roles of NK cells, T cells, and IFN-gamma in the rapid clearance of VV-HA-IL-2, by using specific mAb. Depletion of NK cells with mAb significantly enhanced VV-HA-IL-2 but not VV-HA-TK titers 3 days after infection. NK cells alone could not account for rapid viral clearance, because VV-HA-IL-2 titers in NK cell-depleted mice were not comparable to VV-HA-TK titers. Treatment with a mAb to IFN-gamma completely abrogated the IL-2-induced mechanism(s) of VV-HA-IL-2 clearance, and titers of the IL-2-encoding virus were comparable to control virus titers. In addition, the elimination of CD4+ but not CD8+ T cells resulted in significant increases in VV-HA-IL-2 titers.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T cell-deficient hosts infected with Mycobacterium tuberculosis

Although it is known that IFN-gamma-secreting T cells are critical for control of Mycobacterium tuberculosis infection, the contribution of IFN-gamma produced by NK cells to host resistance to the pathogen is less well understood. By using T cell-deficient RAG(-/-) mice, we showed that M. tuberculosis stimulates NK cell-dependent IFN-gamma production in naive splenic cultures and in lungs of infected animals. More importantly, common cytokine receptor gamma-chain(-/-)RAG(-/-) animals deficient in NK cells, p40(-/-)RAG(-/-), or anti-IFN-gamma mAb-treated RAG(-/-) mice displayed significantly increased susceptibility to M. tuberculosis infection compared with untreated NK-sufficient RAG(-/-) controls. Studies comparing IL-12 p40- and p35-deficient RAG(-/-) mice indicated that IL-12 plays a more critical role in the induction of IFN-gamma-mediated antimycobacterial effector functions than IL-23 or other p40-containing IL-12 family members. The increased susceptibility of IL-12-deficient or anti-IFN-gamma mAb-treated RAG(-/-) mice was associated not only with elevated bacterial loads, but also with the development of granulocyte-enriched foci in lungs. This tissue response correlated with increased expression of the granulocyte chemotactic chemokines KC and MIP-2 in NK as well as other leukocyte populations. Interestingly, depletion of granulocytes further increased bacterial burdens and exacerbated pulmonary pathology in these animals, revealing a compensatory function for neutrophils in the absence of IFN-gamma. The above observations indicate that NK cell-derived IFN-gamma differentially regulates T-independent resistance and granulocyte function in M. tuberculosis infection and suggest that this response could serve as an important barrier in AIDS patients or other individuals with compromised CD4+ T cell function.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Basic fibroblast growth factor and interleukins 4 and 6 stimulate the release of IFN-gamma by individual NK cells.

Both the secretory and cytotoxic activity of natural killer (NK) cells are known to be regulated by such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In the present study we have used the reverse hemolytic plaque assay to investigate either the direct effects of the protein kinase activator, phorbol myristate acetate (PMA), or exposure to recombinant human interleukins 2, 4, and 6 (IL-2, IL-4, and IL-6) tumour necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) on the release of IFN-gamma by individual, immunoidentified NK cells isolated from peripheral blood. This sensitive immunoassay was adapted and coupled with immunocytochemistry not only to immunophenotype and enumerate cells secreting IFN-gamma in a given cell population, but also to quantify the amount of this cytokine released per individual cell. These studies have confirmed mononuclear cells with the morphology of large granular lymphocytes and the immunophenotype of CD3-/CD16+ NK cells to be the predominant source of spontaneously released IFN-gamma in vitro. In contrast to this, fewer than 2% of the CD3+ T cells secreted detectable levels of this cytokine during the assay, irrespective of the stimulus applied. Whilst TNF-alpha had no significant effect on IFN-gamma release by NK cells, a 6-hr exposure to IL-2 or PMA stimulated an increase in the amount secreted per single cell. Furthermore, bFGF and interleukins 4 and 6 elicited a marked, dose-dependent stimulation of IFN-gamma secretion by this cell type. However, exposure to these cytokines did not alter the number of cells capable of releasing detectable levels of IFN-gamma during the assay. These studies demonstrate that (i) both the spontaneous and stimulated release of IFN-gamma by NK cells can be visualized and quantified at the single-cell level using this sensitive immunoassay, and (ii) bFGF and interleukins 2, 4, and 6, but not TNF-alpha, are potent stimulants of IFN-gamma secretion by CD3-/CD16+ NK cells.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.