Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes

Interleukin (IL)-12, a natural killer (NK) cell stimulatory factor, is a heterodimeric cytokine that is known to be a potent activator of non-major histocompatibility complex-restricted cytotoxicity by peripheral blood-derived NK cells. NK cells (CD3-CD16+/CD56+) represent approximately 15% of human umbilical cord blood mononuclear cells (HUCB MNCs) and are known to be highly sensitive to activation by IL-2. In the present study, we monitored the effect of IL-12 on the cytotoxic activity, proliferation, and phenotypic expression of HUCB-derived resting and IL-2-activated cytotoxic cells and compared these parameters with those of bone marrow (BM)-derived cells. Lymphocytes were separated from HUCB by 3% gelatin sedimentation and incubated with IL-12 and/or IL-2 for 18 hours. At effector:target ratios of 40:1 and 20:1, IL-12 (50 U/mL) significantly increased both resting and IL-2-activated NK cell-mediated cytotoxicity in a standard 51Cr-release assay against both NK-sensitive (K562) and NK-resistant (Colo-205) cell lines. In addition, resting and IL-2-activated cytotoxic cells derived from HUCB exhibited superior cytolytic ability compared with BM-derived cells. This increase was observed in resting cells as well as in those that were preincubated with IL-12. Moreover, HUCB-derived cells were found to be more sensitive to IL-12 activation than cytotoxic cells from BM. To evaluate the involvement of accessory cells, NK cells were purified from HUCB using immunomagnetic beads, and these cells were found to have a lower response to treatment with IL-12 than unpurified populations. HUCB MNCs exhibited a nonsignificant increase in proliferation after IL-12 treatment and were better able to respond to IL-12 activation than BM MNCs. Following an 18-hour incubation, IL-12 was able to cause upregulation of CD25 and CD69 activation antigens, whereas no significant change in expression of CD16 and CD56 NK cell surface antigens, CD3 on T cells, or IL-12 receptor was observed. Similarly, IL-12 did not affect NK cell:target cell conjugation as assessed by fluorescence-activated cell sorting. Our results indicate that HUCB-derived NK-mediated cytotoxic capabilities can be increased by IL-12, a finding that may have clinical relevance.     

High lytic activity against human leukemia cells after activation of allogeneic NK cells by IL-12 and IL-2

IL-12 is a novel cytokine with interesting features regarding its potential usefulness in peripheral blood stem cell transplantation and leukemia immunotherapy. We used cryopreserved leukemia cells of 18 patients with acute myelogenous (n= 14) or lymphocytic (n= 4) leukemia to investigate the effect of IL-12, alone or in combination with IL-2, on the cytolytic activity of NK cells against human leukemia targets. Effector cells were peripheral blood mononuclear cells from healthy donors which were depleted from CD3+ T cells by immunomagnetic separation. CD3-negative effector cells (mainly CD56+ NK cells) were treated for 24 h with various concentrations of IL-2 (100 U/ml to 1000 U/ml) and IL-12 (1 U/ml to 100 U/ml). Cytotoxicity was measured in a 4 h 51Cr-release assay. Whereas a two-fold enhancement of cytotoxic activity was observed after incubation with optimal doses of IL-2 or IL-12, the combination of both cytokines (500 U/ml IL-2, 100 U/ml IL-12) increased the lytic activity more than six-fold. This effect was accompanied by increased expression of cellular adhesion molecules (CD2, CD18) and CD25 on CD56+ effector cells. Of 18 leukemias investigated, five were completely resistant to lysis by effector cells activated with IL-2 or IL-12 alone. In three of these five cases, however, high cytolytic activity was observed after coincubation with IL-2 and IL-12. In comparison to allogeneic NK cells, autologous cells of three patients in remission demonstrated significantly lower cytotoxic activity. No killing of nonmalignant cells (PHA blasts) by allogeneic NK cells was observed. Our data demonstrate that IL-12 can enhance or even induce MHC-unrestricted cytotoxicity of IL-2-activated allogeneic natural killer cells. Since IL-12 has also been shown to have stem-cell mobilizing capacities, it could be used for the recruitment of both stem cells and antileukemic effector cells in the context of peripheral blood stem cell transplantation.     

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer

BACKGROUND: Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. METHODS: NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. RESULTS: PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. CONCLUSION: This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.     

Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosin alpha1

The effects of prothymosin alpha1 (Pro alpha1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro alpha1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro alpha1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN gamma secretion. No significant effects on the low level of TNF alpha secretion was noted. By flow cytometry, Pro alpha1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro alpha1 was equally effective by increasing the expression of CD18 and CD11a on lymphocytes from the patients and from normal controls. In conclusion, Pro alpha1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro alpha1 or related thymic peptides in selected immunotherapies against colorectal tumors.     

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.     

Combination chemotherapy and IL-15 administration induce permanent tumor regression in a mouse lung tumor model: NK and T cell-mediated effects antagonized by B cells

Previous studies have demonstrated that IL-15 administration after cyclophosphamide (CY) injection of C57BL/6J mice bearing the i.m. 76-9 rhabdomyosarcoma resulted in a significant prolongation of life. In the present study, we investigated the immune response against the 76-9 experimental lung metastases after CY + IL-15 therapy. Administration of CY + IL-15, but not IL-15 alone, induced prolongation of life and cures in 32% of mice bearing established experimental pulmonary metastases of 76-9 tumor. The CY + IL-15 therapy resulted in increased levels of NK1.1+/LGL-1+ cells, and CD8+/CD44+ T cells in PBL. In vitro cytotoxic assay of PBL indicated the induction of lymphokine-activated killer cell activity, but no evident tumor-specific class I-restricted lytic activity. Survival studies showed that the presence of NK and T lymphocytes is necessary for successful CY + IL-15 therapy. Experiments using knockout mice implied that either alphabeta or gammadelta T cells were required for an antitumor effect induced by CY + IL-15 therapy. However, mice lacking in both alphabeta and gammadelta T cells failed to respond to combination therapy. Cured B6 and alphabeta or gammadelta T cell-deficient mice were immune to rechallenge with 76-9, but not B16LM tumor. B cell-deficient mice showed a significant improvement in the survival rate both after CY and combination CY + IL-15 therapy compared with normal B6 mice. Overall, the data suggest that the interaction of NK cells with tumor-specific alphabeta or gammadelta T lymphocytes is necessary for successful therapy, while B cells appear to suppress the antitumor effects of CY + IL-15 therapy.     

AK-5 tumor-induced expression of interleukin-12: role of IL-12 in NK-mediated AK-5 regression

Spontaneous regression of AK-5, a histiocytic tumor, is mediated by CD3-, CD8+ NK cells through ADCC. The onset of AK-5 regression is associated with the induction of humoral immune response and the augmentation of effector function. The mechanism of tumor cell death involves both necrosis and apoptosis. Interleukin-12, a 75-kDa heterodimeric cytokine, has multiple effects on T and NK cells. We have investigated the role of IL-12 in the NK cell-mediated AK-5 tumor regression process. Subcutaneous transplantation of AK-5 tumor induced the expression of IL-12 (p35 and p40) message by Day 6-8 in the splenocytes of syngenic rats. Similarly, analysis of serum samples from tumor-bearing animals showed the presence of circulating IL-12 around the same time. Interaction of immune cells with antibody-tagged AK-5 cells in vitro also triggered the expression of IL-12 message and protein by 3 hr. The circulating IL-12 in the sera of tumor-rejecting animals, as well as rIL-12, stimulated NK cell proliferation, expression of CD16 and CD25, and the activation of NK cells function. These observations suggest that the ability of the AK-5 tumor to induce the endogenous production of IL-12 may be responsible for keeping the NK cells constantly in an activated state, thus demonstrating an efficient mechanism for the complete regression of the tumor.     

Induction of the 2B9 antigen/dipeptidyl peptidase IV/CD26 on human natural killer cells by IL-2, IL-12 or IL-15

Activation of human natural killer (NK) cells involves sequential events including cytokine production and induction of cell surface molecules, resulting in the enhancement of cytolytic activity. To delineate the activation process of NK cells, we generated murine monoclonal antibodies (mAbs) against YT, a human large granular lymphocyte/natural killer (LGL/NK) cell line. Among the mAbs reactive with YT cells, one mAb, termed 2B9, was noted because of the lack of reactivity with most of the human T- and B-cell lines tested. In fresh peripheral blood mononuclear cells (PBMC), however, the majority of cells expressing this antigen (Ag) were T cells but not CD16+ nor CD56+ NK cells. Since YT cells showed an activated phenotype expressing interleukin-2 (IL-2) receptor alpha chain, we examined whether 2B9 Ag could be induced on normal human peripheral blood NK cells by cytokines known to activate NK cells. The 2B9 Ag was induced on NK cells by IL-2, IL-12 or IL-15 while no induction was observed by interferon-gamma (IFN-gamma). Biochemical analysis showed that anti-2B9 mAb recognized a 115 kDa molecule in YT cells. A cDNA clone encoding the 2B9 Ag was isolated from a cDNA expression library of YT cells and its sequence was identical to CD26 cDNA although it was not of full length. Transient expression of the 2B9 cDNA on COS-7 cells revealed that this cDNA encodes the antigenic epitope(s) recognized by anti-2B9 mAb as well as Ta1, an anti-CD26 mAb. These results showed that the 2B9 Ag is identical to CD26, and demonstrated that CD26 is an activation antigen on CD16+ CD56+ NK cells inducible by IL-2, IL-12 or IL-15.     

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.     

Human natural killer cell development from bone marrow progenitors: analysis of phenotype, cytotoxicity and growth

We have developed a culture system allowing for generation of NK cells from human CD34+ bone marrow progenitors. The appearance of NK cells expressing CD56+, CD3- phenotype and large granular lymphocyte morphology was observed after 2-3 weeks of culture with IL-2. The NK cell appearance coincided with development of lytic activity. NK cells generated in bone marrow cultures proliferated actively (expansion index ranged from 2- to 200-fold). The phenotype of NK cells generated from CD34+ bone marrow deviated from peripheral blood NK cells in that a lower percentage of the former cells expressed CD16, CD2, CD7, and CD8 antigens. NK cells were also generated from CD34+ populations depleted of the CD34+, CD33+ subset indicating that myeloid-committed progenitors are not required for NK cell development. The dose of IL-2 was not important for generation of NK cells; however, only high doses of IL-2 supported development of optimal NK cell cytotoxic potential. Addition of TNF-alpha facilitated IL-2-dependent NK cell generation. These data showed that NK cells can develop from early bone marrow progenitors and that this system may be instrumental in studies on NK cell lineage and differentiation.     

Influence of sulfur oxyanions on reductive dehalogenation activities in Desulfomonile tiedjei

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.     

IL-18 up-regulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor

IL-18 is a powerful inducer of IFN-gamma production, particularly in collaboration with IL-12. IL-18, like IL-12, also augments NK activity. Here we investigated the molecular mechanism underlying the up-regulation of killing activity of NK cells by IL-18. IL-18, like IL-12, dose dependently enhanced NK activity of splenocytes. This action was further enhanced by costimulation with IL-12. Treatment with anti-IL-2R Ab did not affect IL-18- and/or IL-12-augmented NK activity, and splenocytes from IFN-gamma-deficient mice showed enhanced NK activity following stimulation with IL-12 and/or IL-18. Splenocytes from the mice deficient in both IL-12 and IL-18 normally responded to IL-18 and/or IL-12 with facilitated NK activity, suggesting that functional NK cells develop in the absence of IL-12 and IL-18. IL-18R, as well as IL-12R mRNA, was constitutively expressed in splenocytes from SCID mice, which lack T cells and B cells but have intact NK cells, and in those from IL-12 and IL-18 double knockout mice. NK cells isolated from SCID splenocytes expressed IL-18R on their surface. IL-18, in contrast to IL-12, did not enhance mRNA expression of perforin, a key molecule for exocytosis-mediated cytotoxicity. However, pretreatment with concanamycin A completely inhibited this IL-18- and/or IL-12-augmented NK activity. Furthermore, IL-18, like IL-12, failed to enhance NK activity of splenocytes from perforin-deficient mice. These data suggested that NK cells develop and express IL-12R and IL-18R in the absence of IL-12 or IL-18, and that both IL-18 and IL-12 directly and independently augment perforin-mediated cytotoxic activity of NK cells.     

Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10

Interleukin-12 (IL-12) induces differentiation of T helper 1 (Th1) cells, primarily through its ability to prime T cells for high interferon-gamma (IFN-gamma) production. We now report that the presence of IL-12 during the first several days of in vitro clonal expansion in limiting dilution cultures of polyclonally stimulated human peripheral blood CD4+ and CD8+ T cells also induces stable priming for high IL-10 production. This effect was demonstrated with T cells from both healthy donors and HIV+ patients. Priming for IL-4 production, which requires IL-4, was maximum in cultures containing both IL-12 and IL-4. IL-4 modestly inhibited the IL-12-induced priming for IFN-gamma, but almost completely suppressed the priming for IL-10 production. A proportion of the clones generated from memory CD45RO+ cells, but not those generated from naive CD45RO- CD4+ T cells, produced some combinations of IFN-gamma, IL-10, and IL-4 even in the absence of IL-12 and IL-4, suggesting in vivo cytokine priming; virtually all CD4+ clones generated from either CD45RO(-) or (+) cells, however, produced high levels of both IFN-gamma and IL-10 when IL-12 was present during expansion. These results indicate that each Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-10) cytokine gene is independently regulated in human T cells and that the dichotomy between T cells with the cytokine production pattern of Th1 and Th2 cells is not due to a direct differentiation-inducing effect of immunoregulatory cytokines, but rather to secondary selective mechanisms. Particular combinations of cytokines induce a predominant generation of T cell clones with anomalous patterns of cytokine production (e.g., IFN-gamma and IL-4 or IFN-gamma and IL-10) that can also be found in a proportion of fresh peripheral blood T cells with "memory" phenotype or clones generated from them and that may identify novel Th subsets with immunoregulatory functions.     

Clinical and pharmacokinetic observations on fluconazole in the management of cryptococcal meningitis

Natural killer (NK) cell activity, the autologous mixed lymphocyte reaction (AMLR) and proportions of T cell subpopulations (CD3+/CD4+ and CD3+/CD8+) and NK cells (CD16+) were studied in 21 patients with bilateral primary breast cancer (BBC), 10 patients with single-breast cancer (SBC) and 20 healthy controls. All patients studied had no evidence of disease and had been off radiotherapy and/or chemotherapy for at least 1 year. Ten patients with BBC were also treated with tamoxifen. Patients with SBC had NK cell activity, AMLR responses and T cell subpopulations that were comparable to those of normal controls. In patients with BBC, a significant (P < 0.01) increase in NK activity compared to that in normal controls (42 +/- 13% versus 21 +/- 10%, effector-to-target cell ratio, 25:1) and a significant (P < 0.05) decrease in CD4+ T cell proportions (30 +/- 15% versus 49 +/- 13%) and absolute numbers (472 +/- 82/mm3 versus 953 +/- 131/mm3) were found. However, the proliferative response of BBC patients' T lymphocytes in AMLR was in the range of the normal controls. Lymphocytes derived from 10 BBC patients treated with tamoxifen exhibited NK cell activity that was comparable to that of normal controls and patients with SBC, and was significantly (P < 0.01) reduced compared to the pretreatment period. BBC patients who received tamoxifen also show a reduction in the proportion of CD4+ T cells and in AMLR proliferative responses, which decreased compared to levels in normal controls. Taken together, these results indicate that long-term tamoxifen treatment modulates immune responses in BBC patients.     

Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12

Interleukin (IL)-2 IL-7 and IL-12 stimulate the generation of lymphokine-activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56+ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL-7-stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon-gamma (IFN-gamma) mRNA than did IL-2, and IL-7 only weakly influenced the IFN-gamma expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-gamma, whereas high amounts of IFN-gamma and low levels of GM-CSF were detected in supernatants from IL-12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.     

Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection

Human NK cells have been shown to produce cytokines (e.g., IFN-gamma and TNF-alpha) and the chemokine macrophage inflammatory protein (MIP)-1alpha following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1alpha, MIP-1beta, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1alpha, MIP-1beta, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely > or = 90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1(NFN-SX) and T cell-tropic HIV-1(NL4-3) replication in vitro, but not dual-tropic HIV-1(89.6). Importantly, the C-C chemokines MIP-1alpha, MIP-1beta, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2-type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross-linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.