Role of CD2 in regulation of CD3- LGL function.

CD2 is a differentiation marker present on T cells and NK cells. Cytotoxic T lymphocytes (CTL) can be activated by antibodies directed against the CD3/T-cell receptor complex and CD2 structures; however, the role of CD2 in regulation of CD3- large granular lymphocyte (LGL) functions has only recently been studied. Anti-CD2 monoclonal antibodies (mAbs) may be either augmenting or inhibitory and T-cell activation via the CD2 molecule occurs only when mAb binds defined combinations of the CD2 epitopes. Since LGL can be activated by a single stimulus (e.g., IL-2) to proliferate, produce IFN gamma, and increase their cytolytic potential, these functions were chosen to examine the effects of the anti-CD2 mAb and its combinations. Anti-CD2 mAb (D66, GT2, and X11-1) were incubated with LGL for various times in the absence or presence of IL2 and IFN gamma production was monitored. Single anti-CD2 mAb treatment demonstrated minimal augmentation of IFN gamma production. However, combinations of anti-CD2 (9.6) and the other anti-CD2 mAb resulted in a significant, synergistic enhancement of the IFN gamma production. Anti-CD2 mAb treatment appeared to inhibit production generated by optimal doses of IL-2 (1,000 U/ml). The effect of anti-CD2 mAb on IFN gamma production parallel their effects on LGL NK and LAK activity. These data suggested that mAb against the CD2 molecule were important in regulating LGL functions in the absence of a functional CD3 receptor in LGL.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.     

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.     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

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.     

Promotion of human T lymphocyte proliferation by IL-4

The capacity of human rIL-4 to support the proliferation of mitogen-stimulated T cells directly as well as by increasing IL-2 production or enhancing IL-2 responsiveness was investigated. IL-4 augmented proliferation of T cells stimulated with PHA, Con A, immobilized mAb to the CD3 molecular complex (OKT3), or PMA. IL-4 increased the number of mitogen-stimulated cells entering the cell cycle as well as enhancing ongoing proliferation of mitogen-activated lymphoblasts. Facilitation of initial activation by IL-4 was not inhibited by mAb to the p55 component of the IL-2R, anti-Tac, and, therefore, was not dependent on endogenous IL-2 activity. However, IL-4-mediated enhancement of ongoing T cell proliferation stimulated by PHA or OKT3 was partially but not completely blocked by anti-Tac. Analysis of the supernatants from PHA-stimulated T cell cultures indicated that IL-4 increased the production of IL-2 by mitogen-activated cells. Moreover, IL-4 increased the amount of IL-2 mRNA that accumulated in mitogen-stimulated T cells. In addition, IL-4 markedly augmented IL-2R expression by PHA-stimulated T cells. Although IL-4 promoted ongoing DNA synthesis of mitogen-stimulated T cells in an IL-2-dependent manner, it was also able to sustain their proliferation directly. Thus, IL-4 supported proliferation of PMA-activated T cells in a manner that was not inhibited by anti-Tac. Furthermore, IL-4 could augment proliferation and IL-2R expression of T cells stimulated with PHA in the presence of cyclosporin A, which blocks endogenous cytokine production or anti-Tac. Finally, IL-4 was noted to enhance proliferation of both CD4+ and CD8+ T cell subsets. The results indicate that IL-4 enhances proliferation of mitogen-activated human T cells by a number of mechanisms, including the direct promotion of cell cycle entry and subsequent DNA synthesis, enhanced production of IL-2, and increased responsiveness to IL-2 in part by up-regulation of IL-2R expression.     

IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19+ cells, and expression of IL-2R(p55) on either CD3+ or Leu-19+ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19+ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19+ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.     

Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of IL-12-activated natural killer cells

The importance of cell adhesion molecules such as ICAM-1 is emphasized in cell-to-cell interactions that are critical in the generation of effective immune reactions. In this study, the involvement of ICAM-1 in natural killer (NK) cell activities was characterized in IL-12-activated human NK cells. To address the question of whether ligation of ICAM-1 molecules can modulate NK cell cytolytic activities, a 4-h (51)Cr-release assay was performed after pretreatment of NK cells with R6.5 mAb (anti-human ICAM-1 mAb). Ligation of membrane ICAM-1 molecules significantly inhibited IL-12-enhanced NK cytotoxicity against K562, and the pretreatment of neutralizing soluble ICAM-1 with R6.5 mAb blocked this inhibitory effect. The involvement of Ca(2+)-dependent granular exocytosis was evaluated. BLT esterase assay demonstrated that the ligation of ICAM-1 molecules inhibited granular exocytosis of NK cells. Additionally, the ICAM-1-mediated inhibition of Ca(2+) flux in NK cells was detected using Fluo-3AM, while the pretreatment of NK cells with R6.5 mAb did not affect conjugate formation between NK and K562 cells. Collectively, these results suggest that the signals transduced from ICAM-1 molecules might be sufficient to induce inhibitory effects on NK cells.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.