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.     

Lectin-dependent cytotoxicity of human peripheral blood lymphocytes

The evaluative technique of lymphocyte cytolytic activity in human peripheral blood has been designed. The target cells, lectin (Con A) concentration and incubation time for measuring cytolytic activity of lymphocytes pre-purified from adherent cells have been selected. The mean values of lectin-dependent cytotoxicity in peripheral blood of 50 healthy donors are presented.     

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

Differential regulation of fibronectin receptor subunit gene and cell surface expression in human peripheral blood T lymphocytes.

Members of the beta 1 subfamily of heterodimeric integrins, such as the fibronectin receptors alpha 5 beta 1 and alpha 4 beta 1, are expressed on human T lymphocytes. The presence of these two adhesion receptors on T lymphocytes suggests an involvement in cell-cell and cell-extracellular matrix interactions that may be important for the development of immune and inflammatory reactions. We have examined the cell surface expression of alpha 5, alpha 4, and beta 1 subunits on purified peripheral blood T lymphocytes before and after activation with Con A and PMA. Freshly isolated T lymphocytes contained distinct fractions expressing high or low levels of alpha 5 and beta 1. Only a high expressing T lymphocyte population was present after 72-h culture with Con A and PMA. Time course analysis indicated that the shift in alpha 5 and beta 1 expression occurred during the first 24 h after addition of activating agents and occurred in the absence of proliferation. In contrast to alpha 5 and beta 1, essentially all freshly isolated T lymphocytes expressed high levels of alpha 4. After 72-h culture with Con A and PMA, a wide distribution of alpha 4 expression was observed. Further experiments showed that after activation, a proportion of CD4-positive cells decreased their surface expression of alpha 4, but increased their surface expression of alpha 5 and beta 1. In contrast, most CD8-positive cells increased their surface expression of alpha 5, beta 1, and alpha 4 upon activation. An examination of mRNA levels in pan-T lymphocyte cultures after activation indicated that alpha 5 and alpha 4 mRNA expression decreased, whereas beta 1 mRNA expression was unchanged, in Con A/PMA-activated cells as compared to those cultured in medium alone. Our results indicate that T lymphocyte activating agents may differentially affect the expression of alpha 5 beta 1 and alpha 4 beta 1, thus providing a mechanism for the selective regulation of binding interactions that occur at sites of immune reactions.     

Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes.

The membrane potential of human PBMC was modulated in culture by isotonic high extracellular K+ (K+e), or the K+ channel blocker, charybdotoxin (ChTX), to determine the effect of depolarization on stimulated proliferation, IL-2 elaboration, and gene expression. In serum-free cultures, ChTX and high [K+]e induced a specific dose-dependent decrease in IL-2 production. ChTX inhibited proliferation of PBMC and purified T cells, decreased IL-2 elaboration 15 h after stimulation by 78.4 +/- 5.3% (n = 5), and decreased IL-2 mRNA steady-state levels by 80% between 8 and 10 h after stimulation. The IC50 for ChTX-inhibition of IL-2 elaboration and IL-2 mRNA were both 1 nM. Similarly, high [K+]e inhibited proliferation with an IC50 of 38.9 +/- 1.1 mM (n = 13), decreased IL-2 elaboration with an IC50 of 21.3 +/- 1.2 mM (n = 6), and decreased IL-2 mRNA steady-state levels with an IC50 of 18 mM. The sensitivities of both IL-2 production and proliferation to depolarization were substantially reduced by calcium, serum, and exogenous rIL-2. From these findings we conclude that membrane potential may contribute to the control of immune responsiveness in vivo.     

Enhancement of cytotoxicity of human peripheral blood lymphocytes by interferon

Human lung carcinoma cells persistently infected with mumps virus (Pc-10/MpV) were lysed with human peripheral blood mononuclear leukocytes (PBML) obtained from seropositive donors who had anti-mumps virus-neutralizing antibody in their sera. This cellular cytotoxicity was due not to the cytotoxic T lymphocytes but mainly to the non-T, non-B cells, possibly related to natural killer (NK) cells. Moreover, it was concerned not with antibody against mumps virus antigens but with alpha-interferon (IFN-alpha) produced in the mixture of human PBML and Pc-10/MpV cells, since this cellular cytotoxicity was suppressed by anti-human IFN-alpha rabbit serum. Exogeneous IFN-alpha augmented the cytotoxicity of non-T, non-B cells, not T cells, for the uninfected Pc-10 cells. IFN-gamma that had been induced by heat-killed Listeria monocytogenes in PBML had the same capacity to augment NK activity did IFN-alpha.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

Studies on the production and regulation of interleukin,IL-13, IL-4 and interferon-gamma in human Schistosomiasis mansoni

The production and regulation of interleukin (IL) IL-13, IL-4 and interferon-gamma was evaluated in different clinical forms of human schistosomiasis. The mechanisms of immune regulation are apparently different in the various clinical stages of the disease, some of them being antigen specific.     

Autocrine regulation of IL-21 production in human T lymphocytes

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common gamma-chain. IL-21 is mostly produced by CD4(+) T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4(+) T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3(+) T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common gamma-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3(+) lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3(+) lamina propria lymphocytes isolated from patients with Crohn's disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.     

IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.     

Neoantigen of the complement membrane attack complex of cytotoxic human peripheral blood lymphocytes.

Neoantigenic determinants (neoAg) specific for the assembling membrane attack complex (MAC) of complement were detected by immunofluorescence microscopy on the surface of cytotoxic lymphocytes during the antibody-dependent cellular cytotoxicity (ADCC) reaction. This study employed antibody-sensitized chicken erythrocytes as target cells, human peripheral blood lymphocytes as effector cells, and RITC-conjugated rabbit F(ab')2-anti-neoAg. NeoAg was present on 60% of ADCC plaque-forming lymphocytes (PFL). Eight out of 182 neoAg-positive PFL were observed in direct contact with their target cells. In these cases MAC-specific neoAg was visualized at the zone of contact between the cells. Anti-neoAg Ig was found to inhibit ADCC plaque assays up to 62%; and 51Cr-release assays up to 79%. Stimulation of lymphocytes by PHA or mixed lymphocyte culture increased the expression of neoAg. In the case of PHA, increased neoAg expression was correlated with an increased incorporation of 14C-leucine into C5, C6, C7, and C8 antigens, which was detected by immunodiffusion and autoradiography.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.