Lymphokine-activated killer cell regulation of T-cell-mediated immunity to Candida albicans.

Monocytes are important accessory cells in the activation of T cells for specific antigen recognition yet little is known of their regulation. We demonstrated here that interleukin-2 (IL-2)-induced human lymphokine-activated killer (LAK) cells can inhibit monocyte antigen presentation, depending on the state of differentiation of the monocytes. Adherent monocytes cultured for 4 days in medium or granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to equally process and present intact Candida albicans to autologous Percoll gradient-isolated T cells, as measured by [3H]thymidine uptake. However, only the GM-CSF-cultured monocytes were functionally inhibited by autologous 4-day IL-2-induced LAK cells. Even soluble candidal cell wall mannoprotein antigens could not be presented by these monocytes after exposure to LAK cells. Pretreatment of these monocytes with LAK cells for 1 h, followed by subsequent removal of the nonadherent LAK cells, was sufficient to cause significant inhibition, with maximal inhibition observed after 4 h. Northern (RNA) blot analysis indicated that mRNA expression for IL-1 alpha and IL-1 beta in response to C. albicans stimulation was also down-regulated in GM-CSF-cultured monocytes exposed to LAK cells. Interestingly, freshly isolated, Percoll gradient-purified large granular lymphocytes did not suppress antigen presentation in GM-CSF-treated monocytes. Another important finding was the inability of LAK cells to suppress the ability of freshly isolated or gamma interferon-cultured monocytes, which are resistant to LAK cell-mediated lysis, to present antigen to T cells. In contrast, IL-3 was similar to GM-CSF in inducing LAK cell susceptibility in monocytes. Taken together, these results indicated that IL-2 can induce LAK cells to down-regulate antigen presentation function in a select set of monocytes that have been activated by colony-stimulating factor (GM-CSF and IL-3) but not by gamma interferon. LAK cells may therefore play an important role in regulation of monocytes and their function, depending on their differentiation state.     

小鼠血小板/T细胞活化抗原1的表达分布与功能

目的研究小鼠血小板／Ｔ细胞活化抗原１（ＰＴＡ１）在小鼠组织及各种细胞系的表达分布与诱导,及其在杀伤性Ｔ细胞和ＬＡＫ细胞诱导中的作用。方法应用流式细胞仪技术分析ＰＴＡ１在小鼠组织及细胞系中的表达及诱导;用同种异体抗原诱导杀伤性Ｔ细胞,用ＩＬ－２诱导ＬＡＫ细胞,分别研究ＰＴＡ１特异性单克隆抗体对其分化的影响。结果小鼠ＰＴＡ１主要在Ｔ细胞系呈诱导性表达,参与杀伤性Ｔ细胞的分化,但不参与ＬＡＫ细胞的诱导。结论小鼠ＰＴＡ１与人ＰＴＡ１有相似的分布特点与功能,是一种进化中高度保守的白细胞分化抗原     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.     

Lyt-Phenotype Conversion of Cytotoxic T Lymphocytes Specific for the A and E Class II Major Histocompatibility Complex Molecules

The Lyt-1+ (high) Lyt-2+/- (low) primary cytotoxic T lymphocytes (CTL) specific for A(A alpha A beta) molecules and the Lyt-1+Lyt-2+ primary E(E alpha E beta)-specific CTL are both shown to become Lyt-1 Lyt-2+ effector cells after secondary in vitro stimulation. Thus CTL specific for class II major histocompatibility complex molecules exhibit the same Lyt-phenotype shift as class-I-specific CTL do. The data suggest that either both class-I-specific and class-II-specific CTL follow the same differentiation pathway or regulatory cellular interactions allow only Lyt-1-Lyt-2+ cells to differentiate to secondary CTL.     

Allospecific proliferative human T-cell clones acquire the cytotoxic effector function after three months in culture, in IL-2 conditioned medium

Allostimulated T lymphocytes were cloned by micromanipulation and expanded in IL-2 conditioned medium. Three T3+,T4+,T8-, clones called BJ1, BJ4, and BJ37, were extensively studied. The BJ1 cells were able to proliferate and kill the specific target. The BJ4 and BJ37 cells were able to proliferate with the specific restimulator but could not kill even in lectin-dependent cell-mediated cytotoxic assay; however, they acquired the specific cytolytic activity in the 6-day culture when fresh irradiated autologous peripheral blood mononuclear cells as feeder cells were added to the specific irradiated Epstein-Barr virus transformed cell line, in the presence of recombinant IL-2. This observation strongly suggested that the culture conditions could be involved in the differentiation of proliferative clones into cytotoxic T lymphocyte (CTL) clones, by the lymphokines, either present in the IL-2 conditioned medium or secreted by the mixed allogeneic irradiated feeder cells. Moreover, it was shown that the acquisition of the cytolytic function could be blocked by the monoclonal antibody LeoA1, previously described and which recognized the TLiSA1 structure involved in the CTL differentiation.     

Functionally involved cell surface antigens on murine lymphokine activated killer cells

To investigate the possible involvement of some cell surface structures on lymphoid cells in the functional activity of lymphokine activated killer (LAK) cells, a number of monoclonal antibodies (Mab) against such structures was studied for their ability to inhibit LAK activity in a standard cytotoxicity assay against the natural killer-insensitive target cell EL-4. Almost complete inhibition of LAK activity resulted from incubation with antibodies to the LFA-1 antigen, while blocking of the Lyt 2 antigen reduced cytotoxic activity about 50%. Mab to T-200 gave a weak and inconsistent inhibitory activity, while antibodies to Thy 1, L3T4, IL-2 receptor and MHC class I antigens were without effect. Mab to LFA-1 and Lyt 2 inhibited LAK activity towards EL-4, YAC-1 and differentiated F-9 teratocarcinoma cells, but did not affect LAK-mediated killing of undifferentiated F-9 cells. Experiments with separate preincubation of effector and target cells revealed that both LFA-1 and Lyt 2 inhibited LAK activity at the effector cell level only.     

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.     

Interleukin-2 modulates the expression of lymphocyte function-associated antigen-one (LFA-1) and p150,95 during the generation of lymphokine-activated killer (LAK) cells

Lymphocyte function-associated antigen-one (LFA-1), Mac1 and p150,95 represent a family of heterodimeric cell surface molecules with a common beta subunit and distinct alpha subunits. LFA-1 is known to be functionally important in cell-cell interactions between immune cells. In the present study, a mouse monoclonal antibody (mAb), RH1-38, which recognizes an epitope on the beta-chain of LFA-1 was used to study the function and expression of LFA-1 on lymphokine-activated killer (LAK) cells. This mAb has been shown previously to block, in the absence of complement, cytolytic activity mediated by natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and a monocyte-like cell (phorbol diester-stimulated HL-60 cells). LAK cells were generated by culturing in vitro human peripheral blood lymphocytes (PBL) in the presence of human recombinant interleukin-2 (rIL-2), and cytotoxic activity was measured by a 51Cr-release assay using the human NK-resistant Daudi cell line. Addition of RH1-38 ascites supernatant, purified RH1-38 mAb, or F(ab')2 fragment of RH1-38 markedly reduced (>80) LAK cytolytic activity, whereas NS-1 (parent hybridoma) ascites supernatant, normal mouse IgG, and monoclonal anti-HLA had no effect on LAK-mediated killing. Equivalent inhibition of NK and CTL activity by purified RH1-38 required 10-100-fold more antibody. Appreciable inhibition occurred if the mAb was added up to 2 hr after LAK cells were mixed with targets. Indirect immunofluorescence flow cytometry and immunoprecipitation studies revealed that LFA-1 and p150,95 expression were dramatically enhanced in PBL populations cultured with rIL-2 compared with PBL cultured without rIL-2; Daudi cells expressed no detectable LFA-1 family heterodimers. Time-course experiments demonstrated that during culture of PBL in the presence of rIL-2, development of enhanced expression of LFA-1 and p150,95 correlated closely with LAK cytolytic activity. These studies (i) demonstrate that LFA-1 and/or p150,95 are functionally important effector cell surface molecules expressed by LAK cells and that some homology to NK and CTL mechanisms of cell-mediated lysis may exist; and (ii) suggest that enhanced LFA-1 and/or p150,95 expression are important for development of the fully differentiated LAK effector cell in the presence of rIL-2.     

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T alpha 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in T alpha 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and T alpha 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and T alpha 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1 beta) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/inducer (Th, CD4+) and cytotoxic/suppressor (Tc, CD8+) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying T alpha 1 immunoregulatory properties.     

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.     

The cytotoxic T lymphocyte response in reticuloendotheliosis virus-infected chickens is mediated by alpha beta and not by gamma delta T cells.

We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.     

IL-15 induces unspecific effector functions in human peptide-specific CD8+ T-cell cultures

Antigen (Ag)-specific CD8+ T cells are a major host defence against viral infections. In the present study, we generated human CD8+ T-cell lines specific towards influenza matrix peptide (IMP)-pulsed Ag-presenting cells. We compared the effect of interleukin-2 (IL-2) and IL-15 on the proliferation and cytotoxic activity of primary and secondary IMP-specific cytotoxic T lymphocyte (CTL) culture. In primary CTL cultures, IL-15-induced cell expansion was considerably reduced as compared with IL-2-induced cell expansion, and IL-15 favoured the outgrowth of CTLs without peptide specificity in these cultures. Secondary IMP-specific CD8+ T cells were generated by the addition of IL-2 during two cycles of restimulation. From the third restimulation, identical CTL cultures were expanded with either IL-2 or IL-15 in parallel. Cell expansion as well as Ag specificity was considerably reduced after a 5 day culture period in the presence of IL-15. No or low CD69 expression was observed in IL-15-cultured CTLs, whereas IL-2-cultured CTLs contained high fractions of CD69+ cells. Furthermore, a high fraction of these latter cells coexpressed the cytotoxic marker CD56. However, IL-15-cultured CTLs exhibited cytotoxic activity without detectable expression of CD56, suggesting that CD56 is not essential for cytotoxic activity. Thus, the results presented suggest that IL-15 favours the outgrowth of unspecific cytotoxic effector T cells.     

Signal transduction via phosphorylated adhesion molecule, LFA-1{beta} (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

It is well known that IL-2 stimulates natural killer (NK) cellsto express lymphokine activated killer (LAK) activity and thatthis stimulation prompts the acquisition of the ability to lysepreviously insensitive target cells. The possible role of adhesionmolecules in the IL-2 activation process was probed by focussingon a lymphocyte function-associated antigen (LFA)-1-dependentmodel system. A mAb to the LFA-1ß chain abrogatedLAK activity, but only moderately suppressed NK activity, suggestinga differential role for LFA-1ß In LAK compared withNK mediated lysis. Orthophosphate labeling demonstrated thatthe LFA-1ß chain was strongly phosphorylated in LAKbut not NK cells; in contrast, the chain was phosphorylatedsimilarlyin both effector cell types. At least a portion ofthe phosphorylation of the ß chain was on tyrosineresidues, as shown by Western blotting with anti-phosphotyrosineantibody of LFA-1ß immunoprecipitates. Crosslinkingof the LFA-1ß chain with plastic-adhered antibodystimulated Ca²⁺-dependent release of cytoplasmic lytic granulesand induced phosphatidyl inositol turnover in LAK but not NKcells. We conclude that the IL-2-induced phosphorylation oftheß chain of the LFA-1 adhesion molecule in LAK cellsand associated alteration in signal transduction may be importantin the stimulation of LAK cell activity in NK cells.     

Allosensitization in IL-2-containing limiting dilution cultures generates cytotoxic T lymphocytes (CTL) rather than LAK cells reactive with a syngeneic nonimmunogenic murine tumor

We have previously demonstrated that high frequency (1/20) of potent cytotoxic cells reactive with the nonimmunogenic lymphoma PIR-2 of C57BL/6 (B6, H-2b) origin, can be obtained by allosensitization of syngeneic B6 splenocytes against BALB/c (H-2d) splenocytes in limiting dilution cultures (LDC). Since a high concentration (250 U/ml) of exogenous interleukin 2 (IL-2), sufficient for the elicitation of lymphokine-activated killer (LAK) cells, was used in the LDC, and because the LDC-derived cytotoxic cells were active against a wide spectrum of target cells, we investigated whether the anti PIR-2 effector cells are LAK cells or cytotoxic T lymphocytes (CTL). We found that depletion from the B6 responder cell population of Lyt2+ (CTL precursors), but not of asialo GM1+ (LAK cell precursors), prior to LDC, results in the ablation of anti PIR-2 activity. When B6 splenocytes were plated in LDC with IL-2, in the absence of allogeneic stimulating cells, the resulting anti PIR-2 activity was greater than 10- to 500-fold lower than that obtained in LDC in the presence of allogeneic stimulating cells and IL-2. These and other observations suggest that the cytotoxic response against syngeneic tumors elicited by alloantigens in LDC is mediated by CTL rather than LAK cells, and that allogeneic sensitization in LDC can provide a means for the generation of CTL against syngeneic, nonimmunogenic tumors.     

Lymphocyte activation markers in idiopathic myositis: changes with disease activity and differences among clinical and autoantibody subgroups

We studied the immunologic correlates of disease activity and differences among subgroups of patients with idiopathic inflammatory myopathy by analysing phenotypic and activation marker expression on peripheral blood mononuclear cells (PBMC). Compared with controls, myositis patients with clinically active disease (n = 51) had significantly lower proportions of CD8+ cells and higher proportions of PBMC that expressed DR, CD3- DR, CD14- DR, interleukin-2 receptors, and the late T cell activation markers CD26 and TLiSA1. TLiSA1 expression, a marker for cytotoxic differentiation, correlated significantly with both clinical activity indices and serum levels of muscle-associated enzymes. In serial studies of seven patients, the proportion of PBMC expressing MHC class II antigen and late T cell activation markers decreased as myositis disease activity decreased, independent of type of therapy. Among the clinical subgroups, polymyositis (n = 21) and inclusion body myositis (n = 11) were virtually indistinguishable; dermatomyositis patients (n = 19) showed decreased proportions of CD3+DR+ and TLiSA1+ cells, and increased proportions of CD20+ and CD20+DR+ cells compared with the other two groups. Patients with autoantibodies to histidyl-tRNA synthetase (Jo-1 antigen, n = 11) had significantly lower proportions of CD3+ and CD4+ cells, lower CD4/CD8 ratios, and higher proportions of CD+ cells expressing CD20, compared with patients without anti-Jo-1 antibodies. These findings support the concept that activated lymphocytes, especially cells undergoing anamnestic responses and cytotoxic differentiation, are important in the pathogenesis of idiopathic myositis. Moreover, taken together with other studies, these data suggest that groups of patients segregated by clinical or autoantibody status have different mechanisms of systemic immune activation and immunopathology.     

肺巨细胞癌不同转移亚克隆生长因子表达及反应性差异的探讨

目的研究肺巨细胞癌高转移亚系ＰＧｂＥ１和中度转移亚系ＰＧＬＨ７细胞之间部分生长因子的表达及反应性差异。方法利用ＲＴ－ＰＣＲ技术检测了生长因子ＴＧＦα、ＴＧＦβ１、ＩＬ－６、ＩＬ－８、ｂＦＧＦ和ＡＮＧ及受体ＥＧＦＲ、ＩＬ－６Ｒ和ＩＬ－８Ｒ的表达状况;其次采用３Ｈ－ＴｄＲ掺入法观察了重组ＴＧＦα、ＴＧＦβ１和ＩＬ－６对此两个细胞生长的影响。结果ＰＧｂＥ１细胞中ＴＧＦα、ＥＧＦＲ、ＩＬ－６和ＩＬ－６Ｒ的表达水平明显高于ＰＧＨ７细胞,而ＴＧＦβ１、ｂＦＧＦ、ＩＬ－８、ＩＬ－８Ｒ和ＡＮＧ的表达在两个细胞间无明显差别;重组ＴＧＦα和ＩＬ－６对两个细胞均具有生长刺激作用;ＴＧＦβ１具有双重效应。结论ＴＧＦα、ＴＧＦβ１、ｂＦＧＦ、ＩＬ－６、ＩＬ－８、和ＡＮＧ等生长因子可能以自分泌方式参与肺巨细胞癌的生长增殖调控,而ＴＧＦα和ＩＬ－６在肺巨细胞癌的转移中发挥更为重要的作用。