IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

New HLA-A2 variants defined by monoclonal antibodies and cytotoxic T lymphocytes

Three HLA-A2 variants, A2-DW, A2-KC, and A2-Lee, were identified in three Chinese donors using a panel of monoclonal antibodies. A2-DW was negative with two of the ten HLA-A2 monoclonal antibodies tested, whereas A2-KC was negative with five of the ten and A-2 Lee was negative with one.Epstein-Barr virus-specific cytotoxic T cells generated from the A2-DW donor recognized and killed target cells prepared from the A2-KC donor, but did not recognize target cells from HLA-A2.1, –A2.2, or –A2.4 donors. In isoelectric focusing studies, A2-DW and A2-KC focus in identical positions more acidic than the other HLA-A2 antigens tested.     

Murine monoclonal antibodies cytotoxic to human glioma cells in vitro.

Six monoclonal antibodies (mABs) against human glioma cells (T2) were produced. T2 cells grown as solid tumors in nude mice, were dissociated and used to immunize Balb/c mice. After fusion of splenocytes with myeloma cells, eight hybrids secreting mABs were selected according to their ability to react immunohistochemically with T2 cells, but not with normal adult human brain. Cytotoxicity of mABs was tested using (3H)-thymidine incorporation assays in vitro. Four mABs showed complement-mediated cytotoxicity for T2 cells, other human glioma cells (T1), and a human melanoma cell line. Incubation with one antibody, mAb2A1, lowered (3H)-thymidine incorporation in the T2 and T1 cells to ca. 10%, and in melanoma cells to ca. 35% of control levels. Another antibody, mAb3B2, displayed a similar cytotoxicity for T2 and T1 cells, but did not show measurable cytotoxicity for melanoma cells and rat primary astrocyte cultures. Moreover, this antibody did not crossreact with haematopoietic cells from patients bearing CNS tumors or normal subjects. MAb3B2, therefore, appears to recognize and epitope associated to human gliomas, will be a useful glioma tumor marker and may have some potential therapeutical value.     

Depletion of NK cells with the lysosomotropic agent L-leucine methyl ester and the in vitro generation of NK activity from NK precursor cells

Human natural killer (NK) cell activity in peripheral blood lymphocytes (PBL) is totally inhibited by pretreatment of the effector cells with a lysosomotropic agent, L-leucine methyl ester (LeuOMe). This treatment specifically eliminates cells expressing the NK cell markers HNK-1, OKM1, B73.1, or Leu-11b, but has minimal effect on viability of cells with T cell markers Leu-1, OKT3, Leu-2a, or Leu-3a. LeuOMe also drastically decreased the proportion of K562 target-binding lymphocytes among PBL. PBL pretreated with LeuOMe respond normally in thymidine uptake to stimulation by phytohemagglutinin or allogeneic lymphocytes as long as irradiated autologous accessory cells are provided, indicating that the treatment is not toxic to T cells. NK activity can be regenerated in the NK cell-depleted PBL population by incubation with IL 2 or by mixed lymphocyte cultures, but not by alpha-interferon. Cells responsible for regeneration of such NK activity are probably large agranular lymphocytes, because they are resistant to LeuOMe treatment but have the same low buoyant density as NK cells in Percoll density gradient separation. The in vitro-generated NK is still sensitive to LeuOMe inhibition, but a higher concentration of the reagent is required to achieve total inhibition of the activity.     

Stimulation of human T lymphocytes by PHA-activated autologous T lymphocytes: Analysis of the role of Ia-like antigens with monoclonal antibodies

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.     

The presence of NK alloantigens on cloned cytotoxic T lymphocytes

A panel of sera raised against NK-1.1 and NK-2.1 alloantigens was tested for reactivity against a panel of cloned antigen-dependent CTL lines. By using indirect immunofluorescence and flow cytofluorimetry, weak, but clear and consistent, reactivity was found on all CTL. Concordant with the genetics of NK alloantigens, C57BL/6-derived clones were reactive with anti-NK-1.1 and anti-NK-2.1 sera, whereas CBA-derived clones were reactive with anti-NK-2.1 sera but not with anti-NK-1.1 sera. Cloned CTL lines were also able to partially and specifically absorb the antibodies from NK alloantiserum that reacted with splenic NK cells. These results indicate that cloned CTL lines express at least some of the NK alloantigen determinants present on splenic NK cells and have important implications regarding the relationship of CTL and NK cells.     

Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen-presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide.     

Separation and isolation of rat natural killer (NK) cells from T cells with monoclonal antibodies

It has been difficult to clearly differentiate rat NK cells from cytotoxic T cells. In this study we have shown that rat NK cells do not express the T cell protein defined by the OX 19 antibody. By using the FACS we have isolated the OX 19- OX 8+ lymphocyte subset that contains virtually all the NK activity. The simultaneous use of the OX 19 and OX 8 antibodies allows the separation of NK cells from T cells.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Epinephrine-induced mobilization of natural killer (NK) cells and NK-like T cells in HIV-infected patients

HIV infection is known to cause changes in phenotype and function of natural killer (NK) cells. The aim of this study was to characterize the NK cells mobilized from peripheral reservoirs in human immunodeficiency virus (HIV)-infected patients and controls. Seventeen HIV-infected patients and eight age- and sex-matched controls received a 1-h epinephrine infusion. Epinephrine induced mobilization of high numbers of NK-like T cells with no difference between HIV-infected patients and controls. Interestingly, all subjects mobilized NK cells containing increased proportions of perforin, in particular the CD3(-)CD16(+)CD56(+) NK cell subset. The HIV-infected patients mobilized CD3(-)CD16(-)CD56(+) and CD3(-)CD16(+)CD56(+) NK cells to a lesser extent than did controls. In contrast, the HIV-infected patients mobilized relatively more CD3(-)CD16(+)CD56(-) NK cells independent of antiretroviral treatment. It is suggested that these cells represent an immature NK cell subpopulation possibly resulting from an impaired cytokine tissue environment in HIV-infected patients.     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

The Aspergillus toxin restriction is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells

The protein toxin restriction, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restriction toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites interanalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.     

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.     

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.     

Effects of cyclic AMP on in vivo cytotoxic T lymphocytes generation

Chronic schistosomiasis mansoni is associated with a modulation of schistosome egg-associated cell-mediated granuloma formation. In this study the injection of a cell-free sonicate from either spleen or thymus cells from chronic Schistosoma mansoni-infected mice (> 15 weeks) passively modulated granuloma size in acutely infected (6–8 weeks) syngeneic mice. Cell-free sonicates from age-matched uninfected mice did not suppress granuloma formation. The chronic extracts also suppressed the soluble schistosome egg antigenic preparation-elicited delayed skin test reactivity in 7–8 week infected animals, but failed to suppress the unrelated antigen-elicited reactivity to bovine serum albumin (BSA) of BSA-sensitized animals.     

Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors. This revised version was published online in August 2006 with corrections to the Cover Date.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Successful generation of cytotoxic T lymphocytes specific for self immunoglobulin-determinants on B lymphocytes

Cytotoxic T lymphocytes (CTL) specific for self immunoglobulin (Ig) determinants on B lymphocytes were successfully generated. These CTL were induced with stimulator B hybridoma cells bearing self Ig determinants in responders compatible with the stimulators in H-2 antigens but disparate in minor H antigens. Their specificities were directed toward the idiotype and probably the allotype structures of Ig receptors on stimulator B cells. However, no CTL specific for Ig structures other than the idiotype and the allotype were detected. These data indicate the presence of specific precursor CTL for self B cell clones and suggest the possible development and function of these CTL in certain situations.