IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.     

IL-2-PE40 is cytotoxic for activated T lymphocytes expressing IL-2 receptors

IL-2-PE40 is a chimeric molecule in which IL-2 is attached to the amino end of modified Pseudomonas exotoxin molecule lacking cell recognition domain. This molecule was extremely toxic for Con A-stimulated spleen cells from mice. Moreover, IL-2-PE40 has suppressive effect against Ag-activated cells; it inhibits the generation of cytotoxic T lymphocyte activity in a MLC. IL-2-PE40 could be a useful agent in IL-2R targeting therapy including immunosuppressive therapy for allograft rejection or some autoimmune diseases.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Pregnancy ameliorates induction and expression of experimental autoimmune uveitis

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-gamma and IL-12 p40, but unchanged levels of TNF-alpha, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-gamma production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-beta, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-beta.     

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apopotosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-γ release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a “per-cell” basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

The interaction of recombinant IL-2 with human resting lymphocytes: blocking effects of monoclonal antibodies to IL-2 receptors

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin-2 receptors (IL-2.R). Recombinant IL-2 (rIL-2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon-gamma (IFN-gamma) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool-passed non-T, non-B cells ("null cells" or T3- cells); in response to rIL-2, only Leu 11+T3- cells showed enhanced NK activity, and both Leu 11+T3- and Leu11-T3- cells showed predominantly AK activity, proliferation and production of IFN-gamma. These findings suggest that the T3- fraction (null cell fraction) contains predominantly cells expressing IL-2.R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL-2.R, including anti-Tac antibody at any dilution. These results indicate that IL-2.R on the resting T3- cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3- cells possess higher affinity IL-2.R than activated T or B cells. Other possibilities are also discussed.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Bradykinin B2 Receptors of dendritic cells, acting as sensors of kinins proteolytically released by Trypanosoma cruzi, are critical for the development of protective type-1 responses

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R-/- mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R-/- heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-gamma-producing CD4+ and CD8+ T cells in the spleen of B2R-/- and wild-type mice. However, production of IFN-gamma by effector T cells isolated from B2R-/- heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-gamma-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R-/- mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R-/- mice was linked to upregulated secretion of IL-17 and TNF-alpha by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R-/- mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R-/- mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.     

Activated lymphocytes during acute Epstein-Barr virus infection

Activated lymphocytes, as identified by HLA-DR expression, associated with acute Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) were shown to be a heterogeneous population containing significantly elevated cytotoxic/suppressor (CD8) T cells, natural killer (CD16) cells and helper (CD4) T cells. CD8 T cells were the primary activated population representing 24.5% of the total lymphocyte population. The activated CD4 T cells and natural killer cells accounted for 6.7% and 3.5% of the total lymphocyte population, respectively. Analysis of serum soluble interleukin 2 receptors (IL-2R) demonstrated significantly (p less than 0.001) elevated levels in the serum of acute IM patients compared with normal controls. Elevated levels of serum IL-2R were correlated (r = 0.67) with increased percentages of Leu 2a+/HLA-DR+T cells (i.e., activated CD8 T cells). Patients with X-linked lymphoproliferative syndrome and virus-associated hemophagocytic syndrome, two syndromes associated with severe acute EBV infections, demonstrated the most dramatic increase in serum IL-2R levels. These data demonstrate that EBV is associated with intense immune stimulation and that during acute IM activated lymphocytes, other than the CD8 T cells, may contribute to the immune response to EBV.     

Induction of interleukin-2 receptor α-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes

Abstract. The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the α (p55) and β (p75). The α chain, unlike the β, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R α-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R α-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([³H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R α-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.     

RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T lymphocytes. Patients selected for treatment had malignant pleural or peritoneal effusions. Treatment consisted of isolating autologous peripheral blood lymphocytes, ex vivo activation, incubation with bispecific monoclonal antibodies and injection at the effusion site of these BIS-1-redirected lymphocytes. To evaluate the effects of the bispecific monoclonal antibody, five patients received treatments with activated lymphocytes without bispecific antibodies. Effusion samples taken before and at various times after treatment were analysed by immunocytology and for the presence of the soluble factors carcinoembryonic antigen (CEA), interleukin-6 (IL-6), tumour necrosis factor (TNF), C-reactive protein and soluble CD8. In this way both immune activation and anti-tumour activity could be monitored. Conjugate formation between tumour cells and activated lymphocytes was seen as soon as 4 h after injection of BIS-1-redirected activated lymphocytes, followed by a disappearance or reduction of tumour cells after 24–48 h. In parallel with this, the soluble tumour marker CEA decreased in the effusion fluid following injection with the BIS-1-redirected lymphocytes. Furthermore, a steep increase in local granulocyte numbers was observed in the effusion fluid, which reached a maximum 24–48 h after the start of the treatment. Also levels of IL-6 and TNF were greatly elevated. The data suggest tha the treatment induces both antitumour activity and a strong local inflammatory reaction. This is accompanied by no or only minor local and systemic toxicity, i.e. mild fever, which disappeared as the local inflammatory reaction diminished 48–72 h after treatment.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Augmentation versus inhibition: effects of conjunctional OX-40 receptor monoclonal antibody and IL-2 treatment on adoptive immunotherapy of advanced tumor.

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 x 10(6) activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 x 10(6) cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 x 10(6) L-selectin(low) T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 x 10(6) T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.