(E)-4-Hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vγ9Vδ2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells

Vγ9Vδ2 T cells respond to pyrophosphate antigens and display potent antitumour activity in vitro. We have investigated the potential of the most potent phosphoantigen known to activate Vγ9Vδ2 T cells, (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), as an adjuvant for dendritic cell (DC)-based vaccines. A single stimulation of peripheral blood mononuclear cells with HMB-PP and IL-2 was sufficient to generate lines of effector memory Vγ9Vδ2 T cells that retained their cytolytic and cytokine secretion activities. These cells induced differentiation of DC into semi-mature antigen-presenting cells expressing CD86, CD11c, CD54, HLA-DR, CD83 and CD40, which secreted low levels of bioactive IL-12 but no IL-10. Vγ9Vδ2 T cells also strongly costimulated IL-12 release but inhibited IL-10 production by lipopolysaccharide (LPS)-stimulated DC. When substituted for Vγ9Vδ2 T cells, IFN-γ did not induce full DC maturation but it augmented IL-12 and inhibited IL-10 release by LPS-stimulated DC, in a manner similar to HMB-PP-activated Vγ9Vδ2 T cells. Our findings indicate that Vγ9Vδ2 T cells, stimulated with nanomolar concentrations of HMB-PP, strongly promote T helper type 1 (Th1) responses through their ability to induce DC maturation and IL-12 secretion. This adjuvant activity may prove useful in DC-based cancer therapies.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-γ producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-γ production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-γ production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-γ production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.     

Biotic and abiotic initiators for rishitin formation and accumulation in tomato

γ-Ray irradiation of pre-sowing seeds of tomato did not trigger the formation of the phytoalexin “rishitin” in either leaves or fruits of tomato plants through different growth seasons. Application of copper sulfate initiated rishitin formation in both leaves and fruits. Increasing of γ-ray dose was accompanied by decreasing rishitin accumulation in the presence of copper sulfate. Rishitin of tomato leaves was found to be reduced significantly, concomitant with increasing the disease incidence for late and early blight, andFusarium wilt disease, after applying γ-irradiation, in the case of biotic initiatorsPhytophthora infestans, Alternaria solani orFusarium oxysporum alone or together with the abiotic inducer copper sulfate. Shelf-extending γ-ray doses of 1.0, 1.5 and 2.0 kGy decreased rishitin amounts in tomato fruits treated with copper sulfate alone or infected withPhytophthora infestans. Also, the amount of formed rishitin was reduced by extending the storage period.     

Haplotypes of IL12B promoter polymorphisms condition susceptibility to severe malaria and functional changes in cytokine levels in Thai adults

Polymorphic variability in immune response genes, such as IL12B, encoding the IL-12p40 subunit is associated with susceptibility to severe malaria in African populations. Since the role of genetic variation in conditioning severe malaria in Thai adults is largely unexplored, the functional association between IL12B polymorphisms [i.e. IL12Bpro (rs17860508) and IL12B 3′ UTR T/G (rs3212227)], severe malaria and cytokine production was examined in patients with Plasmodium falciparum infections (n = 355) recruited from malaria endemic areas along the Thai–Myanmar border in northwest Thailand. Circulating IL-12p40 (p = 0.049) and IFN-γ (p = 0.051) were elevated in patients with severe malaria, while only IL-12p40 was significantly higher in severe malaria patients with hyperparasitaemia (p = 0.046). Carriage of the IL12Bpro1.1 genotype was associated with enhanced severity of malaria (OR, 2.34; 95% CI, 0.94–5.81; p = 0.066) and hyperparasitaemia (OR, 3.42; 95% CI, 1.17–9.87; p = 0.025) relative to the IL12Bpro2.2 genotype (wild type). Individuals with the IL12Bpro1.1 genotype also had the lowest IL-12p40 (p = 0.002) and the highest IFN-γ (p = 0.004) levels. Construction of haplotypes revealed that carriage of the IL12Bpro-2/3′ UTR-T haplotype was associated with protection against severe malaria (OR, 0.51; 95% CI, 0.29–0.90; p = 0.020) and reduced circulating IFN-γ (p = 0.06). Thus, genotypic and haplotypic variation at IL12Bpro and IL12B 3′ UTR in this population influences susceptibility to severe malaria and functional changes in circulating IL-12p40 and IFN-γ levels. Results presented here suggest that protection against severe malaria in Thai adults is associated with genotypic variants that condition enhanced IL-12p40 and reduced IFN-γ levels.     

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the T_H1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.     

Immune enhancement and anti-tumour activity of IL-23

Immunotherapy, including the use of cytokines and/or modified tumour cells immune stimulatory cytokines, can enhance the host anti-tumour immune responses. Interleukin-23 (IL-23) is a relative novel cytokine, which consists of a heterodimer of the IL-12p40 subunit and a novel p19 subunit. IL-23 has biological activities similar to but distinct from IL-12. IL-23 can enhance the proliferation of memory T cells and the production of IFN-γ, IL-12 and TNF-α from activated T cells. IL-23 activates macrophages to produce TNF-α and nitric oxide. IL-23 can also act directly on dendritic cells and possesses potent anti-tumour and anti-metastatic activity in murine models of cancer. IL-23 can also induce a lower level of IFN-γ production compared with that induced by IL-12. This may make IL-23 an alternative and safer therapeutic agent for cancer, as IL-12 administration can lead to severe toxic side effects because of the extremely high levels of IFN-γ it induces.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.