Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma.

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.     

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.     

Acquired pMHC I Complexes Greatly Enhance CD4~+ Th Cell’s Stimulatory Effect on CD8~+ T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4+ helper T (Th) cells play pivotal roles in induction of CD8+ CTL immunity. However, the mechanism of CD4+ T cell help delivery to CD8+ T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DCOVA) to activate OT-II mouse CD4+ T cells, and then studied the help effect of these CD4+ T cells on CD8+ cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet β cell destruction which led to diabetes in wild-type C57BL/6 mice and transgenic rat insuli...     

IFN-gamma-independent synergistic effects of IL-12 and IL-15 induce anti-tumor immune responses in syngeneic mice

IL-15 and IL-12 display anti-tumor activity in different models and IFN-gamma has been reported as a secondary mediator of both IL-12 and IL-15 effects. TS/A murine adenocarcinoma cells were engineered to secrete IL-12, IL-15 or both cytokines. TS/A cells secreting IL-15 (TS/A-IL-15) displayed a reduced tumorigenicity (50%) when implanted subcutaneously in syngeneic mice, while both TS/A-IL-12 and TS/A-IL-12/IL-15 were rejected by 100% of animals. In contrast, TS/A-IL-15 and TS/A-IL-12 were tumorigenic in syngeneic IFN-gamma knockout mice (100% and >90% of take rate, respectively), but TS/A-IL-12/IL-15 were completely rejected by 90% of these mice. All IFN-gamma-deficient mice rejecting TS/A-IL-12/IL-15 developed protective immunity against wild-type TS/A, as indicated by re-challenge experiments. Immunohistochemical analysis of the TS/A-IL-12/IL-15 tumor rejection area in IFN-gamma-deficient mice showed a marked reactive cell infiltration constituted of CD8+ cells, granulocytes, NK cells, macrophages and dendritic cells associated with the expression of IL-1beta, TNF-alpha, GM-CSF, MCP-1 and MIP-2. In vivo depletion experiments showed that rejection of TS/A-IL-12/IL-15 cells required CD8+ T lymphocytes and also involved granulocytes, while CD4+ and NK cells played a minor role. These data show IFN-gamma-independent synergistic anti-tumor effects of IL-12 and IL-15, involving CD8+ cells and secondary chemokines and cytokines, such as TNF-alpha.     

Unexpected role of TNF-alpha in graft versus host reaction (GVHR): donor-derived TNF-alpha suppresses GVHR via inhibition of IFN-gamma-dependent donor type-1 immunity

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation, leading to significant morbidity and mortality. Host-derived TNF-alpha play a role in the induction of allo-reactive donor T cell activation and the pathogenesis of GVHD. On the other hand, the precise role of donor-derived TNF-alpha in GVHD remains unclear. To elucidate this issue, we designed an acute GVHD model using (B6 x D2) F1 recipient mice transferred with spleen cells derived from either wild-type or TNF-alpha(-/-) C57BL/6 mice. Surprisingly, we found that spleen cells from TNF-alpha(-/-) mice induce more severe graft versus host reaction (GVHR) than wild-type spleen cells upon transfer into B6D2F1 mice. Transplantation of TNF-alpha(-/-) mouse spleen cells was associated with enhanced anti-host CTL generation and augmented deletion of host cells. Moreover, mice receiving TNF-alpha(-/-) cells showed significantly higher levels of serum IFN-gamma, which was mainly produced by donor CD8+ T cells. We also demonstrated that TNF-alpha deficiency in donor spleen cells caused a marked elevation of TNF-alpha producing capacity by LPS-stimulated host macrophages. Such enhanced GVHR was completely prevented by using TNF-alpha(-/-)IFN-gamma(-/-) splenic cells. Our findings demonstrate, for the first time, that donor-derived TNF-alpha suppress GVHR by inhibiting IFN-gamma-dependent donor type-1 immunity which is essential for host TNF-alpha elevation.     

Gamma interferon induces Fas-dependent apoptosis of Peyer's patch T cells in mice following peroral infection with Toxoplasma gondii.

Since we previously observed a remarkable decrease in the numbers of T cells in the Peyer's patches of the small intestines in C57BL/6 mice following peroral infection with Toxoplasma gondii, we performed studies to examine the mechanism(s) whereby this decrease in numbers of the T cells occurs. We found that apoptotic cell death of CD4+ and CD8+ alphabeta T cells occurred in Peyer's patches following infection. Upregulation of Fas expression was observed in these T cells. C57BL/6-background mutant mice which lack functional Fas antigen did not develop apoptosis in their Peyer's patches following infection. Treatment of infected C57BL/6 mice with anti-gamma interferon (IFN-gamma) monoclonal antibodies prevented the upregulation of Fas on their Peyer's patch T cells and inhibited the occurrence of apoptosis of these T cells. These results indicate that IFN-gamma induces Fas-dependent apoptosis in CD4+ and CD8+ alphabeta T cells in Peyer's patches in C57BL/6 mice following peroral infection with T. gondii.     

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.     

Interleukin-12 amplifies the M. leprae hsp65-cytotoxic response in the presence of tumour necrosis factor-alpha and interferon-gamma generating CD56+ effector cells: interleukin-4 downregulates this effect

Interleukin-12 (IL-12) is a major immunomodulatory cytokine that represents a functional bridge between the early resistance and the subsequent antigen specific adaptive immunity. TNF-alpha and IFN-gamma have an important role in the generation of hsp65 specific cytotoxic T lymphocytes (CTL) that lyse hsp65-pulsed autologous macrophages (hsp65 CTL). Since a positive feedback mechanism between TNF-alpha, IFN-gamma and IL-12 has been described, we undertook to evaluate the role of IL-12 on the hsp65 CTL generation in leprosy patients. Our results show that the presence of IL-12 during the first 24 h of the in vitro antigen stimulation amplifies the hsp65 cytotoxic response whenever both IFN-gamma and TNF-alpha are present. The addition of these three cytokines (CKs) was able to abrogate the inhibitory effect of IL-10 on hsp65 CTL in cells from paucibacillary patients (PB) but not that of IL-4 in PB and normal controls (N). Both IL-12 or anti IL-4 enhanced the cytotoxic activity in cells from multibacillary patients (MB). Anti IL-4 upregulated the binding of IFN-gamma and did not modify that of TNF-alpha so the low CTL activity could be as a result of IL-4 by a decrease of the IFN-gamma binding on MB cells. Cells from those MB patients taking thalidomide (MB-T) did neither bind IFN-gamma nor TNF-alpha even when antigen or anti-IL-4 were added, demonstrating that thalidomide inhibits either the in vitro binding or receptor expression of both TNF-alpha and IFN-gamma. Development of CD56 effector cells during the hsp65 stimulation was observed in PB and N by the addition of IL-12 plus TNF-alpha and IFN-gamma, while in MB and MB-T anti IL-4 was also required. So, the inhibitory effect of IL-4 on either production of IFN-gamma, TNF-alpha and/or IL-12 or their receptors could be the mechanism underlying the lack of the hsp65 CTL generation in cells from MB.     

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.     

Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.     

Contrasting effects of myeloid dendritic cells transduced with an adenoviral vector encoding interleukin-10 on organ allograft and tumour rejection

Mouse bone marrow-derived myeloid dendritic cells (DC) propagated in granulocyte-macrophage colony-stimulating factor and transforming growth factor-beta1 (TGF-beta1) (so-called 'TGF-beta DC') are phenotypically immature, and prolong allograft survival. Interleukin-10 (IL-10) has been shown to inhibit the maturation of DC by down-regulating surface major histocompatibility complex (MHC) class II, co-stimulatory and adhesion molecule expression. Genetic engineering of TGF-beta DC to overexpress IL-10 might enhance their tolerogenic potential. In this study, adenoviral (Ad) vectors encoding the mouse IL-10 gene were transduced into B10 (H2b) TGF-beta DC. Transduction with Ad-IL-10 at a multiplicity of infection (MOI) of 50-100 resulted in a modest reduction in the incidence of DC expressing surface MHC class II, CD40, CD80 and CD86. Paradoxically, Ad-IL-10 transduction enhanced the allostimulatory activity of DC in mixed leucocyte reactions and cytotoxic T lymphocyte assays, and increased their natural killer cell stimulatory activity. Systemic injection of normal C3H recipients with Ad-IL-10-transduced B10-DC 7 days before organ transplantation, exacerbated heart graft rejection and augmented circulating anti-donor alloantibody titres. Contrasting effects were observed in relation to tumour growth. All mice preimmunized with Ad-IL-10-transduced, tumour antigen (B16F10)-pulsed DC developed palpable tumours, associated with significant inhibition of splenic anti-tumour cytotoxic T lymphocyte generation. Animals pretreated with control Ad-LacZ-transduced, B16F10-pulsed DC however, remained tumour free. These findings are consistent with the multifunctional immunomodulatory properties of mammalian IL-10.     

CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model

We previously reported that both E7 and CpG-oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7-associated tumour challenge. Here we investigate dendritic cells (DC)-based approach in this protection. In the study, we isolated bone marrow-derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin-12, as compared to that with E7 or ODN alone. Further injection with E7+ODN-stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7-specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon-gamma (IFN-gamma) production from CD4+ T cells and a more significant production of IFN-gamma from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN-gamma staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T-cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7-associated tumour challenge.     

Mechanisms of cytokine synergy essential for vaccine protection against viral challenge.

The ability of cytokines to steer CD4(+) T(h) cell responses toward a T(h)1 or T(h)2 phenotype and enhance the magnitude of both CD8(+) cytotoxic T lymphocytes (CTL) and antibody responses has clearly been demonstrated by our lab and others, but the influence of cytokines on protective immune responses is much less clear. Here we show an essential role for CD4(+) T(h)1 helper cell induction and IFN-gamma production in protection from viral challenge with a recombinant vaccinia virus expressing HIV-1MN viral envelope glycoprotein gp160. Complete protection from viral challenge is achieved only when the triple combination of exogenous cytokines granulocyte macrophage colony stimulating factor (GM-CSF), IL-12 and tumor necrosis factor (TNF)-alpha are co-administered with the peptide vaccine. In vivo depletion of CD4(+) cells or immunization of IFN-gamma-deficient mice abrogates protection. GM-CSF, IL-12 and TNF-alpha also synergize for the enhanced induction of CTL; however, adoptive transfer of a CD8(+) CTL line afforded only partial protection in this viral challenge model. As a possible mechanism of in vivo protection we show that GM-CSF increases the percentage and activity of antigen-presenting dendritic cells in draining lymph nodes where the immune response is initiated. We further demonstrate synergy between IL-12 and the proinflammatory cytokine TNF-alpha in driving IFN-gamma production. Thus, a combination of IL-12 and TNF-alpha is essential for the optimal development of T(h)1 responses and help for CTL induction in BALB/c mice, and is complemented by a third cytokine, GM-CSF, which enhances antigen presentation.     

Critical role of the liver CD8+ CD122+ T cells in the generalized Shwartzman reaction of mice

We examined the role of mouse CD8+ CD122+ T cells, which increase in number with age, in the generalized Shwartzman reaction. This reaction was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in mice of various ages (4-50 weeks of age). Although most young mice (4 or 6 weeks of age) survived, mortality essentially increased with increasing age of the mice, and all mice of 20 weeks of age or older died within 48 h. Serum TNF-alpha levels after LPS challenge also increased age dependently. The neutralization of either IL-12-induced IFN-gamma or LPS-induced TNF-alpha improved the survival of middle-aged (25-week-old) mice. Both IFN-gamma production after IL-12 priming and TNF-alpha production from the liver mononuclear cells after LPS challenge were also prominent in the middle-aged mice. CD8+CD122+ T cells cultured with IL-12 produced a much larger amount of IFN-gamma than CD8+CD122- T cells. Although the depletion of NK/NK T cells did not decrease the IFN-gamma or TNF-alpha production in the Shwartzman reaction of the middle-aged mice, an additional depletion of CD8+CD122+ T cells did decrease such production and also improved mouse survival. Furthermore, young mice transferred with CD8+CD122+ T cells from aged B6 nude mice showed an enhanced Shwartzman reaction.     

IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL

Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i) higher IFN-gamma secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL.     

Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor

We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1-dominated B cell and CD4(+) T cell responses. In this study we show that vaccination with DC pre-pulsed ex vivo with CT-conjugated OVA (OVA-CT) gives rise to OVA-specific CD8(+) T cells that produce IFN-gamma and are cytotoxic for OVA-expressing E.G7 tumor cells both in vitro and in vivo. The induction of specific CD8(+) CTL by OVA-CT-treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC. Vaccination of mice with OVA-CT-pulsed DC resulted in rejection of already established MHC class I-positive, MHC class II-negative, OVA-expressing E.G7 tumors in an antigen-specific, CD8(+) T cell-dependent fashion and was associated with high numbers of tumor-infiltrating CD8(+) T cells. Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor-binding B subunit and induced strong activation-maturation signals through the biologically active A subunit. These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.     

Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） for the initiation of antigen （Ag）-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide （LPS）. However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli （IL-1β, IFN-γ, TNF-α CpG and LPS） in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin （OVA）-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8^＋ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 ceils may prove to be useful in the study of DC biology and antitumor immunity.     

Apoptotic cells induce dendritic cell-mediated suppression via interferon-gamma-induced IDO

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-gamma expression by DC in association with apoptotic environments. The specific generation of IFN-gamma by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-gamma and IDO blockade demonstrated a role for IFN-gamma and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-gamma-dependent. Blocking transforming growth factor-beta (TGF-beta) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-gamma-induced IDO and TGF-beta.     

Photodynamic therapy with recombinant adenovirus AdmIL-12 enhances anti-tumour therapy efficacy in human papillomavirus 16 (E6/E7) infected tumour model

Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. Here we investigated the utility of adenoviral delivery of interleukin-12 (AdmIL-12) as an adjuvant for PDT in mouse tumour challenge model. PDT was performed by irradiating Radachlorin in C57BL/6 mice transplanted with TC-1 cells. PDT plus AdmIL-12 treatment for tumour suppression as well as specific immune responses were evaluated with the following tests: in vitro and in vivo tumour growth inhibition, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) assay, and cytotoxic T lymphocyte (CTL) assay. Direct intratumoral injection of AdmIL-12 resulted in a significant suppression of tumour growth compared to the control group. Treatment of PDT along with AdmIL-12 further enhanced antitumour effects significantly higher than either AdmIL-12 or PDT alone. This combined treatment resulted in complete regression of 9-mm sized tumour in every animal. We also evaluated immune responses induced by these treatments. Combined treatment significantly increased the production level of IFN-gamma and TNF-alpha compared with that by AdmIL-12 or PDT alone. PDT plus AdmIL-12 enhanced antitumour immunity through increased expansion of the CTL subset mediated by CD8+ T cells. Taken together, these results indicate that the high anti-cancer activity of PDT with AdmIL-12 is a powerful tool against cancer therapy and is a promising subject for further investigation.