Endogenous IFN-gamma Research Articles

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3’-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

Mechanisms of induction and expression of anti-tuberculous immunity

The induction of anti-tuberculous immunity highly depends on the cytokines produced endogenously at the initial stage of immunization. Among several cytokines, IFN-gamma appears to be the most important to generate antigen-specific Th1 type of protective T cells in mice. IL-12 and IL-18, which are produced by macrophages in response to virulent mycobacteria, are responsible for stimulating NK cells to produce IFN-gamma. Once antigen-specific Th1 cells are generated, Th1-dependent macrophage activation was effective in the elimination of infected bacteria through enhanced production of reactive oxygen intermediates and reactive nitrogen intermediates. In Listeria monocytogenes, one of the intracellular bacteria, listeriolysin O (LLO) appeared to be responsible for the induction of endogenous IFN-gamma from NK cells. The possible mechanisms operating in the induction and expression of anti-tuberculous immunity are discussed with special reference to cytokine responses. An application of LLO to the induction of protective immunity is also discussed.

Endogenous Interferon Directly Regulates Neural Precursors in the Non-Inflammatory Brain

Although a number of growth factors have been shown to be involved in neurogenesis, the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly, however, these effects were mirrored in vivo, with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis, as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore, NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs, as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen, a downstream marker of IFNgamma-mediated activation. Moreover, the inhibitory effect was ameliorated in the presence of SVZ-derived microglia, with their removal resulting in almost complete inhibition of NP proliferation. Interestingly, in contrast to the results obtained in the adult, exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain, an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain.

Alpha-galactosylceramide promotes killing of Listeria monocytogenes within the macrophage phagosome through invariant NKT-cell activation.

alpha-Galactosylceramide (alpha-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by alpha-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mphi) participate in the control of microbial pathogens. In the present study, we examined the participation of Mphi in immune intervention in infection by alpha-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mphi from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of alpha-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-gamma) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with alpha-GalCer and in culture supernatants of peritoneal Mphi from mice treated with alpha-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mphi by alpha-GalCer was abrogated by endogenous IFN-gamma neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mphi was increased after alpha-GalCer treatment. Our results suggest that amelioration of listeriosis by alpha-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mphi activated by IFN-gamma from iNKT cells residing in an organ(s) other than the peritoneal cavity.

Human osteoarthritic chondrocytes are impaired in matrix metalloproteinase-13 inhibition by IFN-γ due to reduced IFN-γ receptor levels

Human osteoarthritic (OA) cartilage type-II collagen is preferentially cleaved by the proinflammatory cytokine-induced matrix metalloproteinases-13 (MMP-13). Interferon-gamma (IFN-gamma) potently inhibits interleukin-1 (IL-1)-induced MMP-13 expression in healthy chondrocytes. Our goal was to study the previously unknown impact of IFN-gamma on MMP-13 in OA and compare the levels and functional activity of IFN-gamma receptor (IFN-gammaR1) in healthy and OA chondrocytes. Chondrocytes were obtained from OA patients and non-arthritic control subjects and treated with IL-1+ or- IFN-gamma. MMP-13 mRNA and protein expression were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. IFN-gammaR1 expression was assessed by flow cytometry, immunoprecipitation and immunohistochemistry with fluorescein-labeled antibody. IFN-gammaR1 was neutralized with its antibody and signal transducer and activator of transcription 1 (STAT1) phosphorylation analyzed by Western blotting. OA chondrocytes were also transfected with control and IFN-gammaR1 expression vectors. OA chondrocytes displayed a drastically impaired MMP-13 suppression by IFN-gamma compared to control cells. IFN-gammaR1 levels were significantly decreased in OA chondrocytes as assessed by flow cytometry, immunoprecipitation and immunohistochemistry. Consequently, IFN-gamma-stimulated STAT1 phosphorylation mediated by IFN-gammaR1 was also considerably reduced in OA patient chondrocytes. IFN-gammaR1 overexpression in OA cells restored MMP-13 suppression by IFN-gamma. Ability of IFN-gamma to suppress IL-1-induced MMP-13 expression is diminished in OA chondrocytes due to decreased IFN-gammaR1 levels, activity and impaired downstream signal transduction. Therefore, IFN-gammaR1 modulation and weakened endogenous IFN-gamma response may be important mechanisms in OA pathogenesis and cartilage degradation.

Role of Endogenous IFN-γ in Macrophage Programming Induced by IL-12 and IL-18

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.

Transposon-based interferon gamma gene transfer overcomes limitations of episomal plasmid for immunogene therapy of glioblastoma

Despite improvements in gene delivery technology, transient expression of plasmid DNA has limited the efficacy of nonviral vectors applied to cancer gene therapy. We previously developed plasmid DNA vectors capable of transgene integration and long-term expression in human glioblastoma cells by utilizing the Sleeping Beauty (SB) transposable element. In this study, we compared the efficacy of interferon gamma (IFN-gamma) immunogene therapy using episomal or SB vectors in a syngeneic GL261 glioma model. Gene delivery was achieved by intratumoral convection-enhanced delivery of DNA/polyethylenimine complexes. Only mice treated with SB transposase-encoding DNA to facilitate chromosomal integration exhibited a significant increase in survival (P<0.05). SB-mediated intratumoral gene transfer caused sustained IFN-gamma expression assessed by reverse transcription-polymerase chain reaction, of both vector-derived and endogenous IFN-gamma, whereas expression following episomal plasmid gene transfer was undetectable within 2 weeks. Median survival was enhanced further when SB-mediated IFN-gamma gene transfer was combined with CpG oligodeoxynucleotides as adjuvant therapy. Prolonged survival positively correlated with tumor regression measured by in vivo bioluminescent imaging, and enhanced T-cell activation revealed by the ELISPOT assay. SB appears to improve the efficacy of cytokine gene therapy using nonviral vectors by enhancing the duration of transgene expression.

Effects of antifibrotic agents on TGF- β1, CTGF and IFN- γ expression in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a deadly disease, largely unresponsive to treatment with corticosteroids and immunosuppressives. The aim of this randomized, prospective, open-label study was to characterize the molecular effects of IFN-gamma-1b and colchicine, on biomarkers expression associated with fibrosis (TGF-beta, CTGF) and immunomodulatory/antimicrobial activity (IFN-gamma), in the lungs of patients with IPF. Fourteen (14) patients with an established diagnosis of IPF received either 200 microg of IFN-gamma-1b subcutaneously three times per week, or 1mg of oral colchicine per day, for 24 months. Using RT-PCR assay, we evaluated the transcription levels of transforming growth factor beta1 (TGF-beta1), connective-tissue growth factor (CTGF), and interferon-gamma (IFN-gamma) genes in lung tissue before and after treatment with IFN-gamma-1b or colchicine. Marked mRNA expression of TGF-beta1 and CTGF, but complete lack of interferon-gamma was detected in fibrotic lung tissue at entry. After treatment, both groups exhibited increased expression of IFN-gamma gene at 6 months that was sustained at 24 months. The expression of CTGF and TGF-beta1 remained almost stable before and after treatment, in the IFN-gamma-1b group, while TGF-beta1 was statistically decreased after therapy, in the colchicine group (p=0.0002). Significant difference in DLCO (% pred), was found between the two treatment groups in favor of IFN-gamma-1b group (p=0.04). In addition, the IFN-gamma-1b group showed stability in arterial PO2 while the colchicine group significantly deteriorated (p=0.02). In conclusion, we report the effect of antifibrotic agents (IFN-gamma-1b and colchicine) in TGF-beta, CTGF, and endogenous IFN-gamma gene expression, in human fibrosis. However, extended studies are needed to verify the pathophysiological consequences of these findings.

In Vitro Restoration of Post-Operatively Decreased IFN-Gamma Levels After Cardiac Surgery and Its Effect on Pro- and Anti-Inflammatory Mediators

A decreased synthesis of interferon gamma (IFN-gamma) by TH 1 lymphocytes after cardiac operations with cardiopulmonary bypass (CPB) is part of the inflammatory response to local operative and systemic traumas. The consequences of this mechanism on the release of pro- and anti-inflammatory cytokines remain unclear. To evaluate the role of IFN-gamma, we added recombinant IFN-gamma to peripheral blood mononuclear cells (PBMCs) on the first post-operative day in an attempt to restore pre-operative values and then measured the release of pro- and anti-inflammatory cytokines in vitro. PBMCs of 10 patients scheduled for elective coronary artery bypass grafting (CABG) were obtained pre-operatively (d0) and on the first (d1) and third (d3) post-operative days. The release of IL-6, IL-8, TNF-alpha, IFN-gamma, IL-10, IL-2, and IL-4 was studied after stimulation (48 h) with PHA (phytohemagglutinin) and LPS (lipopolysaccharide) in the absence or presence of recombinant human IFN-gamma. Endogenous IFN-gamma synthesis was suppressed on d1. Adding exogenous IFN-gamma restored IFN-gamma levels to normal on d1 and doubled IFN-gamma levels on d0 and d3. The addition of IFN-gamma increased TNF-alpha levels up to 250% on d1 and IL-2 synthesis by 75% on d1 and d3; the IL-2 levels, however, were still significantly depressed. The addition of recombinant IFN-gamma did not affect the synthesis of IL-6, IL-8, IL-10, and IL-4. Contrary to our expectations, the in vitro release of IL-6 and IL-8 as well as IL-10 and IL-4 was not influenced by the addition of IFN-gamma. However, TNF-alpha production in isolated PBMC cultures increased significantly on the first post-operative day. This may indicate a hyper-reactivity of PBMCs to IFN-gamma and suggests that the decrease in IFN-gamma synthesis might prevent an excessive stimulation of the non-specific immune system by high TNF-alpha levels after cardiac surgery.

Mycobacterium tuberculosis Induces Interleukin-32 Production through a Caspase- 1/IL-18/Interferon-γ-Dependent Mechanism

BackgroundInterleukin (IL)–32 is a newly described proinflammatory cytokine that seems likely to play a role in inflammation and host defense. Little is known about the regulation of IL-32 production by primary cells of the immune system.Methods and FindingsIn the present study, freshly obtained human peripheral blood mononuclear cells were stimulated with different Toll-like receptor (TLR) agonists, and gene expression and synthesis of IL-32 was determined. We demonstrate that the TLR4 agonist lipopolysaccharide induces moderate (4-fold) production of IL-32, whereas agonists of TLR2, TLR3, TLR5, or TLR9, each of which strongly induced tumor necrosis factor α and IL-6, did not stimulate IL-32 production. However, the greatest amount of IL-32 was induced by the mycobacteria Mycobacterium tuberculosis and M. bovis BCG (20-fold over unstimulated cells). IL-32-induced synthesis by either lipopolysaccharide or mycobacteria remains entirely cell-associated in monocytes; moreover, steady-state mRNA levels are present in unstimulated monocytes without translation into IL-32 protein, similar to other cytokines lacking a signal peptide. IL-32 production induced by M. tuberculosis is dependent on endogenous interferon-γ (IFNγ); endogenous IFNγ is, in turn, dependent on M. tuberculosis–induced IL-18 via caspase-1.ConclusionsIn conclusion, IL-32 is a cell-associated proinflammatory cytokine, which is specifically stimulated by mycobacteria through a caspase-1- and IL-18-dependent production of IFNγ.

Interferon-γ decreases replication of primary R5 HIV-1 isolates in thymocytes

As interferon-gamma (IFN-gamma) is produced at lower levels by neonatal than adult T cells, we determined whether IFN-gamma influences HIV-1 replication in thymocytes. IFN-gamma significantly decreased replication of R5 but not X4 viruses, and reduced depletion of CD3(hi)CD27 (mature) thymocytes, the preferential targets for R5 HIV-1. Thus infection and depletion of functionally mature thymocytes that can produce endogenous IFN-gamma may mutually contribute to HIV-1 replication in the thymus and to reduced T-cell output.

Mechanisms of strain‐dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.

Reduced interleukin-18 secretion in Brucella abortus 2308-infected murine peritoneal macrophages and in spleen cells obtained from B. abortus 2308-infected mice

Th1 immune responses in which gamma interferon (IFN-gamma) production predominates are associated with protective immunity against intracellular bacteria. Following infection, interleukin-18 (IL-18) may contribute, in association with IL-12, to optimal IFN-gamma production. In this study, the secretion of IL-18 following intracellular infection with virulent Brucella abortus 2308 in CD-1 cultured peritoneal macrophages and splenocyte cultures was investigated. The production of IL-18 was reduced in both CD-1 mouse peritoneal macrophages infected with B. abortus 2308 and splenocyte cultures obtained from B. abortus 2308-infected mice at 3, 6 and 10 days post-infection (p.i.). In contrast, splenocyte cultures obtained from B. abortus 2308-infected mice at 3 days p.i. secreted significant amounts of IFN-gamma. Stimulation of these cells with recombinant IL-18 (rIL-18) and/or rIL-12 did not significantly increase IFN-gamma secretion at the splenocyte level. These data suggest that once the infection has been established, B. abortus 2308 selectively limits IL-18 secretion without affecting endogenous IFN-gamma production.

Transforming growth factor‐β1 enhances the interferon‐γ‐dependent, interleukin‐12‐independent pathway of T helper 1 cell differentiation

Transforming growth factor (TGF)-beta, a pleiotropic cytokine that has multiple effects on immune responses, has been shown to inhibit interleukin (IL)-4/GATA-3 expression as well as T helper 2 (Th2) differentiation. Consistent with these reports, we found that priming T cells from DO11.10 transgenic mice with antigen in the presence of TGF-beta inhibited GATA-3 expression and the development of IL-4-producing T cells. Unexpectedly, the inhibition of Th2 development was accompanied by a substantial increase in the number of interferon-gamma (IFN-gamma)-producing cells. T cells primed with TGF-beta secreted IFN-gamma in response to both T-cell receptor ligation and IL-12/IL-18 stimulation, and expressed high levels of T-bet and low levels of GATA-3. The TGF-beta-mediated enhancement of T helper 1 (Th1) priming was independent of IL-12 and signal transducer and activator of transcription (STAT)-4, but required endogenous IFN-gamma. TGF-beta-mediated enhancement of the IFN-gamma-dependent, IL-12-independent pathway of Th1 priming was mediated primarily by the inhibition of IL-4 produced by memory/activated T cells in the unfractionated CD4+ responder population. Nevertheless, TGF-beta did not inhibit this pathway of Th1 differentiation when purified naive CD4+ T cells were used as responders. These data have important implications for strategies being considered for the use of TGF-beta-producing T cells for the treatment of autoimmune disorders.

Expression of a dominant negative IFN‐γreceptor on mouse oligodendrocytes

The interferon-gamma (IFN-gamma) receptor is expressed by all nucleated cells, and binding of its cognate ligand, IFN-gamma, induces a wide variety of biological functions. Transgenic mice expressing a dominant negative IFN-gamma receptor 1 (IFN-gammaR1DeltaIC) on oligodendrocytes under control of the myelin proteolipid protein promoter are described. The mRNA encoding the transgene was only detected in the nervous system and protein expression was confirmed by immunohistochemistry. Transgenic receptor expression does not alter myelination and the mice exhibited no clinically apparent phenotype. Consistent with the restricted nervous system expression of the transgene, no alterations in peripheral immune responses were detected. Flow cytometric analysis demonstrated constitutive expression of both the IFN-gammaR1DeltaIC transgene and the endogenous IFN-gamma receptor 2 at high levels on oligodendrocytes derived from the transgenic mice. These oligodendrocytes also exhibited decreased STAT1 phosphorylation in response to IFN-gamma, confirming dominant negative transgene function. Transgenic mice in which oligodendrocytes have a diminished ability to respond to IFN-gamma showed delayed virus clearance from oligodendroglia compared with wild-type mice. This model will allow evaluation of oligodendrocyte responses to this critical cytokine during CNS inflammation.

Combination Nonviral Interleukin-2 Gene Immunotherapy For Head and Neck Cancer: From Bench Top to Bedside

Intralesional delivery of cytokine genes has emerged as a promising therapeutic strategy for the treatment of cancer. In addition to the therapeutic effect of the delivered cytokine gene, the components of the gene delivery system also have been shown to induce beneficial immune responses. On the basis of these principles, we hypothesized that a molecular therapy could be developed that would provide synergistic antitumor activity by way of intralesional expression of interleukin (IL)-2 from a recombinant plasmid combined with induction of endogenous interferon (IFN)-gamma and IL-12 cytokines by immunostimulatory DNA. Our objective in these studies was to create and optimize a novel formulation of cationic lipid and DNA that generates local production of IL-2 protein within a targeted tumor environment with concomitant induction of the antitumor cytokines IFN-gamma and IL-12. Prospective laboratory drug development plan that would produce human clinical trials. Engineered bacterial plasmids containing a cytomegalovirus promoter (CMV)-IL-2 expression cassette were specifically formulated with cationic lipids and optimized for antitumor effect in a floor of mouth murine tumor model. The treated tumors were assayed for local expression of IL-2 and concurrent expression of secondary cytokines IFN-gamma and IL-12. Established tumors in C3H/HeJ mice were treated with various IL-2 gene formulations, and clinical and immunologic responses were evaluated. Immunologic studies were performed and included cytolytic T-cell assays and cytokine expression profiles. For human clinical trials, a phase I 10 patient formulated IL-2 gene therapy study was completed. Subsequently, two large scale, phase II multi-institutional and multi-international studies were initiated comparing non-viral IL-2 gene therapy to palliative methotrexate chemotherapy or in combination with cisplatin. In the preclinical stage, maximum tumor inhibition in animal models was obtained using IL-2 plasmid formulated with 1,2-dioleyloxypropyl-3-trimethyl ammonium chloride (DOTMA):cholesterol (1:1 mol:mol) at a plasmid:lipid charge ratio of 1:0.5 (-/+). Cationic lipid formulated IL-2 plasmid significantly inhibited tumor growth compared with formulated control plasmid (P < .01) or vehicle (lactose; P < .01). Consistent with previously reported studies of the immunostimulatory activity of DNA of bacterial origin, treatment of tumors with control plasmid in cationic lipid formulation induced production of endogenous IFN-gamma and IL-12 but not IL-2. Treatment of tumors with formulated IL-2 plasmid produced IL-2 protein levels that were 5-fold over background and increased IFN-gamma by 32-fold (P < .001) and IL-12 by 5.5-fold (P < .001) compared with control plasmid formulations. The phase I human trial demonstrated dose escalation safety, which was its primary objective, and there was one anecdotal reduction in tumor size. The phase II studies have been initiated and focus on either comparing the novel nonviral IL-2 gene immunotherapy formulation alone to methotrexate or comparing IL-2 gene therapy in combination with cisplatin in recurrent or unresectable patients with head and neck squamous cell carcinoma. The preclinical data provided proof of principle for matching a delivered IL-2 transgene with an immunostimulatory nonviral formulation to enhance intralesional production of therapeutic cytokines for the maximization of antitumor response. Human clinical trials have demonstrated this novel therapy to be safe in the human clinical setting. Phase II trials have been initiated to assess efficacy and feasibility as a single or combination therapy for head and neck cancer.

Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-γ.

Regulation of pulmonary fibrosis by chemokine receptor CXCR3.

CXC chemokine receptor 3 (CXCR3) is the receptor for the IFN-gamma-inducible C-X-C chemokines MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. CXCR3 is expressed on activated immune cells and proliferating endothelial cells. The role of CXCR3 in fibroproliferation has not been investigated. We examined the role of CXCR3 in pulmonary injury and repair in vivo. CXCR3-deficient mice demonstrated increased mortality with progressive interstitial fibrosis relative to WT mice. Increased fibrosis occurred without increased inflammatory cell recruitment. CXCR3 deficiency resulted in both a reduced early burst of IFN-gamma production and decreased expression of CXCL10 after lung injury. We identified a relative deficiency in lung NK cells in the unchallenged CXCR3-deficient lung and demonstrated production of IFN-gamma by WT lung NK cells in vivo following lung injury. The fibrotic phenotype in the CXCR3-deficient mice was significantly reversed following administration of exogenous IFN-gamma or restoration of endogenous IFN-gamma production by adoptive transfer of WT lymph node and spleen cells. Finally, pretreatment of WT mice with IFN-gamma-neutralizing Ab's enhanced fibrosis following lung injury. These data demonstrate a nonredundant role for CXCR3 in limiting tissue fibroproliferation and suggest that this effect may be mediated, in part, by the innate production of IFN-gamma following lung injury.

Hypothermia in systemic inflammation: role of cytokines.

Hypothermia is a thermoregulatory response to systemic inflammation that is often regarded as maladaptive to the host. However, rodents show regulated hypothermia (that is, a selection of cool ambient temperature) during systemic inflammation that correlates with enhanced survival, supporting an adaptive value to this response. The mechanisms regulating hypothermia are not fully understood, but cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukins (ILs) and interferon-gamma have been shown to induce or modulate hypothermia. A review of the literature suggests that TNF-alpha functions as an endogenous cryogen (i.e., induces hypothermia), whereas IL-10 modulates TNF-alpha production and/or release as a mechanism of hypothermia attenuation. IL-1beta and IL-6 are typically regarded as endogenous pyrogens, but may induce hypothermia during viral and bacterial inflammation. A role for endogenous IFN-gamma in hypothermia has not been demonstrated, but injection of this cytokine potentiates hypothermia through augmented production of other cytokines. It is clear that additional research is required in this area. Suggested areas for future research include a determination of the final mediator of hypothermia and its specific anatomical site of action as well as the role of cytokines in the regulation of hypothermia under non-inflammatory conditions.

Up-regulation of granulomatous inflammation in interleukin-6 knockout mice infected with Rhodococcus aurantiacus.

After intravenous injection of Rhodococcus aurantiacus normal mice develop non-necrotic granulomas, the formation of which is dependent on endogenous interferon-gamma (IFN-gamma). In the early phase of R. aurantiacus infection a high level of endogenous interleukin-6 (IL-6) is detected in the spleen extracts, though its importance is unknown. Using IL-6 knockout (IL-6-/-) mice, we studied the role of IL-6 in granulomatous inflammation induced by R. aurantiacus. The size of granulomas generated in IL-6-/- mice was significantly larger than that of wild-type (IL-6+/+) mice at 2 weeks postinjection (p.i). Moreover, central necrosis of the granuloma was observed in IL-6-/- mice but not in IL-6+/+ controls. Titres of endogenous IFN-gamma and tumour necrosis factor-alpha (TNF-alpha) were markedly increased in the spleens and livers of IL-6-/- mice in comparison with IL-6+/+ mice at days 1 through 3 p.i. In vivo administration of either an anti-IFN-gamma monoclonal antibody (mAb) or anti-TNF-alpha mAb to IL-6-/- mice reduced the number and size of granulomas, and prevented formation of necrotic granulomas. In addition, the production of endogenous IFN-gamma and TNF-alpha in the early phase of R. aurantiacus infection by IL-6-/- mice was suppressed by treatment with recombinant IL-6 (rIL-6). This suppression of IFN-gamma and TNF-alpha production was followed by a reduction in the number and size of central necrotic granulomas at 2 weeks p.i. These findings suggest that overproduction of IFN-gamma and TNF-alpha induces central necrotic granuloma formation in IL-6-/- mice, and that IL-6 down-regulates granulomatous inflammation reaction in response to R. aurantiacus infection by modulating production of IFN-gamma and TNF-alpha.