Immune signatures in cutaneous melanoma correlate with survival independently of immunotherapy treatment

Immune checkpoint inhibitors (ICIs) have fundamentally improved survival from advanced cutaneous melanoma. Significant efforts have been made to understand the ICI response to identify ways to further improve outcomes. One such approach has been to investigate gene expression associated with response to ICI, which has identified various immune-related mRNA signatures, including a six-gene IFN-γ signature (IFN-γ⁶), an expanded immune signature (IFN-γ¹⁸), an effector T-cell gene signature (T_eff), and a T_eff-associated and IFN-γ-associated gene signature (T_eff + IFN-γ). Given that these signatures appear to reflect expression from T cells and the level of tumour-infiltrating immune cells has been associated with survival, we hypothesised that the prognostic value of the signatures is not limited to ICI treatment and investigated if they were associated with survival also in patients who never received ICI. The signatures were not present in melanoma cell lines when compared with tumour samples, confirming that the signatures were likely derived from the samples' non-tumour (immune) components. We acquired expression and survival data from five melanoma cohorts with a wide range of disease stages, treatments and metrics for survival, and correlated the expression signatures with survival. All four signatures were significantly associated (p < .05) with survival in four of five cohorts, with hazard ratios ranging from 0.69 to 0.92. We conclude that these immune signatures' association with survival is not specific to ICI-treated patients, but present in a number of settings.     

Thyroid hormone analogues potentiate the antiviral action of interferon-γ by two mechanisms

L-thyroxine (L-T₄) potentiates the antiviral activity of human interferon-γ (IFN-γ) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN-γ (1.0 IU/ml), 2) for 24 h cotreatment with IFN-γ, 3) for 4 h, after 20 h cell incubation with IFN-γ, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN-γ. The antiviral effect of IFN-γ was then assayed. L-T₄ potentiated the antiviral action of IFN-γ by a reduction in virus yield of more than two logs, the equivalent of a more than 100-fold potentiation of the IFN's antiviral effect. 3,3′,5-L-triiodothyronine (L-T₃) was as effective as L-T₄ when coincubated for 24 h with IFN-γ but was less effective than L-T₄ when coincubated for only 4 h. D-T₄, D-T₃, 3,3′,5-triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5-diiodothyronine (T₂) were inactive. When preincubated with L-T₄ for 24 h prior to IFN-γ treatment, tetrac blocked L-T₄ potentiation, but, when coincubated with L-T₄ for 4 h after 20 h IFN-γ, tetrac did not inhibit the L-T₄ effect. 3,3′,5′-L-triiodothyronine (rT₃) also potentiated the antiviral action of IFN-γ, but only in the preincubation model. Furthermore, the effects of rT₃ preincubation and L-T₃ coincubation were additive, resulting in 100-fold potentiation of the IFN-γ effect. When L-T₄, L-T₃, or rT₃, plus cycloheximide (5 μg/ml), was added to cells for 24 h and then removed prior to 24 h IFN-γ exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN-γ potentiation by 4 h of L-T₄ or L-T₃ coincubation was not inhibited by cycloheximide (25 μg/ml). These studies demonstrate two mechanisms by which thyroid hormone can potentiate IFN-γ's effect: 1) a protein synthesis-dependent mechanism evidenced by enhancement of IFN-γ's antiviral action by L-T₄, L-T₃, or rT₃ preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis-independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L-T₄ or L-T₃, but not rT₃, with IFN-γ. The protein synthesis-dependent pathway is responsive to rT₃, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN-γ can be regulated has not previously been described. © 1996 Wiley-Liss, Inc.     

Molecular characterisation and expression analysis of interferon gamma in Atlantic cod (Gadus morhua)

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. In this study, the cDNA and genomic sequences of Atlantic cod IFN-γ were cloned and found to encode a putative protein containing 194 amino acids with a 24 amino acid signal peptide sequence. The gene is composed of four exons and three introns similar to IFN-γ genes of other vertebrates. The cod IFN-γ showed only 14–29% amino acid identity with other fish IFN-γ and 9–17% identity with IFN-γ from higher vertebrates. However, cod IFN-γ possesses the typical IFN-γ motifs in the C-terminal end of the protein and displays an alpha-helix structure similar to mammalian IFN-γ. The promoter region contains a putative ISRE element indicating up-regulation by type I IFNs and dsRNA. Real time RT-PCR analysis confirmed that IFN-γ gene expression was up-regulated in organs of cod injected with the dsRNA polyinosinic:polycytidylic acid (poly I:C), which is a strong inducer of type I IFNs. Injection of cod with formalin-killed Vibrio anguillarum also increased IFN-γ expression in head kidney, but to a much lesser extent than poly I:C. The gene expression results thus indicate a role for IFN-γ in innate immune response against both virus and bacteria in Atlantic cod.     

共表达猪繁殖与呼吸综合征病毒GP5基因及猪γ-干扰素基因的腺病毒载体的构建与鉴定

利用PCR方法分别扩增猪繁殖与呼吸综合征病毒全长GP5基因（E蛋白）,EMCV的核糖体介入位点（IRES）序列及猪γ-干扰素（IFN-γ）基因全长序列,序列测定正确后用DNA重组法将三者串联后插入pAdenoVator-CM V5-IRES-GFP穿梭质粒中,形成的穿梭质粒plRES-GP5-IFN-γ用PmeⅠ线性化后,与腺病毒骨架载体pAdEasy-1共转化感受态大肠埃希氏菌BJ5183,经同源重组,构建成含有GP5基因和IFN-γ基因的重组腺病毒载体,pacⅠ酶切线性化充分暴露反向末端重复序列后,脂质体转染HEK293A细胞,借助GFP的表达可以在转染后的2～3天观察到包装病毒rAdeno-GP5-IFN-γ产生,7～10天出现病毒蚀斑。经PCR法及酶切证实各中间过程载体及最终的包装病毒中携带有目的基因,western-blot证实两基因在腺病毒中得到了表达。大肠杆菌内同源重组法能有效和较为方便的构建出含有目的基因的腺病毒载体rAdeno-GP5-IFN-γ,重组子能够在HEK293细胞中稳定扩增,病毒包装的成功为进一步研究PRRSVE蛋白的免疫效果及IFN-γ的作用奠定了基础。     

印度尼西亚源食蟹猴干扰素-γ基因的克隆和序列分析

目的获得印度尼西亚食蟹猴的干扰素-γ基因,为常用实验猕猴干扰素-γ的基因工程生产奠定基础。方法根据GenBank上公布的恒河猴干扰素-γ基因序列设计特异性引物,从印度尼西亚食蟹猴的外周血液中分离单核淋巴细胞,利用Trizol试剂,提取淋巴细胞的总RNA,通过RT-PCR的方法获得干扰素-γ基因片段,并对该片段进行克隆、鉴定和序列分析。结果扩增到一498bp的目的片段,经序列测定证实为印度尼西亚食蟹猴的干扰素-γ基因,与恒河猴、人及狒狒的干扰素-γ基因相比,同源性分别为100%、96%、99%。结论常用的两种实验猕猴食蟹猴与恒河猴的干扰素-γ基因完全相同。     

IFN-γ Receptor-Deficient Donor T Cells Mediate Protection from Graft-versus-Host Disease and Preserve Graft-versus-Tumor Responses after Allogeneic Bone Marrow Transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. It has been previously reported that lung GVHD severity directly correlates with the expansion of donor Th17 cells in the absence of IFN-γ. However, the consequence of Th17-associated lung GVHD in the presence of IFN-γ has not been well characterized. In the current study, T cells from IFN-γ receptor knockout (IFN-γR(-/-)) mice, capable of producing IFN-γ but unable to signal in response to IFN-γ, have been used to elucidate further the role of IFN-γ in GVHD. We found the transfer of donor T cells from either IFN-γR(-/-) or IFN-γ knockout (IFN-γ(-/-)) mice resulted in significant increases in donor Th17 cells in the lung. Marked increases in IL-4-producing Th2 cells infiltrating the lungs were also observed in the mice of donor IFN-γR(-/-) T cells. Notably, despite the presence of these cells, these mice did not show the severe immune-mediated histopathological lung injury observed in mice receiving donor IFN-γ(-/-) T cells. Increases in lung GVHD did occur in mice with donor IFN-γR(-/-) T cells when treated in vivo with anti-IFN-γ demonstrating that the cytokine has a protective role on host tissues in GVHD. A survival benefit from acute GVHD was also observed using donor cells from IFN-γR(-/-) T cells compared with control donors. Importantly, tumor-bearing mice receiving IFN-γR(-/-) T cells versus wild-type donor T cells displayed similar graft-versus-tumor (GVT) effects. These results demonstrate the critical role of IFN-γ on host tissues and cell effector functions in GVHD/GVT.     

Heterogeneous and tissue-specific regulation of effector T cell responses by IFN-gamma during Plasmodium berghei ANKA infection

IFN-γ and T cells are both required for the development of experimental cerebral malaria during Plasmodium berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4(+) and CD8(+) T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN-γ(-/-) mice during P. berghei ANKA infection. The expansion of splenic CD4(+) and CD8(+) T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ, but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN-γ(-/-) mice; however, the migration to, and accumulation of, effector CD4(+) and CD8(+) T cells in the lung, liver, and brain was altered in IFN-γ(-/-) mice. Interestingly, activation and accumulation of T cells in various nonlymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN-γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended on active IFN-γ-dependent environmental signals--leading to T cell apoptosis--rather than upon intrinsic alterations in T cell programming. To our knowledge, this is the first study to fully investigate the role of IFN-γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.     

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H₂O₂), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H₂O₂ together did not protect them from the H₂O₂-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H₂O₂ toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.     

Up-regulation and sustained activation of Stat1 are essential for interferon-gamma (IFN-gamma)-induced dual oxidase 2 (Duox2) and dual oxidase A2 (DuoxA2) expression in human pancreatic cancer cell lines

Dual oxidase 2 is a member of the NADPH oxidase (Nox) gene family that plays a critical role in the biosynthesis of thyroid hormone as well as in the inflammatory response of the upper airway mucosa and in wound healing, presumably through its ability to generate reactive oxygen species, including H2O2. The recently discovered overexpression of Duox2 in gastrointestinal malignancies, as well as our limited understanding of the regulation of Duox2 expression, led us to examine the effect of cytokines and growth factors on Duox2 in human tumor cells. We found that exposure of human pancreatic cancer cells to IFN-γ (but not other agents) produced a profound up-regulation of the expression of Duox2, and its cognate maturation factor DuoxA2, but not other members of the Nox family. Furthermore, increased Duox2/DuoxA2 expression was closely associated with a significant increase in the production of both intracellular reactive oxygen species and extracellular H2O2. Examination of IFN-γ-mediated signaling events demonstrated that in addition to the canonical Jak-Stat1 pathway, IFN-γ activated the p38-MAPK pathway in pancreatic cancer cells, and both played an important role in the induction of Duox2 by IFN-γ. Duox2 up-regulation following IFN-γ exposure is also directly associated with the binding of Stat1 to elements of the Duox2 promoter. Our findings suggest that the pro-inflammatory cytokine IFN-γ initiates a Duox2-mediated reactive oxygen cascade in human pancreatic cancer cells; reactive oxygen species production in this setting could contribute to the pathophysiologic characteristics of these tumors.     

Gamma interferon controls mouse polyomavirus infection in vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.     

Dissociation of human interferon-gamma-like activity from migration-inhibition factor

Supernatants harvested from concanavalin A-stimulated human peripheral mononuclear cells after 24 hr of incubation contain one interferon species similar to human interferon-gamma (IFN-γ) with a pI of 4.6–5.3 (first day pH 5 IFN-γ). In contrast, during the subsequent 24 hr of incubation two species with properties of IFN-γ are produced with pI of 3.6–4.0 (second day pH 4 IFN-γ) and 4.6–5.6 (second day pH 5 IFN-γ), respectively. First day pH 5 IFN-γ and second day pH 5 IFN-γ have been found to differ on the basis of trypsin sensitivity. This pattern of polymorphism is similar to the pattern previously described for human migration-inhibitory factor (MIF) which can be separated into first day pH 5 MIF, second day pH 3 MIF, and second day pH 5 MIF. However, IFN-γ-like species can be differentiated from MIF biochemically and antigenically. Fractions with second day pH 4 IFN-γ have no MIF activity and fractions with second day pH 3 MIF contain no IFN activity. In addition, first and second day pH 5 MIF, which also contain IFN-γ activity, can be separated from the latter by precipitation as well as neutralization with polyclonal and monoclonal anti-human MIF antibodies.     

The effects of γ-interferon on human peripheral blood monocyte/macrophage-mediated bone particle degradation

γ-Interferon (IFN-γ) has recently been demonstrated to inhibit the ability of mononuclcar phagocytes to degrade bone particles. We have further addressed the specificity, potency and mechanism of this activity using human recombinant IFN-γ. Adherent peripheral blood mononuclear leukocytes from normal human volunteers were cultured with washed, sieved (⩽75 μm) ⁴⁵Ca-labelled rat bone particles for 3 days, after which bone particle degradation (7.1 ± 1.6%, n = 11) was calculated from the fraction of⁴⁵Ca released into the medium. As little as 5 U/ml IFN-γ significantly suppressed bone particle degradation and 50 U/ml was associated with consistent marked suppression (74.0 ± 3.5% inhibition, P < 0.001, n = 11). IFN-γ was not suppressive if added to cells 24 h or more after exposure to bone particles. Addition of indomethacin (10 μM) did not reverse the effect of IFN-γ, suggesting that it was not prostaglandin-mediated. In addition, 1,25(OH)₂D₃ (10 nM) did not remove the inhibitor, effect of IFN-γ.Contact of mononuclear phagocytes with bone particles and secretion of soluble factors from these cells have both been demonstrated to play a role in their ability to degrade bone particles. IFN-γ (50 U/ml) inhibited monocyte/macrophage interaction with another unopsonized surface, i.e., one μm fluorescent latex particles, decreasing the number of internalized particles from 12.6 ± 2.9 per cell to 5.9 ± 1.4 per cell (P < 0.01, n = 15), as measured using flow cytometry. However, binding of bone particles by the cells was not diminished by IFN-γ. Exogenous α-imerferon and human recombinant IL-1β, TNF-α, and lymphotoxin did not alter bone particle degradation. In addition, endogenous IL-1β release from human monocyte/macrophages exposed to bone particles was negligible and unaffected by IFN-γ.We conclude that IFN-γ is a potent and specific inhibitor of monocyte/macrophage-mediated bone particle degradation, and that this activity does not appear to be due to effects on the ability of monocytes to bind bone particles or to release IL-1 in response to the particles     

Adoptive transfer of CD4+Foxp3+ regulatory T cells to C57BL/6J mice during acute infection with Toxoplasma gondii down modulates the exacerbated Th1 immune response

Infection of C57BL/6J mice with the parasite Toxoplasma gondii triggers a powerful T_h1 immune response that is detrimental to the host. During acute infection, a reduction in CD4⁺Foxp3⁺ regulatory T cells (Treg) has been reported. We studied the role of Treg during T. gondii infection by adoptive transfer of cells purified from transgenic Foxp3^EGFP mice to infected wild type animals. We found a less severe weight loss, a significant delayed mortality in infected Treg-transferred mice, and reduced pathology of the small intestine that were associated with lower IFN-γ and TNF-α levels. Nevertheless, higher cyst number and parasite load in brain were observed in these mice. Treg-transferred infected mice showed reduced levels of both IFN-γ and TNF-α in sera. A reduced number of CD4⁺ T cells producing IFN-γ was detected in these mice, while IL-2 producing CD4⁺ T cells were restored to levels nearly similar to uninfected mice. CD25 and CD69 expression of CD4⁺ T cells were also down modulated. Our data show that the low Treg cell number are insufficient to modulate the activation of CD4⁺ T cells and the production of high levels of IFN-γ. Thus, a delicate balance between an optimal immune response and its modulation by Treg cells must exist.