Perspective Chapter: The Role of Interferon Gamma in Clinical Medicine

Abstract

Interferon gamma (IFN-γ) is one of the key factors of both innate and adaptive immune response that promotes differentiation of naive CD4+ cells into effector Th1 T cells producing the main mediators of cellular immunity against viral and intracellular bacterial infections, and specific cytotoxic immunity through the interaction of T cells with antigen-presenting cells and macrophage activation. The clinical importance of IFN-γ includes its medical use to treat and prevent various viral and bacterial infections. IFN-γ has a direct antiviral effect on infected cells, activates local infiltrating dendritic cells, macrophages and NK cells, modulates the differentiation and maturation of T and B cells, and enhances inflammation and antiviral functions. Immunoregulatory effect of IFN-γ plays one of the essential roles in the regulation of adaptive immune response in patients with tuberculosis infection and cancer. Producing IFN-γ by T cells increases the efficiency of infiltrated phagocytic cells, by stimulating NO and maintaining local host defense during tuberculosis infection. The direct antitumor effect of IFN-γ revealed in several experimental models has numerous mechanisms for the effect of development. IFN-γ has crucial potential for enhancing any antiviral, antimycobacterial, and specific antitumor therapies.

1. Introduction

Interferon gamma (IFN-γ) is the only member of the type II interferon family, which was discovered and described by E. Frederick Wheelock in 1965 as produced in vitro by leukocytes after their stimulation with phytohemagglutinin (Phaseolus vulgaris extract) and inducing antiviral activity. The physicochemical and biological properties of this virus inhibitor are similar to those of interferon induced by the Newcastle disease virus, except for instability at pH 2 and 10 and at 56°C [1]. IFN-γ is a protein encoded by the IFNG gene, consisting of two polypeptide chains linked in an antiparallel manner [2]. In peripheral blood, IFN-γ is present in three fractions with different molecular weights. One fraction is the active free form of IFN-γ and the other two are mature IFN-γ molecules. A fully synthesized protein is glycosylated at amino acid sites where the level of glycosylation determines the final weight of certain fractions [3, 4]. Glycosylation prevents the degradation of IFN-γ by proteinases increasing its half-life and prolonging the effects mediated by IFN-γ [5]. “Immune” interferon, also called IFN-γ, is a highly pleiotropic cytokine secreted not in response to infection, but indirectly by mitogen-activated T cells and natural killer (NK) cells, the primary producers of IFN-γ during both innate, and adaptive phases of the immune response.

2. General production and signaling pathways

2.1 Adaptive or innate immunity

IFN-γ is produced by NK and natural killer T cells (NKT) of innate immunity, gamma-delta T cells, and B cells. CD8+ and CD4+ T cells are the main paracrine sources of IFN-γ during the adaptive immune response [6]. Professional antigen presenting cells (APCs) have also been proven to secrete IFN-γ. The production of IFN-γ by monocytes/macrophages, dendritic cells acting locally, is important in cell activation [7, 8]. Normally, in the early stages of the host immune response, IFN-γ production by NK cells, CD4 + T (Th1) cells, and CD8 + T cells is aimed at improving antigen recognition in APCs, such as macrophages and dendritic cells.

IFN-γ is one of the key cytokines that promote differentiation of naive CD4+ cells into effector Th1 T cells that produce the main mediators of cellular immunity against viral and intracellular bacterial infections [9]. IFN-γ is the main product of Th1 cells and drives Th1 effector mechanisms: a) innate cell-mediated immunity (through activation of NK cell effector functions); b) specific cytotoxic immunity (through the interaction of T cells with APCs); c) activation of macrophages.

IFN-γ increases the content of lymphocytes and leads to their long-term persistence in the tissue, induces the activation of the cascade of complement components and an acute phase response, plays a role in switching the production of the IgG class, and has a direct antiviral effect [7]. When activated, almost all CD8+ T cells, NK cells, and Th1 lymphocytes produce IFN-γ, stimulating cytokine activity and increasing the expansion of low avid NK cells. Among all the interferons/cytokines of the Th1 response, IFN-γ correlates most strongly with the Th1 response.

CD4+ Th1 are the main source of IFN-γ, determined by the secretion of IL-12, IL-2 and IFN-γ, as well as the expression of T-bet, which is a transcription factor of the T-box family, encoded by the TBX21 gene, and plays the role of a promoter of IFN-γ synthesis [10, 11]. The expression level of T-bet correlates with the production of IFN-γ in Th1 and NK cells. Thus, IFN-γ is produced in response to multiple stimulants from the tissue-specific environment.

Classical inducers of IFN-γ production are IL-12 and IL-18. They activate IFN-γ production by NK cells and T cells [12, 13]. IL-12 has a powerful immunomodulatory effect on innate and adaptive immune cells. IL-12 is secreted as a biologically active 70 kDa heterodimer, consisting of disulfide-linked alpha (p35) and beta (p40) subunits [14]. Gene expression of IL-12 and IFN-γ is coordinated (i.e., IL-12 induces IFN-γ and IFN-γ induces IL-12). Binding of IL-12 to its relative heterodimeric receptor, IL12RB1/2, induces signaling through Jak-mediated phosphorylation of STAT4. Signaling through STAT4 induces IFN-γ expression [15]. Many of the effects mediated by IL-12 are due to the inducible expression of IFN-γ and the shift of CD4 + T cells towards the Th1 phenotype. Synergism between IL-12 and IL-18 has been shown to significantly induce IFN-γ in B cells [16]. IL-12 and other cytokines enhance T cell CTL activity by increasing sensitivity to weak or self-antigens. Together, IFN-γ and IL-12 generate a very strong Th1 response. Th1 cell-mediated cellular immunity and Th2 cell-mediated humoral immunity are modulated by IFN-γ, which influences the differentiation of naive T cells into Th1 or Th2 cells. Induction of IFN-γ in T cells initiates a positive feedback loop, as a result APCs sensitive to IFN-γ are primed to produce additional amounts of IL-12 [17]. IFN-γ blocks the production of IL-4, an inducer of Th2 cell differentiation and proliferation. The synergistic effect of IL-21, IL-18 and IL-15 enhances the production of IFN-γ. IL-15 is the strongest regulator of IFN-γ production compared to IL-21 in human NK and T cells. The cytokines IL-15 and IL-18 are produced by macrophages, while IL-21 is mainly produced by activated T cells. IL-24 or MDA-7 (Melanoma differentiation associated 7) can also activate the production of IFN-γ secreted by activated T-lymphocytes and monocytes and belongs to the IL-10 family [18]. IFN-γ increases the expression of HLA (major histocompatibility complex) class I and II antigen by increasing the expression of subunits increasing the expression and activity of proteasomes, and resulting in increased host sensitivity to an infectious pathogen and ability to identify and respond to this pathogen [19].

2.2 Interferon gamma crossroads

IFN-γ triggers antiviral and adaptive immune responses through the Janus kinase (Jak) and signal transducer and transcriptional activator (STAT) (Jak–STAT) signaling pathway which are the most studied intracellular signaling pathway. After IFN-γ binding and receptor dimerization, Jak1 and Jak2 are activated, which increases their catalytic activity and phosphorylation of the main target, STAT1. Phosphorylated STAT molecules get dimerized and transported to the nucleus, where they bind to the corresponding regulatory gene sequences and trigger their transcription [20]. In this canonical signaling pathway, IFN-γ get dimerized and binds to both IFN-γ receptors, which consist of two different ligand-binding chains: a high-affinity and highly expressed IFN-γ-R1 (α), and two signal-transforming low-affinity IFN-γR2 (β) with corresponding signaling mechanisms. The IFN-γR1 and IFN-γR2 chains belong to the family of class II cytokine receptors. The ligand-binding IFN-γ subunit IFN-γR1 and the assisting subunit IFN-γR2 correspond to chromosomes 6 and 21 in humans [21]. These subunits are intracellularly associated with the kinases of the Jak family, Jak1 and Jak2, respectively. Jak-1 interacts with the IFN-γR1 receptor subunit and Jak-2 interacts with the IFN-γR2 subunit of the IFN-γ receptor. The IFN-γR2 chain limits sensitivity to IFN-γ, and the IFN-γR1 chain is usually in excess. But the expression level of IFN-γR2 can be tightly regulated depending on the state of cell differentiation or activation. Receptors are expressed on the surface of almost all cell types. The expression level is determined by the cell type and its activation status. Initially, IFN-γ binds to IFN-γR1, and the formed IFN-γ:IFN-γR1 complex facilitates its binding to IFN-γ-R2, then events of the downstream signaling pathway are initiated [22].

Transcriptional activation of IFN-γ genes occurs through several mechanisms. The most studied response to IFN-γ is mediated by the STAT-1-containing transcription factor GAF (gamma-activated factor), which is activated by the action of tyrosine kinases Jak1 and Jak2 and binds to the GAS (Gamma Activating Sequence), present in the promoter regions of many genes. As a result of gene activation, the formation of a cellular immune response to a viral infection begins [23]. The JAK/STAT pathway is the main signaling pathway initiated by IFN-γ stimulation. Further, IFN-γ, together with one of its receptor subunits IFN-γR1 and pSTAT1, translocates to the cytoplasmic domain in combination with endocytosis and induces gene expression by binding to GAS elements in the promoter region of inducible IFN genes [24].

Activation of receptor-associated JAKs leads to subsequent phosphorylation, activation, and dimerization of latent cytoplasmic STAT transcription factors. The IFN-γ signaling pathway is negatively regulated by SHP-phosphatases (Shp2) or proteins from the cytokine signaling suppressor family (SOCS), mainly SOCS1 and SOCS3 in the cytoplasm, which are involved in the innate and subsequent adaptive immune responses. SOCS-1 binds to Jak1/2, interfering with tyrosine kinase activity and inhibiting further IFN-γ signaling [25]. This pathway can be inhibited by a protein-based inhibitor of activated STATs, which prevents gene transcription by inducing STAT1 dephosphorylation and DNA release [3]. IFN-γ induces genes called interferon-stimulated genes (ISGs) that are both positive and negative regulators of inflammatory signaling [26].

IFN-γ stimulated cells overexpress interferon regulatory factor-1 (IRF-1), a member of the IRF family, which induces the expression of multiple genes involved in biological processes, such as cell cycle regulation, apoptosis, tumor growth inhibition, activation of the synthesis of related molecules, associated with HLA class I, which increases the sensitivity of cells, exposed to IFN-γ, to cytotoxic attacks on T cells [27].

When viruses inhibit the activation of STAT1 molecules, IFN-γ can independently induce a non-canonical signaling pathway [28] in which IFN-γ is able to induce gene expression in bone marrow STAT1−/− macrophages, suggesting that IFN-γ acts independently of STAT-1 or in an alternative non-canonical manner. As a rule, the activation of the non-canonical pathway occurs later, after the activation of STAT-1. However, there is evidence that the non-canonical pathway can be activated in the absence or presence of STAT-1 in a dependent manner [29]. The IFN-γ and IFN-α/β signaling pathways intersect at several levels, partially overlapping, which makes it possible for certain functions to cross-talk within the cell. This crossover mechanism is relevant because in vivo cells are not stimulated in isolation by a single cytokine, but rather a cytokine cocktail induces gene expression through the integration of multiple signaling pathways.

3. Mechanisms of interferon gamma action

Viruses are intelligent living organisms because they have the ability to invade intracellular organelles and infect host cells [30]. IFN-γ has a direct antiviral effect on infected cells, activates local infiltrating dendritic cells, macrophages and NK cells, modulates the differentiation and maturation of T and B cells, enhances inflammation and antiviral functions [31]. Some of the well-studied antiviral functions of IFN-γ are largely devoid of a specific antiviral mechanism. For example, IFN-γ is a potent inducer of indolamine-2,3-dioxygenase (IDO) and nitric oxide synthase (NOS) [32]. Tryptophan depletion and nitric oxide (NO) production due to IDO and NOS expression, exhibit pronounced antiviral effects, the molecular details of which are mostly unclear [33]. The suppression of any stage of viral life cycle can lead to the inhibition of viral genome replication. IFN-γ is a potent antiviral cytokine that interferes with various stages of the viral life cycle in stimulated cells using the following mechanisms: 1. Inhibits the penetration of virus, both at the extracellular and intracellular stages, by controlling the expression and/or distribution of receptors necessary for the penetration of virus. 2. Inhibits viral replication by disrupting viral replication niche. 3. Disrupts gene expression, preventing translation. 4. Violates stability by interfering with the assembly of the nucleocapsid. 5. Violates the release of virus by breaking the disulfide bond of the required site for cellular interaction. 6. Changes virus reactivation by suppressing the main regulator of viral transcription. 7. Inhibits the penetration of virus at the stage of the invasive viral transfer from endosome to cytoplasm [31]. IFN-γ can also exhibit non-cytolytic antiviral activity against certain viruses. However, the specific targets and effector proteins of the IFN-γ-dependent antiviral response are largely unknown [34].

Tuberculosis (TB) is a chronic infection accompanied by complex changes in both specific and nonspecific reactivity in a patient’s body. During TB infection at an early stage (several hours), type I and II interferons are produced as the first line of immune defense in order to attract the largest number of dendritic cells and macrophages to the site of the lesion, that trigger active phagocytosis and inactivate the pathogen. The optimal immune response is formed a few days after infection. Each form of the tuberculous inflammatory process is characterized by an individual pattern of immunological changes. In patients with tuberculomas, there is a decrease in the phagocytic and functional-metabolic activity of monocytes and NK cells, an increase in the number of CD11b + and CD11c + adhesion molecules on granulocytes, and the number of T-lymphocytes. Infiltrative tuberculosis is accompanied by an increase in the population of monocytes with intensive expression of HLA-DR on them, granulocytes are characterized by the growth of the expression of CD11b + and CD11c + adhesion molecules, the number of T-lymphocytes falls [35]. CD4 + Th1 cells and macrophages play the main regulatory role in the development of the immune response in TB. Quantitative and qualitative imbalance of regulatory subpopulations of T-lymphocytes is accompanied by interleukin-dependent immune disorders and other pronounced changes in the cytokine system directing the TB process along a productive or exudative, caseous pathway. CD4+ and CD8+ effector T-cells are sent to the affected area, and begin to induce type II interferons (IFN-γ) greatly shifting the balance towards this class of cytokines and reducing the risk of developing an active TB process. These T-cells, by producing IFN-γ, increase the efficiency of infiltrated phagocytic cells, especially polymorphonuclear neutrophils [36], by stimulating NO and thereby maintaining local host defense [37].

IFN-γ acts as inhibitor of continuous IL-1β production and recruitment of neutrophils, preventing tissue damage. This adaptive immune response allows suppression of innate inflammatory pathways during the development of persistent TB infection. IFN-γ and IFN-γ-dependent NO plays an extremely important role not only in boosting the resistance to Mtb due to antimicrobial activity, but also in the survival of the macroorganism during this chronic infection [38].

Genetics is also very important for resistance and susceptibility to TB. In a study by Sérgio C. A. et al. the populations of lung dendritic cells derived from genetically different hosts have been studied in terms of their role in the size and function of CD4+ populations. At 30 days after infection with H37Rv M. Tuberculosis, C57BL/6 mice, which generate a stronger IFN-γ-mediated immune response than BALB/c mice, showed a higher number of CD11c + CD11b-CD103+ cells in the lungs compared to BALB/c mice that showed a high frequency of CD11c + CD11b + CD103 cells. CD11c + CD11b-CD103+ cells purified from the lungs of C57BL/6 of infected mice induced higher concentrations of CD4+ IFN-γ producing cells. This pattern of immune response seems to be related to the genetic characteristics of the host. The authors of the work concluded that genetic differences can reveal immunological biomarkers for the development of tests that predict the progression of TB infection [39].

Therefore, if there is a defect (genetically determined or not) in the immune mechanism, recruited macrophages can facilitate infection by providing the microorganism with an opportunity for intracellular growth and spread. Type I IFN in this case will contribute to the development of TB disease by inducing IL-10 and deactivating macrophages. The first level of immune protection will be broken and the cellular response to the antigen blocked, as a result a latent form of tuberculosis may develop. At the same time, mycobacteria tuberculosis (MBT) can suppress the synthesis of endogenous IFN-γ by secreting zinc metalloprotease (ZmpA) inhibiting the production of IL-1β by the host cell. It suppresses the synthesis of PI(3)P, slows down the maturation of phagosomes, and contributes to the development of tuberculosis disease. During the development of TB infection IFN-γ provides the relationship between the two most important links in the immune response of the macroorganism enhancing the antigen-dependent immune response and stimulating the work of phagocytes. IFN-γ activating macrophages attracts them to the focus of infection, increases their ability to destroy absorbed mycobacteria, induces the release of nitric oxide - there is the only inducer of the synthesis of the MHC class II protein in the cell (APC for extracellular pathogens). To kill the mycobacteria survived in the phagosome under the influence of IFN-γ an autophagosome is formed in the macrophage cytosol, the bilayer membrane of it captures the phagosome with mycobacteria and merges it with the lysosome destroying the MBT by lysosome enzymes. The productive type of inflammation in TB is observed with the predominance of the immune response by cell type. It is characterized by a relatively high level of CD4, CD8 lymphocytes, CD4/CD8 index, adequate production of IL-1β, IL-2, IL-12, TNF-a, IFN-γ [40].

Tumor is the result of a complex mechanism of interaction between genetic and epigenetic changes that leads to a dysregulation of intercellular relationships and intracellular signaling pathways. The heterogeneous cellular composition of the tumor and the microenvironment altered by the tumor both limit the effectiveness of standard chemotherapy due to internal, already existing or acquired drug resistance, as well as due to the suppression of apoptosis [41]. One of the recent studies shows that resistance to immunotherapy with checkpoint inhibitors is attributed to defects in the IFN-γ signal [42].

The role of IFN-γ in modulating immune responses is enormous [43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59]. IFN-γ is considered a key component in the immune control of cancer, stimulation of antitumor immunity, and aiding in the recognition and elimination of tumors [43, 59, 60, 61, 62, 63, 64]. In addition to activating APCs, enhancing the expression of a number of cytokines (IL-12 and IL-18) leading to the differentiation of Th-1 cells into cytotoxic lymphocytes, induction of a signal cascade in T cells to ensure their effector functions and activation of the expression of molecules of HLA, that is, the realization of cytotoxicity against the tumor, IFN-γ also causes regression of the vascular system of the tumor. Thus, it is possible that IFN-γ slows down tumor growth by inducing its ischemia [45, 65].

The direct antitumor effect of IFN-γ was revealed in several experimental models, however, the mechanisms for the development of this effect were different. So, colorectal cancer cells, it caused apoptosis associated with autophagy by induction of reactive oxygen species by mitochondria. In the T98G glioblastoma line, the induction of apoptosis is due to the suppression of the PI3K/AKT pathway, while the apoptosis of another glioblastoma cell line (U87MG) occurred independently of the PI3K/AKT signaling pathway, through the activation of NF-kB. In human pancreatic carcinoma cells, IFN-γ induces apoptosis in a caspase-1-dependent manner [56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68]. IFN-γ can induce the activation of some micro-RNAs that have an antitumor effect. Thus, it has been shown on melanoma cell lines that activation of miR-29a/b via STAT-1 by IFN-γ leads to an increase in the rate of IFN-γ with other molecules to implement the antitumor effect [63, 69]. However, the response of myeloid cells and other hematopoietic cells to IFN-γ was insufficient for tumor regression, while the effect of IFN-γ on endothelial cells provided a significant antitumor effect. That is, for the development of the antitumor effect, the action of IFN-γ directly on tumor cells and tumor-infiltrating lymphocytes is not enough, but its effect on stromal cells is also necessary [70, 71, 72, 73].

The mechanism of the complex immune response to cancer may depend on tumor microenvironment [74]. Unfortunately, the tumor can avoid exposure to endogenous IFN-γ due to the loss of expression of molecules of HLA I class, due to metabolic stress. In several studies, the loss of MHC I expression in cancer cells correlated with the resistance to checkpoint blockade or adoptive immunotherapy. However, in some cancers with low levels of MHC I, it was possible to increase its expression via exogenous interferon therapy [75, 76, 77]. The mechanism of how exogenous IFN restores MHC I expression has not been studied in detail. Thus, the answer to the question of how IFN-γ induces signaling pathways that initiate and propagate the apoptotic cascade remains to be seen [78, 79]. One of studies showed that IFN causes increased histone acetylation, demethylation of DNA promoters of TAP genes and immunoproteasomes [77]. Possibly IFN induces IRF1 [80] or stimulates NK cells, which self-disinhibit the launch of their effector mechanisms through the expression of killer inhibitory receptors (KIRs), when they encounter cells with an abnormally low level of MHC I. This is just one of the possible mechanisms described [79, 81].

One more significant fact is that IFN-γ in combination with TNF-α induces the expression of MUC16, a mucin involved in carcinogenesis in breast, ovarian and endometrioid tumors [82].

4. Clinical importance of interferon gamma

Numerous studies are published on the clinical efficacy of IFN-γ in herpesvirus infections (herpes simplex virus type 1 and 2, varicella-zoster virus, Epstein-Barr virus) [78, 83, 84, 85, 86, 87]. The use of IFN-γ has been studied in viral complications after organ transplantation, in purulent-septic diseases of newborns, postnatally acquired cytomegalovirus infection, mumps, multiple sclerosis and various bacterial diseases [88, 89, 90, 91, 92]. IFN-γ has been used in the complex treatment of patients with human papillomavirus infection, according to the published study results, decreases in virus titer, improvement in the condition of patients, a decrease in the duration and severity of relapses, and faster clinical recovery of patients [93, 94]. Urological community [95, 96] has shown a positive effect of the use of recombinant human IFN-γ on chronic prostatitis therapy, expressed in a decrease in pain syndrome, difficulty urinating and improving the quality of life of the patient. IFN-γ has also been used to inhibit Ebola virus infection in macrophages, an early cellular target of infection [97]. The successful treatment of persistent urethroprostatitis with the identified association of sexually transmitted infections is described. Positive dynamics of the most important immunological parameters was noted after complex treatment with the use of IFN-γ [98, 99].

The inclusion of recombinant IFN-γ in therapy of influenza contributes to a more rapid relief of catarrhal and respiratory symptoms both in adults and children [100]. It was found that the universal risk factor for the development of complications in influenza in children is a low blood level of IFN-γ both in the acute period and in dynamics [101, 102]. IFN-γ exhibits pronounced antiviral activity against various strains of influenza virus, including avian and swine types. The use of exogenous IFN-γ in inhalation combined with subsequent narrow-band optical radiation in pediatrics for acute bronchitis induced by virus infections, including adenovirus, rhinosyncytial (RS) virus, parainfluenza virus with underlying persistence of Epstein Barr virus (EBV), cytomegalovirus (CMV), S. aureus, S. pneumoniae and other microorganisms in the lower respiratory tract helped to avoid bacterial complications. Given the fact that influenza viruses can suppress the production of type 1 IFN, the use of type 2 IFN for the prevention and treatment of influenza is advised. The combined use of two types of interferons (alpha and gamma) for the treatment of influenza has also been shown to be promising [103, 104, 105, 106]. A comparative, open, randomized study with COVID-19 patients using IFN-γ in terms of changes in the levels of lactate dehydrogenase and C-reactive protein, blood oxygen saturation and other vital functions in the period of inpatient treatment, as well as survival criterium [107, 108].

A systematic review published by J. Ghanavi et al. in 2021 showed that IFN-γ and its receptor (IFN-γR) play a key role in the formation of immunity against MTB and non-tuberculous mycobacteria [30]. The authors emphasized that there is increasing evidence of IFN-γ’s important role in host defense against these intracellular pathogens by activating macrophages. Studies confirm that IFN-γ is an integral part of various antibacterial “defenses”, including granuloma formation and phagosome-lysosome fusion leading to the death of intracellular mycobacteria. The absence or deficiency of IFN-γ correlates with the overgrowth of intracellular bacteria and the development of tuberculosis infection with mycobacteriosis. New approaches to the treatment of mycobacterial infections are closely related to cell and gene therapy based on the modulation of IFN-γ and IFN-γR.

Meta-analysis on the impact of recombinant IFN-γ on TB patients performed with a number of randomized controlled clinical trials [109] proved its clinical efficacy including for combination of TB with HIV infection. Statistically significant benefits of treatment with recombinant IFN-γ were shown by the results of sputum conversion and X-ray examination of patients. The pooled relative risk (RR) for conversion was 1.97 (95% CI: 1.20–3.24; p = 0.008) after 1 month of treatment, 1.74 (95% CI 1.30–2.34; p = 0.0002) after 2 months of treatment, 1.53 (95% CI 1.16–2.01; p = 0.003) after 3 months of treatment, 1.57 (95% CI 1.20–2.06; p = 0.001) after 6 months of treatment and 1.55 (95% CI 1.17–2.05; p = 0.002) at the end of treatment. The pooled RR for radiographic progression was 1.38 (95% CI 1.10–1.17, p = 0.006) at the end of treatment. Comprehensive treatment with the use of IFN-γ leads to a significant improvement in the indicators of “sleep-rest”, “spirituality”, “everyday affairs”, a decrease in dependence on drugs and medical care. For intramuscularly administered IFN-γ, the meta-analysis included three studies that showed a significant improvement in sputum conversion rates after 2 months of treatment. A randomized controlled trial with aerosolized and subcutaneously administered IFN-γ found a significant reduction in the symptoms of fever, wheezing and night sweats compared with the control group after 1 month of treatment. Meta-analysis suggests that adjuvant therapy using IFN-γ, especially in aerosol form, is effective for patients with TB. IFN-γ within the complex therapy of respiratory TB can significantly increase the effectiveness of anti-tuberculosis therapy (accelerate the cessation of bacterial excretion and closure of cavities in the lungs), prime the immune system and the quality of life of patients. In addition, IFN-γ aerosol may be particularly useful in preventing the development of mycobacterial infections in HIV-infected patients with significantly reduced CD4 cell counts [110].

The results of experimental studies and clinical trials conducted mainly in patients with multi-drug resistant TB, made it possible to propose IFN-γ not only to shorten the long-term standard chemotherapy regimen, however to prevent the latent TB [111]. A. Fortes et al. in 2005 [112] showed that the patients infected with an antibiotic-resistant MBT strain are characterized by a reduced level of endogenous IFN-γ compared to normal patients, and the additional exposure to exogenous IFN-γ in the first months of treatment may lead to the induction of immune system. In this case the appointment of exogenous IFN-γ becomes, in fact, a replacement therapy that can compensate for the endogenous deficiency of the cytokine.

A lot of data has been accumulated regarding the role of IFN-γ in tumor therapy. Antitumor activity of exogenous IFN-γ seems promising for the subsequent development of immunotherapeutic strategies for the complete eradication of cancer. At the same time, it is critical also for the further use of interferon drugs in cancer patients. Clinical studies have shown the effectiveness of IFN-γ therapy in combination with cyclophosphamide and cisplatin, which provided a significant increase in progression-free survival in ovarian cancer. Thus, in a randomized controlled trial 148 patients undergoing primary surgery for stage IC-IIIC ovarian cancer received subcutaneous IFN-γ. In the control group, women received 100 mg/m2 cisplatin and 600 mg/m2 cyclophosphamide, the experimental group included the above regimen with IFN-γ 0.1 mg subcutaneously on days 1, 3, 5, 15, 17, and 19 every 28-day cycle. Progression-free survival (PFS) at 3 years improved from 38% in the control group to 51% in the treatment group, corresponding to median progression times of 17 and 48 months (P = 0.031, relative risk of progression 0.48, CI 0.28–0.82). Overall three-year survival was 58% and 74%, respectively (not significant, median not yet reached). Complete clinical responses were observed in 68% with IFN-γ compared to 56% in controls (not significant). Toxicity was comparable in both groups, with the exception of a mild flu-like syndrome, which was observed in most patients after administration of IFN-γ. Thus, with acceptable toxicity, the inclusion of IFN-γ in first-line ovarian cancer chemotherapy has the advantage of prolonging progression-free survival. This study showed that IFN-γ in combination with carboplatin and paclitaxel is safe as a first-line treatment in patients with advanced ovarian cancer [113]. In an early study by Pujade-Lauraine et al. [114], human recombinant IFN-γ was administered intraperitoneally to patients with stage IIb, IIc, III epithelial ovarian cancer when peritoneal involvement was detected at laparotomy. The study involved 108 patients who received IFN-γ at a dose of 20 × 10 IU/m2 intraperitoneally twice a week for 3–4 months in the absence of clinical manifestations of the disease. IFN-γ response was assessed by exploratory laparotomy. Of 98 patients, 31 (32%) achieved a surgically confirmed response, including 23 patients (23%) with a complete response (CR). Significant prognostic factors for response to IFN-γ were age and size of the residual tumor: a CR rate of 41% was observed in 41 patients younger than 60 years of age and with a residual tumor size of less than 2 cm. The probability of response was independent of previous response to first-line chemotherapy. The median duration of response was 20 months and the 3-year survival rate was 62%. IFN-γ response was the most significant predictor of survival in patients with residual disease. Side effects included fever, flu-like symptoms, neutropenia, and abnormal liver enzyme levels. No significant peritoneal fibrosis was noted. Thus, this work conclude that intraperitoneal administration of IFN-γ promote antitumor response in ovarian cancer [114]. In the study performed by Schmeler et al. [115], human recombinant IFN-γ was administered as subcutaneous injection before and after intravenous carboplatin to patients with recurrent, platinum-sensitive ovarian, fallopian tube and primary peritoneal cancer. The study enrolled 59 patients who received IFN-γ at a fixed dose of 100 mcg on the fifth and seventh day of each 7-day cycle of GM-CSF. IFN-γ response was assessed using the modified World Health Organization Response Evaluation Criteria in Solid Tumors (RECIST). Of the 54 evaluable patients, 9 (17%) achieved a complete response, 21 patients (39%) with a partial response. The overall response rate was 56%. No patients showed treatment-related deaths [115]. Marth et al. [116] in a phase I/II trial tested whether IFN-γ was safe to use it in combination with current standard of care, paclitaxel and carboplatin, in patients with ovarian cancer. Thirty-four patients with newly diagnosed advanced stage III/IV epithelial ovarian cancer were treated with six to nine cycles of paclitaxel (175 mg/m2) and carboplatin ([AUC] 5) every 3 weeks. IFN-γ was administered in increasing doses from 6 days/cycle 0.025 mg SC to 9 days/cycle 0.1 mg SC. As expected, IFN-γ administration was associated with flu-like symptoms. Grade 3/4 neutropenia was observed in 74% (25 of 34) of patients. Other side effects, in particular peripheral neuropathies, were within the previously observed ranges for the combination of paclitaxel + carboplatin. The overall response rate in patients who received either six or nine doses (0.1 mg) of IFN-γ/cycle (n = 28) was 71%. Thus, this study demonstrated the safety of using IFN-γ in combination with carboplatin and paclitaxel for the first-line treatment of patients with ovarian cancer [116].

Intravesical IFN-γ instillations in bladder cancer have been shown to be effective in preventing recurrence. The study included 123 patients with stage Ta, T1, grade 2 tumors who underwent transurethral tumor resection. In group A, 60 patients received IFN-γ (1.5 × 10(7) IU/instillation), while 63 patients from control group B received mitomycin C (40 mg/instillation). During the year of therapy, the following regimen was used: 8 weeks weekly, then four times every two weeks, and then eight monthly instillations for both regimens. The immunophenotypes of intratumoral and intramural leukocytes were also analyzed by immunohistochemical methods and using flow cytometry. As a result of the treatment, relapse was not observed in group A in 44 of 60 (73.4%) patients and in group B in 36 of 63 (57.2%) during a mean follow-up period of 26.5 months (range 3–49 months). After IFN-γ instillations, tissue samples and bladder washes showed a statistically significant increase in the number of T cells: T-helpers, T-cytotoxic cells, natural killer cells and total leukocytes, as well as the number of B cells expressing MHC I, and total leukocytes expressing HLA-DR [117].

Effects of IFN-γ during the adjuvant treatment of radically operated patients with lung adenocarcinoma were evaluated by Pyltsin SP et al. [118] according to the dynamics of the immune status. The study enrolled 63 patients with morphologically verified stages I-IIIA of lung adenocarcinoma. Radical extended pneumonectomy was performed in 17 (26.9%) patients, extended lobectomy - in 42 (66.7%), sublobar resections - in 4 (7.9%). Radically operated patients were randomized into two groups, comparable in terms of the main anthropometric and clinical criteria. By the 21st day after the operation, there were no significant differences in the parameters of cellular immunity in patients of the compared groups, however, significant differences were observed when comparing the parameters of radically operated patients and healthy individuals. Obviously, the transient nature of secondary induced immunosuppression in a tumor process was at least of a prolonged nature (up to 3 months or more) possibly due to the surgical intervention. However, the lack of a trend towards normalization of indicators suggests an increase in immune deficiency, provided by sufficiently aggressive and prolonged adjuvant cytotoxic therapy. After the 1st course, there were no statistically significant differences between the groups, the indicators of both the main and control groups remain lower than in healthy individuals. The changes detected in both groups demonstrated, as before the start of adjuvant treatment, a certain lack of cellular immunity in the form of suppression of T-helpers (CD3 + CD4+) and depression of natural killers (CD56+). The study of the further dynamics of the state of the cellular link of immunity showed that, as a result of IFNγ use after the second course of adjuvant drug therapy, statistically significant differences were detected. In the study group, compared with the control group, the number of T-helper lymphocytes significantly increased (34.1 ± 0.7% versus 31.8 ± 0.8%; p < 0.05). The opposite dynamics was observed in relation to cytotoxic T-lymphocytes, the level of which in the study group got statistically significantly lower compared with the control group (26.2 ± 0.6% and 29.8 ± 0.8%, respectively, p < 0.05). At the same time, there was found a statistically significant increase in the ratio of CD4+ to CD8+, equal to 1.21 ± 0.04 in the study group compared with 1.04 ± 0.016 in the control group (p < 0.05). Depression of NK persisted, both in the study and in the control group, against the background of an increase in the relative number of cytotoxic T cells (CD8+ lymphocytes), especially in the control group. Such changes can develop as a result of compensation for the reduced functional activity of NK by cytotoxic T-lymphocytes. Thus, the administration of exogenous IFNγ leaded to favorable dynamics of the T-helper-inductor link in the patients of the study group, that suggests the immunomodulatory effect of IFNγ on immunocompetent cells with the CD4+ phenotype, which are the main producers of this cytokine when they are differentiated by the Th1 type. Conducting three courses of chemoimmunotherapy to patients of the study group caused the most significant increase in the number of CD4+ lymphocytes, that reached 36.6 ± 0.5% compared with the control group (30.6 ± 0.7%, p < 0.05). The opposite dynamics was noted in the content of CD8+ cells, the level of which gradually decreased in the patients of the study group and increased in the control group; at the end of the 3rd course, it was 25.2 ± 0.6% and 31.9 ± 0.6%, respectively (p < 0.05). All these changes occurred against the background of a statistically significant increase in the level of T-lymphocytes in the study group (54.5 ± 0.7% vs. 52.5 ± 0.7% in the control group; p < 0.05). Conducting adjuvant chemoimmunotherapy with the use of recombinant IFNγ made it possible to achieve a stable correction of the immune status of patients, characterized by the normalization of the main indicators of cellular immunity, with the exception of persistent suppression of the activity of CD56+ cells - NK, effectors of innate immunity, which was also constantly observed during polychemotherapy. In the opinion of the authors, the most important manifestation of the immune action of IFNγ was the leveling of suppression of CD4+ lymphocytes. The full functioning of subpopulations of T-lymphocytes of helpers ensures the regulation of the adaptive cellular immune response, which is very necessary for effective control of the micrometastatic phase of a tumor disease [118].

Early work by Tamura et al. [119] describes the use of recombinant IFN-γ in the treatment of T-cell leukemia in adults. The study involved 5 patients. The drug was administered intramuscularly or intravenously in increasing dosage from 1x10(6) to 8x10(6) JRU (Japan Reference Unit) per day. As a result of the therapy, 1 patient had a complete response, 2 had a partial response, the disease continued to progress in 1 patient, and 1 patient died during the study from pneumonia [119].

The published experience of local application of IFN-γ for the treatment of melanoma of the conjunctiva is represented by several successful clinical cases. The patients received combined treatment with mitomycin C, subconjunctival injections of IFN-γ, and brachytherapy with strontium ophthalmic applicators. IFN-γ at a dose of 500,000 IU was administered under local instillation anesthesia directly under the tumor daily for 10 days. After tumor reduction, brachytherapy was performed. In the first clinical case, after 4 months, a residual radiation reaction was observed in the form of conjunctival hyperemia. Melanoma nodes regressed, areas of flat melanosis remained without progression. In the second clinical case, after combined treatment, a complete regression of the tumor was observed with a follow-up period of 8 months. In the third clinical case, with the follow-up period of 64 months after the first treatment in the area of ​​the tumor cicatricial-modified conjunctiva was found. On the mucosa of the upper eyelid in the center of the scar, there was residual avascular, poorly pigmented tissue. Of the adverse reactions, the researchers observed only local pain at the site of subconjunctival injection of the drug, hyperemia and slight swelling of the conjunctiva and eyelid skin. These side effects did not lead to the continuation of treatment. Thus, the use of IFN-γ can expand the possibilities of organ-sparing treatment of conjunctival melanoma [120].

Clinical experience of IFN-γ application in the treatment of radiation cystitis accompanied with hematuria described by Kaprin AD et al. [121] concerned a study group of 12 patients (vs. 12 in a control group) with late radiation complications from the lower urinary tract. The drug was administered at a dose of 500,000 IU subcutaneously once a day every other day for 20 days (10 injections in total). The effectiveness of the treatment was assessed based on the dynamics of IPSS data (International Prostatic Symptom Score), the degree of pain syndrome, the time of relief of hematuria, and the restoration of urine sterility. The use of IFN-γ made it possible to increase the effectiveness of anti-inflammatory treatment of patients with radiation cystitis: urine sterility was restored in 65% of cases; 4.5 days earlier than in the control group [121].

S.N. Kazakova et al. [122] reported a clinical case of successful rehabilitation approach with local IFN-γ (Ingaron®) use combined to physical therapy in women with postradiation complications as a result of prior endometrial cancer [122].

The summary of antitumor evidence on the clinical efficacy of IFN-γ approaches in oncology is presented in Table 1. Current studies are primarily focused on the effects of IFN-γ in oncogynecology, breast cancer and solid tumors.

Cancer type	Therapy conditions	IFN-γ introduction	Results
Ovarian	First-line	Subcutaneous injection	148 women enrolled. CR were observed in 68%.; The median duration of response was 48 months (p = 0.031, RR of progression 0.48, CI 0.28–0.82) and the 3-year survival rate was 74% (n.s., median not yet reached). [113]
Ovarian	Second-line	Intraperitoneal injection	108 patients enrolled. 32% achieved a surgically documented response, including 23% of patients with a CR. A 41% CR rate was observed in patients younger than 60 y.o. and with residual tumor less than 2 cm. The median duration of response was 20 months and the 3-year survival rate in responders was 62%. [114]
Ovarian, fallopian tube and primary peritoneal	Before and after intravenous carboplatin	Subcutaneous injection	59 patients enrolled. Overall response rate was 56% (95% CI: 41–69%) with median time to progression of 6 months. [115]
Ovarian	First-line	Subcutaneous injection	28 patients enrolled. Overall response rate (CR or PR) was 71%. [116]
Bladder	Prevention of recurrence	Intravesical instillation	123 patients enrolled. CR was 73.4% during the median follow-up period of 26.5 months. [117]
Lung adenocarcinoma	Post-surgery adjuvant therapy	Intravenous injection	63 patients enrolled. Improvement of 3-year event-free survival rate by 19 percent (p = 0.06607). [118]
Leukemia	Adjuvant therapy	Intravenous infusion	11 patients enrolled. Overall response rate (CR or PR) was 60% during the median follow-up period of 4 months. [119]
Melanoma of conjunctiva	Local therapy	Subconjunctival injection	3 patients enrolled. Overall response rate (CR or PR) was 60% during the median follow-up period of 5 to 62 months. [120]

Table 1.

Summary on clinical evidence of antitumor effects of IFN-γ.

CI – confidence interval.

CR – complete response.

PR – partial response.

RR – relative risk.

Undoubtedly, IFN-γ can have direct antiviral and antitumor effect, besides immunomodulating one. However, for a wide clinical use of IFN-y, it is necessary to carry out further experimental research of the mechanisms and conditions that will provide full clinical value and reveal yet hidden potential of this natural pleiotropic molecule for successful health implementation.

5. Conclusion

IFN-γ occupies a special place in the interferon family. Besides antiviral, it has a strong immunoregulatory effect and plays one of the key roles in the regulation of adaptive immune response in patients with tuberculosis infection, and cancer. Due to its complex action, IFN-γ is quite important for enhancing any viral, mycobacterial and specific tumor clearance.