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Major histocompatibility complex (MHC) class I and class II molecules play critical roles in the activation of the adaptive immune system by presenting antigens to CD8+ and CD4+ T cells, respectively. Although it has been well known that CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, as a master regulator of MHC class II gene expression, the mechanism of MHC class I gene transactivation was unclear. Recently, another NLR protein, NLRC5 (NLR family, CARD domain-containing 5), was identified as an MHC class I transactivator (CITA). NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway. CITA/NLRC5 plays a crucial role in human cancer immunity through the recruitment and activation of tumor killing CD8+ T cells. Here, we discuss the molecular function and mechanism of CITA/NLRC5 in the MHC class I pathway and its role in cancer.

Cancer cells need to escape immune surveillance for successful tumor growth. Loss of MHC class I has been described as a major immune evasion strategy in many cancers. MHC class I transactivator (CITA), NLRC5 [nucleotide-binding domain and leucine-rich repeats containing (NLR) family, caspase activation and recruitment domain (CARD) domain containing 5], is a key transcription coactivator of MHC class I genes. Recent genetic studies have revealed that NLRC5 is a major target for cancer immune evasion mechanisms. The reduced expression or activity of NLRC5 caused by promoter methylation, copy number loss, or somatic mutations is associated with defective MHC class I expression, impaired cytotoxic T cell activation, and poor patient prognosis. Here, we review the role of NLRC5 in cancer immune evasion and the future prospects for cancer research.

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of β2 -microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. © 2016 BioFactors, 42(4):349-357, 2016.

A series of novel bis-heterocycles encompassing isoxazolines and triazoles were synthesized through a novel one-pot procedure that involves in situ generation of nitrile oxide and its 1,3-dipolar cycloaddition to variedly substituted acrylates. The synthesized bis-heterocycles (8a-l) were subjected to in vitro lymphocyte proliferation assays followed by in vivo studies of the more active compounds (8g and 8h) to assess their influence on various aspects of the immune system like ex vivo splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titer), delayed-type hypersensitivity reaction, T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4), NO (macrophage) production, and toxic effects. The results from the in vitro and in vivo studies establish that the tested compounds 8g and 8h possess excellent immunopotentiating activity.

MHC class I and MHC class II plays a major role in adaptive immune responses through activation of CD8+ T cells or CD4+ T cells respectively, by presenting intracellular or extracellular antigens. The MHC class I transactivator (CITA), NLRC5 was recently identified as a key transcriptional regulator of MHC class I and related genes. Although promoters of both MHC class I and class II genes share similar cis-regulatory elements, the specificity of NLRC5 to MHC class I gene transactivation remains unclear. To delineate the specificity of NLRC5 in NLRC5-dependent MHC classs I transactivation, we performed co-immunoprecipitation with different deletion mutants of IRF1. The results were further confirmed in mice lacking Irf1 and Nlrc5. We found that the transcription factor IRF1 associates with NLRC5, enabling efficient transactivation of MHC class I and related genes. Double deficiency of Irf1 and Nlrc5 resulted in the severe reduction of MHC class I and related gene expression in vitro and in vivo. Therefore, IRF1 is a key component that determine the specificity of NLRC5-dependent MHC class I gene transactivation, and their involvement in the CITA enhanceosome is critical for MHC class I-dependent immune responses. Further studies on the interaction of IRF1 and NLRC5 would shed light on the potential therapeutic intervention on the MHC class I related diseases such as auto-immune disorders, cancer immunity and in organ transplantation

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we describe insights that have emerged from a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis revealed diverse host proteins that were differentially phosphorylated following fungal infection, indicating global reprogramming of host kinase signaling during this process. Notably, phagocytosis of the pathogen activated the host autophagy initiation complex (AIC) as well as regulatory components that reside upstream of this complex. Cn-containing vacuoles (CnCVs) were found to be decorated with the cell surface marker CD44, which colocalized with components of the AIC complex. Taken together, these findings suggest that associations between CD44 and AIC proteins confer susceptibility to infection, thereby implicating novel host mechanisms in regulating fungal intracellular parasitism.

Herein, we report the isolation and immunomodulatory activity of 11 phytoconstituents, viz. 7 flavonoids, 3 pentacyclic triterpenes and 1 phytosterol of an unexplored plant Actinocarya tibetica Benth. Three flavones, 5-methoxy-6,7-methylenedioxyflavone (6), mosloflavone (7) and negletein (8), showed promising anti-inflammatory activity via inhibition of TNF-α and IL-1β with IC50 values of 0.22, 0.71, 16.4 μM and 10.8, 7.8, 6.4 μM, respectively. These flavones also showed dose-dependent inhibition of TNF-α, IL-1β and iNOS levels in the supernatant of mouse macrophage cell line J774A. Molecular modelling studies revealed orientation and interactions of flavones 6-8 in the active site of TNF-α. These flavones can be used as a starting point to discover lead structures for treatment of inflammatory and immunomodulatory diseases.

Novel lipidated analogs of iridoids viz., Agnuside and Negundoside with different chain length were synthesized and tested for immune adjuvant activity in the presence of weak antigen ovalbumin. Based on in vitro assay, 6-O-palmitoyl Agnuside (AG-3) was further taken up for detailed in vivo activity and found to significantly enhance the production of anti OVA IgG titer, neutralizing antibody (IgG1 and IgG2a) titer as well as soluble mediators of a Th1 (IL-2, IFN-γ)/Th2 response (IL-4) and proliferation of T lymphocyte subsets (CD4/CD8) and co stimulatory molecules CD80/CD86. Furthermore, the SAR studies revealed that presence of acyl group at aglycon moiety of these iridoid glycosides is crucial for immune adjuvant activity.

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice. Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.

In order to evaluate the role of ethyl acetate fraction (PNRS-EtOAC) obtained from the Prunus cerasus fruit in the modulation of immune responses, detailed studies were carried out using a panel of in vivo assays. Oral administration of PNRS-EtOAC (25–100 mg/kg) stimulated the IgM and IgG titre expressed in the form of hemagglutination antibody (HA) titre. Further, it elicited a dose related increase in the delayed type hypersensitivity reaction (DTH) after 24 and 48 h in BALB/c mice. Besides augmenting the humoral and cell mediated immune response, the concentration of cytokines (IFN-γ, IL-4, and TNF-α) in serum with respect to T cell interactions, i.e. the proliferation of lymphocytes were significantly increased at 50 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of PNRS-EtOAC in a dose-dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF-α production as a mode of action.

Bacterial lipoproteins and their synthetic analogs are strong immune modulators of the early host responses. In view of the strong adjuvanticity of bacterial lipopeptide mimics bearing lysine residues, a focused library of lipidated dipeptides and tripeptides has been synthesized with a view to understand the pattern of activity vis a vis the site and extent of lipidation. Compounds 4, 5 and 14 stimulate OVA specific IgG titer, neutralization of antibodies (IgG1 and IgG2a), T lymphocyte sub-sets (CD4/CD8) and its production of soluble mediators for Th1 (IFN-γ)/Th2 (IL-4) cytokines and costimulatory molecules (CD80/CD86) which are ideal traits of immune adjuvants. The results support lipidated lysine dipeptides as potent enhancers of humoral and cell mediated immune responses and thus might become promising immune-adjuvants for self adjuvanted vaccines.

The acylated analogs of picroside-II were synthesized and tested for immune-adjuvant activity in the presence of weak antigen ovalbumin found to stimulate anti-OVA IgG titer, neutralizing antibody (IgG1 and IgG2a) titer as well as the production of soluble mediators of a Th1 response (IL-2 and IFN-γ) and Th2 response (IL-4) and proliferation of T lymphocytes sub-sets (CD4/CD8). Furthermore, these modified analogs of picroside-II were able to elicit a substantial increase in anti-OVA IgG when compared with OVA alone. These results support the use of acylated analogs particularly PK-II-3 and PK-II-4 as potent enhancer of antigen-specific Th1 and Th2 immune responses and thus are promising immune-adjuvant candidate for vaccines.

The genotoxic potential of anti-inflammatory/anti-arthritic and anticancer plant based drug molecule Boswelic acids (BA) was studied by in vivo system. Systematic literature survey revealed that studies on the genotoxicity of BA are not available. Although reports on genotoxicity of Boswellia serrata dry extract and modified 3-O-acetyl-11-keto-β-boswelic acid are available and these studies were conducted in in vitro systems. The earlier general toxicity study of BA has been conducted by us, revealed it to be non toxic. The genotoxicity was carried out in Wistar rats using different cytogenetic assay system-abnormalities viz. chromosomal aberrations; sperm morphology, micronuclei and comet assays. Six groups of animals, each comprised of five rats, were taken for each study. Group1-4 received BA at 125, 250, 500 and 1000 mg/kg p.o., respectively prepared as 2% gum acacia suspension, fifth group received a positive control cyclophosphamide (CP) 40 mg/kg p.o. or metronedazole (MTZ) 130 mg/kg p.o. or mercuric chloride (HgCl2) 0.864 mg/kg p.o. (as per the experiment requirement) whereas the sixth group kept as vehicle control. The results on the bases of the data obtained revealed that BA is quite safe as it did not show any genotoxicity at any dose level up to 1000 mg/kg. The positive controls used in different experiments showed highly significant abnormal cytogenetic changes in comparison to the control group.

Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of β2 -microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. © 2016 BioFactors, 42(4):349-357, 2016.

A series of novel bis-heterocycles encompassing isoxazolines and triazoles were synthesized through a novel one-pot procedure that involves in situ generation of nitrile oxide and its 1,3-dipolar cycloaddition to variedly substituted acrylates. The synthesized bis-heterocycles (8a-l) were subjected to in vitro lymphocyte proliferation assays followed by in vivo studies of the more active compounds (8g and 8h) to assess their influence on various aspects of the immune system like ex vivo splenocyte proliferation (T- and B-cell proliferation), antibody production (HA titer), delayed-type hypersensitivity reaction, T-cell subtypes (CD4 and CD8), cytokine production (IL-2, IFN-γ, and IL-4), NO (macrophage) production, and toxic effects. The results from the in vitro and in vivo studies establish that the tested compounds 8g and 8h possess excellent immunopotentiating activity.

In the present investigation, adjuvant potential of two novel lipidated tripeptide lysine derivatives (KKSM and KKSMB) was evaluated using various in vitro and animal-derived models of humoral and cell-mediated immune events in response to hepatitis B surface antigen (HBsAg). The results were compared with alum adjuvanted with HBsAg. Both these molecules were found to stimulate anti-HBsAg IgG and neutralizing (IgG1 and IgG2a) antibody titres in mice sera. The two molecules stimulated the proliferation of T-lymphocyte sub-sets (CD4/CD8) as well as the production of soluble mediators of Th1 (IL-2 and IFN-γ) and Th2 response (IL-4) in spleen cell culture supernatant. Furthermore, the two lipidated tripeptides enhanced the CD4, CD8, CD3 and CD19 cell populations as well as CD4/CD8 derived IL-2, IL-4, IFN-γ and TNF-α in whole blood of treated mice. There was found to be the significant enhancement in the release of IL-12, IFN-γ and nitrite content in macrophage supernatant. Moreover, the two lipidated tripeptides enhanced the population of CD80 and CD86 in spleen-derived macrophages and did not show any hemolytic effect on rabbit RBCs. Taken together, these results suggest that both these molecules are the potent enhancers of anti-HBsAg immune response via augmenting Th1/Th2 response in a dose dependent manner.

In order to explore the possible role of macrophages and other necessary immune competent (T and B) cells in the modulation of immune responses, an attempt was made to study the immunomodulatory effect of an irridoid glycoside (RLJ-NE-299A) isolated from the roots of Picrorhiza kurroa. Both in vitro and in vivo studies were used to evaluate the effect of RLJ-NE-299A on humoral, cellular, and phagocytic activity of macrophages. The data obtained in the present study showed that RLJ-NE-299A significantly increased sheep red blood cell (SRBC) and induced antibody (IgM and IgG) titer and delayed type hypersensitivity (DTH) reaction in mice. Besides augmenting the humoral and cell-mediated immune response, it induced macrophage phagocytosis and stimulated cytokine-induced macrophage activation and nitric oxide (NO) production, which resulted in a high degree of protection against Candida albicans and Salmonella typhimurium infections. Flow cytometric analysis indicated the enhanced expression of co-stimulatory surface molecules CD80 and CD86. The ability of RLJ-NE-299A to upregulate these cell surface antigens involved in antigen presentation may provide an explanation for the increased T-cell mediated immunity involving macrophages. Taken together this in vitro and in vivo preclinical data suggests that RLJ-NE-299A acts as an effective immunomodulator specifically to improve macrophage function during infections. The effects of this agent on these cells at concentrations relevant to in vivo therapy support its immunopharmacologic application to modify cellular immunity.

Therapeutic immunoadjuvants have been derived through a straightforward glycosylation of various alcohols with unprotected and non-activated monosaccharides, under solvent free conditions using ammonium chloride as a catalyst. Sugar acids such as glucuronic acid can be glycosylated without esterification and after acetylation yielded a novel 3,6-anhydro derivative. All the synthesized sugar glycosides were screened for immunomodulatory activities to be classified as immunostimulator, immunosuppressor, or immunoadjuvant against the weak antigen oval albumin (OVA). Most of the compounds revealed immunosuppressive (↓) or immunostimulatory (↑) activity with reference to lymphocyte proliferation and possess potential as vaccine adjuvants.

This study was conducted to characterize and explore the endophytic fungi of selected plants from the Western Himalayas for their bioactive potential. A total of 72 strains of endophytic fungi were isolated and characterized morphologically as well as on the basis of ITS1-5.8S-ITS2 ribosomal gene sequence acquisition and analyses. The fungi represented 27 genera of which two belonged to Basidiomycota, each representing a single isolate, while the rest of the isolates comprised of Ascomycetous fungi. Among the isolated strains, ten isolates could not be assigned to a genus as they displayed a maximum sequence similarity of 95% or less with taxonomically characterized organisms. Among the host plants, the conifers, Cedrus deodara, Pinus roxburgii and Abies pindrow harbored the most diverse fungi, belonging to 13 different genera, which represented almost half of the total genera isolated. Several extracts prepared from the fermented broth of these fungi demonstrated strong bioactivity against E. coli and S. aureus with the lowest IC50 of 18 μg/ml obtained with the extract of Trichophaea abundans inhabiting Pinus sp. In comparison, extracts from only three endophytes were significantly inhibitory to Candida albicans, an important fungal pathogen. Further, 24 endophytes inhibited three or more phytopathogens by at least 50% in co-culture, among a panel of seven test organisms. Extracts from 17 fungi possessed immuno-modulatory activities with five of them showing significant immune suppression as demonstrated by the in vitro lymphocyte proliferation assay. This study is an important step towards tapping the endophytic fungal diversity from the Western Himalayas and assessing their bioactive potential. Further studies on the selected endophytes may lead to the isolation of novel natural products for use in medicine, industry and agriculture.