The Runx1 transcription factor cooperates with or antagonizes other transcription factors and plays essential roles in the differentiation and function of T lymphocytes. Previous works showed that Runx1 is expressed in peripheral CD4(+) T cells which level declines after T cell receptor (TCR) activation, and artificial deletion of Runx1 causes autoimmune lung disease in mice. The present study addresses the mechanisms by which Runx1 contributes to the maintenance of peripheral CD4(+) T cell quiescence. Microarray and quantitative RT-PCR analyses were employed to compare the transcriptome of Runx1 -/- CD4(+) T cells to those of unstimulated and TCR-stimulated Runx1 +/- cells. The results identified genes whose expression was modulated similarly by Runx1 deletion and TCR activation. Among them, genes encoding cytokines, chemokines, and Jak/STAT signaling molecules were substantially induced. In Runx1-deleted T cells, simultaneous increases in Il-17A and Rorγc, a known master gene in TH17 differentiation, were observed. In addition, we observed that the loss of Runx1 reduced the transcription of genes encoding quiescence-associated transcription factors, including Foxp1, Foxo1, and Klf2. Interestingly, we identified consensus Runx1 binding sites at the promoter regions of Foxp1, Foxo1, and Klf2 genes, which can be enriched by chromatin immunoprecipitation assay with an anti-Runx1 antibody. Therefore, we suggest that Runx1 may activate, directly or indirectly, the expression of quiescence-associated molecules and thereby contribute to the maintenance of quiescence in CD4(+) T cells.
BACKGROUND: Asthma is a complicated network of inflammatory reactions. It is classified into mild, moderate, and severe persistent asthma. The success of asthma therapy relies much on understanding the underlying mechanisms of inflammation at each stage of asthma severity. The aim of this study was to explore the differences in apoptotic potential, CD4/CD8 ratio, memory compartment, and T- helper (Th) 1 and 2 profile of peripheral blood lymphocytes (PBL) in patients with mild intermittent asthma and severe persistent asthma during exacerbation periods.
RESULTS: Four research lines were investigated and compared among mild asthmatics, severe asthmatics, and healthy groups by applying immunocytochemical staining of PBL. Antiapoptotic and proapoptotic proteins with Bcl-2/Bax ratio, CD4, CD8 markers with CD4+/CD8+ ratio, CD45RO+, CD45RA+ markers with memory/naive ratio (CD45RO+/CD45RA+). Th2/Th1 cytokines balance represented by IL-4/IFN-gamma ratio was measured by enzyme-linked immunosorbent assay (ELISA) for in vitro PBL cytokine synthesis. It was found that Bcl-2/Bax ratio was higher in severe than in mild asthmatics which in turn was higher than in healthy group. And memory/naive ratio of PBL was higher in severe than in mild asthmatics. Moreover, memory cells, CD45RO+ and CD45RO+/CD45RA+ ratio were correlated directly with Bcl-2/Bax, in severe and mild asthma patients. In contrast, CD4+/CD8+ ratio was not changed significantly among healthy group, mild and severe asthmatics. However, CD8+ cells were correlated directly with memory cells, CD45RO+, in severe asthmatics only. Interestingly, the dominant profile of cytokines appeared to change from T helper 2 (Th2) in mild asthmatics to T helper 1 (Th1) in severe asthmatics where the lowest in vitro IL-4/IFN-gamma ratio and highest IFN-gamma were found.
CONCLUSION: It was concluded that the underlying mechanisms of inflammation might vary greatly with asthma stage of severity. Mild intermittent asthma is mainly Th2 allergen-oriented reaction during exacerbations with good level of apoptosis making the inflammation as self-limiting, while in severe persistent asthma, the inflammatory reaction mediated mainly by Th1 cytokines with progressive loss of apoptosis leading to longer exacerbations, largely expanded memory cells, CD45RO+, leading to persistent baseline inflammation.
The Plasmodium falciparum serine repeat antigen (SERA) is one of the promising blood-stage malarial vaccine candidates. In this study, recombinant Mycobacterium bovis bacille Calmette-Guerin (rBCG) expressing the 22 kDa protein (SE22) from the 47 kDa Nterminal domain of serine repeat antigen (SERA), generated in favour of mycobacterium codon usage, elicited specific immune response in BALB/c mice with a mixed Th1/Th2 profile. Immunized sera containing high levels of specific IgG1 and IgG2a against the epitope (as determined by ELISA) were reactive with fixed P. falciparum merozoites as demonstrated by indirect immunofluorescence assay (IFA). Furthermore, the lymphocyte proliferative response to SE22 antigen from rBCG-immunized mice was higher than that of controls. The expression of intracellular cytokines (IL-2, IL-4 and IFNγ) in CD4+- and CD8+-cells was also enhanced following in-vitro stimulation with SE22. These findings indicate that a rBCG-based vaccine candidate expressing a blood-stage antigen of P. falciparum could enhance both humoral and cellular immune responses, thus paving the way for the rational use of rBCG as a vaccine candidate against malaria.
Hepatitis B virus (HBV) infection is a major cause of chronic liver disease that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses represent the key determinants of HBV clearance or persistence. Here, we investigated the role of the early activation marker, CD69 and effector cytokines, granzyme B (GrB) and IFN-γ in the exhaustion of innate-like TCR Vα7.2+CD4+T cells, in 15 individuals with chronic HBV (CHB) infection where six were HBV DNA+ and nine were HBV DNA-. The percentage of cytokine-producing T cells and MAIT cells were significantly perturbed in HBV patients relative to healthy controls (HCs). The intracellular expression of GrB and IFN-γ was significantly reduced in MAIT cells derived from HBV-infected patients as compared to HCs, and the levels correlated with the percentage and levels [mean fluorescence intensity (MFI)] of CD69 expression. The total expression of CD69 (iMFI) was lower in CHB patients as compared to HCs. The frequency of CD69+ cells correlated with the levels of cytokine expression (MFI), particularly in CHB patients as compared to HCs. In summary, the polyfunctionality of peripheral T cells was significantly reduced among CHB patients, especially in the TCR Vα7.2+CD4+T cells, and the levels of cytokine expression correlated with functional cytokine levels.
Tamm-Horsfall glycoprotein (THP) and uromodulin are the most abundant glycoproteins in non-pregnant women's/men's and pregnant women's urine, respectively. However, the bioactivities of these glycoproteins are still unclear.
Rhaphidophora korthalsii (Araceae) is a root-climber plant which has been widely used in Chinese traditional medicine for cancer and skin disease treatment. Previous reports have recorded its immunomodulatory effects on mice splenocyte and human peripheral blood. This study investigated the potential immunostimulatory effect of Rhaphidophora korthalsii on human PBMC enriched NK cell.
Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4(+) and CD8(+) cells were measured. MSC equally inhibit CD4(+) and CD8(+) subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.
Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing the malarial epitopes F2R(II)EBA and (NANP)3 as well as two T cell epitopes of the M. tuberculosis ESAT-6 antigen, generated in favour of mycobacterium codon usage elicited specific immune response against these epitopes. Immunised Balb/c mice demonstrated an increase in almost all of the IgG subclasses against both malarial epitopes and enhanced splenocyte proliferative response against the malarial epitopes as well as selected peptides of ESAT-6. Furthermore, flow cytometric analyses showed elevated numbers of CD4+ lymphocytes expressing IFN-gamma and IL-2 against the ESAT-6 peptides, suggesting a specific Th1-mediated response. This study demonstrated that expressing malarial and TB epitopes in a single rBCG construct induced appropriate humoral and cellular immune response against immunogenic epitopes from both organisms.
A case of chronic mucocutaneous candidiasis in a Malaysian child who subsequently developed disseminated tuberculosis and toxoplasmosis is described. The phenotype of her peripheral blood mononuclear cells showed discordance for her T cell markers. The presence of a subpopulation of CD2-/CD3+ mononuclear cells leading to an immunodeficiency state is consistent with failure of activation of CD2-mediated alternative pathway resulting in immunodeficiency. Such abnormal CD2-/CD3+ subpopulations have been described in lepromatous leprosy and foetal abortuses.
Peptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9-11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients' T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.
Although monocytes represent an essential part of the host defence system, their accumulation and prolonged stimulation could be detrimental and may aggravate chronic inflammatory diseases. The present study has explored the less-understood immunomodulatory effects of mesenchymal stem cells on monocyte functions. Isolated purified human monocytes were co-cultured with human umbilical cord-derived mesenchymal stem cells under appropriate culture conditions to assess monocytes' vital functions. Based on the surface marker analysis, mesenchymal stem cells halted monocyte differentiation into dendritic cells and macrophages and reduced their phagocytosis functions, which rendered an inability to stimulate T-cell proliferation. The present study confers that mesenchymal stem cells exerted potent immunosuppressive activity on monocyte functions such as differentiation, phagocytosis and Ag presentation; hence, they promise a potential therapeutic role in down-regulating the unwanted monocyte-mediated immune responses in the context of chronic inflammatory diseases.
Standardized extract of Phyllanthus amarus has previously been shown to have a strong inhibitory effect on phagocytic activity of human neutrophils. The current study was carried out to evaluate the effects of constituents of the extract of P. amarus on nitric oxide (NO) production as well as lymphocyte proliferation and cytokine release from phagocytes. Three compounds, ethyl 8-hydroxy-8-methyl-tridecanoate, 7β,19α dihydroxy-urs-12-ene, and 1,7,8-trihydroxy-2-naphtaldehyde, together with seven known compounds were isolated from the whole plant of P. amarus. The isolated compounds and reference standards, ie, gallic acid, ellagic acid, corilagin, and geraniin, which were quantitatively analyzed in the extracts, were evaluated for their effects on immune cells. Among the compounds tested, the lignans, especially phyltetralin and phyllanthin, showed strong inhibition on lymphocyte proliferation with half maximal inhibitory concentration (IC50) values of 1.07 μM and 1.82 μM, respectively. Ethyl 8-hydroxy-8-methyl-tridecanoate and 1,7,8-trihydroxy-2-naphtaldehyde exhibited strong inhibition on nitric oxide production with IC50 values of 0.91 μM and 1.07 μM, respectively. Of all the compounds, corilagin was the strongest inhibitor of tumor necrosis factor-α release with an IC50 value of 7.39 μM, whereas geraniin depicted the strongest inhibitory activity on interleukin-1β release with an IC50 value of 16.41 μM. The compounds constituting the extract of P. amarus were able to inhibit the innate immune response of phagocytes at different steps.
The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC.
The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats. Rats (24-26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation. Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutrophils that could potentially harm multiple biological systems in aged individuals.
A proportion of HIV patients beginning antiretroviral therapy (ART) develop immune restoration disease (IRD). Immunological characteristics of IRD were investigated in a cohort of HIV patients beginning therapy in Kuala Lumpur, Malaysia.
To examine the relationships between blood CD4 natural regulatory T (Treg) cells, plasma HIV RNA level, CD4 T-cell count and immune activation in untreated HIV-infected patients and immunodeficient patients beginning antiretroviral therapy (ART), using a novel phenotype to define Treg cells (CD25CD127CD4). Data were compared with established Treg cell markers (FoxP3, CTLA-4 and GITR).
Phyllanthus amarus (family: Euphorbiaceae) is of immense interest due to its wide spectrum of biological activities. In the present study, the standardized 80% ethanol extract of P. amarus was investigated for its modulatory activity on various cellular immune parameters, including chemotaxis of neutrophils, engulfment of Escherichia coli by neutrophils, and Mac-1 expression, in leukocytes isolated from treated/nontreated Wistar-Kyoto rats. The detailed cell-mediated activity of P. amarus was also investigated, including analysis of the effects on T- and B-cell proliferation and CD4(+) and CD8(+) T-cell subsets in splenic mononuclear cells, and estimation of serum cytokine production by activated T-cells. The main components of the extract, phyllanthin, hypophyllanthin, corilagin, geraniin, ellagic acid, and gallic acid were identified and quantitatively analyzed in the extracts, using validated reversed-phase high-performance liquid chromatography (HPLC) methods. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophils isolated from rats administered with the extract of P. amarus, at doses ranging from 100 to 400 mg/kg for 14 days, revealed a significant dose-dependent reduction in neutrophil migration (P<0.05). Similar patterns of inhibition were also observed in phagocytic activity and in fMLP-induced changes in expression of β2 integrin polymorphonuclear neutrophils. The results in P. amarus-treated rats also demonstrated a dose-dependent inhibition of both lipopolysaccharide-stimulated B-cell proliferation and concanavalin A-stimulated T-cell proliferation as compared with sensitized control. At a dose of 400 mg/kg (P<0.01), there was a significant decrease in the (%) expression of CD4(+) and CD8(+) in splenocytes and in serum cytokines of T helper (Th1) (IL-2 and IFN-γ) and Th2 (IL-4). In conclusion, P. amarus showed effective immunosuppressive activities in cellular immune response, by various immune regulatory mechanisms, and may be useful for improvement of immune-related disorders.
The role of T-cell immunosenescence and functional CD8(+) T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8(+) T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8(+) T cells, besides diminished expression of CD127 (IL-7Rα) on CD57(+)CD4(+) T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8(+) T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8(+) T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8(+) T cells.
A cross-sectional study on the expression of 6 lymphocyte markers was carried out on 481 patients with human immunodeficiency virus (HIV) and 79 normals after stratification based on absolute CD4 counts. The data were stratified according to the following groups: (I) 1201 to 1600, (II) 801 to 1200, (III) 401 to 800 and (IV) 0 to 400 (x 10(6) CD4 cells per mm3). The mean percentages of the subsets before stratification showed that HIV patients had increased percentages of CD3+ (75.7 against 66.9), CD3+CD8+ (52.2 against 32.3) and CD3+HLA-DR+ (36.1 against 14.4) cells and lower percentages of CD19 (10.3 against 13.3) and natural killer cells (13.7 against 20.4) when compared to controls in the same group. A definite trend, however, was only seen in CD3+CD8+ (47.4, 50.0, 54.0, 57.5 for groups I, II, III and IV respectively) and CD3+HLA-DR+ (29.1, 32.9, 38.4, 43.9 for groups I, II, III and IV respectively).