MicroRNA regulation in human CD8+ T cell subsets – cytokine exposure alone drives miR-146a expression
Hilary M. SheppardDaniel J. VerdonAnna E. S. BrooksVaughan FeisstYu-Yu HoNatalie LorenzVicky FanNigel P. BirchAlicia J. DidsburyP. Rod Dunbar
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microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status.We treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a.We find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells.miR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.Abstract Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.
CD4-CD8 Ratio
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Summary: In the present work, two important negative regulators of T cell responses in rats were examined. At the molecular level, rat CTLA-4, a receptor important for deactivating T cell responses, was examined for the expression pattern and in vitro functions. For this purpose, anti-rat CTLA-4 mAbs were generated. Consistent with the studies in mice and humans, rat CTLA-4 was detectable only in CD25+CD4+ regulatory T cells in unstimulated rats, and was upregulated in all activated T cells. Cross-linking rat CTLA-4 led to the deactivation of anti-TCR- and anti-CD28 stimulated (costimulation) T cell responses such as reduction in activation marker expression, proliferation, and cytokine IL-2 production. Although T cells stimulated with the superagonistic anti-CD28 antibody alone without TCR engagement also increased their CTLA-4 expression, a delayed kinetics of CTLA-4 upregulation was found in cells stimulated in this way. The physiological relevance of this finding needs further investigation. At the cellular level, rat CD25+CD4+ regulatory T cells were examined here in detail. Using rat anti-CTLA-4 mAbs, the phenotype of CD25+CD4+ regulatory T cells was investigated. Identical to the mouse and human Treg phenotype, rat CD25+CD4+ T cells constitutively expressed CTLA-4, were predominantly CD45RC low, and expressed high level of CD62L (L-selectin). CD25+CD4+ cells proliferated poorly and were unable to produce IL-2 upon engagement of the TCR and CD28. Furthermore, rat CD25+CD4+ cells produced high amounts of anti-inflammatory cytokine IL-10 upon stimulation. Importantly, freshly isolated CD25+CD4+ T cells from naive rats exhibited suppressor activities in the in vitro suppressor assays. In vitro, CD25+CD4+ regulatory T cells proliferated vigorously upon superagonistic anti-CD28 stimulation and became very potent suppressor cells. In vivo, a single injection of CD28 superagonist into rats induced transient accumulation and activation of CD25+CD4+ regulatory T cells. These findings suggest firstly that efficient expansion of CD25+CD4+ cells without losing their suppressive effects (even enhance their suppressive activities) can be achieved with the superagonistic anti- CD28 antibody in vitro. Secondly, the induction of disproportional expansion of CD25+CD4+ cells by a single injection of superagonistic anti-CD28 antibody in vivo implies that superagonistic anti-CD28 antibody may be a promising candidate in treating autoimmune diseases by causing a transient increase of activated CD25+CD4+ T cells and thus tipping ongoing autoimmune responses toward selftolerance.
CTLA-4
ZAP70
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CD28 interaction with B7 molecules, expressed on the membranes of antigen‐presenting cells, costimulates cytokine production, T‐cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4 + and CD8 + lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28‐bearing CD4 + and CD8 + cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non‐AIDS patients. At the same time, an increase in the absolute CD8 + CD28 − cell number (greater in stage A than in stage B and C subjects) was observed in HIV‐infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV‐infected patients.
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To investigate the relationship between expression of Fas-FasL and apoptosis of peripheral blood CD8+CD28-and CD8+CD28+T lymphocytes in patients with systemic lupus erythematosus(SLE),the rates of Fas-FasL expression and apoptosis of CD8+CD28-and CD8+CD28+ T cells were measured by three antibodies labeling flow cytometry(FCM) analysis in 22 active cases,18 inactive cases of SLE and 20 controls.The results showed that:(1)Compared to controls and inactive SLE,apoptosis rates of CD8+ T cells was significantly higher in active SLE group(P0.05),and the expression of Fas in CD8+CD28-T cells and FasL in CD8+CD28+T cells were higher in active SLE group(P0.05).The expression rate of Fas of CD8+CD28+ T cells in SLE patients was featured by the fact that active SLE group inactive SLE groupcontrol group,and there was a significant difference in statistics in these 3 groups(P0.05).(2) The activity of SLE was positively correlated with the apoptosis rate of CD8+T cells,but there was no correlation with expression of Fas、FasL in CD8+CD28-and CD8+CD28+ T cells.(3) The expression of Fas、FasL were correlated with apoptosis,and expression of Fas was significant correlated with expression of FasL in CD8+CD28+ T cells.The expression of Fas was not significantly correlated with expression of FasL in CD8+CD28-Tcells.It is concluded that the apoptotic rate of the peripheral blood CD8+T cells is abnormally increased and the activity of SLE was positively correlated with the apoptotic rate of CD8+T cells.The difference of expression in Fas and FasL between active and inactive cases of SLE may induce the disability of immune tolerance.
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SUMMARY To evaluate whether vertical HIV infection interferes with the expression of CD28 on T lymphocytes, 25 HIV-infected children and 29 seroreverted children born to HIV+ mothers were studied. The percentage of CD28− cells among CD8+ T lymphocytes was higher in HIV-infected children than in controls (P < 0.001). In fact, in HIV-infected children, this percentage was elevated from the first year of life, while in healthy seroreverted children, the proportion of CD28− cells among CD8+ cells rose progressively with age (r = 0.49; P = 0.008). In HIV+ children, the CD8+ CD28−, but not CD8+ CD28+ cell proportion was significantly correlated with immunological markers of disease progression, such as CD4+ cell loss (r = −0.65; P < 0.001) and the level of in vitro spontaneous lymphocyte apoptosis (r = 0.53; P = 0.03).
CD4-CD8 Ratio
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Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2- and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4(+)CD25(-) and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4(+)CD25(+) T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4(+)CD25(+)FoxP3(+) regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.
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Objective To study the expressions of CD25 in CD4+ T cells and CD28 in CD8+ T cells in the peripheral blood of HIV/AIDS. Methods CD25 in CD4+ T cells and CD28 in CD8+ T cells in the peripheral blood of 35 HIV/AIDS and 41 normal control were detected by flow cytometr. Results It shows that in patients with HIV/AIDS and the normal subjects,the expression percent of CD25 in CD4+T cells(27.51±4.23)%,(44.41±9.17)%、CD4+25+T cells(2.00±1.42)%,(16.62±4.60)%、CD4+25-T cells(5.16±3.37)%,(21.03±6.19)%、CD28 in CD8+T cells(25.12±6.33)%,(44.24±8.61)%, CD8+28-T cells(36.85±8.98)%,(13.33±4.58)%had significantly different (P0.01);CD8+28+T cells(12.31±4.14)%,(10.51±3.71)%had no significantly different (P0.05). Conclusions The low expression of CD25 in CD4+ T cells and CD28 in CD8+ T cells in HIV infection patients was closely associated with Immunodeficiency by HIV infection, the increase of CD8+28-T cells help to promote HIV/AIDS patients with inflammatory response and immune activation.
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Aplastic anemia
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Objective To investigate the variation of T lymphocyte subsets in peripheral blood of the patients with tumor and their clinical significance.Methods The T lymphocyte subsets in peripheral blood from patients with tumor n = 27 and healthy controls n = 25 were detected with flow cytometry.Results The CD3+ T cells,CD4+ T cells and CD4+/CD8+ ratio in patients with tumor were significantly lower than that in the control group.Comparied with the control group the CD8+ T cells in patients with tumor were significantly higher P 0.01 .Conclusion The immunological function of patients with tumor was decreased.The flow cytometry is a rapid,sensitive and accurate measure to detect the immunological function of patients with tumor.It can play an important role in evaluating the curative effect and prognosis of the disease.
Cytometry
Peripheral blood lymphocyte
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