Impact of in vitro HIV infection on human thymic regulatory T cell differentiation

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment

Generalized Liver- and Blood-Derived CD8⁺ T-Cell Impairment in Response to Cytokines in Chronic Hepatitis C Virus Infection

<div><p>Generalized CD8⁺ T-cell impairment in chronic hepatitis C virus (HCV) infection and the contribution of liver-infiltrating CD8⁺ T-cells to the immunopathogenesis of this infection remain poorly understood. It is hypothesized that this impairment is partially due to reduced CD8⁺ T-cell activity in response to cytokines such as IL-7, particularly within the liver. To investigate this, the phenotype and cytokine responsiveness of blood- and liver-derived CD8⁺ T-cells from healthy controls and individuals with HCV infection were compared. In blood, IL-7 receptor α (CD127) expression on bulk CD8⁺ T-cells in HCV infection was no different than controls yet was lower on central memory T-cells, and there were fewer naïve cells. IL-7-induced signalling through phosphorylated STAT5 was lower in HCV infection than in controls, and differed between CD8⁺ T-cell subsets. Production of Bcl-2 following IL-7 stimulation was also lower in HCV infection and inversely related to the degree of liver fibrosis. In liver-derived CD8⁺ T-cells, STAT5 activation could not be increased with cytokine stimulation and basal Bcl-2 levels of liver-derived CD8⁺ T-cells were lower than blood-derived counterparts in HCV infection. Therefore, generalized CD8⁺ T-cell impairment in HCV infection is characterized, in part, by impaired IL-7-mediated signalling and survival, independent of CD127 expression. This impairment is more pronounced in the liver and may be associated with an increased potential for apoptosis. This generalized CD8⁺ T-cell impairment represents an important immune dysfunction in chronic HCV infection that may alter patient health.</p></div

IL-7-induced proliferation is not impaired while production of Bcl-2 is reduced in blood-derived CD8⁺ T-cells from HCV⁺ individuals.

<p>Cell proliferation in response to IL-7 (10ng/ml) and/or PHA (0.2mg/ml) was measured as CFSE dilution of CD8⁺ T-cells from (A) HCV^- (n = 8) and (B) HCV⁺ individuals (n = 8), with markers indicating the proportion (%) of CFSE^low (dividing) cells. (C) Proliferation of isolated CD8⁺ T-cells induced by IL-7 + suboptimal PHA was significantly increased in both groups (control p = 0.0001 and HCV⁺ p<0.0001, unpaired Student’s <i>t</i>-test), yet there was no difference between these groups (n.s. = not significant). (D) Bcl-2 expression of blood-derived CD8⁺ T-cells was measured. (E) Bcl-2 expression (MFI) of unstimulated CD8⁺ T-cells is summarized (control n = 4, HCV⁺ n = 5) and (F) Bcl-2 production in response to IL-7 after 48 hours is summarized as MFI (<i>t</i> p = 0.0006, non-linear regression, control n = 8, HCV⁺ n = 9). (G) The IL-7-induced expression of Bcl-2 in blood-derived CD8⁺ T-cells from HCV⁺ individuals with low fibrosis (F0-F2) was compared to that of high fibrosis (F3-F4). Values are expressed relative to medium alone (* p = 0.02, unpaired Student’s <i>t</i>-test, error bars represent ±S.D.).</p

Baseline Characteristics of Study Participants.

<p>Baseline Characteristics of Study Participants.</p

The proportion of CD8⁺ T _CM cells is increased, while T_N and T_EMRA cells are decreased in the liver in HCV infection and CD127 expression does not differ between IH- and blood-derived CD8⁺ T-cells.

<p>Representative dot plots of subset distribution are shown for (A) Blood-derived and individually matched (B) IH-CD8⁺ T-cell subsets, as analysed by flow cytometry which distinguished between subsets on the basis of CD45RA and CCR7 expression. (C) The means of these observations are summarized in a bar graph (* T_N p = 0.03, T_CM p = 0.01, T_EMRA p = 0.03, unpaired Student’s <i>t</i>-test, n = 3, error bars represent ± S.D.). Membrane CD127 expression on bulk and CD8⁺ T-cells subsets did not differ between locations, as measured by (D) percentage expression nor (E) intensity of expression (MFI).</p

HCV⁺ individuals have fewer blood-derived naïve CD8⁺ T-cells and a lower expression of CD127 on central memory cells than HCV^- controls.

<p>CD8⁺ T-cell subset distribution was determined by CD45RA and CCR7 staining in (A) controls (n = 10) and (B) HCV infection (n = 12). (C) Subset distribution data for controls and HCV-infected individuals are graphically represented as means (T_N p = 0.006). (D) The expression of CD127 was measured on blood-derived bulk CD8⁺T-cells (control n = 30, HCV⁺ n = 50) and their subsets (control n = 10, HCV⁺ n = 12) and presented as (E) percentage (T_CM p = 0.02, unpaired Student’s <i>t</i>-test) and (F) mean fluorescence intensity of CD127 expressing cells (error bars represent ±S.D.).</p

IL-7-induced signaling of blood-derived CD8⁺ T-cells is impaired in HCV infection.

<p>(A) Phosphorylation of STAT5 was measured as mean fluorescence intensity (MFI) as shown in a representative histogram. (B) The expression of pSTAT5 was significantly increased by increasing concentrations of IL-7 (0.01–10 ng/ml) in blood-derived CD8⁺ T-cells from controls (p < 0.001, n = 10) or chronically infected HCV⁺ individuals (p = 0.003, n = 9) is summarized, as assessed by ANOVA, yet responses of the latter group were significantly less pronounced than controls (<i>t</i>: p = 0.005, non-linear regression analysis). (C) The expression of pSTAT5 in CD8⁺ T-cell subsets were distinguished by CD45RA and CCR7 expression, with significance in T_CM and T_N subsets (<i>t</i>: p<0.0001 for each subset, non-linear regression, control n = 7, HCV⁺ n = 5). Error bars in the graphs represent ± S.D.</p

Image_1_Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.tiff

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment.</p

Data_Sheet_1_Impact of in vitro HIV infection on human thymic regulatory T cell differentiation.docx

BackgroundThe differentiation and function of immunosuppressive regulatory T cells (Tregs) is dictated by the master transcription factor FoxP3. During HIV infection, there is an increase in Treg frequencies in the peripheral blood and lymphoid tissues. This accentuates immune dysfunction and disease progression. Expression of FoxP3 by thymic Tregs (tTregs) is partially controlled by TGF-β. This cytokine also contributes to Treg development in the peripheral blood and lymphoid tissues. Although TGF-β mediates lymphoid tissue fibrosis and peripheral Treg differentiation in HIV-infected individuals, its role in the induction and maintenance of Tregs within the thymus during HIV infection remains unclear.MethodsThymocytes were isolated from fresh human thymic tissues obtained from pediatric patients undergoing cardiac surgery. Infection by both R5- and X4-tropic HIV-1 strains and TGF-β treatment of human thymocytes was performed in an in vitro co-culture model with OP9-DL1 cells expressing Notch ligand delta-like 1 without T cell receptor (TCR) activation.ResultsDespite high expression of CCR5 and CXCR4 by tTregs, FoxP3 +  CD3highCD8- thymocytes were much less prone to in vitro infection with R5- and X4-tropic HIV strains compared to FoxP3-CD3highCD8- thymocytes. As expected, CD3highCD4+ thymocytes, when treated with TGF-β1, upregulated CD127 and this treatment resulted in increased FoxP3 expression and Treg differentiation, but did not affect the rate of HIV infection. FoxP3 expression and Treg frequencies remained unchanged following in vitro HIV infection alone or in combination with TGF-β1.ConclusionFoxP3 expression and tTreg differentiation is not affected by in vitro HIV infection alone or the combination of in vitro HIV infection and TGF-β treatment.</p