The Immune Synapses Reveal Aberrant Functions of CD8 T Cells During Chronic HIV Infection

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise

Magnetic resonance imaging and velocimetry of ethane

This study investigates the experimental conditions required for magnetic resonance imaging (MRI) of thermally polarized hydrocarbon gas, focusing on ethane. The nuclear magnetic resonance (NMR) spectra and relaxation properties of ethane were analysed at different pressures in the range from 1.5 to 6 bar at 7 T using 1H NMR spectroscopy. The spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) were measured, and their dependence on the pressure was determined, showing that both relaxation times increase with pressure. Using the estimated relaxation times, we adjusted parameters for imaging of static ethane using rapid acquisition with relaxation enhancement (RARE) and fast low-angle shot (FLASH). The signal-to-noise ratio (SNR) of ethane images was evaluated and compared to the calculation for the given range of pressures. Then, we imaged flowing gas using a 2D velocity-encoded pulse sequence, which is usually used for liquid flow studies. The MRI-measured flow rates are compared to those pre-set with a pump, showing good agreement in the slow flow range. Overall, the results provide insights into the feasibility of 1H MRI for imaging and flow measurements of thermally polarized ethane

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues

Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues

Functional characteristics of polyclonal and virus-specific effector CD4+ T cell responses in HIV-infected LNs and blood.

<p><b>(A)</b> Flow cytometry plots (HIV ART+ subject) and plots for matched HIV-Gag or–Env-specific CD4+ T cell responses in HIV-infected LN and PB. <b>(B)</b> Flow cytometry plots (HIV-infected CP) showing the negative control (NC) and Gag-specific response of LN CD4+ T cell response. The high abundance of CD107a (red) that is not co-expressed with IFNγ or TNF is illustrated in this example. SPICE analysis of functional combination between LN (red) and PB (red-gray) Gag-specific CD4+ T cell responses for HIV-infected CPs and ART+ subjects. <b>(C)</b> Flow plots (HIV-infected CP) of Gag-specific CD4+ T cell response (red) from LN and PB in relation to CD27 and perforin expression. Graphs represent the frequency of <b>(C)</b> perforin+ and <b>(D)</b> T-bet^hi cells between LN and PB Gag-specific CD4+ T cells. <b>(E)</b> Flow plots (HIV-infected CP) of MIP-1α <i>versus</i> IFNγ and TNF production for LN and PB SEB stimulated CD4+ T cells. Corresponding plots showing the frequency of MIP-1α+ SEB stimulated CD4+ T cells (top) and MIP-1α+ of IFNγ/TNF/CD107a/MIP-1α+ SEB stimulated CD4+ T cells (bottom). <b>(F)</b> Flow plots (HIV-infected CP) of MIP-1α <i>versus</i> IFNγ and TNF production for LN and PB CMV-specific CD4+ T cells and corresponding graphs showing the frequency of MIP-1α+ CMV-specific CD4+ T cells (top) and MIP-1α+ of IFNγ/TNF/CD107a/MIP-1α+ CMV-specific CD4+ T cells (bottom). Median and IQR are shown for all bar plots. Permutation test was performed between the pie charts. Wilcoxon matched-pairs single rank tests were performed to compare differences between two matched groups; *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. All data-points are derived from the Mexico cohort.</p

Expression of effector molecules by Gag-specific CD4+ T cells from HIV elite controller LNs and blood.

<p><b>(A)</b> Flow cytometry plots (HIV elite controllers) illustrating the distribution of LN (red) and PB (blue) IFNγ+ Gag-specific CD4+ T cell responses between the T-bet^dimEomes- and T-bet^hiEomes+ compartment. Corresponding scatterplots showing the frequencies of T-bet^dimEomes- (left) and T-bet^hiEomes+ (right) cells of Gag-specific CD4+ T cells between LN and PB for HIV elite controllers. Flow plots (HIV elite controllers) of IFNγ+ Gag-specific CD4+ T cell response from LN and PB in relation to <b>(B)</b> perforin and <b>(C)</b> MIP-1α expression. Graphs represent the frequency of <b>(B)</b> perforin+ and <b>(C)</b> MIP-1α+ Gag-specific CD4+ T cells between LN and PB. <b>(D)</b> Flow plots and graphs demonstrating the expression pattern of perforin+Granzyme B+ memory CD4+ T cells in acute/early seroconverters (left graph). Right graphs show the frequency perforin+Granzyme B+ or T-bet expression out of total memory Ki-67+ CD4+ T cells. Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between groups; *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. All data-points are derived from the North-American cohort.</p

Phenotypic, cytolytic and transcriptional differences between LN and blood CD4+ T cells in HIV-infected and -uninfected individuals.

<p><b>(A)</b> Flow cytometry plots (HIV-infected CP) and scatter plots for naïve and memory subsets of LN and peripheral blood (PB) CD4+ T cells in HIV-infected and -uninfected subjects. <b>(B)</b> Flow cytometry plots (HIV-infected CP) showing the lack of T-bet^hiEomes+ CD4+ T cells in LNs. Corresponding scatter plots demonstrating the frequency of T-bet^hi cells of memory (non-naïve) CD4+ T cells (top) and frequency of Eomes+ cells of T-bet^hi CD4+ T cells (bottom) for matched LN and PB. <b>(C)</b> Flow plots (HIV-infected CP) showing the lack of Granzyme B+perforin+ CD4+ T cells in LNs and scatter plots with the frequency of LN and PB perforin+ cells of memory CD4+ T cells (top). Frequencies of Granzyme B+ cells of perforin+ CD4+ T cells (bottom) for matched LN and PB. <b>(D)</b> Flow plots (HIV-infected CP) and scatter plots showing the distribution of CD27+ cells within the Granzyme B+ CD4+ T cell compartment for matched LN and PB. <b>(E)</b> The distribution of different populations in the tSNE space is based on 30.000 live CD4+ T cells that were merged from LN and PB from a HIV-infected CP with detectable levels of cytolytic cells in the PB and LN Tfh cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme B, perforin, CXCR5 and PD-1 expression on gated bulk CD4+ T cells. The naïve cluster (green) is based on high CCR7 and low CD45RO intensity; the Tfh cluster (red) on high intensity of PD-1 and CXCR5; and the effector cluster (orange) on high T-bet and perforin expression intensity. After separating out the merged LN and PB single CD4+ T cell data, a lack of Tfh cells was apparent in PB and effector CD4+ T cells in the LN (lower right tSNE plots). Median and IQR are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between two unmatched groups, and Wilcoxon matched-pairs single rank tests between matched samples; *<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. All data-points are derived from the North-American and Mexico cohort.</p

Cytolytic CD4+ T cells express high levels of T-bet and Eomes in blood.

<p><b>(A)</b> Representative flow cytometry plots of Granzyme B and perforin expression in CD4+ T cells for an HIV-infected and–uninfected subject. The distribution of Granzyme B+perforin+ (red) and Granzyme B-perforin- (blue) CD4+ T cells are shown for T-bet and Eomes expression. <b>(B)</b> Frequency of perforin+ CD4+ T cells within the T-bet^hiEomes+ and T-bet^dim/- population (left) and T-bet^hiEomes+ within the perforin+ or perforin- population for HIV-infected and–uninfected subjects. <b>(C)</b> Correlation between the frequency of perforin+ and T-bet^hi CD4+ T cells. <b>(D)</b> Imagestream analysis on T-bet^hi and T-bet^dim CD4+ T cells. Overlays of fluorescent channels for DAPI (nuclear) and T-bet, showing where in the cells T-bet are localized. The frequency of nuclear, nuclear/cytoplasmic and cytoplasmic localization for T-bet^hi and T-bet^dim CD4+ T cells are shown in the before-after graphs. <b>(E)</b> tSNE plots based on 30,000 live CD4+ T cells that were merged from three HIV-uninfected subjects with detectable cytolytic CD4+ T cells. The tSNE clustering is based on CD45RO, CD27, CCR7, T-bet, Eomes, Granzyme A, Granzyme B and perforin expression intensity. The red gate indicates the identified “effector” cluster with overlapped expression of cytolytic markers as well as T-bet and Eomes. <b>(F)</b> Flow plots of MIP-1α production using media (NC) and aCD3-CD28 stimulations for T-bet^hi and Eomes+ CD4+ T cells, as well as correlation between the frequency of T-bet^hiEomes+ and MIP-1α+ CD4+ T cells following aCD3-CD28 stimulations. Median and IQR are shown for all scatter plots and Mann-Whitney tests were performed to compare differences between groups; ***<i>P</i> < 0.001. A non-parametric Spearman test was used for the correlations analysis. All data are derived from the North-American cohort.</p