Additional file 3: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S2. Effects of astrocytes cytokine neutralization on B cell survival and activation. B cells from HC were either cultured alone, or with stimulated astrocyte conditioned-medium (ACM), or with ACM pre-treated with neutralizing antibodies to IL-6 (a, b; anti-IL6: aIL-6), IL-15 (c, d; anti-IL-15: aIL-15) or BAFF (e, f; anti-BAFF: aBAFF); or pre-treated with corresponding isotype control antibodies. After 2 days of culture B cell viability was assessed using ANNEXIN V and 7AAD staining, and CD86 expression was measured by flow cytometry (representative experiment). (TIFF 4226 kb

Additional file 4: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S3. IFNγ production by proliferative T-cells. Human B cells were cultured in transwell as described previously, either alone or with stimulated or unstimulated astrocytes. Following 2 days in culture, B cells were harvested, thoroughly washed and co-cultured with human T cells from allogeneic donors at a B-cell:T-cell ratio of 1:4. Conditioned media of B-cell:T-cell co-culure was collected and IFNγ was measured using ELISA (representative experiment). (TIFF 7670 kb

Additional file 2: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S1. Confirming activation of human astrocytes. Astrocytes were cultured for 24 h and were either left unstimulated or were stimulated with IFNγ (10 ng/ml) and IL-1β (10 ng/ml). After 24 h, the astrocytes were washed thoroughly and fresh medium was added. After an additional 24 h in culture, at which time cultures were imaged and supernatants were collected for subsequent measurement of astrocyte-secreted IL-6 by ELISA. Compared to unstimulated astrocytes (a), stimulated astrocytes exhibited activated morphology (b) and significantly-enhanced production of IL-6 (c; p = 0.0016; paired t-test). (TIFF 3951 kb

Tim-3 Negatively Regulates Cytotoxicity in Exhausted CD8⁺ T Cells in HIV Infection

<div><p>Cytotoxic CD8⁺ T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell ‘exhaustion’ is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8⁺ T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3⁺ CD8⁺ T cells to make perforin and 2) the direct ability of Tim-3⁺ CD8⁺ T cells to kill autologous HIV infected CD4⁺ target cells. Surprisingly, Tim-3⁺ CD8⁺ T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8⁺ T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4⁺ T cells and d) their ability to suppress HIV infection of CD4⁺ T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8⁺ T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8⁺ T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.</p> </div

Additional file 1: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Table S1. (DOCX 68 kb

Perforin content of Tim-3⁺ CD8⁺ T cells as determined by two antibody clones.

<p>In <i>a</i>), a representative experiment showing Tim-3 expression on <i>ex vivo</i> CD8⁺ T cells from a treatment naïve chronically HIV infected subject showing perforin expression by two antibodies detecting different conformations of perforin on Tim-3⁺ or Tim-3<b>⁻</b>CD8⁺ T cells. The δG9 clone has been proposed to predominantly detect stored (granule associated) perforin and clone B-D48 detects stored perforin plus majority of other perforin conformations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040146#pone.0040146-Hersperger2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040146#pone.0040146-Hersperger3" target="_blank">[29]</a>. In <i>b</i>) a representative figure showing the relationship between two different conformations of perforin and Tim-3 expressions after gating out the naïve and terminally differentiated CD8⁺ T cells by gating in all CD45RA^- memory subsets. In <i>c),</i> summary of data for all 9 chronically HIV infected individuals stratifying perforin antibodies and Tim-3 expression on total CD8⁺ T cells. In <i>d</i>) summary of data for 5 chronically HIV infected individuals showing the perforin expression with two clones and Tim-3 expressions of memory subsets of CD8⁺ T cells (<i>p</i> value based on two tailed paired t test).</p

Tim-3 pathway blocking increases the antigen specific CD8⁺ T cells cytotoxicity and HIV suppression in chronic HIV infection.

<p>The effect of HIV Gag-specific CD8⁺ T cells after blocking the Tim-3 pathway with a Tim-3 blocking antibody (clone 2E2) on eliminating HIV-infected CD4⁺ T cells was tested in a virus suppression assay. Autologous CD4⁺ T cells (targets) are infected with a primary HIV virus isolate. Autologous CD8⁺ T cells (effectors) are added in 1∶1 ratio at the time of infection with 2E2 antagonistic anti-Tim-3 antibody or isotype at 10 µg/ml. The co-culture is incubated at 37°C for three days. The final readout is the percentage of HIV (p24⁺) positive target cells on day three examined by intracellular flow cytometry. In <i>a</i>) are autologous CD4⁺ T cells in the absence of autologous CD8⁺ T cells taken from an HIV infected individual after exogenous infection by HIV in the presence of Tim-3 antibody or isotype. Tim-3 blockade had no effect on total and infected CD4⁺ numbers. In <i>b</i>) shown are CD4⁺ T cells from an HIV uninfected normal volunteer infected with exogenous HIV and co-cultured with autologous CD8⁺ T cells (1∶1 ratio). Again we could not appreciate any difference in survival of CD4⁺ T cells. Tim-3 blockade had no effect on CD8⁺ mediated suppression of HIV Infection. In <i>c</i>), is a representative experiment showing the percentage of infected p24⁺ CD4⁺ T cells in the two different conditions in a chronic HIV infected individual and in <i>d)</i> is a representative experiment showing the percentage of infected p24⁺ CD4⁺ T cells in the two different conditions in a viral controller, Shown in <i>e</i>), are summary data for four chronically HIV infected individuals and in <i>f</i>) are summary data for three viral controllers. Each solid circle represents the average of three independent experiments from each individual showing the percentage of p24⁺ CD4⁺ T cells (<i>p</i> value based on two tailed paired t test).</p

Tim-3 pathway blocking increases the ability of CD8⁺ T cells to degranulate upon further in vitro re-stimulation.

<p><i>Ex vivo</i> PBMC from HIV infected subjects were stimulated for 6 hours with a pool of HIV Gag peptides in the presence of sTim-3 (final concentration of 2 µg/ml) or just medium alone, and CD107a expression was measured. In a), a representative experiment showing CD107a expression with or without sTim-3 added to the cell culture from the same individual, In b), are summary of data for all 11 subjects (<i>p</i> value based on two tailed paired t test).</p

Tim-3 pathway blocking improves the perforin release in antigen specific CD8⁺ T cells only in chronically HIV infected individuals.

<p>2×10⁵ negatively sorted CD8⁺ T cells from <i>ex vivo</i> PBMC from 5 untreated chronically infected (Fig <i>a</i>) and four untreated viral controllers (Fig <i>b)</i> were stimulated with a pool of HIV Gag peptides (final concentration of 2 µg/ml/peptide) for 6 h. Perforin released in supernatant was measured in an ELISA experiment in pg/ml. (αTim-3  =  2E2 Tim-3 blocking antibody, Cntr  =  IgG1 isotype control antibody) (<i>p</i> value based on two tailed paired t test).</p

Total and antigen specific Tim-3⁺ CD8⁺ T cells have higher frequencies of perforin expression.

<p><i>Ex vivo</i> PBMC from untreated chronically HIV infected subjects were stained for perforin (clone B-D48) and Tim-3. In <i>a),</i> shown are FMO staining for Tim-3, and B-D48 perforin antibodies of a representative sample, in <i>b),</i> representative gating strategy for Tim-3, and B-D48 perforin and a representative experiment showing the percentage of perforin and Tim-3 expressions on total CD8⁺ CD3⁺ T cells. Summary of data for 22 individuals showing perforin content of Tim-3⁺ and Tim-3<b>⁻</b> CD8⁺ T cells as a % in <i>c</i>) and MFI in <i>d</i>). In <i>e</i>) a representative experiment where <i>ex vivo</i> CD8⁺ T cells are stained with a pool of HIV specific tetramers (see Methods) and perforin expression on gated Tim-3⁺ and Tim-3<b>⁻</b> T cells are shown, with a summary shown in <i>f</i>) for 5 chronically infected individuals. Each symbol represents a single individual. Red histogram represents FMO. In <i>g</i>) <i>Ex vivo</i> PBMC from 7 chronic progressors were stained for HIV-specific tetramers and examined for Tim-3 and T-bet expression. The individual in <i>e)</i> was not examined in <i>g</i>) (<i>p</i> value based on two tailed paired t test).</p