Anti-pneumococcal CD4 T cells proliferative responses in adult tonsils and blood during <i>in vitro</i> pneumococcal peptide antigen challenge.

<p>(<b>a</b>) A typical FACS plot for CFSE staining within CD4⁺ cells post simulation with flu or SPNT compared to unstimulated (media alone) cells. (<b>b</b>) Purified tonsil MNCs (<i>n</i> = 8) were stimulated over 9 days with flu or recombinant Ply peptides, or D39 bacterial SPNT or media. CD4⁺ cells identified by FACS staining were assessed for their proliferative responses by CFSE staining. Percent of proliferating CD4⁺ cells post flu, Ply or SPNT stimulation were all significantly higher than media control (*  = <i>p</i> <0.05). (<b>c</b>) Greater proliferative responses to pneumococcal peptides by tonsil compared to blood CD4⁺ cells. Tonsil MNCs and PBMCs from the same individuals (<i>n</i> = 5), were purified and stimulated <i>in vitro</i> with flu, Ply or SPNT and CD4⁺ cell proliferation assessed after 9 days. No significant difference was observed between tonsil (open bars) and blood (filled bars) CD4⁺ responses to flu but were significant to SPNT (*  =  <i>p</i> <0.05) and almost significant (#  =  <i>p</i> 0.06) for Ply. Values were calculated with the background (i.e. media alone) proliferation subtracted. Error bars show the SEM.</p

Effect of blockage of CTLA-4 and PDL-1 on CD25^hi cells their suppression of anti-pneumococcal proliferative responses by CD4⁺ cells.

<p>Purified CD25^hi cells were preblocked with anti-human CTLA-4 or anti-human PDL-1 blocking antibodies or isotype control (IgG) antibodies and added back to CD25^hi depleted MNCs (i.e. CD25^- cells) at the same original proportion and then stimulated with SPNT over 8 days and CD4⁺ cells proliferation analysed. Graph shows the mean percentage of proliferating CD4⁺ cells post SPNT stimulation in the anti-human CTLA-4 (<b>a</b>) or anti-human PDL-1 preblocked CD25^hi cells (<b>b</b>) groups (<i>n</i> = 5 each group). (*  = <i>p</i> <0.05).</p

Inhibitory action of anti-pneumococcal responses by regulatory T cells.

<p>Inhibition of mucosal CD4⁺ T cell anti-pneumococcal responses to Ply (<b>a</b>) and SPNT (<b>b</b>) but not to flu (<b>c</b>) by CD25^hi regulatory T cells in subjects above 16 years old as indicated by increased cell proliferation following depletion of CD25^hi cells from tonsil MNC population is observed. Subjects (<i>n</i> = 50) were grouped into those aged less than 17 yrs, 17 to 25 yrs and >25 yrs. Individual subject's proliferative response pre and post CD25^hi cell depletion are shown with a connecting dashed grey line, while solid black bars and black dashed line represent mean proliferative values for undepleted and CD25^hi cell depleted populations. (*  = <i>p</i> <0.05).</p

Effect of restoration/addition of Tregs (CD25^hi) cells back into CD25^hi cell depleted MNC samples on proliferative responses by pneumococcal specific CD4⁺ T cells.

<p>Tonsil MNCs were depleted of CD25^hi cells and left or CD25^hi cells added back at the original proportion (∼10%) or at three fold the original proportion (i.e. 30%). Cells were obtained from individuals (<b>a</b>) >16years (<i>n</i> = 5) and stimulated with SPNT, (<b>b</b>) <17 years (<i>n</i> = 3) and stimulated with SPNT or individuals (<b>c</b>) >16 years and stimulated with flu (<i>n</i> = 5). Percentage of proliferating CD4⁺ cells are shown for each individual (open circles) as well as mean (black bars) proliferation in undepleted or CD25^hi depleted or CD25^hi depleted with CD25^hi added back at the original proportion or CD25^hi depleted with CD25^hi added back at three times the original proportion. (*  = <i>p</i> <0.05).</p

Mucosal CD4 T cell responses to pneumococci during aging and its relation with CAP rates.

<p>(<b>a</b>) Mucosal CD4 T cell responses to pneumococcal peptide antigen display gradual age-related increases from early childhood until mid-20's and remain relatively constant until mid-life. Tonsil MNCs from subjects (<i>n</i> = 80) between the ages of 2 to 39 years and grouped into five age groups were assessed for their CD4 T cell proliferative responses to <i>Strep. Pneumoniae</i> Ply (circles, solid grey line of best fit), SPNT (square, dashed black line of best fit) and flu (triangle, black line of best fit) peptides, error bars show SEM. (<b>b</b>) Graph for data observed by Myles <i>et al</i> showing the trend (black crosses, dashed black line of best fit) of incidence rates of CAP per person/year in the UK at different age groups between 1991-2003 (<i>n</i> = 56332, R² = 0.97) in relation to the trend (grey circles, solid grey line of best fit) of mean total (to Ply and to SPNT) anti-pneumococcal CD4 T cell proliferative responses between the ages of 2 to 39 years old (R² = 0.93). Graph for CAP data was generated with permission from P.R. Myles.</p

Detection of Ply specific CD127^low/- FoxP3⁺ CD4⁺ Treg cells in CD25 enriched tonsil and blood MNC.

<p>CD25 enriched MNC were stained with anti- CD127 and anti FoxP3 to allow identification of Tregs and with (<b>a</b>) strepavidin-PE, (<b>b</b>) negative control Ply tetramer-PE and (<b>c</b>) Ply tetramer-PE and analysed by FACS. A typical example of a FACS plot is shown. While the CD127^low/- FoxP3⁺ CD4⁺ Treg cells show low level staining at 0.0% with strepavidn-PE (<b>a</b>) and negative control Ply-tetramer at 0.05% (<b>b</b>), a significantly higher percentage of Treg cells are bound by Ply-tetramers at 1.96%(<b>c</b>).</p

Assessment of CD4+ T Cell Responses to Glutamic Acid Decarboxylase 65 Using DQ8 Tetramers Reveals a Pathogenic Role of GAD65 121–140 and GAD65 250–266 in T1D Development

<div><p>Susceptibility to type 1 diabetes (T1D) is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02). Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65) using DQ8 tetramers. We demonstrated that GAD65_121–140 and GAD65_250–266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65_121–140 and GAD65_250–266 carried a Th1-dominant phenotype, with some of the GAD65_121–140-specific T cell clones producing IL-17. GAD65_250–266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs) revealed that GAD65_250–266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65_121–140 and GAD65_250–266 epitopes and implicate their possible contribution to the progression of T1D.</p></div

Putative beta-cell autoantigenic peptides.

a<p>Zinc transporter.</p>b<p>Preproinsulin.</p>c<p>Islet-specific gluose-6-phosphatase catalytic subunit-related protein.</p>d<p>Glutamic Acid Decarboxylase 65.</p>e<p>Dystrophia Myotonica Kinase.</p>f<p>Islet protein tyrosine phosphates.</p

Prevalence of GAD65-specific T cells in T1D and healthy subjects.

<p>Not all peptides were tested on each subject.</p><p>Statistical analysis was performed using two-tailed Fisher's exact tests.</p><p>Prevalence of GAD65-specific T cells in T1D and healthy subjects.</p

Direct ex vivo analysis of GAD65-specific T cells.

<p>Unmanipulated PBMCs were stained with DQ8/GAD65_250–266 PE-tetramer. Antigen-specific CD4+ T cells were enriched, stained with antibodies against surface markers of interest, and analyzed on a Calibur multi-color flow cytometer. (<b>A</b>) Representative ex vivo analysis of the surface memory marker CD45RO for GAD65_250–266. The frequency of GAD65_250–266-specific CD45RO+CD4+ T cells was 4.4 per million CD4+ T cells for the T1D patient (left panel) and 0.6 per million for the healthy subject (right panel). (<b>B</b>) Ex vivo co-staining of GAD65_250–266-specific cells with DQ8/GAD65_250–266 PE- and DQ8/GAD65_250–266 APC-tetramers. Cells were stained with PE-labeled DQ8/GAD65_250–266 tetramers first. After enrichment, tetramer-positive cells were stained again with APC-labeled DQ8/GAD65_250–266 tetramers at 37°C for 1 h. (<b>C</b>) Cumulative total CD4+ and CD45RO+CD4+ T cell frequencies for GAD65_250–266 in controls (open circles, n = 10) and T1D patients (closed circles, n = 10). <b>***</b> P<0.001, <b>**</b> P<0.01, as evaluated by Mann-Whitney U-test.</p