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

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

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

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

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

Significantly higher lymphoproliferative responses to HIV-1 recombinant antigens mounted by HIV controllers (HIC) compared to HIV-1⁺ viraemic chronic progressors (vCP) and aviraemic chronic progressors (aCP) despite comparable IFN-γ release to HIV-1 peptide pools.

<p>Broad IFN-γ and proliferative responses to HIV-1 Gag p24 in HICs. Box plots show the median with IQR indicated. Whiskers represent the 10^th–90^th percentiles. The Mann-Whitney test was used to compare responses between patient groups, and the Wilcoxon signed rank test to compare paired responses from the same patient. *p<0.05, **p<0.01, ***p<0.001. (a) Lymphoproliferative response to rTat, rRev, rNef, rp24 (Gag) and rgp120 (Env), from HICs (n = 6), vCPs (n = 6) and aCPs (n = 8). The threshold for positivity (SI ≥3) is marked. (b) IFN-γ release (spot forming cells per million PBMC), from HICs (n = 6), vCPs (n = 6) and aCPs (n = 8), in response to stimulation with Tat, Rev, Nef and Gag p24 overlapping peptides. Data represent mean values of triplicate wells with <10% variation among triplicates. The threshold of 20 SFC/10⁶ PBMC is marked. (c) Summation of IFN-γ release in response to stimulation with individual overlapping Gag p24 peptides (1–22) compared to the response to the peptide pool and rp24 protein. Data from two representative HICs is shown. (d) Summation of lymphoproliferative responses, of two representative HICs, to stimulation with individual overlapping Gag p24 peptides (1–22) compared to the response to rp24 protein.</p

Follow up of 41 “LTNPs” over 132 months since original identification using clinical criteria in 1996.

<p>(a) All available pre-HAART data of CD4 T-cell counts for the cohort of 41 LTNPs since identification (normal range 450–1650 cells/µl blood, marked with horizontal line). (b) All available pre-HAART data of plasma viral load for the cohort of 41 LTNPs since identification. DL = detection limit of assay. Data from each individual are plotted with different symbols and regression lines indicate slopes. Once the patient initiates anti-retroviral therapy their characteristics are no longer shown on the graphs.</p

Detailed description of characteristics of the study cohorts.

<p>- , data not available; P values between *HIC and vCP, ^#HIC and aCP, †vCP and aCP; bold face indicates statistically significant difference measured to a 95% confidence interval by the Mann-Whitney test; ^‡2^nd or ^§3^rd visit respectively.</p

Longitudinal analysis of T-cell mediated responses, to HIV-1 antigens and peptides, by two HIV controllers over a 79 and 85 month period.

<p>Data is shown from two HICs; HIC 5 and HIC 2, from two and three time points respectively. Data represent mean values of triplicate wells with <10% variation among triplicates. (a) IFN-γ release (spot forming cells/10⁶ PBMC) in response to stimulation with Tat, Rev, Nef and Gag p24 overlapping peptides. The threshold of 20 SFC/10⁶ PBMC is marked. (b) Lymphoproliferative response to rTat, rRev, rNef, rp24 (Gag) and rgp120 (Env). The threshold for positivity (SI ≥3) is marked.</p