Acquisition of pneumococci specific effector and regulatory Cd4 + T cells localising within human upper respiratory-tract mucosal lymphoid tissue

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Abstract

The upper respiratory tract mucosa is the location for commensal Streptococcus (S.) pneumoniae colonization and therefore represents a major site of contact between host and bacteria. The CD4 + T cell response to pneumococcus is increasingly recognised as an important mediator of immunity that protects against invasive disease, with data suggesting a critical role for Th17 cells in mucosal clearance. By assessing CD4 T cell proliferative responses we demonstrate age-related sequestration of Th1 and Th17 CD4 + T cells reactive to pneumococcal protein antigens within mucosal lymphoid tissue. CD25 hi T cell depletion and utilisation of pneumococcal specific MHCII tetramers revealed the presence of antigen specific Tregs that utilised CTLA-4 and PDL-1 surface molecules to suppress these responses. The balance between mucosal effector and regulatory CD4 + T cell immunity is likely to be critical to pneumococcal commensalism and the prevention of unwanted pathology associated with carriage. However, if dysregulated, such responses may render the host more susceptible to invasive pneumococcal infection and adversely affect the successful implementation of both polysaccharide-conjugate and novel protein-based pneumococcal vaccines. © 2011 Pido-Lopez et al.

Figures

  • Figure 1. Anti-pneumococcal CD4 T cells proliferative responses in adult tonsils and blood during in vitro pneumococcal peptide antigen challenge. (a) A typical FACS plot for CFSE staining within CD4+ cells post simulation with flu or SPNT compared to unstimulated (media alone) cells. (b) Purified tonsil MNCs (n = 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 (* = p ,0.05). (c) Greater proliferative responses to pneumococcal peptides by tonsil compared to blood CD4+ cells. Tonsil MNCs and PBMCs from the same individuals (n = 5), were purified and stimulated in vitro 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 (* = p ,0.05) and almost significant (# = p 0.06) for Ply. Values were calculated with the background (i.e. media alone) proliferation subtracted. Error bars show the SEM. doi:10.1371/journal.ppat.1002396.g001
  • Figure 2. Mucosal CD4 T cell responses to pneumococci during aging and its relation with CAP rates. (a) 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 midlife. Tonsil MNCs from subjects (n = 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 Strep. Pneumoniae 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) Graph for data observed by Myles et al 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 (n = 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. doi:10.1371/journal.ppat.1002396.g002
  • Figure 3. Inhibitory action of anti-pneumococcal responses by regulatory T cells. Inhibition of mucosal CD4+ T cell anti-pneumococcal responses to Ply (a) and SPNT (b) but not to flu (c) by CD25hi regulatory T cells in subjects above 16 years old as indicated by increased cell proliferation following depletion of CD25hi cells from tonsil MNC population is observed. Subjects (n = 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 CD25hi 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 CD25hi cell depleted populations. (* = p ,0.05). doi:10.1371/journal.ppat.1002396.g003
  • Figure 4. Effect of restoration/addition of Tregs (CD25hi) cells back into CD25hi cell depleted MNC samples on proliferative responses by pneumococcal specific CD4+ T cells. Tonsil MNCs were depleted of CD25hi cells and left or CD25hi cells added back at the original proportion (,10%) or at three fold the original proportion (i.e. 30%). Cells were obtained from individuals (a) .16years (n = 5) and stimulated with SPNT, (b) ,17 years (n = 3) and stimulated with SPNT or individuals (c) .16 years and stimulated with flu (n = 5). Percentage of proliferating CD4+ cells are shown for each individual (open circles) as well as mean (black bars) proliferation in undepleted or CD25hi depleted or CD25hi depleted with CD25hi added back at the original proportion or CD25hi depleted with CD25hi added back at three times the original proportion. (* = p ,0.05). doi:10.1371/journal.ppat.1002396.g004
  • Figure 5. Cytokine profile of tonsil MNCs following S. pneumoniae antigen stimulation. Cytokine (IL-10, IFNc, TNFa, IL-2 and IL-17) production by tonsil MNCs following stimulation with Ply (grey bars, SEM are shown by error bars)) was assessed by quantifying cytokine levels in cell culture supernatants (n = 8) at 7 days post cell stimulation by Luminex assay. Mean values are shown for each subject’s cytokine levels post Ply stimulation with background cytokine levels in media alone subtracted, individuals with ,7% proliferating CD4+ cells post Ply stimulation were not assessed (a). Cytokine producing CD4+ cells were analysed by intracellular cytokine FACS analysis for (IL-10, IFNc, TNFa and IL-17) to determine cytokine production specifically by CD4+ cells post Ply stimulation (n = 10). Filled bars show mean percentage of cytokine expressing CD4+ cells with the background (media alone) percentage values subtracted (b). Cytokine production by CD4+ cells post Ply stimulation in undepleted (open bars) and CD25hi cell depleted (filled bars) tonsil MNCs (n = 13) was compared by intracellular FACS analysis. Similarly, values represent the mean percent of CD4+ cells that are expressing cytokine, with the background (media alone) values subtracted. Only the subjects showing .5% increased proliferation post CD25hi cell depletion were assessed (c). All error bars represent SEM (* = p ,0.05 and # = p 0.06). doi:10.1371/journal.ppat.1002396.g005
  • Figure 6. Effect of blockage of CTLA-4 and PDL-1 on CD25hi cells their suppression of anti-pneumococcal proliferative responses by CD4+ cells. Purified CD25hi cells were preblocked with anti-human CTLA-4 or anti-human PDL-1 blocking antibodies or isotype control (IgG) antibodies and added back to CD25hi 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 (a) or anti-human PDL-1 preblocked CD25hi cells (b) groups (n = 5 each group). (* = p ,0.05). doi:10.1371/journal.ppat.1002396.g006
  • Figure 7. Detection of Ply specific CD127low/- FoxP3+ CD4+ Treg cells in CD25 enriched tonsil and blood MNC. CD25 enriched MNC were stained with anti- CD127 and anti FoxP3 to allow identification of Tregs and with (a) strepavidin-PE, (b) negative control Ply tetramer-PE and (c) Ply tetramer-PE and analysed by FACS. A typical example of a FACS plot is shown. While the CD127low/- FoxP3+ CD4+ Treg cells show low level staining at 0.0% with strepavidn-PE (a) and negative control Ply-tetramer at 0.05% (b), a significantly higher percentage of Treg cells are bound by Ply-tetramers at 1.96%(c). doi:10.1371/journal.ppat.1002396.g007
  • Figure 8. Frequencies of Ply specific Tregs in the tonsil compared to blood. CD127low/- FoxP3+ CD4+ Treg cells in CD25 enriched blood and tonsil MNC populations were stained with Ply tetramer-PE and assessed by FACS for percentages of Treg cells specific for Ply in the two compartments. Graph shows the mean % of Tregs bound by Ply tetramer in blood (black bars) and in tonsils (grey bars) with % of Tregs stained with control tetramer subtracted (n = 3 for blood group and = 5 for tonsil group). doi:10.1371/journal.ppat.1002396.g008

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Pido-Lopez, J., Kwok, W. W., Mitchell, T. J., Heyderman, R. S., & Williams, N. A. (2011). Acquisition of pneumococci specific effector and regulatory Cd4 + T cells localising within human upper respiratory-tract mucosal lymphoid tissue. PLoS Pathogens, 7(12). https://doi.org/10.1371/journal.ppat.1002396

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