Skip to main content
NCBI home page
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2018 Oct 31;195(2):213–225. doi: 10.1111/cei.13225

Th17 responses to pneumococcus in blood and adenoidal cells in children

E Oliver 1,✉, C Pope 1, E Clarke 2, C Langton Hewer 3, A D Ogunniyi 4, J C Paton 5, T Mitchell 6, R Malley 7, A Finn 1
PMCID: PMC6330644  PMID: 30325010

Summary

Pneumococcal infections cause a large global health burden, and the search for serotype‐independent vaccines continues. Existing conjugate vaccines reduce nasopharyngeal colonization by target serotypes. Such mucosal effects of novel antigens may similarly be important. CD4+ Th17 cell‐dependent, antibody‐independent reductions in colonization and enhanced clearance have been described in mice. Here we describe the evaluation of T helper type 17 (Th17) cytokine responses to candidate pneumococcal protein vaccine antigens in human cell culture, using adenoidal and peripheral blood mononuclear cells. Optimal detection of interleukin (IL)‐17A was at day 7, and of IL‐22 at day 11, in these primary cell cultures. Removal of CD45RO+ memory T cells abolished these responses. Age‐associated increases in magnitude of responses were evident for IL‐17A, but not IL‐22, in adenoidal cells. There was a strong correlation between individual IL‐17A and IL‐22 responses after pneumococcal antigen stimulation (P < 0·015). Intracellular cytokine staining following phorbol myristate acetate (PMA)/ionomycin stimulation demonstrated that  > 30% CD4+ T cells positive for IL‐22 express the innate markers γδT cell receptor and/or CD56, with much lower proportions for IL‐17A+ cells (P < 0·001). Responses to several vaccine candidate antigens were observed but were consistently absent, particularly in blood, to PhtD (P < 0·0001), an antigen recently shown not to impact colonization in a clinical trial of a PhtD‐containing conjugate vaccine in infants. The data presented and approach discussed have the potential to assist in the identification of novel vaccine antigens aimed at reducing pneumococcal carriage and transmission, thus improving the design of empirical clinical trials.

Keywords: cytokines, human, T cells, vaccination

Introduction

Streptococcus pneumoniae (pneumococcus) remains a significant global cause of morbidity and mortality from diseases, including pneumonia, meningitis, sepsis and otitis media, and is a particular challenge in developing countries. Pneumococcus accounts for 11% of all deaths in children under 5 years of age, resulting in up to a million childhood deaths every year 1, 2. Of growing global concern are the emergence of non‐vaccine serotypes and antibiotic‐resistant strains of pneumococcus 3, 4.

Pneumococcus is a commensal of the human upper respiratory tract, with more than 90 capsular serotypes described. Asymptomatic colonization of the nasopharynx with pneumococcus is more common in young children than in older children and adults 5, 6, 7. In contrast, the development of invasive pneumococcal disease is, relatively speaking, rare. Transmission between children and to other family members sustains the bacteria within a community 8. The introduction of multivalent pneumococcal conjugate vaccines (PCV) has resulted in overall decreases in the incidence of pneumococcal disease, but overall colonization rates in children have changed little as non‐vaccine serotypes replace formerly dominant vaccine types and also cause some replacement disease, limiting the overall efficacy of the vaccines in some settings 9, 10, 11. Thus, the development of serotype‐independent pneumococcal vaccines is a priority in the fight against pneumococcus.

If, like PCVs, such vaccines are to impact disease by reducing carriage and transmission, understanding of naturally acquired mucosal immune responses to pneumococcus and how they affect pneumococcal colonization could guide antigen selection and vaccine formulation. Antibody‐independent CD4+ T cell‐dependent reduction of pneumococcal colonization has been demonstrated in mice 12, 13, 14. A role for CD4+ T helper type 17 (Th17) cells, which can kill and clear pneumococci by recruiting neutrophils to the site of infection, has been proposed 15, 16. Th17 cells exist in adults and children 15, and produce both interleukin (IL)‐17A and IL‐22) 17, 18. The stimulation of production of IL‐17A and IL‐22 by candidate pneumococcal vaccine antigens could indicate their capacity to influence pneumococcal colonisation either by preventing acquisition or promoting clearance.

In this study we describe the measurement and characteristics of Th17 responses in human primary cell cultures from blood and adenoidal tissue (nasal associated lymphoid tissue) of children, and the use of this technique to screen potential pneumococcal vaccine candidate antigens. In particular, we show that for an antigen that recently failed to reduce carriage in a clinical trial, no demonstrable Th17 responses could be seen.

Materials and methods

Subjects and samples

With informed consent, adenoids were collected from children aged 1–14 years, who were undergoing routine adenoidectomy or adenotonsillectomy at the Bristol Royal Hospital for Children. Up to 10 ml of anti‐coagulated peripheral blood was additionally collected from some children. Children were healthy at the time of surgery. Exclusion criteria were: recent/current antibiotics, known immunodeficiency or immunosuppressive treatment within 2 weeks of surgery. Clinical information is provided in detail in Supporting information, Table S1. Almost without exception, they had previously received seven valent pneumococcal conjugate vaccines, and it should be noted that the majority of children were aged 3 years and above. The total number of children studied was 75, and the numbers in the comparisons described varied between three and 38. Among those from whom a nasal swab was obtained and analysis completed (n = 61), the pneumococcal carriage rate by culture was 39%. Ethical approvals were obtained as appropriate from the North Somerset and South Bristol and PATH Research Ethics Committees.

Antigens

The whole‐cell killed unencapsulated pneumococcal antigen (WCA) was made as described in 19 at a concentration of killed bacteria representing 1 × 106 colony‐forming units (CFU)/ml, as determined in previous experiments (data not shown). Different WCA preparations have been shown to be similar with respect to specific antigen content and induction of immunological responses in laboratory animals. Recombinant proteins choline binding protein A (CbpA), pneumococcal surface antigen A (PsaA) and pneumococcal surface protein A (PspA) were purified from recombinant Escherichia coli  expressing the respective cloned genes 20, 21. Recombinant CbpA, PsaA and PspA were assessed to be > 95% pure by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and Coomassie brilliant blue staining. Recombinant protein pneumococcal histidine triad protein D (PhtD) was produced as previously described 22, and isothermal calorimetry was performed to confirm zinc binding with one‐dimensional nuclear magnetic resonance spectroscopy (ID NMR) to show associated changes in protein structure. All proteins were used at a concentration of 8 µg/ml to stimulate optimal CD4+ T cell proliferation as determined in previous experiments (as shown in Supporting information, Fig. S1). The duration of cell stimulation varied between experiments and is shown in the respective figure legends.

Cell isolation and culture

Adenoidal tissue was collected into Hanks’s balanced salt solution (HBSS)/2% HEPES (Thermo Scientific/Life Technologies, Waltham, MA, USA and Sigma‐Aldrich Company Ltd, Poole, UK) with 10% penicillin/streptomycin (Sigma‐Aldrich Company Ltd, Poole, UK), and processed fresh within 24 h. Peripheral blood was processed within 6 h of collection and was prepared by diluting it 1 : 1 with HBSS/2% HEPES. Mononuclear cells from adenoids and peripheral blood were separated on Ficoll‐density gradients as previously described 23. Cells were cultured in 48‐well culture plates at 1 × 106 cells/ml in a 1‐ml volume, in either complete RPMI‐1640 (Thermo Scientific/Life Technologies) containing 20 mM HEPES, 2 mM L‐glutamine, 1% penicillin/streptomycin (Sigma‐Aldrich Company Ltd) with 10% fetal bovine serum (Sigma‐Aldrich Company Ltd) for cytokine analysis, or in complete RPMI media/2% human serum (Sigma‐Aldrich Company Ltd) for intracellular cytokine analysis.

Cell depletions

Memory T cells (CD45RO+) were depleted from the mononuclear cell population using positive selection magnetic‐activated cell sorting (MACS) according to the manufacturer’s guidelines (Miltenyi Biotech, Bergisch Gladbach, Germany). A positive control (‘add back’) was made by mixing the depleted cells with the positively selected cells retained on the magnet during the cell separation procedure. The purity of these cell suspensions (CD45RO– and CD45RO+) was confirmed by immunofluorescence staining [CD4‐allophycocyanin (APC), CD45RO‐fluorescein isothiocyanate (FITC) and CD45RA‐phycoerythrin‐cyanin 7 (PE‐Cy7); BD Biosciences, Oxford, UK] and flow cytometry using a FACS Canto II (BD Biosciences, UK), analysed with FlowJo software (Tree Star, Inc., Ashland, OR, USA) analysis, was conducted by gating on lymphocyte cells identified by their forward‐/side‐scatter profile, gating the CD4+ population and finally gating on CD45RO and CD45RA cell populations. CD45RO and CD45RA cells were remixed at a 1 : 1 ratio (proportion of CD45RO cells before depletion was 16–38% and < 1% after depletion).

IL‐17A and IL‐22 immunoassays

Cells were incubated with or without antigen, and IL‐17A and IL‐22 was measured in the cell supernatant collected between 1 and 15 days using a human IL‐17A or an IL‐22 enzyme‐linked immunosorbent assay (ELISA) Ready‐Set‐Go kit, according to the manufacturer’s instructions (Affymetrix eBiosciences, San Diego, CA, USA). In most cases the same cell supernatant was used for both cytokine assays, except for the experiments shown in Figs. 1 and 2. Supernatants were stored at –20oC for short‐term and at –80oC for long‐term storage.

Intracellular cytokine production

Intracellular cytokine staining was conducted on day 7 of cell culture with or without antigen stimulation. Cells were restimulated with antigen on day 6 to boost their cell‐specific cytokine responses. On day 7, phorbol myristate acetate (PMA) 0·05 µg/ml, ionomycin 1 µg/ml (Sigma‐Aldrich Company Ltd, UK) and Golgistop (BD Biosciences, UK) were added for 5 h. Cell viability staining, cell surface staining and the intracellular cytokine staining processes were carried out using a BD Cytofix/Cytoperm Fixation/Permeabilization kit, according to the manufacturer’s instructions (BD Biosciences, UK). Fixable viability dye eFluor780 (Affymetrix eBiosciences) was used to assess cell viability. Cell surface antibody markers CD4 Alexa Fluor700 (BD Biosciences, UK), CD56 PE‐Cy7/ Brilliant Violet 510 (BD Biosciences, UK/Biolegend, San Diego, CA, USA), T cell receptor (TCR) yδ FITC/ PE‐Cy5.5 (BD Biosciences, UK/ Beckman Coulter, Brea, CA, USA) and intracellular antibodies IL‐17A PE/Brilliant Violet 605 (Affymetrix eBiosciences/Biolegend) and IL‐22 eFluor660 (Affymetrix eBiosciences) were used and concentrations had been predetermined by titrations. The fixed and stained cells were left overnight at 4oC to reduce autofluorescence before being analysed on a LSR II flow cytometer (BD Biosciences, UK), where 20 000 live cells in the lymphocyte gate were collected per stimulation. Analysis was carried out using the software program FlowJo, and only live cells in the lymphocyte gate were analysed. Analysis was conducted by gating on lymphocyte cells identified by their forward‐/side‐scatter profile, live cells were gated based on the live/dead cell marker, then either IL‐17A+ or IL‐22+ populations were identified, followed by the CD4+ population and finally the CD56+ or TCR+ γδ cell populations were gated on.

Statistical analysis

Significance of differences between groups was analysed using a paired t‐test. The relationships between age and cytokine responses were compared using linear regression analysis. Pearson’s correlation was used to investigate correlation in an individual’s IL‐17A and IL‐22 cytokine response to an antigen. Group mean cytokine responses to the panel of antigens were compared by one‐way analysis of variance (anova). Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA).

Results

Optimization of assay system and identification of responding cells

In order to determine optimal methodology for detecting responses, time–course experiments were initially conducted using peripheral blood mononuclear cells (PBMC) and adenoidal mononuclear cells (AMNC) stimulated with WCA. The Th17 cytokines IL‐17A (Fig. 1a) and IL‐22 (Fig. 1b) responses, when present, were consistently strong at days 7 and 11, respectively, and these timings were used in subsequent experiments.

Figure 1.

Figure 1

Open in a new tab

Detection of T helper type 17 (Th17) cytokines. (a) Interleukin (IL)‐17A and (b) IL‐22 in three children’s peripheral blood mononuclear cells (PBMC), and in one child’s adenoidal mononuclear cells (AMNC) after whole cell antigen (WCA) stimulation over a 15‐day time–course. Each data point represents a child’s cytokine response with the background signal subtracted. Each supernatant was taken from a different well of cells at each time‐point. The PBMC IL‐17A response for child 3 was anomalous at days 9 and 11; however, the response at day 7 fitted the broader trend.

To identify the phenotype of responding cells, two experiments showed that depletion of memory T cells (CD45RO+) from PBMC almost entirely eliminated detectable IL‐17A responses (Fig. 2a), which were restored by their replacement (Fig. 2b). This finding was confirmed in four additional children whose undepleted PBMC IL‐17A responses were median (range) = 40·5 (6·3–65·6) pg/ml on day 5 and CD45RO‐depleted (99% purity in all) IL‐17A responses 0 (0–0·4) pg/ml (data not shown).

Figure 2.

Figure 2

Open in a new tab

Interleukin (IL)‐17A responses to whole cell antigen (WCA) over 9 days (a) in undepleted peripheral blood mononuclear cells (PBMC) and memory T cell‐depleted (CD45RO–) PBMC, and (b) in memory T cell‐depleted PBMC with the memory T cells added back. CD45RO‐depleted cells were 99·93 and 99·78% pure (data not shown). For child 1 the add back experiment was only conducted between days 5 and 9 due to limited cells numbers. At each time‐point supernatants from different wells were used and values are shown with unstimulated cells signals subtracted.

Characterization of cytokine responses

Experiments were performed to establish the key characteristics of these responses. There was a correlation between age and the IL‐17A responses to WCA in AMNC, but little evidence that age influenced the IL‐22 response in AMNC or the IL‐17A or IL‐22 response in PBMC (Fig. 3). There was a strong positive correlation in the magnitude of IL‐17A and IL‐22 responses to WCA and CbpA in individual subjects in both PBMC and AMNC (Fig. 4).

Figure 3.

Figure 3

Open in a new tab

The association between age and interleukin (IL)‐17A and IL‐22 responses (above background) to whole cell antigen (WCA) in (a) peripheral blood mononuclear cells (PBMC) and (b) adenoidal mononuclear cells (AMNC). Each data point represents a child’s IL‐17A or IL‐22 response at day 7 or 11, respectively, to WCA; supernatants at both time‐points were collected from the same well of cells; n = 37–39. Linear regression lines are shown with corresponding r 2 and P‐values.

Figure 4.

Figure 4

Open in a new tab

Individual day 11 interleukin (IL)‐22 responses to whole cell antigen (WCA) (left) and choline binding protein A (CbpA) (right) plotted against corresponding day 7 IL‐17 responses in (a) peripheral blood mononuclear cells (PBMC) and (b) adenoidal mononuclear cells (AMNC). Values are shown with unstimulated background values subtracted and supernatants at both time‐points were collected from the same well of cells; n = 26–38. Pearson’s correlation coefficients and two‐tailed t‐test P‐values are shown.

Intracellular cytokine staining was used to investigate further the phenotype of cells producing IL‐17A and IL‐22. For intracellular cytokine staining, IL‐22 detection was measured after 7 days rather than the optimal day 11 (Fig. 5), as there were limited available cells. Live lymphocyte gating (based on forward‐ and side‐scatter parameters) in flow cytometric staining for intracellular IL‐17A and IL‐22 showed strong evidence of increases of 2% or more above the background following stimulation with the recombinant pneumococcal antigen CbpA (P = 0·0021, paired t‐test) (Fig. 5a).

Figure 5.

Figure 5

Open in a new tab

Intracellular cytokine staining analysis to identify interleukin (IL)‐17A+ and IL‐22+ expressing adenoidal mononuclear cells (AMNC) in response to whole cell antigen (WCA) and choline binding protein A (CbpA) at day 7 and to determine their CD4+ expression level. (a) The percentage of live lymphocytes producing IL‐17A or IL‐22. The average percentage of live cells in the lymphocyte gate was 59·5%, n = 14 (data not shown). (b) Comparison of the percentage of cytokine‐producing live lymphocytes expressing CD4+ in response to WCA and CbpA. Statistical analysis was conducted using a paired t‐test with the P‐values shown.

Fifty per cent or more IL‐17A+ live lymphocytes were CD4‐positive, with a somewhat lower percentage of IL‐22+ AMNC expressing CD4 (Fig. 5b). Following stimulation with WCA there was strong evidence of an expansion in the CD4+ IL‐17A+ AMNC expression compared to cultured cells that were stimulated with only PMA/ionomycin.

There was very strong evidence that a much higher proportion of IL‐22+ CD4+ AMNC expressed one, the other or both of the innate cellular markers CD56 and TCR‐γδ than IL‐17A+ CD4+ AMNC, both before and after antigen stimulation (Fig. 6).

Figure 6.

Figure 6

Open in a new tab

Expression of innate cellular markers on CD4+ interleukin (IL)‐17A+ and IL‐22+ adenoidal mononuclear cells (AMNC) at day 7. Each bar represents the mean percentage of live CD4+ cytokine‐producing cells expressing combinations of the cell surface markers CD56 and T cell receptor (TCR) γδ in response to whole cell antigen (WCA) and to choline binding protein A (CbpA); n = 14. A paired t‐test was used to compare the IL‐17A+ cells expressing combinations of the innate cellular markers, with live IL‐22+ cells expressing combinations of the innate cellular markers. The P‐values shown relate to the proportions of cells expressing either combination of markers (CD56 or TCR γδ or both). Standard deviations for the combined innate marker percentages for the six bars are, respectively, 15·5, 12·3, 11·9, 21·1, 22·1 and 21·3. There are no significant differences between conditions.

IL‐17A and IL‐22 responses to a panel of pneumococcal antigens

In order to compare responses to different candidate vaccine antigens, cytokine release by PBMC and AMNC following stimulation with pneumococcal antigens, including three additional proteins previously investigated as candidate vaccines, were measured. Results showed significant variation between antigens (Fig. 7), and in particular there was little apparent response to the surface protein PhtD in PBMC, while responses to the additional proteins PsaA and PspA were weak in AMNC.

Figure 7.

Figure 7

Open in a new tab

Interleukin (IL)‐17A and IL‐22 responses to whole cell antigen (WCA) and to a panel of pneumococcal antigens – choline binding protein A (CbpA), pneumococcal surface antigen A (PsaA), Pneumococcal surface protein A (PspA) and pneumococcal histidine triad protein D (PhtD) – in (a) peripheral blood mononuclear cells (PBMC) and (b) adenoidal mononuclear cells (AMNC). Each data point represents a child’s IL‐17A or IL‐22 response at days 7 or 11, respectively. All PBMC or AMNC samples were tested for all antigens. The background is shown (media) for reference; however, the background has been subtracted from the data points showing the responses to each of the stimulations. Supernatants at both time‐points were collected from the same well of cells. PBMC n = 16 and AMNC n = 14. The bar represents the mean. Comparing group means (excluding the media background) was conducted by one‐way analysis of variance (anova) with the P‐values shown.

Additional post‐hoc analysis, added during the peer review process (Supporting information, Fig. S3 and Fig. S4), indicated apparent significant differences between WCA and the recombinant proteins tested, apart from between WCA and CbpA in adenoids and differences between PhtD and PspA and PsaA in blood, but not clearly in adenoids.

Discussion

We have defined IL‐17A and IL‐22 responses to pneumococcal antigens in adenoidal and peripheral blood mononuclear cells, and thus propose a tool with which to evaluate Th17 cellular immune responses to candidate pneumococcal vaccine antigens. This may assist prioritization for assessment of their effects on colonization and their potential prevention of transmission.

Previous Th17 experiments in mice found day 3 to be optimal to detect IL‐17A responses, and IL‐17A can also be detected from human pharyngeal tonsil mononuclear cells after 3 days 15. However, we have previously shown that CD4+ T cell proliferation in response to pneumococcal antigens in children is optimal after 7 days of stimulation in culture 24 and similarly in the experiments presented here, 7 days of culture was optimal for IL‐17A detection while 11 days was the best for IL‐22 detection (Fig. 1). We have previously demonstrated clearer and more reliable mucosal responses in adenoidal than tonsillar cells 25. Although these relatively slow response rises might suggest that these are not memory responses following previous exposure, depletion of CD45RO+ cells resulted in their almost complete disappearance (Fig. 2), confirming that they are anamnestic, albeit not extremely rapid, in concordance with previous studies of other aspects of these mucosal cellular immune responses 24.

Both rates of colonization and invasive disease due to pneumococcus drops rapidly with increasing age in young children 5, 6, 7, suggesting progressive rises in specific immunity either in response to exposure, or through immune maturation or both. Both we 5 and others have shown evidence of emergence of specific B cell immunity to pneumococcal antigens, particularly during the second year of life 7, 26. IL‐17A responses to pneumococcal antigens in blood leucocytes are higher in adults than children and vary between children in different populations, which is likely to reflect different levels of exposure to pneumococcus 15, 27. In the results presented here there was little evidence of increasing Th17 responses with age, apart from rises in adenoidal cellular elaboration of IL‐17A in response to pneumococcal stimulation (Fig. 3). The IL‐22 responses of individual children in blood and adenoids are not significantly correlated with each other, but IL‐17A responses are (Fig. S2), and close inspection of the data (Fig. 3b) suggests that the reason the IL‐17A age effect is not seen in blood lymphocytes is the more consistently detectable responses in the younger children studied. It is also possible that clearer age‐dependency was not seen in this study in the context of wide variation between individuals owing to other potentially confounding factors, including timing of recent exposure and colonization. As noted, previous studies by our group in this paediatric population showed a consistent steep rise in antibodies to a range of pneumococcal antigens during the second year of life 5, suggesting that few, if any, of the children reported here are likely never to have been colonized or that serology performed in this group would be likely to predict or explain the differences we observed. Alternatively, or in addition, much of any cytokine increase may occur by the end of the second year of life, as seen for antibodies to pneumococcal antigens 5, while the children studied here were nearly all aged 2 years or older.

The classic Th17 pathway shows production of IL‐17A and IL‐22 to be from CD4+ T cells under the influence of transforming growth factor (TGF)‐β, IL‐6 and IL‐23 15, 28, 29, 30. Mucosal immunity to colonization by pneumococcus can be antibody‐independent and CD4+ T cell‐dependent in mice 14, 15, and CD4+ T cells may be important in protection of humans against pneumococcal colonization and disease; for example, in HIV‐infected individuals with reduced numbers of CD4+ T cells and high risk of this infection 31. The question then arises of whether CD4+ T cells are an important cellular source of IL‐17A and IL‐22. While it was clear that both cytokines were elaborated by cells falling within the lymphocyte scatter gate, our results suggest that CD4+ T cells are a source of IL‐17A following stimulation with pneumococcal antigens, but this was not clearly demonstrated for IL‐22 under the conditions we used (Fig. 5). Nevertheless, we showed evidence of strong correlation at an individual level between the size of IL‐17A and IL‐22 responses measured after pneumococcal antigen stimulation both in AMNC and PBMC (Fig. 4). Further characterization of CD4+ T cells expressing the two cytokines both before and after pneumococcal antigen stimulation showed that those producing IL‐22 were much more likely to be expressing innate cell phenotypes (Fig. 6). Both innate natural killer (NK) T cells 32, 33 and TCR γδ cells 34, 35 have previously been shown to be sources of IL‐17A and IL‐22, and both these cell types have been shown to recruit neutrophils to the pneumococcal infection site 36, 37. Our results extend these observations to include CD4+ T cells expressing innate markers as a potential source of IL‐22, particularly in the human upper respiratory tract. In this context, it would be of interest to conduct further CD45RO depletion experiments to examine the effect on IL‐22 production.

We and others have previously investigated mucosal and systemic B cell and T cell CD4+ responses to several pneumococcal antigens, including WCA, CbpA, PsaA and PspA 5, 12, 23, 24, 38. These antigens have also been shown to have protective effects in murine models of pneumococcal colonisation and infection 39, 40, 41. There has been strong interest in PhtD as a vaccine candidate antigen 22. The IL‐17A and IL‐22 responses we demonstrate here to these antigens in primary human cell cultures, notwithstanding wide interindividual variation, demonstrate significance differences between antigens as well (Fig. 7). The relatively larger responses seen in blood than in adenoidal cells occur in the context of a much lower background cytokine production by unstimulated cultures and a known lower T regulatory environment, and has previously been observed by us and others 42, 43; however, further investigation is required to further understand this observed difference. Of particular note were the relative lack of responses to PhtD evident in blood, an antigen which has recently been shown to lack efficacy against pneumococcal colonization in children, although when injected parenterally with aluminium rather than a T cell adjuvant 44. Although it has been proposed that this and related pneumococcal proteins, which are released extracellularly by the bacterium in large quantities, might act as a sink for potentially opsonophagocytosing antibodies 45, our data suggest that PhtD may also fail to induce cellular immune responses.

In this study we describe Th17 responses to pneumococcal antigens in human cell cultures in detail. This approach not only allows detailed description of the immunological responses to pneumococcus in the upper respiratory tract of children, but also has potential to guide antigen selection for candidate vaccines aiming to impact upon carriage and transmission. Future studies should seek to elucidate whether such responses reliably predict protection against acquisition or clearance of carriage in children having repeated evaluation of colonization over time.

Disclosures

R. M. is a named inventor on a patent describing the use of the pneumococcal whole cell vaccine, and is the scientific founder as well as member of the board of directors of Affinivax, a company that is developing a pneumococcal vaccine. J. C. P. is a co‐founder and director of GPN Vaccines Pty Ltd, a company that is developing a pneumococcal vaccine.

Author contributions

E. O. and C. P. conducted the experiments. A. F., E. C., C. P. and E. O. designed the experiments. C. L. H. assisted providing samples. A. O., J. C. P., T. M. and R. M. provided the antigens. E. O. and A. F. wrote the paper with assistance of all the other authors.

Supporting information

Table S1. Demographic data and history of chronic, recent illnesses and infections in the study population.

Click here for additional data file. (24.4KB, docx)

Fig. S1. Human nasal associated lymphoid tissue optimal/sub maximal CD4+ proliferation responses to (a) CbpA, (b) PsaA, (c) PspA and (d) PhtD at day 7. The background unstimulated responses have been subtracted. Bars represent the mean with the standard deviation shown.

Click here for additional data file. (47.5KB, png)

Fig. S2. Individual PBMC and AMNC (a) IL 17A and (b) IL 22 responses to WCA. Data points represent a child’s IL 17A or IL 22 response at day 7 and 11 respectively. The background unstimulated responses have been subtracted. Supernatants at both time points were collected from the same well of cells. n = 20. Pearson’s correlation coefficients and two tailed t test P values are shown.

Click here for additional data file. (31.3KB, png)

Fig. S3. IL 17A and IL 22 responses to WCA and to a panel of pneumococcal antigens – CbpA, PsaA, PspA and PhtD – in (a) PBMC and (b) AMNC. Each data point represents a child’s IL 17A or IL 22 response at day 7 or 11 respectively. All PBMC or AMNC samples were tested for all antigens. The background is shown (media) for reference, however the background has been subtracted from the data points showing the responses to each of the stimulations. Supernatants at both time points were collected from the same well of cells. PBMC n = 16 and AMNC n = 14. The bar represents the mean. Wilcoxon rank sum test was conducted between WCA and each recombinant protein, P values shown.

Click here for additional data file. (111.2KB, png)

Fig. S4. IL 17A and IL 22 responses to WCA and to a panel of pneumococcal antigens – CbpA, PsaA, PspA and PhtD – in (a) PBMC and (b) AMNC. Each data point represents a child’s IL 17A or IL 22 response at day 7 or 11 respectively. All PBMC or AMNC samples were tested for all antigens. The background is shown (media) for reference, however the background has been subtracted from the data points showing the responses to each of the stimulations. Supernatants at both time points were collected from the same well of cells. PBMC n = 16 and AMNC n = 14. The bar represents the mean. Wilcoxon rank sum test was conducted between PhtD and PsaA or PspA, P values shown.

Click here for additional data file. (130.2KB, png)

Acknowledgements

This study was supported by funding provided by PATH. We thank all the children who donated samples to us, the staff at the Bristol Royal Hospital for Children, and our nurses Phoebe Moulsdale, Clare Harrison and Jo Jenkins for recruiting the children to the study. We also acknowledge the assistance of Dr Andrew Herman and the University of Bristol Faculty of Biomedical Sciences Flow Cytometry Facility.

References

  • 1. O’Brien KL, Wolfson LJ, Watt JP et al Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 2009;374:893–902. [DOI] [PubMed] [Google Scholar]
  • 2. Pneumococcal conjugate vaccine for childhood immunization – WHO position paper. Wkly Epidemiol Rec 2007;82:93–104. [PubMed] [Google Scholar]
  • 3. Reinert RR. The antimicrobial resistance profile of Streptococcus pneumoniae . Clin Microbiol Infect 2009;15(Suppl 3):7–11. [DOI] [PubMed] [Google Scholar]
  • 4. Kim L, McGee L, Tomczyk S, Beall B. Biological and epidemiological features of antibiotic‐resistant streptococcus pneumoniae in pre‐ and post‐conjugate vaccine eras: a United States perspective. Clin Microbiol Rev 2016;29:525–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Zhang Q, Bernatoniene J, Bagrade L et al Serum and mucosal antibody responses to pneumococcal protein antigens in children: relationships with carriage status. Eur J Immunol 2006;36:46–57. [DOI] [PubMed] [Google Scholar]
  • 6. Hogberg L, Geli P, Ringberg H, Melander E, Lipsitch M, Ekdahl K. Age‐ and serogroup‐related differences in observed durations of nasopharyngeal carriage of penicillin‐resistant pneumococci. J Clin Microbiol 2007;45:948–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Lipsitch M, Whitney CG, Zell E, Kaijalainen T, Dagan R, Malley R. Are anticapsular antibodies the primary mechanism of protection against invasive pneumococcal disease? PLOS Med 2005;2:e15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Bogaert D, De Groot R, Hermans PW. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis 2004;4:144–54. [DOI] [PubMed] [Google Scholar]
  • 9. Waight PA, Andrews NJ, Ladhani NJ, Sheppard CL, Slack MP, Miller E. Effect of the 13‐valent pneumococcal conjugate vaccine on invasive pneumococcal disease in England and Wales 4 years after its introduction: an observational cohort study. Lancet Infect Dis 2015;15:535–43. [DOI] [PubMed] [Google Scholar]
  • 10. PHE . Pneumococcal disease infections caused by serotypes not in Prevenar 13 vaccine 2017 [updated 27th March 2017]. Available at: https://www.gov.uk/government/publications/pneumococcal-disease-caused-by-strains-not-covered-by-prevenar-13-vaccine/pneumococcal-disease-infections-caused-by-serotypes-not-in-prevenar-13-vaccine (accessed 19 October 2018).
  • 11. Weinberger DM, Malley R, Lipsitch M. Serotype replacement in disease after pneumococcal vaccination. Lancet 2011;378:1962–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, Lipsitch M. CD4+ T cells mediate antibody‐independent acquired immunity to pneumococcal colonization. Proc Natl Acad Sci USA 2005;102:4848–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Malley R, Srivastava A, Lipsitch M et al Antibody‐independent, interleukin‐17A‐mediated, cross‐serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide. Infect Immun 2006;74:2187–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Basset A, Thompson CM, Hollingshead SK et al Antibody‐independent, CD4+ T‐cell‐dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins. Infect Immun 2007;75:5460–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Lu YJ, Gross J, Bogaert D et al Interleukin‐17A mediates acquired immunity to pneumococcal colonization. PLOS Pathog 2008;4:e1000159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Zhang Z, Clarke TB, Weiser JN. Cellular effectors mediating Th17‐dependent clearance of pneumococcal colonization in mice. J Clin Invest 2009;119:1899–909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Harrington LE, Hatton RD, Mangan PR et al Interleukin 17‐producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005;6:1123–32. [DOI] [PubMed] [Google Scholar]
  • 18. Liang SC, Tan XY, Luxenberg DP et al Interleukin (IL)‐22 and IL‐17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med 2006;203:2271–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Malley R, Lipsitch M, Stack A et al Intranasal immunization with killed unencapsulated whole cells prevents colonization and invasive disease by capsulated pneumococci. Infect Immun 2001;69:4870–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Ogunniyi AD, Folland RL, Briles DE, Hollingshead SK, Paton JC. Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae . Infect Immun 2000;68:3028–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Ogunniyi AD, Woodrow MC, Poolman JT, Paton JC. Protection against Streptococcus pneumoniae elicited by immunization with pneumolysin and CbpA. Infect Immun 2001;69:5997–6003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Rioux S, Neyt C, Di Paolo E et al Transcriptional regulation, occurrence and putative role of the Pht family of Streptococcus pneumoniae . Microbiology 2011;157:336–48. [DOI] [PubMed] [Google Scholar]
  • 23. Zhang Q, Choo S, Finn A. Immune responses to novel pneumococcal proteins pneumolysin, PspA, PsaA, and CbpA in adenoidal B cells from children. Infect Immun 2002;70:5363–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Zhang Q, Bagrade L, Bernatoniene J et al Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007;195:1194–202. [DOI] [PubMed] [Google Scholar]
  • 25. Pope C, Oliver EH, Ma J, Langton Hewer C, Mitchell TJ, Finn A. Genetic conjugation of components in two pneumococcal fusion protein vaccines enhances paediatric mucosal immune responses. Vaccine 2015;33:1711–8. [DOI] [PubMed] [Google Scholar]
  • 26. Rapola S, Jantti V, Haikala R et al Natural development of antibodies to pneumococcal surface protein A, pneumococcal surface adhesin A, and pneumolysin in relation to pneumococcal carriage and acute otitis media. J Infect Dis 2000;182:1146–52. [DOI] [PubMed] [Google Scholar]
  • 27. Lundgren A, Bhuiyan TR, Novak D et al Characterization of Th17 responses to Streptococcus pneumoniae in humans: comparisons between adults and children in a developed and a developing country. Vaccine 2012;30:3897–907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Fossiez F, Djossou O, Chomarat P et al T cell interleukin‐17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996;183:2593–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Mangan PR, Harrington LE, O’Quinn DB et al Transforming growth factor‐beta induces development of the T(H)17 lineage. Nature 2006;441:231–4. [DOI] [PubMed] [Google Scholar]
  • 30. Bettelli E, Carrier Y, Gao W et al Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235–8. [DOI] [PubMed] [Google Scholar]
  • 31. Klugman KP, Madhi SA, Feldman C. HIV and pneumococcal disease. Curr Opin Infect Dis 2007;20:11–5. [DOI] [PubMed] [Google Scholar]
  • 32. Rachitskaya AV, Hansen AM, Horai R et al Cutting edge: NKT cells constitutively express IL‐23 receptor and RORgammat and rapidly produce IL‐17 upon receptor ligation in an IL‐6‐independent fashion. J Immunol 2008;180:5167–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Goto M, Murakawa M, Kadoshima‐Yamaoka K et al. Murine NKT cells produce Th17 cytokine interleukin‐22. Cell Immunol 2009;254:81–4. [DOI] [PubMed] [Google Scholar]
  • 34. Lockhart E, Green AM, Flynn JL. IL‐17 production is dominated by γδ T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006;177:4662–9. [DOI] [PubMed] [Google Scholar]
  • 35. Ness‐Schwickerath KJ, Jin C, Morita CT. Cytokine requirements for the differentiation and expansion of IL‐17A‐ and IL‐22‐producing human Vgamma2Vdelta2 T cells. J Immunol 2010;184:7268–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Nakasone C, Yamamoto N, Nakamatsu M et al Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil‐mediated host defense against pneumococcal infection. Microbes Infect 2007;9:251–8. [DOI] [PubMed] [Google Scholar]
  • 37. Kawakami K, Yamamoto N, Kinjo Y et al Critical role of Valpha14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection. Eur J Immunol 2003;33:3322–30. [DOI] [PubMed] [Google Scholar]
  • 38. Zhang Q, Bernatoniene J, Bagrade L et al Regulation of production of mucosal antibody to pneumococcal protein antigens by T‐cell‐derived gamma interferon and interleukin‐10 in children. Infect Immun 2006;74:4735–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Briles DE, Hollingshead SK, Nabors GS, Paton JC, Brooks‐Walter A. The potential for using protein vaccines to protect against otitis media caused by Streptococcus pneumoniae. Vaccine 2000;19(Suppl 1):S87–95. [DOI] [PubMed] [Google Scholar]
  • 40. Briles DE, Ades E, Paton JC et al Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae . Infect Immun 2000;68:796–800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Trzcinski K, Thompson C, Malley R, Lipsitch M. Antibodies to conserved pneumococcal antigens correlate with, but are not required for, protection against pneumococcal colonization induced by prior exposure in a mouse model. Infect Immun 2005;73:7043–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Pido‐Lopez J, Kwok WW, Mitchell TJ, Heyderman RS, Williams NA. Acquisition of pneumococci specific effector and regulatory Cd4+ T cells localising within human upper respiratory‐tract mucosal lymphoid tissue. PLOS Pathog 2011;7:e1002396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Zhang Q, Leong SC, McNamara PS, Mubarak A, Malley R, Finn A. Characterisation of regulatory T cells in nasal associated lymphoid tissue in children: relationships with pneumococcal colonization. PLOS Pathog 2011;7:e1002175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Odutola A, Ota MO, Antonio M et al Efficacy of a novel, protein‐based pneumococcal vaccine against nasopharyngeal carriage of Streptococcus pneumoniae in infants: a phase 2, randomized, controlled, observer‐blind study. Vaccine 2017;35:2531–42. [DOI] [PubMed] [Google Scholar]
  • 45. Plumptre CD, Ogunniyi AD, Paton JC. Surface association of Pht proteins of Streptococcus pneumoniae . Infect Immun 2013;81:3644–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Demographic data and history of chronic, recent illnesses and infections in the study population.

Click here for additional data file. (24.4KB, docx)

Fig. S1. Human nasal associated lymphoid tissue optimal/sub maximal CD4+ proliferation responses to (a) CbpA, (b) PsaA, (c) PspA and (d) PhtD at day 7. The background unstimulated responses have been subtracted. Bars represent the mean with the standard deviation shown.

Click here for additional data file. (47.5KB, png)

Fig. S2. Individual PBMC and AMNC (a) IL 17A and (b) IL 22 responses to WCA. Data points represent a child’s IL 17A or IL 22 response at day 7 and 11 respectively. The background unstimulated responses have been subtracted. Supernatants at both time points were collected from the same well of cells. n = 20. Pearson’s correlation coefficients and two tailed t test P values are shown.

Click here for additional data file. (31.3KB, png)

Fig. S3. IL 17A and IL 22 responses to WCA and to a panel of pneumococcal antigens – CbpA, PsaA, PspA and PhtD – in (a) PBMC and (b) AMNC. Each data point represents a child’s IL 17A or IL 22 response at day 7 or 11 respectively. All PBMC or AMNC samples were tested for all antigens. The background is shown (media) for reference, however the background has been subtracted from the data points showing the responses to each of the stimulations. Supernatants at both time points were collected from the same well of cells. PBMC n = 16 and AMNC n = 14. The bar represents the mean. Wilcoxon rank sum test was conducted between WCA and each recombinant protein, P values shown.

Click here for additional data file. (111.2KB, png)

Fig. S4. IL 17A and IL 22 responses to WCA and to a panel of pneumococcal antigens – CbpA, PsaA, PspA and PhtD – in (a) PBMC and (b) AMNC. Each data point represents a child’s IL 17A or IL 22 response at day 7 or 11 respectively. All PBMC or AMNC samples were tested for all antigens. The background is shown (media) for reference, however the background has been subtracted from the data points showing the responses to each of the stimulations. Supernatants at both time points were collected from the same well of cells. PBMC n = 16 and AMNC n = 14. The bar represents the mean. Wilcoxon rank sum test was conducted between PhtD and PsaA or PspA, P values shown.

Click here for additional data file. (130.2KB, png)

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES