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Immune cell activation and cytokine release after stimulation of whole blood with pneumococcal C-polysaccharide and capsular polysaccharides

  • Marianne Sundberg-Kövamees
    Correspondence
    Corresponding author. Karolinska Institutet, Department of Medicine Solna, Respiratory Medicine Unit L4:01 Karolinska University Hospital Solna 171 76 Stockholm, SWEDEN Office phone +46 8 517 70665; Fax: +46 8 517 75451.
    Affiliations
    Respiratory Medicine Unit, Department of Medicine Solna and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

    Lung Allergy Clinic, Karolinska University Hospital, Stockholm, Sweden
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  • Johan Grunewald
    Affiliations
    Respiratory Medicine Unit, Department of Medicine Solna and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden

    Lung Allergy Clinic, Karolinska University Hospital, Stockholm, Sweden
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  • Jan Wahlström
    Affiliations
    Respiratory Medicine Unit, Department of Medicine Solna and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
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Open AccessPublished:July 16, 2016DOI:https://doi.org/10.1016/j.ijid.2016.07.004

      Highlights

      • CWPS and the three pneumococcal capsular polysaccharides tested (type 3, type 9 and type 23) activated all leukocyte cell types tested but to different degrees.
      • After stimulation, the highest relative MFI values of CD69 were observed for NK cells followed by CD56+ T cells, monocytes, CD4- T cells and finally CD4+ T cells.
      • Stimulation with CWPS, type 3, type 9 and type 23 capsules induced the release of cytokines IL-8, TNF, IL-10 and IFNγ, compared to unstimulated cells.
      • Capsule type 23 induced the strongest CD69 expression and the highest cytokine release followed by type 9 and type 3.

      Abstract

      Streptococcus pneumonia is a major cause of morbidity and mortality in children and adults worldwide. Lack of fully effective pneumococcal vaccines is a problem. Streptococcus pneumoniae exposes on its surface C-polysaccharide (cell wall polysaccharide, CWPS) and serospecific capsular polysaccharides, used in pneumococcal vaccines. We investigated the effect of CWPS and individual capsular polysaccharides, with regard to activation of subsets of immune cells of healthy controls. Three different capsular polysaccharides, CWPS and LPS were used for in vitro stimulation of whole blood. Cell activation (CD69 expression) was assessed in CD4+ and CD4- T cells, NK-like T cells, NK cells and monocytes by flow cytometry. Cytokine levels in supernatants were quantified by Cytometric Bead Array (CBA). CWPS and the capsules activated immune cell subsets, but to different degrees. NK cells and NK-like T cells showed the strongest activation, followed by monocytes. Among the three capsules, capsule type 23 induced the strongest activation and cytokine release, followed by type 9 and type 3. This study increases the understanding of how the human immune system reacts to pneumococcal vaccine components.

      Keywords

      1. Introduction

      Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide. Invasive pneumococcal disease (IPD) is a serious health problem in children and adults, and causes almost 1 million childhood deaths worldwide every year. Streptococcus pneumoniae usually colonizes the nasopharynx of healthy children but is less frequently found as a colonizer in adults.
      • Munoz-Almagro C.
      • Bautista C.
      • Arias M.T.
      • Boixeda R.
      • Del Amo E.
      • Borras C.
      • et al.
      High prevalence of genetically-determined mannose binding lectin deficiency in young children with invasive pneumococcal disease.
      According to figures from WHO, 1.6 million deaths were caused by this agent annually. In Europe and the USA the annual incidence of invasive pneumococcal disease ranges from 10 to 100 cases per 100 000 population. Streptococcus pneumoniae is an encapsulated gram-positive bacterium. On the surface, the bacteria are covered with a capsular polysaccharide that is specific for each serotype. Totally over 94 different serotypes have been described. However, the C-polysaccharide (cell wall polysaccharide, CWPS), another polysaccharide on the pneumococcal surface, has the same structure in all described pneumococcal serotypes, thereby being a common denominator within this species.
      There are two different types of pneumococcal vaccines on the market today. Whereas one type contains only polysaccharides, the other contains proteins in addition to polysaccharides (conjugated vaccine). The polysaccharide vaccine contains 23 of the most common pathogenic capsular pneumococcal polysaccharides while the protein conjugated vaccines contain 7 or 13 different pneumococcal capsular polysaccharides. It is known that polysaccharides are T-cell independent antigens in their capacity to generate B-cell responses to produce antibodies.
      • Defrance T.
      • Taillardet M.
      • Genestier L.
      T cell-independent B cell memory.
      In contrast, when the capsule polysaccharides are conjugated to a protein they generate a T-cell response with memory T cells in addition to B cells and antibodies.
      Because of the evolving antibiotic resistance and the lack of highly effective pneumococcal vaccines covering all pathogenic serotypes, it is of vital importance to investigate processes involved in the pathogenicity of Streptococcus pneumoniae.
      • Perez-Dorado I.
      • Campillo N.E.
      • Monterroso B.
      • Hesek D.
      • Lee M.
      • Paez J.A.
      • et al.
      Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1.
      We hypothesized that the capsular polysaccharides may interact with several types of immune cells, and that the cell-stimulatory capacity will vary between different serotypes of these polysaccharides, which may in turn affect the serotype-specific response induced by vaccination. To investigate the effect of the individual capsular polysaccharides and C- polysaccharide on the immune cells and cytokines, we performed whole blood stimulation using type 3-, type 9- and type 23-polysaccharides (all included in the 23-valent polysaccharide vaccine). Subsequently, the rate of CD69 expression on immune cell subsets was measured as a marker of activation. We also measured the concentration of IL-8, IL-10, TNF and INFγ in supernatants of in vitro stimulated blood cells.

      2. Materials and methods

      2.1 Subjects and blood samples

      The persons included in this study were healthy adult non-pneumococcal vaccinated volunteers, median age 48.5 years, range 42 to 59. Peripheral blood cells were obtained by vein puncture from ten different non-smoking women. All blood samples were drawn in early morning. Blood was collected using Vacutainer TM tubes (Becton-Dickinson Vacutainer Systems, Franklin Lakes, NJ, U.S.A.) containing sodium heparin anticoagulant. All participants signed a written informed consent. The study was approved by the Regional Ethical Review Board, Stockholm, Sweden.

      2.2 In vitro stimulation

      Each polysaccharide was added to 500 μl whole blood from healthy subjects. Pneumococcal capsular polysaccharides type 3, type 9 (9N) and type 23 (23F), (randomly chosen) were purchased from ATCC, (American Type Culture Collection, USA). CWPS, also known as CPS (pneumococcal C- polysaccharide) was purchased from Statens Serum Institute, Copenhagen, Denmark. LPS (E.Coli) was purchased from Sigma-Aldrich, Schnelldorf, Germany. Whole blood alone was used as negative control. LPS was used as positive control. The final concentration of each polysaccharide, LPS and CWPS was 10 μg/ml of blood. Samples were incubated for 4 h (eight persons) and 12 h (nine persons) respectively in 12-well microtiter plates in 37°C in humidified atmosphere of 5% CO2.

      2.3 Antibodies and flow cytometry

      In order to evaluate the activation of different leukocyte subsets, cells were labelled with the following fluorochrome-conjugated monoclonal antibodies: anti-CD3-Pacific Blue, anti-CD56-PE, anti-CD4-APC H7, anti-CD14-PerCP and anti-CD69-FITC. All antibodies were purchased from BD Biosciences, San Jose, CA, USA.
      Antibodies were added to 50 μl aliquots of blood after in vitro stimulation. Remaining blood was centrifuged and supernatants were collected and stored at -20°C before analysis of cytokines. The samples were then incubated with antibodies for 20 min in darkness at room temperature (RT). Lysing of red blood cells, fixation and stabilization of white blood cells was achieved according to the manufacturer's instructions using COULTER Multi-Q-prep (Coulter Electronics Inc., Hialeah FL, U.S.A.) Cells were analysed by flow cytometry (FACS Canto TM II flow cytometer) (BD). Data was processed using Diva 6.1.2 software (BD).
      In order to gate on lymphocytes and select subsets, forward scatter (FSC) and side scatter (SSC) in combination with the following patterns of cell surface marker expression were used (CD3 is a marker of all T cells): CD3+CD4+, CD3+CD4-, CD3+CD56+ (NK-like T cells) and CD3-CD56+ (NK cells). FSC and SSC in combination with CD14+ were used in order to gate monocytes.
      Although limitations of the flow cytometer available for this study did not allow us to include staining also for CD8, in order to determine the proportion of CD8+ cells in the CD4- T cell population we have investigated this in blood samples from four individuals, using the same stimuli and at two timepoints (4 h and 8 h). CD8+ T cells constituted on average 86% of the CD4- T cells (range 77-90%), and this was not altered by the various stimuli.
      Cell activation was assessed as expression of CD69 in the respective subsets. Data are presented as mean fluorescence intensity (MFI) for CD69, or as relative mean fluorescence intensity (MFI) for CD69 (i.e. MFI of CD69 for stimulated cells divided by MFI of CD69 for unstimulated cells).

      2.4 Quantification of secreted cytokines

      The Cytometric Bead Array (CBA) flex set, a multiplex assay allowing simultaneous detection of several analytes in a small sample volume, (BD, Franklin Lakes, NJ, USA) was used to quantify levels of secreted cytokines in whole blood cell supernatants. Briefly, these tests contain micro-particles to which different anti-cytokine antibodies can be coated. After incubation with supernatants followed by labelling with a second fluorescently labelled anti-cytokine antibody, the amount of cytokines can be analysed. The cytokines measured were TNF, IL-8, IL-10 and IFNγ. 30 μl of all samples were prepared as described in the protocol and the cytokines were detected within a range of 10-2500 pg/ml. The assays were performed according to the manufacturer's instructions and the data were analysed using the BD Cytometric Bead Array (BD Biosciences) software, in a FACS Canto TM II flow cytometer (BD). The data were analysed with FCAP Array Software version 1-01 (Soft Flow, Inc., St. Louis Park, MN, USA).

      2.5 Statistical methods and data management

      Multiple comparisons of continuous data were performed by non-parametric analysis of variance. Statistical comparisons in order to test differences between two dependent observations were performed by use of the non-parametric Wilcoxon signed-rank test. In addition to that, descriptive statistics and graphical methods were used to characterize the data.
      The study employs multiple hypotheses testing, where each hypothesis was analysed separately and the existence of patterns in and the consistency of the results were considered in the analysis. All analyses were carried out by use of the SAS system 9.3, (SAS Institute Inc., Cary, NC, USA) and the 5, 1 and 0.1% levels of significance were considered.
      Graphs were drawn using Graph Pad PRISM 5 (GraphPad Software, Inc. San Diego, CA, USA). P-values < 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, NS (non significant).

      3. Results

      3.1 CD69 expression after stimulation of whole blood

      CWPS as well as the three capsules tested (type 3, type 9 and type 23) activated all leukocyte cell types investigated to increased CD69 expression, but to different degrees (table 1). The cell subsets analyzed were CD4+ T cells, CD4- T cells, NK-like (CD56+) T cells, NK cells and monocytes (fig. 1, fig. 2)
      Table 1CD69 expression was determined as a measure of cell activation after in vitro stimulation.
      4 h stimulation
      CD4+PCD4-PCD3+CD56+PNKPMonocytesP
      Unstim.165259361394473
      LPS1385**1873**3228**5193**1426**
      CWPS201*375**663**1204**711**
      T3170*289**410*562*582**
      T9194*356**600**940**638*
      T23194*368**637**1041**746**
      12 h stimulation
      CD4+PCD4-PCD3+CD56+PNKP
      Unstim.175312348284
      LPS1195**2505**3815**6788**
      CWPS205**495**783**1047**
      T3193NS329NS374NS377**
      T9201NS399**631*671**
      T23226**488**799**915**
      Whole blood from healthy non-pneumococcal vaccinated subjects were stimulated for 4 h or 12 h with pneumococcal C- polysaccharide (CWPS) and pneumococcal capsular polysaccharides type 3, type 9 and type 23. Unstimulated whole blood was used as negative control and stimulation with LPS as positive control. Subsets of CD3+CD4+ T cells (T-helper), CD3+CD4- T cells (“T-cytotoxic”), CD3+CD56+ (NK-like T cells), CD3-CD56+ (NK cells) and CD14+ cells (monocytes) were identified and analysed for CD69 expression using flow cytometry. Results are expressed as median values for healthy subjects of mean fluorescence intensity (MFI) values. P values refer to comparisons with unstimulated cells. *p < 0.05, **p < 0.01, ***p < 0.001, NS (non significant).
      Figure thumbnail gr1
      Figure 1Results from gating of whole blood of healthy controls, (a) gating on lymphocytes, (b) gating on NK cells, (c) gating on T cells, (d) gating on CD4+ T cells in the upper box and CD4- T cells in the lower box, (e) gating on NK-like T cells.
      Figure thumbnail gr2
      Figure 2Cell activation was assessed as expression of CD69 by flow cytometric analysis. The histograms show results after whole blood of healthy controls was stimulated by CWPS and three different pneumococcal capsular polysaccharides. The figure depicts CD69 expression on NK cells where (a) shows CWPS and negative control, (b) shows type 3, type 9 and type 23 capsules and negative control and (c) shows LPS and negative control. CWPS; pneumococcal cell wall polysaccharide, LPS; lipopolysaccharide. (Number of individuals included, N = 9).
      We analyzed CD69 expression after 4 h (fig. 3 part A) and after 12 h (fig. 3 part B). After stimulation with CWPS the highest CD69 relative MFI values were observed for NK cells followed by CD56+ T cells, monocytes, CD4- T cells and finally CD4+ T cells, which exhibited the lowest values. Overall, the same pattern (with NK cells having the highest relative CD69 MFI values) was also observed after stimulation with the capsules. In general, CWPS was a stronger stimulator than the capsules, although CWPS was included as a positive control and statistical comparisons were only carried out between the capsules.
      Figure thumbnail gr3a
      Figure 3(A and B) CD69 expression in cell subsets. CD69 expression was determined as a measure of cell activation after in vitro stimulation. Whole blood from healthy non-pneumococcal vaccinated subjects was stimulated for 4 h (Figure part A, N = 8) or 12 h (Figure part B, N = 9) with pneumococcal C-polysaccharide (CWPS) and pneumococcal capsular polysaccharides type 3, type 9 and type 23. Unstimulated whole blood was used as negative control and stimulation with LPS as positive control (data not shown). Graphs show CD69 expression in stimulated versus unstimulated cells, expressed as relative MFI, for the respective cell subsets indicated above each graph. Statistical comparisons were only carried out between the three capsular polysaccharides (type 3, type 9 and type 23).
      Figure thumbnail gr3b
      Figure 3(A and B) CD69 expression in cell subsets. CD69 expression was determined as a measure of cell activation after in vitro stimulation. Whole blood from healthy non-pneumococcal vaccinated subjects was stimulated for 4 h (Figure part A, N = 8) or 12 h (Figure part B, N = 9) with pneumococcal C-polysaccharide (CWPS) and pneumococcal capsular polysaccharides type 3, type 9 and type 23. Unstimulated whole blood was used as negative control and stimulation with LPS as positive control (data not shown). Graphs show CD69 expression in stimulated versus unstimulated cells, expressed as relative MFI, for the respective cell subsets indicated above each graph. Statistical comparisons were only carried out between the three capsular polysaccharides (type 3, type 9 and type 23).
      We compared the capacity of the three capsules to stimulate the above mentioned five cell subsets after 4 h, and four cell subsets after 12 h; these are the nine tests referred to below. Among the investigated capsules, type 23 was the strongest stimulator followed by type 9 and type 3 respectively. A statistically significant difference was detected between type 3 and type 23 in eight out of nine (8/9) tests and between type 3 and type 9 in seven of nine (7/9) tests. Between type 9 and type 23 a significant difference was shown only in three out of nine (3/9) tests. Results were consistent, with small variations, at 4 and 12 hours.

      3.2 Secreted cytokines

      In contrast to unstimulated cells, stimulation with CWPS, type 3, type 9 and type 23 capsules, as well as the additional positive control LPS, induced the release of cytokines IL-8, TNF, IL-10 and IFNγ (Figure 4), except for type 3 with regard to IFNγ and IL-10, and for type 9 with regard to IFNγ.
      Figure thumbnail gr4
      Figure 4Cytokine concentrations of IL-8, TNF, IL-10 and IFNγ in cell culture supernatants after 12 h in vitro stimulation of whole blood from healthy individuals. Cells were stimulated with three different capsular polysaccharides (type 3, type 9, type 23), pneumococcal C-polysaccharide (CWPS), LPS and media alone (unstimulated). Bars show mean values. Wilcoxon two sample test, *p < 0.05, **p < 0.01, ***p < 0.001, NS (non significant). Statistical comparisons were only carried out between the three capsular polysaccharides (type 3, type 9 and type 23). (Number of individuals included, N = 9).
      After CWPS stimulation the highest cytokine concentration was observed for IL-8, followed by IL-10, TNF and INFγ. The capsules type 9 and type 23 were shown to have a somewhat different relative cytokine pattern. Both induced IL-8 to the highest concentration, followed by TNF, IL-10 and IFNγ. Type 3 capsule exhibited a similar pattern but yielded a higher value for IL-10 than TNF.
      Generally, capsules type 9 and type 23 stimulated to a much larger cytokine release than capsule type 3. Release of IL-8, TNF and IL-10 differed significantly between type 3 and type 23 capsules and between type 3 and type 9 capsules respectively. No such significant difference was noted between capsules type 9 and type 23. For IFNγ, no significant differences between the capsules were observed.

      4. Discussion

      In this study, we investigated the effects of in vitro stimulation of whole blood from healthy individuals with pneumococcal C-polysaccharide (CWPS) and three different capsular types of Streptococcus pneumoniae, all of which are included in the 23-valent polysaccharide vaccine. We analyzed the degree of activation (CD69 expression) of monocytes, T-cell subsets and NK cells as well as cytokine secretion (TNF, IL-8, IL-10 and IFNγ). Our results show that CWPS as well as all included individual capsules (serotypes 3, 9 and 23) induced activation of monocytes, T cells and NK cells with few exceptions. CWPS provided the strongest activation, compared to the examined capsules, both for monocytes, T-cells and NK cells. In general (both for CWPS and the capsules), NK cells were activated to the highest degree, followed by CD56+ T cells and monocytes. The lowest grade of activation was evoked in CD4- T cells and CD4+ T cells. Among the capsules type 23 activated the immune cells to the highest degree, followed by type 9 and type 3 capsules, and the same order of potency was observed with regard to cytokine release.
      Since both CWPS and the capsular polysaccharides were able to stimulate a variety of immune cells, the question arises about which mechanisms could be mediating this. CWPS is also known as teichoic acid
      • Fischer W.
      • Behr T.
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      • Peter-Katalinic J.
      • Egge H.
      Teichoic acid and lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures. A reinvestigation of teichoid acid (C polysaccharide).
      , structurally similar (especially in S. pneumoniae) to lipoteichoic acid and a well-known ligand for TLR2
      • Sen G.
      • Khan A.Q.
      • Chen Q.
      • Snapper C.M.
      In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands.
      . Toll-like receptors (TLRs) are part of the innate immune system and recognize conserved molecular patterns on various pathogens. LPS (lipopolysaccharide) is an example and a ligand for TLR4, and was used in this study as an additional positive control. CWPS also has the property of being a so-called zwitter-ionic polysaccharide (ZPS), i.e. it has a pattern of positive and negative charges in repeating units, enabling it to directly stimulate T cells.
      • Malley R.
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      • Watkins C.
      • Tzianabos A.
      • et al.
      Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide.
      ZPS also act as ligands for TLR2, which can explain the indirect effect on T cells via action on antigen-presenting cells that has been shown
      • Wack A.
      • Gallorini S.
      Bacterial polysaccharides with zwitterionic charge motifs: Toll-like receptor 2 agonists, T cell antigens, or both?.
      . CWPS has several identical subunits which cross-links the B cell receptor and elicits B cell proliferation.
      Capsular polysaccharides are T cell independent B cell antigens, and their capacity to induce protective antibody responses are the reason they are used for vaccination. It is less clear how the capsular polysaccharides may stimulate the immune cells investigated in this study, since they have not been shown to be ligands for TLRs
      • Sen G.
      • Khan A.Q.
      • Chen Q.
      • Snapper C.M.
      In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands.
      • Snapper C.M.
      Differential regulation of protein- and polysaccharide-specific Ig isotype production in vivo in response to intact Streptococcus pneumoniae.
      . Only one of them, serotype 1 (not included in this study), is a ZPS, and was shown to act both as a TLR2 ligand and a direct T cell stimulator via a mechanism involving MHC class II molecules on antigen-presenting cells
      • Wack A.
      • Gallorini S.
      Bacterial polysaccharides with zwitterionic charge motifs: Toll-like receptor 2 agonists, T cell antigens, or both?.
      • Velez C.D.
      • Lewis C.J.
      • Kasper D.L.
      • Cobb B.A.
      Type I Streptococcus pneumoniae carbohydrate utilizes a nitric oxide and MHC II-dependent pathway for antigen presentation.
      . From studies in mice, it has been shown that the polysaccharide vaccine contains both TLR2 and TLR4 ligands.
      • Sen G.
      • Khan A.Q.
      • Chen Q.
      • Snapper C.M.
      In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands.
      . It is known that even highly purified pneumococcal capsular polysaccharides contain some amount of CWPS
      • Xu Q.
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      • Ng A.S.
      • Sturgess A.W.
      • Harmon B.J.
      • Hennessey Jr., J.P.
      Characterization and quantification of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharide preparations.
      . Thus it seems reasonable that CWPS may be an important component involved in the activation and cytokine secretion observed here. The amount of bound CWPS may vary depending on the serotype. The CWPS also occurs in two forms, containing one or two phosphorylcholine residues in the repeating unit.
      • Karlsson C.
      • Jansson P.E.
      • Skov Sorensen U.B.
      The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.
      Direct TLR stimulation can only be expected to account for the activation of some of the cells studied, however. Monocytes express TLRs, and so do NK cells
      • Adib-Conquy M.
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      • Cavaillon J.M.
      • Souza-Fonseca-Guimaraes F.
      TLR-mediated activation of NK cells and their role in bacterial/viral immune responses in mammals.
      , as well as NK-like T cells
      • Saikh K.U.
      • Lee J.S.
      • Kissner T.L.
      • Dyas B.
      • Ulrich R.G.
      Toll-like receptor and cytokine expression patterns of CD56+ T cells are similar to natural killer cells in response to infection with Venezuelan equine encephalitis virus replicons.
      , but not “ordinary” (CD56-) CD4- T cells or CD4+ T cells.
      An indirect activation e.g. by cytokines from other directly activated cells may instead explain the stimulation of T cells not expressing TLRs. During vaccination or a live S. pneumoniae infection TLR-expressing cells such as dendritic cells and macrophages present in the tissues but not in peripheral blood are likely to be activated in a way mirroring that of monocytes in the present study. Most likely, a combination of direct and indirect (via cell-cell contact or secreted mediators) activation of immune cells occurs.
      Today two different types of pneumococcal vaccines are available. One contains 23 different pneumococcal capsular polysaccharides. The capsules crosslink the B cell receptors, triggering antibody production. Antibodies to the capsules are highly protective against lethal pneumococcal infections. The other vaccine type, conjugated vaccine, contains 7 respectively 13 different capsular polysaccharides linked to a protein. The protein is broken down to peptides which are presented by the B cell to T cells, thereby inducing T cell help to generate plasma cell differentiation and memory cell generation
      • Pollard A.J.
      • Perrett K.P.
      • Beverley P.C.
      Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines.
      . The conjugated vaccine, in contrast to the capsular–only containing vaccine, induces different immunoglobulin IgG subclass patterns as a result of the influences from T cell cytokines
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      Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM(197) conjugate vaccines in a murine model.
      . Intriguingly, however, immunoglobulin class switching from IgM to IgG also occurs in response to the unconjugated polysaccharide vaccine. This is not expected from a T cell independent (type 2 thymus-independent) antigen. Therefore we speculate that the different potency of capsular polysaccharides to stimulate potentially class switch promoting T cells as well as non-T cells such as NK cells and monocytes affects the outcome of vaccination with the conjugated as well as the unconjugated vaccines. Among T cells, in particular the NK-like T cells (CD56+) were stimulated by the capsules or CWPS. Furthermore, the capacity to induce IL-10 which could negatively affect the impact of vaccination differs between the capsules. Thus, the composition of the mixture of capsules used in the conjugate vaccine may affect the levels of protective antibodies generated by vaccination. Failure to develop significant increases in antibody concentration against specific serotypes after vaccination has been linked to pneumococcal pneumonia
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      . Interestingly, a previous study found that the T cell cytokine profile and IgG subclass response to conjugate vaccines was dependent on the serotype of S. pneumoniae
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      Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM(197) conjugate vaccines in a murine model.
      .
      Although antibody-producing B cells provide the main protection induced by vaccination, other immune cells may contribute to the defense against S. pneumoniae either directly or indirectly by promoting B cell responses.
      Studies have shown a role for CD8+ T cells to promote protective antibody responses against pneumococci
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      . Mice lacking CD8 cannot produce pneumococcal polysaccharide antibodies
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      Cutting edge: antibody production to pneumococcal polysaccharides requires CD1 molecules and CD8+ T cells.
      . The activation by CWPS and the capsules of the CD4- T cell subset indicates that these agents activate CD8+ T cells, since almost 90% of CD4- T cells were CD8+.
      NK cells were strongly activated both by CWPS and by the capsules. Several studies have shown that NK cells stimulate B cells to antibody secretion and isotype switch and this has also been observed after pneumococcal vaccination
      • Snapper C.M.
      Differential regulation of protein- and polysaccharide-specific Ig isotype production in vivo in response to intact Streptococcus pneumoniae.
      • Gray J.D.
      • Horwitz D.A.
      Activated human NK cells can stimulate resting B cells to secrete immunoglobulin.
      • Yuan D.
      • Bibi R.
      • Dang T.
      The role of adjuvant on the regulatory effects of NK cells on B cell responses as revealed by a new model of NK cell deficiency.
      . In the case of NK cell deficiency, B cells cannot switch serotype production
      • Yuan D.
      • Bibi R.
      • Dang T.
      The role of adjuvant on the regulatory effects of NK cells on B cell responses as revealed by a new model of NK cell deficiency.
      .
      The cytokines analyzed in this study were chosen to give a broad overview of pro- and anti-inflammatory mediators. We found that the capsules differed sharply in their capacity to induce cytokine secretion from blood cells. It is likely that such differences between capsules apply also to other cytokines. In the in vitro stimulation in our experiments using whole blood, TNF was probably produced by a variety of cell types such as monocytes, neutrophils, CD4 + T cells and NK cells. TNF has many pro-inflammatory effects and is involved in acute phase reactions. INFγ can be produced by activated T cells and NK cells and activates macrophages to increased phagocytosis and cytokine synthesis. IL-8 can be produced by monocytes and attracts neutrophils. IL-10 is produced by e.g. T cells and monocytes. It is an anti-inflammatory cytokine, which inhibits e.g. T cell responses. In vivo other cells such as macrophages (derived from monocytes) may also be triggered by pneumococcal components to cytokine secretion.
      Generally, the efficacy of vaccine responses is evaluated by measurement of the total IgG concentration in serum. In the present study, we have performed a more detailed analysis on various capsular influences on different immune cells and cytokines. Immune cell subsets were activated by CWPS and the investigated capsules, but to different degrees, with NK cells and NK-like T cells, followed by monocytes, showing the strongest signs of activation. Among the three capsules, capsule type 23 induced the strongest activation, followed by type 9 and type 3. This may be due to the different capsuleś inherent different abilities to activate the immune cells. The cytokine release followed the same pattern of relative potency among the capsules. Since the three capsules were randomly chosen, we believe such differences in immunostimulatory capacity would be observed also for other capsules.

      5. Conclusion

      By differentiating the various immune responses to different vaccine components, the results presented in this study increase the understanding of how human immune cells react upon pneumococcal vaccination and upon exposure to pneumococci. The results shows that monocytes, NK cells, NK-like T cells, CD4+ T cells and CD4- T cells are activated by exposure to capsular polysaccharides, but to different degrees and that there are variations in stimulatory capacity of vaccine capsular components. These cell types may contribute to the immune defence against pneumococcal infection by helping B cells produce antibodies, and by inducing immunoglobulin class switching, a puzzling phenomenon in the context of T-cell independent antigens such as capsular polysaccharides, but which our data may help explain. These results may also help explain variations in efficacy of vaccine capsular components (serotype-specific responses) and contribute to future vaccine development against pneumococcal and other diseases.

      Acknowledgements

      This study was supported by The Stockholm County Council, The Swedish Research Council, The Swedish Heart Lung Foundation, The Mats Kleberg Foundation and Karolinska Institutet. We thank Heléne Blomqvist, Margitha Dahl and Gunnel de Forest for obtaining blood samples and Lotta Müller-Suur for skillful technical assistance regarding flow cytometry, all at the Department of Medicine, Respiratory Medicine Unit, Karolinska Institutet. We also thank Per Näsman for assistance in statistics evaluation.
      The study was approved by the Regional Ethical Review Board, Stockholm Sweden.

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