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. 2019 Jun 27;8(3):90.
doi: 10.3390/pathogens8030090.

Impact of Superantigen-Producing Bacteria on T Cells from Tonsillar Hyperplasia

Fiona J Radcliff  1 , Sharon Waldvogel-Thurlow  2 , Fiona Clow  3 , Murali Mahadevan  2 , James Johnston  2 , Gen Li  3 , Thomas Proft  3 , Richard G Douglas  2 , John D Fraser  3
Affiliations

Impact of Superantigen-Producing Bacteria on T Cells from Tonsillar Hyperplasia

Fiona J Radcliff et al. Pathogens. .

Abstract

Staphylococcus aureus and Group A Streptococcus (GAS) are common occupants of the tonsils and many strains produce potent exotoxins (mitogens) that directly target T cells, which could be a driver for tonsillar hyperplasia. Tonsil tissues from 41 patients were tested for these bacteria in conjunction with profiling of B and T cells by flow cytometry. S. aureus and GAS were detected in tonsil tissue from 44% and 7%, respectively, of patients by bacteriological culture; immuno-histology showed bacteria in close proximity to both B and T lymphocytes. The presence of tonsillar S. aureus did not alter B or T cell populations, whereas peripheral blood mucosal-associated invariant T (MAIT) cells were significantly increased in S. aureus culture positive individuals (p < 0.006). Alterations of tonsil CD4+ TCR Vβ family members relative to peripheral blood were evident in 29 patients. Three patients had strong TCR Vβ skewing indicative of recent exposure to superantigens, their tonsils contained mitogenic bacteria, and supernatants from these bacteria were used to partially recapitulate the skewing profile in vitro, supporting the notion that superantigens can target tonsillar T cells in situ. Tonsils are a reservoir for superantigen-producing bacteria with the capacity to alter the composition and function of key immune cells.

Keywords: Group A Streptococcus; Staphylococcus aureus; Streptococcus pyogenes; TCR Vβ; mucosal-associated invariant T cells; obstructive sleep apnea; recurrent tonsillitis; superantigen; tonsillar hyperplasia.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
S. aureus and Group A Streptococcus (GAS) are located within tonsillar tissue. Bacteria were detected in paraffin-embedded tonsil tissue with antibodies specific for S. aureus or GAS (green), combined with a nuclear stain (DAPI, blue). Examples include S. aureus sparsely distributed in tonsillar tissue at 100× (A), clusters of bacteria at 100× (B) and at 10× (C); for GAS, occasional cocci are shown (D,E) and a large cluster of bacteria (F), all at 100× magnification.
Figure 2
Figure 2
Presence of sag genes and mitogenicity of tonsil S. aureus isolates. A representative S. aureus isolate from each culture positive patient was tested for the presence of genes encoding for common Staphylococcal superantigens by multiplex PCR. Mitogenicity of supernatants from each of these isolates was determined using peripheral blood leucocytes (PBL) from three healthy donors and assigned a grade, with +++ being highly mitogenic. S. aureus from 5 recurrent tonsillitis (RT) and 13 hyperplasia patients were profiled; each line is a representative isolate from an individual patient.
Figure 3
Figure 3
S. aureus and GAS are located in close proximity to B and T cells in the tonsils. Clusters of S. aureus (A) or GAS (B) detected by immuno-labelling and confocal microscopy in paraffin-embedded tonsillar tissue. Green = S. aureus or GAS; blue = DAPI; yellow = CD3+ T cells; red = CD20+ B cells. 100× magnification.
Figure 4
Figure 4
Characterization of mucosal-associated invariant T (MAIT) cells and the effect of S. aureus in the tonsils. A comparison of CD3+ MAIT and non-MAIT cells in blood and tonsil tissue in patients identified as culture positive (filled circle) or negative (open circle) for S. aureus. MAIT cells were identified as CD3+ CD161++Vα7.2+ (A) and the remaining CD3+ population classified as non-MAIT cells. These cell populations were quantified in blood or tonsil tissue (B) and then assessed for CD4 and CD8 (C), CD69 (D), TCR Vβ2 (E), and TCR Vβ13.1 (F). Values are expressed as a percentage of CD3+ cells. Each point is a value from an individual patient and the horizontal line is the median. The two patient groups were compared with a two-tailed Mann–Whitney test or a Kruskal–Wallis test with Dunn’s multiple comparison test applied.
Figure 5
Figure 5
Tonsil TCR Vβ profiles normalized to peripheral blood for all patients in the study. Tonsil TCR Vβ profiles were normalized to peripheral blood profiles and data from all 41 patients combined to highlight any consistent differences for both CD4+ (A) and CD8+ (B) T cells; family members with a mean value of ≥1.2 are highlighted in red, those with a mean value of ≤0.8 are highlighted in blue. Data are mean + standard deviation.
Figure 6
Figure 6
Comparison of TCR Vβ profiles from tonsils and healthy PBL stimulated with bacterial supernatants. Individual profiles showing strong localized TCR Vβ skewing in the tonsil (LHS) aligned with TCR Vβ profiles of healthy PBL after stimulation with culture supernatant from S. aureus or GAS isolated from these individuals (RHS); red bars identify TCR Vβ family members expanded in both sets of cells. Results are from three patients who underwent surgery to treat RT who were positive for S. aureus (A), GAS emm28 (B), or GAS emm89 (C) respectively. Tonsil data are normalized to peripheral blood profiles from the same individual. PBL data are shown as fold change relative to unstimulated PBL, incorporating mean values plus standard deviation from two healthy donors.
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