Research ArticlePEDIATRICS

Recurrent group A Streptococcus tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant TFH cells

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Science Translational Medicine  06 Feb 2019:
Vol. 11, Issue 478, eaau3776
DOI: 10.1126/scitranslmed.aau3776
  • Fig. 1 RT children have fewer GC-TFH cells in their tonsils.

    Immunophenotyping analysis of cohort 1 of patients with and without RT. (A) Number of RT episodes in RT children (n = 23) and non-RT children (n = 11). (B) Flow cytometry of GC-TFH (CXCR5hiPD-1hiCD45RO+CD4+), mTFH (CXCR5+PD-1+CD45RO+CD4+), and non-TFH (CXCR5CD45RO+CD4+) cells. (C) GC-TFH cell frequencies in RT tonsils (n = 26) and non-RT tonsils (n = 39), quantified as percentage of total CD4+ T cells. (D) GC-TFH cells by age. (E) Flow cytometry of BGC cells (CD38+CD20+CD19+), plasma cells (PC; CD38hiCD20+CD19+), and memory (CD27hiCD20+CD19+)/naïve (CD27CD20+CD19+) B cells. (F) BGC cell frequencies in RT and non-RT tonsils, quantified as percentage of total B cells. (G) BGC cells by age. (H) Representative Ki67-stained sections from RT and non-RT tonsils. m.(I) Quantitation of GC areas (in μm2) in RT tonsils (n = 21) and non-RT tonsils (n = 16). Each data point represents an individual GC. (J) Staining of BGC cells (Ki67) and GC-TFH cells [programmed cell death protein 1 (PD-1)]. Insets: Enlarged versions of representative GCs stained for Ki67 or PD-1. II and III show PD-1+ GC-TFH cells in representative GCs from a non-RT tonsil and an RT tonsil, respectively. ****P < 0.0001, ***P < 0.001. Statistical significance was determined by Mann-Whitney tests (A to C, E, F, and I) and multivariate analysis of covariance (ANCOVA; D and G). DZ, dark zone; LZ, light zone.

  • Fig. 2 RT children have lower circulating anti-SpeA IgG titers.

    Serological and immunophenotyping analysis of cohort 2 of patients with and without RT. (A) Plasma anti-SLO IgG titers in RT children (n = 23), non-RT children (n = 16), and normal healthy adults (n = 14). LOD, limit of detection; RU, relative units. (B) Plasma anti-SpeA IgG titers in RT children (n = 42), non-RT children (n = 45), and normal healthy adults (n = 17). (C) GC-TFH cell frequencies in RT tonsils (n = 40) and non-RT tonsils (n = 41), quantified as percentage of total CD4+ T cells. (D) GC-TFH cells by age. (E) BGC cell frequencies in RT and non-RT tonsils, quantified as percentage of total B cells. (F) BGC cells by age. *P < 0.05, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by Mann-Whitney test.

  • Fig. 3 RT tonsils have reduced GAS-specific GC-TFH cells.

    (A) Identification of GAS-specific CD4+ T cells (CD45RA) and GAS-specific GC-TFH cells (CD45RACXCR5hiPD-1hi) using OX40+CD25+ AIM (AIM25). Tonsil cells were left unstimulated or stimulated with antibiotic-killed L. lactis (a nonpathogenic Gram-positive bacteria that served as a negative control; 10 μg/ml), heat-inactivated, antibiotic-killed GAS (10 μg/ml), or staphylococcal enterotoxin B [SEB (positive control); 1 μg/ml] for 18 hours. (B) GAS-specific GC-TFH cell frequencies. (C) GAS-specific CD45RACD4+ T cell frequencies, quantified as percentage of total CD4+ T cells, in RT tonsils (n = 31) and non-RT tonsils (n = 35). (D) Fraction of GAS-specific GC-TFH cells (CXCR5hiPD-1hi) among total GAS-specific CD4+ T cell (AIM25+ CD45RA) in RT tonsils (n = 31) and non-RT tonsils (n = 35). ****P < 0.0001, *P < 0.05. Statistical significance was determined by paired t test (B) and Mann-Whitney tests (C and D).

  • Fig. 4 HLA class II associations identified in RT children.

    (A) Family history of tonsillectomy (RT = 71 and non-RT = 63). (B) HLA DQB1*06:02 allelic frequency in non-RT children (gray bar, n = 192), RT children (white bar, n = 138), ethnically matched adults from the SD GP (black bar, n = 242), and non-RT children and GP (blue bar, n = 434). NS, not significant. (C) Left: HLA DRB1*01:01 and HLA DRB1*07:01 allelic frequencies in RT tonsils with the lowest quartile of GC activity, defined as the lowest combined frequencies of GC-TFH and BGC cells [green dots, n = 15 (GClo samples); blue dots, n = 46 (GClo samples); red dot, mean of non-RT GC activity]. Right: HLA DRB1*01:01 and HLA DRB1*07:01 allelic frequencies in non-RT tonsils (gray bar, n = 190), GP (black bar, n = 246), and GP and non-RT tonsils (blue bar, n = 436). RT children HLA allele counts (white bar, n = 30). ***P < 0.001, *P < 0.05. Statistical significance was determined by Fisher’s exact test (A to C).

  • Fig. 5 HLA class II associations identified in RT and non-RT children segregate on the basis of preferential GAS superantigen SpeA binding.

    (A) Comparison of AIM+ GC-TFH cells after stimulation with either antibiotic-killed (AK) GAS (10 μg/ml), antibiotic-killed, heat-inactivated (AK HI) GAS (10 μg/ml), or antibiotic-killed SpeA-deficient (AK ΔSpeA) GAS (10 μg/ml); n = 10, including RT and non-RT donors. Heat-inactivation of antibiotic-killed GAS inactivates GAS superantigens. (B) SpeA-responsive GC-TFH cells in tonsils from patients with RT with risk HLA alleles (n = 12) compared to non-RT patients with protective HLA alleles (n = 12). Tonsils were stimulated with SpeA (1 μg/ml) for 18 hours and background-subtracted as determined with unstimulated cells. (C) Histogram flow cytometric quantitation of SpeA binding by HLA allele using HLA cell lines. n = 3 experiments. (D) Proliferation of total CD4+ T cells from peripheral blood mononuclear cells (PBMCs) of HLA DQB1*06:02+ donors cocultured with recombinant SpeA (rSpeA) and a cell line expressing HLA DQB1*06:02 and of CD4+ T cells from PBMCs of HLA DRB1*07:01+ donors cocultured with rSpeA and a cell line expressing HLA DRB1*07:01. n = 4 experiments. **P < 0.01, *P < 0.05 (D). Statistical significance was determined by Mann-Whitney test. CTV, cell trace violet.

  • Fig. 6 SpeA stimulation of GC-TFH cells from RT tonsils induces GzmB.

    (A) Volcano plot showing fold change of genes in SpeA-stimulated GC-TFH cells from RT tonsils (n = 5) compared to SpeA-stimulated GC-TFH cells from non-RT tonsils (n = 5). Red dots denote genes with a fold change of <0.5 or >2. (B) Frequency of intracellular GzmB expression in GC-TFH cells by flow cytometry. Tonsil cells were stimulated with SpeA (1 μg/ml) for 24 hours (top). Backgating of the GzmB+ GC-TFH cells among total CD45RA CD4+ T cells (bottom). (C) GzmB+ GC-TFH cells in RT tonsils (n = 20) and non-RT tonsils (n = 17) after SpeA stimulation. (D) Fluorescence-activated cell–sorted GC-TFH cells and autologous B cells were cultured ± SpeA for 5 days and stained for GzmB and perforin expression. n = 3 donors. (E) ImageStream cytometry plot of GzmB+ GC-TFH cells after SpeA stimulation. GC-TFH cells were gated as CXCR5hiPD-1hi of live CD45RACD4+ T cells. n = 1 donor. (F) ImageStream imaging of GC-TFH cells after SpeA stimulation, showing representative GzmB and GzmB+ cells. (G) Confocal microscopy of a GzmB+ CD4+ T cell in a GC in an RT tonsil (*). A GzmB+ CD8+ T cell is also shown for reference (<). m.n = 8 donors. (H) GC-TFH cells (CXCR5hiPD-1hiCD45RACD4+) were cocultured with autologous CTV-labeled B cells (CD19+CD38). Killing was quantified as outlined in Materials and Methods, with controls shown in fig. S7 (J to L). n = 15 and 11 (RT and non-RT donors, respectively). (I) GzmB expression (percentage) by GC-TFH cells from healthy LNs and from RT and non-RT tonsils. GzmB expression after SpeA stimulation of GC-TFH cells from RT tonsils (n = 11), non-RT tonsils (n = 11), or healthy LNs (n = 4). **P < 0.01, *P < 0.05. Statistical significance was determined by Mann-Whitney test (C, H, and I).

  • Table 1 Study participant demographics for cohort 1.
    RT (n = 26)Non-RT (n = 39)P value
    Gender (%)0.21*
    Female65.4%48.7%
    Male34.6%51.2%
    Age (mean years)118.310.0092

    *P value determined by Fisher’s exact test using R.

    P value determined by Mann-Whitney U test.

    • Table 2 Study participant demographics for cohort 2.

      RT (n = 40)Non-RT
      (n = 41)
      P value
      Gender (%)0.0058*
      Female77.5%46.3%
      Male22.5%53.7%
      Age (mean years)9.658.390.21

      *P value determined by Fisher’s exact test using R.

      P value determined by Mann-Whitney U test.

      • Table 3 Study participant demographics for entire combined cohort.

        RT (n = 66)Non-RT
        (n = 80)
        P value
        Gender (%)0.0055*
        Female72.7%47.5%
        Male27.3%52.5%
        Age (mean years)10.188.350.0024

        *P value determined by Fisher’s exact test using R.

        P value determined by Mann-Whitney U test.

        Supplementary Materials

        • www.sciencetranslationalmedicine.org/cgi/content/full/11/478/eaau3776/DC1

          Materials and Methods

          Fig. S1. RT and non-RT tonsillar immunophenotyping of cohort 1.

          Fig. S2. RT and non-RT tonsillar immunophenotyping of cohort 2.

          Fig. S3. GAS-specific CD4+ T cells by AIM assay.

          Fig. S4. HLA typing of entire tonsillar cohort.

          Fig. S5. SpeA-responsive GC-TFH cells.

          Fig. S6. SpeA-responsive GC-TFH cells by AIM assay.

          Fig. S7. SpeA induced GzmB production.

          Fig. S8. SpeA-responsive GC-TFH cells by AIM assay.

          Table S1. RNA-seq analysis.

          Table S2. Flow cytometry antibodies for fresh tonsil stain.

          Table S3. Flow cytometry antibodies for AIM assay.

          Table S4. Flow cytometry antibodies for PBMC proliferation assay.

          Table S5. Flow cytometry antibodies for GzmB detection.

          Table S6. Flow cytometry antibodies used for sorting GC-TFH and non-BGC cells for GzmB expression after 5-day in vitro culture.

          Table S7. Flow cytometry antibodies for GzmB detection from sorted GC-TFH cells.

          Table S8. Flow cytometry antibodies used for sorting for cytotoxicity assay.

          Data file S1. Primary data.

          References (5970)

        • The PDF file includes:

          • Materials and Methods
          • Fig. S1. RT and non-RT tonsillar immunophenotyping of cohort 1.
          • Fig. S2. RT and non-RT tonsillar immunophenotyping of cohort 2.
          • Fig. S3. GAS-specific CD4+ T cells by AIM assay.
          • Fig. S4. HLA typing of entire tonsillar cohort.
          • Fig. S5. SpeA-responsive GC-TFH cells.
          • Fig. S6. SpeA-responsive GC-TFH cells by AIM assay.
          • Fig. S7. SpeA induced GzmB production.
          • Fig. S8. SpeA-responsive GC-TFH cells by AIM assay.
          • Table S1. RNA-seq analysis.
          • Table S2. Flow cytometry antibodies for fresh tonsil stain.
          • Table S3. Flow cytometry antibodies for AIM assay.
          • Table S4. Flow cytometry antibodies for PBMC proliferation assay.
          • Table S5. Flow cytometry antibodies for GzmB detection.
          • Table S6. Flow cytometry antibodies used for sorting GC-TFH and non-BGC cells for GzmB expression after 5-day in vitro culture.
          • Table S7. Flow cytometry antibodies for GzmB detection from sorted GC-TFH cells.
          • Table S8. Flow cytometry antibodies used for sorting for cytotoxicity assay.
          • References (5970)

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          Other Supplementary Material for this manuscript includes the following:

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