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

Abstract

"Strep throat" is highly prevalent among children, yet it is unknown why only some children develop recurrent tonsillitis (RT), a common indication for tonsillectomy. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric group A Streptococcus (GAS) RT and non-RT tonsils from two independent cohorts. GAS RT tonsils had smaller germinal centers, with an underrepresentation of GAS-specific CD4⁺ germinal center T follicular helper (GC-T_FH) cells. RT children exhibited reduced antibody responses to an important GAS virulence factor, streptococcal pyrogenic exotoxin A (SpeA). Risk and protective human leukocyte antigen (HLA) class II alleles for RT were identified. Lastly, SpeA induced granzyme B production in GC-T_FH cells from RT tonsils with the capacity to kill B cells and the potential to hobble the germinal center response. These observations suggest that RT is a multifactorial disease and that contributors to RT susceptibility include HLA class II differences, aberrant SpeA-activated GC-T_FH cells, and lower SpeA antibody titers.

Fig. 1.. RT children have fewer GC-T_FH 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-T_FH (CXCR5^hiPD-1^hiCD45RO⁺CD4⁺), mT_FH (CXCR5⁺PD-1⁺CD45RO⁺CD4⁺), and non-T_FH (CXCR5⁻CD45RO⁺CD4⁺) cells. (C) GC-T_FH cell frequencies in RT tonsils (n = 26) and non-RT tonsils (n = 39), quantified as percentage of total CD4⁺ T cells. (D) GC-T_FH cells by age. (E) Flow cytometry of B_GC cells (CD38⁺CD20⁺CD19⁺), plasma cells (PC; CD38^hiCD20⁺CD19⁺), and memory (CD27^hiCD20⁺CD19⁺)/naive (CD27⁻CD20⁺CD19⁺) B cells. (F) B_GC cell frequencies in RT and non-RT tonsils, quantified as percentage of total B cells. (G) B_GC 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 B_GC cells (Ki67) and GC-T_FH 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-T_FH 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-T_FH cell frequencies in RT tonsils (n = 40) and non-RT tonsils (n = 41), quantified as percentage of total CD4⁺ T cells. (D) GC-T_FH cells by age. (E) B_GC cell frequencies in RT and non-RT tonsils, quantified as percentage of total B cells. (F) B_GC 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-T_FH cells.

(A) Identification of GAS-specific CD4⁺ T cells (CD45RA⁻) and GAS-specific GC-T_FH cells (CD45RA⁻CXCR5^hiPD-1^hi) 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-T_FH cell frequencies. (C) GAS-specific CD45RA⁻CD4⁺ 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-T_FH cells (CXCR5^hiPD-1^hi) 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-T_FH and B_GC cells [green dots, n = 15 (GC^lo samples); blue dots, n = 46 (GC^lo 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-T_FH 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-T_FH 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-T_FH cells from RT tonsils induces GzmB.

(A) Volcano plot showing fold change of genes in SpeA-stimulated GC-T_FH cells from RT tonsils (n = 5) compared to SpeA-stimulated GC-T_FH 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-T_FH cells by flow cytometry. Tonsil cells were stimulated with SpeA (1 μg/ml) for 24 hours (top). Backgating of the GzmB⁺ GC-T_FH cells among total CD45RA⁻ CD4⁺ T cells (bottom). (C) GzmB⁺ GC-T_FH cells in RT tonsils (n = 20) and non-RT tonsils (n = 17) after SpeA stimulation. (D) Fluorescence-activated cell-sorted GC-T_FH 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-T_FH cells after SpeA stimulation. GC-T_FH cells were gated as CXCR5^hiPD-1^hi of live CD45RA⁻CD4⁺ T cells. n = 1 donor. (F) ImageStream imaging of GC-T_FH 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-T_FH cells (CXCR5^hiPD-1^hiCD45RA⁻CD4⁺) 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-T_FH cells from healthy LNs and from RT and non-RT tonsils. GzmB expression after SpeA stimulation of GC-T_FH 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).