Detection of live bacteria promotes T FH cell differentiation

In order to assess the contribution of innate immune signals to the differentiation of human T FH cells, we co-cultured human CD14+CD16– monocytes (as APCs) with autologous naive CD4+ T cells isolated by negative selection of CD45RO+CD4+ T cells. APCs were stimulated with either live avirulent thymidine-auxotrophic (thyA–; replication-defective) Escherichia coli (called ‘EC’ here)7 or a heat-killed version of the same E. coli (HKEC). We chose avirulent auxotrophic bacteria to selectively analyze the effect of bacterial viability without the confounding effects of virulence factors and bacterial replication7. Following 90 min of stimulation of human CD14+CD16– monocytes with EC or HKEC, naive CD4+ T cells were added, together with antibiotics, and helper T cell differentiation was assessed 5 d later. Notably, EC-stimulated APCs induced naive CD4+ T cells to produce large amounts of IL-21 and interferon-γ (IFN-γ), which are characteristic cytokines of T FH cells and the T H 1 subset of helper T cells, respectively (Fig. 1a,b). This response was almost completely absent in cultures with APCs stimulated with HKEC or medium alone (Fig. 1a,b). T cell proliferation rates were similar in all conditions, and IL-17 was produced in moderate amounts regardless of bacterial viability (Fig. 1a,b). In line with the increased IL-21 production, stimulation with EC promoted the expression of the T FH cell markers CXCR5, ICOS and PD-1, but stimulation with HKEC did not15,16,17 (Fig. 1c,d and Supplementary Fig. 1a).

Fig. 1: The recognition of live bacteria by the innate immune system promotes T FH cell differentiation. a, Flow-cytometry analysis of the IL-21 expression and proliferation of autologous naive CD4+ T cells cultured for 5 d, in the presence of staphylococcal enterotoxin B (T cell antigen receptor stimulus in all T cell conditions), with human monocytes (as APCs) previously stimulated with medium (Ctrl), live EC or HKEC (above plots); proliferation was assessed as dilution of the division-tracking dye CFSE. b, Frequency of cytokine-positive CD4+ T cells in co-cultures as in a (key). c,d, Flow-cytometry analysis of the expression of CXCR5 (c), ICOS and PD-1 (Supplementary Fig. 1a) by T cells cultured as in a (above plots), and frequency of T cells positive for all three in such cultures (key) (d). e,f, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by cells cultured as in a (above plots) (e), and frequency of IL-21+BCL-6+ cells in such cultures (key) (f). g, Fluorescent hybridization-based multiplex assay measuring the expression of genes encoding IL-21 (IL21) and the transcription factors BCL-6 (BCL6), Maf (MAF), T-bet (TBX21), GATA-3 (GATA3) and RORγt (RORC) in CD4+ T cells on day 1, 3 or 5 (key) of culture as in a (horizontal axis) (n = 6 donors), presented as corrected fluorescence intensity (FI), calculated by subtraction of the fluorescence intensity of the control sample at the same time point. h,i, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by cells cultured as in a (above plots) but with CD1c+ myeloid DCs as APCs (h), and frequency of IL-21+BCL-6+ cells in such cultures (key) (i). j–l, Flow-cytometry analysis of the expression of CD27 and CD38 by tonsillar memory B cells cultured for 7 d alone (left) or with CD4+CD45RA–CXCR5+ T FH cells from day 5 of culture with EC-stimulated APCs as in a (left) or autologous naive CD4+CD45RA+ T cells from same donor (middle) (j), frequency of CD38+CD27hi plasma cells in cultures as in j (key) (k) and ELISA of IgG in supernatants of cultures as in j (key) (l). Numbers adjacent to or in outlined areas (a,c,e,h,j) indicate percent cells in each throughout. Each symbol (b,d,f,i,k,l) represents an individual donor (n = 9 (IL-21), 9 (IFN-γ) or 7 (IL-17) (b); n = 13 (d); n = 9 (f); n = 3 (i); or n = 9 (CXCR5+ T cells), 9 (Naive T cells) or 3 (No T cells) (k,l)); error bars indicate maximum and minimum (g,l). NS, not significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (one-way analysis of variance (ANOVA) with post-hoc correction for multiple comparisons). Data are representative of one experiment per donor. Full size image

The transcription factor BCL-6 is required for successful development of T FH cells11,12. APCs stimulated with EC induced co-expression of BCL-6 and IL-21 in CD4+ T cells, as assessed by flow cytometry, whereas APCs stimulated with killed bacteria failed to do so (Fig. 1e,f). The expression of mRNA encoding the transcription factors T-bet and GATA-3 was downregulated in T cells incubated with APCs stimulated with EC or HKEC, relative to such expression in T cells activated with unstimulated APCs, whereas expression of mRNA encoding the transcription factors RORγt and Maf was slightly increased by the incubation of T cells with APCs stimulated with either EC or HKEC, as measured by hybridization-based multiplexed gene-expression quantitation (Fig. 1g and Supplementary Fig. 1b). The specific induction of T FH cells upon sensing of live EC by the innate immune system was not specific to CD14+CD16– monocytes, as similar results were obtained with primary human CD1c+ myeloid dendritic cells (DCs) as APCs (Fig. 1h,i).

In order to assess the functionality of newly generated T FH cells as true helpers of B cells, we sorted CD4+CD45RA–CXCR5+ T FH cells from the APC–T cell co-culture system at day 5 and compared them with naive CD4+CD45RA+CXCR5– T cells sorted from the blood of the same donor, assessing their ability to promote plasma-cell differentiation when co-cultured with heterologous tonsillar memory B cells. CXCR5+ T FH cells induced by EC-stimulated APCs promoted robust differentiation of CD27hiCD38+ plasma cells, while naive CXCR5– T cells from the same donor failed to do so (Fig. 1j,k). In addition, CXCR5+ T FH cells stimulated robust production of immunoglobulin G (IgG), which was not observed with naive T cells or in B cell–only cultures (Fig. 1l). CXCR5– T cells sorted from the co-cultures at day 5 provided weaker help than did CXCR5+ T cells (Supplementary Fig. 1c–f). Collectively, these results indicated that the recognition of bacterial viability by human APCs of the innate immune system elicited potent differentiation signals for the generation of fully functional T FH cells.

Recognition of bacterial viability modulates the cytokine profile of human APCs

In order to characterize the innate immune signals that control T FH cell programming after the recognition of bacterial viability, we compared the transcriptome of CD14+CD16– monocytes in response to live bacteria with that in response to dead bacteria. Detection of either EC or HKEC led to similar regulation of 1,051 transcripts in human monocytes (Fig. 2a), which indicated substantial similarity between the two stimuli, both of which contain an abundance of PAMPs and thereby engage a multitude of pattern-recognition receptors7. We detected a set of 193 genes that were regulated differentially in CD14+CD16– monocytes in response to EC relative to their regulation in response to HKEC, including genes encoding the inflammatory cytokines TNF (TNF) and IL-12p40 (IL12B) (Fig. 2a,b and Supplementary Table 1). Accordingly, we detected release of IL-12 and TNF (assessed by ELISA) nearly exclusively in response to EC, not in response to HKEC, whereas the cytokines IL-6, IL-10, IL-23 and GM-CSF were produced regardless of bacterial viability (Fig. 2c).

Fig. 2: Detection of viable bacteria skews the cytokine profile of human monocytes. a, Genome-wide transcriptional analysis of human CD14+CD16– monocytes (n = 4 donors) stimulated for 6 h with medium (control (Ctrl)), EC or HKEC, presented as the mean signal log ratio (SLR) of each gene in EC-treated cells relative to that in control cells (EC vs Ctrl) plotted against that of HKEC-treated cells relative to that of control cells (HKEC vs Ctrl); red symbols indicate genes with a difference in SLR of >2 in EC-treated cells relative to that in HKEC-treated cells. b, Heat map of 193 genes with a change in SLR of >2 or ≤2 (key) in EC–versus–Ctrl compared with HKEC–versus–Ctrl as in a (below plot). c, Concentration of cytokines secreted by APCs (n = 3–6 donors) left untreated (Ctrl) or stimulated for 18 h with EC or HKEC (key). d, Concentration of cytokines secreted by APCs (n = 4 donors) stimulated with EC or HKEC (key) at an increasing multiplicity of infection (MOI) (horizontal axis). e, Concentration of cytokines secreted by APCs (n = 2–5 donors (top row) or n = 4 donors (bottom row)) that were left untreated (Ctrl) or stimulated with live B. subtilis (BS) or heat-killed B. subtilis (HKBS) (key) (top row) or with live M. bovis strain BCG (BCG) or heat-killed M. bovis strain BCG (HKBCG) (key) (bottom row). f, Flow-cytometry analysis of the surface expression of various markers on APCs (n = 5 donors) at 18 h after treatment as in c (key). *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA (c,e) or multiple t-tests with post-hoc correction for multiple comparisons (d)). Data are representative of four independent experiments (one per donor; a,b), three to six experiments (one per donor: n = 6 (IL-1β and IL-12p70), n = 5 (IL-6, IL-10, IL-12p40 and TNF), n = 4 (IL-23) or n = 3 (GM-CSF); (c), eight experiments (d; error bars, mean ± s.e.m.) or five experiments (top row) or four experiments (bottom row) (e) (error bars (c,e), maximum and minimum). Full size image

Our finding of differential expression of IL-12 and TNF was in contrast to published observations of mouse macrophages and DCs, which produce large amounts of TNF and IL-12 in response to either live bacteria or killed bacteria or purified bacterial cell-wall components7. Similar to results obtained for mouse macrophages and DCs7, release of IL-1β was induced by EC, but not by HKEC, in human monocytes (Fig. 2c), indicative of inflammasome activation. Production of TNF and IL-12 could not be restored by higher doses of HKEC (Fig. 2d). Other bacterial species, including avirulent Gram-positive Bacillus subtilis, as well as BCG (an attenuated strain of Mycobacterium bovis and widely used live vaccine against tuberculosis (TB)), elicited comparable cytokine patterns (Fig. 2e), which indicated that the response to bacterial viability was conserved and was largely independent of features specific to the bacterial species. Both EC and HKEC induced similar upregulation of the expression of maturation markers such as CD40 (Fig. 2f), which indicated intact innate recognition of both stimuli. Thus, human CD14+CD16– monocytes discriminated precisely between live bacteria and dead bacteria independently of virulence or bacterial species, and they responded to bacterial viability with a distinct transcriptional program and cytokine profile.

‘Viability-induced’ T FH cell responses are mediated by APC-derived IL-12

Next, we investigated whether APC-derived cytokines induced by the detection of live bacteria were driving the T FH cell differentiation. CD4+ T cells (isolated by negative selection from peripheral blood) showed high expression of IL-21 and BCL-6 and of CXCR5, ICOS and PD-1 when activated via CD3 and CD28 in the presence of supernatants of EC-stimulated CD14+CD16– monocytes (Fig. 3a,b). Supernatants of HKEC-stimulated APCs did not induce a T FH cell phenotype in CD4+ T cells (Fig. 3a,b), while proliferation rates and IL-17 production were induced similarly by supernatants of EC-conditioned APCs and supernatants of HKEC-conditioned APCs (Supplementary Fig. 2a–c). These results indicated a dominant role for APC-derived soluble factors in the initial stages of T FH cell differentiation in response to live EC.

Fig. 3: IL-12 is a critical signal for ‘viability-induced’ T FH cell differentiation. a,b, Frequency of IL-21+BCL-6+ cells (left) or CXCR5+ICOS+PD-1+ cells (right), assessed by flow cytometry (a), and ELISA of IL-21 (left) and IFN-γ (right) in supernatants (b) of CD4+ T cells polyclonally activated by plate-bound antibody to CD3 and soluble antibody to CD28 in the presence of supernatants collected from APCs left unstimulated (Ctrl) or stimulated for 18 h with EC or HKEC (key) (n = 19 donors (IL-21+BCL-6+) or 8 donors (CXCR5+ICOS+PD-1+) (a); or n = 11 donors (b)). c, Linear-regression analysis of the concentration of IL-21 produced by CD4+ T cells versus that of IL-12p70 (top), TNF (middle) or IL-6 (bottom) in supernatants of APCs (samples from nine donors in five independent experiments; n = 45 (IL-12p70), 21(TNF) or 21 (IL-6)) in cultures as in a. d–g, Flow-cytometry analysis of the expression of IL-21 and BCL-6 (d), frequency of IL-21+BCL-6+ cells, assessed as in d (e) (n = 14), and ELISA of IL-21 in supernatants (f,g) (n = 7) of T cells cultured as in a,b with supernatants of EC-stimulated APCs in the presence or absence of the irrelevant control antibody IgG or neutralizing antibodies (anti-) or supernatants of control APCs supplemented with recombinant (r) cytokines (above plots (d) or horizontal axis (e–g)). Each symbol (a,e) represents an individual donor. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA with post-hoc correction for multiple comparisons). Data are representative of 19 experiments (a,b), 45 experiments (c), 14 experiments (d,e) or 7 experiments (f,g) (error bars (b,f,g), maximum and minimum). Full size image

Various cytokines and cytokine combinations, including IL-21, IL-6, IL-27 and type I interferons, have been reported to promote T FH cell differentiation in mice9,18,19, whereas the differentiation of human T FH cells is mediated mainly by the cytokines IL-12, IL-23 and TGF-β10,20. The cytokine requirements for vita-PAMP–induced human T FH cell differentiation remain unclear. We found a strong correlation between the amount of IL-12 and TNF in supernatants of EC-stimulated APCs and the production of IL-21 by the activated T cells, whereas the amount of IL-6 in supernatants of APCs did not correlate with the production of IL-21 by T cells (Fig. 3c). The addition of a neutralizing antibody against IL-12 to the supernatants of EC-induced APCs almost completely abolished T FH cell differentiation without affecting T cell proliferation rates (Fig. 3d–f and Supplementary Fig. 2a,d–f). Neutralization of IL-6 or IL-27 had no significant effect, whereas blockade of TNF partially inhibited T FH cell differentiation, relative to such differentiation after the addition of isotype-matched control antibodies (Fig. 3d–f). Conversely, supplementing supernatants of unstimulated (control) APCs with recombinant IL-12 restored T FH cell differentiation (Fig. 3d,e,g). Supplementation with recombinant TNF alone did not promote T FH cell differentiation (Fig. 3g), which indicated that TNF might have a minor role or act in concert with IL-12 or other APC-derived factors. Neutralization of IL-1β in the supernatants of EC-induced APCs partially diminished T FH cell differentiation, while supplementation of supernatants of unstimulated (control) APCs with recombinant IL-1β alone was insufficient to support T FH cell differentiation (Supplementary Fig. 2d–f); this indicated that IL-1β might have additive effects in humans, consistent with published observations20. Blocking IFN-β or supplementation with recombinant IFN-β did not alter the differentiation of human T FH cells (Supplementary Fig. 2d–f). Although membrane-bound mediators such as ICOSL14 and OX40L21 contribute to different stages of T FH cell differentiation in vivo, we found no major difference in their surface expression on APCs stimulated with EC relative to that on APCs stimulated with HKEC (Fig. 2f), an observation that does not exclude the possibility of a role at later stages of differentiation. Thus, IL-12 was the critical innate immune signal produced in response to live bacteria to instruct early priming of T FH cells in humans.

TLR8 is a vita-PAMP receptor that mediates IL-12 production and T FH cell differentiation

In order to identify potential targets for T FH cell–skewing adjuvants, we next investigated the nature of the innate immune receptor(s) and the ligands that elicited ‘viability-induced’ T FH cell differentiation signals. We supplemented HKEC with various PAMPs and compared subsequent cytokine responses in CD14+CD16– monocytes. Of the five different PAMPs tested here, only ligands of the endosomal single-stranded-RNA receptors TLR7 and TLR8 restored the production of IL-12 and TNF to levels comparable to those induced by EC (Fig. 4a). Inhibition of actin polymerization and phagocytosis via cytochalasin D, as well as blockade of endolysosomal acidification with bafilomycin A, abolished the EC-induced production of IL-12, but not that of IL-6 (Supplementary Fig. 3a), indicative of a role for endosomal receptors in the sensing of viable bacteria.

Fig. 4: APCs sense live bacteria via TLR8. a, ELISA of IL-12p70 (top), TNF (middle) and IL-6 (bottom) in supernatants of monocytes (n = 3 donors) left untreated (Ctrl) or exposed to EC or HKEC or the TLR2 ligand Pam 3 CSK 4 (P3CSK4), the TLR3 ligand poly(I:C) (pI:C), the TLR7 and TLR8 ligand CL075 or the TLR9 ligand CpG (horizontal axis) alone or exposed to HKEC supplemented with those TLR ligands (key). b, ELISA of cytokines as in a in supernatants of monocytes (n = 4 donors) left unstimulated (Ctrl) or stimulated with EC, HKEC, bacterial RNA (RNA) or CL075 (horizontal axis). c,d, ELISA of cytokines as in a in supernatants of human monocytes (n = 3 donors) treated with control siRNA with a scrambled sequence (Ctrl siRNA) or one of three siRNAs (-1, -2, -3; key) directed against TLR8 (c) or MyD88 (d) and stimulated with EC or HKEC (below plot). **P < 0.01 and ***P < 0.001 (one-way ANOVA (a,b) or two-way ANOVA (c,d) with post-hoc correction for multiple comparisons). Data are representative of three experiments (a,c,d) or four experiments (b) (error bars, maximum and minimum). Full size image

Since human monocytes expressed TLR8 but had only low expression of TLR7 (Supplementary Fig. 3b), and TLR8 recognizes bacterial RNA22,23, we investigated whether TLR8 was the primary human vita-PAMP receptor for live bacteria. Endosomal delivery of bacterial RNA fully restored the production of TNF and IL-12 to levels comparable to those induced by EC and synthetic agonists of TLR7 and TLR8 (Fig. 4b) and induced upregulation of the expression of CD40, CD80 and other maturation markers (Supplementary Fig. 3c). Conversely, silencing the expression of the gene encoding TLR8 or the gene encoding its signaling adaptor MyD88 by RNA-mediated interference (with small interfering RNA (siRNA)) in CD14+CD16– monocytes abolished the release of IL-12p70 and TNF in response to EC (Fig. 4c,d). The production of IL-6 was not affected by the silencing of either of those genes (Fig. 4c,d). In line with the proposed critical role for TLR8 as a vita-PAMP receptor, we found that ligation of TLR8 in APCs by the synthetic agonists CL075 or R848 potently promoted the differentiation of IL-21+BCL-6+ T FH cells (Fig. 5a–c). In contrast, all other TLR ligands tested, including the licensed vaccine adjuvants MPLA (monophosphoryl lipid A; a TLR4 agonist) and CpG DNA (a TLR9 agonist), did not promote T FH cell responses, even at high concentrations (Fig. 5a–c). Similar to its activation by live bacteria, the activation of TLR8 by purified bacterial RNA resulted in the robust differentiation of T FH cells and their production of IL-21 (Fig. 5d,e), which demonstrated that the recognition of bacterial RNA by the innate immune system was a potent stimulator of T FH cell–differentiation signals. Silencing TLR8 expression in CD14+CD16– monocytes diminished their ability to promote T FH cell differentiation in response to EC, relative to the corresponding ability of EC-stimulated monocytes treated with control siRNA with a scrambled sequence (Fig. 5f,g). Collectively, these results identified TLR8 as the key sensor for bacterial viability in human APCs and a critical driver of IL-12 production and subsequent T FH cell responses.

Fig. 5: TLR8 senses bacterial viability and drives T FH cell differentiation. a, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by CD4+ T cells (n = 7 donors) stimulated in the presence of supernatants of APCs previously left unstimulated (Ctrl) or stimulated with EC or HKEC or various TLR ligands (above plots). b, Frequency of IL-21+BCL-6+ cells in cultures as in a (horizontal axis). c, Frequency of IL-21+BCL-6+ T cells (assessed by flow cytometry) following co-culture with APCs (as in Fig. 1) left unstimulated (Ctrl) or stimulated with EC, HKEC or HKEC plus CL075 or with increasing concentrations (wedges) of CL075 (0.1, 0.5 or 1 μg/ml), MPLA (0.1, 0.5 or 1 μg/ml) or CpG (0.1, 1 or 2.5 μM) (horizontal axis) . d, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by CD4+ T cells stimulated by supernatants of APCs previous left unstimulated (Ctrl) or stimulated with EC, HKEC, bacterial RNA, HKEC plus bacterial RNA, or CL075 (above plots). e, Frequency of IL-21+BCL-6+ cells in cultures of CD4+ T cells stimulated by supernatants of APCs previously left unstimulated (Ctrl) or stimulated with EC or HKEC (horizontal axis) and the polycationic polypeptide poly-l-arginine (pLa) or bacterial RNA (key) (left), and ELISA of IL-21 in supernatants of those cultures (right). f,g, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by CD4+ T cells stimulated by supernatants of APCs previously left unstimulated (Ctrl) or stimulated with EC or HKEC (above plots) and treated with control or TLR8-specific siRNA (left margin) (f), frequency of IL-21+BCL-6+ cells in cultures as in f (key) (g, left) and ELISA of IL-21 in supernatants of cultures as in f (key) (g, right). Each symbol (b,c,e,g) represents an individual donor (n = 7 (b), n = 7 (c), n = 4 (e) or n = 8 (g)). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (one-way ANOVA (b,c) or two-way ANOVA (e,g) with post-hoc correction for multiple comparisons). Data are representative of seven experiments (a–c), four experiments (d,e) or eight experiments (f,g) (error bars (e,g, right), maximum and minimum). Full size image

Recognition of bacterial viability is conserved in porcine APCs

Domestic pigs (Sus scrofa domestica) are increasingly used for biomedical and pharmaceutical studies due to the substantial analogies between porcine physiology and human physiology24,25. The porcine and human immune systems also share many similarities25, including the expression and function of TLR826. Thus, we next assessed T FH cell differentiation in response to viable bacteria in pigs. Porcine CD172+CD14+ monocytes and CD172+CD14– DCs were sorted from spleen samples of domestic pigs and were stimulated with live and dead bacteria. We used the thymidine-auxotrophic E. coli (EC) or a live attenuated strain of Salmonella enterica serovar Typhimurium (ST) (distributed under the trade name ‘Salmoporc-STM’) as a live Salmonella vaccine for pigs27. Salmoporc-STM bacteria are histidine–adenine auxotrophs, leading to severe growth attenuation. Porcine CD172+CD14+ monocytes and CD172+CD14– DCs secreted large amounts of IL-12 in response to live EC and ST and the TLR8 agonist CL075 but not after stimulation with heat-killed ST (HKST) or HKEC (Fig. 6a,b). Secretion of IL-6 was induced similarly by live and dead EC and ST (Fig. 6a,b). Selective induction of IL-12 by live EC and ST was consistently observed (Fig. 6a–c), yet statistical testing did not reveal significant differences in this, due to limited sample size and high inter-experimental variation in cytokine production. Purified EC RNA also promoted increased secretion of IL-12p40 by porcine CD14+ monocytes, as assessed by ELISA, a result that was not observed for ligands of TLR2 and TLR4 (Supplementary Fig. 4a). To determine whether the mechanisms of ‘viability recognition’ are conserved between human APCs and porcine APCs, we used RNA-mediated interference to silence the expression of the gene encoding TLR8 in porcine CD14+ monocytes. Knockdown of TLR8 abolished the expression of IL-12p40 in response to live ST, whereas the production of IL-6, which is induced independently of bacterial viability, was unaffected (Fig. 6c). We next assessed the effect of bacterial viability on porcine T FH cell differentiation. Pig splenocytes (populations that included APCs and CD4+ T cells) were stimulated for 1 h with varying doses of ST or HKST, followed by the addition of antibiotics to prevent residual bacterial growth, plus concanavalin A to induce polyclonal T cell proliferation. We observed a dose-dependent increase in the frequency of CD4+IL-21+BCL-6+ T FH cell–like cells in response to ST, compared with the frequency of such cells in response to stimulation with HKST (Fig. 6d,e). Next we compared the ability of soluble PAMPs to induce a T FH cell phenotype in pig splenocytes in vitro. The TLR8 agonists bacterial RNA and CL075 induced CD4+IL21+BCL-6+ T cells, but the TLR4 agonist LPS did not (Fig. 6f). Thus, recognition of bacterial viability via TLR8 constituted a critical stimulus of porcine T FH cell differentiation.

Fig. 6: Detection of viable bacteria via TLR8 by porcine APCs promotes T FH cell differentiation. a,b, Multiplex bead array of IL-12p40 (left) and IL-6 (right) in cultures of porcine CD14+CD172+ monocytes (a) or CD14–CD172+ DCs (b) sorted from spleen samples and stimulated with medium (Ctrl), EC, HKEC, ST, HKST or CL075 (horizontal axis). c, ELISA of IL-12p40 (top) and IL-6 (bottom) in supernatants of porcine splenic CD14+ monocytes (n = 3 pigs) treated with control siRNA or siRNA directed against porcine TLR8 (key) and stimulated with medium (Ctrl), ST, HKST or CL075 (horizontal axis). d, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by CD4+ T cells among porcine splenocytes stimulated for 4 d with concanavalin A in the presence of increasing doses (above plots) of ST (top) or HKST (bottom) (left margin). e, Frequency of IL-21+BCL-6+ cells in cultures as in d (n = 3 pigs). f, Frequency of IL-21+BCL-6+ cells among CD4+ T cells in cultures of porcine splenocytes stimulated for 4 d with medium (Ctrl), CL075, LPS or bacterial RNA (horizontal axis) in the presence of concanavalin A. Each symbol (a,b,f) represents an individual pig (n = 3 (IL-6 (a,b)) or 2 (IL-12p40 (b)) (a,b), or n = 3 (f)). *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA (a,b,f) or two-way ANOVA (c, e) with post-hoc correction for multiple comparisons). Data are representative of three experiments (error bars, maximum and minimum (c) or mean ± s.e.m. (e)). Full size image

Bacterial viability promotes T FH cell differentiation in vivo

To directly assess the role of the detection of bacterial viability by the innate immune system in T FH cell responses in vivo, we vaccinated domestic pigs with live attenuated ST (Salmoporc-STM), an equivalent dose of HKST or solvent (saline; as a control). The frequency of CD4+IL-21+BCL-6+ T FH cell–like cells was greater in the draining lymph nodes (dorsal superficial cervical nodes) and spleen of pigs immunized with Salmoporc-STM than in that of pigs immunized with HKST or saline (Fig. 7a,b) Other markers of effector T cell differentiation, including T-bet and FoxP3, were similarly altered in T cells from the ST-vaccinated or HKST-vaccinated groups (Supplementary Fig. 4b–e). We observed an increased number of PAX5+ B cell follicles in the spleen of ST-vaccinated pigs relative to the number of such follicles in the spleen of saline-treated pigs, a result that was not observed for the HKST-vaccinated group compared with the saline-treated group (Fig. 7c,d), although the difference between the ST-vaccinated group and the HKST-vaccinated group did not reach statistical significance. B cell follicles in the spleen of ST-vaccinated pigs showed considerable enrichment for Ki67+PAX5+ B cells (Fig. 7e and Supplementary Fig. 5a), indicative of active germinal centers, but B cell follicles were negative for BCL2 (Supplementary Fig. 5b), which ruled out the possibility of malignant transformation. We also found an increased frequency of antibody-forming cells and plasma cells, which can be further separated into CD3–CD8–SLAII+IgM+CD2+CD21– effector and CD3–CD8–SLAII+IgM+CD2−CD21– resting antibody-forming cells and plasma cells28, in ST-vaccinated pigs relative to the number of such cells in HKST-vaccinated pigs (Fig. 7f). Notably, higher levels of Salmonella-binding serum IgG were detected after vaccination with live ST than after vaccination with HKST (Fig. 7g), which demonstrated enhanced humoral immunity in response to the live vaccine. These results indicated that the recognition of bacterial viability was an essential driver of vaccine-induced T FH cell and antibody responses in vivo.

Fig. 7: A live attenuated vaccine promotes T FH cell differentiation in swine. a, Flow-cytometry analysis of the expression of IL-21 and BCL-6 by CD4+ T cells from the draining lymph nodes (LN) or spleen of 5-week-old domestic piglets on day 30 after subcutaneous immunization with saline (Ctrl), ST or HKST (above plots). b, Frequency of IL-21+BCL-6+ cells in lymph nodes or spleen (above plots) of pigs as in a (key). c, Microscopy of paraffin-embedded sections of spleen tissues from pigs as in a (above images), stained for the transcription factor PAX5. Scale bars, 5 mm (top) or 500 μm (bottom). d, Morphometric quantification of PAX5+ follicles in spleen sections of pigs as in c (key). e, Microscopy of co-immunofluorescence staining of PAX5 (red) and Ki67 (green) on sections of spleen from pigs vaccinated with ST; cell nuclei were stained with the DNA-binding dye DAPI (blue). Each row shows a follicle from a separate pig. Scale bar, 50 μm. f, Frequency of antibody-forming cells (AFC) and plasma cells (PC) among lymphocytes in spleen samples of pigs as in c (key), assessed by flow cytometry. g, ELISA of anti-Salmonella IgG in serum samples obtained from pigs as in c (key) before vaccination (day 0) and on day 14 and 21 after vaccination (horizontal axis), presented as the optical density at 450 nm (OD 450 ). Each symbol (b,d,f) represents an individual pig (n = 5 per group (b) or (n = 3 per group (f)). *P < 0.05 and **P < 0.01 (one-way ANOVA (b,d) or two-way ANOVA (g) with post-hoc correction for multiple comparisons). Data are representative of one experiment with various numbers of pigs in each panel (as indicated in legend above; error bars (g), mean ± s.e.m.). Full size image

A TLR8 polymorphism is associated with vaccine protection in humans

Several functional polymorphisms in TLR8 in humans have been described29,30. The TLR8 single-nucleotide polymorphism (SNP) TLR8-A1G (rs3764880; called ‘TLR8-G’ here) alters the start codon ATG into a GTG triplet29, which shifts the signal peptide by three amino acids, with a second in-frame ATG (M4) being used as an alternative start codon. According to in silico modeling predictions based on the published crystal structure of TLR831, the amino-acid truncation leads to significant structural alterations to the protein (Supplementary Fig. 6 and Supplementary Note 1). The increased disorder, free energy and increased flexibility of the protein encoded by TLR8-G (Supplementary Fig. 6) probably make the receptor better adapted to side-chain rearrangement and dimerization. The larger volume of clefts and cavities on the surface of the protein encoded by TLR8-G than on that encoded by TLR8-A (the protein encoded by TLR8 without the SNP rs3764880) might increase its potential for ligand binding, whereas functional pockets and nests are slightly decreased (Supplementary Fig. 7). These models suggest altered receptor functionality, which might cause a gain of function for the receptor encoded by the TLR8-G variant. In line with those predictions, APCs from individuals expressing the TLR8-G variant showed a slightly enhanced release of IL-12 in response to stimulation of TLR8, but not in response to the TLR4 agonist LPS, relative to the response of APCs derived from carriers of TLR8-A (Supplementary Fig. 8a,b). We also studied HEK293T human embryonic kidney cells expressing a reporter gene to measure actvity of the transcription factor NF-κB. Such cells stably expressing the TLR8-G variant showed higher NF-κB-reporter activity in response to TLR8 ligands than that of their TLR8-A-expressing counterparts (Supplementary Fig. 8c), in support of the proposed gain-of-function phenotype of the TLR8-G variant.

Carriage of the TLR8-G allele has been associated with slower progression of infection with human immunodeficiency virus29 and protection against pulmonary TB (PTB)32. Here we assessed distribution of the TLR8-G allelein 293 patients with confirmed TB and 165 of their healthy household contacts (control subjects; Supplementary Table 2). Significantly more control subjects (53.9%) than patients with TB (41.3%) were homo- or hemizygous carriers of TLR8-G (Fig. 8a and Supplementary Table 3). The TLR8-A allele was associated with significantly increased odds for infection with M. tuberculosis (odds ratio = 1.94 [95% confidence interval, 1.194–3.156]; P = 0.007), and similar results were obtained for the subgroup of patients with PTB (Fig. 8a and Supplementary Table 3), indicative of a protective effect of TLR8-G against PTB, as has been reported32. Further subgroup analysis revealed that distribution of the TLR8 allele was in fact significantly different only for subjects who had previously received the live BCG vaccine against TB (P = 0.002), whereas its distribution was not significantly different for unvaccinated subjects (P = 0.754) (Fig. 8a and Supplementary Table 4). In this study, vaccination with BCG was associated with significant risk protection in carriers of the TLR8-G allele (odds ratio = 0.280 [95% confidence interval, 0.105–0.742]) but not in carriers of the TLR8-A allele (Fig. 8b and Supplementary Table 4). These epidemiological results indicated that TLR8-G was associated with improved BCG vaccine–mediated protection without affecting susceptibility to PTB itself, and they linked the function of TLR8 to protective immunity in response to a live bacterial vaccine in a large human cohort.