Specific gut commensal flora locally alters T cell tuning to endogenous ligands

Abstract

Differences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells.

Figure 1. SFB colonization of host gut-commensal flora enhances the severity of autoimmune disease in a T cell transfer model of arthritis

(A) Weekly arthritis score and (B) serum autoantibody titers in RF- or MPF-housed mPCC, Cd3e^−/− hosts at indicated time points after naïve 5C.C7 T cell transfer. Data (mean±SEM) pooled from two (A) and four (B) independent experiments (n=9 and 18 mice per group respectively). (C–D) One group of RF-housed mPCC, Cd3e^−/− mice was cohoused with MPF-housed hosts for 3 weeks prior to T cell transfer (RF(MPF)). (C) Bi-weekly arthritis scores and (D) autoantibody titers at day 31. Data (mean±SEM) pooled from two independent experiments (n=9 RF, n=10 RF(MPF) and n=8 MPF). (E) Various antibiotic formulations were given in the drinking water of MPF-housed mPCC, Cd3e^−/− mice starting 3 weeks prior to T cell transfer and maintained for the length of the experiment. “All antibiotics” formulation contains ampicillin, neomycin, vancomycin and metronidazole. Data (mean±SEM) pooled from two independent experiments (n=10 mice per group). (F) Amount of SFB 16sRNA in cecum contents of indicated mPCC, Cd3e^−/− hosts 8 weeks post T cell transfer (n=5 mice per group, mean±SEM). (G) Same as (C) with an additional group of RF-housed mPCC, Cd3e^−/− mice cohoused with SFB-monocolonized GF mice 3 weeks prior to T cell transfer (RF(SFB)). (n=4 RF and RF(SFB), n=6 RF(MPF) and n=5 MPF, mean±SEM). See also Figure S1.

Figure 2. SFB-containing flora promotes the differentiation of arthritis-inducing Th1 cells

(A–C) 4 days post transfer of naïve 5C.C7 T cells into mPCC, Cd3e^−/− hosts, the cells were stained for intracellular IL-17A and IFN-γ following a 3h stimulation with PMA and ionomycin. (A) Representative IFN-γ and IL-17A expression profiles, gated on live, CD4⁺Vβ3⁺ T cells and (B) frequencies of IFN-γ⁺ or IL17A⁺ CD4⁺Vβ3⁺ T cells in the indicated organs. (C) Frequencies of IFN-γ⁺ in CD4⁺Vβ3⁺ T cells in mLN of indicated host. Data (mean±SEM) pooled from 3 independent experiments each (n=7 (A–B) and n=5–6 (C) mice per group). (D) 10 days post transfer of naïve CD45.1⁺ 5C.C7 T cells into 8–12 wk. old mPCC, Cd3e^+/+ hosts, the cells were stained for intracellular IL-17A and IFN-γ following a 5.5h stimulation with PMA and ionomycin. Frequencies of IFN-γ⁺ or IL17A⁺ in CD45.1⁺ CD4⁺Vβ3⁺ T cells in indicated organs. Data (mean±SEM) pooled from 5 independent experiments, each representing one pool of 6 RF-housed or 2 cohoused-RF(MPF) mice. RF(MPF) hosts were cohoused for 2 weeks with MPF-housed mPCC, Cd3e^−/− mice prior to T cell transfer. (E) Bi-weekly arthritis scores of MPF-housed mPCC, Cd3e^−/− mice post transfer of naïve 5C.C7 WT or Ifng^−/− 5C.C7, Rag2^−/− T cells. Data (mean±SEM) pooled from two independent experiments (n=10 mice per group). See also Figure S2.

Figure 3. SFB-containing flora sustains chronic proliferation of self-reactive CD4⁺ T cells

(A–D) 14 days post naïve 5C.C7 T cell transfer, mPCC, Cd3e^−/− hosts were injected with EdU and sacrificed 1hr. later. (A) Absolute number of CD4⁺Vβ3⁺ T cells in spleen, mLN, Peyer’s patches (PP) and lamina propria (LP) of the small intestine. (B) Representative EdU and Ki-67 expression profiles and (C–D) Frequency of EdU⁺Ki-67^hi cells in live, CD4⁺Vβ3⁺ T cells isolated from indicated organs of RF- or MPF-housed hosts (B–C) or mLN of indicated hosts (D). Data (mean±SEM) pooled from 3 independent experiments each ((A–C) n=8 and (D) n=6–7 mice per group). (E) Representative CD25 and Ki-67 expression profiles, gated on CD4⁺Vβ3⁺ T cells and (F) representative phosphorylated S6 ribosomal protein (pS6) expression profile in CD25− (open, blue line) and CD25⁺ (open, red line) CD4⁺Vβ3⁺ T cells isolated from mLN of indicated hosts at day 14 and compared to naive 5C.C7 T cells (closed, grey). (G–I) 10 days post transfer of naïve CD45.1⁺ 5C.C7 T cells into 4–5 wk. old mPCC, Cd3e^+/+ hosts, the recipient mice were injected with EdU and sacrificed 1 hr. later. (G) Absolute number of CD45.1⁺CD4⁺Vβ3⁺ T cells recovered at day 10 from indicated organs. (H) Representative EdU and Ki-67 expression profiles and (I) frequency of EdU⁺Ki-67^hi cells in live, CD45.1⁺CD4⁺Vβ3⁺ T cells isolated from mLN and PP. Data (mean±SEM) pooled from 6 (G) and 4 (H–I) independent experiments. (n=6 pools of 1 to 3 mice per group and 4 pools of 3 mice per group respectively). (J) Same as (I) in 8–12 wk. old RF-housed mPCC, Cd3e^+/+ mice cohoused for 2 weeks with MPF-housed mPCC, Cd3e^−/− mice prior to T cell transfer. Data (mean±SEM) pooled from 5 independent experiments, each representing one pool of 6 RF-housed or 2 cohoused-RF(MPF) mice. (G–J) All EdU and Ki-67 stainings were performed on purified CD4⁺ T cells. See also Figure S3.

Figure 4. SFB-containing flora locally modulates T cell intrinsic tuning of TCR activation threshold to endogenous antigen

(A and E) CD69 expression and (C) 3H-Thymidine incorporation in CD4⁺Vβ3⁺ T cells purified from spleen or mLN of mPCC, Cd3e^−/− hosts 7 days post naïve 5C.C7 T cell transfer and cultured for 16h (A and E) or 84h (B) with fresh Cd3e^−/− splenocytes and various concentrations of MCC peptide (A and C) or plate-bound anti-CD3 (2C11) and soluble anti-CD28 in the absence of added APC (E). 3H-Thymidine was added to the culture for the last 24h. One representative experiment out of 4 (pooled triplicates) or 3, respectively. (B and D) Summary of EC50 for each independent T cell population tested. Data (mean±SEM) pooled from (A–B) 5 and (C–D) 3 independent experiments, * P<0.05, student t test. (F) Frequencies of IL-2⁺ cells in live, CD4⁺Vβ3⁺ T cells isolated from spleen or mLN of mPCC, Cd3e^−/− hosts at the indicated time points post T cell transfer and restimulated for 3h with PMA and ionomycin or 3μM MCC peptide. (G) Representative expression profiles and (H) tuning ratios, as described in experimental procedures, for IL-2 in live, CD4⁺Vβ3⁺ T cells isolated at day 14 in mLN of indicated host. Data (mean±SEM) pooled from 5 independent experiments (n=2 to 7 mice per time point) (F) and 3 independent experiments (n=6–7 mice per group) (H). See also Figure S4.

Figure 5. Modulation of T cell tuning by commensal flora broadly affects self-reactive CD4⁺ Th1 and Th17 subsets

(A) Representative expression profiles at day 28 and (B) Frequencies of IFN-γ⁺ and IL17A⁺cells in live, CD4⁺Vβ3⁺ T cells isolated from mLN of mPCC, Cd3e^−/− hosts at the indicated time points post naïve 5C.C7 T cell transfer and restimulated for 3h with PMA and ionomycin or 3μM MCC peptide. (C) Tuning ratios, as described in experimental procedures, for IFN-γ and IL17A in live, CD4⁺Vβ3⁺ T cells isolated from mLN of indicated hosts 14 days post T cell transfer and restimulated for 3h with PMA and ionomycin or 3μM MCC peptide. Data (mean±SEM) pooled from 3 independent experiments (n=6–7 mice per group). (D–E) 17 or 26 days post T cell transfer, MPF-housed mPCC, Cd3e^−/− hosts were injected with EdU and sacrificed 1hr later. (D) Representative EdU and IFN-γ expression profiles in live, CD4⁺Vβ3⁺ T cells isolated from mLN at day 17. (E) Tuning ratio for IFN-γ in live, EdU⁺ versus EdU⁻ CD4⁺Vβ3⁺ T cells. Data (mean±SEM) pooled from 2 independent experiments (n=3 mice per group).

Figure 6. Host microbiota dynamically regulates self-reactive T cell tuning

(A) Two cohorts of mPCC, Cd3e^−/− mice were injected simultaneously with either naïve CD45.2⁺ 5C.C7 T cells or CD45.1⁺ 5C.C7 T cells. 7 days later, spleen and mLN were harvested from hosts injected with CD45.2⁺ T cells. Purified T cells (10⁶) were further reinjected into the hosts previously transferred with CD45.1⁺ T cells. mLN cells were stained at day 14 for Ki-67 expression or for intracellular IL-2 and IFN-γ following 3h stimulation with 3μM MCC peptide (MCC). (B) Frequency of Ki-67⁺ (top), IFN-γ⁺ (middle), or IL-2⁺ (bottom) in live, CD4⁺Vβ3⁺ T cells in the CD45.2⁺ donor (left) or the CD45.1⁺ recipient (right) fractions. Data (mean±SEM) pooled from 3 independent experiments. See also Figure S5.

Figure 7. In vivo IL-12p40 blockade restores optimal T cell tuning in MPF-housed hosts

(A) IL-12p40, IL-6, G-CSF and IL-1Ra levels in sera of mPCC, Cd3e^−/− hosts 14 days post naïve 5C.C7 T cell transfer. Data (mean±SEM) pooled from 2 independent experiments (n=5 mice per group). (B–E) Starting at day 7, MPF-housed mPCC, Cd3e^−/− mice were further injected twice daily i.p. with anti-IL12p40 or a corresponding isotype control mAb. At day 14, mice were injected with EdU and sacrificed 1hr later. (B) Representative EdU and Ki67 expression profiles and (C) frequency of EdU⁺Ki-67^hi cells in live, CD4⁺Vβ3⁺ T cells isolated from mLN (n=3 mice per group, mean±SEM). * P<0.05, student t test. (D) Representative IL-17A and IFN-γ expression profiles and (E) tuning ratio for IL-17A (left), IFN-γ (middle) and IL-2 (right) production in live, CD4⁺Vβ3⁺ T cells isolated from mLN at day 14 and restimulated for 3h with PMA and ionomycin or 3μM MCC peptide. Data (mean±SEM) pooled from 4 independent experiments (n=9–10 mice per group). See also Figure S6.