IL-23 drives differentiation of peripheral γδ17 T cells from adult bone marrow-derived precursors

Abstract

Pro-inflammatory interleukin (IL)-17-producing γδ (γδ17) T cells are thought to develop exclusively in the thymus during fetal/perinatal life, as adult bone marrow precursors fail to generate γδ17 T cells under homeostatic conditions. Here, we employ a model of experimental autoimmune encephalomyelitis (EAE) in which hematopoiesis is reset by bone marrow transplantation and demonstrate unequivocally that Vγ4⁺ γδ17 T cells can develop de novo in draining lymph nodes in response to innate stimuli. In vitro, γδ T cells from IL-17 fate-mapping reporter mice that had never activated the Il17 locus acquire IL-17 expression upon stimulation with IL-1β and IL-23. Furthermore, IL-23R (but not IL-1R1) deficiency severely compromises the induction of γδ17 T cells in EAE, demonstrating the key role of IL-23 in the process. Finally, we show, in a composite model involving transfers of both adult bone marrow and neonatal thymocytes, that induced γδ17 T cells make up a substantial fraction of the total IL-17-producing Vγ4⁺ T-cell pool upon inflammation, which attests the relevance of this novel pathway of peripheral γδ17 T-cell differentiation.

Keywords: IL‐17; IL‐23; T‐cell differentiation; experimental autoimmune encephalomyelitis; γδ T cells.

Figure 1. Peripheral differentiation of γδ17 T cells upon EAE inflammation

1. Experimental setup: bone marrow chimeras (BMCs) were generated by injecting total bone marrow cells from wild‐type (WT) Thy1.1⁺ donor mice into TCRδ^−/− (Thy1.2⁺) hosts. After 8 weeks, these BMCs were immunized s.c. in both flanks with 125 μg of MOG^(35–55) peptide emulsified in CFA solution; additionally, BMCs were given 200 ng of PTx i.v. on days 0 and 2 p.i. for additional adjuvant effect. Mice were sacrificed at day 14 p.i., and brain, draining lymph nodes (dLN), cervical lymph nodes (cLN), and spleen were harvested.

2. Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from naïve or EAE‐induced Thy1.1:TCRδ^−/− BMCs.

3. Mice were observed daily and scored for clinical signs of EAE.

4. Frequencies of IL‐17A⁺ cells within the Thy1.1⁺CD3⁺TCRδ⁺ population in the different organs analyzed. Each symbol represents an individual BMC.

5. Flow cytometry analysis of TCR‐Vγ4 and TCR‐Vγ1 expression in Thy1.1⁺CD3⁺TCRδ⁺IL‐17A⁺ cells isolated from EAE‐induced Thy1.1:TCRδ^−/− BMC.

6. Flow cytometry analysis of intracellular RORγt (top panel) and T‐bet (bottom panel) expression in Thy1.1⁺CD3⁺TCRδ⁺IL‐17A⁺ cells isolated from the spleen of EAE‐induced Thy1.1:TCRδ^−/− BMC.

7. Flow cytometry analysis of IL‐1RI expression in IL‐17A⁺ (blue), IFN‐γ⁺ (red) or IL‐17A⁻IFN‐γ⁻ (purple) cells within Thy1.1⁺CD3⁺TCRδ⁺Vγ4⁺ cells isolated from the spleen of EAE‐induced Thy1.1:TCRδ^−/− BMC. FMO refers to Fluorescence Minus One (FMO) controls (without anti‐IL‐1R1 antibody) on the same cell population.

8. Flow cytometry analysis of CD44 and IL‐17A expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from the spleen of EAE‐induced Thy1.1:TCRδ^−/− BMC.

Data information: (B–D) “naïve” refers to non‐immunized BMCs. (C, D) Data pooled from two independent experiments (n = 4–10 mice per group). (E–H) Data representative of at least two independent experiments. Each symbol represents an individual BMC. (C–H) Error bars represent mean ± SD. (D) *P < 0.05 **P < 0.01 (Mann–Whitney U‐test). (G) *P < 0.05 (nonparametric one‐way ANOVA, Kruskal–Wallis test).

Figure EV1. Bone marrow chimeras contain IFN‐γ⁺ but not IL‐17⁺ γδ T cells

Flow cytometry analysis of intracellular IL‐17A (blue bars) or IFN‐γ (red bars) expression among gated Thy1.1⁺CD3⁺TCRδ⁺ cells after stimulation with PMA and ionomycin. Each symbol represents one Thy1.1:TCRδ^−/− bone marrow chimera (BMC), and error bars represent mean ± SD.

Figure 2. Peripheral γδ17 T‐cell differentiation occurs in draining lymph nodes

1. Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from naïve or EAE‐induced Thy1.1:TCRδ^−/− BMCs (n = 5–6 mice per group), established as in Fig 1A.

2. Frequencies of IL‐17A⁺ cells within the Thy1.1⁺CD3⁺TCRδ⁺ population in the draining LN of the BMCs in (A).

3. Flow cytometry analysis of intracellular IL‐17A and Ki67 expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from EAE‐induced Thy1.1:TCRδ^−/− BMCs (as in A).

4. Frequencies of IL‐17A⁺ cells within the Thy1.1⁺CD3⁺TCRδ⁺ population in the organs of the BMCs depicted in (A).

Data information: (A–D) Error bars represent mean ± SD. Data pooled from two independent experiments. Each symbol represents an individual BMC. (A, B) “Naïve” refers to non‐immunized BMCs. (A–D) n = 4–9 mice per group. (B) ****P < 0.0001 (Mann–Whitney U‐test). (D) **P < 0.01 ***P < 0.001 (nonparametric one‐way ANOVA, Kruskal–Wallis test).

Figure 3. Peripheral γδ17 T‐cell differentiation does not require myelin antigens or cell‐intrinsic TLR recognition

1. A

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from the dLN of Thy1.1:TCRδ^−/− BMCs injected subcutaneously with IFA or CFA followed or not by PTx administration.

2. B

   Frequencies of Thy1.1⁺CD3⁺TCRδ⁺IL‐17A⁺ cells within the Thy1.1⁺CD3⁺TCRδ⁺ population in the dLN of the BMCs in (A).

3. C, D

   Thy1.1:TCRδ^−/− or MyD88^−/−:TCRδ^−/− BMCs were injected subcutaneously with CFA and given 200 ng of PTx i.v. on days 0 and 2 p.i. for additional adjuvant effect. (C) Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in CD3⁺TCRδ⁺ cells isolated at day 7 p.i. Data are representative of two independent experiments. (D) Frequencies of IL‐17A⁺ cells within the CD3⁺TCRδ⁺ population in the dLN.

Data information: (A–D) Error bars represent mean ± SD. n.s., not significant. Data pooled from two independent experiments. Each symbol represents an individual BMC. (A) “Naïve” refers to non‐immunized BMCs. (A, B) n = 4–10 mice per group; (C, D) n = 6–7 mice per group. (B) **P < 0.01 (nonparametric one‐way ANOVA, Kruskal–Wallis test). (D) Mann–Whitney U‐test.

Figure EV2. Induction of IL‐17 expression in peripheral γδ T cells does not occur in the spleen and is independent on cell‐intrinsic TLR signaling

1. A

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated from the dLN of Thy1.1:TCRδ^−/− BMCs injected subcutaneously with IFA or CFA followed or not by PTx administration.

2. B

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated at day 7 p.i. from the spleens of Thy1.1:TCRδ^−/− or MyD88^−/−:TCRδ^−/− BMCs immunized subcutaneously with CFA and given 200 ng of PTx i.v. on days 0 and 2 p.i.

3. C, D

   Flow cytometry analysis and frequencies of intracellular IL‐17A and IFN‐γ expression in Thy1.1⁺CD3⁺TCRδ⁺ cells isolated at day 3 p.i. from the dLN and spleens of Thy1.1:TCRδ^−/− BMCs injected subcutaneously 50 μg of each individual TLR agonist (Pam₃CSK₄, Poly(I:C), LPS or CpG).

Data information: (A–D) Data pooled from two independent experiments. Each symbol represents one individual BMC. Error bars represent mean ± SD.

Figure 4. IL‐23 drives peripheral γδ17 T‐cell differentiation

1. A–C

   CD3⁺TCRδ⁺eYFP⁻ cells were FACS‐sorted from the peripheral LN and spleen of Il17a ^Cre R26R ^eYFP mice and cultured in vitro for 72 h in the presence of IL‐1β (10 ng/ml), IL‐23 (10 ng/ml), IL‐6 (10 ng/ml), TGF‐β (10 ng/ml), and plate‐bound anti‐CD3 mAb (10 μg/ml) combined as shown in (A). All conditions also included IL‐7 and IL‐21 (10 ng/ml each), except condition I, which contained IL‐7 (10 ng/ml) only. Data pooled from two independent experiments (n = 7 mice per experiment). (A) Flow cytometry analysis of eYFP expression in CD3⁺TCRδ⁺ cells after 72 h under the conditions depicted. Data are representative of two independent experiments. (B) Frequency and (C) mean fluorescence intensity (MFI) of eYFP⁺ in CD3⁺TCRδ⁺ cells (as in A).

2. D

   WT (Thy1.1⁺) and IL‐23R^−/− (Thy1.2⁺) or IL‐1R1^−/− (Thy1.2⁺) bone marrow total cells were mixed at 1:1 ratio to reconstitute lethally irradiated TCRδ^−/− hosts. After 8 weeks, mice were injected subcutaneously with CFA and given 200 ng of PTx i.v. on days 0 and 2 p.i. for additional adjuvant effect. “Naïve” refers to non‐immunized BMCs.

3. E

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in CD3⁺TCRδ⁺ cells isolated at day 7 p.i. from the dLN of the Thy1.1:IL‐23R^−/− mixed BMCs (D).

4. F

   Frequencies of IL‐17A⁺ cells within the CD3⁺TCRδ⁺ population in the dLN of naïve (white bar) or CFA‐immunized (gray bar) Thy1.1:IL‐23R^−/− mixed BMCs (as in D).

5. G

   Flow cytometry analysis and frequencies of IL‐23R^+/+ (Thy1.1⁺Thy1.2⁻; white bar) and IL‐23R^−/− (Thy1.1⁻Thy1.2⁺; black bar) within CD3⁺TCRδ⁺IL‐17A⁺ cells from the dLN of CFA‐immunized Thy1.1:IL‐23R^−/− mixed BMCs (as in D).

6. H

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in CD3⁺TCRδ⁺ cells isolated at day 7 p.i. from the dLN of Thy1.1:IL‐1R1^−/− mixed BMCs (D).

7. I

   Frequencies of IL‐17A⁺ cells within the CD3⁺TCRδ⁺ population in the dLN of naïve (white bar) or CFA‐immunized (gray bar) Thy1.1:IL‐1R1^−/− mixed BMCs (as in D).

8. J

   Flow cytometry analysis and frequencies of IL‐1R1^+/+ (Thy1.1⁺Thy1.2⁻; white bar) and IL‐1R1^−/− (Thy1.1⁻Thy1.2⁺; black bar) within CD3⁺TCRδ⁺IL‐17A⁺ cells from the dLN of CFA‐immunized Thy1.1:IL‐1R1^−/− mixed BMCs (as in D).

Data information: (A–J) Each symbol represents an individual BMC. Error bars represent mean ± SD. (E–G) Data pooled from two independent experiments (n = 3–8 mice per group). (H, J) n = 4–5 mice per group. (F, G, I, J). *P < 0.05, ***P < 0.001 (Mann–Whitney U‐test).

Figure EV3. γδ17 T‐cell composition in naïve versus immunized mixed BMCs

WT (Thy1.1⁺) and IL‐23R^−/− (Thy1.2⁺) or IL‐1RI^−/− (Thy1.2⁺) bone marrow total cells were mixed at 1:1 ratio to reconstitute lethally irradiated TCRδ^−/− hosts. After 8 weeks, mice were injected subcutaneously with CFA and given 200 ng of PTx i.v. on days 0 and 2 p.i. for additional adjuvant effect. “Naïve” refers to non‐immunized controls.

1. Flow cytometry analysis of IL‐23R^+/+ (Thy1.1⁺Thy1.2⁻) and IL‐23R^−/− (Thy1.1⁻Thy1.2⁺) within total CD3⁺TCRδ⁺ cells. Data are representative of two independent experiments.

2. Frequencies of IL‐23R^+/+ (Thy1.1⁺Thy1.2⁻; white bar) and IL.23R^−/− (Thy1.1⁻Thy1.2⁺; black bar) within CD3⁺TCRδ⁺ cells from the dLN of naïve or CFA‐immunized BMCs.

3. Flow cytometry analysis of IL‐1RI^+/+ (Thy1.1⁺Thy1.2⁻) and IL‐1RI^−/− (Thy1.1⁻Thy1.2⁺) within total CD3⁺TCRδ⁺ cells. Data are representative of two independent experiments.

4. Frequencies of IL‐231^+/+ (Thy1.1⁺Thy1.2⁻; white bar) and IL.1RI^−/− (Thy1.1⁻Thy1.2⁺; black bar) within CD3⁺TCRδ⁺ cells from the dLN of naïve or CFA‐immunized BMCs.

Data information: (A, B) Data pooled from two independent experiments (n = 3–8 mice per group). (C, D) n = 4–5 mice per group. (B, D) Error bars represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 (Mann–Whitney U‐test).

Figure 5. Induced γδ17 T cells make a large contribution to the total γδ17 T‐cell pool in EAE

1. A

   Neonatal thymocytes (NeoThy; Thy1.1⁺Thy1.2⁺) and bone marrow cells (BM; Thy1.1⁺Thy1.2⁻) were injected into lethally irradiated TCRδ^−/− hosts (Thy1.1⁻Thy1.2⁺). After 8 weeks, these NeoThy+BM chimeras were immunized s.c. in both flanks with 125 μg of MOG^(35–55) peptide emulsified in CFA solution; additionally, BMCs were given 200 ng of PTx i.v. on days 0 and 2 p.i. for additional adjuvant effect. Mice were sacrificed on day 14 p.i., at the peak of the disease, and brain, spinal cord, dLN, cLN, and spleen were harvested.

2. B

   Flow cytometry analysis of intracellular IL‐17A and IFN‐γ expression in CD3⁺TCRδ⁺ cells isolated from naïve or EAE‐induced NeoThy+BM chimeras.

3. C

   Frequencies of IL‐17A⁺ cells within the CD3⁺TCRδ⁺ population in the different organs analyzed from naïve (black bar) and EAE‐immunized (red bar) NeoThy+BM chimeras.

4. D, E

   Flow cytometry analysis (D) and frequencies (E) of NeoThy (Thy1.1⁺Thy1.2⁺)‐ versus BM (Thy1.1⁺Thy1.2⁻)‐derived cells within CD3⁺TCRδ⁺IL‐17A⁺Vγ4⁺CD44^hi (left panels) and CD3⁺TCRδ⁺IL‐17A⁺Vγ4⁻CD44^hi (right panels) cells.

Data information: (A–E) Each symbol represents an individual BMC. Error bars represent mean ± SD. Data pooled from two independent experiments (n = 7 mice per group). (C, E) *P < 0.05, **P < 0.01, ***P < 0.001 (Mann–Whitney U‐test)

Figure EV4. γδ17 T cells in naïve NeoThy + BM chimeras are mostly of neonatal thymic origin

1. Flow cytometry analysis of the fraction of Thy1.1⁺Thy1.2⁻ (BM; white) or Thy1.1⁺Thy1.2⁺ (NeoThy; gray) cells among Vγ4⁺ (left) or Vγ4⁻ (right) subsets of CD3⁺TCRδ⁺IL‐17A⁺ lymph node cells from lethally irradiated mice transplanted with both neonatal thymocytes and bone marrow (n = 7 mice). *P < 0.05 (Student's t‐test). Data are representative of two independent experiments.

2. Mice were observed daily and scored for clinical signs of EAE.

3. Pie chart distribution of TCR‐Vγ chain usage of γδ17 T cells from different organs of naïve (left) or EAE‐immunized (right) NeoThy+BM chimeras, as determined by flow cytometry analysis of TCR‐Vγ1 and TCR‐Vγ4 expression within the CD3⁺TCRδ⁺IL‐17A⁺ population.

4. Frequencies of Vγ4⁺CD44^hi cells within the CD3⁺TCRδ⁺IL‐17A⁺ population determined by flow cytometry in different organs of naïve (black bar) or EAE‐immunized (red bar) NeoThy + BM chimeras.

Data information: (A–D) Data pooled from two independent experiments. (D) Error bars represent mean ± SD. **P < 0.01; (Mann–Whitney U‐test).