Novel Th17 Lymphocyte Populations, Th17.1 and PD1+Th17, are Increased in Takayasu Arteritis, and Both Th17 and Th17.1 Sub-Populations Associate with Active Disease

Figures & data

Figure 1 Representative flow cytometry plots. The presented plots denote gating for lymphocytes (A), gating for CD4+ lymphocytes (B), Th1 lymphocytes (C), Th2 lymphocytes (D), Th17 lymphocytes (E), Th17.1 lymphocytes (F), PD1+ CD4+ T lymphocytes (G), PD1+ Th17 lymphocytes (H) and T regulatory lymphocytes (I). The red markings indicate gating for lymphocytes on forward scatter vs side scatter plot. The blue boxes indicate gating for CD4+ lymphocytes within the lymphocyte gate.

Abbreviations: CD, Cluster of differentiation, FoxP3, Forkhead box P3, IFN-γ, Interferon gamma, IL, Interleukin; PD1, Programmed cell death 1, Th, T helper; UL, Upper left quadrant; UR, Upper right quadrant, LL, Lower left quadrant, LR, Lower right quadrant.

Table 1 Characteristics of Patients with Takayasu Arteritis

Characteristic	Number (%) (n=57)
Clinical features at presentation
Systemic features	22 (39)
Hypertension	44 (77)
Stroke	8 (14)
Dizziness or syncope	11 (19)
Vascular bruits	46 (81)
Pulse inequality	34 (60)
Pulse loss	41 (72)
Limb claudication	18 (32)
Carotidodynia	7 (12)
Aortic regurgitation	3 (5)
Heart failure	3 (5)
Angiographic subtype (Numano’s)^Citation41
I	11 (19)
IIa	1 (2)
IIb	5 (9)
III	0 (0)
IV	7 (12)
V	33 (58)
Treatment-naive	24 (42)
Active disease	30 (53)
C-reactive protein (mg/dL) [median (interquartile range)]	0.69 (0.32–1.79)
ITAS2010 [median (interquartile range)]	0 (0–6)
ITAS-A CRP [median (interquartile range)]	2 (0–6)
Immunosuppressive medications at study recruitment	Corticosteroids (n=27) Methotrexate (n=14) Tacrolimus (n=5) Mycophenolate mofetil (n=5) Azathioprine (n=3) Hydroxychloroquine (n=1)

Abbreviations: ITAS2010, Indian Takayasu Clinical Activity Score; ITAS-A CRP, Indian Takayasu Clinical Activity Score adjusted to include C-reactive protein.

Figure 2 Comparison of T helper lymphocyte populations between patients with Takayasu arteritis (TAK), healthy controls (HC) and disease control group of sarcoidosis (SAR). (A) Th1 lymphocytes (B) Th2 lymphocytes (C) Th17 lymphocytes (D) T regulatory lymphocytes (Treg) (E) Th17.1 lymphocytes (F) PD1+CD4+ T lymphocytes (G) PD1+Th17 lymphocytes (H) Th1 lymphocytes normalised to Tregs (I) Th2 lymphocytes normalised to Tregs (J) Th17 lymphocytes normalised to Tregs (K) Th17.1 lymphocytes normalised to Tregs (L) PD1+CD4+ T lymphocytes normalised to Tregs (M) PD1+Th17 lymphocytes normalised to Tregs. Data on T helper lymphocyte populations are presented for 57 patients with TAK [except for Th17.1 lymphocytes (n= 56) and PD1+CD4+ and PD1+Th17 T lymphocytes (n=52)], 21 healthy controls and 11 patients with sarcoidosis. Comparisons are presented between TAK and HC, and between TAK and SAR (p values presented for Mann–Whitney test after Bonferroni-Sidak correction). p values <0.05 are highlighted in bold.

Abbreviations: CD, Cluster of differentiation; PD1, Programmed cell death 1; Th, T helper; Treg, T regulatory.

Figure 3 Comparison of cytokines in serum and in culture supernatant of peripheral blood mononuclear cells following stimulation with anti-CD3/ anti-CD28 between patients with Takayasu arteritis (TAK), healthy controls (HC) and disease control group of sarcoidosis (SAR). (A) IFN-γ in serum (B) IFN-γ in culture supernatant (C) IL-4 in serum (D) IL-4 in culture supernatant (E) IL-17A in serum (F) IL-17A in culture supernatant (G) IL-6 in serum (H) IL-6 in culture supernatant (I) IL-23 in serum (J) IL-23 in culture supernatant (K) TGF-β1 in serum (L) TGF-β1 in culture supernatant (M) IL-10 in serum (N) IL-10 in culture supernatant) (O) TNF-α in serum (P) TNF-α in culture supernatant (Q) IL-1β in serum (R) IL-1β in culture supernatant The cytokines IFN-γ, IL-4, IL-17A, IL-23, IL-1β and IL-10 were undetectable in serum in most patients and controls. In such instances, the lower limit of the detectable range for the ELISA kit was used for graphical representation of data. Data on serum cytokines presented for 56 patients with TAK, 21 healthy controls and 11 patients with sarcoidosis. Data on cytokines in culture supernatant are presented for 52 patients with TAK, 21 healthy controls and 11 patients with sarcoidosis. Comparisons are presented between TAK and HC, and between TAK and SAR (p values presented for Mann–Whitney test after Bonferroni-Sidak correction). p values <0.05 are highlighted in bold.

Abbreviations: IFNγ, Interferon gamma; IL, Interleukin; TGF-β1, Transforming growth factor beta 1; TNF-α, Tumor necrosis factor alpha.

Table 2 Comparison of T Helper Lymphocyte Profiles and Cytokines Between Patients with Takayasu Arteritis with Active and Inactive Disease

	Active Takayasu Arteritis [Median (Interquartile Range)]	Inactive Takayasu Arteritis [Median (Interquartile Range)]	p value (Mann Whitney Test)	p value Corrected for Multiple Testing
C-reactive protein (mg/dL)	0.83 (0.34–4.45) (n=30)	0.63 (0.15–1.44) (n=27)	0.162	0.162
T helper lymphocyte populations	n=30	n=27
Th1 (% CD4+)	62.30 (53.48–68.01)	50.02 (39.80–62.30)	0.034	0.214
Th2 (% CD4+)	45.95 (26.10–56.05)	38.44 (26.64–46.65)	0.241	0.854
Th17 (% CD4+)	2.06 (1.10–2.95)	1.10 (0.60–1.71)	<0.001	0.006
Th17.1 (% CD4+ IL17+)	0.96 (0.38–1.45) (n=29)	0.21 (0.08–0.78)	0.004	0.030
PD1+CD4+ (% CD4+)	34.00 (26.01–44.26) (n=25)	27.40 (24.27–37.80)	0.148	0.673
PD1+Th17 (% CD4+ IL17+)	1.10 (0.38–2.83) (n=25)	0.64 (0.41–1.02)	0.086	0.467
Treg (% CD4+)	3.10 (2.21–4.41)	3.11 (2.30–4.10)	0.902	1.000
Th1: Treg	16.94 (13.26–24.07)	14.65 (8.88–22.58)	0.189	0.715
Th2: Treg	12.59 (9.10–19.36)	12.91 (9.15–15.66)	0.531	0.989
Th17: Treg	0.49 (0.35–0.97)	0.31 (0.18–0.52)	0.002	0.012
Th17.1: Treg	0.27 (0.17–0.36) (n=29)	0.10 (0.03–0.33)	0.016	0.091
PD1+CD4+: Treg	9.90 (6.77–13.06) (n=25)	9.22 (6.91–11.36)	0.856	1.000
PD1+Th17: Treg	0.28 (0.18–0.81) (n=25)	0.23 (0.16–0.39)	0.143	0.603
Serum cytokines	n=29	n=27
IFNγ (pg/mL)	9.35 (9.35–9.35)	9.35 (9.35–9.35)	0.612	0.999
IL-4 (pg/mL)	31.30 (31.30–31.30)	31.30 (31.30–31.30)	0.052	0.383
IL-17 (pg/mL)	15.60 (15.60–15.60)	15.60 (15.60–15.60)	0.360	0.982
IL-6 (pg/mL)	455.50 (270.00–697.25)	478.50 (304.50–769.50)	0.476	0.997
IL-23 (pg/mL)	125.00 (125.00–125.00)	125.00 (125.00–125.00)	0.612	0.999
TGF-β1 (pg/mL)	1680.00 (1439.50–1929.29)	1478.57 (1024.33–1610.00)	0.018	0.151
IL-10 (pg/mL)	31.25 (31.35–31.25)	959.00 (67.00–1644.00)	0.582	0.999
TNF-α (pg/mL)	1016.00 (421.50–1806.50)	67.00 (15.62–959)	0.354	0.980
IL-1β (pg/mL)	3.91 (3.91–3.91)	3.91 (3.91–3.91)	0.817	1.000
Cytokines in culture supernatant after stimulation with anti-CD3/anti-CD28	n=25	n=27
IFNγ (pg/mL)	1104.50 (236.75–1277.25)	987.00 (334.50–1126.00)	0.121	0.687
IL-4 (pg/mL)	31.30 (31.30–150.50)	31.30 (31.30–31.30)	0.016	0.138
IL-17 (pg/mL)	163.00 (15.60–1226.00)	701.00 (15.60–1137.00)	0.641	0.999
IL-6 (pg/mL)	556.50 (472.00–648.50)	521.50 (459.50–586.00)	0.234	0.909
IL-23 (pg/mL)	165.00 (125.00–545.75)	125.00 (125.00–145.00)	0.012	0.102
TGF-β1 (pg/mL)	1732.85 (366.43–1932.07)	1012.24 (254.29–1702.85)	0.165	0.803
IL-10 (pg/mL)	319.00 (174.71–516.86)	417.57 (110.43–613.29)	0.824	1.000
TNF-α (pg/mL)	1745.00 (637.50–1910.00)	1874.00 (1110.00–2087.00)	0.184	0.840
IL-1β (pg/mL)	186.33 (158.67–250.00)	196.00 (139.67–244.83)	0.831	1.000

Notes: p values <0.05 are highlighted in bold.

Abbreviations: CD, Cluster of differentiation; IFNγ, Interferon gamma; IL, Interleukin; PD1, Programmed cell death 1; TGF-β1, Transforming growth factor beta 1; Th, T helper; Treg, T regulatory; TNF-α, Tumor necrosis factor alpha.

Table 3 Logistic Regression Analysis for Associations of Disease Activity in Takayasu Arteritis

Covariates	Odds Ratio (95% Confidence Intervals)	p value
Model 1 (including Th17 lymphocyte population) (n=57)
CRP	1.43 (0.96–2.12)	0.077
Th1	1.01 (0.98–1.05)	0.446
Th2	0.99 (0.94–1.04)	0.785
Th17	2.89 (1.32–6.36)	0.008
Treg	0.87 (0.51–1.47)	0.604
Model 2 (including Th17.1 lymphocyte population) (n=56)
CRP	1.47 (0.95–2.26)	0.081
Th1	1.01 (0.98–1.05)	0.425
Th2	1.00 (0.96–1.05)	0.860
Th17.1	1.97 (0.82–4.74)	0.130
Treg	0.91 (0.55–1.48)	0.689
Model 3 (including PD1+ Th17 lymphocyte population) (n=52)
CRP	1.50 (0.97–2.33)	0.071
Th1	1.02 (0.98–1.05)	0.335
Th2	0.99 (0.94–1.04)	0.693
PD1+ Th17	1.71 (0.91–3.24)	0.098
Treg	1.03 (0.60–1.75)	0.922
Model 4 (including cytokines in serum) (n=56)
CRP	1.56 (0.96–2.51)	0.072
TGF-β1	1.00 (0.99–1.00)	0.212
TNF-α	1.00 (0.99–1.00)	0.362
IL-6	0.99 (0.99–1.00)	0.852

Notes: p values <0.05 are highlighted in bold.

Abbreviations: CRP, C-reactive protein; IL, Interleukin; PD1, Programmed cell death 1; TGF-β1, Transforming growth factor beta 1; Th, T helper; TNF-α, Tumor necrosis factor alpha; Treg, T regulatory.

Table 4 Comparison of T Helper Lymphocyte Profiles and Cytokines Between Patients with Takayasu Arteritis Who Were Immunosuppressive Treatment-Naïve and Those on Immunosuppressive Therapy

	Takayasu Arteritis – Treatment-Naïve [Median (Interquartile Range)]	Takayasu Arteritis – on Treatment [Median (Interquartile Range)]	p value (Mann Whitney Test)	p value Corrected for Multiple Testing
C-reactive protein (mg/dL)	0.66 (0.33–3.49) (n=24)	0.75 (0.27–1.78) (n=33)	0.764	0.764
T helper lymphocyte populations	n=24	n=33
Th1 (% CD4+)	57.03 (42.75–64.65)	60.10 (41.11–70.61)	0.605	0.999
Th2 (% CD4+)	42.45 (26.28–55.76)	40.00 (26.37–51.70)	0.583	0.998
Th17 (% CD4+)	1.60 (0.80–2.85)	1.59 (0.89–2.07)	0.668	0.999
Th17.1 (% CD4+ IL17+)	0.90 (0.31–1.40) (n=23)	0.62 (0.09–1.04)	0.244	0.859
PD1+CD4+ (% CD4+)	28.38 (24.27–37.73) (n=20)	32.33 (24.80–39.18) (n=32)	0.592	0.998
PD1+Th17 (% CD4+ IL17+)	0.68 (0.40–2.10) (n=20)	0.91 (0.44–1.11) (n=32)	0.618	0.999
Treg (% CD4+)	2.75 (2.20–4.11)	3.20 (2.55–4.15)	0.251	0.868
Th1: Treg	16.68 (13.11–24.21)	14.66 (10.30–23.29)	0.408	0.957
Th2: Treg	12.81 (10.44–16.25)	12.30 (8.02–15.76)	0.271	0.849
Th17: Treg	0.42 (0.30–0.79)	0.46 (0.26–0.65)	0.680	0.999
Th17.1: Treg	0.23 (0.12–0.34) (n=23)	0.18 (0.03–0.36)	0.225	0.783
PD1+CD4+: Treg	9.69 (6.88–12.17) (n=20)	8.73 (6.90–12.84) (n=32)	0.918	1.000
PD1+Th17: Treg	0.29 (0.19–0.55) (n=20)	0.26 (0.13–0.40) (n=32)	0.280	0.861
Serum cytokines	n=24	n=32
IFNγ (pg/mL)	9.35 (9.35–9.35)	9.35 (9.35–9.35)	>0.999	1.000
IL-4 (pg/mL)	31.30 (31.30–31.30)	31.30 (31.30–31.30)	0.484	0.997
IL-17 (pg/mL)	15.60 (15.60–15.60)	15.60 (15.60–15.60)	0.714	1.000
IL-6 (pg/mL)	450.75 (259.63–611.38)	491.50 (317.13–772.88)	0.143	0.750
IL-23 (pg/mL)	125.00 (125.00–125.00)	125.00 (125.00–125.00)	0.171	0.815
TGF-β1 (pg/mL)	1550.94 (1050.75–1813.54)	1577.86 (1343.93–1893.93)	0.684	1.000
IL-10 (pg/mL)	31.25 (31.25–31.25)	31.25 (31.25–31.25)	>0.999	1.000
TNF-α (pg/mL)	819.00 (82.75–1594.00)	1228.50 (157.75–1798.75)	0.202	0.869
IL-1β (pg/mL)	3.91 (3.91–3.91)	3.91 (3.91–3.91)	0.300	0.959
Cytokines in culture supernatant after stimulation with anti-CD3/CD28	n=20	n=32
IFNγ (pg/mL)	1058.00 (114.25–1245.88)	1029.50 (548.00–1181.63)	0.952	1.000
IL-4 (pg/mL)	31.30 (31.30–62.25)	31.30 (31.30–37.83)	0.425	0.993
IL-17 (pg/mL)	136.50 (15.60–929.50)	836.00 (15.60–1299.00)	0.173	0.819
IL-6 (pg/mL)	533.25 (440.13–643.50)	530.25 (463.38–602.63)	0.720	1.000
IL-23 (pg/mL)	135.00 (125.00–408.63)	125.00 (125.00–193.75)	0.363	0.983
TGF-β1 (pg/mL)	1116.64 (332.86–1990.29)	1137.14 (278.57–1803.20)	0.439	0.995
IL-10 (pg/mL)	334.71 (132.93–580.43)	319.71 (110.79–609.71)	0.834	1.000
TNF-α (pg/mL)	1810.00 (1206.00–2028.75)	1777.00 (1014.00–2036.25)	0.967	1.000
IL-1β (pg/mL)	204.50 (158.25–250.00)	188.25 (139.04–235.46)	0.408	0.991

Abbreviations: CD, Cluster of differentiation; IFNγ, Interferon gamma; IL, Interleukin; PD1, Programmed cell death 1; TGF-β1, Transforming growth factor beta 1; Th, T helper; Treg, T regulatory; TNF-α, Tumor necrosis factor alpha.

Figure 4 Cytokine production following in-vitro stimulation of peripheral blood mononuclear cells from patients with Takayasu arteritis with anti-CD3/anti-CD28 alone, with tacrolimus, with tadalafil or with a combination of tacrolimus and tadalafil. Cytokines presented in each graph (A) IFN-γ (B) IL-4 (C) IL-17A (D) IL-23 (E) TGF-β1 (F) IL-10 (G) IL-6 (H) IL-1β (I) TNF-α Wherever cytokines were undetectable, the lower limit of the detectable range for the ELISA kit was used for graphical representation of data. Data on cytokines in culture supernatant are presented for 52 patients with TAK. Comparisons are presented between unstimulated and post-stimulation with anti-CD3/anti-CD28, post-stimulation with anti-CD3/anti-CD28 alone or along with tacrolimus, post-stimulation with anti-CD3/anti-CD28 alone or along with tadalafil, and post-stimulation with anti-CD3/anti-CD28 with tacrolimus or with tacrolimus and tadalafil (p values presented for Wilcoxon matched-pairs signed rank test after Bonferroni-Sidak correction). p values <0.05 are highlighted in bold.

Abbreviations: CD, Cluster of differentiation; IFNγ, Interferon gamma; IL, Interleukin; TGF-β1, Transforming growth factor beta 1; TNF-α, Tumor necrosis factor alpha.

Figure 5 T helper lymphocyte profiles in patients with TAK before and after immunosuppressive therapy. (A) Th1 lymphocytes (B) Th2 lymphocytes (C) Th17 lymphocytes (D) T regulatory lymphocytes (Treg) (E) Th17.1 lymphocytes (F) PD1+CD4+ T lymphocytes (G) PD1+Th17 lymphocytes (H) Th1 lymphocytes normalised to Tregs (I) Th2 lymphocytes normalised to Tregs (J) Th17 lymphocytes normalised to Tregs (K) Th17.1 lymphocytes normalised to Tregs (L) PD1+CD4+ T lymphocytes normalised to Tregs (M) PD1+Th17 lymphocytes normalised to Tregs. Data on Th1, Th2, Th17 and Treg are presented for 16 pairs, Th17.1 for 15 pairs, PD1+CD4+ T lymphocytes and PD1+Th17 lymphocytes for 12 pairs. Comparisons are present before and after treatment (p values presented for Wilcoxon matched pairs signed rank test after Bonferroni-Sidak correction). p values <0.05 are highlighted in bold. Median duration of follow-up 0.21 (0.13–2.52) years. All patients had been treated with corticosteroids (ongoing in 15/16 at follow-up sample collection). 9 were on tacrolimus alone, 2 were on tacrolimus and methotrexate, one was on methotrexate alone. At the time of follow-up sample collection, disease activity had ceased clinically in 14/16 patients.

Abbreviations: CD, Cluster of differentiation; PD1, Programmed cell death 1; Th, T helper; Treg, T regulatory.

Figure 6 Cytokine profiles in patients with TAK before and after immunosuppressive therapy. (A) IFN-γ in serum (B) IFN-γ in culture supernatant (C) IL-4 in serum (D) IL-4 in culture supernatant (E) IL-17A in serum (F) IL-17A in culture supernatant (G) IL-6 in serum (H) IL-6 in culture supernatant (I) IL-23 in serum (J) IL-23 in culture supernatant (K) TGF-β1 in serum (L) TGF-β1 in culture supernatant (M) IL-10 in serum (N) IL-10 in culture supernatant) (O) TNF-α in serum (P) TNF-α in culture supernatant (Q) IL-1β in serum (R) IL-1β in culture supernatant. Wherever cytokines were undetectable, the lower limit of the detectable range for the ELISA kit was used for graphical representation of data. Data are presented for 16 pairs for serum cytokines and 12 pairs for culture supernatant for IFN-γ, IL-4, IL-17A, IL-6, IL-23, TGF-β1, IL-10, TNF-α; for 13 pairs for serum cytokines and 13 pairs for culture supernatant for IL-1β. Comparisons are present before and after treatment (p values presented for Wilcoxon matched pairs signed rank test after Bonferroni-Sidak correction). p values <0.05 are highlighted in bold. Median duration of follow-up 0.21 (0.13–2.52) years. All patients had been treated with corticosteroids (ongoing in 15/16 at follow-up sample collection). 9 were on tacrolimus alone, 2 were on tacrolimus and methotrexate, one was on methotrexate alone. At the time of follow-up sample collection, disease activity had ceased clinically in 14/16 patients.

Abbreviations: IFNγ, Interferon gamma; IL, Interleukin; TGF-β1, Transforming growth factor beta 1; TNF-α, Tumor necrosis factor alpha.

Figure 7 Putative mechanisms by which Th17 lymphocytes play a role in the pathogenesis of Takayasu arteritis. When compared with the native arterial wall, the arterial wall in large vessel vasculitis is thickened, with intimal proliferation, thickening of the media due to inflammatory cell infiltrate and adventitial fibrosis. T helper 17 (Th17) lymphocytes secrete the cytokine interleukin 17A (IL-17A) which attracts neutrophils to the media where they degranulate and release enzymes resulting in tissue destruction. Th17.1 lymphocytes secrete interferon gamma (IFN-γ) which contributes towards granuloma formation. Th17.1 lymphocytes also express p-glycoprotein and are resistant to corticosteroids. Programmed cell death 1 (PD1) positive Th17 lymphocytes secrete transforming growth factor beta 1 (TGF-β1) which in turn drives adventitial fibrosis.

Jenkins DG, Quintana-Ascencio PF. A solution to minimum sample size for regressions. PLoS One. 2020;15(2):e0229345. doi:10.1371/journal.pone.0229345

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