Table S1. Baseline characteristics of patients with SLE ... · Baseline characteristics of patients...

Table S1. Baseline characteristics of patients with SLE receiving low-dose rhIL-2 (n=40).

Characteristic Value

Age, year, median (range) 31 (18-60)

Female/Male 37/3

Weight, kg, median (range) 54 (42-80)

Height, cm, median (range) 160 (150-175)Area, m2, median (range) 1.61 (1.36-1.92)Duration of SLE, year, median (range) 5 (0.5-20)

Prednisone dose, mg/day, median (range) 30 (0-60)

Use of concomitant agents (no. of patients)

Hydroxychloroquine 37

Cyclophosphamide 6

Azathioprine 2

Cyclosporine 0

Methotrexate 0

Mycophenolatemofetil 14

Leflunomide 1

Thalidomide 1

Nature Medicine: doi:10.1038/nm.4148

Table S2. Baseline characteristics of patients with SLE treated with conventional therapy (n=15).

Characteristic Value

Age, year, median (range) 30 (18-62)

Female/Male 13/1

Weight, kg, median (range) 55 (44-78)

Height, cm, median (range) 161 (155-175)Area, m2, median (range) 1.59 (1.32-1.89)Duration of SLE, year, median (range) 5 (0.5-18)

Prednisone dose, mg/day, median (range) 30 (10-60)


Hydroxychloroquine 12

Cyclophosphamide 2

Azathioprine 2

Cyclosporine 1

Methotrexate 0

Mycophenolatemofetil 10

Thalidomide 0


Characteristics Baseline week 12 P value

SELENA-SLEDAI, median (range) 11 (8-19) 5 (2-9) 0.002

Prednisone, mg, median (range) 30 (10-60) 25 (20-40) <0.001

Rash, n (%) 5 (33.3) 2 (16.7) 0.208

Oral ulcers, n (%) 1 (6.7) 0 (0) 0.326

Serositis, n (%) 1 (6.7) 0 (0) 0.326

Raynaud’s, n (%) 2 (16.7) 2 (16.7) 1

Alopecia, n (%) 4 (26.7) 2 (16.7) 0.477

Arthritis, n (%) 1 (6.7) 0 (0) 0.326

Leukopenia, n (%) 4 (26.7) 0 (0) 0.032

Thrombocytopenia, n (%) 2 (13.3) 0 (0) 0.153

IgA, g/L, median (range) 2.6 (0.76-4.35) 2.63 (0.7-5.06) 0.0347

IgG, g/L, median (range) 15.2 (5.2-37.5) 10.57(4.1-16.2) 0.0178

IgM, g/L, median (range) 0.94( 0.12-2.42) 0.967 (0.152-1.52) 0.2896

C3, g/L, median (range) 0.62 (0.333-1.18) 0.835 (0.61-1.16) 0.0018

C4, g/L, median (range) 0.157 ( 0.09-0.58) 0.192 (0.072-0.318) 0.2752

Anti-dsDNA, IU/mL, median (range) 698.8 (10-1223.9) 220.3 (2.0-299.2) <0.001

UrinePRO, g/24h, median (range), n=10 2.18 (0.08-12.08) 0.51 (0.0-0.99) 0.0339

Platelet, x1012/L, median (range) 176 (9-429) 227 (70-305) 0.2793

WBC, x109/L, median (range) 4.85 (2.02-12.83) 6.33 (2.92-11.14) 0.3829

Lymphocyte, x109/L, median (range) 1.12 (0.31-3.59) 1.62 (0.66-4.79} 0.0475

Monocyte, x109/L, median (range) 0.34 (0.11-0.77) 0.53 (0.28-1.13) 0.0310

Neutrophil, x109/L, median (range) 3.43 (1.3-9.19) 4.21 (1.87-8.85) 0.6594

Eosinophil, x109/L, median (range) 0.02 (0-0.08) 0.03 (0-0.07) 0.0552

Table S3. Clinical characteristics of patients with SLE during 12 weeks of conventional therapy (n=15).


Table S4. Clinical characteristics before and after low-dose rhIL-2 (n=38).

Characteristics Baseline week 12 P value

SELENA-SLEDAI, median (range) 11 (8-23) 4 (0-12) <0.001

Prednisone, mg, median (range) 30 (0-60) 17.5�0-30� <0.001

Rash, n (%) 24 (63.2) 4 (10.5) <0.001

Oral ulcers, n (%) 3 (7.9) 0 (0) 0.079

Serositis, n (%) 5 (13.2) 0 (0) 0.021

Raynaud’s, n (%) 6 (15.8) 5 13.2) 0.794

Alopecia, n (%) 14 (36.8) 1 (2.6) 0.034

Arthritis, n (%) 11 (28.9) 1 (2.6) 0.013

Leukopenia, n (%) 19 (50.0) 1 (2.6) <0.001

Thrombocytopenia, n (%) 4 (10.5) 0 (0) <0.001

IgA, g/L, median (range) 2.6 (0.81-6.5) 2.76 (1.1-6.0) 0.016

IgG, g/L, median (range) 14.2 (5.4-35.9) 11.3 (3.6-22.5) <0.001

IgM, g/L, median (range) 0.94( 0.12-2.42) 0.967 (0.152-1.52) 0.217

C3, g/L, median (range) 0.42 (0.118-1.46) 0.809 (0.48-1.23) <0.001

C4, g/L, median (range) 0.075 ( 0.017-0.54) 0.158 (0.072-0.38) <0.001

Anti-dsDNA, IU/mL, median (range) 417.9 (10-3987.5) 175.9 (3.8-2413.2) <0.001

UrinePRO, g/24h, median (range), n=18 2.71 (0.68-21.78) 0.83 (0.0-5.8) 0.005

Platelet, x1012/L, median (range) 161 (62-337) 218 (119-301) <0.001

WBC, x109/L, median (range) 3.95 (1.65-11.74) 8.1 (2.56-10.47) <0.001

Lymphocyte, x109/L, median (range) 0.81 (0.17-2.22) 1.60 (0.54-2.89} <0.001

Monocyte, x109/L, median (range) 0.31 (0.09-0.81) 0.49 (0.29-0.91) 0.034

Neutrophil, x109/L, median (range) 2.34 (0.79-10.57) 4.86 (0.12-8.52) 0.001

Eosinophil, x109/L, median (range) 0.02 (0-0.11) 0.03 (0-0.17) <0.001


Table S5. Adverse events during low-dose rhIL-2 administration (n=38)

Adverse events No. of Patients

Injection-site reaction 5

Fatigue 1

Fever 1Infection 0


!

Table S6. Clinical responses to rhIL-2 treatment in patients with SLE (including clinical benefits and adverse effects).

SN Response in clinical

manifestations

SLEDAI changes pre and

post therapy

Response description Description of rhIL-2 related adverse effects

1 Remission in proteinuria and alopecia 10-6=4

1. Proteinuria decreased from 1.43 to 0.82g/d and the hairs became thick in 3 courses of treatment. 2. Laboratory variables: anti-dsDNA antibodies decreased from 229.48 to 125.67 IU/ml; AnuA decreased from 62.54 to 34.07 IU/ml; C3 increased from 0.643 to 0.819 g/l; C4 increased from 0.072 to 0.13 g/l in 3 courses.

Weak local reactions at

injection sites.

2 Remission in facial rash, oral ulcers and alopecia.

10-4=6

1. Decreased facial rash, heal of oral ulcers and thick hairs achieved in three courses. 2. Laboratory variables: anti-dsDNA decreased from 347.69 to 233.12IU/ml; AnuA decreased from 66.85 to 23.22 IU/ml; C3 increased from 0.622 to 0.719 g/l; C4 increased from 0.165 to 0.179 g/l; Acl antibodies decreased from 36.4 to 22 U/ml in 10 weeks.

None.

3 Remission in fever, arthritis leucopenia and anemia.

10-2=8

1. Fever reduced to normal rapidly in one injection. Arthralgia relieved in 2 courses of treatment. 2. Laboratory variables: WBC and RBC recovered to normal after 1 week of treatment. Anti-dsDNA decreased from 287.01 to 130.73 IU/ml; AnuA decreased from 85.63 to 28.9 IU/ml; C3 increased from 0.298 to 0.895 g/l; C4 increased from 0.065 to 0.205 g/l in 3 courses.


injection sites during the first 2

injections.


!

4 Remission in fever, rash , arthralgia, proteinuria, thrombocytopenia and cough.

16-2=14

1. Fever recovered to normal since the second day of the rhIL-2 treatment. Malar rash disappeared and PLT increased from 70 to 157×109/L in one course. Arthralgia and subjective breathing improved in 3 courses . 2. Proteinuria decreased from 0.87 to 0.38 g/d in 3 courses. 3. Laboratory variables: anti-dsDNA decreased from 189.14 to 42.35 IU/ml; AnuA decreased from 127.83 to 8.7 IU/ml; C3 increased from 0.118 to 0.763 g/l; C4 increased from 0.113 to 0.171 g/l in 10 weeks.

None.

5 Remission in fever, headache, alopecia, rash and proteinuria. 21-2=19

1.! The treatment caused a rapid reduction in fever and improvement in headache after one week. Rash decreased and partially thick hairs achieved in 10 weeks.

2.! Proteinuria decreased from 0.83 to 0.1 g/d. 3.! Laboratory variables: anti-dsDNA decreased from 618.46 to 300.67 IU/mL;

AnuA decreased from 489.8 to 169.7 IU/ml; C3 increased from 0.37 to 0.88 g/l; C4 increased from 0.076 to 0.32 g/l in 3 courses.

None.

6 Remisson in headache, edema, oliguria and hypertension.

14-6=8

1. The headache recovered in one course of treatment. Edema, oliguria and hypertension improved in 8 weeks of treatment. No diuretics were needed. 2. Laboratory variables: proteinuria decreased from 4.97 to 1.76 g/d; serum albumin increased from 24.7 to 36.9 g/l; C3 increased from 0.534 to 0.829 g/l; C4 increased from 0.159 to 0. 282 g/l in 3 courses.

None.

7 Remission in proteinuria

8-4=4

1. Proteinuria decreased from 20.08 to 3.2 g/d and serum albumin increased from 20.9 to 25 g/l in 3 courses. 2. Laboratory variables: AnuA decreased from 46.21 to 6.4 IU/ml; C3 increased from 0.305 to 0.973 g/l; C4 increased from 0.087 to 0.219 g/l in 2 courses. 3. The patient changed to belimumab treatment due to personal preference.

None. The patient

discontinued because he was

willing to accept Belimumab


!

treatment. 8 Remission in fever,

rash, alopecia, headache, oral ulcers and leukopenia

20-4=16

1. Headache and fever were controlled and WBC increased from 3.37 to 8.63×109/L in one course. The hairs became thick and rash decreased with a continuation of improvement at 10 weeks compared with 2 weeks after treatment. 2. Laboratory variables: anti-dsDNA decreased from 1277.88 to 328.62 IU/ml; AnuA decreased from 1991.9 to 66.9 IU/ml; C3 increased from 0.239 to 0.721 g/l; and C4 increased from 0.032 to 0.13 g/l in 10 weeks.

None and the patient decided to continue rhIL-2

treatment.

9 Remission in rash, edema, hypertension, polyserositis, anemia and oliguria.

16-6=10

1.! The rash, edema, hypertension and polyserositis improved gradually, and completely remitted in two courses.

2.! Hb increased from 78 to 118 g/l and serum albumin increased from 25.2 to 38.3 g/l in one course.

3.! Laboratory variables: proteinuria decreased from 3.51 to 1.5g/d; anti-dsDNA decreased from 748.86 to 215.6 IU/ml; AnuA decreased from 135.4 to 85.4 IU/ml; C3 increased from 0.212 to 0.723 g/l; and C4 increased from 0.042 to 0.147 g/l in 3 courses.

None.

10 Remission in alopecia, proteinuria and leukopenia 15-8=7

1.! The weakened hairs became thick in two courses of treatment. 2.! WBC increased from 2.35 to 3.52×109/L in 2 weeks. Proteinuria decreased

from 2.82 to 1.53 g/d and serum albumin increased from 26.1 to 37.6 g/d after 4 weeks.

3.! Laboratory variables: anti-dsDNA decreased from 1489.22 to 433.05 IU/ml; AnuA decreased from 1918.3 to 313.2 IU/ml; C3 increased from 0.396 to 0.556 g/l; and C4 increased from 0.073 to 0.139 g/l in 3 courses.

None.

11 Remission in rash, alopecia, oral and foot ulcers and

19-2=17 1.! The rash faded gradually with treatment. Moreover, oral and foot ulcers

shrinked and alopecia improved in 3 courses. 2.! WBC increased from 1.65 to 6.16×109/L and albumin increased from 28.2 to

None.


!

leukopenia 40.9 g/l in 2 weeks. 3.! Laboratory variables: anti-dsDNA decreased from 1027.11 to 126.67 IU/ml;


12 Remission in rash, proteinuria, edema, hypertension, and polyserositis. 10-6=4

1.! Edema, hypertension, polyserositis and oliguria relieved and rash disappeared in 10 weeks.

2.! Proteinuria decreased from 21.78g to 7.82 g/d and albumin increased from 19.1 to 29.6 g/l.

3.! Laboratory variables: anti-dsDNA decreased from 22 to 4.5 IU/ml; AnuA decreased from 12.13 to 7.88 IU/ml; C3 increased from 0.407 to 0.594 g/l; C4 increased from 0.173 to 0.333 g/l in 3 courses of treatment.

None.

13 Remission in fever, proteinuria, polyserositis and thrombocytopenia. 16-5=11

1.! Fever reduced to normal rapidly and PLT increased from 37 to 219×109/L in one course. Polyserositis relieved after 2 courses of treatment.

2.! Proteinuria decreased from 12.14 to 0.18 g/d and albumin increased from 25.4 to 41.4 g/l.

3.! Laboratory variables: anti-dsDNA decreased from 275.13 to 138.62 IU/ml; AnuA decreased from 105.3 to 32.97 IU/ml; C3 increased from 0.265 to 0.448 g/l; and C4 increased from 0.054 to 0.105 g/l in 3 courses of treatment.

None.

14 Remission in rash and leukopenia.

8-4=4

1. The facial rash faded in one course of treatment. WBC increased from 2.21 to 9.2×109/L in 2 weeks. 2. Laboratory variables: anti-dsDNA decreased from 781.97 to 179.11 IU/ml; AnuA decreased from 324.2 to 23.75 IU/ml; C3 increased from 0.385 to 0.836 g/l; C4 increased from 0.036 to 0.105 g/l in 3 courses.

None.

15 No remisson in proteinuria.

8-8=0 1. Proteinnuria barely improved. 2. Laboratory variables: anti-dsDNA decreased from 202.48 to 116.01 IU/ml and

None.


!

AnuA decreased from 65.7 to 21.02 IU/ml in 3 courses of treatment. 16 Remission in rash,

alopecia, edema, oliguria proteinuria and polyserositis.

18-4=14

1. Rash disappeared in 1 course. Edema, polyserositis and oliguria relieved in 6 weeks of treatment, as well as and alopecia. 2. Proteinuria decreased from 11.47 to 3.95 g/d after 3 courses. Serum albumin increased from 21.2 to 27.4 g/l in 2 weeks. 3. Laboratory variables: anti-dsDNA decreased from 267.27 to 54.77 IU/ml; AnuA decreased from 166.34 to 15.4 IU/ml; C3 increased from 0.193 to 0.809 g/l; C4 increased from 0.123 to 0.357 g/l; Acl antibodies decreased from 16.2 to 3.2 U/ml in 3 courses of treatment.

None.

17 Remission in fever, rash, arthritis, polyserositis, proteinuria and pancytopenia. 19-6=13

1.! The fever reduced to normal and rash faded in 1 course of treatment. 2.! Arthritis and polyserositis relieved; proteinuria decreased from 0.68 to

0.14g/d; WBC increased from 2.91 to 3.78×109/L; Hb increased from 81 to 111g/l; and PLT increased from 21 to 147×109/L in 8 weeks.

3.! Laboratory variables: anti-dsDNA decreased from 463.22 to 331.23 IU/ml; AnuA decreased from 1117.5 to 124.89 IU/ml; C3 increased from 0.394 to 0.845 g/l; C4 increased from 0.159 to 0.19 g/l in 2 courses of treatment.

4.! The patient was changed to cyclophosphamide treatment due to her wish to reduce the frequency of hospital visits.

None. Discontinued at the

beginning of the third course.

18 Remission in fever, rash, arthritis and leukopenia

10-4=6

1.! A rapid reduction in fever achieved and WBC increased from 2.43 to 12.96 ×109/L after 2 weeks of treatment.

2.! Laboratory variables: anti-dsDNA decreased from 846.5 to 278.5 IU/ml; AnuA decreased from 868.2 to 106.04 IU/ml; C3 increased from 0.438 to 0.594 g/l; C4 increased from 0.148 to 0.178 g/l; Acl antibodies decreased from 33.1 to 6.8 U/mL; and β2-GPI decreased from 17.3 to 8.59 RU/mL in 3 courses of treatment.

None.


!

19 Remission in proteinuria, alopecia, temporary spasm and pancytopenia.

20-8=12

1.! Temporary spasm did not re-attack. The weakened hairs got thick in 3 courses of treatment.

2.! WBC and PLT changed to normal in 2 courses. Proteinuria decreased from 0.98 to 0.5 g/d in 3 courses.


None.

20 Complete remission in rash, arthritis and leucopenia.

11-0=11

1.! Rash disappeared; arthritis relieved; and WBC increased from 2.57 to 9.04×109/L in 1 course.

2.! Laboratory variables: anti-dsDNA decreased from 199.14 to 10 IU/ml; AnuA decreased from 36.82 to 2 IU/ml; C3 increased from 0.541 to 0.827 g/l; C4 increased from 0.138 to 0.204 g/l in 3 courses of treatment.

None.

21 Remission in fever, rash and arthritis.

11-2=9

1.! Fever reduced rapidly since the second day of treatment and rash disappeared in 1 course of treatment. Arthritis relieved in 2 courses.

2.! Laboratory variables: anti-dsDNA decreased from 423.5 to 105.079 IU/mL; AnuA decreased from 200 to 17.89 IU/mL; C3 increased from 0.532 to 0.995 g/l; C4 increased from 0.073 to 0.137 g/l in 3 courses of treatment.

None.

22 Remission in arthritis and leukopenia. 11-4=7

1.! Arthritis relieved in 3 courses of treatment, but malar rash still existed. 2.! The titer of related antibodies was high at baseline. Anti-dsDNA decreased

from 423.5 to 105.079 IU/ml; AnuA decreased from 200 to 17.89 IU/ml; C3 increased from 0.532 to 0.995 g/l; C4 increased from 0.073 to 0.137 g/l in 3 courses.

None.

23 Remission in fever and leucopenia, and partial

12-6=6 1.! Fever recovered to normal until two weeks of treatment. The patient still had

rash and arthritis (slight improvement) after 3 courses. 2.! WBC increased from 2.56 to 6.67×109/L in two weeks.

Mild fever and weak local reactions at


!

remission in rash and arthritis.

3.! The laboratory variables did not improved significantly. Anti-dsDNA decreased from 1235.5 to 1021.84 IU/ml; AnuA fluctuated from 238.57 to 245.54 IU/ml; C3 increased from 0.423 to 0.475 g/l; C4 increased from 0.057 to 0.132 g/l in 3 courses.

injection sites happened in the

first course.

24 Remission in proteinuria and pancytopenia. 12-4=8

1.! WBC increased from 2.63 to 4.6×109/L and PLT increased from 90 to 100×109/L in 1 course. Protein decreased from 3.3 to 1.33 g/d.

2.! Laboratory variables: anti-dsDNA decreased from 386.57 to 112.606 IU/ml; AnuA decreased from 176.04 to 12.74 IU/ml; C3 increased from 0.607 to 1.13 g/l; C4 increased from 0.071 to 0.2 g/l in 3 courses.

None.

25 Remission in proteinuria and leucopenia. 9-6=3

1.! WBC increased from 3.09 to 8.99×109/L in 1 course. Protein decreased from 1.61 to 1.17 g/d in 3 courses.


None.

26 Remission in fever, rash, alopecia and leukopenia. 10-2=8

1.! Fever recovered to normal; rash disappeared; and WBC increased from 2.67 to 7.55×109/L in 1 course. The weakened hairs became thick in 3 courses.

2.! Laboratory variables: anti-dsDNA decreased from 1586.97 to 305.86 IU/ml; AnuA decreased from 594.85 to 48.86 IU/ml; C3 increased from 0.294 to 1.09 g/l; C4 increased from 0.155 to 0.374 g/l after 3 courses of treatment.

None.

27 Partial remission in leucopenia, not in rash, alopecia and proteinuria.

11-8=3

1.! WBC increased from 2.67 to 3.5 in 1 course of treatment. Rash and alopecia and proteinuria (from 1.7 to 2.27g) did not improved.

2.! Laboratory variables: C3 increased from 0.385 to 0.684 g/l; C4 increased from 0.06 to 0.118 g/l in 3 courses. The titer of anti-dsDNA and AnuA varied slightly.

None.

28 Remission in rash 8-4=4 1.! Rash disappeared in one course and alopecia relieved in 3 courses. None.


!

and alopecia. 2.! Laboratory variables: anti-dsDNA decreased from 344.47 to 253.597 IU/ml; AnuA decreased from 19.48 to 15.87 IU/ml; C3 increased from 0.751 to 0.757 g/l; C4 increased from 0.084 to 0.117 g/l after 3 courses of treatment.

29 Remission in alopecia and proteinuria. 10-6=4

1.! The thick hairs achieved in 2 courses of treatment. Proteinuria decreased from 2.6 to 2.0 g/d in 3 courses.


None.

30 Remission in rash and alopecia.

8-4=4

1.! Rash disappeared and alopecia relieved in 1 course of treatment. 2.! Laboratory variables: anti-dsDNA decreased from 638.11 to 369.475 IU/ml;

AnuA decreased from 151.846 to 82.91 IU/ml; Complement slightly fluctuated (C3 from 0.839 to 0.744 g/l and C4 from 0.202 to 0.213 g/l, respectively).

None.

31 Remission in fever, malar rash, alopecia and leucopenia.

8-4=4

1.! Fever recovered to normal and malar rash faded in 1 course. Alopecia became thick and rash completely disappeared in 3 courses.

2.! WBC increased from 3.86 to 9.54×109/L in 1 course. 3.! Laboratory variables: anti-dsDNA decreased from 513.74 to 352.933 IU/ml;


None.

32 Remission in fever, rash, proteinuria and anemia. 15-2=13

1.! Fever recovered to normal and rash faded in 1 course. Proteinuria decreased from 1.96 to 0.19 g/d and Hb increased from 86 to 100 g/l in 3 courses.


Weak local reactionsat

injection sites in first 3 injections.

33 Remission in fever, 8-2=6 1.! The hairs became thick and malar rash faded in 1 course of treatment. None.


!

malar rash and leucopenia.


34 Remission in rash, alopecia and arthritis. 12-0=12

1.! Rash disappeared in 1 course. Alopecia and arthritis relieved in two courses. 2.! Laboratory variables: anti-dsDNA decreased from 116.06 to 26.35 IU/ml;

AnuA decreased from 25.54 to 2.16 IU/ml; Acl antibodies decreased from 15.8 to 5.7 U/ml; C3 increased from 0.497 to 0.809 g/l; C4 increased from 0.067 to 0.123 g/l in 3 courses.


injection sites.

35 Remission in edema and oliguria.

8-6=2

1.! Edema and oliguria relieved in one course. Protein decreased from 3.49 to 1.23g/d in 3 courses.

2.! Laboratory variables: the titer of anti-dsDNA and complement fluctuated slightly.

None.

36 Remission in fever, leucopenia and proteinuria. 8-3=5

1.! The temperature changed to normal in 1course. WBC increased from 3.11 to 3.36×109/L in 1 course and fluctuated in this level in 3 courses.

2.! Laboratory variables: Proteinuria decreased from 0.42 to 0.2 g/d; anti-dsDNA decreased from 280.5 to 182.83 IU/ml; AnuA decreased from 70.49 to 43.87 IU/ml; the complement was nearly stable.

Fatigue in the first course of treatment.

37 Remission in arthritis.

8-2=6

1.! Arthritis relieved in 1 course of treatment. 2.! Laboratory variables: anti-dsDNA decreased from 158.23 to 110.37 IU/ml;

C3 increased from 0.593 to 0.799 g/l; C4 increased from 0.104 to 0.14 g/l in 3 courses of treatment.

None.

38 Remission in alopecia and leucopenia.

9-4=5

1.! Malar rash disappeared in 1 course, but emerged in 3 courses. WBC increased from 2.7 to 7.08×109/L in 1 course. Alopecia relieved in 2 courses.

2.! Laboratory variables: anti-dsDNA decreased from 144.53 to 53.31 IU/ml; AnuA decreased from 19.56 to 6.11 IU/ml; C3 increased from 0.382 to 0.654

None.


!

g/l; C4 increased from 0.065 to 0.106 g/l in 3 courses. 39 Remission in fever,

malar rash, alopecia and leucopenia

10-4=6

1.! The temperature recovered to normal in 1 week. Malar rash disappeared and thick hairs achieved in three courses.

2.! WBC increased from 3.02 to 6.09×109/L in one course. 3.! Laboratory variables: anti-dsDNA decreased from 584.11 to 433.97 IU/ml;

AnuA decreased from 188.06 to 87.6 IU/ml in 3 courses.

None.

40 Remission in fever, malar rash and arthritis.

11-4=7

1.! Malar rash decreased and arthritis relieved in 2 courses. 2.! Laboratory variables: anti-dsDNA decreased from 1514.04 to 336.703 IU/ml;


None.

SLEDAI: Systemic Lupus Erythematosus Disease Activity Index (Safety of Estrogens in Lupus Erythematosus National Assessment version). WBC: white blood cells; Hb: hemoglobin; PLT: platelet; AnuA: Anti-nucleosome Antibody; C3: complement C3; C4: complement C4; Acl: anticardiolipin.


Table S7. Comparison of baseline characteristics between the group undergoing T cell immunological profiling (n=23) and the group without undergoing such profiling (n=15) among patients with SLE receiving low-dose IL-2 therapy.

CharacteristicWith T cell

immunologicalprofiling (n=23)

Without T cell immunological

profiling (n=15)P value

Age, year, median (range) 31 (18-60) 29(21-50) 0.361

Female/Male 21/2 14/1 0.826

Duration of SLE, year, median (range) 5 (0.5-15) 3(0.5-20) 0.577

Prednisone dose, mg/day, median (range) 30 (0-50) 35(15-60) 0.372


Hydroxychloroquine 23 12 ---

Cyclophosphamide 1 4 ---

Azathioprine 1 1 ---

Cyclosporine 1 4 ---

Methotrexate 0 0 ---

Mycophenolatemofetil 10 4 ---

Leflunomide 1 0 ---

Thalidomide 0 1 ---


Table S8. Comparison of clinical characteristics between the group with undergoing T cell immunological profiling (n=23) and the group without undergoing such profiling (n=15) among patients with SLE before and after receiving low-dose IL-2 therapy With T cell immunological

profiling (n=23) Without T cell immunological

profiling (n=15) P value

before after before after before after

SELENA-SLEDAI, median (range) 12 (8-23) 4 (0-12) 11 (8-18) 4(0-6) 0.840 0.770

Prednisone, mg (range) 30 (0-50) 17.5�0-30� 40 (15-60) 17.5 (10-30) 0.309 0.591

Rash, n (%) 15 (65.2) 3 (13.0) 9 (60.0) 1 (6.7) 0.753 0.544

Oral ulcers, n (%) 0 (0) 0 (0) 3 (20.0) 0 (0) 0.025 1

Serositis, n (%) 0 (0) 0 (0) 5 (33.3) 0 (0) 0.002 1

Raynaud’s, n (%) 4 (17.4) 3 (13.0) 2 (13.3) 2 (13.3) 0.746 0.980

Alopecia, n (%) 7 (30.4) 1 (4.35) 7 (46.7) 0 (0) 0.407 0.427

Arthritis, n (%) 8 (34.8) 1 (4.35) 3 (20.0) 0 (0) 0.339 0.427

Leukopenia, n (%) 13 (56.5) 1 (4.35) 6 (40.0) 0 (0) 0.333 0.427

Thrombocytopenia, n (%) 2 (8.7) 0 (0) 2 (13.3) 0 (0) 0.659 1

IgA, g/L, median (range) 2.65 (1.39-6.5) 2.86 (1.1-6.0) 2.6 (0.81-5.35) 2.76 (1.43-3.6) 0.191 0.322

IgG, g/L, median (range) 16.1 (8.2-35.9) 11.8 (5.8-22.5) 13.9 (5.4-21.5) 11.3 (3.6-15.7) 0.218 0.156

IgM, g/L, median (range) 0.967 (0.152-1.52) 0.967 (0.152-1.52) 0.94 (0.12-2.42) 0.940 ( 0.12-2.42) 0.217 0.217

C3, g/L, median (range) 0.46 (0.193-1.46) 0.809 (0.48-1.13) 0.37 (0.118-0.806) 0.814 (0.52-0.987) 0.787 0.150

C4, g/L, median (range) 0.090 ( 0.017-0.54) 0.178 (0.072-0.38) 0.072 (0.032-0.224) 0.160 ( 0.081-0.312) 0.542 0.823

Anti-dsDNA, IU/mL, median (range) 417.9 (71.4-3987.5) 233.1 (10-2413.2) 347.7 (10-1489.2) 123.2 (3.8-717.9) 0.459 0.239

UrinePRO, g/24h, median (range), n=18 1.70 (0.68-11.47) 1.20 (0.0-3.95) 2.71 (0.68-21.78) 1.53 (0.6-7.9) 0.204 0.578

Platelet, x1012

/L, median (range) 160 (62-326) 213 (110-298) 162 (69-337) 161 (62-337) 0.494 0.380

WBC, x109/L, median (range) 3.70 (1.93-7.87) 7.1 (2.56-10.4) 4.75 (1.65-11.74) 8.2 (2.65-10.74) 0.308 0.146

Lymphocyte, x109/L, median (range) 0.79 (0.17-2.22) 1.60 (0.46-2.65) 0.95 (0.25-2.06) 1.75 (0.6-2.41) 0.160 0.176

Monocyte, x109/L, median (range) 0.27 (0.09-0.71) 0.44 (0.29-0.91) 0.47 (0.15-0.81) 0.65 (0.21-0.85) 0.112 0.153

Neutrophil, x109/L, median (range) 2.31 (0.9-9.53) 3.81 (0.12-8.52) 2.53 (0.79-10.57) 4.54 (0.51-10.04) 0.168 0.158

Eosinophil, x109/L, median (range) 0.02 (0-0.10) 0.03 (0-0.17) 0.02 (0-0.11) 0.04 (0-0.11) 0.656 0.574

!


!

Table S9. Clinical responses to conventional treatment in patients with SLE (including clinical benefits and adverse effects).

SN Response in clinical

manifestations

SLEDAI changes pre and

post therapy

Response description Description of related adverse

effects

1 Remission in proteinuria and alopecia

10-4=6 1. Proteinuria decreased from 12.08 to 0.99g/d and alopecia improved. 2. Laboratory variables: anti-dsDNA antibodies decreased from 626.05 to 45.27 IU/ml; C3 increased from 0.552 to 0.809 g/l.

None

2 Remission in headache and proteinuria.

16-8=8

1. Headache recovered after high-dose corticosteroid therapy. Proteinuria decreased over three months. 2. Laboratory variables: anti-dsDNA decreased from 593.22 to 100.54 IU/ml; C3 increased from 0.395 to 0.771 g/l; C4 increased from 0.038 to 0.196 g/l.

None.

3 Remission in leucopenia.

9-5=4

1. Laboratory variables: WBC recovered to normal in 3 months. Anti-dsDNA decreased from 248.8 to 224.55 IU/ml; AnuA decreased from 44.35 to 26.2 IU/ml; C3 increased from 0.627 to 0.767g/l; C4 increased from 0.106 to 0.169g/l in 3 months.

Upper respiratory tract infection

4 Slightly decrease in proteinuria. 8-8=0

1. Proteinuria decreased slightly in 3 months.. 2. Laboratory variables: there were no significant changes in the level of anti-dsDNA, ANA and complement.

Urinary tract infection

5 Remission in fever, polyserositis , leucopenia and

12-4=8 1.! The treatment caused a rapid reduction in fever in 2 weeks. The polyserositis

was improved at 3 months. 2.! Proteinuria decreased from 0.61 to 0.31 g/d.

None.


!

proteinuria. 3.! Laboratory variables: WBC increased from 2.77×10^9/l to 10.51×10^9/l; anti-dsDNA decreased from 761.58 to 214.3 IU/mL; C3 increased from 0.357 to 0.776 g/l; C4 increased from 0.064 to 0.192 g/l in 3 months.

6 Remisson in fever, headache, pancytopenia, arthritis.

19-2=17

1. Headache and fever were controlled after one course of glucosteroid-pulse therapy. Arthritis was improved at 3 months. 2. Laboratory variables: WBC and PLT increased to normal; anti-dsDNA decreased from 172.52 to 94.59 IU/ml; C3 increased from 0.59 to 1.0 g/l; C4 increased from 0.217 to 0. 382 g/l in 3 months.

None.

7 Remission in fever, rash, alopecia, proteinuria

14-9=5

1. The temperature recovered to normal in one week. The rash faded gradually with treatment. Alopecia improved during 3 months. However, WBC did not improved. 2. Proteinuria decreased from 1.12 to 0.62 g/d at 3 months. 3. Laboratory variables: anti-dsDNA decreased from 1828 to 256.12 IU/ml; C3 increased from 0.382 to 0.652 g/l; C4 increased from 0.058 to 0.094 g/l at 3 months.

None.

8 Remission in fever, rash, alopecia, oral ulcers and leukopenia

10-2=8

1. Leukopenia and fever were controlled and WBC increased from 1.8 to 4.8×109/L in one course of glucosteroid-pulse therapy. Alopecia improved and rash decreased with a continuation of improvement at the third month. 2. Laboratory variables: anti-dsDNA decreased from 677.9 to 299.17 IU/ml; C3 increased from 0.33 to 1.14 g/l; and C4 increased from 0.08 to 0.19 g/l in 3 months.

Skin fungi, Upper respiratory

tract infection

9 Remission in thrombocytopenia. 5-5=0

1.! PLT increased from 17 to 70 ×109/L at 3 months. 2.! Laboratory variables: there were no significant changes in the level of

anti-dsDNA, ANA and complement Herpes zoster

10 Remission in rash 10-6=4 1.! Rash faded gradually with treatment. Moreover, proteinuria decreased from Skin fungi


!

and proteinuria. 0.52 to 0.29 g/d at 3 months. 2.! Laboratory variables: anti-dsDNA decreased from 336.45 to 142.98 IU/ml;

C3 increased from 0.564 to 0.628 g/l; and C4 increased from 0.154 to 0.241g/l at 3 months.

11 Remission in arthritis and proteinuria 12-9=4

1.! Arthralgia relieved gradually with treatment. Moreover, proteinuria decreased from 1.38 to 0.6 g/d at 3 months.

2.! Laboratory variables: anti-dsDNA decreased from 1223.39 to 620 IU/ml; C3 increased from 0.464 to 0.528 g/l; C4 increased from 0.024 to 0.065 g/l at 3 months.

None.

12 Remission in rash, proteinuria and leukopenia.

13-8=4

1.! Rash disappeared in 4 weeks. 2.! Proteinuria decreased from 1.01 g to 0.6 g/d and WBC increased from 3.22 to

5.23×109/L at 3 months. 3.! Laboratory variables: anti-dsDNA decreased from 92.61 to 65.42 IU/ml;

ANA decreased from 44.15 to 10.68 IU/ml; C3 increased from 0.452 to 0.693 g/l; C4 increased from 0.063 to 0.147 g/l at 3 months.

None.

13 Remission in rash and proteinuria.

10-8=2

1.! The rash faded gradually with the treatment by 2 months. 2.! Proteinuria decreased from 0.91 to 0.53 g/d at 3 months. 3.! Laboratory variables: anti-dsDNA decreased from 752.07 to 423.1 IU/ml; C3

increased from 0.449 to 0.61 g/l; and C4 increased from 0.09 to 0.122 g/l at 3 months.

None.

14 Remission in thrombocytopenia. 3-0=3

1. PLT increased from 9.0 to 279×109/L in 3 months. 2. Laboratory variables: anti-dsDNA decreased from 212.5 to 87.2 IU/ml; serum complement was stable.

None.

15 Rremisson in fever, rash, arthritis and

13-6=7 1. The temperature recovered to normal in 1 week. Rash and arthritis were relieved at 3 months.

None.


!

proteinuria. 2. Proteinuria decreased from 2.38 to 0.56 g/d at 3 months. 3. Laboratory variables: anti-dsDNA decreased from 435.9 to 216.01 IU/ml in 3 months. There was no significant change in the level of complement.

SLEDAI: Systemic Lupus Erythematosus Disease Activity Index (Safety of Estrogens in Lupus Erythematosus National Assessment version). WBC: white blood cells; Hb: hemoglobin; PLT: platelet; AnuA: Anti-nucleosome Antibody; C3: complement C3; C4: complement C4. Note:! Patients! with! conventional! treatment were allowed to increase the dosage of corticosteroids and add immunosuppressants after enrollment according to their disease activities. !!!!!!!!!!!!


Table S10. Antibodies used in flow cytometric analysis.

Antibody Parameter Clone Reactivity Validation

B220 APC-Cy7 RA3-6B2 mouse by antibodypedia

CD4 PE-Cy7 RM4-5 mouse by 1DegreeBio

CD44 FITC IM7 mouse by antibodypedia

IL-17a PE TC11-18H10 mouse by 1DegreeBio

IFN-g APC XMG1.2 mouse by 1DegreeBio

GL-7 Alexa Fluor 488 GL7 mouse by 1DegreeBio

CD25 V450 PC61 mouse by 1DegreeBio

Foxp3 PE-Cy5.5 FJK-16s mouse by 1DegreeBio

Bcl-6 APC 7D1 mouse by antibodypedia

CXCR5 PE 2G8 mouse by 1DegreeBio

CCR4 PerCP-Cy5.5 TG6 human by antibodypedia

CCR6 PE G034E3 human by antibodypedia

CCR7 PE-Cy7 G043H7 human by antibodypedia

CXCR5 Alexa Fluor 647 RF8B2 human by 1DegreeBio

CD4 Alexa Fluor 700 RPA-T4 human by antibodypedia

CD45RA APC-Cy7 HI100 human by antibodypedia

CXCR3 Brilliant Violet 421 G025H7 human by antibodypedia

CD8 Brilliant Violet 510 RPA-T8 human by antibodypedia

CD127 Brilliant Violet 605 A019D5 human by antibodypedia

CD3 Brilliant Violet 650 OKT3 human by antibodypedia

PD-1 Brilliant Violet 711 EH12.2H7 human by antibodypedia

CD14 Brilliant Violet 785 M5E2 human by antibodypedia

CD19 Brilliant Violet 785 HIB19 human by antibodypedia


!

!

!

Figure S1. Schematic of study design and criteria for patient enrollment.

SLE patients who fulfilled the inclusion and exclusion criteria administered 3 cycles of rhIL-2

subcutaneously at a dose of 1 million IU every other day for 2 weeks (a total of 7 doses), followed by

a 2-week break. Clinical and laboratory data were measured at baseline and every 2 weeks thereafter

until week 12.

Figure S2. Phenotypic characterization of T-cell subsets by Flow cytometry

(a) The lymphocyte gating strategy for identification of CD4+ T cell subsets is indicated. Within

these T cells, Treg cells were identified as CD25high CD127low (shown in the truncated ectangle, c).

Subordinately, within the rest of CD4+ T cells (effector T cells, c), TFH cells were defined as

CXCR5+ PD1+CCR7low (i), TH1 as CXCR3+ CCR6- CCR4- CCR7low (h), TH2 as CXCR3- CCR6-

CCR4+ CCR7low (g), and TH17 as CXCR3- CCR6+ CCR4+ CCR7low (d). The complete markers for

each subset are detailed in the lower table.

Figure S3. Changes in the suppressive activity of Treg cells after low-dose rhIL-2 administration.

Ex vivo suppression assays were performed to examine the suppressive activity of Treg cells obtained

from 8 patients before and after 2 weeks of rhIL-2 treatment. Carboxyfluorescein succinimidyl ester

(CFSE)-labelled CD25- effector CD4+ T cells from healthy donors were stimulated and co-cultured

with Treg cells from each patient. (a) Representative histograms showed the proliferation of effector

CD4+ T cells with or without Treg cells before (left) or after (right) 2 weeks’ rhIL-2 treatment. n = 8.

(b, c) Treg cell-mediated suppression of effector CD4+ T cell proliferation was measured using

percentages of divided cells (b) and division index (c). n = 8, P < 0.05. Paired-sample t-tests were

used to compare results before and after IL-2 administration.

Figure S4. Changes in DN T cells during low-dose rhIL-2 administration.

Changes in percentages of double-negative T cells (CD4-CD8-CD3+ TCRαβ+) within αβ T cells at

baseline and after twelve weeks of rhIL-2 administration, as well as median and interquartile range

values at indicated time points were shown. The median values at each indicated time point are


!

!

!

linked by the trend line. n = 23, P < 0.001. Paired-sample t-tests were used to compare percentages

of DN T cell between baseline and week 12.

Figure S5. Immunological changes in responses to conventional therapies.

(a) Comparison of CD4+ T cell subsets at baseline between patients treated with low-dose rhIL-2

administration (n = 23) and patients treated with conventional therapies (n = 15). Two-tailed t-tests

were used to compare percentages of Treg, TFH, TH17, TH1 and TH2 cells between the IL-2 treatment

and conventional treatment group). P > 0.05. (b) Changes in percentages of CD4+ T cell subsets at

baseline and after 12 weeks of conventional therapies. Paired-sample t-tests were used to compare

percentages of Treg, TFH, TH17, TH1 and TH2 cells between baseline and week 12. n = 15, P > 0.05.

Figure S6. Experimental design to evaluate the immunological effects of rhIL-2 in immunized

mice.

Eight to twelve weeks old mice were intraperitoneally immunized with 50µg ovalbumin (OVA) in

Complete Freud’s Adjuvant (CFA). Different doses of subcutaneous rhIL-2 (10,000IU, 30,000IU or

100,000IU) and PBS were administrated daily from day 3 to day 9. Splenocytes were analyzed by

flow cytometry on day 10. Flow cytometric plots show the phenotypic characterization of Treg, TFH,

TH17, TH1 and GC B cells. The biologic markers for these subsets are indicated in the lower table.

Figure S7. Immunological response to rhIL-2 in mice.

Statistical analysis (n = 5 mice per group) of data from individual mice and the average value of each

group. (a-e) Changes and comparison in number of Treg, GC B, TH17, TFH, and TH1 cells within per

106 splenocytes are shown. The results represent two independent experiments. Paired sample t-tests

were used to compare PBS and different doses of IL-2.

Figure S8. Effect of rhIL-2 on the suppressive function of Treg cells.


!

!

!

Eight to twelve weeks old mice (n = 3) were subcutaneously injected with rhIL-2 (30,000IU) or PBS

daily for 7 days. Ex vivo suppression assays were performed to examine the suppressive activity of

Treg cells. Cell Trace Violet-labelled splenocytes from naïve mice were stimulated and co-cultured

with Treg cells from rhIL-2 or PBS treated mice as indicated ratios. The suppression of proliferation

of CD4+ T cells (a) and CD8+ T cells (b) was measured using percentages of divided cells and

division index and analysed using two-tailed t-tests.

Figure S9. Influence of background therapies on clinical and laboratory measures during low-

dose rhIL-2 therapy.

Patients receiving rhIL-2 administration were divided into three groups according to background

therapy: G + HCQ (black line, n=17), G + HCQ + MMF (blue line, n=13) and G + HCQ + CTX

(green line, n=7). Comparison of improvement in clinical and laboratory parameters among three

groups is indicated from baseline to 12 weeks, including SELENA-SLEDAI (a), anti-dsDNA

immunoglobulin G (b), complement C3 concentrations (c) and complement C4 concentrations (d).

Data is presented as median and interquartile range and the median values at each indicated time are

linked by the trend line. G, glucocorticoid; HCQ, hydroxycloroquine; MMF, mycophenolate mofetil;

CTX, cyclophosphamide.

Figure S10. Immunological responses in low- and high-responders to low-dose rhIL-2.

Twenty responder patients, defined by SELENA-SLEDAI scores < 8 after 12-week low-dose IL-2

administration, were classified into low responder group (final SLEDAI 4 - 7, n=10) and high

responder group (final SLEDAI score < 4, n=10). Paired-sample t-tests were used to compare

changes in percentages of Treg (a), TFH (b), and TH17 cells (c), and the TFH + TH17/Treg ratio (d),

between week 0 and week 12 in each group. P > 0.05.


0 2 4 6 8 10 12Low*dose.IL*2.therapy.(n=40,.including.23.with.consent.for.immunological.analysis). PBMC.for.

immunological.analysis

Tx

Tx Tx Tx

Conventional.therapy.(n=15,.all.with.consent.for.immunological.analysis).

Eligible.patients.with.moderate.to.severe.SLE(SELENA*SELDAI≥8)

Figure S1. Schematic of study design and criteria for patient enrollment.

Time.(weeks)

Time.(weeks)

0 2 4 6 8 10 12

a

b Inclusion criteria:

• Age:18*65 years• Meet the 1997 American College of Rheumatology criteria for the diagnosis of SLE.• At least 4 weeks of stable background treatment with corticosteroids (≤1.0 mg/kg/d prednisone or

equivalent) and/orwith antimalarials, nonsteroidal anti*inflammatory drugs or immunosuppressants• Background treatment failed to control flares or to permit prednisone tapering• SELENA*SLEDAI ≥8• Negative HIV test.• Negative for hepatitis B and C virus.

Exclusion.criteria:• Severe chronic liver, kidney, lung or heart dysfunction] (heart failure (≥ grade III NYHA), hepatic

insufficiency (transaminases > 2 times of the upper limit of the normal range), renal insufficiency(creatinine clearance ≤30ml/min)

• Serious infection such as bacteremia, sepsis]• Cancer or history of cancer cured for less than five years (except in situ carcinoma of the cervix or

Basocellular carcinoma)]• High*dose steroid pulse therapy (>1.5mg/kg) or IV bolus of corticosteroids in the last 2 months.• History of administration of rituximab or other biologics]• Purified protein derivative (tuberculin) >10mm• Pregnancy or lactation in females• Active infection (hepatitis B orC virus, Epstein*Barr virus, HIV orMycobacterium tuberculosis)• Mental disorder or any other chronic illness or drug abuse that could interfere with the ability to comply

with the protocol or to give information]• Inability to comply with IL*2 treatment regimen.

(Aug%2013*Aug%2014)

(Oct%2015*Jan%2016)


DN.(CD4*CD8*) CD4* CD8.*CD3+.TCRαβ+.CD14* CD19* 7AAD*

Treg CD25high.CD127low.CD4+.CD8.*CD3+.TCRαβ+.CD14* CD19* 7AAD*

TH1 CXCR3+.CCR6* CCR4* CCR7low.CD4+.CD8.*CD3+.TCRαβ+.CD14* CD19* 7AAD*

TH2 CCR4+.CXCR3* CCR6* CCR7low.CD4+.CD8.*CD3+.TCRαβ+.CD14* CD19* 7AAD*

TH17 CCR4+.CCR6+.CXCR3* CCR7low.CD4+.CD8.*CD3+.TCRαβ+.CD14*CD19* 7AAD*

TFH CXCR5+PD*1+.CCR7low.CD4+.CD8.*CD3+.TCRαβ+.CD14* CD19* 7AAD*

Figure S2. Phenotypic characterization of T cell subsets by flow cytometry.

7AAD

TCRαβ

CD14/CD19

CD3

CD4

CD8

CD127

CD25

CD45RA

CCR7

CXCR3

CXCR5

CCR4

CCR7

CCR4

CCR7

PD*1

CCR7

CCR4

CCR7

CXCR3

CCR6

Treg

TFH

TH1

TH2

TH17

a

b c

d e f

g h i

DN


0

10

20

30

0

10

20

30

40

0

20

40

60

80

100

Before.

Dividedcells.(%)

Suppression.(%)

No.Treg +.Treg No.Treg +.Treg Before After

0.0

0.1

0.2

0.3

0.4

0.0

0.1

0.2

0.3

0.4

0.5

0

20

40

60

80

100

Suppression.(%)

Before.

DivisionIndex

No.Treg +.Treg No.Treg +.Treg Before After

P =.0.016 P =.0.004 P"=.0.042

P =.0.025 P =.0.003 P =.0.017

4+.321....04+.321....0 4+.321....0 4+.321....0

CFSE CFSE

Before

Cell.count

400

300

200

100

0

300

200

100

0

No.Treg +.Treg No.Treg +.Treg

After

Cell.count

Figure S3. Changes in the suppressive activity of Treg cells after low-dose rhIL-2 administration.

a

b

c

After

Dividedcells.(%)

After

DivisionIndex


0

2

4

6 P <.0.001

120Time.(weeks)

0 2....4.....6.....8 10 120

1

2

3

4

CD4*CD8*αβT.cells.

In.αβT.cells.(%)

Figure S4. Changes in DN T cells during low-dose rhIL-2 administration.

Time.(weeks)


0

4

8

12

16

0

2

4

6

8

10P =.0.669

0

5

10

15

20

25

120

T regin.CD4+T.cells.(%)

T FHin.CD4+T.cells.(%)

T H17.in.CD4+T.cells.(%)

P =.0.850 P"=.0.416

IL*2 Conv0

5

10

15

20T regin.CD4+T.cells.(%) P =.0.063

0

5

10

15

20

IL*2 ConvT FHin.CD4+T.cells.(%) P =.0.471

0

5

10

15

IL*2 ConvT H17.in.CD4+T.cells.(%)

P =.0.369

0

10

20

30

40

50

IL*2 Conv

T H1.in.CD4+T.cells.(%) P =.0.787

0

5

10

15

IL*2 Conv

T H2.in.CD4+T.cells.(%) P"=.0.282

Figure S5. Immunological changes in responses to conventional therapies.a

b

0

10

20

30

40

50

0

2

4

6

8P =.0.246 P =.0.817

T H1.in.CD4+T.cells.(%)

T H2.in.CD4+T.cells.(%)P =.0.978

0

0.5

1.0

1.5

2.0

2.5

Ratio.of.T

FH+T

H17/Treg

Time.(weeks)

120Time.(weeks)

120Time.(weeks)

120Time.(weeks)

120Time.(weeks)

120Time.(weeks)


CD4

B220

Bcl6

CXCR5CD25

Foxp3

Bcl6

GL7

GC

Treg

TFH

CD44

IFNγ

TH1

CD44

IL*17

TH17

Treg Foxp3+ CD25+ CD4+ B220-

TH1 IFNγ+ CD44high CD4+ B220-

TH17 IL-17+ CD44high CD4+ B220-

TFH Bcl6+ CXCR5high Foxp3- CD4+ B220-

GC Bcl6+ GL-7+ B220+ CD4-

Day.1 Day.3 Day.9

Immunisation.With.OVA.in.CFA

Day.10

Treatment.with.IL*2.(Dose:.I0,000,.30,000.or.100,000.IU)

Analysis

Analysis

Figure S6. Experiment design to evaluate the immunological effects of low-dose IL-2 in immunized mice.


0

5000

10000

15000

20000

25000

0

400

800

1200

0

3000

6000

9000

0

50

100

150

200

250

0

400

800

1200

PBS 10,00030,000 100,000IL*2.(IU)

PBS 10,00030,000 100,000IL*2.(IU)

PBS 10,00030,000 100,000IL*2.(IU)

PBS 10,00030,000 100,000IL*2.(IU)

P"=.0.04

P =.0.03P =.0.01

P <.0.01P =.0.02

P"<.0.01

P =.0.04P >.0.05.(N.S.)

P =.0.03

P >.0.05.(N.S.)

P <.0.01P <.0.01

T regcells

Cell.number.per.

106splenocytes

T H17.cells.

Cell.number.per.

106splenocytes

T FHcells

Cell.number.per.

106splenocytes

GC.B.cells

Cell.number.per.

106splenocytes

a

PBS 10,00030,000 100,000IL*2.(IU)

P">.0.05.(N.S.)P">.0.05.(N.S.)

P">.0.05.(N.S.)

T H1.cells

Cell.number.per.

106splenocytes

b

c d

e

Figure S7. Immunological response to rhIL-2 in mice.


5+...4321.0

5+...4321.05+...4321.0

5+...4321.05+...4321.0

5+...4321.0

100

80

60

40

20

0100

80

60

40

20

0100

80

60

40

20

0

5+...4321.0100

80

60

40

20

0

No.Treg.control

Cell.count

CellTrace.Violet

PBS IL*2

60

40

20

0

60

40

20

0

60

40

20

0

0.0

0.3

0.6

0.9

1.2

0.0

0.3

0.6

0.9

1.2

0.0

0.3

0.6

0.9

1.2

Division.index

Ratio.1:1

Ratio1:2

WithTreg

Ratio.1:0.5

6+.5 4321.0 6+.5 4321.0

6+.5 4321.06+.5 4321.0

6+.5 4321.0 6+.5 4321.0

CellTrace.Violet

6+.5 4321.0

Ratio.1:1

Ratio1:2

Ratio.1:0.5

300

200

100

0

150

100

50

0150

100

50

0150

100

50

0

0

20

40

60

80

0

20

40

60

80

0

20

40

60

80

Cell.count

3

2

1

0

Suppression.of.CD4+ T.cells

Suppression.of.CD8+ T.cells

a

b

3

2

1

0

3

2

1

0

No.Treg.control

P >.0.05.(N.S.)

P">.0.05.(N.S.)

P >.0.05.(N.S.)

P >.0.05.(N.S.)

P <.0.01 P <.0.01

P =.0.03

P""=.0.05

P" =.0.04

P" =.0.05

P <.0.01 P <.0.01

Figure S8. Effect of rhIL-2 on the suppressive function of Treg cells.

PBS.IL*2 PBS.IL*2Divided.cells.(%)

PBS.IL*2 PBS.IL*2

Divided.cells.(%)

Division.index


0

5

10

15

20

SELENA*SLEDAI.Score

Anti*dsDNAIgG

(x100.IU/mL�

0

5

10

15

Time.(weeks)0. 2......4.......6......8 10....12

Complement.C4.(g/L)

0.0

0.1

0.2

0.3

0.4

0.0

0.3

0.6

0.9

1.2

Complement.C3.(g/L)

Time.(weeks)

0. 2......4.......6......8 10....12

Time.(weeks)0. 2......4.......6......8 10....12

Time.(weeks)

0. 2......4.......6......8 10....12

a b

c d

Figure S9. Influence of background therapies on clinical and laboratory measures during low-dose rhIL-2 therapy.

G+HCQ.�n=17�

G+HCQ+MMF.(n=13)

G+HCQ+CTX.(n=7)


0

10

20

30

40

0

5

10

15

20

0

5

10

15

0.0

0.5

1.0

1.5

2.0

T regin.CD4+T.cells.(%) P">.0.05/N.S.

(↑ x.1.4)P">.0.05/N.S.(↑ x.1.3)

W0 W12Low.responder High.responder

W0 W12

T FHin.CD4+T.cells.(%) P">.0.05/N.S.

(↓ x.1.4)P">.0.05/N.S.(↓ x.1.8)

P">.0.05/N.S.(↓ x.1.2)

P">.0.05/N.S.(↓ x.1.2)

T H17.in.CD4+T.cells.(%

)

P">.0.05/N.S.(↓ x.1.6)

P"<.0.01(↓ x.2.0)

Ratio.of.T

FH.+.TH17/Treg


W0 W12


W0 W12 W0 W12Low.responder High.responder

W0 W12

Figure S10. Immunological responses in low- and high- responders to low-dose rhIL-2

a b

c d


Table S1. Baseline characteristics of patients with SLE ... · Baseline characteristics of patients...

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