Accepted Manuscript

Effect of IL6 and IL23 on double negative T cells and anti ds-DNA in Systemiclupus Erythematosus Patients

Asmaa S. Shaltout, Douaa Sayed, Mohamed S. Badary, Amany M. Nafee, MonaH. El Zohri, Rania Bakry, Shabaan H. Ahmed

PII: S0198-8859(16)30116-1DOI: http://dx.doi.org/10.1016/j.humimm.2016.06.007Reference: HIM 9775

To appear in: Human Immunology

Received Date: 6 May 2015Revised Date: 27 May 2016Accepted Date: 7 June 2016

Please cite this article as: Shaltout, A.S., Sayed, D., Badary, M.S., Nafee, A.M., El Zohri, M.H., Bakry, R., Ahmed,S.H., Effect of IL6 and IL23 on double negative T cells and anti ds-DNA in Systemic lupus Erythematosus Patients,Human Immunology (2016), doi: http://dx.doi.org/10.1016/j.humimm.2016.06.007

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1

Original article

Effect of IL6 and IL23 on double negative T cells and anti ds-DNA in Systemic

lupus Erythematosus Patients

Asmaa S. Shaltout1, Douaa Sayed 2*, Mohamed S. Badary 1, Amany M. Nafee 1,

Mona H. El Zohri3, Rania Bakry2, Shabaan H. Ahmed1

1 Medical Microbiology & Immunology, Faculty of Medicine Assiut University,

Assiut, Egypt

2 Department of Clinical pathology, South Egypt Cancer Institute, Assiut University,

Egypt

3 Internal Medicine , Rheumatology unit, Assiut University, Assiut, Egypt.

Word count:

Running title: Effect of IL6 & IL23 on DN T cells in SLE

*Correspondence to: Douaa Sayed, MD. Director of Flow Cytometry Lab. Clinical

pathology Department-South Egypt Cancer Institute-Assiut University.

Mail: Clinical pathology Department-South Egypt Cancer Institute-Assiut University.

Assiut- Egypt

E-mail: douaa_sayed@hotmail.com ; douaasayed@aun.edu.eg

Tel: +201006261987

Fax: +2/88/2348609

2

Abstract

Several evidences suggest that DN T cells, IL23 and IL6 play a role in the

pathogenesis of SLE. This study aimed to evaluate the frequency of DN T cells in

SLE patients and the relation to their activity also to assess the possible role of IL6

and IL23 on DN T cells. Thirty patients with SLE and sixteen healthy blood donor

females were enrolled. There was a significant increase in DN T cells in patients than

controls (P=0.001). These cells had a significant positive correlation with SLEDAI (r

= 0.486, P= 0.006). DN T cells from SLE patient samples were expanded when

stimulated in vitro with RhIL6 or RhIL23 in patients than controls. Furthermore, anti

ds-DNA level was found to be increased in supernatant of PBMCs when stimulated

by these cytokines in different concentrations. Our findings suggest that IL6 and IL23

may play role in SLE pathogenesis through their effect on DN T cells and anti ds-

DNA.

Key words: DN T cells; SLE; IL23

3

1. Introduction:

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune

disease that affects many organs as the kidneys, joints, nervous system and skin.

Characterized by immune system abnormalities as production of different auto

antibodies, activation of complement and deposition of immune-complex also tissues

infiltration by lymphocytes resulting in organ damage. The etiology and pathogenesis

of SLE are still unclear. A combination of genetic, hormonal and environmental

factors with a dominant autoimmune component may be result in SLE [1].

Double negative (DN) T cells express TCRαβ but do not express CD4 or CD8

[2]. A subset of natural killer T cells (NK T) expresses those three markers [3, 4]. In

human NK T cell marker (CD56) was known to be expressed so it was used to

distinguish these two subsets [5].

Several evidences suggest that DN T cells play a role in the pathogenesis of

SLE, it infiltrates the kidneys in lupus nephritis patients and produces IL17 [6] which

activates proinflammatory cytokines and recruits neutrophils to tissues [7] also DN T

cells help B cells to produce pathogenic auto antibodies [8].

Cytokines play a major role in the pathogenesis of SLE; interleukin 23 (IL23)

is considered an important cytokine in the pathogenesis of that disease, as its level

increases in SLE patients and correlates with SLE disease activity index (SLEDAI)

[9]. A study was done by Kyttaris et al on lupus prone mice that lacked the IL23

receptor (IL23R), they found that IL23R−/−

B6/lpr mice had a significant decrease in

the number DN T cells among lymph node cells when compared with wild type

B6/lpr mice [10].

In SLE T and B cell abnormalities may be linked to abnormal increase in IL6

also the blocking of IL6 receptors (IL6R) leads to decrease of pathogenic trafficking

of lymphocytes [11]. Anti-double stranded DNA (anti ds-DNA) antibodies levels

increase and it is considered highly specific for SLE [12].

The aim of this study was to evaluate the frequency of DN T cells in SLE

patients and the relation to their activity also to assess the possible role of IL6 and

IL23 on DN T cells. In addition the effect of different concentrations of these

cytokines on levels of anti-ds-DNA antibodies was investigated.

4

2. Patients and methods:

2.1. Study participants

This analytical case-control study included thirty patients with SLE who fulfilled at

least four criteria of SLE according to American College of Rheumatology [13]. All

patients were newly diagnosed and hadn't received steroids or other immune

suppressive drugs. All of them were females admitted to Rheumatology Unit of

Internal Medicine Department in Assuit University Hospital from September 2013 to

September 2014. Table 1 illustrates the clinical characteristics of the enrolled SLE

patients.

The study controls were sixteen, age matched, healthy blood donor females. They

were attending blood bank of South Egypt Cancer Institute during the study period.

The study was approved by the Ethical Review Board of Assiut Faculty of Medicine

and all women gave written informed consent.

2.2. Separation and culture of PBMCs

From each subject 8ml venous blood were obtained by clean venupuncture and

collected into sterile tubes containing sodium heparin anticoagulant. It was diluted

with an equal volume of cold phosphate buffer saline (PBS). Blood-PBS mix was

carefully layered over 10.5ml of Lymphocyte Separation Medium (Lonza

Walkersville, Inc.,USA) and PBMCs were separated by Ficol density gradient

centrifugation. The upper plasma layers pipetted and kept in -20°C for further

measurement of anti-ds-DNA. PBMCs interface was counted using 0.4% trypan blue;

cell preparations were typically composed of > 90% viable cells.

1×106 cells were suspended in 0.5 ml RPMI 1640 medium supplemented with

10% fetal calf serum, 1% penicillin and streptomycin. Cells were added to seven wells

of a 24well culture plate. Recombinant human IL6 (RhIL6) (US biological, New

England) was added to reach a final concentrations of 10 ng/ml, 50 ng/ml and 100

ng/ml in three wells. Recombinant human IL23 (RhIL23) (US biological, New

England) was added at the same end concentrations in another three wells. The last

well was contained 1×106/ml cells only with no cytokines. Then the plate was

incubated for 24 hours at 37°C in presence of 5% CO2. Following the incubation

period the cultured cells were collected in sterile eppindorfs labeled with each

concentration then eppindorfs were centrifuged at 2500 rpm for 2 minutes and

5

supernatants were collected and kept at -20°C for enzyme-linked immunosorbent

assays (ELISA) analysis. Cell pellet was resuspend to be measured by the flow

cytometry.

2.3. Flow cytometry

Both freshly isolated PBMCs and those activated with each cytokine at 10, 50

and 100 ng/ml were analyzed by flow cytometry for 3 samples. The best proliferation

result was after incubation at 50 ng/ml. 100 ng/ml of cytokines gave the same result

but with less viability (figure 1). For all samples; both freshly isolated PBMCs and

those activated with each cytokine at 50 ng/ml were analyzed by flow cytometry Cells

were stained by incubation with anti-CD4 FITC, anti-CD8 PE, anti-TCRαβ APC and

anti-CD56 PerCP (all purchased from EXBIO, Czech Republic) for 15 minutes in

dark. Then 2 ml PBS were added to the FACS tubes and cells were centrifuged at

2500 rpm for 3 minute. The supernatant was discarded and cells were collected for

analysis. Appropriate isotype controls were also prepared and processed in a similar

manner. Each monoclonal antibody was titrated using serial dilutions starting with the

concentration recommended by the manufacture.

Acquisition of PBMCs was performed in a four-colour FACSCalibur flow

cytometer (Becton Dickinson, USA). A minimum of 200,000 events were collected for

each sample analysis. Cells were selected by drawing a region1 (R1) around PBMCs

population on TCRαβ+ /side scatter (SSC) dot plot and cells were reanalyzed in TCRαβ

vs CD8 plot. (R2) was drawn to select TCRαβ+ CD8- T cells and cells in this region

were reanalyzed in TCRαβ vs CD4 plot. (R3) was drawn to select TCRαβ

+ CD4

- T cells

(based on discrimination between populations) and again these cells were reanalyzed in

TCRαβ vs CD56 plot to quantify percentage of TCRαβ

+CD8

-CD4

-CD56

- as shown in

(figure 1) Analysis was performed with CellQuest software (Becton Dickinson).

2.4. Detection of anti-ds-DNA IgG

In order to determine dose dependent effect of IL6 or IL23 on anti-ds-DNA

IgG, both supernatants of activated PBMCs with different cytokine concentrations and

with no cytokine activation in 12 patients were analyzed for anti-ds-DNA IgG using

ELISA kit (DiaMetra, Italy). The procedure was performed according to

manufacturer's instruction.

6

2.5. Statistical analyses of the data

Statistical package for social sciences (SPSS) version 16 was used for data

analysis. All quantitative data were expressed as mean ± standard error (SE).

Differences in mean between the different groups of subjects were calculated using

the independent t test. Differences in mean between the same groups were calculated

using paired t test. Differences in mean of anti-ds-DNA levels between the same

groups were calculated using wilcoxon signed ranks test. P-value <0.05 was

considered to be significant.

3. Results:

3.1. Age and laboratory data of study participants and patients' activity:

Age of study participants ranged from 17-30 years. Mean age for both patients

and controls was (22 ± 3.80 and 22.25 ± 1.61 years) respectively. No significant

difference was found between the patient and control groups as regard age (P = 0.80).

Patients SLEDAI ranged from 5 to 27 and most of them was high and very high

activity as shown in Table (2). The baseline laboratory data is shown in Table (3).

3. 2. Analysis of freshly isolated double negative T cells (TCRαβ+CD4

-CD8

-CD56

-)

and their relation to clinical and laboratory data.

We evaluated DN T cells percentage (from freshly isolated PBMCs) and found that it

was significantly higher in SLE patients (2.34 ± 0.17 %) in comparison to controls

(0.64 ± 0.08%) with p- value (P=0.001) as shown in figure (2).

The percentage of these cells in SLE patients has a significant positive correlation

with SLEDAI and serum creatinine (r = 0.486, P= 0.006 and r=0.576, P=0.024

respectively). There were fair positive correlations between frequency of DN T cell

and all of the followings: protein in 24hr urine, blood urea and anti ds-DNA with no

statistical significance (r=0.490, P=0.064; r=0.441, P=0.1 and r=0.358, P=0.190

respectively). There is no significant difference in frequency of DN T cells in relation

to clinical manifestations and other laboratory data.

3. 3. Analysis of double negative T cells (TCRαβ +CD4

-CD8

-CD56

-) after activation

with IL6

7

DN T cells percentage in SLE patient samples was significant increased after

activation with IL6 than before activation (P=0.001), there was no significant difference

in activated control samples than before activation as shown in table (4) and figures (3

& 4). The increase in DN T cells percentage was significantly higher in activated SLE

patient samples with IL6 in comparison to activated control samples (P=0.001).

3. 4. Analysis of double negative T cells (TCRαβ +CD4

-CD8

-CD56

-) after activation

with IL23

Significant increase in DN T cells percentage in SLE patient samples after

activation with IL23 than before activation (P=0.001) as shown in table (4) and figure

(4 & 5). The increase in DN T cells percentage was significantly higher in activated

SLE patient samples with IL23 in comparison to activated control samples (P=0.001).

3. 5. Analysis of anti-ds-DNA IgG in 12 studied SLE patient samples

Level of anti-ds-DNA (IgG) after stimulation with 10 ng IL6 was significantly

increased than non-stimulated (P=0.002). Level of anti-ds-DNA (IgG) after

stimulation with 50 ng/ml IL6 was insignificantly different in comparison to

non-stimulated (P=0.840) and level of anti-ds-DNA (IgG) after stimulation

with 100 ng/ml IL6 was significantly increased than in non-stimulated

(P=0.012) as shown in table (5) and figure (6).

Level of anti-ds-DNA (IgG) after stimulation with 10 ng/ml IL23 was significantly

increased than in non-stimulated (P=0.049). Level of anti-ds-DNA (IgG) after

stimulation with 50 ng/ml and 100 ng/ml IL23 was insignificantly different in

comparison to non-stimulated with P value (P=0.307) and (P=0.157)

respectively as shown in table (5) and figure (7).

4. Discussion

This study showed that the percentage of circulating TCRαβ+CD4

- CD8

-CD56

-

T cells within PBMCs was significantly higher in SLE patients than in controls. This

is consistent with the findings described by Anand et al. who stated that the

percentage of TCRαβ+CD4

-CD8

- T cells was found to be significantly higher in

patients with SLE as compared with healthy controls [14], but they didn't use a

marker to exclude the presence of NK T cells. In another study which done by Crispín

8

et al. they stained NK T cells with an anti-TCR Vα24 and found that the number of

Vα24+ DN T cells was negligible [6]. We found the same results when cells stained

with CD56 were analyzed (data not shown). Our results are not matched with Shirota

et al. as they found that no differences in DN T cells isolated from PBMCs between

SLE patients and controls [11]. This may be due to their study was done on only 15

patients with mild to moderate activity but our study was done on 30 patients; 73.3%

with high and very high activity. DN T cells percentage in SLE patients was

correlated with disease activity and the laboratory data which indicates renal

affection; these data indicates that, DN T cells may play a role in the pathogenesis of

the disease and its activity.

Our study showed significant effect on DN T cells percentage after activation

with IL6. To the best of our knowledge no study assessed effect of activation by IL6

on DN T cells. However these results are supported by Ohteki et al. who stated that

IL6 which produced by hepatic mononuclear cells in the liver of MRL/lpr mice may

be responsible for the first step in the proliferation of DN T cells [15].

Our in-vitro study results were also supported with invivo study done by

Shirota et al. to evaluate the effect of in-vivo blockage of IL6R on peripheral

lymphocyte subsets by treatment with tocilizumab (a humanized anti IL6R antibody)

and they found that no change of DN T cells between SLE patients and controls after

tocilizumab treatment [11].

In addition our study showed significant effect on DN T cells percentage after

activation with IL23 and this effect wasn't assessed before. These results supported by

Kyttaris, who stated that in the absence of IL23R, the DN T cell population did not

expand, suggesting that IL23 may play a role in the generation and/or maintenance of

this T cell population [16]. The effect of IL6 and IL23 on percentage of DN T cells

was significantly higher in SLE patient samples in comparison to control samples.

In our study we found significant increase in anti-ds-DNA (IgG) when

stimulated with 10 ng/ml IL6. This is consistent with the result of Richards et al.

their study was done on IL6 deficient (IL6-/-) and intact (IL6+/+) mice these were

treated with pristane to induce lupus they found that pristane induced high levels of

IgG anti ds-DNA, in IL6+/+

, but not IL6-/-

mice by ELISA [17].

9

There was significant increase in anti-ds-DNA (IgG) when stimulated with 10

and 100 ng/ml IL6 but no increase at concentration 50 ng/ml IL6 was occurred. These

results may be explained by Müller-Newen et al. who stated that the presence of

soluble IL6 receptors (sIL6R and sgp130) has been detected in body fluids to

modulate cytokine response. They found that IL6/sIL6R complexes can be trapped by

sgp130 in the soluble high affinity ternary complexes and are thereby IL6 efficiently

neutralized before they bind to the cell surface receptors. They concluded that sgp130

and sIL6R are present in the body to inhibit systemic IL6 responses. They found that,

this effect is more pronounced at higher concentrations of IL6, sIL6R and sgp130

[18]. This explains why IL6 concentration at 10 ng/ml produced significant increase

in anti-ds-DNA IgG while 50 ng/ml did not produce increase in anti-ds-DNA IgG.

The reappearance of increased ds-DNA IgG production at 100 ng/ml is due to the

presence of high concentration beyond the antagonizing effect of sgp130 so IL6

become free from inhibitory effect of sgp130.

In our study we found significant increase in anti-ds-DNA (IgG) when

stimulated with IL23 at 10 ng/ml. This is supported by Kyttaris et al. who found

lower anti ds-DNA antibodies production in IL23R deficient lupus prone mice

(IL23R−/−

B6/lpr) compared with control B6/lpr mice [10].

No significant increase in anti-ds-DNA (IgG) when stimulated with 50 ng/ml

and 100 ng/ml IL23. Which can be explained by that 10 ng/ml is the optimum

concentration for stimulation. This may be in agreement with Cheng et al. they found

that there was no significant correlation between concentration of IL23 and levels of

anti-ds-DNA antibodies in 45 SLE patients using Spearman rank correlation test [9].

5. Conclusions

DN T cell may play a role in the pathogenesis of SLE as it increases in those

patients and correlate with disease activity. There is obvious effect of IL6 and IL23 on

DN T cells percentage and anti-ds-DNA levels as they increase after stimulation with

each of them. Taken together the ability of IL6 and IL23 to increase the frequency of

DN T cells and level of anti-ds-DNA suggests that they play an important role in

pathogenesis of SLE. However; more studies are needed to confirm dose dependent

increase of DN T cell after IL-6 and 23 stimulation to reinforced our hypothesis.

10

Conflict of interest

All authors declare that they have no conflict of interest.

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Table (1): Clinical characteristics in the enrolled SLE patients

% N Clinical data

86.7 26 Fever

Skin and mucous membrane:

60 18 Malar rash

56.7 17 Photosensitivity

40 12 Fall of hair

46.6 14 Oral ulcers

Vasculitis

6.7 2 Periungal infarction

6.7 2 Leg ulceration

6.7 2 Pericarditis

13.3 4 Arthritis

63.3 19 Arthralgia

Neurological manifestations

13.3 4 Headache

13.3 4 Fits

6.7 2 Psychosis

Renal affection manifestations

66.7 20 Positive proteinuria

86.7 26 Increase serum creatinine above normal level

14

Table (2): SLEDAI in 30 studied patients of SLE:

N=30 %

No activity 0 0 0

Mild activity (1-5) 2 6.6

Moderate activity (6-10) 6 20

High activity (11-19) 8 26.66

Very high activity (≥ 20) 14 46.66

SLEDAI: systemic lupus erythematosus disease activity index.

N= number ,%= percentage.

15

Table (3): Heamatological parameters, anti ds-DNA and parameters of kidney

function in 30 studied patients with SLE and 16 controls:

Urine analysis:

0.273 2.75 ± 1.03 3.13 ± 0.81 Pus cells /HPF

0.076 2.50 ± 0.53 3.06 ± 0.82 RBCs /HPF

0.024* 0.134 ± 0.04 2.90 ± 3.29 Proteins in 24 hr urine

(gm)

0.001* 3.51 ± 0.80 8.48 ± 3.69 Urea (mmol/L)

0.009* 70.75 ± 10.26 150.73 ± 80.69 Creatinine (umol/L)

p-value

Controls

(n=16)

Patients

(n=30)

Parameters

Mean ±±±± SD Mean ±±±± SD

0.001* 11.78 ± 0.60 8.67 ± 1.96 Heamoglobin (gm/dl)

0.533 6.05 ± 0.69 5.26 ± 3.46 WBCs (103/ul)

0.005* 248.12 ± 40.17 144.73 ± 94.15 Platelet (103/ul)

0.001* 18.35 ± 5.83 118.95 ± 65.14 Anti-dsDNA (IU/ml)

16

Table (4) Percentage of DN T cells (before and after activation with IL6 and

IL23) in SLE patients and controls:

Percentage of DN T cells in

patients’ samples

(30)

Percentage of DN T cells in

controls’ samples

(16)

Before

activation

After

activation

p-value Before

activation

After

activation

p-value

50 ng/ml IL6 1.46 ± 0.16 * 3.46± 0.40* 0.001 1.10 ±0.23* 1.16 ± 0.34* 0.805

50 ng/ml IL23 1.46 ± 0.16* 3.16 ± 0.36* 0.001 1.10 ±0.23* 1.12 ±0.29* 0.908

(*): percentage of DN T cells expressed as mean ± SEM

IL: interlukein

17

Table (5) Anti-ds-DNA IgG level in supernatant of PBMCs (before and after

activation with IL6 and IL23) in 12 studied patients with SLE:

Anti-ds-DNA IgG level in supernatant of PBMCs in patients, samples

Non

stimulated

Stimulated

with 10ng/ml

P-

value

Stimulated

with 50ng/ml p-value

Stimulated

with 100ng/ml

p-

value

IL6 22.27 ± 3.99* 40.29 ± 7.59* 0.002 21.61± 4.47* 0.480 37.42 ± 7.61* 0.012

IL23 22.27 ± 3.99* 35.36 ± 7.51* 0.049 20.77±3.47* 0.307 27.20 ± 4.93* 0.157

(*): level of anti-ds-DNA IU/ml expressed as mean ± SEM

IL: interlukein

18

(A) (B)

(C) (D)

(E) (F) (G)

19

Figure (1) Gating strategy showing DN T cells as TCRαβ +CD4-CD8-CD56- in SLE

patients. (A) Cells were selected by drawing a region (R1) around PBMCs population

on TCRαβ+ /side scatter (SSC) dot plot. (B) Cells in R1 were reanalyzed in TCRαβ vs

CD8 plot. and (R2) was drawn to select TCRαβ+ CD8- T cells. (C) Cells in R2 region

were reanalyzed in TCRαβ vs CD4 plot and (R3) was drawn to select TCRαβ+ CD4- T

cells (D) cells in R3 were reanalyzed in TCRαβ vs CD56 plot to quantify percentage

of TCRαβ+CD8-CD4-CD56-. (E, F, G) DN T cells after incubation with 10, 50 and

100 ng/ml respectively (dose depending effect).

Figure (2) Mean percentage of freshly isolated DN T cells in SLE patients and

controls.

0.64 ± 0.08

2.35 ± 0.17

0

0.5

1

1.5

2

2.5

3

control SLE

% o

f D

N T

ce

lls

P= 0.001

0.64 ± 0.08

20

Figure (3) Mean percentage of DN T cells before and after stimulation with 50 ng /ml

IL6 in SLE patient and control samples ● P=0.001. Mean percentage of DN Tcells in

activated patient samples is significantly higher than activated control samples ■

P=0.001.

SLE Control

(A)

(B)

1.10 ±0.23

1.46 ± 0.16

3.46± 0.40

1.16 ±0.34

0

0.5

1

1.5

2

2.5

3

3.5

4

control SLE

% o

f D

N T

ce

lls

unstimalted

stimulated

● P=0.001

■ P=0.001

21

(C)

(D)

(E)

(F)

Figure (4) frequency of double negative T cell (TCRαβ +CD4

-CD8

-CD56

-) in PBMCs of

SLE patient and control. Figure (A&B) showing frequency of non-stimulated SLE patient and

control sample (0.84% and 0.34%) respectively. Figure (C&D) showing frequency of

stimulated SLE patient and control sample with 50ng/ml interlukein 6 (3.65% and 0.36%)

respectively. Figure (E&F) showing frequency of stimulated SLE patient and control sample

with 50ng/ml interlukein 23 (3.38% and 0.27%) respectively.

22

Figure (5) Mean percentage of DN T cells before and after stimulation with 50 ng/ml

IL23 in patient and control samples ● P=0.001 . Mean percentage of DN T cells in

activated patient samples is significantly higher than activated control samples ■

P=0.001.

1.46 ± 0.16

1.10 ±0.23

3.16 ± 0.36

1.12 ±0.29

0

0.5

1

1.5

2

2.5

3

3.5

4

control SLE

% o

f D

N T

cell

sunstimulated

stimulated

● P=0.001

■ P=0.001

Figure (6) Anti ds-DNA level (IgG) before and after stimulation with different

concentrations of interleukin 6.

Figure (7) Anti ds-DNA level (IgG) before and after stimulation with different

concentrations of interleukin 23.

0

5

10

15

20

25

30

35

40

45

none 10 ng/ml 50ng/ml 100 ng/ml

stimulation with IL6

An

ti d

s-D

NA

(Ig

G)

IU/m

l

P=0.002 P=0.012

0

5

10

15

20

25

30

35

40

45

none 10 ng/ml 50ng/ml 100 ng/ml

Stimulation with IL23

An

ti d

s-D

NA

(Ig

G)

IU/m

l P=0.049