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Regulation of the Function of T Follicular Helper Cells and B Cells in Type 1

Diabetes Mellitus by the OX40/OX40L Axis

Xuan Du*

, Yan Zhu*

, Wen Lu

, Nannan Fu

, Qin Wang

2,#

, Bimin Shi

1,#

Xuan Du and Yan Zhu

contributed equally to this study.

Department of Endocrinology and Metabolism, First Affiliated Hospital of Soochow University, Suzhou,

Jiangsu, China

Department of Immunology, Suzhou Medical College of Soochow University, Suzhou, Jiangsu, China

Keywords

: T1DM, Tfh, OX40, OX40L

#Co-corresponding authors

Qin Wang: wangqin78@suda.edu.cn

Bimin Shi:

SDshibimin@163.com

Funding

: This study was supported by grants from the Suzhou Science and Technology Fund

(SKY2022125) and the Key University Science Research Project of Jiangsu Province (20KJA180002).

Declarations of interests

: The authors report no conflicts of interest.

ORCID number of the corresponding author

: 0009-0006-7688-2283 (BM.Shi)

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Regulation of the Function of T Follicular Helper Cells and B Cells in Type 1

Diabetes Mellitus by the OX40/OX40L Axis

Abstract

Objective/Main Outcome

: To study the expression of OX40 on T follicular helper (Tfh) cells and the

ligand OX40L on antigen-presenting cells (APCs) in peripheral blood of patients with Type 1 diabetes

mellitus (T1DM) and the role of OX40 signaling in promoting Tfh cells to assist B-cell differentiation.

Design

: Cross-sectional study.

Setting

: Endocrinology department of a university hospital.

Participants

: Twenty-five patients with T1DM and 35 with newly diagnosed T2DM from January 2021–

December 2021 (39 males, 21 females; mean age: 31.0 ± 4.5, range: 19 –46 years).

Interventions

: None.

Methods

: The peripheral blood proportion of CD4

CD25

−

CD127

CXCR5

PD1

Tfh cells in patients with

T1DM or T2DM and the OX40L expression in CD14

monocytes and CD19

B cells were analyzed by

flow cytometry. The OX40 signal effect on Tfh-cell function was analyzed by co-incubating B cells with

Tfh cells under different conditions. Flow cytometry detected the ratio of CD19

−

CD138

plasmacytes.

Results

：

The Tfh cells ratio and intracellular IL-21 expression in peripheral blood was significantly

higher in patients with T1DM than with T2DM, and the OX40 expression in peripheral Tfh cells and

OX40L expression in APC were significantly higher in T1DM. After adding OX40L protein, the

CD19

−

CD138

-plasmacytes percentage was significantly increased and higher in T1DM. Blocking of

anti-OX40L monoclonal antibodies significantly reduced the plasmacytes ratio.

Conclusions

: The peripheral Tfh cells proportion increased and the OX40 expression in peripheral Tfh

cells was upregulated in patients with T1DM versus patients with T2DM. OX40/OX40L signaling

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enhanced the Tfh-cell function to assist B-cell differentiation, which may contribute to the pathogenesis

of T1DM.

Introduction

Type 1 diabetes mellitus (T1DM) is an organ-specific chronic autoimmune disease. Characterized by

insulin deficiency due to islet-β cell destruction, T1DM patients depend on exogenous insulin to maintain

glucose metabolism.

1,2

Although environmental factors are implicated, immunological dysfunction has a

crucial role in the pathogenesis of T1DM. However, the exact mechanisms of autoimmune responses

remain unclear. Abnormal functions of Tfh and B cells have important roles in development of T1DM.

The differentiation process of B cells requires the help of Tfh cells, which are important regulators of

humoral immune responses. The inflammatory cytokine interleukin -21 (IL-21) secreted by Tfh cells

promotes activation of B cells and secretion of other cytokines, such as IL-17, thereby aggravating

inflammation. In peripheral lymphoid tissues, following activation by DCs, naïve T cells rapidly

upregulate CXCR5 and arrive at the T-cell–B-cell border under the chemotaxis of CXCL13 secreted by B

cells and then enter the lymphoid follicle to become Tfh cells, which help naïve B cells differentiate into

plasma cells by secreting IL-21 and help B cells produce antigen-specific antibodies to induce humoral

immune reponses.

3-5

Hence, enhancement of Tfh cells may have an important role in the pathogenesis of

T1DM.

OX40 molecules are mainly expressed on the membranes of activated CD4

and CD8

T cells.

Synergizing with ICOS/ICOSL, OX40/OX40L signaling induces activation of antigen -specific CD4

cells and upregulates CXCR5 expression, which promotes T-cell migration to lymphoid follicles and GC

formation.

6-8

As an independent subset of CD4

T cells, Tfh cells have been confirmed to also express

OX40 molecules. However, there are no published reports on OX40

Tfh cells in T1DM. Furthermore, the

potential mechanisms underlying the function of OX40

Tfh cells in T1DM have not been elucidated.

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Our study aimed to detect the expression of OX40/OX40L and Tfh cells in the peripheral blood of

patients with T1DM and explore the potential mechanisms of Tfh and B cell differentiation regulated by

OX40/OX40L signaling in T1DM, which could provide potential therapeutic targets for developing novel

therapies for T1DM.

Materials and Methods

Patients and Laboratory Analysis

A total of 25 patients living with T1DM and 35 patients with newly diagnosed T2DM between January

2021 and December 2021, including 39 males and 21 females (mean age: 31.0 ± 4.5 years, range: 19 –46

years), in the Department of Endocrinology of the First Affiliated Hospital of Soochow University were

included in this cross-sectional study. The study was approved by the Ethics Committee of the First

Affiliated Hospital of Soochow University (Suzhou, China). Patients and controls provided written

informed consent. The diagnostic criteria for T1DM were according to the “The T1DM Exchange Clinic

Registry,” and the patients with T2DM were diagnosed according to the 1999 WHO diagnostic criteria.

9,10

Patients with secondary diabetes, acute or chronic inflammatory diseases, infectious diseases, additional

autoimmune disorders, and cancers were excluded. In addition, body height and weight were measured.

Based on the measurements, the body mass index (BMI) was calculated. Blood samples from an

antecubital vein were collected in a quiet state in the morning after fasting overnight for 12 h, and fasting

blood glucose (FBG), glycosylated hemoglobin A1c (HbA1c), C-peptide (CP), C-peptide after a 2-h meal

(2hCP), glutamic acid decarboxylase (GAD)Ab, ZnT8A, ICA, and IAA levels were measured. The

glucose oxidation method was used to measure the blood glucose levels, with an automatic biochemical

analyzer (7600, HITACHI Company, Japan). HbA1c was measured by high-performance liquid

chromatography and a Bio-Rad Variant II analyzer. (HLC-723G8, TOSOH Company, Japan). Serum CP

and 2hCP levels were measured by chemiluminescent immunoassay (AIA-2000ST; TOSOH Company,

Japan). GADA, ZnT8A, ICA, and IAA (iFLASH test panel, YHLO Company, China) levels were

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measured by chemiluminescence immunoassay analyzer (iFLASH 3000, YHLO Company, China).

Cell Staining and Flow Cytometry

To assess the expression of OX40 on Tfh cells and OX40L on CD14

monocytes and CD19

B cells,

heparinized peripheral venous blood was obtained from the study subjects. Flow cytometry ( Beckman

Coulter, USA) was performed on peripheral blood mononuclear cells (PBMCs), separated by

centrifugation of blood over a Ficoll–Conray gradient (Dayou Co, China), and incubated with

fluorochrome-labeled monoclonal antibodies (mAbs) for 30 min. Anti-CD4-BV605 (Catalog # 300556,

RRID: AB_2564391), anti-CD25-BV421 (Catalog # 562442, RRID: AB_11154578), anti-PD1-FITC

(Catalog # 557860, RRID: AB_2159176), anti-OX40-Percpcy5.5 (Catalog # 350010, RRID:

AB_10719224), anti-CD19-BV605 (Catalog # 562653, RRID: AB_2722592), and anti-OX40L-PE

(Catalog # 558164, RRID:

AB_647195) mAbs were obtained from Becton Dickinson, USA. Anti-

CXCR5-APC (Catalog # 12-9185-42, RRID: AB_11219877) was obtained from eBioscience, USA, and

anti-CD14-PEvivo-770 was obtained from Miltenyi Biotec, German.

To detect intracellular IL-21 produced by Tfh cells, PBMCs isolated from DM patients were cultured with

anti-CD3 mAb (200 ng/ml, Catalog #

BE0001-2, RRID:

AB_1107632, Bio Cell, USA) at 37°C, 5% CO

and 95% humidity. The next day, phorbol-12-myristate-13-acetate (50 µg/ml,

Sigma-Aldrich, USA),

ionomycin (750 ng/ml, BioVision, USA), and GolgiStopTM (Catalog # 51-2092KZ, RRID: AB_2869009,

BD, USA) were added to the plates. Anti-CD4-BV605, anti-PD1-FITC, anti-CXCR5-APC, and anti-

OX40-Percpcy 5.5 were added and labeled for 30 min. Then, after Fixations/Permeation Buffer (Catalog

# 51-2092KZ, RRID: AB_2869009, BD, USA), Perm/Wash

buffer (Catalog # 51-2092KZ, RRID:

AB_2869009, BD, USA) and anti-IL-21-BV421 (Catalog # 564755, RRID:

AB_2738933) configured

with Perm/Wash

buffer were respectively added, they were measured by flow cytometry.

Purification of CD4

CXCR5

Tfh Cells

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Following the manufacturer’s instructions, a REAlease CD4 Micro Beads kit (Miltenyi Biotec, German)

was used to separate CD4

T cells from PBMCs. After labeling with anti-CXCR5-APC for 30 min,

CD4

CXCR5

Tfh cells were isolated from the purified CD4

T cells after co-culture with 20-L Anti-APC

Micro Beads (Miltenyi Biotec, German) according to the manufacturer’s instructions. The purity of the

separated cells was monitored by flow cytometry. Purity >90% was acceptable.

Co-culture of Tfh and B Cells

We performed co-culture assays using CD4

CXCR5

Tfh cells and B cells that were separated from

PBMCs by negative selection using a B-cell isolation kit (Miltenyi Biotec, German) at a ratio of 1:1 (1 ×

:1 × 10

). The co-cultures were incubated for 72 h at 37°C, 5% CO

, and 95% humidity. The

concentrations of SEBAnti-CD3 mAb (200 ng/ml, clone: OKT3; Bio Cell, USA), sOX40L (R&D, USA),

and anti-OX40L (Catalog # MAB10541, RRID: AB_2272152) were 100, 200, 100, and 500 ng/ml,

respectively. The experimental groups included B+Tfh, B+Tfh+sOX40L/anti-OX40L, and B cells alone.

After 72-h incubation, the cocultured cells were collected. After anti-CD19-BV605 and anti-CD138-APC

were added and labeled for 30 min, they were measured by flow cytometry.

Statistical analysis

Graph Pad Prism 9.0 software (San Diego, Calif, USA) was used for data analysis. The Shapiro–Wilk test

was performed to determine data normality. To examine differences between the two groups, Student’s

tests (two-tailed) were performed. To assess the ratio of plasmacytes in the cocultured cells between the

T1DM and T2DM groups, a two-way ANOVA test was performed. Statistical significance was set at

0.05. Data are expressed as the mean ± standard deviation (SD).

Ethics

This study was approved by the Biomedical Ethics Committee of the First Affiliated Hospital of Soochow

University (2022180). All participants signed the informed consent forms.

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Results

Clinical and Biochemical Characteristics of the Patients

Twenty-five patients with T1DM and 35 newly diagnosed T2DM were recruited. Their clinical and

biochemical characteristics are shown in Table 1. The levels of FCP and 2hCP were significantly lower in

the T1DM group than in the T2DM group (

p <

0.01), whereas the levels of GADA, ZnT8A, ICA, and

IAA were significantly higher in the T1DM group (

< 0.01). There were no significant differences in the

age, sex, FBG, and HbA1c levels between the two groups (

> 0.05).

Increased Circulating Tfh and IL-21

Tfh Cells in T1DM

To measure the ratio of the Tfh-cell subset in T1DM, PBMCs from patients with T1DM and T2DM were

detected by flow cytometry. The gating strategy for flow cytometric analysis of Tfh

(CD4

CD25

−

CD127

CXCR5

PD1

) cells is shown in Figure 1A. In contrast to T2DM (7.63% ± 0.87%),

the percentage of circulating Tfh cells increased in T1DM (11.35% ± 1.38%), (

< 0.05, Figure 1A, B).

IL-21, produced by Tfh cells, is critical for GC formation as well as Tfh-cell generation. We further tested

the expression of IL-21 in the Tfh cells. The expression of IL-21 was significantly higher in the T1DM

group (53.08% ± 4.53%) than in the T2DM group (37.04% ± 3.37%), (

< 0.01, Figure 1C, D). The

results indicated that the elevated level of IL-21 may be associated with the increased ratio of the Tfh-cell

subset in patients with T1DM.

Upregulation of Tfh-cellular OX40 Expression in T1DM

To explore the role of OX40 in Tfh cells, the expression of OX40 in Tfh cells was further analyzed. The

gating strategy for the flow cytometric analysis of OX40

effector T cells and OX40

Tfh cells is shown in

Figure 2. Compared with that in T2DM (5.42% ± 1.28%), The OX40 expression of effector T cells

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increased in T1DM (12.02% ± 3.34%) (

< 0.05, Figure 2A–B). Moreover, the expression of OX40 in Tfh

cells was significantly higher in T1DM (6.38% ± 1.29%) than in T2DM (2.98% ± 0.58%) (

< 0.01,

Figure 2C–D). Thus, OX40 was mainly expressed in Tfh cells.

Upregulation of OX40L Expression in APCs in T1DM

We further determined the expression of OX40L in CD14

monocytes and CD19

B cells in T1DM and

T2DM. The percentage of OX40L

CD14

monocytes was higher in T1DM (14.25% ± 3.59%) than in

T2DM (6.12% ± 1.29%) (

< 0.05). The expression of OX40L in CD19

B cells was higher in T1DM

(11.68% ± 2.05%) than in T2DM (4.04% ± 0.60%) (

p <

0.01). Overall, the expression of OX40L in APCs

was obviously higher in T1DM than in T2DM. Therefore, we inferred that OX40L expressed in T1DM

APCs might bind to OX40 expressed in Tfh cells to promote the differentiation and activation of Tfh cells

(Figure 3).

Correlation of OX40

Tfh with T1DM-related Antibodies

GADAb, ZnT8A, ICA, and IAA, all of which are proteins associated with secretory granules in β-cells,

are biomarkers of T1DM-associated autoimmunity and can be used to identify T1DM. Thus, the

relationship between the percentage of OX40

Tfh cells and the levels of serum T1DM-related antibodies,

such as GADA, ZnT8A, and ICA, was further analyzed. We found that the percentage of OX40

Tfh cells

in T1DM was positively associated with the levels of GADA (

p <

0.05) (Figure 4A), but not with ZnT8A

and ICA levels, suggesting that Tfh cells have a close relationship with GADA during the development of

T1DM by OX40/OX40L signaling.

Differentiation of B Cells Induced by Tfh Cells Through the OX40 Signal

In Vitro

To further identify the potential roles of Tfh cells in regulating B cells, peripheral B cells and Tfh cells

from T1DM or T2DM were cocultured for 72 h

in vitro

. Next, the ratio of CD19

−

CD138

plasmacytes

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were analyzed by flow cytometry. As shown in Figures 5A and B, there was a higher percentage of

plasmacytes in T1DM than in T2DM. When B cells were cocultured with Tfh cells, the percentage of

plasmacytes was upregulated. Furthermore, after sOX40L protein was added, the percentage of

plasmacytes was significantly increased in the T1DM group (Figure 5C and D). These data indicate that

OX40 may have an important role in the differentiation of B cells by promoting Tfh -cell differentiation.

To further determine the potential role of OX40 during the differentiation of

cells stimulated by Tfh

cells in vitro, anti-OX40L antibodies were added to the Tfh–B-cell co-culture system for 72 h. Compared

with the group without anti-OX40L antibodies, these antibodies significantly downregulated the

percentage of plasmacytes in T1DM. Therefore, blockade of OX40/OX40L signaling inhibited B -cell

differentiation induced by Tfh cells

in vitro

. Together, these data indicated that OX40/OX40L may

promote the differentiation of B cells stimulated by Tfh cells.

Discussion

As an important member of the tumor necrosis factor receptor, the OX40 co-stimulatory molecule has

an important role in promoting the proliferation and activation of CD4

T cells. Kurata I

et al

. detected

high expression of OX40 in Tfh cells, especially Tfh17 cells, in patients with rheumatoid arthritis (RA)

and in RA mouse models.

Jacquemin

et al.

found that OX40/OX40L signaling was involved in the

pathogenesis of systemic lupus erythematosus by promoting the differentiation of Tfh cells.

However,

the definite mechanism by which OX40/OX40L regulates Tfh cells in the pathogenic processes of T1DM

remains unclear.

Our study indicated that abnormal differentiation of Tfh cells regulated by OX40 contributed to the

immunopathological process in T1DM. Compared with patients with T2DM, patients with T1DM had

larger numbers of OX40

effector T cells, which was consistent with a previous finding,

indicating that

effector T cells were abnormally proliferated in patients with T1DM. Soroosh

et al

. found that the number

of effector T cells in OX40-knockout mice was significantly reduced, whereas the number of TCM cells

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remained normal,

suggesting that OX40/OX40L has an important role in the accumulation of effector T

cells. Furthermore, the results showed an increased number of CD4

CD25

–

CD127

CXCR5

PD1

Tfh

cells, IL-21

Tfh cells, and OX40

Tfh cells gated on CD4

CD25

–

CD127

effector T cells in T1DM.

Reportedly, Tfh cells promote the onset of T1DM through CXCR5 and/or IL-21. In peripheral lymphoid

tissues, following activation by DCs, naïve T cells rapidly upregulate CXCR5 and arrive at the T–B-cell

border under the chemotaxis of CXCL13 secreted by B cells and then enter the follicle to become Tfh

cells, which assist naïve B cells to differentiate into plasma cells by secreting IL-21. The pathogenesis of

T1DM is thought to involve T-cell and B-cell–mediated destruction of β-cells. The islet-targeting

autoantibodies of GADA, ZnT8A, ICA, and IAA, all of which are proteins associated with secretory

granules in β-cells, are biomarkers of T1DM-associated autoimmunity and can be used to identify and

study individuals at risk of developing T1DM.

Studies have demonstrated that B cells are crucial APCs in

the pathogenic T-cell response to GADA and for overcoming a checkpoint in T-cell tolerance to B-cell

Ags. Although proinflammatory Tfh cells are considered to be the functional mediators of cell destruction

in the pancreatic islets, the exact role of B cells in the disease process of T1DM remains unclear.

15-19

importantly, the increased number of OX40

Tfh cells in patients with T1DM was positively associated

with the level of GADA, a marker of autoantibodies for T1DM. GAD is an intracellular enzyme fairly

expressed in insulin-secreting pancreatic β cells and neurons. The physiologic function of GAD is

decarboxylation of glutamate to gamma-aminobutyric acid.

20-23

Responses to immunodominant GAD65

peptides were also absent in B-cell–deficient non-obese diabetic mice, suggesting that B cells are crucial

for the autoimmune response in T1DM. Therefore, OX40

Tfh cells are closely associated with the

immunopathological process of T1DM. In addition, flow cytometry analysis demonstrated a significant

increase in OX40L expression in CD14 monocytes and CD19 B cells in T1DM, consistent with the trend

of OX40 expression. Thus, OX40 molecules expressed in Tfh cells may combine with OX40 ligands

expressed in APCs to influence Tfh differentiation and function and induce the development of T1DM.

To further define the function of OX40 in Tfh cells in T1DM, a Tfh–B-cell co-culture system was used

in vitro. The ratio of CD19

−

CD138

plasmacytes increased when B cells were cocultured with Tfh cells

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isolated from T1DM. Adding sOX40L protein further increased the proportion of CD19

−

CD138

plasmacytes, which indicated that OX40/OX40L signaling could enhance Tfh-cell function and promote

B-cell activation and antibody secretion. Furthermore, the OX40/OX40L pathway was blocked by anti-

OX40L Abs in the Tfh–B-cell co-culture system, and an obviously decreased ratio of CD19

−

CD138

plasmacytes was detected, which indicated inhibition of B-cell differentiation when OX40 signaling was

weakened. These data suggest that the mechanisms of B-cell activation and differentiation induced by

OX40

Tfh cells exist in two aspects. One aspect is the combination of OX40 expressed in Tfh cells and

OX40L expressed in B cells, which delivers signaling to enhance the function of Tfh cells and production

of IL-21, which in turn induces plasma cell differentiation. The other aspect is reverse signaling, which is

delivered from OX40 expressed in Tfh cells to OX40L expressed in B cells to promote B -cell

differentiation directly. In summary, OX40/OX40L signaling between Tfh cells and B cells is

bidirectional, which could affect B cells directly or indirectly by enhancing Tfh function.

Previous studies have suggested that Tfh cells have an important role in the pathogenesis of various

autoimmune diseases, such as T1DM, SLE, and RA. These diseases result from the inability of the

immune system to suppress autoreactive T-cell responses and autoantibody production.

24-27

Thus, the main

function of Tfh cells is to assist antigen-specific B-cell proliferation, antibody classification conversion

and promote the differentiation of B lymphocytes into plasmacytes and memory B cells. Tfh cells assist B

cells mainly by secreting cytokines and by direct contact in the GC. IL-21, produced by Tfh cells, can

induce B cells to differentiate into plasmacytes, which can secrete antibodies.

28-30

Tfh cells assist B cells,

which are indispensable for high-affinity antibody production and memory B-cell yield.

31-35

In summary, our study demonstrated that the function of Tfh cells and B cells is regulated by the

OX40/OX40L axis in T1DM. However, there are still some problems related to this issue that remain

unresolved in this study. Given that blocking OX40/OX40L signaling has previously shown great

therapeutic effects in some mouse models of autoimmune diseases, targeting OX40/OX40L is promising

as a new therapeutic approach for these diseases.

Rituximab, an anti-CD20 monoclonal antibody, represses Tfh differentiation by decreasing IL-21,

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which inhibits the differentiation of Tfh cells by distinct mechanisms and thus exert therapeutic effect on

T1DM. Rocatinlimab, an anti-OX40 antibody, was evaluated in a clinical trial in patients with atopic

dermatitis; however, its therapeutic effect on T1DM is unknown. The efficacy data from clinical trials

that targeted OX40/OX40L and T1DM are limited. In addition, the exact mechanism by which the

OX40/OX40L axis affects Tfh also remains unknown at present. Therefore, additional studies are needed

to evaluate the biological characteristics of these cells and their mechanisms involved in the prevention or

treatment of T1DM and other autoimmune diseases.

Conclusion

Compared with patients with T2DM, the proportion of peripheral Tfh cells increased and the expression

of OX40 in peripheral Tfh cells was upregulated in patients with T1DM. The OX40/OX40L signal can

enhance the function of Tfh cells to assist B-cell differentiation and autoantibody production, a finding

that potentially can be used in new approaches for preventing or treating T1DM.

Acknowledgments

Address all correspondence and requests for reprints to:

Qin Wang, M.D., Ph. D., Department of

Immunology, Suzhou Medical College of Soochow University, Renai Road 199, Suzhou, 215123, Jiangsu

Province, China. E-mail: Qin Wang: wangqin78@suda.edu.cn

Bimin Shi

，

M.D., MD, PhD, Department

of Endocrinology and Metabolism, the First Affiliated Hospital of Soochow University, Shizi Road 188,

Suzhou, 215129 Jiangsu, China. E-mail: SDshibimin@163.com

Funding

This study was supported by grants from the Suzhou Science and Technology Fund (SKY2022125)

and

the Key University Science Research Project of Jiangsu Province (20KJA180002)

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Disclosures

The authors report no conflicts of interest.

Data Availability

All datasets generated during and analyzed during the current study are not publicly available but are

available from the corresponding author on reasonable request.

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Table 1. Clinical and biochemical characteristics of the patients

T2DM group

(n = 35)

T1DM group

(n = 25)

value

Age (year)

41.31 ± 2.45

36.432.95

0.055

Male/Female

25/10

14/11

0.276

BMI (kg/m

)

29.29 ± 0.83

22.43 ± 0.90

<0.01

FBG (mmol/L)

8.90 ± 0.49

8.77 ± 0.95

0.894

HbA

(%)

10.76 ± 0.38

10.99 ± 0.64

0.745

FCP (ng/ml)

1.97 ± 0.21

0.46 ± 0.14

<0.01

2hCP (ng/ml)

5.26 ± 0.48

1.06 ± 0.25

<0.01

GADAb (IU/ml)

1.68 ± 0.34

83.36 ± 35.09

<0.01

ZnT8A (AU/ml)

0.60 ± 0.13

1.43 ± 0.59

<0.01

ICA (JDF units)

0.06 ± 0.00

0.79 ± 0.28

<0.01

IAA (JDF units)

0.17 ± 0.05

1.19 ± 0.48

0.011

< 0.05 compared with the control group;

< 0.01 compared with the control group;

BMI, body mass index; FPG, fasting plasma glucose; HbA

, glycosylated hemoglobin; FCP, fasting

C-peptide; 2hCP, C-peptide after 2-h meal; GADA, glutamic acid decarboxylase antibodies; ZnT8A,

zinc transporter-8 autoantibodies; ICA, islet-cell antibodies; IAA, insulin autoantibody

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Figure 1. Flow cytometry analysis of the percentage of circulating Tfh cells and their IL-21 expressions in

patients with T1DM and T2DM. PBMCs (5 × 10

/tube) were isolated from individual subjects and stained

with anti-CD4, anti-CD25, anti-CD127, anti-PD1, anti-CXCR5, and intracellular anti-IL-21. The cells

were characterized by flow cytometry analysis by gating initially on living lymphocytes and then on

CD4

CD25

−

CD127

effector T cells and CD4

CD25

−

CD127

CXCR5

PD1

Tfh cells. Subsequently, the

numbers of different subsets of Tfh cells and IL-21

Tfh cells were calculated according to the frequency

of effector T cells and Tfh cells. A. Flow cytometry analysis of Tfh cells. B. The numbers of

CD4

CD25

−

CD127

CXCR5

PD1

Tfh cells in T1DM and T2DM. A significant increase in the frequency

of Tfh cells was observed in T1DM (11.35% ± 1.38%) compared to T2DM (7.63% ± 0.87%). C. Flow

cytometry analysis of IL-21

Tfh cells. D. Percentage of IL-21

Tfh cells in T1DM and T2DM. The IL-21

level iss significantly higher in the T1DM group (53.08% ± 4.53%) than in the T2DM group (37.04% ±

3.37%). The experimental data are expressed as x ± SD. *:

< 0.05; **:

< 0.01.

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Figure 2. Flow cytometry analysis of the expression of OX40 in effector T and Tfh cells in patients with

T1DM and T2DM. PBMCs (5 × 10

/tube) were isolated from individual subjects and stained with anti-

CD4, anti-CD25, anti-CD127, anti-PD1, anti-CXCR5, and anti-OX40. The cells were characterized by

flow cytometry analysis by gating initially on living lymphocytes and then on CD4

CD25

−

CD127

effector T cells or CD4

CD25

−

CD127

CXCR5

PD1

Tfh cells. Subsequently, the numbers of OX40

effector T and OX40

Tfh cells were calculated according to the frequency of effector T and Tfh cells. A.

Flow cytometry analysis of OX40

effector T cells. B. Number of OX40

effector T cells in T1DM and

T2DM. Compared with that in T2DM (5.42% ± 1.28%), OX40 expression in effector T cells increased in

T1DM (12.02% ± 3.34%). C. Flow cytometry analysis of OX40

Tfh cells. D. Numbers of OX40

Tfh

cells in T1DM and T2DM. The expression of OX40 was significantly higher in Tfh cells in the T1DM

group (6.38% ± 1.29%) than in the T2DM group (2.98% ± 0.58%).The experimental data are expressed

as x ± SD. *:

< 0.05; **:

< 0.01.

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Figure 3. Flow cytometry analysis of the expression of OX40L in CD14

monocytes and CD19

B cells in

T1DM and T2DM patients. PBMCs (5 × 10

/tube) were isolated from individual subjects and stained with

anti-CD14, anti-CD19, and anti-OX40L. The cells were characterized by flow cytometry analysis by

initially gating living monocytes and B cells. Subsequently, the numbers of OX40L

CD14

monocytes

and OX40L

CD19

B cells were calculated. A. Flow cytometry analysis of OX40L

CD19

B cells. B.

Numbers of OX40L

CD19

B cells in T1DM and T2DM. The percentage of OX40L

CD14

monocytes

(14.25% ± 3.59%) was higher in T1DM than in T2DM (6.12% ± 1.29%). C. Flow cytometry analysis of

OX40L

CD14

monocytes. D. Numbers of OX40L

CD14

monocytes in T1DM and T2DM. The

expression of OX40L in CD19

B cells was higher in T1DM (11.68% ± 2.05%) than in T2DM (4.04% ±

0.60%). The experimental data are expressed as x ± SD. *:

< 0.05; **:

< 0.01.

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Figure 4. Correlation between serum levels of GADA, ZnT8A, and ICA and the ratio of OX40

Tfh cells

in T1DM. A. Correlation of serum levels of GADA with the ratio of OX40

Tfh cells in T1DM. B.

Correlation of serum ZnT8A levels with the ratio of OX40

Tfh cells in T1DM. C. Correlation of serum

ICA levels with the ratio of OX40

Tfh cells in T1DM. The percentage of OX40

Tfh cells in T1DM is

positively associated with GADA levels (

< 0.05), but not with ZnT8A and ICA levels.

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Figure 5. OX40 promoted B-cell differentiation induced by Tfh cells

in vitro.

Peripheral blood Tfh cells

were obtained from 6 T1DM and 4 T2DM cells incubated with B cells with or without sOX40L.

Cocultured cells were collected and stained with anti-CD19 and anti-138 antibodies. The cells were

characterized by flow cytometry analysis by initially gating B cells. Subsequently, the numbers of

CD19

−

CD138

plasmacytes was calculated. The experimental groups included anti-CD3+SEB+B, anti-

CD3+SEB+B+Tfh, and anti-CD3+SEB+B+Tfh+sOX40L. A. Flow cytometry analysis of CD19

CD138

plasmacytes. B. Percentages of plasmacytes in the different cocultured groups. Compared with T2DM, a

higher percentage of plasmacytes was detected when B cells were cocultured with Tfh cells in T1DM.

Furthermore, after sOX40L protein was added, the percentage of plasmacytes was significantly increased

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in the T1DM group. Blockade of OX40/OX40L signaling inhibits B-cell differentiation induced by Tfh

cells

in vitro.

Peripheral blood Tfh cells obtained from 3 T1DM and 2 T2DM cells incubated with B cells

with or without anti-OX40L antibodies. Subsequently, the numbers of CD19

−

CD138

plasmacytes was

calculated. Experimental groups included anti-CD3+SEB+B, anti-CD3+SEB+B+Tfh, and anti-

CD3+SEB+B+Tfh+anti-OX40L. C. Flow cytometry analysis of CD19

−

CD138

plasmacytes. D.

Percentages of plasmacytes in the different cocultured groups. Compared with the group without anti -

OX40L antibodies, these antibodies significantly downregulated the percentage of plasmacytes in T1DM.

Therefore, blockade of OX40/OX40L signaling inhibits B-cell differentiation induced by Tfh cells in

vitro. The experimental data are expressed as x ± SD. *:

< 0.05.

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