Whole blood exchange-lymphoplasmapheresis combined transfusion as an immunotherapy in systemic lupus erythematosus

Summary

Systemic lupus erythematosus (SLE) remains a therapeutic challenge. This study aims to evaluate the efficacy and safety of whole blood exchange-lymphoplasmapheresis combined transfusion (WLCT) in SLE. This retrospective study evaluated the clinical and immunological impact of WLCT in 84 patients with refractory SLE, compared to 61 controls receiving conventional therapy (CT). WLCT significantly attenuated disease activity, evidenced by reductions in Systemic Lupus Erythematosus Disease Activity Index scores, serum creatinine, uric acid, proteinuria, and glucocorticoid dependence. WLCT restored hemoglobin levels in autoimmune hemolytic anemia and suppressed pathogenic immunoglobulins (IgG, IgA, IgM). Flow cytometry showed expanded regulatory T cells and reduced Th17 frequencies, along with decreased IL-17A and TNF-α and increased TGF-β. Longitudinal follow-up demonstrated sustained benefits, including reduced proteinuria and neutrophil-driven inflammation at 12 months. These findings suggest that WLCT may offer therapeutic potential in controlling disease flares and mitigating organ damage in refractory SLE.

Graphical abstract

Highlights

• •WLCT associates with reduced disease activity and glucocorticoid dependence in refractory SLE
• •WLCT modulates T cell subsets by expanding Tregs and reducing Th17 cells
• •WLCT downregulates proinflammatory cytokines (IL-17A and TNF-α) and enhances TGF-β
• •WLCT reduces proteinuria and neutrophil-driven inflammation at 12 months of follow-up

Health sciences

Introduction

Systemic lupus erythematosus (SLE) is a complex and debilitating autoimmune disorder characterized by widespread immune dysregulation, leading to significant morbidity and mortality.¹ The pathogenesis of SLE involves both innate and adaptive immune responses, with the polyclonal activation of B and T lymphocytes being a hallmark feature.² Despite considerable advancements in therapeutic strategies, the heterogeneous nature of immune dysregulation in SLE results in variable patient responses to treatment. A subset of patients remains refractory to current state-of-the-art therapies, placing them at high risk for severe complications, including organ failure and death. Furthermore, the delayed onset of therapeutic efficacy and the associated risks of infections, hepatotoxicity, and other adverse effects pose significant challenges in SLE management.³

Therapeutic plasmapheresis (TPE) has been proposed as an intervention for severe SLE due to its capacity to rapidly remove autoantibodies, proinflammatory cytokines, and other pathogenic mediators from circulation, thereby providing prompt control of disease activity.^4,5 However, the clinical efficacy of TPE remains controversial, as some studies have reported limited or no improvement in patients with SLE undergoing this procedure.⁶ Building on the conventional TPE approach, our institution has pioneered the use of lymphoplasmapheresis (LPE) combined with whole-blood exchange (WBE) for the treatment of severe SLE. This innovative strategy not only targets circulating soluble pathogenic factors, such as autoantibodies, complement components, cytokines, and adhesion molecules, but also depletes immunocompetent cells, including sensitized T and B lymphocytes, as well as sensitized red blood cells (RBCs).

Emerging evidence suggests that lymphocytes play a pivotal role in both the initiation and perpetuation of the inflammatory cascades underlying SLE.² Additionally, studies demonstrated that SLE erythrocytes retain mitochondria due to defective mitophagy, and these mitochondria-retaining erythrocytes (MREs) drive type I IFN production through the activation of monocytes.^7,8 Given that LPE effectively depletes lymphocytes and WBE removes whole blood, including pathogenic RBCs and MREs, the combination of LPE and WBE may represent a promising alternative to current second-line therapies for refractory SLE. Our prior work in SLE-associated autoimmune hemolytic anemia (AIHA) provided the rationale to extend this approach to broader refractory SLE populations.

To evaluate the efficacy and safety of this combined therapeutic strategy, we conducted a retrospective study involving patients with severe SLE. By analyzing real-world clinical data, we aim to provide robust evidence supporting the application of WLCT as a viable treatment option for severe SLE, particularly in cases where conventional therapies have failed. This study seeks to address the unmet clinical need for more effective and rapid interventions in the management of this formidable autoimmune disease.

Results

The baseline clinical information of patients with SLE in the CT and WLCT groups

Patients with SLE in the WLCT group exhibited significantly higher glucocorticoid doses and elevated C-reactive protein (CRP) levels compared to those in the CT group. Additionally, the WLCT group showed lower hemoglobin (Hb) and platelet (PLT) counts than the CT group. However, no significant differences were observed between the WLCT and CT groups in terms of age, gender, or Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores, which are critical indicators of disease severity (Table 1). These findings suggest that while the WLCT group may experience more pronounced inflammatory responses and hematological abnormalities, the overall disease severity, as indicated by demographic and clinical parameters, remains comparable between the two groups.

Table 1.

Baseline clinical characteristics and treatment condition of patients from 2 groups

	CT (n = 61)	WLCT (n = 84)	p value
Age (years)	39.41 ± 1.47	35.63 ± 1.61	0.099
Female [n (%)]	54(88.52%)	71 (84.52%)	0.494
SLEDAI	15.25 ± 0.48	16.24 ± 1.15, n = 46, NA = 38	0.384
GC therapy
Glucocorticoid pulse [n (%)]	60 (100%, n = 60), NA = 1	65 (100%, n = 65), NA = 19	/
Total prednisone dose(mg) (from Hb baseline until Hb ≥ 60 g/L)	58.59 ± 5.85	103.8 ± 13.92	0.004∗∗
Second-line treatment	n = 61	n = 56, NA = 28
IVIG [n (%)]	11 (18.03%)	24 (42.86%)	/
RTX [n (%)]	7 (11.48%)	2 (3.57%)	/
Immunosuppressive agent	n = 61	n = 56, NA = 28	/
Cyclosporin A [n (%)]	1 (1.64%)	5 (8.93%)	/
Tacrolimus [n (%)]	6 (9.84%)	9 (16.07%)	/
Cyclophosphamide [n (%)]	26 (42.6%)	28 (50.00%)	/
Mycophenolate Mofetil [n (%)]	26 (42.6%)	13 (23.21%)	/
BRT	n = 54, NA = 7	n = 83, NA = 1
Hb baseline (g/L)	101.07 ± 3.03	68.82 ± 2.47	<0.0001∗∗∗∗
PLT (10ˆ9/L)	171.26 ± 10.54	121.84 ± 11.59	0.004∗∗
WBC count(10ˆ9/L)	5.79 ± 0.39	7.79 ± 0.48	0.003∗∗
Lymphocyte
Count (10ˆ9/L)	0.84 ± 0.05	0.88 ± 0.07	0.344
Propotion (%)	17.35 ± 1.17	13.77 ± 1.14	0.075
Neutrophil
Count (10ˆ9/L)	4.39 ± 0.35	6.30 ± 0.42	0.002∗∗
Proportion (%)	72.88 ± 1.47	77.95 ± 1.42	0.018∗
Monocyte
Count (10ˆ9/L)	0.44 ± 0.04	0.55 ± 0.05	0.153
Proportion (%)	7.66 ± 0.42	7.30 ± 0.47	0.599
Other clinical indexes
CRP (mg/L)	11.90 ± 2.56, n = 57, NA = 4	23.74 ± 4.43, n = 65, NA = 19	0.027∗
Cre (μmol/L)	119.23 ± 18.32, n = 54, NA = 7	126.37 ± 13.51, n = 47, NA = 37	0.761
BUN (mmol/L)	10.57 ± 1.29, n = 54, NA = 7	14.09 ± 1.43, n = 47, NA = 37	0.071
UA (μmol/L)	440.36 ± 20.48, n = 54, NA = 7	407.60 ± 22.13, n = 40, NA = 44	0.301
Proteinuria (+)	1.69 ± 0.13, n = 54, NA = 7	2.48 ± 0.21, n = 28, NA = 56	<0.001∗∗∗
Immunoglobulin
IgA (g/L)	2.66 ± 0.15	2.33 ± 0.16, n = 75, NA = 9	0.144
IgM (g/L)	0.92 ± 0.06	1.10 ± 0.08, n = 77, NA = 7	0.089
IgG (g/L)	14.29 ± 0.84	17.02 ± 1.06, n = 77, NA = 7	0.054
Complement
C3(g/L)	0.43 ± 0.02	0.37 ± 0.02, n = 78, NA = 6	0.122
C4(g/L)	0.08 ± 0.01	0.09 ± 0.01, n = 78, NA = 6	0.525
Positive rate of autoantibodies		n = 82, NA = 2
dsDNA [n (%)]	53 (86.89%)	63 (76.83%)	/
AHA [n (%)]	59 (96.72%)	78 (95.12%)	/
ANUA [n (%)]	34 (56.67), NA = 1	34 (41.46%)	/
Sm [n (%)]	29 (48.33%), NA = 1	27 (32.93%)	/
Ribosome P [n (%)]	26 (43.33%), NA = 1	32 (39.02%)	/
SSA [n (%)]	39 (65.00%), NA = 1	52 (63.41%)	/
SSB [n (%)]	11 (18.33%), NA = 1	15 (18.29%)	/
Ro52 [n (%)]	38 (63.33%), NA = 1	49 (59.76%)	/
aPL [n (%)]	2 (3.33%), NA = 1	13 (15.85%)	/
Platelet antibody [n (%)]	0 (0.00%), NA = 1	8 (9.76%)	/

CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; SLEDAI, systemic lupus erythematosus disease activity index; GC, Glucocorticoid; IVIG, intravenous immunoglobulin; RTX, rituximab; Hb, hemoglobin; PLT, platelet; WBC, white blood cell; CRP, C-reactive protein; Cre, creatinine; BUN, blood urea nitrogen; UA, uric acid‌; Ig, immunoglobulin; C3, complement 3; C4, complement 4, dsDNA, anti-dsDNA antibody; AHA, antihistone antibody; ANUA, antinucleosome antibody; Sm, anti-Smith antibody; SSA, anti-SSA antibody; SSB, anti-SSB antibody; Ribosome P, anti-ribosome P antibody; Ro52, anti-SSA-RO52 antibody; aPL, antiphospholipid antibody including anticardiolipin antibody, anti-b2-glycoprotein 1, and lupus anticoagulant; NA, Not Available; /, no comparison; Quantitative data are shown as means ± standard error of mean (SEM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

WLCT significantly alleviated SLE disease phenotypes

WLCT exhibited marked efficacy in attenuating a broad spectrum of SLE disease manifestations, as evidenced by significant improvements in serum creatinine (Cre), uric acid (UA), urinary protein, complement components (C3 and C4), CRP, and SLEDAI scores, alongside a reduction in glucocorticoid requirements. In contrast, CT showed no significant impact on these critical SLE biomarkers, including serum Cre, UA, proteinuria, C3 and CRP levels, nor did it alter glucocorticoid dosing (Figure 1). These results collectively establish that WLCT was associated with improvements in controlling disease manifestations and minimizing reliance on corticosteroids.

Figure 1.

WLCT significantly alleviated SLE disease phenotypes

∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, ns = not significant. CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; Cre: creatinine; BUN: blood urea nitrogen; UA: uric acid; C3: complement 3; C4: complement 4; CRP: C-reactive protein; SLEDAI: systemic lupus erythematosus disease activity index.

Effect of WLCT on Hb and immunoglobulin levels in patients with SLE

During the procedure, WLCT does not involve concurrent PLT exchange, resulting in a significant reduction in PLT counts post-treatment. However, the reduction was transient and not associated with bleeding events in any patient. WLCT was associated with elevated Hb levels in patients with SLE (Figure 2A). Comparative analyses showed that the increase in Hb was greater in the WLCT group than in the CT group (Figure 2A), underscoring the WLCT’s superior capacity to counteract erythrocyte destruction.

Figure 2.

WLCT alleviated Hb and immunoglobulin levels in patients with SLE

(A) Anemia-related indicators.

(B) Serum immunoglobulin. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, ns = not significant. CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; PLT: platelet; Hb: hemoglobin; AIHA: autoimmune hemolytic anemia; ITP: immune thrombocytopenia; Ig: immunoglobulin.

Notably, in patients with AIHA, WLCT treatment was associated with increased Hb levels; in patients with immune thrombocytopenia (ITP), WLCT was associated with increased PLT levels. In both subgroups, reductions in anti-IgG and anti-C3 autoantibodies, key drivers of complement-mediated hemolysis in AIHA, were also observed (Figure 2A).

WLCT markedly reduced the secretion of serum immunoglobulins, including IgA, IgM, and IgG. In contrast, the CT group showed a reduction only in IgG levels. (Figure 2B). These results highlight the potent immunomodulatory effects of WLCT in modulating humoral immune responses in SLE.

Effect of WLCT on WBC

WLCT did not exert a significant effect on the total white blood cell count in patients with SLE. However, in comparison to CT, WLCT treatment resulted in a moderate decrease in the absolute counts of lymphocytes, neutrophils, and monocytes. Moreover, WLCT therapy was associated with a reduced relative proportion of lymphocytes within the total leukocyte population (Figure 3).

Figure 3.

Effect of WLCT on blood cells

∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, ns = not significant. CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; WBC: white blood cell.

Regulation of WLCT on T cell subtypes and cytokines

To delineate the specific immune cell subsets implicated in the therapeutic effects of WLCT in SLE, we conducted a comprehensive flow cytometric analysis of PBMCs isolated from patients with SLE pre- and post-WLCT. Our investigations revealed a significant reduction in the CD3⁺ T cell compartment following WLCT treatment, concomitant with a marked expansion of the regulatory T cell (Treg) population. Notably, WLCT intervention elicited a moderate attenuation of Th17 cell frequencies, thereby establishing a more favorable Th17/Treg balance within the immune microenvironment (Figure 4A).

Figure 4.

Regulation of WLCT on T-cell subtypes and cytokines

(A) Representative flow cytometry and quantification of T cell subtypes.

(B) WLCT’s effects on cytokines. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, ns = not significant. Th17: T helper cell 17; Treg: regulatory T cell; IL: interleukin; TNF-α: tumor necrosis factor; TGF-β: transforming growth factor β.

Parallel cytokine profiling studies demonstrated that WLCT exerted profound modulatory effects on the cytokine milieu, characterized by a substantial downregulation of pro-inflammatory mediators, particularly IL-17A and TNF-α. Conversely, WLCT treatment enhanced the production of immunoregulatory cytokines, including IL-10 and TGF-β. Furthermore, our analysis revealed a moderate yet statistically significant suppression of IL-2 and IL-6 secretion following WLCT treatment, suggesting a broader immunomodulatory impact beyond the Th17/Treg axis (Figure 4B).

Effect of WLCT on SLE disease progression in follow-up studies

To investigate the long-term efficacy of WLCT intervention in SLE, we performed a longitudinal, 12-month follow-up study comparing WLCT-treated patients with those receiving CT treatment. At the 3-month interval, WLCT-treated individuals demonstrated a marked reduction in proteinuria and serum IgA levels relative to the CT group, with these therapeutic benefits sustained over the entire observation period (Figure 5).

Figure 5.

Effect of WLCT on SLE disease progression in follow-up studies

Horizontal bars represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; Cre: creatinine; BUN: blood urea nitrogen; UA: uric acid; C3: complement 3; C4: complement 4; CRP: C-reactive protein; Ig: immunoglobulin.

Notably, hematological profiling at the 3-month timepoint revealed a significant decrease in both absolute neutrophil counts and their relative frequency in peripheral blood among WLCT-treated patients compared to CT controls (Figure 6).

Figure 6.

Effect of WLCT on blood cells of patients with SLE in follow-up studies

Horizontal bars represent the mean ± SEM. ∗p < 0.05, and ∗∗p < 0.01. CT: conventional therapy; WLCT: whole blood exchange-lymphoplasmapheresis combined transfusion; WBC: white blood cell.

Safety

Adverse reactions were observed in four cases of the WLCT group, among which two cases were allergic reactions and two cases were hypocalcemia. All adverse events were mild, and patients recovered rapidly. There were no deaths or irreversible complications.

Discussion

SLE is a complex autoimmune disorder characterized by the dysregulation of B and T lymphocytes, leading to the production of numerous autoantibodies.¹ This immune dysregulation often results in severe tissue and organ damage, with potentially fatal consequences.

The therapeutic objectives for SLE include improving long-term survival, preventing organ damage, and minimizing treatment-related toxicity. Hydroxychloroquine is the first-line therapy for most patients, while additional treatments often involve immunosuppressive agents such as glucocorticoids, azathioprine, methotrexate, cyclosporine, and mycophenolate mofetil.⁹ For patients with persistently active or relapsing disease, or those with severe, organ-threatening manifestations, biologic agents such as belimumab and rituximab may be considered.¹⁰ However, these pharmacological treatments are often limited by delayed response times, low response rates, and significant adverse effects associated with prolonged use.⁶ Although B-cell-targeted therapies have shown promise in SLE, many clinical trials evaluating biologic agents targeting specific pathways have failed to meet their primary endpoints.¹¹

Given these challenges, there is an urgent need to explore novel therapeutic approaches, either as monotherapies or in combination with conventional and biologic agents. These approaches should prioritize minimizing glucocorticoid use while achieving both short- and long-term treatment goals. This remains a critical area of research in the quest for more effective and safer management strategies for SLE.

Growing evidence has demonstrated that therapeutic apheresis is a potential technique as either escalation therapy or adjuvant treatment across multiple clinical domains of SLE.^12,13 Nevertheless, current data remain insufficient to establish definitive preferential indications for TPE within SLE management protocols. Notably, our prior investigation has yielded clinically relevant findings, specifically documenting that WBE demonstrates measurable therapeutic efficacy as an adjunctive intervention in refractory severe SLE-associated AIHA, particularly when conventional therapies prove inadequate.^14,15 Emerging evidence shows that erythrocytes from active patients with SLE retain mitochondria, and these MREs drive type I IFN production via monocyte activation. Coupled with the 81% DAT-positive rate in our cohort, these findings provide a mechanistic basis for extending this approach to broader refractory SLE.^7,8

In this study, WLCT treatment significantly attenuated disease phenotype in patients with SLE. Statistical analysis confirmed that the differential effects between WLCT and CT were significant across some evaluated metrics. This suggests that WLCT not only targets the underlying immune dysregulation characteristic of SLE but also offers enhanced protection against end-organ damage. Our 12-month longitudinal study evaluates the long-term efficacy of WLCT in SLE management. WLCT-treated patients exhibited sustained reductions in proteinuria and serum IgA levels compared to the CT group, indicating durable therapeutic benefits in addressing renal and immunological aspects of SLE.

Hematological profiling revealed a significant decline in both absolute and relative neutrophil counts at 3 months. Neutrophils play a key role in SLE pathogenesis through pro-inflammatory cytokine release and NET formation.¹⁶ This reduction may indicate the suppression of neutrophil-driven inflammatory pathways, further supporting WLCT’s role in modulating SLE-related inflammation.

Furthermore, WLCT treatment reduced immunoglobulin levels in patients with SLE, positioning WLCT as a multifaceted immunomodulatory strategy capable of targeting both cellular and humoral components of autoimmune dysregulation.

Emerging evidence highlights the critical involvement of Tregs and Th17 cells in the pathogenesis and progression of SLE.^17,18 Proinflammatory cytokines, such as IL-17A and TNF-α, are known to exacerbate SLE disease activity,^19,20 while anti-inflammatory mediators such as IL-10 and TGF-β exert protective effects by attenuating inflammation.^21,22 However, the role of IL-10 in SLE remains controversial.²³ Following WLCT treatment, a significant increase in Treg populations and a corresponding decrease in Th17 cell proportions were observed. These cellular shifts were paralleled by a marked reduction in proinflammatory cytokines, including IL-17A and TNF-α, and a significant elevation in TGF-β levels.

These findings suggest that WLCT elicits a broad immunomodulatory response in SLE, likely through the concurrent regulation of effector T cell subsets and cytokine networks. By rebalancing proinflammatory and anti-inflammatory pathways, WLCT may facilitate the restoration of immune homeostasis in this complex autoimmune disorder.

Limitations of the study

Several limitations should be acknowledged in the current investigation. Firstly, the single-center design of this case collection may restrict the generalizability of our findings, necessitating future validation through multicenter studies with larger, more diverse patient cohorts. Secondly, the retrospective nature of this research inherently carries the risk of potential selection bias, which could influence the interpretation of clinical outcomes. Furthermore, the absence of a TPE control group represents a critical methodological constraint, precluding direct comparative analysis between WLCT and established TPE protocols. Fourthly, anti-dsDNA antibody testing in our center during the study period was performed only as a qualitative assay (positive/negative), and post-treatment results were not routinely available for most patients due to the retrospective design. Therefore, we could not quantitatively assess changes in this important autoantibody. Furthermore, other key hemolytic markers—such as haptoglobin, lactate dehydrogenase, and peripheral blood film—before and after treatment were not monitored in the clinical patients. Future studies will incorporate these crucial parameters to further substantiate the clinical evidence. These limitations underscore the need for prospective randomized controlled trials to objectively evaluate the therapeutic efficacy and safety profile of WLCT relative to CT treatment.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Ning Li (liningxy@csu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All original data and code are available in this paper’s electronic supplemental information.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 82502192), the Youth Science Foundation of Xiangya Hospital (grant no. 2024Q17), and the Health Research Project of Hunan Provincial Health Commission (grant no. 20256933).

Author contributions

Z.Z., Y.J., and L.L. had the idea for the article; Z.Z. and J.Z. drafted the manuscript. Z.Z., Z.J., S.P., W.Y., and B.L. conducted the experiments. All the other authors critically revised the work.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Human CD3	BioLegend	Cat# 317333
Anti-Human CD4	eBioscience	Cat# 11-0049
Anti-Human CD25	BD Pharmingen	Cat# 557138
Anti-Human Foxp3	eBioscience	Cat# 17-4776
Anti-Human IL-17A	eBioscience	Cat# 12-7179
Human TruStain-FcX (Fc receptor blocker)	BioLegend	Cat# 422301
Chemicals, peptides, and recombinant proteins
Ficoll-Paque PLUS	Cytiva (formerly Cytive)	Cat# 17544202
Cell Stimulation Cocktail (plus protein transport inhibitors)	eBioscience	Cat# 00-4975
Zombie Aqua™ Fixable Viability Kit	BioLegend	Cat# 423101
Fixation Buffer	BioLegend	Cat# 424401
Permeabilization Buffer	BioLegend	Cat# 424401
Critical commercial assays
Human TGF-β1 ELISA Kit	CUSABIO	CSB-E04725h
Human TNF-α ELISA Kit	CUSABIO	CSB-E04740h
Human IL-2 ELISA Kit	CUSABIO	CSB-E04626h
Human IL-6 ELISA Kit	CUSABIO	CSB-E0463h
Human IL-17A ELISA Kit	CUSABIO	CSB-E12819h
Deposited data
Flow cytometry raw data (FCS files)	Mendeley Data	Available upon request from lead contact
Software and algorithms
FlowJo (version 10.0.8)	BD Biosciences	https://www.flowjo.com
GraphPad Prism (version 9.5.1)	GraphPad Software	https://www.graphpad.com
SPSS (version 22.0)	IBM	https://www.ibm.com/spss
Other
Cytek flow cytometer	Cytek Biosciences	Not applicable
COBE Spectra/Spectra Optia cell separator	Terumo BCT	Not applicable

Experimental model and study participant details

Human subjects

Patients admitted to Xiangya Hospital of Central South University between September 2018 and October 2024 were screened for eligibility based on the following inclusion criteria: (1) a confirmed diagnosis of SLE according to established classification criteria, including the American College of Rheumatology (ACR) 1997, Systemic Lupus International Collaborating Clinics (SLICC) 2012, or ACR 2019 guidelines; and (2) administration of WBE combined with LPE as part of their therapeutic regimen. Exclusion criteria were applied to ensure the integrity of the study cohort and included: (1) lack of informed consent; (2) pregnancy or the presence of other major comorbidities; and (3) a life expectancy of less than one year.

A total of 84 SLE patients who had failed or were intolerant to at least two conventional immunosuppressive regimens (glucocorticoids, hydroxychloroquine, and/or immunosuppressive agents) and physician-assessed urgent need for rapid disease control were enrolled to undergo WLCT treatment. All patients provided written informed consent for WLCT treatment. The consent process included: (1) detailed explanation of the experimental nature of the procedure; (2) discussion of alternative treatments including continued conventional therapy or biologic agents; (3) disclosure of potential risks (hypotension, allergic reactions, hypocalcemia, infection, and bleeding); and (4) confirmation that patients could withdraw at any time. For comparative analysis, 61 SLE patients receiving conventional therapy (CT) during the same study period were included as a control group. The treatment protocols and patient allocation are detailed in Figure S1. The baseline of clinical characteristics and treatment condition of patients were shown in the supplementary materials (Tables 1 and S3). Due to the retrospective design and the small number of male patients, we could not statistically assess the influence of sex on treatment outcomes, this limitation is acknowledged. Laboratory tests with incomplete or missing clinical data will be excluded from the comparative analysis of pre- and post-treatment outcomes for both CT and WLCT treatment. This study was conducted in compliance with ethical standards and was approved by the Ethics Committee of Xiangya Hospital (Approval No. 2025101707).

Method details

Procedure of WLCT

The WLCT procedure consists of two sequential steps using a continuous-flow cell separator (COBE Spectra or Spectra Optia), the procedure complied with the instructions previously reported by Li et al. in our group.²⁴ The first step is performing WBE, WBE previously reported by Li et al. and Jiang et al. in our group^14,15 (Figure S2). Briefly, after installing the disposable tubing of Dual-Needle TPE on the machine, connect access and return terminals with bilateral veins or artery and vein. And then run the procedure and turn into manual mode. By clamping the returned RBCs line, patient’s whole blood is removed to the collect bag from removed plasma line. Donor’s leukoreduced suspended red blood cells and fresh frozen plasma were sequential transfused into patient from the return line. When WBE procedure is finished, change into LPE procedure. LPE was performed as previously described,²⁵ the procedure of LPE as follows (Figure S2): Release the returned RBCs line, and then the lymphoid cells were collected using density gradient centrifugation and photoelectricity technology in a manual program based on mononuclear cell collection. Along with peripheral blood mononuclear cells (PBMCs), the lymphocytes and the autologous plasma were removed from removed plasma line, while fresh frozen plasma was reinfused to the patient’s body after the confluence of the patient’s red blood cells from the return line. Anticoagulation was maintained with sodium citrate (ACD-A), maintaining a blood-to-anticoagulant ratio between 10:1 and 12:1. Blood flow rate was 30∼40 mL/min, and total procedure time was 90∼120 min. Prior to the procedure, anti-allergic drugs were given to preventing the anaphylaxis reaction (for example: administrated promethazine at a dosage of 25 mg by intramuscular injection). Sodium citrate was used as anticoagulant. And about 2 g of calcium gluconate was administrated in case of hypocalcemia. All adverse events were recorded.

Flow cytometry

PBMCs were isolated by Ficoll-Paque gradient centrifugation according to the manufacturer’s protocol (Cytive, CAT # 17544202) from lupus patients before and after treatment, the clinical details were shown in the Table S1. To identify Th17 cells, PBMCs were cultured with 1 × Cell Stimulation Cocktail (plus protein transport inhibitors) in 5% carbon dioxide at 37°C for 6–8 h (eBioscience, Cat# 00–4975). Zombie Aquat Fixable Viability Kit (BioLegend, CAT # 423101) was used to exclude the dead cells. Single-cell suspensions were blocked with Human TruStain-FcXTM (BioLegend, CAT # 422301) for 10 min prior to staining with antibodies. The cells were then stained with Anti-Human CD3 (BioLegend, CAT # 317333), Anti-Human CD4 (eBioscience, CAT #11–0049), Anti-Human CD25 (BD Pharmingen, CAT #557138) for 30 min at 4 °C in the dark. For staining the receptor in the nucleus, cells were fixed with fixation buffer at room temperature for 50 min, then washed with permeabilization buffer twice (BioLegend, CAT # 424401). After that, the cells were incubated with the Anti-Human Foxp3 (eBioscience, CAT #17–4776) and Anti-Human IL17A (eBioscience, CAT #12–7179) antibodies in the dark for 50 min at 4 °C, and then washed with staining buffer once. Cells were analyzed using a Cytek flow cytometer. The percentages of CD4⁺CD25⁺Foxp3⁺ (Treg) cells and CD4⁺IL17⁺ (Th17) cells were further analyzed using FlowJo software (version 10.0.8).

ELISA

The serum of TGF-β1 (CUSABIO, CSB-E04725h), TNF-α (CUSABIO, CSB-E04740h), IL-2 (CUSABIO, CSB-E04626h), IL-6 (CUSABIO, CSB-E0463h), and IL-17A (CUSABIO, CSB-E12819h), from the patients with SLE before and after WLCT treatment were detected using ELISA Kits according to the manufacturer’s instructions. The clinical details were shown in the Table S2.

Quantification and statistical analysis

We used the t test for statistical analysis for parametric data and the Mann–Whitney U test for non-parametric data. Oneway analysis of variance was used when there were more than two groups, and then followed by Bonferroni test among different groups. We performed statistical analyses with SPSS22.0 software and GraphPad Prism 9.5.1 and considered a P-value less than 0.05 as significant. Data are shown as means ± standard error of mean (SEM).