Article

Podocyte SIRPa reduction aggravates lupus

nephritis via promoting T cell inflammatory
responses
Graphical abstract Authors
Bin Qian, Rui Lu, Shuya Mao, ...,
Zhihong Liu, Ke Zen, Limin Li

Correspondence
kzen@nju.edu.cn (K.Z.),
liminli@cpu.edu.cn (L.L.)

In brief
Qian et al. show that SIRPa expression is
downregulated in podocytes exposed to
LN. The decrease of SIRPa stimulates
podocyte antigen presentation through
the SHP-1/p-Syk axis. In lupus-prone
mice, podocyte-specific knockout of
SIRPa aggravates T cell immune
response and renal damage, while the
Syk inhibitor GS-9973 effectively
alleviates renal disease progression and
T cell immune response.

Highlights
d Strong reduction of SIRPa in podocytes of lupus nephritis
(LN) patients and lupus-prone mice

d Deletion or knockdown of SIRPa enhances podocyte antigen

presentation

d Deletion of SIRPa activates specific T cell immune response

via promoting Syk activation

d Syk inhibitor GS-9973 alleviates the progression of LN

induced by SIRPa deletion

Qian et al., 2024, Cell Reports 43, 114249

May 28, 2024 ª 2024 The Author(s). Published by Elsevier Inc.
https://doi.org/10.1016/j.celrep.2024.114249 ll
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Article
Podocyte SIRPa reduction
aggravates lupus nephritis via promoting
T cell inflammatory responses
Bin Qian,1 Rui Lu,1 Shuya Mao,1 Yang Chen,1 Miao Yang,1 Wenxuan Zhang,2 Mingchao Zhang,3 Dihan Zhu,1 Zhihong Liu,3
Ke Zen,1,4,* and Limin Li1,5,*
1State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of Life Science and

Technology, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu 211198, China
2School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu 211198, China
3National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002,

China
4State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210093, China
5Lead contact

*Correspondence: kzen@nju.edu.cn (K.Z.), liminli@cpu.edu.cn (L.L.)

https://doi.org/10.1016/j.celrep.2024.114249

SUMMARY

Signal-regulatory protein alpha (SIRPa) has recently been found to be highly expressed in podocytes and is
essential for maintaining podocyte function. However, its immunoregulatory function in podocytes remains
elusive. Here, we report that SIRPa controls podocyte antigen presentation in specific T cell activation via
inhibiting spleen tyrosine kinase (Syk) phosphorylation. First, podocyte SIRPa under lupus nephritis (LN) con-
ditions is strongly downregulated. Second, podocyte-specific deletion of SIRPa exacerbates renal disease
progression in lupus-prone mice, as evidenced by an increase in T cell infiltration. Third, SIRPa deletion or
knockdown enhances podocyte antigen presentation, which activates specific T cells, via enhancing Syk
phosphorylation. Supporting this, Syk inhibitor GS-9973 prevents podocyte antigen presentation, resulting
in a decrease of T cell activation and mitigation of renal disease caused by SIRPa knockdown or deletion.
Our findings reveal an immunoregulatory role of SIRPa loss in promoting podocyte antigen presentation to
activate specific T cell immune responses in LN.

INTRODUCTION immune regulation is considered an initiating factor of LN.6,7

Lupus nephritis (LN) is one of the most common and severe com-
plications of systemic lupus erythematosus (SLE), characterized
by kidney inflammation and eventual loss of renal function.1 It is
estimated that approximately 40%–60% of patients with SLE
experience kidney injury at disease onset, with 10%–15% of
LN cases progressing to end-stage renal disease.2 Conse-
quently, LN results in the highest mortality rate among all organs
and systems affected by SLE. Although current studies have
shown that renal damage in LN primarily stems from immune
complex deposition on glomeruli-circulating or in situ accompa-
nied by inflammatory cell infiltration and local immune inflamma-
tion,3 the regulatory interplay between these pathogenic factors
has not been fully elucidated.
Podocytes are one of the main targets of immune complexes
in LN, which share many elements of innate immune cells and
adaptive immune cells.4,5 Therefore, podocytes may also play
a role in innate and adaptive immune function as immune cells
by actively participating in glomerular injury. However, limited
studies have focused on this aspect, potentially constraining
the understanding of LN pathogenesis. The disruption of T cell
T cells play multiple roles in the pathogenesis of LN, including
participating in the secretion of pro-inflammatory cytokines
and amplifying inflammation, thereby exacerbating renal dam-
age. As such, controlling the migration and infiltration of T lym-
phocytes may serve as a therapeutic strategy for LN.7,8 How-
ever, the relationship between the infiltration of T cells and
renal parenchymal cell injury remains unclear. Studies have
shown that the expression of major histocompatibility molecules
and costimulatory molecules CD80 and CD86 on podocytes
from lupus-prone mice and patients with LN is increased,9 sug-
gesting that podocytes may play a role in T cell activation and
accumulation in the renal parenchyma. However, conclusive
experimental evidence and a molecular mechanism to support
the rationale are lacking.
Signal-regulatory protein alpha (SIRPa) is an important regula-
tory protein that regulates various inflammatory processes such
as leukocyte migration, secretion, and phagocytosis.10 It con-
sists of three extracellular Ig-like domains, a single hydrophobic
transmembrane sequence, and immune receptor tyrosine inhib-
itory motifs (ITIMs). Tyrosine phosphorylation at ITIM sites re-
cruits phosphatases Shp-1 and Shp-2, thereby activating these

Cell Reports 43, 114249, May 28, 2024 ª 2024 The Author(s). Published by Elsevier Inc. 1
This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
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Figure 1. Reduction of SIRPa in podocytes under the conditions of lupus nephritis

(A) The location and expression levels of SIRPa in renal sections from paracancerous tissue of renal cell carcinoma (PCT) and lupus nephritis (LN). Scale bar,
30 mm. The histogram on the right represents the statistical analysis results (n = 5 for each group, with 10 glomeruli counted at random per sample).
(B and C) After being treated with LN serum for 72 h, the protein and mRNA levels of SIRPa were measured by western blotting and RT-qPCR.
(D and E) LN serum was separated into immunoglobulin (Ig) and immunoglobulin-free serum (Ig free) and was used to treat podocytes separately for 72 h. SIRPa
protein levels in HPCs were measured by western blotting.
(F) The protein levels of SIRPa in HPCs with treatment of IFN-a (30 ng/mL), IL-17 (50 ng/mL), or IFN-g (20 ng/mL) for 24 h. Data are represented as mean ± SEM.
*p < 0.05; **p < 0.01; ***p < 0.001, unpaired Student’s t test.

phosphatases and facilitating the transduction of negative regu-
lation signals.11,12 In macrophages, SIRPa inhibits IFN-g or
TLR1/2/3 agonist-stimulated activation of STAT3, PI3K-Akt2,
NK-kB, and MAPK signal pathways. This inhibition leads to the
suppression of pro-inflammatory cytokine production.13 In po-
docytes, SIRPa plays an important role as a slit diaphragm pro-
tein to maintain glomerular filtration structure and function.14 Ka-
jiho et al. identified that SIRPa is highly phosphorylated at site
Y501 in rat glomeruli.15 Furthermore, Takahashi et al., discov-
ered phosphorylated SIRPa recruits and activates Shp-2.16
Our previous study demonstrated that SIRPa plays an important
role in maintaining high levels of autophagic activity, protecting
podocytes from cell injury.17 Therefore, studies have established
that podocytes specifically express SIRPa, which is crucial for
maintaining the filtration structure and function of podocytes.
However, whether SIRPa plays an immunoregulatory function
in podocytes akin to its function in leukocytes is still unclear.
In this study, we observed a reduction of SIRPa levels in podo-
cytes from LN, which activates podocyte antigen presentation
and triggers T cell immune response by promoting spleen tyro-
sine kinase (Syk) phosphorylation. Podocyte-specific knockout
of SIRPa aggravates renal damage and inflammation in lupus-
prone mice, and Syk inhibitor entospletinib (GS-9973) effectively
alleviates renal disease progression. We provide a pathogenic
mechanism of T cell activation by podocytes in LN and under-
score the potential of the SIRPa/Shp-1/Syk axis in podocytes
as a therapeutic target.
RESULTS
Reduction of SIRPa in podocytes under the conditions in
LN
To investigate the immune regulatory effect of SIRPa in podo-
cytes, we compared the expression of SIRPa in renal sections

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Figure 2. Reduction of SIRPa in podocytes of lupus-prone mice

(A) The protein of SIRPa in glomeruli isolated from pristane-induced lupus mice.
(B) The protein of SIRPa in glomeruli isolated from MRL/lpr mice.
(C) Representative immunofluorescence staining results from MRL/lpr mice and pristane-induced lupus mice. SIRPa (green), nephrin (red), and nuclei (DAPI,
blue). Scale bar, 15 mm. The histogram on the right represents the statistical analysis results (n = 5 for each group, with 10 glomeruli counted at random per
sample).
(D) The protein levels of SIRPa in MPCs with treatment of IFN-a (30 ng/mL), IL-17 (50 ng/mL), IFN-g (20 ng/mL), or not (Con) for 24 h. Data are represented as mean ±
SEM. ***p < 0.001, unpaired Student’s t test. Podocyte-specific deletion of SIRPa exacerbates renal disease progress in lupus-prone mice.

from paracancerous tissue of renal cell carcinoma (PCT) and LN
patients. We found a significant reduction of SIRPa in podocytes
of patients with LN (Figure 1A). To explore the reasons for the
downregulation of SIRPa in podocytes, we co-cultured serum
from healthy controls and LN patients with human podocyte
cell line (HPCs) for 72 h.18 Expectedly, we found that the protein
and mRNA levels of SIRPa in HPCs were significantly decreased
under the administration of LN serum (Figures 1B and 1C). Next,
LN patients’ serum was separated into Ig fraction and Ig-free
fraction by Protein A/G Magnetic Beads and then co-cultured
with HPCs for 72 h. Interestingly, the expression of SIRPa in
HPCs was notably reduced under the treatment of serum from
LN patients even after IgG depletion (Figures 1D and 1E). There-
fore, the effect of autoantibodies on the expression of SIRPa
might not be a main factor. Pro-inflammatory cytokines are
pivotal contributors participating in renal damage in LN. The
most common pro-inflammatory cytokines in circulation of LN
patients are interleukin and type I and type II interferon.19 Com-
bined with our previous study,20 IFN-a, IL-17, and IFN-g were
selected for subsequent podocyte injury induction experiments.
After treatment with these three cytokines for 24 h, respectively,
the protein level of SIRPa in HPCs was significantly decreased
(Figure 1F), which is consistent with the results of serum treat-
ment. These results suggested that pro-inflammatory factors
may cause the decreased expression of SIRPa in podocytes un-
der LN conditions.
Next, we used two distinct types of LN mouse models, namely
the spontaneous LN (MRL/lpr) mice and pristane-induced
LN mice, to evaluate the expression of SIRPa in mouse podo-
cytes. As shown in Figures S1A and S1B, both MRL/lpr and

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pristane-induced LN mice exhibited varying degrees of base-
ment membrane thickening and glycogen deposition and the
deposition of IgG, IgA, IgM, and C3 during the course of disease.
Protein levels of SIPRa were decreased in glomeruli isolated
from MRL/lpr and pristane-induced LN mice compared to wild-
type mice (WT) (Figures 2A and 2B). Furthermore, immunofluo-
rescence analysis of frozen kidney sections provided further
indication that SIPRa expression was downregulated in podo-
cytes of mice with LN (Figure 2C). In addition, protein levels of
SIPRa were decreased obviously in mouse podocyte cells
(MPCs) after 24 h of IFN-a, IL-17, or IFN-g stimulation (Figure 2D),
suggesting that SIRPa was downregulated in podocytes by in-
flammatory factors during the pathogenesis of LN.
To elucidate the role of SIRPa in podocytes under lupus con-
ditions, as shown in Figures 3A–3C, we generated podocyte-
specific SIRPa knockout mice (SirpaDpod mice). The mice were
characterized by RT-qPCR (Figure 3A), immunoblot (Figure 3B),
and immunofluorescence analysis (Figure 3C). Since podocin-
Cre has been reported to have potentially toxic effects on
podocytes,21 we compared the phenotypes of WT, Nphs2-Cre,
and SirpaDpod mice, and the results indicated no statistically
significant distinction between Nphs2-Cre and WT mice (Figures
S2A–S2G). Then, we established an LN animal model in Nphs2-
Cre and SirpaDpod mice via intraperitoneal injection of pristane
(Figure 3D). Serum and urine samples were collected from
mice at the ages of 8, 16, and 24 weeks. As shown in Figures
3E–3G, the levels of ratio of urinary albumin and creatinine (Fig-
ure 3E), serum antinuclear antibodies (Figure 3F), and serum
double-stranded DNA (Figure 3G) were increased significantly,
while body weight (Figure 3H) and kidney weight (Figure 3I)
were decreased remarkably in SirpaDpod mice 16 weeks after
intraperitoneal injection of pristane. Moreover, SirpaDpod mice
showed enhanced immune activity (Figures 3J and 3K), charac-
terized by an increase in spleen weight and the proportion of
T cells in the spleen. One of the clinical manifestations of SLE
is lymphatic organ enlargement22; splenomegaly and an in-
crease in the percentage of T cells indicate that SIRPa knockout
aggravated the development of LN in mice. Consistent with this,
SirpaDpod lupus-prone mice exhibited a thickened glomerular
basement membrane and an increased deposition of IgG, IgA,
IgM, and C3 (Figures 3L and 3M) compared to Nphs2-Cre
lupus-prone mice. We also investigated the effect of SIRPa dele-
tion on the ultrastructure of podocytes. The results showed that
the slit diaphragm was blurred, and foot process fusion was
increased in SirpaDpod lupus-prone mice compared with
Nphs2-Cre lupus-prone mice (Figure 3N). Moreover, SirpaDpod
lupus-prone mice had more glomerular infiltration of CD4+ and
CD8+T cells and higher infiltration levels of renal macrophages
and active T cells compared with WT lupus-prone mice
(Figures 3O and 3P). Given that the SirpaDpod lupus-prone
mice exhibited autoimmunity, we conducted an analysis of
apoptosis levels in the kidney and spleen of Nphs2-Cre and
SirpaDpod mice, revealing a marginal increase in apoptosis
among SirpaDpod mice, potentially contributing to heightened
autoimmunity (Figures S3A and S3B). Collectively, these results
suggest that loss of SIRPa in podocytes promotes local renal
inflammation and systemic inflammation and aggravates pris-
tane-induced LN progression in mice.
To explore the relationship between SIRPa and inflammatory
activation in LN, glomeruli were isolated from the pristane-
treated Nphs2-Cre and SirpaDpod mice for RNA microarray anal-
ysis. GO analysis results demonstrated a significant correlation
between SIRPa knockout and antigen presentation process (Fig-
ure 4A; Table S3). Given that podocytes possess many elements
of the innate and adaptive immune cells,9 and that our previous
results also showed that LN-associated inflammatory cytokines,
IFN-g and IL-17, regulate podocyte antigen presentation,17 we
hypothesized that LN-associated inflammatory cytokines might
modulate podocyte antigen presentation by targeting SIRPa.
As shown in Figures 4B and S4A–S4D, podocytes from SirpaDpod
mice expressed high levels of MHC-I, MHC-II, CD80, and CD86,
suggesting that SIRPa knockdown may increase MHC-medi-
ated antigen processing and presentation. Thus, we used
DQ-OVA as an antigen to detect the regulatory effect of SIRPa
on podocyte antigen processing. DQ-OVA is a conjugation of
OVA with BODIPY fluorescent dye (DQ), which remains self-
quenched until OVA protein hydrolysis to generate DQ-green
fluorescence. Our results revealed that podocytes isolated
from SirpaDpod mice have a higher ability to phagocytize and pro-
cess DQ-OVA (Figure 4C). In order to investigate the regulatory
effect of SIRPa on podocyte antigen presentation, we then
co-cultured IL-17/IFN-g-activated podocytes isolated from
Nphs2-Cre or SirpaDpod mice with OVA for 2 h, removed the
OVA from the medium, and detected the antigen peptides pre-
sented on the surface of podocytes the next day. As shown in
Figure 4D, podocytes isolated from SirpaDpod mice presented

Figure 3. Podocyte-specific deletion of SIRPa exacerbates renal disease development in lupus-prone mice
(A–C) Podocyte-specific SIRPa knockout mice (SirpaDpod) were characterized by detecting SIRPa in glomerulus via RT-qPCR (A), western blotting (B), and
immunofluorescence staining (C). SIRPa (green), nephrin (red) and nuclei (DAPI, blue). Scale bar, 15 mm
(D) A schematic diagram of constructing LN mice by pristane.
(E–G) Urinary albumin and creatinine (UACR), serum levels of antinuclear antibodies (ANA), and anti-dsDNA antibodies were detected by ELISA (n = 8).
(H–J) The body weight, kidney weight, and spleen weight of Nphs2-Cre and SirpaDpod lupus-prone mice.
(K) Statistical analysis of the frequencies of CD3+CD4+ T cells, CD3+CD8+ T cells, CD3+CD4 CD8 T cells, and CD4+CD25+Foxp3+ T cells in the spleen of mice.
(L and M) Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining, IgG, IgA, IgM, and C3 deposition in the renal tissues of Nphs2-Cre and SirpaDpod
lupus-prone mice. Scale bar, 20 mm. The histogram on the right represents the statistical analysis results (n = 5 for each group, with minimally 50 glomeruli
counted at random per mouse).
(N) Transmission electron microscopy analysis showed the ultrastructure of the podocytes of the Nphs2-Cre and SirpaDpod lupus-prone mice. Scale bar, 2mm.
(O) Infiltration of CD4+ and CD8+T cells in the glomerulus of Nphs2-Cre and SirpaDpod lupus-prone mice. Scale bar, 20 mm.
(P) Infiltration of CD11b+ and CD69+ T cells in the kidney of Nphs2-Cre and SirpaDpod lupus-prone mice. n = 5; Scale bar, 20 mm. Data are represented as mean ±
SEM. *p < 0.05; **p < 0.01; ***p < 0.001, unpaired Student’s t test. Podocyte-specific SIRPa-deficient mice display greater capacity of antigen presentation and
T cell activation.

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Figure 4. Podocytes with specific SIRPa knockout exhibit enhanced antigen presentation and specific T cell activation ability
(A) Podocytes from Nphs2-Cre and SirpaDpod lupus-prone mice were isolated for RNA microarray analysis. Gene ontology analysis was used to identify
significantly different pathways.
(B) The statistical radio of immunofluorescence intensity of MHC-I, MHC-II, CD80, and CD86 to nephrin in podocytes of Nphs2-Cre and SirpaDpod lupus-prone
mice.

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more OVA257-254 peptides. Therefore, loss of SIRPa promotes
podocytes processing and presenting antigen under LN-associ-
ated inflammatory conditions.
Previous studies by us17 and others9 suggest that podocytes
are non-professional antigen-presenting cells and may actively
participate in the regulation of local inflammatory activation
and amplification in the kidney. Considering that Sirpa deficiency
in podocytes can enhance antigen presentation by podocytes, it
is reasonable to speculate that aggravation of renal inflammation
and tissue damage by podocyte-specific Sirpa deficiency may
be through promoting podocyte antigen presentation to acti-
vating specific T cells. To validate this, we first induced podo-
cytes to present the OVA257-264 antigen peptide and then co-
cultured them for 72 h with naive CD8+T cells from OT-I mice.
Our results showed that the proliferation activity of T cells co-
cultured with the group of podocytes isolated from SirpaDpod
mice significantly increased compared to Nphs2-Cre group of
podocytes (Figures 4E and 4F). Consistent with this, SirpaDpod
lupus-prone mice had higher levels of renal IL-2, IL-17, and
IFN-g compared with Nphs2-Cre lupus-prone mice (Figure 4G).
Taken together, these results suggest that LN-associated in-
flammatory cytokines, such as IFN-a, IFN-g, or IL-17, decrease
SIRPa expression in podocytes, thereby activating podocytes
to process and present antigens, leading to the specific activa-
tion of T cells and amplifying local inflammation of the kidney.
To validate that antigen presentation can also occur in vivo, an
appropriate model was established. Nphs2-Cre and SirpaDpod
were radiated, OVA was injected into the renal cortex, and
CFSE-labeled CD8+ T cells from OT-I mice were injected by
the tail vein (Figure 4H). We found the proliferation of CFSE-
labeled CD8+ T cells in spleen of SirpaDpod mice (Figure 4I).
Moreover, in a glomerulus section from SirpaDpod mice, we
observed CFSE-labeled CD8+ T cells surrounded by the podo-
cytes (Figure 4J).
The extracellular domain of SIRPa contains three Ig-like do-
mains, and the cytoplasmic domain contains two tyrosinase
phosphorylation sites. SIRPa can bind to protein tyrosine phos-
phatases Shp-1 and Shp-2, thereby activating these phospha-
tases.23,24 To elucidate the mechanism by which SIRPa
regulates podocyte antigen presentation, we constructed
Shp-1C453/S453 mutant plasmid with FLAG tag. Mutation at
this site deprived the ability of Shp-1 to dephosphorylate sub-
strates but retained its function of trapping substrates.25 Thus,
we overexpressed Shp-1S453 in MPCs and used FLAG anti-
bodies to pull down the proteins binding to Shp-1S453. By protein
profiling, we obtained a series of proteins binding to Shp-1S453
(Figure 5A). It is widely known that Shp-1 is a negative regulator
(C and D) Representative immunofluorescence images of antigen phagocytosis (C) and presentation (D) of podocytes from Nphs2-Cre and SirpaDpod lupus-prone
mice. DQ-OVA and OVA257-264 (red); nuclei (DAPI, blue). Scale bar, 5 mm.
(E) The proliferation of T cells co-cultured with podocytes from Nphs2-Cre and SirpaDpod mice was measured.
(F) IL-2 secreted by co-cultured T cells was detected.
(G) IL-2, IL-17, and IFN-g levels in the kidney of SirpaDpod mice were detected (n = 4).
(H) The schematic drawing illustrates the experimental setup. More experimental details can be found in the methods.
(I) The proliferation of CD8+ T cells from OT-I mice was analyzed.
(J) In Nphs2-Cre and SirpaDpod mice, CFSE-labeled CD8+ T cells from OT-I mice were examined in the glomerulus. Nephrin (red), CFSE (green), and nuclei (blue,
DAPI). Scale bar, 10 mm. Data are represented as mean ± SEM. **p < 0.01; ***p < 0.001, unpaired Student’s t test. SIRPa regulates podocyte antigen presentation
and immune response by dephosphorylating Syk.
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of signal transduction in innate and adaptive immune cells, which
can inhibit signal transduction by binding to the phosphorylated
ITIM on immune receptors.11,26 Therefore, we further screened
binding proteins with kinase characteristics and finally focused
on Syk. To determine Syk as the downstream substrate of
SIRPa/Shp-1, we repeated the immunoprecipitation experiment
in MPCs and further established that Shp-1 could bind Syk and
SIRPa (Figure 5B). The expression of Syk was also detected in
the kidneys of LN patients and LN mice. As shown in Figures
5C and 5D, phosphorylated Syk (p-Syk) was almost absent in
the glomeruli of paracancerous tissue of renal cell carcinoma
(PCT) and control mice (CTL) but significantly increased in the
glomeruli of LN patients and LN mice. To further clarify the reg-
ulatory relationship between SIRPa and Syk, the expression level
of p-Syk was significantly increased after knocking down SIRPa
in MPCs. GS-9973, a selective Syk inhibitor that is being evalu-
ated in hematopoietic malignancies,27,28 reversed the increase
of p-Syk induced by SIRPa siRNA (Figure 5E). Next, we further
evaluated the effect of GS-9973 on the immune response func-
tion of podocyte induced by SIRPa reduction. As shown in
Figures 5F–5H, S5A, and S5B, compared to the SIRPa knock-
down group, GS-9973 resisted the increase of MHC-I, MHC-II,
CD80, and CD86 expression and inhibited the ability of antigen
processing and presentation induced by SIRPa siRNA.
To further explore the therapeutic effect of GS-9973 in LN, we
evaluated the therapeutic effect on LN mice by administering
10 mg/kg GS-9973 by gavage for 1 week.29 Compared with
SirpaDpod lupus-prone mice without GS-9973 administration
(SirpaDpod), SirpaDpod lupus-prone mice with GS-9973 adminis-
tration (SirpaDpod + GS-9973) showed decreased urinary protein,
serum antinuclear antibody, serum double-stranded DNA,
glomerular glycogen, and IgG/IgA/IgM/C3 deposition (Figures
6A and 6B). Moreover, podocyte ultrastructural examination
showed that GS-9973 reduced SIRPa deletion-induced foot pro-
cess fusion (Figure 6C). Furthermore, there was a significant
decrease in the quantity of renal and spleen T cells (Figures 6D
and 6E) as well as the renal concentrations of IL-2, IL-17, and
IFN-g (Figure 6F). In renal tissue slices, we also noticed close
connections between CD3+ T cells and podocytes labeled with
nephrin (Figure 6G).
The expression of CD80, CD86, MHC-I, and MHC-II on the
surface of podocytes of SirpaDpod lupus-prone mice was also
reduced by GS-9973 (Figures 7A and 7B). We next examined
the renal level of Syk in three groups of mice. Western blotting re-
sults showed that 1-week gavage of GS-9973 resulted in a slight
decrease of p-Syk levels in the glomeruli (Figure 7C). Secondly,
we evaluated the level of antigen processing and presentation by
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podocytes and found that SirpaDpod mice treated with GS-9973
showed decreased ability to process and present foreign antigen
(Figures 7D and 7E). Finally, we examined the effect of GS-9973
on T cell proliferation co-cultured with podocytes from three
groups of mice. Flow cytometry results showed that GS-9973
reduced the proliferative activity of CD8+T cells co-cultured
with podocytes from SirpaDpod mice, and the level of IL-2 was
also significantly reduced in the co-culture supernatant (Figures
7F and 7G). These results indicated that GS-9973 alleviated renal
damage progression and local inflammation potentiated by
SIRPa deletion.
DISCUSSION
In glomerular diseases, podocytes are not only passive target
cells but actively participate in the pathological mechanisms of
many diseases. Goldwich et al., 30 using a mouse model of
anti-glomerular basement membrane (anti-GBM) nephritis,
found that podocytes could ingest soluble and granular anti-
gens, activate CD4+T cells, and cross-present exogenous anti-
gens on MHC class I molecules to CD8+T cells. However, this
study did not clarify the pathological significance and regulatory
mechanism of podocyte antigen presentation. Previous research
by our group found that inflammatory cytokines IFN-g and IL-17
promote podocyte antigen presentation, specifically activate
T cells, and promote podocyte injury and renal inflammatory
response, suggesting the pathological significance of podocyte
antigen presentation. Yet the regulatory mechanism remained
unclear.20 In this study, we discovered that SIRPa expression
in podocytes is decreased in response to IFN-a, IFN-g, and
IL-17. The downregulation of SIRPa enhances podocyte antigen
presentation and specifically activates T cells. LN involves the
activation of local inflammatory reactions, renal damage, and
the release of damage-associated molecular patterns, which
provides favorable conditions for activating T cells through anti-
gen presentation. IFN-a, IFN-g, and IL-17 are important pro-in-
flammatory cytokines involved in LN.31–36 Given this, our study
not only discovered an important regulatory role of SIRPa
actively participating in the acquired immune response of podo-
cytes under the lupus nephritis condition but also revealed that
the molecular mechanism of IFN-a, IFN-g, and IL-17 regulating
podocyte antigen presentation might be by targeting SIRPa.
Immune injury and changes in the expression of podocyte-
specific molecules are two important factors leading to podo-
cyte injury, but the relationship between the two is still unclear.
The reason is that there is no suitable correlation molecule to
study the direct relationship between the two. SIRPa was found
to be located in the slit diaphragm,14 and subsequent studies
confirmed the important role of SIRPa in maintaining the struc-
ture and function of podocytes.15,16 Actually, SIRPa is a signal-
regulatory protein mainly expressed on leukocytes, its typical
function is to inhibit the phagocytosis of phagocytes by interact-
ing with the self-recognition marker CD47.37 Bian et al. found
that SIRPa-deficient macrophages activate tumor-specific
T cells under radiotherapy in tumors.38 Consistently, we found
that SIRPa-deficient podocytes activate specific T cell immune
responses and promote podocyte injury and the progression of
LN-prone mice, suggesting SIRPa may not only play a role as
the structural protein of podocytes, but it also regulates the
macrophage-like function of podocytes. Although we found the
correlation of SIRPa deletion and specific T cell activation and
infiltration in glomerulus of LN mice, there is still a lack of evi-
dence that podocytes directly present antigens to T cells in vivo.
Future studies should use podocyte-specific SirpaDpod 3 OT-I/
OT-II hybrid mice to elucidate this.
To our surprise, we observed systemic inflammation, evi-
denced by spleen enlargement and T cell proliferation, in podo-
cyte-specific SIRPa knockout mice. Previous studies have
shown that B cell-derived extracellular vesicles can present
MHC-II peptides to CD4+ T cells in vitro.39 Additionally, murine
bone-marrow-derived DC cells secrete extracellular vesicles
containing MHC-I and MHC-II, as well as costimulatory mole-
cules that interact with T cells.40 Injection of extracellular vesicles
carrying MHC-II peptides in mice also leads to the activation of
CD4+ T cells.39 It is possible that podocytes activate T cells
not only by direct contact in the glomerulus; instead, under
various stress conditions, podocytes may release extracellular
vesicles to activate T cells in secondary lymphoid organs, thus
promoting systemic inflammation. Moreover, our previous
research has indicated that the splenic red pulp also plays a
role in cell clearance.41 As a result, we analyzed the amounts
of apoptosis in the kidney and spleen of both Nphs2-Cre and
SirpaDpod mice. The results showed that SirpaDpod mice had a
slightly higher amount of apoptosis than Nphs2-Cre mice, which
may have contributed to systemic and local renal inflammation in
SirpaDpod mice (Figure S3). However, whether apoptotic renal
cells utilize this pathway and activate systemic inflammation re-
quires further investigation.
Syk, a member of the protein tyrosine family, is abundantly
present in the cells of hematopoietic lineage.42 Activation of
Syk mediates the activation of B cells and T cells.43–47 An essen-
tial function of phosphorylated syk is to facilitate antigen

Figure 5. SIRPa regulates podocyte antigen presentation by dephosphorylating Syk

(A) Schematic diagram of immunoprecipitation and mass spectrometry analysis.
(B) Co-immunoprecipitation of FLAG-Shp-1 and identification of Syk and SIRPa.
(C) Representative immunofluorescence images of SIRPa and p-Syk expression in renal sections from paracancerous tissue of renal cell carcinoma (PCT) and
lupus nephritis (LN) patients. Scale bar, 30 mm.
(D) Expression of SIRPa and p-Syk in renal tissues of mice without pristane treatment, and Nphs2-Cre and SirpaDpod mice with pristane treatment. (C and D)
SIRPa (green), phosphorylated Syk (red), and nuclei (DAPI, blue). Scale bar, 15 mm.
(E) Expression of p-Syk and total Syk in MPCs with or without SIRPa knockdown and GS-9973 (2 mM) treatment.
(F–G) Statistical analysis of MHC-I, MHC-II (F), CD80, and CD86 (G) expression in MPCs with or without SIRPa knockdown and GS-9973 (2 mM) treatment.
(H) Representative immunofluorescence images of antigen phagocytosis and antigen presentation in MPC with or without SIRPa knockdown and GS-9973 (2 mM)
treatment. DQ-OVA, OVA257-264 (red), and nuclei (DAPI, blue). Scale bar, 5 mm. Data are represented as mean ± SEM. ***p < 0.001, unpaired Student’s t test. Syk
inhibitor alleviates the effect of podocyte-specific SIRPa deletion on renal disease progression in lupus-prone mice.

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Figure 6. Syk inhibitor alleviates the aggravating effect of SIRPa deletion on renal disease progression of lupus-prone mice
(A) H&E and PAS staining, IgG, IgA, IgM, and C3 deposition in the kidney of Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973
(10 mg/kg). Scale bar, 20 mm.
(B) UACR, serum ANA, and anti-dsDNA antibodies levels of Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973.
(C) Ultrastructural analysis of the podocytes in Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973. Scale bar, 2 mm.
(D) Infiltration of CD4+ and CD8+T cells in the glomerulus of Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973. Scale bar, 15 mm.

(legend continued on next page)

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presentation. p-Syk is involved in both BCR-mediated antigen by GS-9973 inhibits podocyte antigen presentation and T cell
presentation and the presentation of freshly produced MHC activation and proliferation and alleviates pristane-induced LN.
class II molecules. Tyrosine residues in the immunoreceptor
tyrosine-based activation motifs of the Fcg receptor become Limitations of the study
phosphorylated during phagocytosis, which causes local Fcg re- Although we found the correlation of SIRPa deletion and specific
ceptor aggregation. This attracts and activates Syk, which in turn T cell activation and infiltration in glomerulus of LN mice, there is
triggers the activation of downstream signaling molecules.48,49 still a lack of evidence that podocytes directly present antigens
Syk is reported to be involved in various glomerular diseases to active T cells in vivo. Future studies should use podocyte-spe-
such as anti-GBM nephritis,50,51 LN,52–54 diabetes nephropa- cific SirpaDpod 3 OT-I/OT-II hybrid mice to elucidate this.
thy,55–57 and IgA nephritis,58–60 and it may be a potential thera-
peutic target for these diseases. For example, studies have sug- STAR+METHODS
gested that Syk can be a potential therapeutic target for LN. Syk
inhibition or Syk deletion reduces the activation of the JNK/p38/ Detailed methods are provided in the online version of this paper and include
the following:
MAPK pathway and significantly reduces leukocyte infiltration,
crescent formation, and fibrosis in the model of nephrotoxic d KEY RESOURCES TABLE
serum nephritis.51 Our study found that Syk is specifically acti- d RESOURCE AVAILABILITY
vated in podocytes under LN conditions, which is regulated by B Lead contact
B Materials availability
SIRPa. In addition, we found that Syk inhibitor GS-9973 inhibits
B Data and code availability
T cell proliferation induced by SIRPa-deficient podocytes and al-
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
leviates pristane-induced LN. GS-9973 is an oral and highly se- B Patients
lective Syk inhibitor currently being studied in clinical trials of B Mice
autoimmune and tumor diseases. In this study, we revealed B Cell culture and treatment

that the Syk inhibitor GS-9973 inhibits podocyte antigen presen- B Primary mouse podocyte isolation and treatment

tation, providing a therapeutic mechanism for targeting Syk in LN d METHOD DETAILS

B Flow cytometry
settings. Thus, our findings not only elucidate the ‘‘brake’’ role of
B Measurement of urine albumin, serum dsDNA and serum ANA
SIRPa in podocyte antigen presentation and T cell activation B Ig and Ig-free serum preparation
through Syk activation but also expand the treatment scope of B Western blotting
GS-9973 in LN. B Histological and immunofluorescence analysis

The disorder of T cell immune regulation is considered the initi- B Quantification of immunostaining

ating factor of LN. T cells play multiple roles in the pathogenesis B mRNA microarray analysis
B Mass spectrometry
of SLE, such as participating in immune tolerance, the secretion
d QUANTIFICATION AND STATISTICAL ANALYSIS
of inflammatory cytokine, assisting in the differentiation of B lym-
phocytes, and producing autoantibody. It is suspected that tar-
SUPPLEMENTAL INFORMATION
geting T cells may be an important therapeutic strategy for SLE
and LN. Currently, clinical drugs targeting LN, such as predni- Supplemental information can be found online at https://doi.org/10.1016/j.
sone, mainly achieve immunosuppressive effects by inhibiting celrep.2024.114249.
the differentiation of B cells or the proliferation of T cells. How-
ever, existing drugs cannot completely cure LN. Some alterna- ACKNOWLEDGMENTS
tive therapies such as anti-type I interferon receptor antibodies
This work was supported by grants from the National Natural Science Founda-
or anti-IFN-g antibodies are also being attempted.61 Our
tion of China (32170897). Kidney samples were obtained from Renal Biobank
research found that IFN-a and IFN-g could downregulate SIRPa
of National Clinical Research Center for kidney Diseases (Jiangsu Provincial
expression, promote Syk activation, and induce T cell prolifera- Science and Technology Resources Coordination Service Platform).
tion, thus providing evidence and mechanisms for targeting
interferon receptors or therapy. AUTHOR CONTRIBUTIONS
In summary, our results indicate that SIRPa plays an important
role in podocyte immune activation, T cell proliferation, and the L.L. and B.Q. conceived and designed the experiments; B.Q., R.L., S.M., Y.C.,
production of inflammatory factors. LN-associated inflammatory and M.Z. performed experiments; B.Q., R.L., S.M., and Y.C. analyzed data;
M.Z., C.Z., X.Z., D.Z., and Z.L. contributed technical or material support;
cytokines caused a reduction of SIRPa, releasing the capture
L.L., K.Z., and B.Q. wrote and reviewed the manuscript.
effect of SHP1 on P-Syk, promoting the activation of Syk,
improving the antigen presentation ability of podocytes, thus DECLARATION OF INTERESTS
inducing the specific activation and proliferation of T cells, and
aggravating the progress of LN. Blocking the activation of Syk All the authors declared no competing interests.

(E) Statistical analysis of the frequencies of CD3+CD4+ and CD3+CD8+ T cells in the spleens of Nphs2-Cre and SirpaDpod lupus-prone mice with or without
treatment of GS-9973.
(F) IL-2, IL-17, and IFN-g levels in kidney of Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973. (n = 4).
(G) In Nphs2-Cre and SirpaDpod lupus-prone mice, CD3 expression was examined in the glomerulus either with or without GS-9973 (10 mg/kg) therapy. Nephrin
(red), CD3 (green), and nuclei (blue, DAPI). Scale bar, 10 mm. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, unpaired Student’s t test.

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(legend on next page)

12 Cell Reports 43, 114249, May 28, 2024

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Received: December 4, 2023
Revised: April 7, 2024
Accepted: May 2, 2024
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Figure 7. Syk inhibitor alleviates the aggravating effect of SIRPa deletion on antigen presentation of podocytes from lupus-prone mice
(A) CD80, CD86, MHC-I, and MHC-II expression in the glomerulus of Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973 (10 mg/kg).
CD80, CD86, MHC-I, and MHC-II (green); nephrin (red); nuclei (DAPI, blue). Scale bar, 15 mm
(B) The statistical analysis of immunofluorescence intensity of MHC-I, MHC-II, CD80, and CD86 to nephrin of Nphs2-Cre and SirpaDpod lupus-prone mice with or
without treatment of GS-9973 (10 mg/kg).
(C) Western blotting analysis of p-Syk and total Syk in glomerulus from Nphs2-Cre and SirpaDpod lupus-prone mice with or without treatment of GS-9973.
(D and E) Antigen phagocytosis (D) and presentation (E) analysis of podocytes isolated from Nphs2-Cre and SirpaDpod mice with or without treatment of GS-9973.
DQ-OVA, OVA257-264 (red), and nuclei (DAPI, blue). Scale bar, 5 mm.
(F) Flow cytometry analysis of T cell proliferation co-cultured with podocytes isolated from Nphs2-Cre and SirpaDpod mice with or without treatment of GS-9973
for 72 h.
(G) Levels of IL-2 secreted by T cells co-cultured with podocytes isolated from Nphs2-Cre and SirpaDpod mice with or without treatment of GS-9973 for 72 h (n = 4).
Data are represented as mean ± SEM. **p < 0.01; ***p < 0.001, unpaired Student’s t test.

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Cell Reports 43, 114249, May 28, 2024 15
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit monoclonal anti-SIRP alpha Abcam Cat# ab191419, RRID: AB_2924335
Rabbit monoclonal anti-ACTB ABclonal Cat# AC028, RRID: AB_2769861
Rabbit polyclonal anti-SHP1 Proteintech Cat# 24546-1-AP, RRID: AB_2879600
Mouse monoclonal anti-SYK Proteintech Cat# 66721-1-Ig, RRID: AB_2882072
Rabbit monoclonal anti-Phospho-Syk (Tyr525/526) (C87C1) Cell Signaling Technology Cat# 5514, RRID: AB_2797615
Rabbit polyclonal anti-GAPDH ABclonal Cat# AC001, RRID: AB_2619673
Goat polyclonal anti-rabbit IgG-(HRP) Cell Signaling Technology Cat# 7074, RRID: AB_2099233
Horse polyclonal anti-mouse IgG-(HRP) Cell Signaling Technology Cat# 7076 (also 7076S, 7076V, 7076P2),
RRID: AB_330924
Alexa Fluor(R) 647 anti-mouse H-2Kb/H-2Db BioLegend Cat# 114612, RRID: AB_492931
PE anti-mouse MHC Class II (I-A/I-E) Thermo Fisher Scientific Cat# 12-5321-82, RRID: AB_465928
FITC anti-mouse CD3e BD Biosciences Cat# 553061, RRID: AB_394594
PE anti-mouse CD4 BD Biosciences Cat# 553730, RRID: AB_395014
APC anti-mouse CD8a BD Biosciences Cat# 553035, RRID: AB_398527
FITC anti-mouse CD80 BD Biosciences Cat# 553768, RRID: AB_395038
PE anti-mouse CD86 Thermo Fisher Scientific Cat# 12-0862-81, RRID: AB_465767
Mouse monoclonal anti-human Synaptopodin (D-9) Santa Cruz Biotechnology Cat# sc-515842, RRID: AB_2921309
Guinea pig polyclonal anti-Nephrin Fitzgerald Industries Cat# 20R-NP002, RRID: AB_1288100
International
Rabbit monoclonal anti-IgG Agilent Cat# F0202, RRID: AB_2335710
Rabbit polyclonal anti-IgA Agilent Cat# F0204, RRID: AB_2335712
Rabbit polyclonal anti-IgM Agilent Cat# F0203, RRID: AB_2335711
Rabbit polyclonal anti-C3 Agilent Cat# F0201, RRID: AB_2335709
Mouse monoclonal anti-CD80 Proteintech Cat# 66406-1-Ig, RRID: AB_2827408
Rabbit polyclonal anti-CD86 Proteintech Cat# 13395-1-AP, RRID: AB_2074882
Rat monoclonal anti-MHC class I Santa Cruz Biotechnology Cat# sc-59199, RRID: AB_1126186
Rat monoclonal anti-MHC class II Santa Cruz Biotechnology Cat# sc-59322, RRID: AB_831551
Mouse monoclonal anti-OVA Sigma-Aldrich Cat# A6075, RRID: AB_258279
Rabbit polyclonal anti-WT1 Proteintech Cat# 12609-1-AP, RRID: AB_2216225
Mouse monoclonal anti-CD69 Santa Cruz Biotechnology Cat# sc-373799, RRID: AB_10915746
FITC anti-mouse CD25 Proteintech Cat# FITC-65137, RRID: AB_2883797
PE anti-mouse Foxp3 Proteintech Cat# PE-65089, RRID: AB_2883883
Mouse monoclonal anti-CD11b Proteintech Cat# 66519-1-Ig, RRID: AB_2881882
Bacterial and virus strains
BSR-LW025-CTRL Syngen N/A
BSR-LW025-SIRPa Syngen N/A
Biological samples
Human LN kidney tissues and their matched Jinling Hospital N/A
paracancerous tissues, see Tables S1 and S2
Human LN serum and their matched normal Jinling Hospital N/A
serum, see Tables S1 and S2
Whole blood from C57BL/6J, Nphs2-Cre, This paper N/A
OT-I and SirpaDpod female mice
Spleen from C57BL/6J, Nphs2-Cre and This paper N/A
SirpaDpod female mice
(Continued on next page)

16 Cell Reports 43, 114249, May 28, 2024

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Kidney from C57BL/6J, Nphs2-Cre, This paper N/A
MRL/lpr and SirpaDpod female mice
Bone marrow from Nphs2-Cre and This paper N/A
SirpaDpod female mice
Chemicals, peptides, and recombinant proteins
DAPI Sigma-Aldrich Cat# D9542
Lipo 3000 Thermo Fisher Scientific Cat# L3000-015
Fetal Bovie Serum (FBS) Gibco Cat# 10091148
Protein A/G Magnetic Beads MedChemExpress Cat# HY-K0202
1640 Gibco Cat# 31870082
GS-9973 MedChemExpress Cat# HY-15968
Pristane Sigma-Aldrich Cat# P2870
DMEM Gibco Cat# 11965092
Collagenase D Sigma-Aldrich Cat# C6885
Protease Sigma-Aldrich Cat# P6911
DNase I Sigma-Aldrich Cat# D5025
HBSS Gibco Cat# 14025092
Type I collagen Corning Cat# 354236
Trypsin-EDTA Gibco Cat# 25200056
IL-17 R&D Systems Cat# C17-ILB-050
IFN-g R&D Systems Cat# 285-IF-100
IFN-a Novoprotein Cat# CK83
OVA Invitrogen Cat# O23021
CFSE Invitrogen Cat# C34554
Critical commercial assays
Urinary albumin ELISA kit Wako Cat# 638-04309
Urine Creatinine ELISA kit Wako Cat# 290-65901
Mouse anti-dsDNA IgG ELISA kit Cusabio Cat# CSB-E11194m
IL-2 ELISA kit Cusabio Cat# CSB-E04627m
Mouse anti-ANA ELISA kit Cusabio Cat# CSB-E12912m
IL-17 ELISA kit Cusabio Cat# CSB-E04608m
IFN-g ELISA kit Cusabio Cat# CSB-E04578m
Tunel kit MedChemExpress Cat# HY-K1079
Deposited data
mRNA microarray data This paper GSE: 240916
Experimental models: Cell lines
HPC Li, Shan et al.20 N/A
MPC Li, Shan et al.20 N/A
Experimental models: Organisms/strains
C57BL/6J mice Beijing Vital River Laboratory N/A
Animal Technology Co., Ltd.
OT-I mice Jackson Laboratory Strain#: 003831
Nphs2-Cre mice This paper N/A
SirpaDpod mice This paper N/A
MRL/lpr mice Jackson Laboratory Strain#: 000485
Oligonucleotides
Specific primers for qRT-PCR, see Table S4 This paper N/A
(Continued on next page)

Cell Reports 43, 114249, May 28, 2024 17

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Software and algorithms
FlowJo Tree Star https://www.flowjo.com
ImageJ Wayne Rasband https://imagej.net
Prism GraphPad https://www.graphpad.com
FV31S Olympus https://www.olympus.com

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Limin Li
(liminli@cpu.edu.cn).

Materials availability
All newly generated materials associated with the paper are available upon request from the lead contact.

Data and code availability

d The mRNA microarray data is deposited in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under accession number
GSE240916. All other data that support the conclusions are available from the authors upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon
request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Patients
Patients with LN (N = 10) were enrolled in this study. All LN patients were diagnosed based on renal biopsies at the National Clinical
Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine. Kidney samples were obtained from
Renal Biobank of National Clinical Research Center of Kidney Diseases, Jiangsu Biobank of Clinical Resources, a part of The Open
Project of Jiangsu Biobank of Clinical Resources (JSRB2021-03). Informed consent was obtained from all patients. Patient exclusion
criteria included obesity, diabetes mellitus, HBV infection, hepatitis or malignant tumors, and participation in continuous renal
replacement therapy. Table S1 lists the demographics/clinical characteristics of LN patients. Table S2 provides other relevant details
of individual patients. All diagnoses were made by expert renal pathologists. The diagnosis of SLE was based on the American Col-
lege of Rheumatology revised classification criteria for SLE.

Mice
Female C57BL/6J (8 weeks, 22–25 g), podocyte-specific SIRPa-knockout (SirpaDpod) mice and OT-I mice were obtained from the
Model Animal Research Center of Nanjing University. In brief, SIRPa-flox homozygous were first generated using the CRISPR/
Cas9 system, and SirpaDpod mice were obtained by mating SIRPa-flox homozygous and Nphs2-Cre mice. All mice were back-
crossed to a C57BL/6 background. All mice were acclimatized for 2 weeks before experiments, then received a single intraperitoneal
(i.p.) injection of 0.5 mL pristane, and monitored for the following 4 months. For in vivo experiments of Syk inhibitor, mice were treated
with pristane for 4 months, followed by daily oral dosing (bid at 12 h intervals) with saline or GS-9973 (10 mg/kg) for one week.62 To
analyze the antigen presentation of podocytes in vivo, Nphs2-Cre and SirpaDpod were radiated with 3 Gy. After one week, we injected
OVA into the renal cortex, as previously published.20 In brief, the mice were anesthetized by intraperitoneal injections of sodium
pentobarbital (30 mg/kg). When the mice were fully anesthetized, the kidney was exposed via a flank incision. A solution containing
1mg IFNg/IL-17 and 200mg OVA was injected into the kidney cortex at four different sites (20 mL solution for each site). Meanwhile, 107
CFSE-labeled CD8+ T cells from OT-I mice were transferred intravenously. In the following experiments, the mice were humanely
sacrificed. The spleen, kidney, urine and blood were harvested.

Cell culture and treatment

Human and mouse podocyte cell lines (HPC and MPC) were gifted by M. Saleem (Children’s Renal Unit, Bristol Royal Hospital for
Children, University of Bristol, Bristol, United Kingdom) and Stuart J. Shankland (University of Washington, Seattle, Washington,
USA), and were cultured as previously described.63,64 To detect the ability of podocytes to present antigens, MPCs were incubated
with DQ-OVA or OVA (10 mg/mL, Invitrogen, Shanghai, PR China, O23021) in the presence or absence of IFN-a (30 ng/mL,

18 Cell Reports 43, 114249, May 28, 2024

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Novoprotein, CK83), IFN-g (30 ng/mL, R&D Systems, 285-IF-100) or IL-17 (50 ng/mL, R&D Systems, C17-ILB-050). To detect podo-
cyte activating specific T cell proliferation, MPCs incubated with OVA were co-cultured with T cells from OT- I mice.

Primary mouse podocyte isolation and treatment

Mouse glomeruli were isolated according to a protocol described previously.65,66 In brief, the renal cortex was cut into small cubes of
1 mm3 and digested with collagenase D (1 mg/mL, Sigma-Aldrich, C6885), protease (1 mg/mL, Sigma-Aldrich, P6911) and DNase I
(1 U/ml, Sigma-Aldrich, D5025) in HBSS for 15 min at 37 C. The digested tissue was screened through a 100 mm filter. Glomeruli
containing Dynabeads were gathered using a magnetic particle concentrator. To obtain primary podocytes, isolated glomeruli
were plated in cell culture flasks that had been treated with type I collagen (Corning, New York, USA, 354 236). Five days later,
the cells were treated with trypsin-EDTA solution and the digested cells were passed through a 40 mm sieve to remove the remaining
glomerular cores.

METHOD DETAILS

Flow cytometry
Flow cytometry was performed using a CytoFLEX S flow cytometer (Beckman Coulter, Miami, USA). The following antibodies were
used: MHC-I (Biolegend, San Diego, USA, 114612), MHC-II (eBioscience, San Diego, USA, 12-5321-82), CD3e (BD Biosciences,
553061), CD4 (BD Biosciences, 553730), CD8 (BD Biosciences, 553035), CD80 (BD Biosciences, 553768), and CD86 (eBioscience,
12-0862-81).

Measurement of urine albumin, serum dsDNA and serum ANA

Urine samples were collected over 24 h from pristane-treated mice once a week and urinary albumin was measured using an ELISA
quantitation kit (Wako, Japan, 638–04309). The serum samples of pristane-treated mice were collected at the beginning and the end
of the observation period. The serum anti-dsDNA IgG and ANA IgG levels were detected by ELISA (Cusabio, Wuhan, China, CSB-
E11194m, CSB-E12912m).

Ig and Ig-free serum preparation

Ig and Ig-free serum preparation were purified from LN patients’ serum pools using the Protein A/G Magnetic Beads (MCE, Shanghai,
China, HY-K0202). Separation Process According to the Manufacturer’s Recommended Protocol.

Western blotting
Western blot analyses were performed as described previously.67 In brief, protein extracts from podocytes were resolved by
SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with blocking solution
for 30 min and then incubated with primary antibodies, followed by three washes then incubation with HRP-conjugated secondary
antibody. The intensity of the signals on each blot was analyzed using ImageJ software. The following antibodies were used: SIRPa
(1:1000, Abcam, Cambridge, UK, 191419), b-actin (1:5000, ABclonal, Wuhan, China, AC028), Shp-1(1:1000, Proteintech, Chicago,
USA, 24546-1-AP), Syk (1:1000, Proteintech, Chicago, USA, 66721-1-Ig), Phospho-Syk (1:1000, CST, Massachusetts, USA, C87C1),
and GAPDH (1:5000, ABclonal, Wuhan, China, AC001)

Histological and immunofluorescence analysis

For the immunohistochemical assay, mouse kidneys were fixed by paraformaldehyde of which the concentration was 4%, and then
embedded in paraffin. Sliced sections of 5 mm thickness were affixed into HistoBond adhesive microscopic slides. These slides were
stained with H&E (hematoxylin and eosin) or PAS (Periodic Acid-Schiff) and then stored at room temperature. For the immunofluo-
rescence (IF) assay, mouse kidneys were fixed by O.C.T, and sliced sections of 5 mm thickness were affixed into HistoBond adhesive
microscopic slides. Then, slides were incubated in blocking buffer (5% FBS in PBST), and primary and secondary antibodies, respec-
tively. Immune-complex (IC) depositions in the mouse kidneys were analyzed and evaluated using a digital fluorescence microscope
(Nikon), the protein expression in mouse kidneys was pictured and analyzed by a confocal laser scanning microscope (FV3000,
Olympus, USA). The following antibodies were used: SIRPa (1:1000, Abcam, Cambridge, UK, 191419), Synaptopodin (1:50, Santa
Cruz, California, USA, sc-515842), Nephrin (1:50, Fitzgerald, Acton, USA, 20R-NP002), IgG (1:50, DAKO, Denmark, F0202), IgA (1:50,
DAKO, Denmark, F0204), IgM (1:50, DAKO, Denmark, F0203), C3 (1:50, DAKO, Denmark, F0201), CD80 (1:50, Proteintech, China,
66406-1-Ig), CD86 (1:50, Sigma, USA, SAB1411654), Phospho-Syk (1:50, CST, USA, C87C1), Syk (1:100, Proteintech, USA,
66721-1-Ig), MHC-I (1:50, Santa Cruz, California, USA, sc-59199), MHC-II (1:50, Santa Cruz, California, USA, sc-59322).

Quantification of immunostaining
The staining of OVA257–264 in podocytes was quantified using ImageJ software. In brief, the cell area and integrated fluorescence
intensity were measured. The image was then converted to 8-bit grayscale by ImageJ software, followed by threshold adjustment
and analysis of the integrated density of the selected cell region. The ratio of fluorescence intensity versus cell area was obtained.

Cell Reports 43, 114249, May 28, 2024 19

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The phagocytic index was determined by normalizing the fluorescence intensity of ingested DQ-OVA against the total area of podo-
cytes using ImageJ. Quantification of focal adhesion was performed by counting the number of paxillin patches.

mRNA microarray analysis

Separate the glomeruli of three pristane-induced 24 weeks Nphs2-Cre and SirpaDpod mice, and extract RNA separately. Agilent mi-
croarray chip was used and experiments were operated at Shanghai Kangcheng Biological Corporation (China).

Mass spectrometry
The co-immunoprecipitation (CO-IP) analyses were carried out in MPCs and stably overexpressed Flag-tagged Shp-1 by using Flag
antibody as the bait. The immunocomplex was treated with trypsin and processed by Shanghai Applied Protein Technology’s Mass
Spectrometry and Proteomics Core facility.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data were analyzed using GraphPad Prism 8.3. These experiments were conducted at least three times. A statistical analysis was
performed using Student’s t-test and differences among multiple groups were calculated using one-way ANOVA or two-way ANOVA
followed by Tukey’s test. p value <0.05 (*), <0.01 (**) and <0.001 (***) were considered statistically significant.

20 Cell Reports 43, 114249, May 28, 2024