cells

Article
IL-2 Therapy Diminishes Renal Inflammation and
the Activity of Kidney-Infiltrating CD4 + T Cells in
Murine Lupus Nephritis
Angelika Rose 1,2 , Caroline von Spee-Mayer 1,2 , Lutz Kloke 3 , Kaiyin W u 4 , Anja Kühl 5 ,
Philipp Enghard 6 , Gerd-Rüdiger Burmester 1 , Gabriela Riemekasten 1,7 and
Jens Y . Humrich 1 , 7 , *
1 Charit é – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Univ ersität
zu Berlin, and Berlin Institute of Health, Department of Rheumatology and Clinical Immunology ,
Charit é platz 1, 10117 Berlin, Germany
2 German Rheumatism Research Center (DRFZ), a Leibniz Institute, Charit é platz 1, 10117 Berlin, Germany
3 Institute of Biotechnology , Department of Medical Biotechnology , T echnical University Berlin,
Gustav-Meyer-Allee 25, 13355 Berlin, Germany
4 Charit é – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Unive rsität
zu Berlin, and Berlin Institute of Health, Department of Pathology , Charit é platz 1, 10117 Berlin, Germany
5 Charit é – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Univ ersität
zu Berlin, and Berlin Institute of Health, Medical Department (Gastroenter ology , Infectious Diseases and
Rheumatology) / Research Center ImmunoSciences (RCIS), Hindenbur gdamm 30, 12200 Berlin, Germany
6 Charit é – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Univ ersität
zu Berlin, and Berlin Institute of Health, Department of Nephrology and Intensive Car e Medicine,
Augustenburger Platz, 13353 Berlin, Germany
7 University Hospital Schleswig-Holstein, Campus Lübeck, Department of Rheumatology and Clinical
Immunology , Ratzeburger Allee 160, 23538 Lübeck, Germany
* Correspondence: [email protected] ; T el.: + 49-451-500-45203; Fax: + 49-451-500-45204
Received: 30 August 2019; Accepted: 7 October 2019; Published: 11 October 2019
       
  

Abstract:
An acquir ed deficiency of interleukin-2 (IL-2) and r elated disturbances in regulatory T cell
(T reg) homeostasis play an important r ole in the pathogenesis of systemic lupus erythematosus (SLE).
Low-dose IL-2 therapy was shown to r estore T reg homeostasis in patients with active SLE and its
clinical e ffi cacy is curr ently evaluated in clinical trials. Lupus nephritis (LN), a challenging or gan
manifestation in SLE, is characterized by the infiltration of pathogenic CD4 + T cells into the inflamed
kidney . However , the role of the T r eg-IL-2 axis in the pathogenesis of LN and the mode of action of
IL-2 therapy in the inflamed kidneys ar e still poorly understood. Using the (NZB
×
NZW) F1 mouse
model of SLE we studied whether intrar enal T r eg are a ff ected by a shortage of IL-2 in comparison with
lymphatic or gans and whether and how intrarenal T cells and r enal inflammation can be influenced
by IL-2 therapy . W e found that intrar enal T r eg show phenotypic signs that ar e reminiscent of IL-2
deprivation in parallel to a progr essive hyperactivity of intrarenal conventional CD4 + T cells (T con).
Short-term IL-2 tr eatment of mice with active LN induced an expansion the intrar enal T r eg population
wher eas long-term IL-2 tr eatment r educed the activity and proliferation of intrar enal T con, which was
accompanied by a clinical and histological amelioration of LN. The association of these immune
pathologies with IL-2 deficiency and their r eversibility by IL-2 therapy provides important rationales
for an IL-2-based immunotherapy of LN.
Keywords: SLE; lupus nephritis; r egulatory T cell; interleukin-2; immunotherapy
Cells 2019 , 8 , 1234; doi:10.3390 / cells8101234 www .mdpi.com / journal / cells

Cells 2019 , 8 , 1234 2 of 16
1. Introduction
Systemic lupus erythematosus (SLE) is a sever e systemic autoimmune disease characterized by
the br each of self-tolerance to nuclear autoantigens leading to inflammation and tissue destruction in
multiple or gan systems [
1
]. Lupus nephritis (LN) is a major contributor to the morbidity and mortality
in SLE [
2
]. As early as 1984, Austin et al. linked r enal inflammation with lymphocyte infiltration in LN
and developed a scoring system for the histological assessment of LN [
3
]. Pr edominantly CD4 + T
cells that belong to the Th1 lineage infiltrate the inflamed kidneys and are a pr edictor for the degree of
r enal inflammation in patients with LN [
4
–
7
]. Infiltrating CD4 + T cells thus play a crucial r ole for the
initiation and perpetuation of inflammatory pr ocesses in LN. Studies that directly addr ess the r ole of
infiltrating immune cells in human or gans are limited. Alternatively , the (NZB
×
NZW) F1 mouse
model can be used as a suitable model to investigate the r ole of immune cells in the pathogenesis of
LN [ 8 ].
Regulatory CD4 + T cells (T reg) that expr ess the transcription factor FoxP3 are crucial for the
contr ol of autoimmunity by suppr essing the activation and di ff erentiation of self-r eactive T cells and
other pathogenic immune cells [
9
–
11
]. Their gr owth and survival fundamentally depend on the
availability of the immunoregulatory cytokine interleukin-2 (IL-2) [
12
,
13
]. In the (NZB
×
NZW) F1
mouse model of lupus and in human SLE patients we found that an acquired deficiency of IL-2 caused
a homeostatic disbalance between pr oliferating T r eg and conventional CD4 + T cells (T con), which was
accompanied by the loss of CD25 expression on T r eg and an accelerated T con hyperactivity in lymphatic
or gans and peripheral blood [
14
,
15
]. These disturbances in the T reg-IL-2 axis wer e associated with
incr eased disease activity , thereby highlighting the importance of IL-2 deficiency in SLE pathogenesis.
Accor dingly exogenous supplementation of IL-2 temporarily r estored CD25 expr ession in T r eg and the
homeostatic balance between T reg and T con, and was capable to r educe disease activity in lupus-prone
mice and also in SLE patients with active disease [ 14 – 18 ].
The coincidence of homeostatic and phenotypic abnormalities of the T reg population due to
IL-2 deficiency with the development of LN pr oposes that an impairment of intrarenal T reg also
contributes to r enal inflammation. On the other hand, the fact that adoptive T reg transfers can delay the
pr ogression of LN [
14
,
19
] suggests that T reg may be dir ectly involved in the regulation of pathogenic
cells that infiltrate the inflamed kidneys. However , it is still unknown whether local or systemic IL-2
deficiency a ff ects tissue-r esident intrarenal T reg in analogy to lymphatic or gans. In addition, the e ff ects
of IL-2 therapy on the composition and phenotype of intrar enal T r eg and T con and the r eversibility of
or gan-related pathologies associated with IL-2 deficiency ar e not well explored.
2. Materials and Methods
2.1. Mice
Female (NZB
×
NZW) F1 mice wer e bred at the Animal Facility of the Charit
é
—University Medicine
Berlin and kept under a special pathogen-fr ee condition at the Deutsches Rheuma-Forschungszentrum
(DRFZ), Berlin, Germany . All mice wer e grouped by age and disease activity and tr eated with
r ecombinant mouse IL-2 (R&D Systems, Minneapolis, MN, USA) or sterile PBS according to institutional
and federal guidelines (Landesamt für Gesundheit und Soziales, LaGeSo, Berlin, Germany).
2.2. Monitoring of Disease Activity and Definition of Disease Stages
Pr oteinuria was determined with Multistix 10 V isual (Siemens Healthcar e Diagnostics Inc.,
T arrytown, NY , USA). The following scoring system was used: Scor e 0 = 0 to 15 mg / dL,
scor e 1 = 30 mg / dL
, scor e 2 = 100 mg / dL, score 3 = 100–300 mg / dL, scor e 4 = 300 mg / dL and
scor e 6 = 2000 mg / dL
. The onset disease stage was defined as having a proteinuria scor e ranging from
0 to 3 and the nephritic stage as having a pr oteinuria score of > 3.

Cells 2019 , 8 , 1234 3 of 16
2.3. Cell Preparation
For the analysis of peripheral blood derived cells, 50–200
µ
L of whole blood was taken fr om the
tail veins of each animal at defined time points and befor e organs wer e harvested. Blood clotting was
pr evented by using heparin (Liquemin, Roche Pharma AG, Gr enzach-W yhlen, Germany) containing
vials. Erythr ocytes were lysed using a standard lysis bu ff er . Animals wer e sacrificed and spleens,
lymph nodes (superficial inguinal and axillary) and one kidney were separately mashed thr ough cell
strainers (mesh size: 70
µ
m; BD Bioscience, Heidelberg, Germany) to obtain single cell suspensions.
Suspensions wer e dissolved in PBS containing 0.2% BSA and 2 mM EDT A and were stained with
fluor escently labeled antibodies.
2.4. Flow Cytometry
Cells wer e stained with indicated antibodies in PBS containing 0.2% BSA. For extracellular staining
the following fluor escently labeled antibodies were used: Anti-CD4-PerCP (GK1.5, eBioscience, Thermo
Fisher Scientific, W altham, MA, USA), anti-CD44-Pacific Blue (IM7, DRFZ), anti-CD69-FITC (H1.2F3,
eBioscience) and anti-CD25-APC (PC61.5, eBioscience). For intracellular staining cells were fixed with
fixation bu ff er and afterwar ds permeabilized with perm bu ff er (Foxp3 / transcription factor staining
bu ff er set, eBioscience) and then stained with anti-FoxP3-PE (FJK16s, eBioscience), anti-Helios-APC
(22F6, Biolegend, San Diego, CA, USA) and anti-Ki67-Pe-Cy7 (B56, BD Bioscience) with the appropriate
bu ff ers accor ding to the manufacturer ’s pr otocol. Cells wer e stored in PBS containing 0.2% BSA and
0.01% sodium azide at 4
◦
C in the dark until measur ement. Flow cytometry was performed using
MACS Quant (Miltenyi Biotech, Bergisch Gladbach, Germany) and data wer e analyzed using FlowJo V
9.6.1 (BD Bioscience).
Gating strategy: 1. L ymphocyte gate in the FSC / SSC plot. 2. Gating either for CD4 + FoxP3 + T reg
or CD4 + FoxP3
−
T con in a CD4 / FoxP3 dot plot. 3. Fr equencies of CD25 + , CD69 + , CD44hi, Ki67 +
and Helios + cells among gated CD4 + FoxP3 + or CD4 + FoxP3
−
cells wer e determined either by using
di ff er ent dot plot combinations or by using histograms.
2.5. Intracellular Cytokine Staining
1
×
10
6
total splenocytes wer e stimulated for 5 h with 20 ng / mL PMA and 1
µ
g / mL ionomycin
(Sigma-Aldrich Chemie GmbH, T aufkirchen, Germany) together with Br efeldin A (Sigma-Aldrich) at a
final concentration of 2
µ
g / mL. After stimulation, cells were stained with anti-CD4-Pe-Cy7 (GK 1.5,
Biolegend) and anti-CD3-APCeFluor780 (17A2, eBioscience) in PBS and were afterwar ds fixed with BD
Bioscience Cytokines Fixation Kit (according to manufactur er ’s protocol). After fixation, cells were
r esuspended in 1
×
Perm bu ff er (BD Bioscience) and stained intracellularly with anti-IFN
γ
-FITC
(XMG1.2, BD Bioscience), anti-IL-2-APC (JES6-5H4, Biolegend) and anti-CD44-Pacific Blue (IM7, DRFZ).
2.6. Histological Analyses
Kidneys wer e fixed for at least 24 h in 4% paraformaldehyde immediately after harvesting.
Afterwar ds, organs wer e dehydrated, embedded in para ffi n and cut into 1–2
µ
m consecutive sections.
Histochemical staining using hematoxylin and eosin (HE) as well as periodic acid Schi ff (P AS) were
performed. Sections were scor ed in a blinded manner by one independent pathologist using the renal
activity index by Austin [
3
]. In detail, the activity index (AI) is the sum of six di ff er entially weighted
histomorphological scor es, which are glomer ular cell proliferation (
×
1), leukocyte exudation (
×
1),
karyorr hexis and / or fibrinoid necrosis (
×
2), cellular crescents (
×
2), hyaline deposits (
×
2) and interstitial
inflammation and tubulitis (
×
1). Each score was scaled fr om 0 to 3 (0 = no changes, 1 = mild changes,
2 = moderate changes and 3 = sever e changes).

Cells 2019 , 8 , 1234 4 of 16
2.7. IL-2 T reatments
Disease activity and age-matched mice wer e injected subcutaneously either with 25 ng (2500 units)
per gram bodyweight (between 500 ng and 1000 ng per injection) of r ecombinant mouse IL-2 (R&D
systems, 402 ML) or with PBS alone every 24 h within the first five days (five times in total, short-term
tr eatment). In the long-term treatments, IL-2 injections wer e continued every four days as maintenance
tr eatment until day 29 (six additional injections). Mice wer e sacrificed and cells from di ff er ent organs
and peripheral blood wer e analyzed 12 h (short-term treatment) or 48 h (long-term tr eatment) after the
last IL-2 injection.
2.8. Statistical Analyses
Graph Pad Prism 5 softwar e (GraphPad Software Inc., La Jolla, CA, USA) was used for the
analysis. Statistical di ff erences between two gr oups were analyzed by using the non-parametric Mann
Whitney U test. Correlation analyses wer e performed by using Spearman’s rank correlation coe ffi cients.
Outliers wer e identified using the r obust regr ession and outlier removal- (ROUT -) test. Cleared data
wer e used for further statistical analyses and graphs. Di ff erences wer e considered significant if p values
wer e less than 0.05.
3. Results
3.1. Progr essive Homeostatic Imbalance between Intrar enal T reg and T con
T o obtain insights into the importance of T r eg in the course of LN, we analyzed longitudinal
changes in the numbers, frequencies and phenotype of intrar enal CD4 + FoxP3 + T reg and of intrar enal
CD4 + FoxP3 − T con during the progr ession of LN.
First, we correlated the quantities of intrar enal T r eg and T con with the corresponding
histomorphological activity index (AI) and with the prot einuria index (PUI) in (NZB
×
NZW)
F1 lupus pr one mice with di ff erent r enal activity . Increases in absolute numbers of total intrar enal CD4 +
T cells, of intrarenal CD4 + FoxP3 + T r eg and of intrarenal CD4 + FoxP3
−
T con significantly correlated
with the AI and also with the PUI (Figur e 1 A,B). Calculation of the ratio between the absolute numbers
of T reg and of T con revealed a mor e pronounced incr ease in T r eg numbers than in T con numbers
in corr elation with the PUI (Figur e 1 C, left). Accordingly , the frequencies of intrar enal FoxP3 + T reg
among CD4 + T cells also corr elated with the PUI (Figure 1 C, middle). In general, the PUI significantly
corr elated with the histomorphological AI (Figure 1 C, right), suggesting that the PUI could be used as
an alternative measur e for the degree of r enal inflammation.
Next we determined the fr equencies of proliferating cells among intrar enal T r eg and T con by
using the pr oliferation marker Ki67. Here we could not observe significant corr elations between the
PUI and the fr equencies of Ki67 + cells neither among T reg nor among T con, although a moderate
decr ease in Ki67 + T reg and a moderate incr ease in Ki67 + T con was apparent (Figur e 1 D). However ,
the calculated ratio between pr oliferating intrarenal Ki67 + T reg and Ki67 + T con, repr esenting a measure
of the homeostatic T reg / T con balance, continuously decreased in corr elation with the PUI (Figure 1 E).
This suggests that a pr ogressive homeostatic imbalance between T reg and T con also develops in the
inflamed kidneys, comparable to findings in lymphatic organs and peripheral blood of these mice
(Figur e S1A) [ 14 ] and to findings in patients with SLE [ 15 ].

Cells 2019 , 8 , 1234 5 of 16
Cells 2019 , 8 , x FO R PE ER R E VIE W 5 of 16

Figure 1. Prog r e ssi ve Treg /Tc o n im balance d u ring prog ression of lu pu s ne phritis. C e ll s fr om k i dney s
o f ( N Z B × N Z W ) F 1 m i c e a t d i f f e r e n t d i s e a s e s t a g e s w e r e a n a l y z e d b y f l o w c y t o m e t r y . ( A , B ) Ab solute
numbers of total intrarenal CD 4+ T cells, of intrarenal CD4+F oxP3+ Treg and of intrarenal
CD4+FoxP3 − Tcon in correlation with the re nal hi stomorphological act i vi ty index (AI) ( A ) and w i th
the proteinuria index ( B ). ( C ) Graphs show t h e correlation of the calculat e d ratio betwee n absolut e
numbers of CD4+FoxP 3+ Treg and CD4+FoxP3 − Tcon ( l eft graph), the cor r elation of the percentages
of FoxP3+ ce lls among CD4+ T cells (middle g r aph) an d the correlation of th e AI with the proteinuria
index (rig ht g r aph). ( D ) The percentage of int r arenal Ki67+ ce lls among CD4+FoxP3+ Treg (left graph)
and among CD4+FoxP3 − Tcon (right grap h) is shown. ( E ) The calculate d ratio betwee n percentages
of Ki67+ Treg a n d of Ki67+ T c on in correlatio n with the proteinuria index is shown. Data are derived
from one kidney of each mo use from two t o five indepen d ent experiments ( n = 10 for correlations
with AI; n = 28 –30 for correlat i ons w i th prote i nuria inde x). Correlation an alyses were pe rformed by
using Spearman’s rank correlation coefficients. Bl a c k line s in dicat e line a r reg r ession c u rves u s ing
Pearson analyses (* p < 0.05 , ** p < 0 . 01 and *** p < 0 . 001).
Next we de te rmined the fr equen c ie s of prolifer at ing cell s am ong i n tra r ena l Tre g and Tcon b y
usin g the pro lif er at ion m a r k er Ki 6 7 . Her e we co uld n o t obse rve s i g n ifi c an t cor r e l a t ion s be twe e n the
PUI a n d t h e f r equenci e s of Ki 67+ cell s nei t her a m ong Treg nor among Tcon, a l t h ough a m o derat e
decrea se in K i 6 7 + Treg and a m o de ra te i n creas e in Ki 67 + Tcon w a s ap p a r e nt (F i gure 1D ). Ho wev e r,
the ca lcu l ate d ra tio b e tw een p r oli f er a t ing int r ar en al K i 6 7 + Tre g and Ki 6 7+ Tcon, rep r ese n ting a

Figure 1.
Progr essive T r eg / T con imbalance during progr ession of lupus nephritis. Cells from kidneys
of (NZB
×
NZW) F1 mice at di ff erent disease stages wer e analyzed by flow cytometry . (
A
,
B
) Absolute
numbers of total intrarenal CD4 + T cells, of intrar enal CD4 + FoxP3 + T reg and of intrar enal CD4 + FoxP3
−
T con in correlation with the r enal histomorphological activity index (AI) ( A ) and with the proteinuria
index (
B
). (
C
) Graphs show the corr elation of the calculated ratio between absolute numbers of
CD4 + FoxP3 + T r eg and CD4 + FoxP3
−
T con (left graph), the correlation of the per centages of FoxP3 +
cells among CD4 + T cells (middle graph) and the correlation of the AI with the pr oteinuria index (right
graph). (
D
) The percentage of intrar enal Ki67 + cells among CD4 + FoxP3 + T r eg (left graph) and among
CD4 + FoxP3
−
T con (right graph) is shown. (
E
) The calculated ratio between per centages of Ki67 + T r eg
and of Ki67 + T con in corr elation with the proteinuria index is shown. Data are derived fr om one kidney
of each mouse from two to five independent experiments ( n = 10 for corr elations with AI; n = 28–30 for
correlations with pr oteinuria index). Corr elation analyses were performed by using Spearman’s rank
correlation coe ffi cients. Black lines indicate linear regr ession curves using Pearson analyses (* p < 0.05,
** p < 0.01 and *** p < 0.001).
3.2. Phenotypic Changes of Intrarenal T r eg
Next, we assessed whether intrar enal T reg develops typical phenotypic alterations that ar e
associated with IL-2 deficiency during pr ogression of LN, such as r educed expression of the IL-2

Cells 2019 , 8 , 1234 6 of 16
r eceptor
α
-chain, i.e., CD25, and increased expr ession of CD44 and CD69 as previously observed in the
lymphatic or gans and peripheral blood of these mice [
14
]. Therefor e we corr elated the frequencies of
CD25, CD44 and CD69 expressing cells among T r eg with the easy accessible PUI as an indicator of
nephritic activity . The frequency of CD25 + cells among intrar enal T reg r emained unchanged during
pr ogression of nephritis fr om the onset stage to the more active stages (Figur e 2 A). However , at the
onset stage the fr equency of CD25 + cells among intrarenal T r eg was significantly lower compared to
T reg fr om the spleens of the same disease stage and was in a similar range than in peripheral blood
(Figur e S1B, left). Of note, T reg fr om spleens and peripheral blood of (NZB
×
NZW) F1 mice at the
onset stage of LN wer e shown to have alr eady a r educed expr ession of CD25 when compar ed to T r eg
fr om age-matched BALB / c mice or to young (NZB
×
NZW) F1 mice [
14
]. Similar to observations in
the spleens of (NZB
×
NZW) F1 mice, 50%–60% of the intrar enal T reg expressed the memory T cell
marker CD44 [
20
] (Figur e S1B, middle), which however did not considerably change in the kidneys
during disease pr ogression (Figur e 2 A, middle). CD69 has been considered to be a marker for r ecently
activated T cells [
21
], however more r ecently it has been shown that CD69 also plays an important
r ole in the retention of T cells in the inflamed or gans [
22
]. The frequencies of CD69 + cells among
intrar enal T reg significantly corr elated with the PUI (Figure 2 A, right) and wer e significantly higher
than in the spleens at the disease onset (Figure S1B), suggesting a r etention of intrarenal T r eg at
the site of inflammation and a high activity of T reg alr eady in the early phase of LN, which further
incr eases during the progr ession of LN. In conclusion, the phenotype of intrarenal T r eg together with
the pr ogressive homeostatic T reg / T con imbalance suggests that a deficiency of IL-2 is also pr esent in the
inflamed kidneys of lupus-pr one mice and such shortage of IL-2 occurs early during the course of LN.
Cells 2019 , 8 , x FO R PE ER R E VIE W 6 of 16
mea s ure of the homeosta ti c Treg/ T con balance, con t inuously dec r ease d in corr elation w i th the PUI
(Figure 1E). This su gges t s th at a pro g r e ssive ho meo s tatic imbalance betw een Treg and Tco n also
develops in t h e in fl amed k i dney s, comp ar able to f i nd ings in l y mphat i c o r g a ns and pe ripher al blood
of th ese m i ce (Fi g ure S 1 A ) [1 4] an d to f i ndings in p a ti ents wi th SLE [ 1 5 ] .
3.2. Phe n otypic Changes of Intrare n al Tre g
Next , we ass e ssed whe t he r in tr aren al Treg d e v e lop s typ i c a l p h e n otyp ic al ter a t i ons th a t a r e
assoc i ate d with IL-2 de fic i ency dur i ng progress io n of LN, such as re duced e x pression o f the IL-2
receptor α -ch a in , i.e., CD25, and incre a sed expression of CD 44 and CD69 as pre v iously ob ser v ed in
the lymph a tic org a ns and p e ripher al blo o d o f these m i ce [14]. Th erefore we cor r elate d the fre q uencies
of C D 2 5 , C D 4 4 an d C D 6 9 e x p r essin g cel l s am ong Tr eg with the e a s y acces s ib l e P U I as an indic a tor of
nep h rit i c ac ti v i ty . The fre q uency o f C D 2 5 + cel l s am on g in tr aren al Treg rem a in ed unch anged d u ring
p r ogress ion o f nep h ri ti s f r o m the onse t s t a g e to the m o re ac tiv e st a g es ( F i g ure 2 A ). Howev e r, at the
onset st ag e th e fre q u e ncy o f C D 2 5 + ce lls am ong intr ar enal Treg w a s signific an tly lower comp ared to
Treg f r om t h e s p l eens of t h e sa me di s e a s e sta g e a n d wa s i n a si mi lar ra ng e t h a n in peri pheral bl ood
(Fi g ure S 1 B , l e ft ). Of note , Treg f r om sp l eens and periphera l blood of (NZB × N Z W ) F 1 m i c e a t t h e
onset stage of LN were sho w n to h a ve already a re du ce d e x pr es si on of CD25 when c o mpa r ed to Tr e g
f r o m a g e - m a t c h e d B A L B / c m i c e o r t o y o u n g ( N Z B × N Z W ) F 1 m i c e [ 1 4 ] . S i m i l a r t o o b s e r v a t i o n s i n
the sple ens of (NZB × NZW) F 1 m i ce, 50%–60% of th e in tr aren al T r eg expressed th e memor y T cell
marker CD 4 4 [2 0] (F igu r e S1B , midd le ), which howe ver did no t c o nsider abl y c h ange in the kidney s
durin g dise ase progression (Figure 2A, m i ddle). CD69 has been con s idered to be a marker for r e cently
act i v a ted T c e ll s [2 1 ] , how e v e r m o re re centl y it h a s b een shown t h at C D 69 als o p l ays an im p o rtan t
role in the re tent ion of T c e ll s in the in f l amed or gan s [ 2 2]. T he f r eq uenci e s of CD6 9+ cell s among
intr aren al T r e g s i gn if ican tl y corre l a te d with th e P U I (Figure 2A, r i ght) and w e r e s i gnific an tly higher
than in the spleens at the d i se ase on set (Fig ure S1B ) , s u gge st ing a r e ten t ion o f in tra r ena l Treg at the
si te o f inf l am m a t i on and a high act i v i ty of Treg a l re ady in th e e a rly p h as e o f LN, which f u rther
incre a ses dur i ng the pro g re ssion o f L N . I n conclu sion , the pheno t yp e of intr aren al Treg toge the r with
the progr e ssive homeostatic Treg /Tcon imbalance sug g ests that a d e fic i en cy o f I L -2 is a l so pre s ent in
the in flamed kidney s of lupus-prone m i ce and such shortage of IL -2 occurs early during th e course
of LN.

Figure 2 . Phen otypic changes of intrarenal Treg and Tcon . Graphs show the percentag e of CD25+,
CD44hi and CD69+ cells a m ong CD4+ Fo xP3+ Treg ( A ) and among CD4+FoxP3– Tcon ( B ) in
correlation wit h the proteinuria index. Data are de ri ved fro m one ki dney of ea ch mouse from two to
five independent experiments ( n = 26). Correlation analyse s were perfor med by using Spearman’s
rank correlatio n coeffi cient s . Black lines ind i cate linear reg r ession cu rves u s ing Pears o n a n aly s es (* p
< 0.05 and ** p < 0.01) .

Figure 2.
Phenotypic changes of intrar enal T r eg and T con. Graphs show the percentage of CD25 + ,
CD44hi and CD69 + cells among CD4 + FoxP3 + T r eg (
A
) and among CD4 + FoxP3– T con (
B
) in correlation
with the pr oteinuria index. Data are derived from one kidney of each mouse fr om two to five
independent experiments ( n = 26). Correlation analyses wer e performed by using Spearman’s rank
correlation coe ffi cients. Black lines indicate linear regr ession curves using Pearson analyses (* p < 0.05
and ** p < 0.01).
3.3. Increased Activation of Intrar enal T con
In parallel to the T reg analyses, we assessed phenotypic changes of intrar enal T con during the
pr ogression of LN. The expr ession of CD25 is induced in T con upon activation independent of IL-2
availability and thus can be used as an activation marker for T con. The frequencies of CD25 + cells
among intrar enal T con considerably correlated with the PUI as an indicator of nephritic activity
(Figur e 2 B). The incr eases in the frequencies of CD25 + cells among T con between the onset stage and
the nephritic stage were in a similar range than in the spleens and peripheral blood of these mice
(Figur e S1C). Corresponding to this, also the frequencies of CD44hi e ff ector / memory cells among

Cells 2019 , 8 , 1234 7 of 16
intrar enal T con markedly correlated with the PUI (Figur e 2 B). The frequencies of CD69 + cells among
intrar enal T con were at the same level than in spleens (Figur e S1C), but did not change considerably
during pr ogression of LN (Figur e 2 B). The increases in the fr equencies of CD44hi cells among intrarenal
T con between the onset and nephritic stage were similar to those in the spleens and peripheral blood
(Figur e S1C). These findings point to a progr essive activation of intrarenal T con and an accumulation
of e ff ector / memory T con in the inflamed kidneys.
3.4. Decrease of Intrar enal IL-2 Pr oducing CD4 + Memory / E ff ector T Cells in Active LN
T o assess whether shortage of IL-2 is present in the inflamed kidneys at a cellular level as suggested
by the data above, we determined the capability of intrarenal CD4 + CD44hi memory / e ff ector T cells
to pr oduce cytokines in comparison to splenic memory / e ff ector T cells of (NZB
×
NZW) F1 mice.
The fr equency of IFN-
γ
pr oducing cells among splenic CD4 + CD44hi memory / e ff ector T cells incr eased
during pr ogression fr om the onset to the nephritic stage (Figure 3 A). In parallel and in line with
pr evious findings in this lupus model [
14
], the fr equency of IL-2 producing cells among splenic
CD4 + CD44hi T cells declined (Figur e 3 B). In nephritic mice, frequencies of IL-2 and of IFN-
γ
pr oducing
cells among intrar enal CD4 + CD44hi T cells were in a similar range than in the spleens of nephritic mice
(Figur e 3 A, B), suggesting that a decrease of IL-2 pr oducing T con and a concomitant pr edominance of
Th1 cells also occurs in the inflamed kidneys.
Cells 2019 , 8 , x FO R PE ER R E VIE W 7 of 16
3. 3. Inc rease d Acti vati on of Intrare n al Tcon
In paralle l to the Treg analyses , we asse ssed pheno t y p ic change s o f in trarenal T c on durin g th e
progression o f L N . The ex pression o f C D 25 is ind u ced in Tcon up on ac tiv a tion independen t of IL -2
av a i l a b i li ty a n d th us can b e use d as an ac tiv a t i on m a rker fo r Tco n . The fre q ue ncies of C D 2 5 + cel l s
am ong int r ar enal T c on co nsider ab l y co rrel a te d w i th the PUI a s a n ind i ca tor o f nep h r i t i c ac tiv i ty
( F ig u r e 2 B ). The i n c r ea se s in t h e f r equ e nc i e s of CD25 + c e ll s a m ong Tc on be t w e e n t h e ons e t s t a g e a n d
t h e ne phrit i c s t a g e we r e i n a si mi la r ra ng e t h a n i n th e spleens and peripher al blood of th ese mice
(Fi g ure S 1 C ) . C o rresp ondi ng to thi s , al so the fr equencies o f CD44hi effe ctor/memory cells amon g
intr aren al Tc on m a r kedl y correl a t e d w i th the PUI (F i gure 2 B ). The freq uenc ies o f C D 69 + c e l l s am ong
intr aren al Tc on were a t th e s a me leve l t h an in sp leen s (Figure S1C ) , b u t d i d not change considerab ly
durin g progr e ssion o f L N (Fi g ure 2B ). The incre a s e s in the fr e q uenc ies o f CD44h i cel l s among
intr aren al T c on b e tween t h e onse t and nep h ri tic s t a g e were s i m i lar to those i n the sp l eens and
peripheral blood (Figure S1 C). These f i n d ings p o int to a p r ogre ssiv e act i v a tion o f intr aren al Tc on and
an accum u lation of effec t or/memor y Tco n in the inflamed kidneys.
3.4. Decrea se o f Intr arenal IL-2 Pro d u c ing CD4+ Memor y/Effector T Cell s in Acti ve L N
To asse ss wh ether shor tag e of IL -2 is p r esen t in the in fl amed k i dneys a t a c e ll ul ar leve l as
sugg es ted b y the da t a ab ov e , we de term in ed th e c a p a b i li ty of in tr a r ena l C D 4+ C D 44h i
me mo r y / e f f e ct o r T c e ll s t o pr o d u c e cy t o k i n e s i n c o mpa r is o n t o s p l e n i c me mo ry / e f f e ct o r T ce l l s o f
(NZB × NZ W) F 1 mice . The freque ncy of IFN- γ producin g cell s among splenic CD 4 + C D44h i
memory/e ffec t or T ce ll s inc r eas e d dur i n g prog ress io n from the ons e t to the n e p h rit i c s t a g e ( F ig ure
3A ). In pa ra ll el and in l i ne wi th prev io us fin d ing s i n th is l u pus model [ 1 4 ] , t h e fr eq uency of IL- 2
producin g c e ll s among splenic CD 4 + CD4 4 hi T c e ll s d e cl ined (F ig ure 3 B ). In nephr i t i c mice ,
frequenc ies o f IL-2 and of IFN- γ pro d u c ing ce ll s am ong in tr aren a l CD4 + CD 44 hi T ce ll s we re in a
similar ran g e than in the spleens o f nephritic mice (Fig ure 3A, B ) , s u gges t in g th at a decre a se o f IL-2
producin g Tc on and a conc omitant pred ominance of Th1 ce lls a l so occurs in the infl amed k i d n eys.

Figure 3. Low frequency of intrarenal IL- 2 producing e ffector/m em ory T cel l s in a c tive lu pu s
nephritis. Sort ed C D 4+ T ce ll s from sing le s p leens and k i d n ey s of indi vi du al m i ce at t h e indicate d
disea s e s t ag e were stim u l ate d with PMA/ io nom y c in for 5 h and the frequ e ncies of IF N- γ ( A ) and IL -
2 ( B ) producing cells among CD3+CD4+CD 44hi memory T ce lls were dete rmined by flo w cytometry
(onset: n = 8 an d nephritic: n = 6). Horizontal lines represent the median. M a nn-Whitney U test wa s
used for statistical analyse s (* p < 0 . 05 and ** p < 0.01) .
3. 5. Sho r t - Ter m I L - 2 Trea tm ent E x pan d s th e In trare n al Tr eg Po pula tion
The data abo v e suggested that IL -2 de fic i ency, eith er loca ll y or systemi c al ly, mi ght cont ri bu te t o
rena l in fl am m a t i on in (N Z B × NZ W ) F 1 m i ce . There f ore, (NZ B × NZ W) F1 m i c e with ac tiv e nep h rit i s
were tre a t e d dai l y wi th a subcu t aneou s injec t ion o f r e combinan t I L -2 (2 5 ng /g body we igh t ) for 5
consecu t iv e d a ys . In tr aren a l C D 4+ FoxP 3 + Tre g and C D 4+ FoxP 3 − Tcon were a n al yzed 12 h a f ter t h e
la st IL - 2 injec t ion and w e re com p ared to age - and d i se ase ac tiv i t y - m atche d PB S- tre a te d con t r o l m i ce .
Ab sol u te n u m b ers o f to ta l int r ar ena l C D 4+ T ce lls, and more pro n ounced of C D 4+FoxP 3+ T r eg
and of C D 4 + F o xP3 − T c on i n creas e d ap p r ox. two- fol d durin g the sh ort- term IL- 2 tre a tm en t co m p ared
to contro l m i ce (Fi gure 4A –C ). The fre q uencie s of Fo xP3 + T r eg am ong intr aren a l C D 4 + T ce ll s we re

Figure 3.
Low frequency of intrar enal IL-2 producing e ff ector / memory T cells in active lupus nephritis.
Sorted CD4 + T cells from single spleens and kidneys of individual mice at the indicated disease stage
were stimulated with PMA / ionomycin for 5 h and the frequencies of IFN-
γ
(
A
) and IL-2 (
B
) pr oducing
cells among CD3 + CD4 + CD44hi memory T cells wer e determined by flow cytometry (onset: n = 8 and
nephritic: n = 6). Horizontal lines r epresent the median. Mann-Whitney U test was used for statistical
analyses (* p < 0.05 and ** p < 0.01).
3.5. Short-T erm IL-2 T reatment Expands the Intrar enal T reg Population
The data above suggested that IL-2 deficiency , either locally or systemically , might contribute to
r enal inflammation in (NZB
×
NZW) F1 mice. Therefor e, (NZB
×
NZW) F1 mice with active nephritis
wer e tr eated daily with a subcutaneous injection of recombinant IL-2 (25 ng / g body weight) for 5
consecutive days. Intrarenal CD4 + FoxP3 + T r eg and CD4 + FoxP3
−
T con were analyzed 12 h after the
last IL-2 injection and wer e compared to age- and disease activity-matched PBS-tr eated control mice.
Absolute numbers of total intrar enal CD4 + T cells, and more pr onounced of CD4 + FoxP3 +
T reg and of CD4 + FoxP3
−
T con increased appr ox. two-fold during the short-term IL-2 tr eatment
compar ed to control mice (Figur e 4 A–C). The frequencies of FoxP3 + T reg among intrar enal CD4 +
T cells wer e significantly higher in IL-2 treated mice compar ed to controls (Figur e 4 D), indicating
that IL-2 pr eferentially expands the T reg population in the inflamed kidney . Although significant
incr eases in the frequencies of CD25 + cells among T reg wer e observed in peripheral blood and spleens
of IL-2 tr eated mice (Figure S3A), the fr equency of CD25 + cells among intrarenal T r eg was una ff ected
(Figur e 4 E, left). Nonetheless, the geometric mean fluorescence intensity (gMFI) of CD25 in intrar enal
CD4 + FoxP3 + CD25 + T reg and of FoxP3 in intrar enal CD4 + FoxP3 + T reg, r epresenting the expr ession of
CD25 and FoxP3 on a per -cell basis, was significantly higher compared to the contr ol group (Figur e 4 E

Cells 2019 , 8 , 1234 8 of 16
right; Figure S2). In addition, the frequencies of Ki67 + CD25 + among intrar enal T reg wer e higher in the
IL-2 tr eated mice compared to contr ol mice (Figure 4 F). This was also observed in peripheral blood but
not in spleens of the same mice (Figure S3C), which suggests that systemically administer ed IL-2 has
di ff ering e ff ects on T reg populations fr om di ff erent or gans or compartments.
Cells 2019 , 8 , x FO R PE ER R E VIE W 8 of 16
sign ifican tly higher in IL -2 tre a te d mice compared to con t ro ls (Fi g ure 4D ), indic a ting th at IL- 2
preferen tially expands the Treg population in the in fl a m ed kidn ey. Al though si g n ifi c an t incre a se s in
the frequencies of CD25+ cells among Treg were ob served in per i pheral blood and sple ens of IL-2
tre a te d m i ce ( F ig ure S 3 A ) , t h e fre q uenc y of C D 2 5+ ce l l s a m ong i n t r arenal Treg was unaf fect ed (Fi g ure
4E, le ft). No netheless, th e geome t r i c mean fluore s c ence in ten s i t y (g MFI) of CD2 5 in in t r aren al
C D 4+ FoxP 3+ C D 25 + Tre g a n d of FoxP 3 i n in tra r ena l C D 4+ FoxP 3+ Treg, r e p r ese n ting th e exp r ess i on
of CD2 5 a n d F o xP 3 on a per- cell ba si s, wa s si g n if i c a n t l y hi g h er compa r ed t o t h e cont rol grou p (F i g u r e
4E r i gh t; F i g u re S 2 ). In ad d i t i on, the f r eq uencie s of K i 67 +C D 2 5 + a m ong int r ar e n al Tre g wer e higher
in the IL-2 tre a te d mice co mpared to co ntrol m i ce (Figure 4F ). Th is was also ob served in per i pheral
blood b u t no t in spleen s o f the same mice (Figure S3 C ) , which sug g ests th at system ically admin i ster ed
IL - 2 has di ff er i n g ef fe c t s on Tr e g popula ti ons from d i fferen t organ s or comp artments.

Figure 4. Short - term IL-2 trea tment expa nds the intrarenal Treg popula tion. Cells from kidneys of
(NZB × NZW) F1 mice at th e disease onse t were analyze d by flow cyto metry 24 h after a 5-day
treatment course w i th daily injections of rIL- 2 and compared w i th PBS-tr eated control mice. ( A – C )
Total numbers of intrarenal CD4+ T cells ( A ), of intrarenal CD4+FoxP3+ Treg ( B ) and of intrarenal
CD4+FoxP3- T c on ( C ) from IL-2 treated mice (IL-2) compared to control m i ce (C TRL) are shown. ( D )

Figure 4.
Short-term IL-2 treatment expands the intrarenal T reg population. Cells from kidneys of
(
NZB × NZW
) F1 mice at the disease onset wer e analyzed by flow cytometry 24 h after a 5-day treatment
course with daily injections of rIL-2 and compared with PBS-tr eated control mice. (
A
–
C
) T otal numbers
of intrarenal CD4 + T cells (
A
), of intrarenal CD4 + FoxP3 + T reg (
B
) and of intrarenal CD4 + FoxP3- T con (
C
)
from IL-2 tr eated mice (IL-2) compared to contr ol mice (CTRL) are shown. (
D
) Representative contou r
plots and scatter plot show the frequencies of FoxP3 + cells among CD4 + T cells in IL-2 tr eated mice
compared to contr ols. (
E
) The frequency of CD25 + cells among CD4 + FoxP3 + T r eg and the geometric
mean fluor escence intensity (gMFI) of CD25 in CD4 + FoxP3 + CD25 + T reg ar e shown. (
F
) The percentage
of intrarenal CD25 + Ki67 + cells among CD4 + FoxP3 + T reg is shown. (
G
) The frequency of CD25 + cells
among CD4 + FoxP3
−
T con and the gMFI of CD25 in CD4 + FoxP3-CD25 + T con are shown. (
H
) The
calculated ratio between percentages of Ki67 + T reg and of Ki67 + T con is shown. Filled squares
indicate PBS treated contr ol mice (CTRL, n = 9) and open squares r epresent IL-2 tr eated mice (IL-2,
n = 10
). Horizontal lines r epresent the median. Mann-Whitney U test was used for statistical analyses
(* p < 0.05).

Cells 2019 , 8 , 1234 9 of 16
Analysis of intrar enal T con revealed higher frequencies of CD25 + cells among T con in short-term
IL-2 tr eated mice compared to contr ol mice (Figure 4 G), which was not observed in spleens or peripheral
blood (Figur e S3B). However , and in contrast to intrarenal T reg, the gMFI of CD25 in intrar enal CD25 +
T con remained una ff ected (Figur e 4 G) and neither changes in the frequencies of intrar enal T con
expr essing Ki67, CD69 or CD44 (data not shown) nor changes in the T reg / T con proliferation ratio
(Figur e 4 H) were observed in short-term IL-2 tr eated mice. Of note, the proliferation ratio of T reg / T con
was significantly higher in the spleens of IL-2 tr eated mice, mainly due to a lower frequency of
pr oliferating cells among T con (Figure S3D). T aken together , intrarenal T r eg of lupus prone mice could
be influenced by a short-term IL-2 treatment by means of numbers, phenotype and proliferation,
wher eas with the exception of an increase in the fr equency of CD25 + T con, intrarenal T con were
almost una ff ected.
3.6. Long-T erm IL-2 T reatment Diminishes the Activity of Intrar enal T con
In addition to the short-term experiments, we next assessed the e ff ects of long-term IL-2 treatments
on intrar enal T reg and T con in comparison to spleens and peripheral blood from (NZB
×
NZW) F1
mice with active LN and whether there ar e di ff er ences in the AI determined by histomorphological
analyses of the kidneys. Mice wer e treated with IL-2 on 5 consecutive days (five injections in total)
followed by a maintenance tr eatment with repetitive IL-2 injections every four days for the duration of
29 days (six additional injections). Quantification of intrarenal CD4 + T cells at day 31 after the start of
the IL-2 tr eatments (48 h after the last IL-2 injection) revealed no significant di ff er ences in the absolute
numbers of total CD4 + T cells, of CD4 + FoxP3 + T r eg or of CD4 + FoxP3- T con between IL-2 treated
and contr ol mice (Figur e 5 A–C). Unexpectedly , the frequency of intrar enal T reg among CD4 + T cells
was significantly lower in the IL-2-tr eated mice compar ed to contr ol mice (Figure 5 D), which was not
observed in spleens or in peripheral blood of these mice (Figur e S4A).
Phenotypical analyses of intrar enal T reg showed lower fr equencies of CD69 + and Ki67 + cells
among T reg in IL-2 tr eated mice, whereas fr equencies of CD44hi cells among T reg wer e una ff ected by
the long-term IL-2 tr eatments (Figure 5 E). Comparable changes in the fr equencies of CD69 + and Ki67 +
T reg could be observed in the spleens but not in the peripheral blood of IL-2 tr eated mice (Figure S4D).
Although fr equencies of CD25 + cells among total intrarenal T reg did not incr ease in IL-2 tr eated mice
(data not shown), which was di ff er ent to findings in the spleens (Figure S4D), we found significantly
higher fr equencies of CD25 + cells among the Helios + FoxP3 + T reg subset, which is consider ed to be of
thymic origin [ 23 ], in the kidneys and also in spleens of IL-2 tr eated mice (Figure 5 E, Figur e S5).
In parallel to the e ff ects on intrar enal T r eg, we observed significantly lower frequencies of CD44hi
e ff ector / memory cells among intrar enal T con in IL-2 treated mice compar ed to controls (Figur e 5 F).
In addition, fr equencies of CD69 + cells and of proliferating, Ki67 + cells among intrar enal T con were
significantly lower in IL-2 treated mice compar ed to the control gr oup (Figur e 5 F). Fr equencies of
CD25 + cells among intrar enal T con did not change significantly under the IL-2 treatment (not shown).
Of note, fr equencies and phenotype of intrarenal T r eg and T con in long-term IL-2 treated mice with
active LN wer e in a comparable range as in (NZB
×
NZW) F1 mice at the onset stage (Figur e 1 ).
Similar , but less pronounced, changes in the fr equencies of CD25 + , CD44hi, CD69 + and Ki67 + T con
wer e observed in spleens, but not in the peripheral blood of long-term IL-2 treated mice (Figur e S4E).
Simultaneously performed histological analyses of the kidneys showed a significantly lower AI in IL-2
tr eated mice compared to contr ol mice (Figure 6 A–C). Complementary to this IL-2 tr eated mice also
had a lower PUI than contr ol mice at day 31 (Figure 6 D).

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Cells 2019 , 8 , x FO R PE ER R E VIE W 10 of 16

Figure 5 . Long-term IL-2 treatment diminishes the a c tiv i ty and prolif eration of intrarenal Treg and
Tcon. Cel l s from ki d n eys of (N ZB × N Z W) F1 mi c e wi th active nephritis were analyzed by flow
cytometry at day 31 after the initiation o f th e IL-2 tr eatments (48 h after the last IL-2 inj e ction; IL-2,
white bars) an d were compar ed to PBS-trea t e d control m i c e (CTRL, bla c k bars). ( A – D ) S c atter plots
show the abso l u te nu m b ers of total C D 4+ T cell s ( A ), of C D 4+FoxP3+ Treg ( B ) and of C D 4+FoxP3 −
Tcon ( C ) and th e perc entage of FoxP 3+ c e ll s among CD4+ T cel l s i n ki d n eys ( D ). ( E – F ) The p e rcentages
of CD25+, CD 44hi, CD69+ a n d of K i 67+ ce lls among intr arenal CD4+FoxP3+ Treg ( E ) and among
intrarenal CD4+FoxP3 − Tcon ( F ) are shown. F illed squ a res i n dicate PB S tre a ted control m i ce (CTRL ;
n = 8) and open squares represent IL-2 treate d mice (IL-2, n = 13). Horizontal lines in dicat e t h e media n
of each group. Data are the su mmary of two to five indepen d ent experiments. Mann-Whitney U te st
was u s ed for st atisti cal analy s es (* p < 0.05 and ** p < 0 . 01).
Phenot yp i c al a n al yses of int r arenal Treg show ed l o wer f r eq uenci e s of CD6 9+ a n d Ki67 + cell s
a m ong Tr eg in IL -2 t r ea t ed mi c e , whe r ea s f r eq ue nc i e s of CD44 hi ce l l s a m ong Tr e g we re u n af f e ct e d
by the long -term IL-2 tr eatments (Figur e 5E ). Co m p a r ab le ch ang e s in th e fr eq ue ncies o f C D 6 9+ and
Ki67+ Treg c o uld be ob se rved in the spleens but n o t in the p e r i p h eral b l ood of IL- 2 tre a te d m i ce
(Fi g ure S4D ) . Al though fre q uenc ies o f C D 25 + cel l s am ong tot a l int r aren al Treg d i d no t incre a se in IL -
2 treated mice (data no t sho w n), which w a s d i fferen t to find ings in th e spleen s ( F ig ure S 4 D ) , we f o und
sign ifican tly higher fr equencies of CD 25+ cell s among the Helios+Fox P 3+ T r eg subset, which is
consider ed to be o f thym ic origin [2 3] , in the k i dne y s a n d a l so in sp l eens o f I L - 2 tr eat e d m i ce (F igur e
5E, Figure S5).
In par a llel to the effects o n intrar enal Treg, we obs e rved s i gn if i c ant l y lower freq uenc ies o f
C D 44h i ef fec t or/m em o r y cell s am ong intr aren al Tc on in IL -2 tr eat e d m i ce c o m p ared to control s
(Fi g ure 5F) . I n add i tion, f r equen c ie s o f C D 69 + ce ll s a n d of p r ol ifer at ing, Ki 6 7 + c e ll s am ong in tra r ena l
Tcon were s i gnif ic ant l y lo wer in IL - 2 t r ea ted mice compared to th e con t ro l group (F ig ur e 5F) .
Freq uenc ies of C D 2 5 + ce l l s am ong in t r aren al Tcon did not change s i gn ificantly und e r the IL-2

Figure 5.
Long-term IL-2 treatment diminishes the activity and pr oliferation of intrarenal T reg and T con.
Cells from kidneys of (NZB
×
NZW) F1 mice with active nephritis were analyzed by flow cytometry
at day 31 after the initiation of the IL-2 treatments (48 h after the last IL-2 injection; IL-2, white bars)
and were compar ed to PBS-treated contr ol mice (CTRL, black bars). (
A
–
D
) Scatter plots show the
absolute numbers of total CD4 + T cells (
A
), of CD4 + FoxP3 + T r eg (
B
) and of CD4 + FoxP3
−
T con (
C
) and
the percentage of FoxP3 + cells among CD4 + T cells in kidneys (
D
). (
E
–
F
) The percentages of CD25 + ,
CD44hi, CD69 + and of Ki67 + cells among intrarenal CD4 + FoxP3 + T reg (
E
) and among intrarenal
CD4 + FoxP3
−
T con (
F
) are shown. Filled squares indicate PBS tr eated control mice (CTRL; n = 8) and
open squares r epresent IL-2 tr eated mice (IL-2, n = 13). Horizontal lines indicate the median of each
group. Data are the summary of two to five independent experiments. Mann-Whitney U test was used
for statistical analyses (* p < 0.05 and ** p < 0.01).

Cells 2019 , 8 , 1234 11 of 16
Cells 2019 , 8 , x FO R PE ER R E VIE W 11 of 16
tre a tm en t (no t shown) . Of note, fre q uen c ies and p h en otype of intrarenal Treg an d Tcon in lon g -term
IL-2 tre a te d m i ce w i th act i v e L N were i n a com p arab le ran g e as in (NZ B × NZ W ) F1 m i ce a t th e onse t
st age ( F i g ure 1) . Sim i l a r, b u t l e ss p r ono u nced, ch ang e s in the fre q uencie s of C D 25 +, C D 44h i, C D 6 9 +
a n d K i 6 7 + T c o n w e r e o b s e r v e d i n s p l e e n s , b u t n o t i n t h e p e r i p h e r a l b l o o d o f l o n g - t e r m I L - 2 t r e a t e d
mi ce ( F i g ure S4 E) . Si mu lt a n eou s ly performed histologic al an al yses of the kidney s sho w ed a
sign if ican t l y l o wer AI in IL -2 tr ea ted mic e compare d t o contro l m i c e (F ig ure 6 A – C ). Complem e nta r y
to this IL- 2 tr eat e d m i ce al so ha d a low e r PUI th an co ntrol m i ce a t day 3 1 (F igur e 6D ).

Figure 6. Long -term IL-2 trea tment reduces nephriti c act i vity at a histo l o g ical leve l. ( A ) T h e re na l
a c t i v i t y i n d e x ( A I ) w a s d e t e r m i n e d i n s i n g l e k i d n e y s f r o m ( N Z B × N Z W ) F 1 m i c e w i t h e s t a b l i s h e d
nephritis at da y 31 after the initiation of the IL-2 tr eatments (48 h after th e la st IL-2 injection; IL-2,
white bars) and compared to PBS-treate d co ntrol mice (CTRL, bla c k bars). ( B ) The six differentially
weig hted histo m orpholog ical scores ac cordin g to the AI are shown for the co ntrol and the IL-2 treated
group. ( C ) Representative i m ages of hem a toxylin and e osin stained k i dney sect ions from either
controls (le ft) o r from IL-2 tr e a ted mice (right) are shown. ( D ) T h e pro t ei nuri a i n d e x o f co nt ro l s a n d
IL-2 treat e d mice at day 31 ar e shown. Data repres ent the mean + SD of t h e score s sum m arized from
two to five inde pendent experiments ( n = 6–17). Mann-Whitney U test wa s used for statistical analyses
(* p < 0.05 and * * p < 0.01) . One outlier in the C T RL group at d0 was i d e n ti fi ed and re mov e d afte r us i n g
the ROUT te st.
4. Disc ussion
An ac qu ired defic i ency o f IL-2 pro f oun d ly af fect s Tr eg biolo gy in lupu s-prone i n divid u a l s an d
sign if ican t l y c o ntrib u te s to the im m u ne p a tho g enes is o f SLE [ 1 4 , 2 4 ] . Low-dose IL - 2 ther ap y cur r entl y
receives m a jo r at ten t ion in medicine as a novel and u n iq ue the r ap eut i c appro a c h , wh ich is d i ffer e nt
from conv ent i ona l tr ea tm e n t st ra teg i es tha t com m o n l y ind u ce g l o b al im m u nos u p p r ess i on [ 2 5 , 2 6 ] .
Low-dose IL - 2 ther ap y in te nds to r e s t ore T r eg hom e os ta si s and ear l y p h a s e c lin ic al t r i a l s s u g g e s t th at

Figure 6.
Long-term IL-2 treatment reduces nephritic activity at a histological level. (
A
) The renal
activity index (AI) was determined in single kidneys from (NZB
×
NZW) F1 mice with established
nephritis at day 31 after the initiation of the IL-2 treatments (48 h after the last IL-2 injection; IL-2,
white bars) and compared to PBS-tr eated control mice (CTRL, black bars). (
B
) The six di ff erentially
weighted histomorphological scores accor ding to the AI are shown for the contr ol and the IL-2 treated
group. (
C
) Representative images of hematoxylin and eosin stained kidney sections fr om either controls
(left) or from IL-2 tr eated mice (right) are shown. (
D
) The pr oteinuria index of contr ols and IL-2 tr eated
mice at day 31 are shown. Data r epresent the mean + SD of the scor es summarized from two to five
independent experiments ( n = 6–17). Mann-Whitney U test was used for statistical analyses (
* p < 0.05
and ** p < 0.01). One outlier in the CTRL grou p at d0 was identified and removed after using the
ROUT test.
4. Discussion
An acquir ed deficiency of IL-2 profoundly a ff ects T reg biology in lupus-pr one individuals and
significantly contributes to the immune pathogenesis of SLE [
14
,
24
]. Low-dose IL-2 therapy currently
r eceives major attention in medicine as a novel and unique therapeutic appr oach, which is di ff erent
fr om conventional treatment strategies that commonly induce global immunosuppr ession [
25
,
26
].
Low-dose IL-2 therapy intends to r estore T reg homeostasis and early phase clinical trials suggest that
this tr eatment is e ffi cacious in large variety of autoimmune diseases including SLE [
17
,
18
,
25
,
27
,
28
].
Although it is undisputable that low-dose IL-2 therapy is capable to expand the T reg population very
e ffi ciently and also quite selectively in virtually every tr eated patient, the mechanisms of action of
low-dose IL-2 therapy beyond T reg expansion ar e still not well understood [
18
,
28
,
29
]. In particular ,
deeper insights into the IL-2 induced immunological changes in the inflamed tissues ar e necessary for
a better understanding of the modes of action of low-dose IL-2 therapy . In this study , we addressed

Cells 2019 , 8 , 1234 12 of 16
whether kidney-infiltrating T reg ar e a ff ected by a shortage of IL-2 and whether immune pathologies in
the inflamed kidneys can be influenced by exogenous administration of IL-2.
W e found that already at the onset stage of LN, intrar enal T r eg showed a reduced expr ession of
CD25, which was even lower than in T reg fr om spleens of these mice. The low expression of CD25 on
T reg r epresents a hallmark of IL-2 deficiency [
13
,
14
] and thus suggests an insu ffi cient availability of
intrar enal IL-2 alr eady at the early phase of LN. Complementary to these abnormalities we found low
fr equencies of IL-2 producing cells among intrar enal e ff ector / memory T con in mice with active nephritis.
The r educed availability of IL-2 in the inflamed kidneys of (NZB
×
NZW) F1 lupus-pr one mice could
ther efore r esult in impaired T reg gr owth and survival ther eby impeding their capability to adequately
contr ol the progr essive hyperactivity of intrarenal e ff ector / memory T con. Based on these findings,
it would be plausible to assume that numbers and fr equencies of intrarenal T reg decline during the
pr ogression of LN. Instead we observed an incr ease in both, the numbers and the frequencies of T r eg
during the course of LN indicating a continuous enrichment or expansion of the T reg population in the
inflamed kidneys. Similar observations wer e made for T r eg in the spleens of these mice and are also
r eported for human SLE patients, where the fr equencies of CD4 + FoxP3 + T r eg in the peripheral blood
corr elated with disease activity [
15
,
30
]. In addition, already at the onset stage a lar ge proportion of
intrar enal T reg expr essed CD69 (a marker for tissue-r esident cells and T cell activation) suggesting
that intrar enal T reg ar e in a highly active state and are r etained in the inflamed kidney . Overall these
observations suggest a counter -regulatory mechanism attempting to dampen cellular hyperactivity in
LN that however may fail due to an insu ffi cient availability of gr owth factors, such as IL-2 [ 14 , 24 ].
Another hallmark of IL-2 deficiency in SLE is the development of an imbalanced proliferation
between T reg and T con in favor of T con in lymphatic tissues and the peripheral blood [
14
,
15
]. Although
the pr oportions of pr oliferating cells among intrarenal T r eg only moderately declined in parallel to
a moderate increase in the pr oportions of proliferating T con, the calculated T r eg / T con proliferation
ratio, as a measur e of the homeostatic balance between T reg and T con, significantly declined during
the pr ogression of LN. This is analogous to findings in lymphatic or gans of these mice [
14
] and
indicates that a pr ogressive homeostatic T reg / T con imbalance develops also in the inflamed kidneys.
This also suggests that intrar enal T reg have a r educed capacity to e ffi ciently control the expansion
of pr o-inflammatory intrarenal T con. Thus, the progr essive increase in fr equencies and quantities
of intrar enal T reg that we observed during the course of LN might be the result of enhanced tissue
r ecruitment in order to compensate for the loss of contr ol over T con activity in a numerical way [
14
,
24
].
Y et, the parallel incr ease in the numbers of kidney-infiltrating T con and their increased activation
pr oposes that during pr ogr ession of LN T reg lose contr ol over T con activation in the inflamed kidneys.
Based on these findings we hypothesized that systemic administration of IL-2 also dir ectly a ff ects
intrar enal T r eg and thereby corr ects the observed T r eg defects in the kidneys. W e found that short-term
IL-2 tr eatment for five consecutive days was capable to increase the numbers and fr equencies of
intrar enal T reg. Simultaneously the expr ession levels of CD25 (gMFI) on T reg and the pr oportion of
pr oliferating CD25 + T r eg were significantly augmented in the kidneys. This indicates that intrarenal
T reg can be tar geted and modulated by IL-2 therapy , although the e ff ects on intrarenal T r eg appeared
to be in part di ff er ent to those observed in spleens and peripheral blood. The r easons for these
or gan-related di ff er ences in T r eg responsiveness ar e unclear , but we suppose that this may be due to
di ff er ences in the distribution and clearance of IL-2 in the organism. It is known that kidneys are the
major site of clearance of cir culating IL-2 in the body , which is mediated by both glomerular filtration
and peritubular extraction [
31
]. Thus, it could be speculated that intrarenal T r eg ar e exposed to higher
levels of IL-2 than T reg in other or gans. The transient increase in the fr equencies of CD25 + cells among
intrar enal T con during the short-term IL-2 treatment is in line with r ecent findings in peripheral blood
of SLE patients tr eated with low doses of IL-2 and may indicate a certain lack of selectivity for T r eg [
18
].
Y et, in consideration of the amelioration of LN and the reduction in activated intrar enal T con that was
observed during the long-term IL-2 tr eatment, such a transient activation of intrar enal T con may be
clinically rather negligible.

Cells 2019 , 8 , 1234 13 of 16
Long-term IL-2 tr eatment led to a decreased activity and pr oliferation of intrarenal T con and in
parallel r esulted in an amelioration of renal inflammation assessed by histomorphological scoring
of the kidney . The fact that the reduction of T con hyperactivity in the kidneys was only observed in
long-term tr eated mice and not in the short-term setting, suggests that multistep immunor egulatory
pr ocesses, most likely mediated by T reg, took place in the kidneys, and that dir ect and rapid e ff ects of
IL-2 on the T con population, such as the induction of activated-induced cell death [
32
,
33
], were not
pr edominant in this setting.
Unexpectedly , and in contrast to findings in peripheral blood and spleens and also di ff er ent
to the short-term tr eatment, the fr equencies and numbers of intrarenal T reg and their pr oliferation
and activation state was lower in long-term IL-2 treated mice compar ed to contr ols. At first view ,
such findings under long-term IL-2 tr eatment seem to be contradictory . However , we suppose that
the r educed quantities and activation state of intrarenal T reg under long term IL-2 tr eatment are
the consequence of the r educed activity of intrarenal T con, leading also to a lesser r equirement of
counteracting T reg in the kidneys. Of note, both, frequencies and absolute numbers of intrar enal T reg
in long-term IL-2-tr eated mice were in the same range as in mice at the onset stage with less active LN.
Overall these findings further support that long-term IL-2 tr eatment is capable to decelerate or reverse
T con activity in the a ff ected organs. Although no increase in the fr equencies of CD25 + among total T r eg
was observable in the kidneys of long-term IL-2 tr eated mice we found a significantly higher frequency
of CD25 + cells among the Helios + T reg subset, suggesting a pr eferential tar geting of thymic-derived
T reg by IL-2 therapy .
The r esults of our study are complementary to r ecent findings in the MRL / lpr mouse model of
SLE wher e it was shown that systemic administration of low doses of IL-2 decreases the numbers of
IL-17- pr oducing, so called, double negative T cells in the spleens and in parallel r educes inflammation
in the kidneys and other a ff ected or gans [
34
]. More r ecently it was also shown in the (NZB
×
NZW) F1
model that tr eatment with IL-2 / anti-IL-2 complexes induced an expansion of the T reg population in the
kidneys, which was accompanied by a reduction of kidney-infiltrating IFN-
γ
+ CD4 + and IL-17A + CD4 +
T cells and an amelioration of LN at a histomorphological and clinical level [
35
]. These data together
with our findings reasonably support the implementation of an IL-2-based immunotherapy in the
tr eatment of LN.
Limitations of our study include that we wer e not able to assess the suppressive function of
intrar enal T reg because of limited amounts of cells that can be isolated and sorted fr om the kidneys.
In addition, more detailed analyses of the e ff ects of IL-2 on intrar enal T cell subsets, including Th1,
Th17, T follicular helper cells and CD8 + T cells or B cell subsets and NK cells ar e requir ed for a deeper
understanding of the complex immunological pr ocesses that occur in the inflamed tissues under IL-2
therapy . Moreover , the diversity of potential suppr essive mechanisms of T reg that take place in the
inflamed kidney including T reg-mediated cytotoxic e ff ects on T con and other harmful immune cells is
well worth to be addr essed in future studies.
In summary , our study pr oposed that a disturbance of the T r eg-IL-2 axis contributed to the
pathophysiology of LN and particularly pr omoted the hyperactivity of intrarenal T con. The reversibility
of these immune pathologies in a ff ected or gans by IL-2 therapy shown here pr ovided valuable insights
into the mechanisms of action of IL-2 therapy at a cellular level and o ff er ed additional rationales for an
IL-2-based immunotherapy of LN.
Supplementary Materials:
The following are available online at http: // www .mdpi.com / 2073- 4409 / 8 / 10 / 1234 / s1 ,
Figure S1: Phenotypic changes of T reg and T con in di ff er ent organs during pr ogression of LN, Figur e S2: Short-term
IL-2 treatment incr eases the expression of FoxP3 in intrar enal T reg, Figur e S3: Phenotypic changes of T reg and
T con in spleens and peripheral blood after short-term IL-2 tr eatment, Figur e S4: Phenotypic changes of T reg and
T con in spleens and peripheral blood after long-term IL-2 tr eatment, Figur e S5: Long-term IL-2 tr eatment increases
CD25 expression in Helios + T reg in the spleen.
Author Contributions:
Conceptualization, G.R. and J.Y .H.; methodology , A.R., K.W ., A.K., P .E. and J.Y .H.; software,
A.R. and C.v .S.-M.; validation, A.R., K.W ., A.K. and J.Y .H.; formal analysis, A.R., C.v .S.-M., L.K., K.W ., P .E. and
J.Y .H.; investigation, A.R., C.v .S.-M., L.K. and K.W .; resour ces, G.-R.B. and G.R.; data curation, A.R. and J.Y .H.;

Cells 2019 , 8 , 1234 14 of 16
writing—original draft preparation, A.R. and J.Y .H.; writing—review and editing, P .E., G.-R.B., G.R. and J.Y .H.;
visualization, A.R. and J.Y .H.; supervision, G.R. and J.Y .H.; project administration, G.R. and J.Y .H.; funding
acquisition, G.R. and J.Y .H.
Funding: This study was supported by grants fr om the German Research Foundation (DFG) (SFB650, TP10 and
Z03) and r esearch grants fr om the Charit
é
— Universitätsmedizin Berlin. W e acknowledge support from the Open
Access Publication Fund of Charit é —Universitätsmedizin Berlin.
Acknowledgments:
W e thank Anika Klaus for technical assistance and T oralf Kaiser and Jenny Kirsch from the
Flow Cytometry Core Facility of the DRFZ for their technical support.
Conflicts of Interest: The authors have no competing financial interests.
Abbreviations
AI, activity index; gMFI, geometric mean fluorescence intensity; IL-2, interleukin 2; LN, lupus nephritis; NZB,
New Zealand Black; NZW , New Zealand White; PUI, proteinuria index; SLE, Systemic Lupus erythematosus;
T con, conventional T cells; T r eg, regulatory T cells.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http: // creativecommons.or g / licenses / by / 4.0 / ).

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