Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance

Abstract

Store-operated Ca2+ entry through calcium release-activated calcium channels is the chief mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx, whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription factor NFAT. T cell-specific ablation of both STIM1 and STIM2 resulted in a notable lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that STIM proteins are required for the development and function of regulatory T cells.

Full text

Dual functions for the endoplasmic reticulum calcium sensors
STIM1 and STIM2 in T cell activation and tolerance
Masatsugu Oh-hora1, Megumi Yamashita2, Patrick G Hogan1, Sonia Sharma1, Ed
Lamperti1, Woo Chung2, Murali Prakriya2, Stefan Feske1,3, and Anjana Rao1
1Harvard Medical School and Immune Disease Institute, Boston, Massachusetts 02115, USA.
2Department of Molecular Pharmacology and Biological Chemistry, Northwestern University,
Feinberg School of Medicine, Chicago, Illinois 60611, USA.
Abstract
Store-operated Ca2+ entry through calcium release–activated calcium channels is the chief
mechanism for increasing intracellular Ca2+ in immune cells. Here we show that mouse T cells and
fibroblasts lacking the calcium sensor STIM1 had severely impaired store-operated Ca2+ influx,
whereas deficiency in the calcium sensor STIM2 had a smaller effect. However, T cells lacking either
STIM1 or STIM2 had much less cytokine production and nuclear translocation of the transcription
factor NFAT. T cell–specific ablation of both STIM1 and STIM2 resulted in a notable
lymphoproliferative phenotype and a selective decrease in regulatory T cell numbers. We conclude
that both STIM1 and STIM2 promote store-operated Ca2+ entry into T cells and fibroblasts and that
STIM proteins are required for the development and function of regulatory T cells.
Calcium is a universal second messenger1. In T cells, mast cells and other cells of the immune
system, Ca2+ entry occurs mainly through the specialized store-operated Ca2+ entry channels
known as calcium release–activated Ca2+ (CRAC) channels2–5. CRAC channels are low-
conductance Ca2+ channels with an unusually high selectivity for Ca2+ and a characteristic
inwardly rectifying current-voltage relationship6,7. CRAC channels open in response to
signaling cascades initiated by immunoreceptors such as T cellantigen receptors (TCRs) and
mast cell Fc receptors. Stimulation of these immunoreceptors induces the recruitment and
activation of protein tyrosine kinases and the formation of large adaptor protein complexes and
ultimately results in tyrosine phosphorylation and activation of phospholipase C-γ (PLC-γ). In
T cells and mast cells, activated PLC-γ1 generates the second messenger inositol-1,4,5-
trisphosphate, which by binding to receptors in the endoplasmic reticulum membrane causes
the release of endoplasmic reticulum Ca2+ stores. In turn, the depletion of endoplasmic
reticulum Ca2+ stores induces the opening of CRAC channels, which permit sustained influx
of Ca2+ into the cell. Increases in the intracellular Ca2+ concentration promote rapid responses
© 2008 Nature Publishing Group
Correspondence should be addressed to A.R. (arao@cbr.med.harvard.edu) or S.F. (stefan.feske@med.nyu.edu)..
3Present address: Department of Pathology, New York University, School of Medicine, New York, New York 10016, USA.
AUTHOR CONTRIBUTIONS M.O. generated the gene-disrupted mice and did the bulk of the experiments; M.Y., W.C. and M.P. were
responsible for all electrophysiology experiments; S.S. established the NFAT translocation assay; E.L. did the immunohistochemistry;
S.F. did the single-cell Ca2+ imaging for T cells and codirected the project with P.G.H. and A.R.; and M.O., S.F., P.G.H. and A.R. wrote
the manuscript together.
Note: Supplementary information is available on the Nature Immunology website.
COMPETING INTERESTS STATEMENT The authors declare competing financial interests: details accompany the full-text HTML
version of the paper at http://www.nature.com/natureimmunology/.
Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions
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such as mast cell degranulation, as well as long-term responses that involve new gene
transcription5,8.
Two chief participants in the pathway connecting the depletion of endoplasmic reticulum stores
to the opening of CRAC channels have been identified. The endoplasmic reticulum Ca2+ sensor
STIM1 (stromal cell—interaction molecule) has been identified by two limited RNA-mediated
interference screens in drosophila and HeLa cells9,10. The drosophila protein Orai (encoded
by olf186-F), identified as an essential regulator of store-operated Ca2+ influx by a series of
genome-wide drosophila RNA-mediated interference screens11–13, and its three human
homologs, ORAI1—ORAI3 (also called CRACM1—CRACM3), are small proteins with four
transmembrane domains whose amino and carboxyl termini are both located in the
cytoplasm11,12,14. Mutational and electrophysiological analyses have identified drosophila
Orai and human ORAI1 as pore subunits of the CRAC channel15–17.
The importance of Ca2+ influx through CRAC channels is emphasized by the existence of at
least three families with severe combined immunodeficiency associated with defects in CRAC
channel function18–20. The underlying genetic defect responsible for the impaired CRAC
channel function in one such family is a cytosine-to-thymine transition leading to a missense
mutation in the gene encoding ORAI1 (ref. 11). T cells from patients of all three families show
almost no store-operated Ca2+ entry or CRAC channel function and, as a result, fail to
proliferate in response to TCR stimulation in vitro18–22 and fail to activate the Ca2+-responsive
transcription factor NFAT or produce appreciable amounts of NFAT-dependent cytokines18,
21,23.
STIM1 and STIM2 are single-pass transmembrane proteins with paired amino-terminal ‘EF
hands’ located in the endoplasmic reticulum lumen and several protein-protein—interaction
domains located in the endoplasmic reticulum lumen and the cytoplasm4,9,10,24. The first EF
hand of each pair binds Ca2+ with low affinity matched to the high concentration of Ca2+ in
the endoplasmic reticulum (200–600 μM)25,26. After store depletion, STIM proteins form
oligomers in the endoplasmic reticulum membrane and then move to regions of endoplasmic
reticulum—plasma membrane apposition that coincide with sites of Ca2+ entry and contain
small clusters (puncta) of STIM1 and ORAI1 localized together10,24,27–30. STIM2 differs from
STIM1 in that it is already partially active at basal endoplasmic reticulum Ca2+ concentrations
and becomes activated earlier during endoplasmic reticulum store depletion, before substantial
decreases in endoplasmic reticulum Ca2+ concentrations24. STIM proteins with EF-hand
alterations that impair Ca2+ binding are constitutively present in puncta, even in resting cells
with full Ca2+ stores10,24,31. The propensity of STIM proteins to undergo a conformational
change and form oligomers when their EF hands are not bound by Ca2+ has also been
demonstrated with short recombinant amino-terminal fragments containing only the EF hands
and the adjacent sterile α-motif domains25.
Here we investigated the physiological functions of STIM1 and STIM2 in mice with
conditionally targeted alleles of Stim1 and Stim2. Each protein promoted store-operated
Ca2+ entry. In T cells, deficiency of either Stim1 or Stim2 impaired sustained Ca2+ influx,
nuclear translocation of NFAT and cytokine production. However, mice lacking both Stim1
and Stim2 in T cells developed a lymphoproliferative syndrome characterized by
splenomegaly, lymphadenopathy, infiltration of leukocytes into many organs and a selective
decrease in the number and function of regulatory T cells (Treg cells) expressing the lineage-
specific transcription factor Foxp3. This syndrome was ameliorated by adoptive transfer of
wild-type Treg cells, and the Treg cell defect was cell intrinsic rather than being the result of
the lack of interleukin 2 (IL-2) production by STIM-deficient T cells32–34. Our findings are
consistent with published studies demonstrating ‘cooperation’ of NFAT and the signal
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transducer Smad3 at the Foxp3 promoter35 and a requirement for NFAT in Foxp3-mediated
transcription and suppressor function36–38.
RESULTS
Conditional ablation of Stim1 or Stim2
As both STIM1 and STIM2 are ubiquitously expressed in vivo39, we generated mice bearing
loxP-flanked alleles of Stim1 and Stim2 (Supplementary Fig. 1 online). We bred these mice
with a ‘CMV-Cre deleter strain’ (expressing a transgene encoding Cre recombinase under
control of the cytomegalovirus promoter)40 to examine the effects of deleting Stim1 and
Stim2 in all tissues. STIM1-deficient mice on the C57BL/6 background were alive at the
expected mendelian ratios at embryonic day 18.5 but died perinatally, with 75% of the pups
born dead and most of the remaining pups dying within 2 d; in contrast, mice lacking STIM2
survived until 4 weeks after birth but showed slight growth retardation and died at 4–5 weeks
of age (Supplementary Table 1a,b online). To ‘rescue’ the STIM1-deficient mice from perinatal
death, we crossed these mice with the outbred ICR mouse strain. Although perinatal death was
still high (38%), about half of the outbred STIM1-deficient offspring survived past day 2 with
severe growth retardation and died of unknown causes within the next 2 weeks (Supplementary
Table 1c).
To examine the effect of STIM deficiency in T cells, we crossed mice with loxP-flanked
Stim1 (Stim1fl/fl) or Stim2 (Stim2fl/fl) with mice expressing a Cre transgene under control of a
Cd4 enhancer-promoter-silencer cassette (CD4-Cre) that causes deletion at the double-positive
(CD4+CD8+) stage of thymocyte development41. Thymic cellularity and T cell development
seemed normal in Stim1fl/fl CD4-Cre+ and Stim2fl/fl CD4-Cre+ mice (data not shown), which
permitted analysis of peripheral CD4+ and CD8+ T cells (Fig. 1). Unless otherwise indicated,
we used T cells from mice in which Stim1 and/or Stim2 were (was) conditionally deleted with
CD4-Cre for T cell experiments, whereas we used mouse embryonic fibroblasts (MEFs) from
Stim1fl/fl CMV-Cre+ and Stim2fl/fl CMV-Cre+ mice for fibroblast experiments.
STIM proteins regulate store-operated Ca2+ influx
STIM1-deficient CD4+ T cells showed almost no Ca2+ influx after passive depletion of
endoplasmic reticulum Ca2+ stores with thapsigargin, an inhibitor of the sarcoplasmic-
endoplasmic reticulum Ca2+-ATPase, or after crosslinking of TCRs with antibody to CD3
(anti-CD3; Fig. 1a). Resting control and STIM1-deficient T cells had similar expression of
surface CD3 and TCRβ and similar depletion of endoplasmic reticulum Ca2+ stores and
expression of the activation markers CD25 and CD69 after stimulation with anti-CD3 or with
anti-CD3 and anti-CD28 (Supplementary Fig. 2a—c online). STIM1-deficient CD4+ T cells
failed to produce IL-2 after stimulation with the phorbol ester PMA and ionomycin (Fig. 1b)
or with anti-CD3 and anti-CD28 (Supplementary Fig. 2d). These results collectively provide
genetic evidence that STIM1 controls store-operated Ca2+ influx and the production of Ca2+-
dependent cytokines in primary mouse T cells.
In contrast, STIM2-deficient primary CD4+ T cells from Stim2fl/fl CMV-Cre+ mice showed
little or no impairment in Ca2+ influx or IL-2 production relative to control CD4+ T cells in
response to treatment with thapsigargin, ionomycin or anti-CD3 (Fig. 1c,d). A possible
explanation for this discrepancy is that naive CD4+ T cells had much lower expression of
STIM2 than STIM1 (Supplementary Fig. 3a online). Although T cell activation led to a
substantial increase in STIM2 expression, which was maintained after 3–7 d of differentiation
into T helper type 1 or T helper type 2 cells, this greater quantity of STIM2 nevertheless
amounted to only a small proportion (3–10%) of total STIM protein (Supplementary Fig. 3a,b).
Consistent with the fact that even in differentiated T cells, STIM2 constitutes a minor fraction
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of total STIM protein, STIM2-deficient helper T cells differentiated for 1 week in nonpolarizing
conditions showed slightly less Ca2+ influx in response to acute low-dose thapsigargin or anti-
CD3 stimulation (Fig. 1e). However, these cells were much less able to produce IL-2 and
interferon-γ (IFN-γ) after sustained stimulation with PMA and ionomycin (Fig. 1f) or anti-CD3
and anti-CD28 (Supplementary Fig. 2e). Likewise, STIM2-deficient T helper type 1 and T
helper type 2 cells had less production of IL-2, IL-4 and IFN-γ (data not shown).
We also examined store-operated Ca2+ influx in MEFs from Stim1fl/fl CMV-Cre+ and
Stim2fl/fl CMV-Cre+ mice. We confirmed that Ca2+ influx induced by treatment of wild-type
MEFs with thapsigargin was due to depletion of endoplasmic reticulum Ca2+ stores, as we
noted no Ca2+ influx in the absence of thapsigargin treatment (data not shown). As in T cells,
STIM1 deficiency abrogated Ca2+ influx; moreover, in MEFs, STIM2 deficiency also resulted
in less Ca2+ influx (Fig. 2a). These experiments collectively show that STIM1 deficiency
results in a complete loss of store-operated Ca2+ entry in T cells and fibroblasts, whereas STIM2
deficiency has a smaller effect.
To confirm that STIM2 is a functional endoplasmic reticulum Ca2+ sensor, we reconstituted
STIM1-deficient T cells and MEFs with Myc-tagged STIM1 or STIM2 then tested Ca2+ influx
and cytokine production (Fig. 2a,b). We used retroviral vectors permitting stable expression
of small amounts of protein to avoid artifacts resulting from overexpression. The introduced
STIM1 and STIM2 were expressed in similar amounts in both cell types, as shown by
immunoblot analysis with anti-Myc (Supplementary Fig. 3b and data not shown). STIM1
robustly reconstituted store-operated Ca2+ influx in STIM1-deficient MEFs (Fig. 2a) and in
STIM1-deficient helper T cells differentiated for 1 week in nonpolarizing conditions and
treated with thapsigargin (Fig. 2b); in contrast, we noted weaker Ca2+ influx in cells
reconstituted with STIM2 (Fig. 2a,b). Nevertheless, STIM2 was unexpectedly effective in
restoring cytokine expression to STIM1-deficient T cells stimulated with PMA and ionomycin
(Fig. 2c). These results collectively indicate that endogenous STIM2 is a positive regulator of
Ca2+ signaling in T cells and MEFs rather than being an inhibitor of STIM1 function, as
proposed before42.
Aborted Ca2+ entry and nuclear transport of NFAT
To reconcile the slightly lower store-operated Ca2+ influx with the (relatively) much lower
cytokine expression noted in STIM2-deficient T cells, we examined Ca2+ influx on a longer
time scale by loading the cells with Fura-PE3, a calcium indicator that is well retained in the
cytoplasm43. STIM2-deficient T cells had less store-operated Ca2+ entry than did wild-type T
cells and attained a lower plateau of sustained intracellular free Ca2+ concentration ([Ca2+]i)
after 20 min (Fig. 3a). To confirm that Ca2+ signaling is lower in STIM2-deficient cells, we
monitored the nuclear translocation of the calcium-dependent transcription factor NFAT1 (ref.
8; A000024). We quantified nuclear translocation with the MetaXpress program
(Supplementary Fig. 4 online) and differentiated helper T cells from wild-type, STIM1-
deficient and STIM2-deficient mice for 1 week in nonpolarizing conditions and then stimulated
them with PMA and ionomycin in the same conditions of stimulation used for the cytokine
assay, so that we could directly compare nuclear translocation of NFAT1 (Fig. 3b,c) and
cytokine expression (Fig. 3d,e).
The results unambiguously showed that in physiological conditions both STIM1 and STIM2
contributed to the sustained Ca2+ influx and nuclear translocation of NFAT required for high
expression of cytokine genes8. In STIM1-deficient helper T cells differentiated for 1 week in
nonpolarizing conditions, NFAT was transiently imported into the nucleus, presumably
because of the transient increase in [Ca2+]i that accompanies the depletion of endoplasmic
reticulum Ca2+ stores, but it was imported into the nucleus in only a fraction of cells and was
rapidly re-exported (Fig. 3b). In contrast, almost as many STIM2-deficient as control cells (70–
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75% versus 85–95%, respectively) had nuclear NFAT1 at 10 min, but this response was
sustained in control but not STIM2-deficient cells (Fig. 3c). These results point to an important
function for STIM2 in T cell signaling and explain the much lower cytokine expression in
STIM2-deficient T cells (Figs. 1f and 3e).
CRAC current impairment in STIM1-deficient cells
We used whole-cell patch-clamp recordings to determine whether deletion of STIM1 and
STIM2 affected the CRAC current (ICRAC). In response to depletion of endoplasmic reticulum
Ca2+ stores by thapsigargin, control mouse CD4+ T cells had Ca2+ currents with properties
similar to those of ICRAC of human T cells (Fig. 4). These properties included an inwardly
rectifying current-voltage relationship with a very positive reversal potential in the presence
of 20 mM Ca2+ (Fig. 4a), fast inactivation in 20 mM Ca2+ (Supplementary Fig. 5c online),
depotentiation of the Na+ current in divalent cation—free solutions (Fig. 4b), low Cs+
permeability (Cs+ permeability/Na+ permeability = 0.2 ± 0.04; n = 14 samples), blockade of
the Na+ current by micromolar concentrations of extracellular Ca2+ (Supplementary Fig. 5b)
and potentiation and inhibition by low and high concentrations of 2-aminoethoxydiphenyl
borate44 (although potentiation by 2-aminoethoxydiphenyl borate in mouse cells did not seem
as robust as that in Jurkat or human T cells; Supplementary Fig. 5d and data not shown).
Furthermore, inclusion of calcium-specific chelator BAPTA in the patch pipette at a
concentration of 10 mM caused the slow development of an inward current in an extracellular
calcium concentration of 20 mM after whole-cell break-in, reminiscent of the development of
ICRAC in response to store depletion (data not shown). These results collectively indicated that
mouse T cells have a Ca2+ current with properties indistinguishable from those of ICRAC.
Consistent with the Ca2+ imaging results, STIM1-deficient T cells showed no detectable
ICRAC in either 20 mM Ca2+ or divalent cationfree medium; in contrast, STIM2-deficient cells
had an ICRAC of slightly smaller magnitude than that of wild-type cells that did not, however,
reach statistical significance with the number of cells examined (Fig. 4c). The properties of
ICRAC were unaltered in STIM2-deficient T cells in terms of Ca2+ and Cs+ selectivity, fast
inactivation, depotentiation and responsiveness to high and low concentrations of 2-
aminoethoxydiphenyl borate (Fig. 4, Supplementary Fig. 5 and data not shown). These results
indicate that endogenous STIM1 is required for ICRAC in primary mouse CD4+ T cells but that
endogenous STIM2 makes little or no contribution to the recorded ICRAC in the same
conditions, possibly because it constitutes a very low fraction of total STIM protein even in
differentiated T cells (Supplementary Fig. 3b).
Complex phenotype of mice with double-knockout T cells
To analyze the consequences of combined deletion of Stim1 and Stim2 in T cells in vivo, we
generated Stim1fl/flStim2fl/fl CD4-Cre+ double-knockout mice. These mice showed no defect
in conventional thymic development, as assessed by thymic cellularity and the numbers and
proportions of CD4–CD8– double-negative cells, CD4+CD8+ double-positive cells and
CD4+ or CD8+ single-positive cells (data not shown). Two possible explanations are that STIM
proteins are long-lived and thus residual STIM1 and STIM2 protein may be present and
functional well after gene deletion has occurred at the double-positive stage, or that thymocytes
use STIM-independent Ca2+ influx mechanisms that differ from those used by peripheral T
cells. The functions of STIM proteins in T cell development and thymic selection remain to
be identified with mice in which Cre expression is initiated early during T cell or hematopoietic
cell development.
As expected from the severely deleterious phenotype of STIM1-deficient T cells, peripheral
CD4+ T cells lacking both STIM proteins showed essentially no Ca2+ influx in response to
stimulation with thapsigargin or anti-CD3 (Fig. 5a and Supplementary Fig. 6 online). The small
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amount of residual Ca2+ influx in the averaged curves of [Ca2+]i (Fig. 5a) and the small residual
ICRAC (Fig. 4c) stemmed from a small number of individual cells with normal Ca2+ influx and
ICRAC (single-cell images, Fig. 5a, right). Of 15 double-knockout cells examined, two had
normal ICRAC in the recordings in Figure 4c and the remaining cells had no ICRAC. These may
have represented contaminating non—CD4+ cells that ‘came through’ the purification
procedure or a small number of CD4+ T cells that escaped Cre-mediated deletion of STIM1
or STIM2 (discussed below). The double-knockout T cells produced almost no IL-2, although
they did produce small amounts of tumor necrosis factor in response to primary stimulation
(Fig. 5b), possibly because of PMA-induced activation of the transcription factor NF-κB, which
proceeded normally in the double-knockout cells (M.O., unpublished data). The double-
knockout T cells did upregulate expression of the activation markers CD69 and CD25, albeit
to a lesser extent than did control T cells (Fig. 5c), and they underwent proliferation, albeit to
a much smaller extent, after TCR stimulation (Fig. 5d,e).
Unexpectedly, double-knockout mice older than 8 weeks of age developed a notable phenotype
of splenomegaly, lymphadenopathy, dermatitis and blepharitis (Supplementary Fig. 7a online
and data not shown). Histological analysis showed infiltration of leukocytes into many organs,
including lung and liver (Fig. 6a and data not shown). Mice lacking STIM1 alone in CD4+ T
cells had a milder version of the lymphoproliferative phenotype (Supplementary Fig. 7a and
data not shown). The double-knockout mice also had more CD4+ T cells with a surface
phenotype characteristic of memory or effector status
(CD62LloCD44hiCD69hiCD45RBloCD5hi), more germinal centers in the spleen, more B cells
with a germinal center phenotype (CD95hiCD38lo) and more differentiated immunoglobulin
E—positive (IgE+) B cells, large numbers of CD11b+IL-5R+ eosinophils or basophils and
much higher serum concentrations of IgG1 (Supplementary Fig. 7b—d and data not shown).
CD4+ T cells from double-knockout mice produced IL-5 but not IL-4 in response to stimulation
with PMA and ionomycin (data not shown). Notably, the phenotype of double-knockout mice
was similar but not identical to that of mice with a substitution (Y136F) in the T cell
transmembrane adaptor Lat that eliminates the docking site for PLC-γ1 and results in lower
Ca2+ influx in response to TCR crosslinking45,46.
STIM in Treg cell differentiation and function
The autoreactive phenotype of the Lat (Y136F) mutant mice described above has been
attributed to impaired negative selection allowing escape of autoreactive T cells into the
periphery47 and to a lower number of Treg cells48. Direct examination of the function of STIM
proteins in positive and negative selection of thymocytes will require mice in which Stim1 and
Stim2 are ablated at an earlier stage of T cell development with Lck-Cre and breeding of these
mice with HY TCR-transgenic mice. Meanwhile, here we documented many fewer Treg cells
in the thymi, spleens and lymph nodes of 5- to 6-week-old Stim1fl/flStim2fl/fl CD4-Cre mice
(Fig. 6b,c and data not shown). The proportion of Treg cells in the spleens and lymph nodes of
double-knockout mice increased with age but nevertheless remained between 10% and 20%
of that in control mice (Fig. 6c); these increases were probably the result of age-dependent
increases in the size of peripheral lymphoid organs. Mice lacking either STIM1 or STIM2 had
normal numbers of CD4+CD25+Foxp3+ Treg cells (data not shown). The number of cells
expressing GITR, another marker of Treg cells, was also lower in double-knockout mice (data
not shown). We noted complete deletion of Stim1 and Stim2 in CD25– and CD25+ T cells from
older (8-week-old) double-knockout mice (Supplementary Fig. 8a online). Like
CD4+CD25– T cells, CD4+CD25+ Treg cells from double-knockout mice showed impaired
Ca2+ influx in response to treatment with thapsigargin or anti-CD3 (Fig. 6d and Supplementary
Fig. 8b).
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The much lower percentages and absolute numbers of Treg cells in double-knockout mice could
have reflected defective Treg cell development, survival in the periphery or both. Given the
finding that double-knockout T cells made very little IL-2, one possibility was that Treg cell
development was defective in part because of a lack of IL-2 production by ‘bystander’ T
cells32–34. To address those possibilities, we generated mixed—bone marrow chimeras. We
reconstituted sublethally irradiated recipient mice deficient in recombination-activating gene
1 (Rag1–/– mice) with T cell—depleted bone marrow from Thy-1.2+ control mice alone or
Thy-1.2+ double-knockout mice alone or bone marrow from Thy-1.2+ double-knockout mice
and Thy-1.1+ wild-type mice mixed at a ratio of 2:1. As expected, mice given only double-
knockout bone marrow had many fewer Treg cells in both thymus and lymph nodes than did
mice reconstituted with control bone marrow (Fig. 7) and developed the same severe
lymphoproliferative phenotype as that of unmanipulated double-knockout mice
(Supplementary Fig. 9a online). In contrast, mixed chimeras reconstituted with both double-
knockout and wild-type bone marrow did not show any signs of lymphoproliferative disease
and remained as healthy as control chimeric mice (Supplementary Fig. 9a). In these chimeric
mice, the wild-type bone marrow gave rise to normal numbers of peripheral Treg cells, whereas
the double-knockout precursors yielded far fewer Treg cells both in thymus and in the periphery
(Fig. 7). These results collectively indicate that the double-knockout mice have a cell-intrinsic
defect in Treg cell development that is not restored by IL-2 produced by ‘bystander’ T cells
derived from wild-type bone marrow32–34.
Next we determined whether we could prevent onset of the lymphoproliferative phenotype of
double-knockout mice by injecting young double-knockout mice with Treg cells from wild-
type mice. Injection of 2-week-old double-knockout mice with wild-type Treg cells prevented
the development of lymphoadenopathy and splenomegaly 8 weeks later, whereas injection with
phosphate-buffered saline or with non–Treg cells did not (Fig. 8a and Supplementary Fig. 9b).
Because double-knockout mice had very few endogenous Treg cells and we transferred only 3
× 105 wild-type CD4+CD25+ T cells into each double-knockout mouse, the injected mice
continued to have very few Treg cells at 8 weeks after injection (Fig. 8b). We distinguished
Thy-1.1+ Treg cells derived from the transferred wild-type Treg population from endogenous
Thy-1.2+ Treg cells by flow cytometry (Fig. 8b,c). As expected, endogenous Thy-1.2+ Treg
cells accounted for most of the CD4+CD25+Foxp3+ cells in double-knockout mice injected
with wild-type CD4+CD25– T cells; these mice showed lymphadenopathy and splenomegaly
at 10 weeks of age (Fig. 8c). In contrast, mice injected with wild-type CD4+CD25+ Treg cells
and ‘cured’ of the lymphopro-liferative phenotype had a Treg cell population derived
approximately equally from Thy-1.1+ donor and Thy-1.2+ endogenous cells (Fig. 8c). These
data indicate that even though some Treg cells develop in double-knockout mice, they function
poorly (if at all) relative to Treg cells from wild-type mice. Moreover, the numbers of
endogenous Treg cells were lower in the presence of transferred wild-type Treg cells (Fig. 8c,
left versus right), which suggested that Treg cells from the double-knockout mice were at a
competitive disadvantage in vivo. We confirmed the defective function of the residual Treg
cells in double-knockout mice with an in vitro suppression assay (Fig. 8d and Supplementary
Fig. 10 online). These data collectively suggest that loss of both STIM1 and STIM2 impaired
the development and function of Foxp3+ Treg cells.
DISCUSSION
Here we have examined the physiological functions of STIM1 and STIM2 in mice with
conditionally targeted alleles of Stim1 and Stim2. We found that STIM1 is a critical activator
of store-operated Ca2+ entry and the function of CRAC channels, in agreement with published
studies in which STIM1 was depleted by RNA-mediated interference9,10. In the absence of
STIM1, T cells and fibroblasts showed almost no Ca2+ influx in response to depletion of
endoplasmic reticulum Ca2+ stores. T cells from STIM1-deficient mice also lacked detectable
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ICRAC and consequently were severely compromised in their ability to produce the cytokines
IL-2, IFN-γ and IL-4 in response to TCR stimulation. These results are consistent with
published analyses of immunodeficient patients with a mutation in the gene encoding the
ORAI1 subunit of the CRAC channel11,21 and confirm the prevailing idea that STIM1 and
ORAI1 are critical components of the pathway of store-operated Ca2+ entry2,3. STIM1 is also
crucial in mediating store-operated Ca2+ entry, degranulation and cytokine production by mast
cells stimulated through the Fc receptor FcεRI (ref. 49).
Our work has also clarified the biological function of endogenous STIM2. STIM2-deficient
fibroblasts had much less store-operated Ca2+ entry, which indicated a ‘positive’ function for
STIM2 in this cell type. STIM2-deficient CD4+ T cells differentiated for 3–7 d in vitro in
nonpolarizing conditions consistently showed slightly less store-operated Ca2+ entry in
response to treatment with low-dose thapsigargin, ionomycin or anti-CD3. Furthermore,
STIM2 compensated partially for the absence of STIM1, as STIM1-deficient T cells and
fibroblasts regained a moderate degree of store-operated Ca2+ influx after ectopic expression
of STIM2. These data collectively show that endogenous STIM2 promotes Ca2+ influx in both
T cells and fibroblasts, consistent with published studies demonstrating coupling of both
STIM1 and STIM2 to store-operated Ca2+ entry in HeLa cells10,24.
The moderately diminished store-operated Ca2+ entry and CRAC channel function noted in
differentiated STIM2-deficient cells contrasts with their more notably diminished cytokine
expression. We traced the cytokine deficiency to the inability of STIM2-deficient cells to
sustain nuclear translocation of NFAT. Many studies have established that optimal cytokine
production by activated T cells requires sustained Ca2+ influx and prolonged nuclear residence
of NFAT5,8,50. Consistent with those findings, the amount of cytokines produced by wild-type,
STIM1-deficient and STIM2-deficient T cells correlated precisely with their ability to sustain
increased [Ca2+]i and to maintain NFAT for long periods in the nucleus. Wild-type T cells
retained substantial NFAT in the nucleus for more than 6 h during continuous stimulation and
had robust production of cytokines. STIM1-deficient T cells showed only a small early transient
increase in nuclear NFAT, which returned to baseline within 30 min even during continuous
stimulation, and they produced almost no cytokines. STIM2-deficient T cells had an almost
normal early phase of NFAT nuclear residence, which presumably reflected the residual
function of STIM1, but were unable to maintain nuclear translocation of NFAT to the extent
that wild-type T cells did and produced much smaller amounts of cytokines than did wild-type
cells. These findings are consistent with the demonstration that STIM2 is active at higher
endoplasmic reticulum Ca2+ concentrations than is STIM1 and therefore would maintain store-
operated Ca2+ entry and NFAT nuclear residence during the late stages of a response when
STIM1 has been inactivated by partial refilling of the endoplasmic reticulum stores24.
Mice with targeted deletion of both STIM1 and STIM2 in T cells had a notable
lymphoproliferative phenotype characterized by splenomegaly, lymphadenopathy, infiltration
of leukocytes into many organs and signs of autoimmunity (or infection secondary to immune
deficiency) in the form of blepharitis and dermatitis. This syndrome was associated with many
fewer Treg cells (approximately 10% of wild-type control numbers), which distinguished it
from other lymphopro-liferative disorders such as autoimmune lymphoproliferative syndrome
(mutation in the gene encoding the cytokine receptor Fas) and X-linked lymphoproliferative
disease (mutation in the gene encoding the adaptor protein SAP)51,52. Mixed—bone marrow
chimera experiments showed that this syndrome was also distinct from Treg cell deficiencies
arising from loss of IL-2 or transforming growth factor-β signaling33,34,53,54. In addition, both
in the mixed—bone marrow chimeras and in double-knockout mice that received wild-type
CD4+CD25+ Treg cells at a young age, the development of splenomegaly and lymphade-
nopathy was mostly prevented, which indicated that the small population of residual Treg cells
in double-knockout mice was functionally impaired. In support of that conclusion, double-
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knockout Treg cells were far less able than wild-type Treg cells to suppress the proliferation of
cocultured CD4+CD25– responder T cells in vitro. These findings indicate that store-operated
Ca2+ entry through the STIM-Orai1 pathway is essential for the development and function of
Treg cells.
We also found that Foxp3-expressing natural Treg cells were fully capable of activating store-
operated Ca2+ influx in response to CD3 crosslinking, which indicated that the steps of PLC-
γ1 activation, generation of inositol-1,4,5-trisphosphate, store-operated Ca2+ influx and
(presumably) nuclear translocation of NFAT were entirely functional in this cell type. On the
basis of those data and published studies indicating NFAT-Foxp3 ‘cooperation’ is essential for
the suppressive function of Foxp3-transduced T cells37,38, we propose that NFAT activation
and possibly NFAT-Foxp3 ‘cooperation’ are also essential for the development and function
of thymus-derived Treg cells. Indeed, it has been shown that when thymi from 1-week-old
heterozygous nude mice (nu/+), treated from birth with the NFAT inhibitor cyclosporin A, are
transplanted into recipient homozygous nude mice (nu/nu), the recipient mice develop diverse
organ-specific autoimmunity55. In this model, autoimmunity can be prevented by inoculation
of the recipient mice with suspensions of normal thymocytes, leading the authors of that study
to suggest that cyclosporin A interferes selectively with thymic production of ‘suppressor T
cells’ whose responsibility it is to control self-reactive T cells. The defect in Treg cell
development noted in that study and here could also reflect the reported ‘cooperation’ between
Smad3 and NFAT at an enhancer element in the Foxp3 locus35. It is notable that Treg cell
development is also impaired in mice with other mutations that affect TCR signaling, including
mice lacking Bcl-10, which show defective activation of NF-κB56.
Aspects of the lymphoproliferative phenotype of the double-knockout mice resembled the
phenotype of mice with other hypomorphic mutations in the Ca2+-NFAT pathway. For
example, mice with a Y136F substitution of the transmembrane adaptor Lat (which when
phosphorylated forms a docking site for PLC-γ1; refs. 57–59) also have a block in Treg cell
development but can be distinguished from STIM double-knockout mice in that they also have
a substantial block in the double-negative—to—double-positive stage of conventional T cell
development45,46. Similarly, mice lacking NFAT1 and NFAT4 show hyperproliferation and
hyperactivation of T cells and B cells60 but normal development and function of Treg cells;
indeed, T cells in these mice are not effectively inhibited by wild-type Treg cells61. Finally,
mice with early Lck-Cre—mediated deletion of the gene encoding calcineurin B1 in the thymus
show a block at the double-positive—to—single-positive stage of thymocyte development, but
autoimmune and/or hyperproliferative syndromes and Treg cell dysfunction have not been
reported62. Further studies are needed to elucidate the complex effects of mutations in the
Ca2+-calcineurin-NFAT signaling pathway on the development of conventional and regulatory
T cells.
METHODS
Animals and conditional gene targeting
Stim1 and Stim2 were targeted by homologous recombination in Bruce-4 embryonic stem cells
derived from C57BL/6 mice as described63. Chimeric mice with targeted Stim1 or Stim2 alleles
were generated by blastocyst injection of heterozygous Stim1neo/+ or Stim2neo/+ embryonic
stem cell clones (Supplementary Fig. 1; ‘neo’ indicates the neomycin-resistance gene).
Stim1–/– or Stim2–/– mice were generated by intercrossing of the progeny of founder
Stim1neo/+ or Stim2neo/+ mice after they were bred with CMV-Cre (‘Cre deleter’) transgenic
mice40. For establishment of Stim1+/- or Stim2+/- mice without the transgene encoding Cre,
Stim1+/- CMV-Cre+ mice or Stim2+/- CMV-Cre+ mice were bred with C57BL/6 mice. For
generation of the conditional Stim1fl/+ or Stim2fl/+ alleles, founder Stim1neo/+ or Stim2neo/+
chimeric mice were bred with ‘Flp deleter’ transgenic mice64 for removal of the neomycin-
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resistance cassette from the targeted Stim1 or Stim2 alleles. Rag1–/– and B6.Cg (Igha, Thy-1.1,
Gpi1a) mice were from the Jackson Laboratory. For the generation of mice with T cell—
specific disruption of Stim1 and/or Stim2, CD4-Cre transgenic mice41 were bred with each
founder Stim1fl/+ or Stim2fl/+ mouse and the progeny were intercrossed. All mice were
maintained in specific pathogen—free barrier facilities at Harvard Medical School and were
used in accordance with protocols approved by the Center for Animal Resources and
Comparative Medicine of Harvard Medical School.
T cell differentiation, retroviral transductions and stimulation
Purification of CD4+ T cells from spleen and lymph nodes, induction of T helper cell
differentiation, stimulation with 10 nM PMA and 1 μM ionomycin or with plate-bound anti-
CD3 (purified from supernatants of the 2C11 hybridoma) and anti-CD28 (37.51; BD
Pharmingen), and assessment of cytokine production by intracellular staining and flow
cytometry were done as described65. Foxp3 expression was assessed by intracellular staining
with anti-Foxp3 (FJK-16s; eBioscience) according to the manufacturer’s protocol and was
analyzed by flow cytometry. Retroviral transduction was done as described37 with KMV
retroviral expression plasmids (a modified moloney murine leukemia virus vector), either
empty or containing Stim1 or Stim2 cDNA, followed by cDNA encoding green fluorescent
prptein (GFP) under control of an internal ribosome entry site. Despite their defect in Ca2+
influx, STIM1-deficient T cells upregulated CD25 (IL-2 receptor α-chain) normally. Thus,
because differentiating cultures were maintained in IL-2, almost equivalent cell numbers were
recovered (total number of differentiated STIM1-deficient cells at 7 d was 60–70% that of
wild-type) and STIM-deficient cells could be retrovirally transduced with the same efficiency
as wild-type cells.
Establishment of MEF cell lines
Stim1-/- and Stim2-/- MEFs were established with standard protocols from embryos at
embryonic day 14.5 obtained by intercrossing of Stim1+/- or Stim2+/- mice. MEFs were
immortalized by retroviral transduction with SV40 large T antigen in a plasmid carrying the
hygromycin-resistance gene, followed by hygromycin selection.
Antibodies and immunoblot
Cell extracts were prepared by resuspension of cells in PBS, followed by lysis in a buffer of
50 mM NaCl, 50 mM Tris-HCl, pH 6.8, 2% (wt/vol) SDS and 10% (vol/vol) glycerol (final
concentrations). Protein concentrations were determined with the BCA Protein Reagent kit
(Pierce), then 2-mercaptoethanol was added to a final concentration of 100 μM and samples
were boiled. Standard protocols were used for immunoblot analysis. Polyclonal anti-STIM1
(Open Biosciences) was generated against a carboxy-terminal peptide of human STIM1
(CDNGSIGEETDSSPGRKKFPLKIFKKPLKK-COOH, in which the cysteine at the amino
terminus was introduced for the purpose of coupling the peptide with a carrier protein) and
was used at a dilution of 1:2,000. Affinity-purified polyclonal antibodies were generated
against a carboxy-terminal peptide of human STIM2 (CKPSKIKSLFKKKSK, in which the
cysteine at the amino terminus was introduced for the purpose of coupling the peptide with a
carrier protein) and were used at a concentration of 2 μg/ml. Polyclonal anti-actin (I-19;
SC-1616; Santa Cruz) was used at a dilution of 1:500. Monoclonal antibody to the Myc epitope
tag was purified from supernatants of 9E10 hybridoma cell lines. All of the following
fluorescence-conjugated antibodies used for flow cytometry were from eBioscience or BD
Pharmingen: Pacific blue—conjugated anti-CD4 (RM4-5); fluorescein isothiocyanate—
conjugated anti-CD8 (53-6.7), anti-Thy-1.1 (HIS51), anti-IgE (R35-72) and anti-CD11c
(M1/70); phycoerythrin-conjugated anti-IL-2 (JES6-5HA), anti-IL-4 (11B11), anti-Foxp3
(FJK- 16s), anti-CD19 (1D3), anti-CD125 (T21.2), anti-CD44 (IM7) and anti-CD95 (Jo2);
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peridinine chlorophyll protein complex—conjugated anti-B220 (RA3-6B2); peridinine
chlorophyll protein complex—cyanine 5.5—conjugated anti-CD4 (RM4-5); phycoerythrin-
indotricarbocyanine—conjugated anti-Thy-1.2 (53-2.1); allophycocyanin-conjugated anti-
IFNγ (XMG1.2), anti-IL-10 (JES5-16E3), anti-body to tumor necrosis factor (anti-TNF; MP6-
XT22), anti-CD25 (PC61.5), anti-TCRβ (H57-597), anti-CD38 (90) and anti-CD62L
(MEL-14); biotinylated anti-CD5 (53-7.3) and anti-CD69 (H1.2F3); and phycoerythrin-
streptavidin.
Single-cell [Ca2+]i imaging
CD4+ T cells were isolated as described65, were incubated overnight in loading medium (10%
(vol/vol) FBS in RPMI 1640 medium) and were loaded for 30 min at 22–25 °C with calcium
indicator Fura-2-AM (1 μM; Invitrogen) at a density of 1 × 106 cells per ml. Before
measurements, T cells were attached for 15 min to poly-L-lysine-coated cover-slips. For anti-
CD3 stimulation, T cells were incubated for 15 min at 22–25 °C with biotin-conjugated anti-
CD3 (5 μg/ml; 2C11; BD Pharmingen), and anti-CD3 crosslinking was achieved by perfusion
of cells with streptavidin (10 μg/ml; Pierce). For long-term Ca2+ imaging, differentiated
CD4+ T cells were loaded with 1 μM Fura-PE3, then were stimulated with 10 nM PMA and
0.5 μM ionomycin in Ringer’s solution containing 2 mM Ca2+ supplemented with 2% (vol/
vol) FCS. During image acquisition, cells were constantly perfused with buffer warmed to 37
°C. [Ca2+]i was measured and analyzed as described11. Ca2+ influx rates were inferred from
the maximum rate of the initial increase in [Ca2+]i in 0.2–2 mM extracellular Ca2+, expressed
as the ratio ‘Δ[Ca2+]i/Δt’, where ‘Δ[Ca2+]i’ is the maximum difference in [Ca2+]i over a 20-
second time interval (Δt) between the readdition of extracellular Ca2+ and the peak of the
Ca2+ influx response. For each experiment, 100–150 T cells or at least 30 individual MEFs
were analyzed with Igor Pro analysis software (Wavemetrics).
NFAT1 nuclear-translocation assay
CD4+ T cells were cultured in nonpolarizing conditions and were collected at day 5, then were
stimulated for various times with 10 nM PMA plus 1 μM ionomycin at a density of 1 × 105
cells per well in a volume of 200 μl in 96-well plates and were attached to poly-L-lysine-coated
wells in 384-well plates (5 × 103 to 8 × 103 cells per well; three wells per sample) by
centrifugation for 3 min at 149g. Cells were fixed with 3% (vol/vol) paraformaldehyde then
were stained with anti-NFAT1 (purified rabbit polyclonal antibody to the ‘67.1’ peptide of
NFAT1)66 and indocarbocyanine-conjugated anti-rabbit (secondary antibody) and
counterstained with the DNA-intercalating dye DAPI (4,6-diamidino-2-phenylindole). Images
were acquired with the ImageXpress Micro automated imaging system (Molecular Devices)
with a 20 × objective and were analyzed with the translocation application module of
MetaXpress software version 6.1 (Molecular Devices). Cytoplasmic-to-nuclear translocation
was assessed by calculation of a correlation of the intensity of indocarbocyanine—anti-NFAT1
staining and DAPI staining; T cells were considered to have nuclear NFAT1 when over 90%
of the indocarbocyanine—anti-NFAT1 staining coincided with the fluorescence signal from
DAPI. Each data point represents an average of at least 300 individual cells per well.
Patch-clamp measurements
CD4+ T cells were cultured in nonpolarizing conditions and were collected at day 5. An
Axopatch 200 amplifier (Axon Instruments) interfaced to an ITC-18 input/output board
(Instrutech) and an iMac G5 computer were used for patch-clamp recordings. Currents were
filtered at 1 kHz with a four-pole Bessel filter and were sampled at 5 kHz. Recording electrodes
were ‘pulled’ from 100-μl pipettes, were coated with Sylgard and were fire-polished to a final
resistance of 2–5 MΩ. ‘In-house’ routines developed on the Igor Pro platform (Wavemetrics)
were used for stimulation and data acquisition and analysis. All data were corrected for the
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liquid-junction potential of the pipette solution relative to that of Ringer’s solution in the bath
(–10 mV) and for leak currents collected in 20 mM extracellular Ca2+ plus 25 μM La3+. The
standard extracellular Ringer’s solution was 130 mM NaCl, 4.5 mM KCl, 20 CaCl2, 1 mM
MgCl2, 10 mM D-glucose and 5 mM Na-HEPES, pH 7.4. In some experiments, 2 mM
CaCl2 was used in the standard extracellular solution and the NaCl concentration was increased
to 150 mM. Standard divalent cation—free Ringer’s solutions were 150 mM NaCl, 10 mM
tetraacetic acid, 1 mM EDTA and 10 mM HEPES, pH 7.4. Charybdotoxin (25 nM; Sigma)
was added to all extracellular solutions to eliminate contamination from Kv1.3 channels. The
standard internal solution was 145 mM cesium aspartate, 8 mM MgCl2, 10 mM BAPTA (1,2-
bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid) and 10 mM Cs-HEPES, pH 7.2.
Averaged results are presented as the mean value ± s.e.m. Curve fitting was done by the least-
squares methods with built-in functions in Igor Pro 5.0. The permeability of Cs+ relative to
that of Na+ was calculated from the bionic reversal potential with the equation
where PCs and PNa are the permeability of Cs+ (the test ion) and Na+, respectively; [Cs]i and
[Na]o are the ionic concentrations; Erev is the reversal potential; F is the Faraday constant (9648
C mol–1); R is the gas constant (8.314 J K–1 mol–1); and T is the absolute temperature.
Hematoxylin and eosin staining
Tissues from 3- to 4-month-old mice were fixed in 10% (vol/vol) formalin. A standard
procedure was used for hematoxylin and eosin staining.
Purification and adoptive transfer of Treg cells
CD4+CD25+ and CD4+CD25– T cells derived from Thy-1.1+ congenic mice were sorted with
a FACSVantage after purification of CD4+ T cells with Dynabeads Mouse CD4 and DETA-
CHaBEAD Mouse CD4 (Invitrogen). Wild-type Thy-1.1+ CD4+CD25+ or CD4+CD25– T cells
(3 × 105) were injected intraperitoneally into 2-week-old mice. Injected mice were analyzed
at 8 weeks after adoptive transfer.
Mixed—bone marrow transfer
T cell—depleted bone marrow from Thy-1.2+ double-knockout mice (3 × 106 cells) was mixed
with that of Thy-1.1+ congenic wild-type mice (1.5 × 106 cells) and the mixture was injected
through the retro-orbital sinus into sublethally irradiated Rag1–/– mice (450 rads).
Reconstituted mice were analyzed 10–12 weeks after bone marrow transfer.
Proliferation and in vitro suppression assays
CD4+CD25– or CD4+CD25+ T cells were positively selected with MACS CD25 microbeads
(Miltenyi Biotec) after purification of CD4+ T cells. Purified CD4+CD25– T cells (2 × 107
cells per ml) were incubated for 10 min at 37 °C with 1.25 μM CFSE (carboxyfluorescein
diacetate succinimidyl diester). Cells were stimulated for 72 h with anti-CD3 and anti-CD28
and the number of cell divisions was assessed by flow cytometry. In vitro suppression assays
were done by coculture of 5 × 104 CFSE-labeled CD4+CD25– T cells at various ratios with
CD4+CD25+ T cells purified from control littermates or double-knockout mice; cells were
cocultured for 72 h at 37 °C in round-bottomed plates in the presence of mitomycin C—treated
T cell—depleted splenocyte samples (5 × 104 cells) and anti-CD3 (0.3 μg/ml; 2C11).
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Accession code
UCSD-Nature Signaling Gateway (http://www.signalinggateway.org): A000024.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
We thank K. Rajewsky and members of the Rajewsky lab for help with blastocyst injection of embryonic stem cells;
M.E. Pipkin and A.Y. Rudensky for comments and discussions; Y. Gwack for purification of anti-STIM2; and B.
Baust for help in establishing the NFAT-translocation assay. Supported by the National Institutes of Health (A.R.,
S.F. and M.P.), Juvenile Diabetes Research Foundation (A.R.), March of Dimes Foundation (S.F.), Uehara Memorial
Foundation (M.O.) and Canadian Institutes of Health Research (S.S.).
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Figure 1.
STIM1 is a predominant effector of store-operated Ca2+ entry into T cells. (a) Store-operated
Ca2+ influx in littermate control (Stim1+/+ CD4-Cre or Stim1fl/fl) (black lines) and Stim1fl/fl
CD4-Cre (gray lines) naive CD4+ T cells in response to 1 μM thapsigargin (TG; left) or
crosslinking with anti-CD3 (α-CD3) followed by 1 μM ionomycin (Iono; right) in the presence
of 0.2 or 2 mM extracellular Ca2+. (b) IL-2 production by naive CD4+ T cells stimulated for
6 h with PMA and ionomycin, assessed by intracellular cytokine staining. CTRL, control
(Stim1+/+ CD4-Cre or Stim1fl/fl). (c) Store-operated Ca2+ entry in response to 1 μM thapsigargin
(top) or crosslinking with anti-CD3 followed by 1 μM ionomycin (center and bottom) in naive
CD4+ T cells from wild-type mice (black lines) and Stim2–/– mice (gray lines), both obtained
by intercrossing of Stim2+/- CMV-Cre– mice. (d) IL-2 production by naive wild-type (WT)
and Stim2–/– CD4+ T cells stimulated for 6 h with PMA and ionomycin, assessed by
intracellular cytokine staining. (e) [Ca2+]i responses of control (Stim2+/+ CD4-Cre or
Stim2fl/fl; black lines) and Stim2–/– (gray lines) helper T cells differentiated for 7 d in vitro in
nonpolarizing conditions, in response to high (1 μM) or low (10 nM) concentrations of
thapsigargin or anti-CD3 followed by ionomycin. (f) Production of IL-2 and IFN-γ by wild-
type and Stim2–/– helper T cells differentiated for 7 d in vitro in nonpolarizing conditions, then
restimulated for 6 h with PMA and ionomycin. T cells from Stim1+/+ or Stim2+/+ CD4-Cre
mice were compared with T cells from Stim1fl/fl or Stim2fl/fl mice in initial experiments to
confirm that Cre expression had no toxic or other deleterious effects on proliferation or cytokine
expression; in subsequent experiments, Stim1+/+ CD4-Cre and Stim1fl/fl mice or Stim2+/+ CD4-
Cre and Stim2fl/fl mice, respectively, were used interchangeably as controls. Data are
representative of at least three independent experiments.
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Figure 2.
Both STIM1 and STIM2 reconstitute store-operated Ca2+ entry and cytokine production in
STIM1-deficient T cells and MEFs. (a) Ca2+ influx into wild-type, Stim1–/– and Stim2–/– MEFs
stimulated with 1 μM thapsigargin in Ringer’s solution containing 20 mM Ca2+ (top), and
reconstitution of store-operated Ca2+ entry by retroviral transduction of Stim1–/– MEFs with
Myc-tagged STIM1 or STIM2 or empty vector (bottom). Expression vectors contain an internal
ribosome entry site–GFP cassette and only GFP+ cells are analyzed here. (b) Ca2+ influx in
response to treatment with 1 μM thapsigargin in control (Stim1+/+ CD4-Cre or Stim1fl/fl) and
Stim1fl/fl CD4-Cre helper T cells differentiated for 7 d in vitro in nonpolarizing conditions and
transduced with retroviral vector encoding Myc-tagged STIM1 or STIM2 or empty vector (only
GFP+ cells are analyzed). (c) Flow cytometry (left) and averaged data (right) of cytokine
production by control and Stim1fl/fl CD4-Cre cells transduced with retroviral vectors (right
margin and below bars); only GFP+ cells are analyzed. Numbers in quadrants (left) indicate
percent cells in each. Stim1+/+ or Stim2+/+ CD4-Cre mice were used initially as controls, after
which both Stim1+/+ CD4-Cre and Stim1fl/fl mice or Stim2+/+ CD4-Cre and Stim2fl/fl mice,
respectively, were used. Data are representative of three independent experiments (error bars,
s.d.).
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Figure 3.
Loss of STIM2 affects sustained Ca2+ influx and the late phase of NFAT1 nuclear localization.
(a) Averaged peak and steady-state (60 min after stimulation) [Ca2+]i and initial rates of change
in [Ca2+]i in control CD4+ T cells (CTRL; n = 8; Stim2+/+ CD4-Cre or Stim2fl/fl) or STIM2-
deficient CD4+ T cells (2KO; n = 7; Stim2fl/fl CD4-Cre) differentiated for 5 d in nonpolarizing
conditions, labeled with Fura-PE3 and stimulated with PMA and ionomycin. P values, unpaired
Student’s t-test. (b,c) STIM1-deficient (b), STIM2-deficient (c) and control cells with nuclear
NFAT1 after stimulation as described in a, calculated with the data in Supplementary Figure
4. (d,e) Expression of IL-2 and IFN-γ by the cells in b (d) and c (e). In b,d, control is
Stim1+/+ CD4-Cre or Stim1fl/fl; in c,e, control is Stim2+/+ CD4-Cre or Stim2fl/fl. Stim1+/+ or
Stim2+/+ CD4-Cre mice were used initially as controls, after which both Stim1+/+ CD4-Cre and
Stim1fl/fl mice or Stim2+/+ CD4-Cre and Stim2fl/fl mice, respectively, were used. Data are
representative of three independent experiments (error bars, s.e.m. (a) or s.d. (b,c)), with at
least 300 cells per well analyzed for each time point (b–e).
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Figure 4.
STIM1-deficient but not STIM2-deficient T cells lack ICRAC. (a) Current-voltage relations
recorded in individual differentiated CD4+ T cells in 20 mM extracellular Ca2+ or divalent
cation—free solution (DVF), assessing the Ca2+ current (left) and monovalent cation current
(right) elicited by 1 μM thapsigargin. (b) Single-cell recordings of depotentiating Na+ current
elicited by replacement of 20 mM Ca2+ in Ringer’s solution (filled bars) with divalent cation
—free solution (open bars), measured during hyperpolarizing pulses to –100 mV applied every
1 s. (c) Summary of peak current densities averaged over all cells analyzed in a,b (n, number
of cells). CTRL, Stim1+/+ CD4-Cre, Stim1fl/fl or Stim2fl/fl, or Stim1fl/flStim2fl/fl; STIM1-KO,
Stim1fl/fl CD4-Cre; STIM2-KO, Stim2fl/fl CD4-Cre; DKO, Stim1fl/flStim2fl/fl CD4-Cre.
Stim1+/+ or Stim2+/+ CD4-Cre mice were used initially as controls, after which both Stim1+/+
CD4-Cre and Stim1fl/fl mice or both Stim2+/+ CD4-Cre and Stim2fl/fl mice, respectively, were
used. Data are representative of individual single-cell measurements (a,b) or are the mean ±
s.e.m. of all single-cell measurements (c).
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Figure 5.
Impaired Ca2+ influx, cytokine production and proliferation in double-knockout T cells. (a)
Store-operated Ca2+ influx in naive CD4+ T cells from littermate control mice (CTRL;
Stim1fl/flStim2fl/fl) and double-knockout mice (DKO; Stim1fl/flStim2fl/fl CD4-Cre), stimulated
with thapsigargin (top) or with anti-CD3 followed by crosslinking with streptavidin (SA;
bottom) in nominally Ca2+-free Ringer’s solution, followed by perfusion with 2 mM Ca2+ in
Ringer’s solution to induce Ca2+ influx (left). Middle, quantification of peak [Ca2+]i in 2 mM
Ca2+ in Ringer’s solution. Right, single-cell ‘false-color’ images showing [Ca2+]i at the peak
of the Ca2+ influx response. Original magnification, ×20. (b) Production of IL-2 and tumor
necrosis factor (TNF) by naive CD4+ T cells from control (Stim1+/+ CD4-Cre or
Stim1fl/flStim2fl/fl) or double-knockout mice, stimulated for 6 h with PMA and ionomycin.
(c) Expression of CD25 and CD69 on naive CD4+ T cells from control (Stim1+/+CD4-Cre or
Stim1fl/flStim2fl/fl) or double-knockout mice, left unstimulated (Unstim; control cells) or
stimulated for 16 h with anti-CD3 and anti-CD28 (control (CTRL) or double-knockout (DKO)
cells). (d) Proliferation of naive CD4+CD25– T cells from control (Stim1fl/flStim2fl/fl) or
double-knockout mice, stimulated for 72 h with anti-CD3 and anti-CD28 and assessed by CFSE
labeling. Open histograms, stimulated cells; shaded histograms, unstimulated cells. Numbers
above bracketed lines indicate number of cell divisions. (e) Cells undergoing zero to five cell
divisions (from data in d). Stim1+/+ or Stim2+/+ CD4-Cre mice were used initially as controls,
after which Stim1+/+ CD4-Cre, Stim2+/+ CD4-Cre and Stim1fl/flStim2fl/fl mice were used. Data
are representative of three (a) or two (b—d) independent experiments (error bars (a), s.e.m.).
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Figure 6.
Double deficiency in STIM1 and STIM2 disrupts peripheral T cell homeostasis. (a)
Hematoxylin and eosin staining of lung sections from a 4-month-old control mouse
(Stim1fl/flStim2fl/fl) and a double-knockout mouse (Stim1fl/flStim2fl/fl CD4-Cre). Scale bar, 100
μm. (b) Staining of CD4, CD25 and Foxp3 in thymocytes and splenocytes from 5- to 6-week-
old (left) or 3-month-old (right) control mice (Stim1+/+ CD4-Cre or Stim1fl/flStim2fl/fl) and
double-knockout mice. Numbers above outlined areas indicate percent cells in gate. (c) Total
cells (left) and CD4+CD25+ Treg cells (right) in thymi and spleens of 5- to 6-week-old control
mice (Stim1+/+ CD4-Cre or Stim1fl/flStim2fl/fl) or double-knockout mice (n = 3 mice). (d) Peak
Ca2+ response induced by CD3 crosslinking (left) or treatment with 1 μM thapsigargin (right)
in CD4+CD25+ cells isolated from control (Stim1fl/flStim2fl/fl) or double-knockout mice.
Measurements were made in Ringer’s solution with 2 mM Ca2+. Stim1+/+ or Stim2+/+ CD4-
Cre mice were used initially as controls to confirm that Cre expression did not result in toxicity
or other adverse effects, after which Stim1fl/flStim2fl/fl mice were used to control for other
factors, such as sex, age and breeding environment. Data are representative of results from two
independent experiments with three control or double-knockout mice (a); three independent
experiments (b,c; mean ± s.d., c); or three (anti-CD3) and two (thapsigargin) independent
experiments with 110–170 cells analyzed in each (d).
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Figure 7.
Absence of STIM1 and STIM2 impairs the development of Treg cells. Flow cytometry of
thymus and lymph node cells from sublethally irradiated Rag1–/– mice reconstituted with T
cell—depleted bone marrow from Thy-1.2+ control littermates (Stim1fl/flStim2fl/fl) alone (3 ×
106 cells), from Thy-1.2+ double-knockout mice alone (3 × 106 cells), or from both
Thy-1.2+ double-knockout mice (3 × 106 cells) and congenic B6 Thy-1.1+ wild-type mice (WT;
1.5 × 106 cells), stained with anti-CD4, anti-Thy-1.1 and anti-Thy-1.2, together with anti-CD25
and anti-Foxp3, at 10–12 weeks after reconstitution. Numbers in quadrants and above outlined
areas indicate percent cells in each. SP, single-positive. As no Cre toxicity was noted in
previous experiments, Stim1fl/flStim2fl/fl mice were used as controls to control for other factors,
such as sex, age and breeding environment. Data are representative of results from two
independent experiments with three mixed chimeric mice.
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Figure 8.
Adoptive transfer of wild-type Treg cells suppresses the lymphoproliferative phenotype of
double-knockout mice. (a–c) CD4+CD25+ (CD25+) or CD4+CD25– (CD25–) Thy-1.1+ T cells
from wild-type mice (3 × 105 cells) were transferred into 2-week-old Thy-1.2+ double-
knockout mice, and cells in recipient mice were analyzed 8 weeks after transfer. CTRL, control
littermates (Stim1fl/flStim2fl/fl) injected with PBS; PBS, double-knockout mice injected with
PBS; CD25–, double-knockout mice injected with CD25– cells; CD25+, double-knockout mice
injected with CD25+ cells. (a) Spleen and lymph node (LN) cells from recipient mice. P values,
paired Student’s t-test. (b) Antibody staining of spleen and lymph node cells from recipient
mice. Numbers above outlined areas indicate percent cells in gate. (c) Staining of cells with
anti-Thy-1.1, anti-Thy-1.2, anti-CD4, anti-CD25 and anti-Foxp3 for analysis of donor cell
engraftment, presented as endogenous Treg cells (endog; Thy-1.2+) and transferred Treg cells
(transf; Thy-1.1+) in mice that received CD25– (Non) or CD25+ T cells. (d) In vitro suppression
assay of CD4+CD25+ T cells purified from control (Stim1fl/flStim2fl/fl) or double-knockout
mice and cultured for 72 h at a ratio of 1:1 with CFSE-labeled responder CD4+CD25– T cells
in the presence of mitomycin C–treated T cell—depleted splenocyte samples and anti-CD3
(0.3 μg/ml). As no Cre toxicity was noted in previous experiments, Stim1fl/flStim2fl/fl mice
were used as controls to control for other factors, such as sex, age and breeding environment.
Data are representative of results from two independent experiments with three mice (a–c;
error bars (a,c), s.d.), or at least three independent experiments (d).
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