PTPN22 Deficiency Cooperates with the CD45 E613R Allele to
Break Tolerance on a Non-Autoimmune Background1
Julie Zikherman,*†‡Michelle Hermiston,§David Steiner,?Kiminori Hasegawa,#Andrew Chan,#
and Arthur Weiss2*†‡¶
Pep and CD45 are tyrosine phosphatases whose targets include the Src-family kinases, critical mediators of Ag receptor signaling.
A polymorphism in PTPN22, the gene that encodes the human Pep orthologue Lyp, confers susceptibility to multiple human
autoimmune diseases in the context of complex genetic backgrounds. However, the functional significance of the R620W risk allele
is not clear. We report that misexpression of wild-type or R620W Pep/Lyp in Jurkat cells, in the context of its binding partner
Csk, unmasks the risk allele as a hypomorph. It has been shown previously that although Pep-deficient mice on the B6 background
have hyperresponsive memory T cells, autoimmunity does not develop. Mice containing a point mutation in the CD45 juxtamem-
brane wedge domain (E613R) develop a B cell-driven, lupus-like disease on the mixed 129/B6 background, but not on the B6
background. We studied the ability of Pep deficiency to act as a genetic modifier of the CD45 E613R mutation on the nonauto-
immune B6 background to understand how complex susceptibility loci might interact in autoimmunity. In this study we report
that double mutant mice develop a lupus-like disease as well as lymphadenopathy, polyclonal lymphocyte activation, and accel-
erated memory T cell formation. Following Ag receptor stimulation, peripheral B cells in the double mutant mice phenocopy
hyperresponsive CD45 E613R B cells, whereas peripheral T cells respond like Pep?/?T cells. These studies suggest that Pep?/?
T cells in the context of a susceptible microenvironment can drive hyperresponsive CD45 E613R B cells to break tolerance. The
Journal of Immunology, 2009, 182: 4093–4106.
relative risk. Modeling and understanding the mechanisms that un-
derlie such interactions in a tractable genetic system such as the
mouse has been challenging.
A polymorphism in the human gene PTPN22 (C1858T/R620W)
confers risk for developing multiple autoimmune diseases, includ-
ing rheumatoid arthritis (RA),3systemic lupus erythematosus
(SLE), and type 1 diabetes (1–3). Indeed, PTPN22 R620W was the
second most significant risk allele identified in two unbiased whole
genome scans for RA, although the odds ratio associated with car-
rying the risk allele is quite modest, with a value of ?2 (4–6).
Nevertheless, human autoimmune disease has a striking genetic
contribution; RA heritability has been estimated at 60% (7).
Clearly, even potent susceptibility loci must interact with one an-
uman autoimmune diseases arise from environmental
challenge in the context of cooperating genetic suscep-
tibility loci, each of which independently confers a small
other to account for this phenomenon. How then can this complex
process be effectively modeled and studied?
PTPN22 encodes Lyp, a hematopoietic phosphatase, the mouse
homologue of which is Pep (PEST domain-enriched tyrosine phos-
phatase) (8, 9). Pep/Lyp negatively regulates TCR signaling by
dephosphorylating the activating tyrosine of the Src family kinase
(SFK) Lck (10, 11). SFKs are critical mediators of signal trans-
duction by ITAM-bearing immunoreceptors such as the TCR (12).
Pep is cytoplasmic but is brought into proximity of its target,
coreceptor-associated Lck, at the plasma membrane via its consti-
tutive association with the cytoplasmic tyrosine kinase Csk (10,
13). Csk phosphorylates the inhibitory tyrosine of the SFKs and is
itself a potent negative regulator of TCR signaling (14). Csk is
recruited to the membrane by the transmembrane adaptor PAG
(phosphoprotein associated with glycosphingolipid-enriched mi-
crodomains), which is phosphorylated under basal conditions (15,
16). Following TCR ligation, PAG is rapidly dephosphorylated,
releasing Csk from the membrane (16). Pep and Csk interact func-
tionally as well as physically to cooperatively inhibit TCR signal-
ing (10, 11). This cooperative inhibition has been shown to depend
upon the C-terminal proline-rich sequence (PRS) of Pep and the
SH3 domain of Csk (10). The PRS of Pep (613–620 PPPLPERT),
containing the critical R620 (murine homolog R619) residue
(boldface and italics), is absolutely required for this association
and for the cooperative inhibition of TCR signaling (13).
To complement in vitro overexpression studies, Pep-deficient
(Pep?/?) mice provide in vivo support for a negative regulatory
role in TCR signaling (17). The phenotype is, however, surpris-
ingly subtle; TCR signaling is enhanced at the CD4?CD8?(dou-
ble positive) stage of thymocyte development and in the effector/
memory compartment of peripheral T cells, but naive T cells and
B cells appear to have no functional abnormality. Although
Pep?/?mice develop an expanded effector/memory T cell com-
partment and increased numbers of spontaneous germinal centers,
*Division of Rheumatology,†Rosalind Russell Medical Research Center for Ar-
thritis,‡Department of Medicine,§Department of Pediatrics,¶Howard Hughes
Medical Institute, and?Medical Scientist Training Program, University of Cali-
fornia, San Francisco, CA 94143; and#Department of Immunology, Genentech,
Inc., South San Francisco, CA 94080
Received for publication October 3, 2008. Accepted for publication January 27, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by National Institutes of Health Grant POI AI
035297 (to A.W. and M.H.) and by a grant from the Arthritis Foundation (to J.Z.).
2Address correspondence and reprint requests to Dr. Arthur Weiss, Department of
Medicine, Division of Rheumatology, Department of Microbiology and Immunology,
513 Parnassus Avenue, Room S-1032C, Box 0795, San Francisco, CA 94143-0795.
E-mail address: email@example.com
3Abbreviations used in this paper: RA, rheumatoid arthritis; DP, double positive; FO,
follicular; LN, lymph node; PRS, proline-rich sequence; SFK, Src family kinase;
SLE, systemic lupus erythematosus; WT, wild type.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
no autoantibodies or frank autoimmune diseases are observed on
the B6 genetic background. One possible explanation for this sub-
tle phenotype is redundancy with the ubiquitously expressed, re-
lated phosphatase PTP-PEST (18).
The R620W polymorphism partially disrupts the association
of Pep/Lyp with Csk (1, 19). These data are consistent with a
critical role for the PRS domain and suggest that the R620W
allele might represent a hypomorphic variant. However, evi-
dence from genetic studies does not clearly identify either a
pure recessive or dominant effect of the polymorphism in RA
(heterozygous odds ratio 1.98; homozygous odds ratio 3.32)
(4). Relatively few in vitro functional studies of the polymor-
phism have been reported. In a Jurkat overexpression study, the
R620W allele of Lyp exerted a more potent inhibition of TCR
signaling than wild type (19). Importantly, the polymorphism
was not studied in the context of its binding partner Csk to
identify its effect on the synergistic inhibition of TCR signaling.
More recently, another group has studied the functional conse-
quences of the R620W variant directly in primary human T cells
from heterozygous and homozygous carriers of the polymor-
phism. These studies found impaired calcium flux and up-reg-
ulation of activation markers in response to TCR ligation in
peripheral blood memory T and B cells. Notably, naive T cell
function was normal and no significant differences in IL-2 pro-
duction or proliferation were detectable (20). Three additional
studies in which primary PBMCs from patients with type I di-
abetes or myasthenia gravis who carry the R620W allele have
been subjected to functional assays report mixed results (19, 21,
The functional consequences of the R620W polymorphism, the
mechanism by which PTPN22 acts as an autoimmune susceptibil-
ity locus, and the genetic contexts in which this might occur re-
main unclear. Given the significance of PTPN22 for human auto-
immunity, we sought to establish systems in which we could
model and study the risk allele in vitro in the context of Csk and
in vivo in the context of a permissive genetic background. To this
end, we studied coexpression of Csk and Pep as well as Lyp vari-
ants in Jurkat cells. To model the genetic cooperation of PTPN22
with other autoimmune susceptibility loci, we took advantage of
CD45 E613R mice in which hyperresponsive B cells, characteris-
tic of human and murine SLE, drive a lupus-like disease only on
certain genetic backgrounds.
CD45 is a receptor-like tyrosine phosphatase expressed at
high levels on all nucleated hematopoietic cells (23). CD45
deficiency in humans and mice leads to SCID phenotypes, re-
vealing a crucial positive role for CD45 in immunoreceptor
signaling and lymphocyte development (24–27). Polymor-
phisms in CD45 that influence regulated splicing are associated
with autoimmune disease in humans (28). CD45 dephosphory-
lates the negative regulatory tyrosine of SFKs, thereby “prim-
ing” cells to respond to signals through immunoreceptors (23).
This positive regulatory role is normally counterbalanced by the
cooperating module of Csk/Pep.
Our laboratory previously identified a critical membrane prox-
imal residue (E613) in the cytoplasmic domain of CD45 that me-
diates dimerization-induced inhibition of phosphatase activity.
Mutation of this site to an arginine ablates such inhibition (29, 30).
Further support for the critical regulatory role of this residue was
provided by the E613R “wedge” (CD45w/w) knockin mouse that
developed lymphoproliferation, polyclonal lymphocyte activation,
autoantibodies, and immune complex glomerulonephritis, a phe-
notype reminiscent of human SLE (31). Genetic deletion of B cells
revealed that the lymphoproliferation was B cell driven (32). At
the cellular level, B cells were extremely hyperresponsive to BCR
signaling, a characteristic shared with spontaneous and engineered
mouse models of lupus as well as B cells from patients with SLE
However, further backcrossing of the original knockin mouse
revealed a remarkable background dependence of the disease
phenotype (M. Hermiston, V. Lam, R. Mills, N. Oksenberg, N.
Cresalia, A. Tam, M. Anderson, and A. Weiss, manuscript in
preparation). B cell hyperresponsiveness was noted on all back-
grounds, but the original disease phenotype was recapitulated
only on the B6/129 F1background. Indeed, CD45 E613R mice
on the pure B6 background develop no autoantibodies or end-
organ disease. Therefore, CD45 E613R effectively functions as
a murine autoimmune susceptibility allele in which “lupus-like”
B cells cooperate with other factors. This result suggested that
the CD45w/wB6 mouse might provide a suitable background
against which to study candidate genetic modifiers such as
In this study we report that misexpression of wild-type (WT)
and R620W Pep and Lyp alleles in the context of Csk in Jurkat
cells unmasks R620W as a hypomorphic allele. We took advantage
of Pep-deficient mice as a model of the hypomorphic human risk
allele, albeit a more extreme allele. We crossed the Pep?/?mice
onto the nonautoimmune CD45w/wB6 background to model how
subtle susceptibility loci might cooperate in human autoimmune
disease. We found that these alleles cooperate to break tolerance
on the nonautoimmune B6 genetic background. Indeed, double
mutant CD45w/w/Pep?/?mice develop lymphadenopathy and
splenomegaly, autoantibodies, glomerulonephritis, and premature
mortality. The cellular phenotype is characterized by polyclonal B
and T cell activation as well as early and progressive expansion of
the memory T cell compartment. Studies of Ag receptor signal
transduction revealed that peripheral B cells in double mutant
mice respond like single mutant CD45w/wB cells, whereas pe-
ripheral T cells resemble single mutant Pep?/?T cells. In con-
trast, thymic signaling reflects cooperation between the two mu-
tant alleles. These studies suggest that Pep?/?T cells in the
context of a susceptible microenvironment drive CD45w/wB
cells to break tolerance.
Materials and Methods
CD45 E613R (CD45w/w) mice were generated as previously described
(31). All experiments described in this manuscript involve mice back-
crossed at least nine generations onto the B6 background. Pep?/?mice
(17) were originally generated on a pure B6 background and obtained from
A. Chan (Genentech). DNase1?/?mice (36) were backcrossed for at least
nine generations onto the B6 background and were obtained from T. Mo ¨ro ¨y
(Institut de Recherches Cliniques de Montre ´al, Montre ´al, Canada). All an-
imals were genotyped by PCR. Mice were used at 6–10 wk of age for all
functional and biochemical experiments. Mice used at older ages for phe-
notypic assessment are indicated in the text. All animals were housed in a
specific pathogen-free facility at the University of California (San Fran-
cisco, CA) according to University and National Institutes of Health
Tissues were fixed in 10% buffered formalin for a minimum of 24 h and
subsequently embedded in paraffin, sectioned, and stained using standard
techniques. Sections were then subjected to H&E staining.
Serum processing and ELISA
Serum was prepared from blood harvested either by cardiac puncture at the
time of sacrifice or via tail bleed with the use of serum separator tubes (BD
Microtainer; BD Biosciences). Serum Ig levels were determined by ELISA
using an Ig isotyping kit and standards (SouthernBiotech) as per the man-
ufacturer’s protocol. IgG Abs to dsDNA in serum from individual mice
were measured by ELISA as described (37). The results were expressed in
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
arbitrary ELISA units relative to a standard positive sample derived from
Antinuclear Ab immunofluorescence staining
Hep2A cell 12-well slides (INOVA Diagnostics) were stained with
mouse serum prepared as above at 1/100 dilution. Slides were incubated
with serum for 30 min, washed, and incubated with FITC-conjugated
goat anti-mouse IgG (Jackson Immunochemicals) for 10 min, washed,
coverslipped, and then visualized using a Zeiss microscope. MRL/lpr
serum was used as a positive control and B6 serum was used as a
Abs and reagents
Abs to murine CD1d, CD3, CD4, CD5, CD8, CD11b, CD19. CD21,
CD23, CD44, CD62L, CD69, CD138, AA4.1, Gr-1, IgD, IgM, or B220
conjugated to FITC, PE-Texas Red, PE, PerCP-Cy5.5, PE-Cy5.5, PE-
Cy7, Pacific Blue, allophycocyanin, or Alexa Fluor 647 were obtained
from eBioscience or BD Biosciences. Abs to intracellular IFN-?, IL-17,
IL-10, and IL-4 conjugated to PE or allophycocyanin were obtained
from BD Biosciences. Erk1 and 2 Abs were obtained from Santa Cruz
Biotechnology. Myc (9B11) and Phospho-Erk Abs used for Western
blotting and flow cytometry were obtained from Cell Signaling. Poly-
clonal Abs to Pep (PEP1 and PEP2 generated against PRS1 and 2 do-
mains, respectively) were compliments of A. Chan (Genentech). The
Csk Ab (C-20 clone) was obtained from Santa Cruz Biotechnology.
Stimulatory anti-CD3? anti-Armenian hamster Abs (2C11 clone) were
obtained from Harlan. Goat anti-Armenian hamster IgG (H and L
chains) Abs, goat anti-mouse IgM (F(ab?)2), and goat anti-rabbit IgG
Abs conjugated to either PE or allophycocyanin were obtained from
Jackson ImmunoResearch Laboratories.
Single cell suspensions were prepared from lymph node (LN), spleen, and
thymus. Fc receptors were blocked with rat anti-CD16/32 (clone 2.4G2;
BD Biosciences). Cells (106) were stained with the indicated Abs and an-
alyzed on a FACSCalibur (BD Biosciences) or CyAN ADP (Beckman
Coulter) flow cytometry system. Forward and side scatter exclusion was
used to identify live cells. Data analysis was performed using FlowJo (ver-
sion 8.5.2) software (Tree Star).
Single cell suspensions were prepared from LN, spleen, and thymus. For
soluble stimulation (whole cell lysates and flow cytometric analysis of
calcium flux or Erk phosphorylation), T cells were stimulated at 37°C in
suspension with anti-CD3? followed after 30 s by cross-linking with goat
anti-Armenian hamster IgG. Plate-bound stimulation of T cells (for acti-
vation marker up-regulation) was conducted by precoating 96-well plates
with anti-CD3? and incubating plates overnight at 4°C. Cells were then
plated at a concentration of 2 ? 106/ml and harvested after 15 h incubation
at 37°C. B cell stimulation, whether soluble or plate bound, was conducted
with soluble goat anti-mouse IgM (F(ab?)2).
Single cell suspensions of thymocytes or lymphocytes were loaded with 4
?g of Fluo4-AM per 107cells (Molecular Probes) for 30 min at 37°C in
complete culture medium. T cells were stimulated (as described above) and
collected at 37°C on a FACSCalibur flow cytometer. Intracellular calcium
concentration was determined using the FlowJo kinetic function. Double
positive thymocytes were gated using a small size gate (?95% purity),
whereas peripheral T cells were gated using dump staining for B cells and
either CD8 or CD4. Cells were stimulated with ionomycin for positive
Intracellular staining for phospho-Erk
Thymocytes, spleen, or LN cells were harvested into serum-free medium.
Single cell suspensions were rested at 37°C for at least 30 min before
stimulation (as described above) and subsequently fixed to terminate stim-
ulation. Cells were permeabilized using 95% ice-cold methanol (EMS) for
30 min, stained with phospho-Erk Ab for 1 h, stained with surface markers
and donkey anti-rabbit-PE or allophycocyanin for 1 h, and fixed. Data were
collected on a FACSCalibur flow cytometer and analyzed using FlowJo
Intracellular staining for Pep
Single cell suspensions from spleen or LN were resuspended in FACS
buffer. Cells were then surface stained, fixed, stained for intracellular Pep
with PEP1 or PEP2 polyclonal Abs (described above) in saponin-based
medium B (Caltag Laboratories), and washed with Perm/Wash (BD Bio-
sciences), followed by secondary staining with goat-anti-rabbit Ab (Jack-
son ImmunoResearch Laboratories) conjugated to PE. Data were collected
on a FACSCalibur and analyzed using FlowJo software.
T cell purification
B cells, CD4 T cells, or CD8 T cells were purified with MACS columns
(Miltenyi Biotec) as per the manufacturer’s protocol and subsequently used
for whole cell lysates.
Whole cell lysates were generated by lysing stimulated or unstimulated
cells in SDS sample buffer. Samples were analyzed by SDS-PAGE and
immunoblotting. Blots were visualized with Western Lightning ECL re-
agent (PerkinElmer Life Sciences) and a Kodak Imaging Station.
Murine Pep construct in a pCMV expression vector was obtained from
the Mammalian Gene Collection Library via Invitrogen. The Lyp1
C1858*R620 allele was cloned from a Jurkat cDNA library. Site-di-
rected mutagenesis using QuikChange (Stratagene) was performed to
introduce the R619W (murine homologue)/R620W residue change into
Pep and Lyp1, respectively. Both alleles were then subcloned into a
pEF expression vector (Invitrogen). Myc-tagged murine Csk in the pEF
expression vector was obtained from J. Schoenborn (University of Cal-
ifornia, San Francisco, CA).
Jurkat cells were grown in RPMI 1640 supplemented with 10% FCS at
a density of 0.1–0.6 ? 106cells/ml without supplementary antibiotics.
Constructs with Csk, WT Pep, R619W Pep, WT Lyp1, R620W Lyp1,
human CD16, and/or GFP under the control of the pEF promoter were
transiently transfected via electroporation. A total of 20 ?g of plasmid
was introduced into 12 ? 106cells per condition. Empty vector was
included to maintain constant plasmid concentration. Functional studies
were performed and lysates were generated 12–15 h after transfection.
Lysates were made with 1% Nonidet P-40 lysis buffer. Csk, Lyp,
and Pep expression levels were probed by Western blotting with Myc,
Csk, and Pep Abs (described above). For functional studies of Erk
phosphorylation, cells were rested in serum-free RPMI 1640 at 37°C for
at least 15 min before stimulation with C305 or PMA for 2 min. Cells
were then immediately fixed and subjected to permeabilization and
staining for phospho-Erk as described above for primary cells. For anal-
ysis of calcium flux, cells were loaded with fluo-3 and fura red (Mo-
lecular Probes) for 30 min at 37°C, surface stained for the transfection
marker hCD16 (BD Biosciences), stimulated with C305, and collected
at 37°C on a FACSCalibur flow cytometer. Intracellular calcium con-
centration was determined using the FlowJo kinetic function and fluo-
3/fura red ratio.
Pep is expressed predominantly in T cells
The ubiquitous expression of CD45 on all nucleated hemato-
poietic cells is well established (23). Pep/Lyp expression is not
as well characterized. Lyp message has been detected in human
thymus and peripheral lymphoid tissue, including B and T cells
(8). Lyp protein expression has been seen in thymocytes and
primary human T cells (8). To identify which immune cell sub-
sets might contribute to autoimmune disease in mice and which
cellular context would serve best to study the functional con-
sequences of the risk allele, we defined the protein expression
pattern of Pep in mice. Due to the presence of nonspecific
bands, we controlled for Ab specificity by using Pep-deficient
cell lysates. Using a combination of Western blotting (Fig. 1A)
and flow cytometric detection of intracellular staining (Fig. 1B)
with two independent polyclonal Abs to Pep, we found that Pep
is expressed at appreciable levels predominantly in T cells. We
detected expression in thymocyte subsets as well as peripheral
T cells and noted that CD8 T cells express considerably more
Pep than CD4 T cells (Fig. 1A and data not shown). We also
4095The Journal of Immunology
noted increased expression in CD69highactivated and CD44high
memory/effector T cells, but were unable to detect significant pro-
tein expression in myeloid subsets, B cells, whole spleen, or bone
marrow (Fig. 1 and data not shown). This is consistent with reports
that Lyp is up-regulated in T cells upon TCR stimulation, as well
as the absence of detectable Lyp message in human bone marrow
or myeloid cell lines (8). This result is furthermore consistent with
the absence of a B cell phenotype in the Pep?/?mice (17).
cells. A, Whole cell lysates from WT or Pep?/?animals
were prepared from bone marrow, LNs, spleen, thymus,
and MACS-purified B cells, CD4 T cells, and CD8 T
cells. Western blots were probed with polyclonal Ab
recognizing Pep. The 105-kDa band corresponding to
Pep is depicted and total Erk 1 and 2 is stained as a
loading control. Analogous results with an indepen-
dently generated Pep polyclonal Ab were obtained. B,
Histograms of LN or splenic subsets stained with poly-
clonal Pep Ab following permeabilization with saponin.
Histograms of WT cells are represented by a black line
whereas Pep?/?cells are represented by shaded gray
histogram in each plot. Data presented are representa-
tive of three independent experiments.
Pep is expressed predominantly in T
functions as a hypomorph in the context of Csk. A, Csk
and WT or R620W Pep were misexpressed in Jurkat
cells together with GFP. Upper panel represents whole
cell lysates of transfected cells probed for either Pep or
Csk. Upper band in Pep blots represents murine Pep
whereas the lower band is nonspecific background. En-
dogenous (endog) Lyp is not detected by this polyclonal
Ab. B, Histograms correspond to samples in A and rep-
resent intracellular phospho-Erk staining of transfected
Jurkat cells stimulated through the TCR by C305 or
with PMA. GFP high, intermediate (MED), and low
gating is shown. Gray histograms represent unstimu-
lated transfectants for comparison. Black-lined histo-
grams represent stimulated transfectants as identified in
A. C, Bar graph corresponding to samples generated and
stimulated as in A and B. The percentage (%) of phos-
pho-Erk-positive cells is defined as the percentage of
cells that falls into the high expressing half of the bi-
modal histogram distributions in B. Error bars represent
SEM. Plots and Western blots are representative of at
least five independent experiments.
The murine Pep R620W polymorphism
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
The R620W allele of PTPN22 is a hypomorph in the context
To clarify the functional consequences of the human R620W
PTPN22 polymorphism, we undertook in vitro studies of the risk
allele in the Jurkat human T cell line. The Jurkat cell line is an
appealing model system in which to pursue these studies because
it has been recently shown that PTP-PEST, a Pep-related phos-
phatase with partially redundant functions, is not expressed in Ju-
rkat cells (and down-regulated in primary effector T cells) (18).
This context supplies an opportunity to unmask Pep-dependent
Overexpression of phosphatases is difficult to evaluate because
of the promiscuity of their target selection. Both mislocalization
and stoichiometric perturbations of overexpression can lead to “ec-
topic” dephosphorylation within the cell and off-target effects that
are not physiologic. It has been shown that under basal conditions,
25–50% of Pep in the cell is bound to Csk (13). Because Csk
targets Pep to its substrate Lck by virtue of the interaction between
the Csk SH3 domain and the Pep PRS domain that contains the
R620 residue, it is critical to study the R620W allele in the context
of Csk. We studied Pep R620W in the context of cooperating Csk
to reduce overexpression and to limit the off-target effects that may
have confounded earlier studies. We used flow cytometric readouts
of Erk phosphorylation to permit cell-specific assessment of a wide
range of construct expression levels to carefully control for over-
expression and dose.
morphism functions as a hypomorph in the context of
Csk. A, Csk and WT or R620W Lyp1 were misex-
pressed in Jurkat cells together with GFP. The upper
panel represents whole cell lysates of transfected cells
probed for either Myc-tagged Lyp1 or Csk. B, Histo-
grams correspond to samples in A and represent intra-
cellular phospho-Erk staining of transfected Jurkat cells
stimulated through the TCR by C305 or with PMA.
GFP high, intermediate (MED), and low gating is
shown. Gray histograms represent unstimulated trans-
fectants for comparison. Black-lined histograms repre-
sent stimulated transfectants as identified in A. C, Bar
graph corresponding to samples generated and stimu-
lated as in A and B. The percentage of phospho-Erk
positive cells are defined as in Fig. 2C. Error bars rep-
resent SEM. D, Intracellular calcium levels of Lyp1/Csk
transfected Jurkat cells (gated for cotransfected CD16).
The fluo3/fura red ratio was monitored by flow cytom-
etry before and after C305 stimulation of fluorophor-
loaded transfectants. Solid line represents Csk plus
Lyp1 R620*C1858 WT allele. Dotted line represents
Csk plus Lyp1 W620*T1858 risk allele. Plots, Western
blots, and calcium flux are representative of at least
three independent experiments.
The human Lyp1 R620W (T1858) poly-
4097 The Journal of Immunology
In cotransfection studies in the Jurkat cell line with Pep and Csk
vectors, we have been able to show that Csk and WT Pep can
cooperate synergistically to inhibit Erk phosphorylation following
TCR stimulation (Fig. 2). In these experiments we consistently
found that WT Pep inhibits Erk phosphorylation more potently
than the R620W allele, both independently and more so in the
context of Csk (Fig. 2). Furthermore, this functional readout cor-
relates with cotransfected GFP levels in a flow-based assay (Fig.
2B). Relative function of the WT and R620W Pep alleles is qual-
itatively unchanged at both high and low GFP levels in our studies
(Fig. 2B and data not shown), suggesting that overexpression ar-
tifact does not play a role here.
Total transfection efficiency was comparable between sam-
ples (data not shown), and Pep as well as Csk protein levels
were comparable between samples (Fig. 2A). By using murine
Pep in our studies, we were able to take advantage of a poly-
clonal Ab to murine Pep that does not recognize endogenous
human Lyp to identify total levels of misexpressed protein. By
using Myc-tagged murine Csk, which migrates more slowly
than endogenous untagged Csk, we could distinguish the two by
Western blotting. We can identify very modest levels of over-
expression and make use of endogenous Csk levels as an ac-
curate loading control (Fig. 2A).
Human Lyp and murine Pep proteins are only ?70% conserved,
although the critical PRS1 domain that mediates Csk association is
completely conserved (8). We therefore performed parallel exper-
iments in Jurkat cells using Myc-tagged Lyp1 C1858*R620 and
T1858*W620 alleles. We observed similar effects of Lyp and Pep
homologues on the inhibition of TCR-induced Erk phosphoryla-
tion (Fig. 3, A–C). The human Lyp1 C1858*R620 WT allele more
potently inhibits TCR signaling than the T1858*W620 risk allele,
both independently and in the context of Csk. As in Pep misex-
pression experiments, transfection efficiency and dose were con-
trolled for by GFP cotransfection as well as Western blotting de-
tection of Myc-tagged Lyp and Csk (Fig. 3, A and B). Furthermore,
in both Pep and Lyp experiments no defect in PMA-induced Erk
develop lymphoproliferation and autoimmunity. Abbre-
viations apply to all subsequent figures: WEDGE,
CD45w/w; PEP, Pep?/?; DNASE1, DNase1?/?; DOU-
BLE, CD45w/w/Pep?/?. A, ELISA for IgG autoantibod-
ies reactive to dsDNA were performed on sera from
CD45w/w/Pep?/?double mutant mice at the indicated
time points. Values represent individual mice and were
normalized to average MRL/lpr serum ? 1000 arbitrary
ELISA units. Inset represents antinuclear Ab staining
pattern for 1/100 dilution of representative CD45w/w/
Pep?/?double mutant serum. B, ELISA for IgG dsDNA
autoantibodies of sera from various genotypes sampled
at 9–10 mo of age, normalized as in A. C, Representa-
tive spleens and LNs from genotypes at 5 mo of age. D,
Lymph node absolute cell counts at 6 mo of age. Values
are the mean of three biological replicates ? SEM. Sim-
ilar results were obtained at multiple time points ranging
from 6 wk to 12 mo of age in at least five independent
experiments, each including three animals per genotype.
E–H, H&E staining of formalin-fixed kidney sections
from WT (E and G) and CD45w/w/Pep?/?double mu-
tant (F and H) animals at 12 mo of age. Representative
specimens at original magnifications of ?10 (E and F)
and ?20 (G and H) are depicted.
CD45w/w/Pep?/?double mutant mice
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
phosphorylation was observed, implying a proximal target for Pep/
Lyp function in TCR signaling (Figs. 2B and 3B).
To assess a distinct functional readout, we assayed the effect of
Lyp/Csk coexpression upon TCR-induced calcium flux by flow
cytometry. By gating on the cotransfected surface marker CD16,
we observed relatively stronger inhibition of calcium entry by the
Lyp1 C1858*R620 WT allele than by the T1858*W620 risk allele
in the context of Csk (Fig. 3D). This effect was dose dependent as
assessed by CD16 gating (data not shown). Because calcium and
Erk signals are similarly perturbed by Lyp/Csk misexpression, we
conclude that Lyp and Csk must cooperatively inhibit TCR sig-
naling at or proximal to the Lat/Slp76 signalosome at which these
signals diverge. This is consistent with known and putative Lyp
substrates, including Lck, Zap70, CD3?, CD3?, and Vav (38).
These flow-based readouts effectively capture the functional phe-
notype of the Pep and Lyp phosphatases. More proximal substrates
such as Lck 394 are difficult to perturb and assay in the context of
From these studies we conclude that the R620W human risk
allele of PTPN22 functions as a hypomorph in the context of TCR
signal transduction, and may be appropriately modeled using the
Double mutant CD45w/w/Pep?/?mice develop a lupus-like
CD45 E613R (CD45w/w) mice, previously backcrossed for more
than nine generations to the B6 background, were crossed to
Pep?/?mice originally generated on a B6 background. Double
mutant CD45w/w/Pep?/?B6 mice homozygous for each of the two
mutant alleles were generated and aged. In a parallel experiment,
DNase1?/?mice were crossed onto the B6 CD45w/wbackground
and aged (CD45w/w/DNase1?/?). DNase1 has also been shown to
be a murine lupus susceptibility locus, but is also not associated
with autoantibodies on the B6 background (36).
The CD45w/w/Pep?/?mice developed IgG autoantibodies to
dsDNA in a partially penetrant fashion, with elevated titers detect-
able by ELISA as early as 3 mo of age and increasing with time
(Fig. 4A). Titers in some mice reached levels comparable to those
seen in the MRL/lpr lupus-prone mouse model (arbitrary ELISA
units ? 1000) by 9 mo of age (Fig. 4, A and B). Autoantibody
specificity was also assayed by indirect immunofluorescence stain-
ing of Hep2A cells and revealed a homogeneous nuclear pattern,
consistent with the original observation in mixed B6/129 back-
ground CD45w/wmice (Fig. 4A). In contrast, CD45w/w/DNase1?/?
CD45w/w/Pep?/?double mutant mice. A, Lymph node
composition at 6 mo of age was determined by staining
single cell suspensions with CD4 (dark gray), CD8
(black), and CD19 (light gray) mAb. B, CD4 T cell/CD8
T cell ratios calculated from LN specimens at 6 mo of
age. Values are the mean of three biological repli-
cates ? SEM. C and E, Representative cytometry plots
of splenic CD4 and CD8 T cells stained with Abs to
CD62L and CD44 at 6 mo of age. Gated fractions rep-
resent memory/effector cells. D and F, Gated splenic T
cell memory fractions depicted in C and E are plotted
over time in D and F, respectively. G and H, Represen-
tative histograms of splenic CD4 T cells (G) and CD8 T
cells (H) stained with CD69 to detect activation status.
Shaded histogram represents WT for comparison.
Animals were assessed at age 6 mo. Data in panels
A–G are representative of at least three independent
experiments performed at varying time points, each
WEDGE; DBL, DOUBLE.
T cell activation and differentiation in
4099The Journal of Immunology
mice developed no significant autoantibody titers up to an age of
10 mo (Fig. 4B). IgG anti-dsDNA production did not reflect a
polyclonal gammopathy, as total IgG levels in all mutants were
comparable to those in WT mice (data not shown).
We noted that as early as 8 wk of age, the CD45w/w/Pep?/?
mice developed lymphadenopathy and splenomegaly that were
progressive with time and greatly exceeded the mild lymphopro-
liferation evident in each of the single mutants (Fig. 4C). LN ab-
solute cell counts were variably elevated among the aging cohort,
with some reaching 10-fold normal levels by 6 mo of age (Fig.
4D). The lymphadenopathy was generally out of proportion to
the degree of splenomegaly observed, suggesting a lymphoid-
predominant dysregulation of the hematopoietic compartment
(Fig. 4C and data not shown). In contrast, LN size, LN cell
number, and spleen weights in CD45w/w/DNase1?/?mice were
comparable to the values in CD45w/wsingle mice (age 10 mo;
data not shown). No excess mortality was observed out to 10
mo of age. Subsequent results and analysis in this paper will be
limited to the CD45w/w/Pep?/?mice.
Although CD45w/w/Pep?/?mice displayed reduced body
weights as early as 6 mo of age, overt morbidity and mortality
were evident only by 10 mo of age. We noted the onset of pro-
teinuria and subsequent mortality in a subset of mice by 10 mo of
age (data not shown). By 12 mo of age, 33% of our initial cohort
of CD45w/w/Pep?/?mice had died (n ? 15). The remaining ge-
notypes had normal life expectancy.
We next attempted to define the cause of death in the CD45w/w/
Pep?/?mice. The surviving CD45w/w/Pep?/?mice at the age of
12 mo were small, had variable degrees of proteinuria, and had
pale and nodular kidneys on gross pathology (data not shown).
Other major organs appeared grossly normal. Histologic examina-
tion of CD45w/w/Pep?/?kidneys revealed marked abnormalities,
including perivascular lymphocytic infiltrates, interstitial lympho-
cytic infiltrates, and hypercellular glomeruli with evidence of seg-
mental sclerosis (Fig. 4, E–H). CD45w/wsingle mutant mice
showed very mild evidence of similar abnormalities in kidney his-
tology. Both CD45w/w/Pep?/?and CD45w/wsingle mutants had
evidence of perivascular infiltrates in lung and liver at 12 mo of
age to a comparable extent, but these infiltrates did not appear to
extend into organ parenchyma (data not shown). We conclude that
renal failure due to glomerulonephritis was the likely cause of
premature mortality in the CD45w/w/Pep?/?mice.
Given the presence of lymphoproliferation, autoantibody pro-
duction, autoimmune kidney pathology, and mortality, we con-
clude that the CD45w/w/Pep?/?mice develop a lupus-like disease
in a partially penetrant fashion that recapitulates the original dis-
ease phenotype observed in CD45 E613R animals on the B6/129
turbed B cell development in double mutant mice. A,
Representative histograms of LN CD19?B cells stained
with CD69 to detect activation status. Shaded histogram
represents WT for comparison. Animals were assessed
at age 6 mo. B, Plasma cell marker CD138 expression
among splenocytes (%) at age 3 mo. C, Representative
cytometry plots of CD19?splenocytes stained with
CD23 and AA4.1 to identify transitional (T1, T2) and
FO mature B cell subsets. D, Quantification of subsets
identified in C. E, Representative cytometry plots of
CD19?splenocytes stained with IgM and IgD to iden-
tify transitional (T1, T2), marginal zone (MZ), and FO
mature B cell subsets (FM). F, Quantification of subsets
identified in E. Animals in C–F were 2 mo of age at the
time of analysis. In B, D, and E the values are the means
of three biological replicates ? SEM. All of the data
presented are representative of at least 3 independent
experiments at varying time points. WDG, WEDGE;
Polyclonal B cell activation and per-
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
mixed background (31). Neither CD45w/wnor Pep?/?single mu-
tant mice developed lupus-like autoimmunity over the course of 12
mo of observation. CD45w/w/DNase1?/?mice displayed no evi-
dence of genetic cooperation or frank autoimmunity.
Double mutant mice develop polyclonal T cell activation and an
expanded effector/memory compartment
To begin to understand the mechanism by which overt autoimmu-
nity arises in the CD45w/w/Pep?/?double mutant mice, we as-
sessed the cellular immune phenotype. The enlarged lymphoid
compartment in double mutant mice was not due to the expansion
of a single hematopoietic lineage. Double mutant LNs contained
relatively preserved ratios of T and B cells (Fig. 5A). The CD4:
CD8 peripheral T cell ratio was increased consistently in the dou-
ble mutants and less so in each single mutant relative to WT in
both LN and spleen (Fig. 5B and data not shown). This ratio shift
was evident as early as 6 wk of age and persisted throughout the
observation period (12 mo).
The most marked cellular phenotype was the progressive ex-
pansion of the CD4 and CD8 T cell memory/effector compart-
ments in double mutant mice as identified by CD44 and CD62L
surface markers (Fig. 5, C and E). A more subtle version of this
phenotype is evident in each single mutant. This phenomenon was
observed in the spleen as well as the LN T cell compartment,
occurred early, and progressed with time (Fig. 5, D and F, and data
not shown). The activation marker CD69 was up-regulated in dou-
ble mutant CD4 and CD8 T cells and to a lesser extent in each
single mutant (Fig. 5, G and H).
B cell development and activation are not influenced by Pep
In contrast to T cells, B cells up-regulated activation markers such
as CD69 as well as CD80/86 in CD45w/wand double mutant mice,
but not in Pep?/?mice. The activation status of B cells at age 2 mo
was matched in CD45w/wand double mutant mice, but by 6 mo of
age double mutant mice further up-regulated B cell activation
markers (Fig. 6A and data not shown). The plasma cell compart-
ment identified by cell surface CD138 expression was expanded in
CD45w/wand double mutant spleens to a similar extent, whereas
Pep?/?mice resembled WT (Fig. 6B).
BCR signaling is critical for B cell development, which is char-
acteristically perturbed in CD45w/wmice (32). Using the markers
CD21, CD23, IgM, IgD, and AA4.1, we found that double mutant
mice precisely phenocopied the CD45w/wsplenic B cell phenotype
with the expansion of newly formed/transitional (T1/T2) B cell
subsets at the expense of the mature follicular (FO) B cell subset
as previously described (Fig. 6, C–F and data not shown) (32).
Consistent with the previously reported expansion of B1 B cells in
CD45w/wmice, double mutant and CD45w/wmice have increased
total serum IgM levels (data not shown) (32). In contrast, the
Pep?/?splenic B cell phenotype resembled WT, consistent with
the absence of Pep expression in B cells.
of time (0, 1, 3, and 5 min) with goat anti-mouse IgM F(ab?)2or, in the bottom panels, PMA. Fixed and methanol-permeabilized cells were then stained
with Ab against phospho-Erk and assessed by flow cytometry. Histograms represent gated B220?B cells. Black solid line, CD45w/w; black dotted line,
double mutant; gray dashed line, Pep?/?; gray shaded histogram, WT. B, Lymph node B cells were stimulated for 15 h with goat anti-mouse IgM F(abp)2
and stained for the activation marker CD69. The graph represents the percentage of total B cells up-regulating CD69 at varied doses of stimulus. PMA responses
in all genotypes were identical. Values were plotted on log2scale with each point representing mean ? SEM of three biological replicates per genotype. Genotypes
are identified as in A. C, Splenic B cells stimulated and stained as described in A were additionally stained with CD23 and CD21 to identify newly formed (NF)
and FO cell populations as depicted in the left panel. The upper histogram depicts FO B cell subsets whereas the lower histogram depicts newly formed (NF) B
cell subsets. Genotypes are identified as in A. All data presented are representative of at least three independent experiments.
Pep?/?does not contribute to CD45w/wB cell hyperresponsiveness. A, Lymph node or splenic B cells were stimulated for varied periods
4101The Journal of Immunology
BCR signaling is not influenced by Pep deficiency
Because double mutant mice uniquely develop a break in B cell
tolerance as indicated by autoantibody production, we first
sought to define the cell-intrinsic contribution of the B cell
lineage to this phenotype by assessing BCR signaling directly.
Double mutant and CD45w/wB cells in spleen and LNs were
found to be hyperresponsive to in vitro BCR stimulation as
assessed by Erk phosphorylation. Notably, Pep?/?B cells re-
sponded like WT B cells, whereas double mutant B cells
responded like CD45w/wB cells with no evidence of unmasked
synergy in the double mutant (Fig. 7A). We identified similar
trends with alternate readouts downstream of BCR ligation such
ence TCR signaling in a developmental stage-specific
manner. A, Graph depicts mean fluorescence intensity
(MFI) for CD5 staining on gated DP thymocytes. Val-
ues are the means of three biological replicates ? SEM
and represent at least five independent experiments,
each involving three animals per genotype. B, Intracel-
lular calcium levels of fluo-4 AM-loaded thymocytes
(gated for small size to identify DP population). Green
fluorescence was monitored by flow cytometry before
and after stimulation with soluble anti-CD3? Ab in the
upper panel or ionomycin in the lower panel. Geno-
types are identified as in A. Plots are representative of at
least four independent experiments. C, DP thymocytes
were stimulated with soluble anti-CD3?. Fixed and
methanol-permeabilized cells were then stained with Ab
against phospho-Erk and assessed by flow cytometry.
Histograms represent gated DP thymocytes. WT DP
thymocytes are represented by the shaded gray histo-
gram in each plot for comparison. PMA responses in all
genotypes were identical. Plots are representative of at
least five independent experiments. WDG, WEDGE;
DBL, DOUBLE. D, LN T cells were stimulated with
anti-CD3 Ab, fixed, and permeabilized with methanol
and subsequently stained with Abs to CD4, CD8, CD44,
and phospho-Erk. Histograms represent staining for
phospho-Erk on cells gated for CD4, CD8, and either
CD44 low or high surface staining to identify naive and
memory subsets. Shaded histograms represent WT for
comparison. Plots are representative of at least three in-
dependent experiments each with three biological rep-
licates per genotype. E, In vitro generated effector CD4
or CD8 T cells were stimulated, fixed, and permeabil-
ized as described above and subsequently stained with
Ab to phospho-Erk. Histograms represent staining for
phospho-Erk. Shaded histograms represent WT for
comparison. Plots are representative of two independent
Pep?/?and CD45 E613R alleles influ-
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
as calcium flux, activation marker up-regulation, and CFSE di-
lution (Fig. 7B and data not shown). These data are compatible
with the absence of detectable Pep expression in B cells and
unperturbed B cell development in Pep?/?mice. Consistent
with these data, a B cell signaling phenotype was not previously
demonstrated in Pep?/?mice (17).
Although LN B cells represent a relatively uniform, predomi-
nantly mature phenotype, splenic B cells include multiple devel-
opmental stages, each with unique signaling thresholds. To define
the precise stage of B cell development at which CD45w/wcon-
tributes to hyperresponsiveness, we assessed BCR-induced Erk
phosphorylation in conjunction with B cell surface staining by
flow cytometry. Surprisingly, signaling was relatively normal in
newly formed B cells of all genotypes, but FO B cells from
CD45w/wand double mutants were markedly hyperresponsive,
consistent with the LN data (Fig. 7C).
We suggest from these data that in double mutant mice, the
CD45 E613R wedge allele contributes to a B cell tolerance break
at the follicular mature stage of development in a cell intrinsic
fashion, whereas Pep deficiency does so in a cell-extrinsic fashion.
Pep deficiency and CD45 E613R cooperate to enhance TCR
signaling in the thymus
To understand the contribution of Pep deficiency to the double
mutant phenotype, we examined the T cell lineage, where Pep is
predominantly expressed. Thymocyte development was grossly in-
tact as assessed by double negative, double positive, and single
positive subset compartment size (data not shown). Double nega-
tive subsets 1–4 as assessed by CD44 and CD25 surface markers
were also unperturbed without evidence of abnormalities in ?-se-
lection (data not shown).
Both Pep?/?and CD45w/wmice have been previously indepen-
dently crossed to TCR transgenes (H-Y, OT-2, and DO11.10), re-
vealing increased positive selection in each background (17, 55);
(M. Hermiston, manuscript in preparation). We therefore charac-
terized the cell surface phenotype of double positive (DP) thymo-
cytes. We noted up-regulation of CD5 and CD69 in double mutant
mice relative to WT and less so in each single mutant (Fig. 8A and
data not shown). These markers are well-established correlates of
TCR signal strength and suggest that CD45w/wand Pep?/?coop-
erate in a cell intrinsic manner at the DP thymocyte stage of T cell
We tested this hypothesis by stimulating the TCR on DP thy-
mocytes in vitro and assessed Erk phosphorylation and calcium
flux by flow cytometry. These studies revealed subtle hyperrespon-
siveness in DP thymocytes of each single mutant and a uniquely
heightened response in double mutant mice (Fig. 8, B and C). This
alteration in signaling was not due to any downstream rewiring, as
PMA and ionomycin produced comparable Erk phosphorylation
and calcium flux, respectively (Fig. 8B and data not shown).
CD45 E613R does not contribute to TCR hyperresponsiveness
in peripheral T cells
Although Pep?/?and CD45w/wcooperate to enhance TCR signal-
ing in double mutant DP thymocytes, such a perturbation should
in theory increase both positive and negative selection, result-
ing in enhanced rather than impaired central tolerance. This
conclusion led us to turn our attention to peripheral T cells to
understand in what cell type and at which stage the two muta-
tions cooperate to break tolerance. Because peripheral T cell sub-
sets have qualitatively distinct signaling properties, we systemat-
ically interrogated TCR signaling in naive, memory, and effector
CD4 and CD8 T cells. We initially hypothesized that the unique
memory/effector phenotype in double mutant mice was due to
uniquely enhanced peripheral T cell signaling in double mutant
mice. To our surprise, we found that this was not the case. We
further discovered that the CD45 E613R allele makes no signifi-
cant contribution to TCR signaling in peripheral T cells.
Because of the unequal size and signaling properties of
CD44highmemory and CD44lownaive T cell subsets in our four
genotypes, we compared responsiveness to TCR ligation sepa-
rately in naive and memory T cell subsets by gating on surface
CD44 expression while evaluating Erk phosphorylation by flow
cytometry. This revealed no substantial differences in naive or
memory T cell responsiveness among the four genotypes, except
among CD8?naive (CD44low) T cells where we observed consis-
tent and equivalent TCR hyperresponsiveness in Pep?/?and dou-
ble mutant mice (Fig. 8D).
We confirmed these observations by examining an event further
downstream of TCR stimulation. We evaluated CD69 up-regula-
tion specifically in CD44low-gated naive T cells. Double mutant
CD8 T cells consistently responded comparably to Pep?/?T cells,
whereas CD45w/wT cells responded comparably to WT (data not
shown). Among CD8 T cells, we observed at most a 2-fold shift in
the dose-response curve to anti-CD3 stimulation, suggesting that
Pep?/?alters signal sensitivity very subtly in the naive T cell
compartment. Other readouts such as calcium flux and CFSE di-
lution failed to reveal consistent differences between the four ge-
notypes, possibly because they rely upon a collective signal from
a heterogeneous pool of T cells. To our surprise, no readout of
TCR signal strength revealed hyperresponsiveness in CD45w/wna-
ive or pooled peripheral T cells.
To explain the accumulation of memory/effector T cells in dou-
ble mutant mice in the absence of a unique double mutant naive T
cell phenotype, we hypothesized a stage-specific influence of
CD45w/wand Pep?/?alleles on effector T cells, rather than naive
or memory T cells. We stimulated the TCR of in vitro generated
CD4 and CD8 effector T cells from all four genotypes and assessed
Erk phosphorylation both by flow and by Western blotting. We
found subtle and comparable hyperresponsiveness in Pep?/?and
double mutant CD4 effectors, but not in CD8 effectors (Fig. 8E).
Consistent with these observations, Hasegawa et al. have previ-
ously characterized the T cell phenotype in Pep?/?mice, reporting
hyperresponsiveness in the “memory/effector” compartment in the
absence of a naive T cell phenotype (17). Again, no impact of the
CD45 E613R wedge allele was observed, implying that the pro-
gressive accumulation of memory/effector T cells in double mutant
animals cannot be accounted for by a purely T cell-intrinsic sig-
Function of PTPN22
Our model is the first reported autoimmune disease phenotype
for PTPN22 in mice and helps to clarify both the mechanism by
which PTPN22 functions as a general autoimmune susceptibil-
ity locus in humans, and the genetic context in which it might
do so. The R620W polymorphism disrupts the critical interac-
tion of Pep/Lyp with Csk and thereby impairs the ability of Pep
to effectively access its target Lck (2). Prior studies of the func-
tional consequences of this polymorphism have led to conflict-
ing results. The R620W variant was reported to have slightly
enhanced enzymatic activity in an vitro phosphatase assay (19).
Studies of primary human cells harboring the risk allele and
prior overexpression studies in Jurkat cells suggest as well that
this variant impairs TCR signaling, implying a gain of function
(19, 20). Several other studies using primary human cells from
patients with autoimmune disease are more difficult to interpret
4103The Journal of Immunology
(19, 21, 22). However, previous Jurkat studies have failed to
define fold overexpression or dose response, and function was
not assessed in the context of Csk (19).
We find that WT murine Pep or human Lyp overexpression in
the context of Csk clearly exhibits cooperative inhibition of
proximal TCR signaling and does so more effectively than the
R620W allele. Our studies reflect a more physiologic stoichi-
ometry than prior work and control explicitly for expression
level and overexpression artifact. These differences may explain
discrepancies with prior reports. Furthermore, we can now ex-
clude the possibility that human and murine orthologues may
exhibit functional differences in this context. It remains a for-
mal possibility that Pep and/or Lyp regulates signaling path-
ways other than those downstream of the Ag receptor and tar-
gets additional and as yet unidentified substrates.
Clues to the functional impact of this polymorphism on disease
pathogenesis can be gleaned from several human genetic studies.
The PTPN22 R620W polymorphism is a risk factor not simply for
autoimmune disease characterized by pathogenic autoantibody
production (such as SLE, Grave’s disease, and myasthenia gravis),
but particularly for autoantibody-positive disease variants (5). The
risk allele is specifically associated with seropositive and anticy-
clic citrullinated peptide Ab-positive RA and not with a seroneg-
ative disease (5, 40, 41). Indeed, there is no positive association
between the risk allele and either inflammatory bowel disease or
multiple sclerosis, neither of which have autoantibodies as a sig-
nificant pathogenic feature (5, 40). Interestingly, the nonrisk allele
of PTPN22 is associated with tuberculosis, not only suggesting a
selective evolutionary pressure to account for the prevalence of the
risk allele, but supplying hints of a mechanism (42). Certainly,
hyperresponsive T cells such as those in Pep?/?mice might plau-
sibly mediate this resistance. The host response of these mice in
the context of infection has not been defined.
Unambiguous resolution of whether the R620W allele is a
hypomorph or a hypermorph must address both the genetic and
environmental heterogeneity that confounds functional studies
of primary human cells and the limitations of phosphatase over-
expression in cell lines. Certainly, a gain of function Pep/Lyp
allele could impair negative selection in the thymus or regula-
tory T cell function in the periphery. Both types of perturbations
can provoke autoimmune disease in mice (and rarely in hu-
mans) (43, 44). Alternatively, we have shown that a hypomor-
phic PTPN22 variant in T cells might serve to drive hyperre-
sponsive B cells to break tolerance in the periphery and produce
autoantibodies, as we see in the CD45w/w/Pep?/?model. The
association of the R620W allele almost exclusively with au-
toantibody-positive diseases and variants could be understood
in light of this mechanism.
Cellular mechanism of disease
Although we have provided evidence that the human risk allele of
PTPN22 is a hypomorph, Pep-deficiency on the B6 background
fails to break tolerance independently. In the present study we
demonstrate that it cooperates with the CD45 E613R genetic back-
ground to provoke overt autoimmune disease. By generating dis-
ease in the context of a bona fide human susceptibility allele, our
model sheds light on the early pathogenesis of human autoimmune
In our analysis of this model, we sought to identify cell-intrinsic
and cell-extrinsic phenotypes to establish direct and indirect
events, respectively, in disease pathogenesis. We reasoned that the
cell-intrinsic effects of the genetic perturbations in our model
ought to directly and primarily influence Ag-receptor signal trans-
duction, because Pep and CD45 reciprocally regulate the SFKs,
critical proximal mediators of these signals.
We have demonstrated enhanced TCR signal sensitivity at the
DP stage of thymic development. The critical central tolerance
mechanism of negative selection remains intact, preventing auto-
reactive clones from escaping to the periphery. As a result of en-
hanced positive selection, the T cell repertoire may be perturbed in
favor of nonautoreactive, weak affinity receptors and expanded
numbers of regulatory T cells. Neither change would be expected
to contribute to a break in self-tolerance. Abnormally selected,
weak affinity TCRs coupled to very subtly hyperresponsive pe-
ripheral T cells ought not to cross the threshold for inappropriate
activation. Indeed, other autoimmune mouse models with altered T
cell repertoires that contribute to disease show evidence of im-
paired rather than enhanced selection in the thymus (43–46).
Therefore, we suggest that the thymic phenotype is not likely to
contribute to the tolerance break we detect in our model.
We have shown that Pep is expressed in T cells and that Pep-
deficiency makes a lone contribution to peripheral TCR hyperre-
sponsiveness in double mutant mice. This suggests that the CD45
E613R allele does not exert a significant cell-intrinsic effect in
peripheral T cells. In contrast, the accumulation of effector/mem-
ory T cells in the double mutant mice is significantly accelerated
relative to either mutation in isolation. We conclude that the
CD45w/wmicroenvironment makes a purely cell-extrinsic contri-
bution to this “indirect” T cell phenotype. The CD45w/wcontribu-
tion could represent the influence of myeloid and/or B cell com-
partments. Other murine models of lupus have demonstrated that
both scenarios are possible (47–49). The non-T cell compartment
may secrete cytokines or provide costimulation to drive T cell
differentiation. It is also possible, although less likely, that an al-
tered T cell repertoire in the double mutant mice contributes to
these “emergent” T cell phenotypes.
BCR signaling in the double mutant mice, in contrast, is influ-
enced exclusively by the CD45w/wgenetic background and is spe-
cifically perturbed at the “final” FO mature stage of B cell devel-
opment. We therefore conclude that the unique B cell phenotypes
seen only in the double mutant mouse such as augmented poly-
clonal activation, B cell compartment expansion, and, of course,
autoantibody production are partially B cell extrinsic in our model,
driven by Pep?/?T cells. Others have demonstrated that memory/
effector T cells have pathogenic potential, possibly by interacting
with B cells via CD40L or ICOS (50). Indeed, Pep?/?mice de-
velop spontaneous germinal centers despite the absence of a B
cell-intrinsic signaling phenotype, suggesting a role for Pep?/?
effector Th cells in promoting B cell differentiation in this model.
Hyperresponsive FO mature CD45w/wB cells are ideally situated
to cooperate productively in such a context. These genetic inter-
actions between Pep deficiency and the CD45w/wbackground by-
pass tight central tolerance mechanisms and lead to a tolerance
break only in the periphery. Indeed previous analysis of CD45w/w
B cells in the context of the Ig HEL (hen egg-white lysozyme)
transgene confirm that central negative selection of B cells is not
only intact, but is enhanced (32).
Numerous spontaneous and engineered models of lupus in mice
have also demonstrated the critical role of hyperresponsive BCR
signaling in disease pathogenesis (34, 35, 51). Substantial evidence
suggests that primary human B cells from patients with SLE are
similarly hyperresponsive and that this phenotype is independent
of disease activity and treatment (33). Recent data from whole
genome association studies of SLE in humans have identified
genes in the BCR signaling pathway, including the B cell-specific
Src family kinase Blk and the adaptor BANK1 (52, 53). In this
PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL
regard, the B cell hyperresponsiveness of the CD45 E613R mice is
a representative model of human lupus pathogenesis.
Our lupus model is characterized by hyperresponsive T and B
cells. Each cellular phenotype taken independently is insufficient
to break tolerance, but in combination emergent phenotypes such
as polyclonal T and B cell activation and frank autoimmune dis-
ease develop. This is reminiscent of the genetic dissection of the
polygenic spontaneous murine lupus model NZB/NZW (New Zea-
land Black/New Zealand White) (54). Given the significance of the
PTPN22 genetic locus in human autoimmunity, our data suggest
that similar analysis may be valid in human disease. Indeed, given
the subtlety of the risk conferred by the R620W allele in human
disease, it is not surprising that cooperating functional phenotypes
such as B cell hyperreactivity may be required to break tolerance.
Although most common human autoimmune diseases are complex
polygenic traits, studies of such processes in mice using reverse
genetics have traditionally involved creating models in which a
single overwhelming genetic lesion provokes disease. These mod-
els are invaluable for understanding late disease pathogenesis and
testing therapeutic interventions. However, as whole genome scans
come of age and identify multiple subtle human genetic polymor-
phisms that cooperate in a very complex fashion, we need a novel
approach to understand how such polymorphisms function and in
In this report we have demonstrated that a loss-of-function
PTPN22 gene product in T cells recapitulates the R620W human
risk allele and can cooperate with hyperresponsive B cells to pro-
voke autoantibody production and a lupus-like autoimmune dis-
ease, reminiscent of its human disease associations.
We thank Al Roque for assistance with animal husbandry and members of
the Weiss Laboratory for helpful discussions. We are grateful to Jaime
Schoenborn for generating Csk constructs and assisting with transfection
experiments, and to Lyn Hsu for help with cloning.
The authors have no financial conflict of interest.
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PTPN22 DEFICIENCY COOPERATES WITH CD45 E613R IN AN SLE MODEL