PTPN22 Deficiency Cooperates with the CD45 E613R Allele to Break Tolerance on a Non-Autoimmune Background

Mice

CD45 E613R (CD45^w/w) mice were generated as previously described (31). All experiments described in this manuscript involve mice backcrossed 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. Möröy (Institut de Recherches Cliniques de Montréal, Montréal, Canada). All animals 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 phenotypic assessment are indicated in the text. All animals were housed in a specific pathogen-free facility at the University of California (San Francisco, CA) according to University and National Institutes of Health guidelines.

Histology

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 manufacturer’s protocol. IgG Abs to dsDNA in serum from individual mice were measured by ELISA as described (37). The results were expressed in arbitrary ELISA units relative to a standard positive sample derived from MRL/lpr mice.

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 negative control.

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-γ, 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. Polyclonal Abs to Pep (PEP1 and PEP2 generated against PRS1 and 2 domains, respectively) were compliments of A. Chan (Genentech). The Csk Ab (C-20 clone) was obtained from Santa Cruz Biotechnology. Stimulatory anti-CD3ε 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.

Flow cytometry

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 analyzed 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 (version 8.5.2) software (Tree Star).

Lymphocyte stimulation

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-CD3ε followed after 30 s by cross-linking with goat anti-Armenian hamster IgG. Plate-bound stimulation of T cells (for activation marker up-regulation) was conducted by precoating 96-well plates with anti-CD3ε 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).

Calcium flux

Single cell suspensions of thymocytes or lymphocytes were loaded with 4 μg of Fluo4-AM per 10⁷ cells (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 control.

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 stimulation. 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 software.

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 Biosciences), followed by secondary staining with goat-anti-rabbit Ab (Jackson 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.

Immunoblotting

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 reagent (PerkinElmer Life Sciences) and a Kodak Imaging Station.

Expression vectors

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-directed 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 California, San Francisco, CA).

Jurkat experiments

Jurkat cells were grown in RPMI 1640 supplemented with 10% FCS at a density of 0.1–0.6 × 10⁶ cells/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 μg of plasmid was introduced into 12 × 10⁶ cells 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 analysis of calcium flux, cells were loaded with fluo-3 and fura red (Molecular 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 concentration 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 hematopoietic 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 subsets might contribute to autoimmune disease in mice and which cellular context would serve best to study the functional consequences 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. 1⇓A) and flow cytometric detection of intracellular staining (Fig. 1⇓B) 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. 1⇓A and data not shown). We also noted increased expression in CD69^high activated and CD44^high memory/effector T cells, but were unable to detect significant protein 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).

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FIGURE 1.

Pep is expressed predominantly in T 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 independently generated Pep polyclonal Ab were obtained. B, Histograms of LN or splenic subsets stained with polyclonal 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 representative of three independent experiments.

The R620W allele of PTPN22 is a hypomorph in the context of Csk

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 phosphatase with partially redundant functions, is not expressed in Jurkat cells (and down-regulated in primary effector T cells) (18). This context supplies an opportunity to unmask Pep-dependent events.

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 “ectopic” 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 overexpression and dose.

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 correlates with cotransfected GFP levels in a flow-based assay (Fig. 2⇓B). Relative function of the WT and R620W Pep alleles is qualitatively unchanged at both high and low GFP levels in our studies (Fig. 2⇓B and data not shown), suggesting that overexpression artifact does not play a role here.

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FIGURE 2.

The murine Pep R620W polymorphism 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. Endogenous (endog) Lyp is not detected by this polyclonal Ab. B, Histograms correspond to samples in A and represent 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 unstimulated transfectants for comparison. Black-lined histograms represent stimulated transfectants as identified in A. C, Bar graph corresponding to samples generated and stimulated as in A and B. The percentage (%) of phospho-Erk-positive cells is defined as the percentage of cells that falls into the high expressing half of the bimodal histogram distributions in B. Error bars represent SEM. Plots and Western blots are representative of at least five independent experiments.

Total transfection efficiency was comparable between samples (data not shown), and Pep as well as Csk protein levels were comparable between samples (Fig. 2⇑A). By using murine Pep in our studies, we were able to take advantage of a polyclonal 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 overexpression and make use of endogenous Csk levels as an accurate loading control (Fig. 2⇑A).

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 experiments 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 phosphorylation (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 misexpression experiments, transfection efficiency and dose were controlled for by GFP cotransfection as well as Western blotting detection of Myc-tagged Lyp and Csk (Fig. 3⇓, A and B). Furthermore, in both Pep and Lyp experiments no defect in PMA-induced Erk phosphorylation was observed, implying a proximal target for Pep/Lyp function in TCR signaling (Figs. 2⇑B and 3⇓B).

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FIGURE 3.

The human Lyp1 R620W (T1858) polymorphism functions as a hypomorph in the context of Csk. A, Csk and WT or R620W Lyp1 were misexpressed 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, Histograms correspond to samples in A and represent 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 unstimulated transfectants for comparison. Black-lined histograms represent stimulated transfectants as identified in A. C, Bar graph corresponding to samples generated and stimulated as in A and B. The percentage of phospho-Erk positive cells are defined as in Fig. 2C. Error bars represent SEM. D, Intracellular calcium levels of Lyp1/Csk transfected Jurkat cells (gated for cotransfected CD16). The fluo3/fura red ratio was monitored by flow cytometry 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.

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. 3⇑D). 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 signaling 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, CD3ε, CD3ζ, and Vav (38). These flow-based readouts effectively capture the functional phenotype of the Pep and Lyp phosphatases. More proximal substrates such as Lck 394 are difficult to perturb and assay in the context of minimal misexpression.

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 Pep-deficient mouse.

Double mutant CD45^w/w/Pep^−/− mice develop a lupus-like disease

CD45 E613R (CD45^w/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 CD45^w/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 CD45^w/w background and aged (CD45^w/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 CD45^w/w/Pep^−/− mice developed IgG autoantibodies to dsDNA in a partially penetrant fashion, with elevated titers detectable by ELISA as early as 3 mo of age and increasing with time (Fig. 4⇓A). 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 staining of Hep2A cells and revealed a homogeneous nuclear pattern, consistent with the original observation in mixed B6/129 background CD45^w/w mice (Fig. 4⇓A). In contrast, CD45^w/w/DNase1^−/− mice developed no significant autoantibody titers up to an age of 10 mo (Fig. 4⇓B). 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).

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FIGURE 4.

CD45^w/w/Pep^−/− double mutant mice develop lymphoproliferation and autoimmunity. Abbreviations apply to all subsequent figures: WEDGE, CD45^w/w; PEP, Pep^−/−; DNASE1, DNase1^−/−; DOUBLE, CD45^w/w/Pep^−/−. A, ELISA for IgG autoantibodies reactive to dsDNA were performed on sera from CD45^w/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 CD45^w/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, Representative 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. Similar 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 CD45^w/w/Pep^−/− double mutant (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.

We noted that as early as 8 wk of age, the CD45^w/w/Pep^−/− mice developed lymphadenopathy and splenomegaly that were progressive with time and greatly exceeded the mild lymphoproliferation evident in each of the single mutants (Fig. 4⇑C). LN absolute cell counts were variably elevated among the aging cohort, with some reaching 10-fold normal levels by 6 mo of age (Fig. 4⇑D). The lymphadenopathy was generally out of proportion to the degree of splenomegaly observed, suggesting a lymphoid-predominant dysregulation of the hematopoietic compartment (Fig. 4⇑C and data not shown). In contrast, LN size, LN cell number, and spleen weights in CD45^w/w/DNase1^−/− mice were comparable to the values in CD45^w/w single 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 CD45^w/w/Pep^−/− mice.

Although CD45^w/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 proteinuria 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 CD45^w/w/Pep^−/− mice had died (n = 15). The remaining genotypes had normal life expectancy.

We next attempted to define the cause of death in the CD45^w/w/ Pep^−/− mice. The surviving CD45^w/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 examination of CD45^w/w/Pep^−/− kidneys revealed marked abnormalities, including perivascular lymphocytic infiltrates, interstitial lymphocytic infiltrates, and hypercellular glomeruli with evidence of segmental sclerosis (Fig. 4⇑, E–H). CD45^w/w single mutant mice showed very mild evidence of similar abnormalities in kidney histology. Both CD45^w/w/Pep^−/− and CD45^w/w single 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 CD45^w/w/Pep^−/− mice.

Given the presence of lymphoproliferation, autoantibody production, autoimmune kidney pathology, and mortality, we conclude that the CD45^w/w/Pep^−/− mice develop a lupus-like disease in a partially penetrant fashion that recapitulates the original disease phenotype observed in CD45 E613R animals on the B6/129 mixed background (31). Neither CD45^w/w nor Pep^−/− single mutant mice developed lupus-like autoimmunity over the course of 12 mo of observation. CD45^w/w/DNase1^−/− mice displayed no evidence 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 autoimmunity arises in the CD45^w/w/Pep^−/− double mutant mice, we assessed 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. 5⇓A). The CD4:CD8 peripheral T cell ratio was increased consistently in the double mutants and less so in each single mutant relative to WT in both LN and spleen (Fig. 5⇓B and data not shown). This ratio shift was evident as early as 6 wk of age and persisted throughout the observation period (12 mo).

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FIGURE 5.

T cell activation and differentiation in CD45^w/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 replicates ± 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 represent 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, Representative 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 involving three animals per genotype. WDG, WEDGE; DBL, DOUBLE.

The most marked cellular phenotype was the progressive expansion of the CD4 and CD8 T cell memory/effector compartments 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 double 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 deficiency

In contrast to T cells, B cells up-regulated activation markers such as CD69 as well as CD80/86 in CD45^w/w and double mutant mice, but not in Pep^−/− mice. The activation status of B cells at age 2 mo was matched in CD45^w/w and double mutant mice, but by 6 mo of age double mutant mice further up-regulated B cell activation markers (Fig. 6⇓A and data not shown). The plasma cell compartment identified by cell surface CD138 expression was expanded in CD45^w/w and double mutant spleens to a similar extent, whereas Pep^−/− mice resembled WT (Fig. 6⇓B).

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FIGURE 6.

Polyclonal B cell activation and perturbed 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 identify 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; DBL, DOUBLE.

BCR signaling is critical for B cell development, which is characteristically perturbed in CD45^w/w mice (32). Using the markers CD21, CD23, IgM, IgD, and AA4.1, we found that double mutant mice precisely phenocopied the CD45^w/w splenic 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 CD45^w/w mice, double mutant and CD45^w/w mice 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.

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 CD45^w/w B cells in spleen and LNs were found to be hyperresponsive to in vitro BCR stimulation as assessed by Erk phosphorylation. Notably, Pep^−/− B cells responded like WT B cells, whereas double mutant B cells responded like CD45^w/w B cells with no evidence of unmasked synergy in the double mutant (Fig. 7⇓A). We identified similar trends with alternate readouts downstream of BCR ligation such as calcium flux, activation marker up-regulation, and CFSE dilution (Fig. 7⇓B 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).

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FIGURE 7.

Pep^−/− does not contribute to CD45^w/w B cell hyperresponsiveness. A, Lymph node or splenic B cells were stimulated for varied periods of time (0, 1, 3, and 5 min) with goat anti-mouse IgM F(ab′)₂ or, 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, CD45^w/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 log₂ scale 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.

Although LN B cells represent a relatively uniform, predominantly mature phenotype, splenic B cells include multiple developmental stages, each with unique signaling thresholds. To define the precise stage of B cell development at which CD45^w/w contributes 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 CD45^w/w and double mutants were markedly hyperresponsive, consistent with the LN data (Fig. 7⇑C).

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 intact as assessed by double negative, double positive, and single positive subset compartment size (data not shown). Double negative subsets 1–4 as assessed by CD44 and CD25 surface markers were also unperturbed without evidence of abnormalities in β-selection (data not shown).

Both Pep^−/− and CD45^w/w mice have been previously independently crossed to TCR transgenes (H-Y, OT-2, and DO11.10), revealing increased positive selection in each background (17, 55); (M. Hermiston, manuscript in preparation). We therefore characterized the cell surface phenotype of double positive (DP) thymocytes. We noted up-regulation of CD5 and CD69 in double mutant mice relative to WT and less so in each single mutant (Fig. 8⇓A and data not shown). These markers are well-established correlates of TCR signal strength and suggest that CD45^w/w and Pep^−/− cooperate in a cell intrinsic manner at the DP thymocyte stage of T cell development (39).

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FIGURE 8.

Pep^−/− and CD45 E613R alleles influence TCR signaling in a developmental stage-specific manner. A, Graph depicts mean fluorescence intensity (MFI) for CD5 staining on gated DP thymocytes. Values are the means of three biological replicates ± SEM and represent at least five independent experiments, each involving three animals per genotype. B, Intracellular 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-CD3ε Ab in the upper panel or ionomycin in the lower panel. Genotypes are identified as in A. Plots are representative of at least four independent experiments. C, DP thymocytes were stimulated with soluble anti-CD3ε. 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 histogram 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 independent experiments each with three biological replicates per genotype. E, In vitro generated effector CD4 or CD8 T cells were stimulated, fixed, and permeabilized 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 experiments.

We tested this hypothesis by stimulating the TCR on DP thymocytes in vitro and assessed Erk phosphorylation and calcium flux by flow cytometry. These studies revealed subtle hyperresponsiveness 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. 8⇑B and data not shown).

CD45 E613R does not contribute to TCR hyperresponsiveness in peripheral T cells

Although Pep^−/− and CD45^w/w cooperate to enhance TCR signaling in double mutant DP thymocytes, such a perturbation should in theory increase both positive and negative selection, resulting 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 mutations cooperate to break tolerance. Because peripheral T cell subsets have qualitatively distinct signaling properties, we systematically 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 significant contribution to TCR signaling in peripheral T cells.

Because of the unequal size and signaling properties of CD44^high memory and CD44^low naive T cell subsets in our four genotypes, we compared responsiveness to TCR ligation separately 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 (CD44^low) T cells where we observed consistent and equivalent TCR hyperresponsiveness in Pep^−/− and double mutant mice (Fig. 8⇑D).

We confirmed these observations by examining an event further downstream of TCR stimulation. We evaluated CD69 up-regulation specifically in CD44^low-gated naive T cells. Double mutant CD8 T cells consistently responded comparably to Pep^−/− T cells, whereas CD45^w/w T 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 dilution failed to reveal consistent differences between the four genotypes, 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 CD45^w/w naive or pooled peripheral T cells.

To explain the accumulation of memory/effector T cells in double mutant mice in the absence of a unique double mutant naive T cell phenotype, we hypothesized a stage-specific influence of CD45^w/w and 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. 8⇑E). Consistent with these observations, Hasegawa et al. have previously 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 progressive accumulation of memory/effector T cells in double mutant animals cannot be accounted for by a purely T cell-intrinsic signaling phenotype.

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 susceptibility locus in humans, and the genetic context in which it might do so. The R620W polymorphism disrupts the critical interaction of Pep/Lyp with Csk and thereby impairs the ability of Pep to effectively access its target Lck (2). Prior studies of the functional consequences of this polymorphism have led to conflicting 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 (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 stoichiometry than prior work and control explicitly for expression level and overexpression artifact. These differences may explain discrepancies with prior reports. Furthermore, we can now exclude the possibility that human and murine orthologues may exhibit functional differences in this context. It remains a formal possibility that Pep and/or Lyp regulates signaling pathways other than those downstream of the Ag receptor and targets 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 anticyclic citrullinated peptide Ab-positive RA and not with a seronegative 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 significant 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 plausibly 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 overexpression in cell lines. Certainly, a gain of function Pep/Lyp allele could impair negative selection in the thymus or regulatory T cell function in the periphery. Both types of perturbations can provoke autoimmune disease in mice (and rarely in humans) (43, 44). Alternatively, we have shown that a hypomorphic PTPN22 variant in T cells might serve to drive hyperresponsive B cells to break tolerance in the periphery and produce autoantibodies, as we see in the CD45^w/w/Pep^−/− model. The association of the R620W allele almost exclusively with autoantibody-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 background to provoke overt autoimmune disease. By generating disease in the context of a bona fide human susceptibility allele, our model sheds light on the early pathogenesis of human autoimmune disease.

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 transduction, 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 autoreactive clones from escaping to the periphery. As a result of enhanced 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 peripheral 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 impaired rather than enhanced selection in the thymus (43, 44, 45, 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 hyperresponsiveness 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/memory T cells in the double mutant mice is significantly accelerated relative to either mutation in isolation. We conclude that the CD45^w/w microenvironment makes a purely cell-extrinsic contribution to this “indirect” T cell phenotype. The CD45^w/w contribution could represent the influence of myeloid and/or B cell compartments. Other murine models of lupus have demonstrated that both scenarios are possible (47, 48, 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 altered 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 influenced exclusively by the CD45^w/w genetic background and is specifically perturbed at the “final” FO mature stage of B cell development. We therefore conclude that the unique B cell phenotypes seen only in the double mutant mouse such as augmented polyclonal 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 develop 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 CD45^w/w B cells are ideally situated to cooperate productively in such a context. These genetic interactions between Pep deficiency and the CD45^w/w background bypass tight central tolerance mechanisms and lead to a tolerance break only in the periphery. Indeed previous analysis of CD45^w/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 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 disease develop. This is reminiscent of the genetic dissection of the polygenic spontaneous murine lupus model NZB/NZW (New Zealand 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.

Conclusion

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 models 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 polymorphisms that cooperate in a very complex fashion, we need a novel approach to understand how such polymorphisms function and in which contexts.

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 provoke autoantibody production and a lupus-like autoimmune disease, reminiscent of its human disease associations.