A Precision B cell-targeted Therapeutic Approach to Autoimmunity Caused by PI3K Pathway Dysregulation

Abstract

The inositol lipid phosphatases, PTEN and SHIP-1 play a crucial role in maintaining B cell anergy and are reduced in expression in B cells from SLE and T1D patients, consequent to aberrant regulation by miRNA-7 and 155. With an eye towards eventual use in precision medicine therapeutic approaches in autoimmunity, we explored the ability of p110δ inhibition to compensate for PI3K pathway dysregulation in mouse models of autoimmunity. Low doses of the p110δ inhibitor Idelalisib, which spare the ability to mount an immune response to exogenous immunogens, are able to block the development of autoimmunity driven by compromised PI3K pathway regulation resultant from acutely-induced B cell-targeted haploinsufficiency of PTEN and SHIP-1. These conditions do not block autoimmunity driven by B cell loss of the regulatory tyrosine phosphatase SHP-1. Finally, we show that B cells in NOD mice express reduced PTEN, and low dose p110δ inhibitor therapy blocks disease progression in this model of T1D. These studies may aid in the development of precision treatments that act by enforcing PI3K pathway regulation in patients carrying specific risk alleles.

Introduction

Multiple mechanisms are involved in the maintenance of B cell tolerance to autoantigens. In the bone marrow, receptor editing and clonal deletion ensure that B cells undergoing high avidity interactions with self-antigens are removed from the repertoire (1–4). However, B cells recognizing lower avidity self-antigens do not undergo receptor editing, but instead are released into the periphery where they are maintained transiently in an unresponsive state called anergy (5–7). Anergy is rapidly reversible, requiring chronic receptor stimulation by self-antigen (8, 9), suggesting maintenance by nondurable biochemical mechanisms. Anergy is therefore a fragile state and these cells represent a pool of autoreactive cells that may participate in pathogenic autoimmune responses under circumstances of immunological stress such as inflammation. Increasing evidence indicates that a number of genetic alleles that confer increased risk of autoimmunity may act by weakening intrinsic mechanisms that maintain the unresponsiveness of anergic B cells (10–16).

Genome-Wide Association (GWAS) and candidate studies have revealed more than 100 genetic polymorphisms that confer increased risk of developing Systemic Lupus Erythematosus (SLE) (17), several of which encode molecules thought to function in regulation of B cell antigen receptor (BCR) signaling (reviewed here: (18). Precise regulation of BCR signaling is key to ensuring that protective responses are mounted against potential pathogens, while preventing responses to self or endogenous antigens. Maintenance of the anergic state of peripheral autoreactive B cells involves multiple regulatory mechanisms that operate proximally in BCR signaling. Among these are inositol lipid phosphatases, PTEN and SHIP-1 that, in anergic cells prevent the BCR mediated accumulation of PI(3,4,5)P3, which is crucial for recruitment and activation of PH-domain-containing signaling intermediaries such as Bruton’s tyrosine kinase (BTK) and phospholipase Cγ (PLCγ) (19–21). Acting in concert with parallel signaling pathways, these effectors function in B cell activation and differentiation. Certain alleles of genes that encode or regulate expression of components of this axis, including PTEN (22), SHIP-1 (23), SHP-1 (24, 25), Csk (16), PTPn22 (10–13) and Lyn (14, 15) have been shown to confer risk of autoimmunity (26). We, and others, have shown that acute deletion of SHIP-1 or PTEN and expression of a constitutively active catalytic subunit of PI3K in anergic B cells leads to immediate loss of anergy followed by cell proliferation, differentiation, and production of autoantibodies, thus demonstrating the importance of these proteins and their regulation of the PI3K pathway in maintaining B cell anergy (19, 27, 28). Importantly, B cells from SLE, Type 1 Diabetes (T1D) and Autoimmune Thyroiditis (AITD) patients express reduced levels of PTEN, consistent with a possible role in autoimmunity (22, 29). The apparent inability to regulate the PI3K pathway in these patients suggests that inhibition of PI3K could, by compensating for reduced inositol lipid phosphatase activity, be an affective therapeutic.

PI3Ks regulate numerous biological functions via generation of inositol lipid second messengers. Class IA PI3Ks are heterodimeric proteins comprised of a regulatory subunit (p85α, p85β or p55γ) and a catalytic subunit (p110α, p110β or p110δ) that function in antigen, costimulatory and cytokine receptor signaling. Class IB PI3Ks consist of a regulatory subunit, p101, and a catalytic subunit, p110γ, and are activated by chemokine receptor signaling. p110δ and p110γ are restricted in expression to the lymphoid compartment with nonredundant, nonoverlapping roles, whereas p110α and p110β are ubiquitously expressed and removal of these subunits results in embryonic lethality (30, 31). There is a growing body of evidence indicating that p110δ is the functionally dominant isoform utilized in BCR signaling (32–34). p110δ deficient mice show marked phenotypic changes in the B cell compartment, with defects in BCR-mediated calcium mobilization, decreased germinal center formation and reduced antibody responses to both T-dependent and T-independent antigens (35). To eliminate potential confounding compensation from other isoforms, Okkenhaug and colleagues introduced a point mutation in p110δ that resulted in an amino acid change, p110δ^D910A, rendering the enzyme catalytically inactive. p110δ^D910A mice have drastically decreased B cell responses, both in vivo and in vitro, with slight reduction in T cell populations (36). There is conflicting evidence regarding the functional importance of p110δ in T cells, as no defects or only mild defects are observed in knockout mice (35–38). This topic is expertly reviewed elsewhere (39). The minimal effect of p110δ deficiency on the T cell compartment is presumably due to the compensation by redundant p110 isoform function, that enables normal T cell function in the absence of p110δ (40). It is noteworthy that p110δ^D910A T cells have a more naïve-like phenotype in the periphery, suggesting T cells develop, but fail to mature normally (33). Conversely, p110γ knockout mice have marked reductions in development and function of the T cell compartment, both in vitro and in vivo, while B cell responses and populations are unaltered (41). Recent in vitro data utilizing isoform specific inhibitors of p110δ, p110γ and dual p110δ and p110γ attempt to tease apart the contribution of individual isoforms in B cell signaling and B cell responses. Inhibition of both p110δ and p110γ did not suppress B cell responses more than inhibition of p110δ alone (42). Additionally, treatment with p110γ inhibitor alone did not have an effect on B cell proliferation, survival or plasmablast formation, suggesting that p110γ plays a minor role in B cell function (43). Thus p110δ is critical for B cell but not T cell function.

We hypothesized the autoimmunity caused by failed regulation of PI(3,4,5)P3 levels in B cells might be corrected by compensatory inhibition of p110δ using low doses of pharmacologic inhibitor which preserve the ability of naïve B cells to mount protective immune responses. Idelalisib, or CAL-101, is a reversible p110δ inhibitor that noncovalently binds the ATP binding pocket of the catalytic subunit (32). Idelalisib targets the p110δ isoform with 110–453 fold more selectivity than other class 1 isoforms. Idelalisib was FDA approved for the treatment of Lymphocytic Lymphoma, Chronic Lymphocytic Leukemia, and Non-Hodgkin’s Lymphoma in 2014 (44), and has completed a Phase I clinical trial for treatment of allergic rhinitis (45). It is noteworthy that doses of Idelalisib (30mg/kg) used in these applications do indeed significantly deplete B cells, and a black box warning has been issued for fatal and/or severe colitis, pneumonitis and infection (46, 47). However, we reasoned that at lower doses Idelalisib may specifically prevent/treat autoimmunity caused by defective PI3K pathway regulation.

Here we demonstrate that compensation of failed PI3K pathway regulation using low doses of PI3K inhibitor is sufficient to delay development of autoimmunity in VH125.NOD mice, a murine model of T1D. Chronically treated animals remain immunocompetent as indicated by production of class switched high affinity antibodies in response to immunization. We show that low dose p110δ inhibition can selectively inhibit participation in autoimmunity of autoreactive B cells that have lost anergy due to defective PI3K pathway regulation, while autoreactive B cells that have lost anergy due to loss of a regulatory tyrosine phosphatase, SHP-1, still develop autoimmunity. We report that low dose Idelalisib treatment does not affect in vitro or in vivo T cell responses. This study supports the principle of effective generation of precision therapies based on predisposing genetic factors, and should provide a precise therapeutic approach for patients that possess risk alleles that compromise PI3K pathway regulation.

Materials and Methods

Mice

Except where otherwise indicated, 6–16 week old mice were used in all experiments. Both male and female mice were used, but experiments were sex matched and both sexes gave identical results, with the exception of only female mice being used in the VH125.NOD experiments as female mice develop accelerated disease. Dr. J.W. Thomas (Vanderbilt) generously provided VH125.NOD animals. hCD20-TamCre animals (48) were intercrossed with mice carrying the rosa26-flox-STOP-YFP allele (49), generating mice in which YFP is expressed in B cells upon Cre activation. These mice were crossed with Ars/A1 (50) BCR transgenic mice to generate hCD20-TamCre × rosa26-flox-STOP-YFP × Ars/A1 mice. B cells from these mice will be referred to as WT Ars/A1. These mice were also crossed with SHIP-1^flox/flox mice {gift from J. Ravetch and S. Bolland, The Rockefeller University, New York, NY; (51)}. hCD20-TamCre × rosa26-flox-STOP-YFP × Ars/A1 mice were also crossed to PTEN^flox/flox mice {gift from R. Rickert, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA; (52)} and SHP-1^flox/flox mice (53) to generate mice in which SHIP-1, PTEN and SHP-1 deletion can be induced in anergic B cells. hCD20-TamCre × rosa26-flox-STOP-YFP × PTEN^flox/flox × Ars/A1 were crossed to hCD20-TamCre × rosa26-flox-STOP-YFP × SHIP-1^flox/flox × Ars/A1 to generate hCD20-TamCre × rosa26-flox-STOP-YFPx PTEN^flow/wt × SHIP-1^flox/wt × Ars/A1 to allow for the double haploinsufficiency of both PTEN and SHIP-1 within B cells. Mice were housed and bred at the Biological Resource Center at National Jewish Health or at the University of Colorado Anschutz Medical Center Vivarium, with the exception of C57BL/6 mice and CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ) which were purchased from Jackson ImmunoResearch Laboratories, Inc. All experiments with mice were performed in accordance with the regulations and approval of National Jewish Health and the University of Colorado Denver Institutional Animal Care and Use Committee.

Adoptive transfers and tamoxifen induction

2 hours before adoptive transfer, C57BL/6 recipient mice received 200 rads irradiation. For MD4 transfers, recipients did not receive prior irradiation. B cells from donor mice were isolated via depletion of CD43+ cells with anti-CD43-conjugated magnetic beads (MACS anti-mouse CD43; Miltenyi Biotec). Alternatively, CD4+ T cells were isolated via CD4 positive selection (MACS anti-mouse CD4 (L3T4) Miltenyi Biotec). Resultant populations were >97% pure based on flow cytometric analyses. Donor B cells were labeled with either CellTrace Violet (Molecular Probes) or CFSE (Molecular Probes) at 5μM for 5 minutes at room temperature prior to transfer. Donor CD4 T cells were labeled with CFSE (Molecular Probes) at 5μM for 5 minutes at room temperature prior to transfer. 1–2×10⁶ donor cells in 200μl PBS were adoptively transferred via IV injection. 24 hours post transfer, tamoxifen was administered to activate Cre. Tamoxifen (T-5648; Sigma-Aldrich) was dissolved in 100% corn oil (Sigma-Aldrich) at 20mg/ml. Recipient mice were injected IP with 100μl (2mg) on two consecutive days.

Manufacturing and administration of Idelalisib containing rodent chow

Idelalisib (LC Laboratories) was shipped to ResearchDiets Inc. for blending the compound homogenously into modified OSD with 24 kcal% protein, 16 kcal% fat, 60% kcal% carbohydrate, 100g of cellulose and 25g inulin. Diet dose (DD) is calculated by multiplying the single daily dose (SD) by the body weight of a mouse (BW) and dividing that by the daily food intake (FI) [DD= (SD × BW)/FI]. Chow used includes base chow as described above with: + 0mg Idelalisib/kg diet (Vehicle Control), + 600mg Idelalisib/kg diet (30mg/kg ingested Idelalisib dose), + 75mg Idelalisib/kg diet (3.75mg/kg ingested Idelalisib dose), and + 18.75mg Idelalisib/kg diet (0.9375mg/kg ingested Idelalisib dose).

For PTEN^fl/wt × SHIP-1^fl/wt and SHP-1^fl/fl adoptive transfers, control chow and Idelalisib containing chow was given to mice on day 7 post-tamoxifen administration. For MD4 and OT-II adoptive transfers, control chow and Idelalisib containing chow was given to mice 2 days post transfer. For VH125.NOD experiments, immediately following weaning, animals are placed on control chow. Following 2 consecutive diabetic blood glucose readings (between 150–200mg/dl) animals either remain on vehicle control chow or are placed on 0.9375mg/kg Idelalisib containing chow and disease progression is monitored. For NP₄Ova-alum experiments, animals were placed on either vehicle control chow or 0.9375mg/kg Idelalisib chow for 28 days and subsequently immunized.

Antigens and immunization

HEL conjugated to SRBCs was used to produce antigen for experiments with MD4 B cells. SRBCs were purchased from the Colorado Serum Company, and stored in Alsever’s solution at 4°C. SRBCs are washed 3 times in PBS prior to use. 1ml of 50mg/ml of the chemical crosslinker N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (Sigma-Aldrich) was added to 1ml packed SRBCs and 15ml of 5mg/ml HEL (Sigma-Aldrich), mixed and rotated at room temperature for 45 minutes. Mice were immunized IP with 200μl of a 5% HEL-SRBC in PBS. For NP₄Ova-Alum immunizations, 10mg/ml alum and 5mg/ml NP₄Ova were mixed to a final concentration of 250μg/ml NP₄Ova and 2.5mg/ml alum and rotated for 3 hours at room temperature. NOD mice were placed either on vehicle control chow or 0.9375mg/kg Idelalisib chow for 28 days. Mice were then immunized with NP₄Ova-alum IP in 200μl/mouse, and responses were measured on day 7 and day 14 post-immunization.

Phenotypic analysis by FACS

Spleens were mechanically disrupted, single-cell suspensions were generated, and red blood cells were lysed with ammonium chloride TRIS. Cells were resuspended in PBS containing 1% FBS and incubated with indicated antibodies. For analysis of cell surface markers, antibodies against the following molecules were used: B220-PE (RA3–6B2; BioLegend), B220-BV786 (RA3–6B2; BD Biosciences), B220-BV510 (RA3–6B2; BioLegend), CD4-BV711 (GK1.5; BioLegend), CD8 BV421 (53–6.7; BioLegend), CD138-PECy7 (281–2; BioLegend), CD69-BV786 (H1.2F3; BD Biosciences), CD86-PerCPCy5.5 (GL-1; BioLegend). After cell surface staining, the cells were fixed and permeabilized with Cytofix/Cytoperm (BD) (as per manufacturer’s instructions) and stained with Dylight650-E4 anti-Ars/A1 idiotype (produced and conjugated in our laboratory) OR HEL-650 (produced and conjugated in our laboratory) and Alexa Fluor 488-anti-GFP (rabbit polyclonal; Life Technologies). Events were collected on a CyAn ADP (Dako) and subsequent analysis using FlowJo software (Tree Star).

Analysis of calcium mobilization

For measurements of intracellular free calcium concentration ([Ca²⁺]_i), RBC-depleted single-cell suspended splenocytes were simultaneously stained with CD8-PE (53–6.7; BD Biosciences), CD4-APC (GK1.5; BioLegend) or B220-PE (RA3–6B2; BioLegend) and loaded with Indo-1 acetoxymethyl (Indo1-AM; Molecular Probes), as described previously (8). For analysis of ([Ca²⁺]_i), cells were suspended at 10×10⁶ cells/ml in warm IMDM + 2% FBS in a 500ul volume. Cells were acquired for 30 seconds to establish a baseline, and then stimulated with 5μg/ml of F(ab’)₂ rabbit anti-mouse anti-IgG (H&L; Invitrogen) +/− indicated doses of Idelalisib (LC Laboratories) and acquired for 3 minutes. For CD4 and CD8 T cells, cells were acquired for 30 seconds to establish a baseline, and then 10μg/ml of anti-CD3-biotin (145–2C11; BD Biosciences) +/− indicated doses of Idelalisib was added, 60 seconds later 20μg/ml streptavidin (Sigma-Aldrich) was added and acquired for 3 minutes. Mean relative ([Ca²⁺]_i) was monitored over time using an LSR Fortessa X-20 (BD) with analysis using FlowJo software (Tree Star).

Analysis of phosphorylated signaling intermediaries

RBC-depleted single-cell suspended splenocytes were suspended at 10×10⁶ cells/ml in serum free IMDM, +/− indicated doses of Idelalisib (LC Laboratories) (as indicated in Figure 4) and rested for 1 hour at 37°C. Cells are then washed 2X in serum free IMDM, and stimulated with 5μg/ml of F(ab’)₂ rabbit anti-mouse anti-IgG (H&L; Invitrogen) or 10μg/ml anti-CD3-biotin (145–2C11; BD Biosciences) + 20μg/ml streptavidin (Sigma-Aldrich) for 2 minutes. Signaling was stopped by addition of 20% PFA to a final concentration of 2%, incubated at 37°C for 15 minutes and resuspended in 100% ice-cold MeOH (directly from −80°C). Cells were then placed on ice for 30 minutes and placed at −20°C for storage. For analysis, cells were stained with B220-BV786 (RA3–6B2; BD Biosciences), CD4-BV711 (GK1.5; BioLegend), CD8-BV421 (53–6.7; BioLegend) and/or pAKT-Alexa Fluor 647 (pS373; M89–61; BD Biosciences), pPLCγ-PE (pY759; K86–689.37; BD Biosciences), pBTK-BV421 (pY180; N35–86; BD Biosciences) pSyk/Zap70-PE (pY352/pY319; BD Biosciences) at room temperature for 1 hour. Cells were washed 3 times and samples were acquired in triplicate on an LSR Fortessa X-20 (BD) with analysis using FlowJo software (Tree Star).

Figure 4. p110δ inhibition suppresses B cell calcium flux and reduces phosphorylation of downstream signaling intermediaries.

(A) Calcium flux of B220+ cells stimulated with anti-H&L with simultaneous addition of 0nM (black line), 15nM (solid grey line), 60nM (grey long dashed line) or 490nM (grey dashed line) Idelalisib. (B) Quantification of area under the curve (AUC) seen in A. (C) Quantification of phosphorylated signaling intermediaries after preincubation with indicated doses of Idelalisib and BCR stimulation. (Idel:= Idelalisib. n = 5/group. Data shown are representative of at least three replicated experiments. Bars in B & C represent mean ± SEM. Student T test was used to calculate statistics in B. One-Way ANOVA and Tukey’s multiple comparisons test was used to calculate statistics in C. *=p<0.05, **= p<0.01, ***=p<0.005, ****=p<0.0001).

Enzyme linked immunosorbent assay

For detection of IgM^a anti-Ars antibodies, microtiter plates were coated with 10μg/ml Ars-BSA16 in PBS and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of IgM^a anti-HEL antibodies, microtiter plates were coated with 10μg/ml HEL in PBS and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of total NP-specific IgM and IgG, microtiter plates were coated with 20μg/ml NP₂₇BSA and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of high affinity NP-specific IgM and IgG, microtiter plates were coated with 20μg/ml NP₂BSA and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. Serial dilutions of mouse serum in PBS were added and incubated overnight at 4°C. Ars/A1-derived IgM^a antibodies and MD4-derived HEL IgM^a antibodies were detected with biotinylated DS.1 anti-IgM^a (BD Pharmingen) in PBS, followed by Streptavidin-HRP (Thermo Fisher Scientific). For NP-specific IgM antibodies were detected using goat-anti-mouse IgM-HRP (SouthernBiotech). For NP-specific IgG antibodies were detected using goat-anti-mouse IgG-HRP (SouthernBiotech). Between all steps, plates were washed 3 times with PBS 0.05% Tween-20. The ELISA was developed with TMB single solution (Invitrogen) and the reaction was stopped with 1M HCl. OD was measured at 450nm using a VERSAMax plate reader (Molecular Devices) and data analyzed with SoftMax Pro6 software.

ELISPOT

For detection of IgM^a anti-Ars antibodies, microtiter plates were coated with 10μg/ml Ars-BSA16 in PBS and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of IgM^a anti-HEL antibodies, microtiter plates were coated with 10μg/ml HEL in PBS and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of total NP-specific IgM and IgG, microtiter plates were coated with 20μg/ml NP₂₇BSA and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. For detection of high affinity NP-specific IgM and IgG, microtiter plates were coated with 20μg/ml NP₂BSA and blocked with 2mg/ml BSA in PBS 0.05% Tween-20. Plates were washed 3 times prior to use with PBS 0.05% Tween-20. RBC-depleted single-cell suspension of splenocytes in complete medium were added in two-fold serial dilutions starting at 1/100^th of a spleen in the first well. Plates were incubated overnight at 37°C. Ars/A1-derived IgM^a antibodies and MD4-derived HEL IgM^a antibodies were detected with biotinylated DS.1 anti-IgM^a (BD Pharmingen) in PBS, followed by Streptavidin-AP (Southern Biotech). For NP-specific IgM antibodies were detected using goat-anti-mouse IgM-AP (SouthernBiotech). For NP-specific IgG antibodies were detected using goat-anti-mouse IgG-AP (SouthernBiotech). Between all steps, plates were washed 3 times with PBS 0.05% Tween-20. The plates were developed by incubated with ELISPOT developing buffer (25μm 5-bromochloro-3-indolyl phosphate p-toluidine, 100nm NaCl, 100mM Tris, and 10mM MgCl₂, pH 9.5) for 1 hour. The reaction was stopped by washing the plates 3 times with PBS 0.05% Tween-20. The number of spots at a cell dilution in the linear range was determined, and the number of ASCs was calculated.

Statistics

Data were analyzed using Prism GraphPad Software. Statistical analyses were performed using the indicated statistical tests in figure legends. P values ≤0.05 were considered statistically significant. Throughout, asterisks used to denote p-values of: * = p≤0.05, ** = p≤0.01, *** = p≤0.005 and **** = p≤0.0001.

Results

Idelalisib prevents T1D progression in VH125.NOD mice

At the outset of our studies we attempted a “home run” experiment, exploring the ability of low doses of p110δ inhibitor to arrest progression of T1D in a genetically complex model of autoimmunity. The most commonly used mouse model of T1D, the non-obese diabetic mouse, reflects disease progression in the human (54). Female NOD mice develop overt diabetes at ~20 weeks of age, with lymphocytic infiltration of the islets and autoantibody production preceding hyperglycemia and diabetes. NOD mice are protected from disease development upon deletion of the B cell compartment (NOD.uMT^−/−) (55, 56), or upon skewing of the BCR repertoire away from insulin reactivity (VH281.NOD) (57), but not upon removal of autoantibody (mIgM NOD) (58). In these studies we utilized the VH125.NOD mouse model of disease in which mice carry an immunoglobulin heavy chain transgene specific for insulin, the dominant autoantigen in T1D (59). Importantly, the transgenic heavy chain can pair with any endogenous light chain, resulting in a frequency of peripheral B cells reactive with insulin of 1–3%. In WT female NOD mice, disease penetrance only reaches ~70%, but skewing of the B cell repertoire towards insulin reactivity leads to 100% penetrance of disease in female mice and earlier disease onset (57).

While multiple insulin-dependent (Type 1) diabetes (Idd) loci contribute to disease development in NODs (60), B cells in these mice exhibit a marked reduction in PTEN levels in both insulin-reactive B cells and total B cells (Smith and Cambier, manuscript in revision) compared to closely related, autoimmunity resistant VH125.C57BL/6-H2g7 mice. On this background, but not VH125.NOD, high affinity insulin-reactive B cells are anergic. NOD mice have increased susceptibility to additional autoimmune diseases, such as Rheumatoid Arthritis (RA), SLE and the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS (61). Reduced B cell expression of PTEN has been reported in lupus patients (22), and we have observed reduced PTEN levels in the B cells of both T1D and AITD patients (29). Thus loss of B cell tolerance in both man and mouse may be driven in part by PI3K pathway dysregulation. We therefore postulated VH125.NOD mice and T1D patients, both of which have PI3K pathway regulation defects, may benefit from low dose Idelalisib, a p110δ inhibitor, to reinstate anergy of autoreactive B cells.

To test this possibility, immediately post-weaning, female VH125.NOD mice were placed on vehicle control chow to allow habituation to the diet. Upon two consecutive blood glucose readings in the pre-diabetic range, some mice were maintained on the vehicle control chow, while others were fed 0.9375mg/kg Idelalisib containing chow (Figure 1A). Disease progression was monitored based on blood glucose levels and visible signs of disease (i.e., hunching, scruffy fur, excessive urination). In mice receiving 0.9375mg/kg Idelalisib, disease progression was significantly delayed, and survival was extended (Figure 1B, Supplemental Figure 1).

Figure 1. Low dose Idelalisib delays disease progression in VH125.NOD mice without compromising response to immunization.

(A) A schematic representation of the survival experimental protocol. (B) Disease incidence as measured by % non-diabetic (two consecutive blood glucose readings <250mg/dl) mice receiving vehicle control chow (black line) or 0.9375mg/kg Idelalisib containing chow (grey line) (n = 25/group). (C) A schematic representation of the immunization experimental protocol. (D) The total NP-specific (NP27 binding) and high affinity NP-specific (NP2 binding) IgM (left) and IgG (right) antibody secreting cell response (ASCs/Spleen) 14 days post immunization of mice receiving vehicle control chow (white bar) or 0.9375mg/kg Idelalisib chow (grey bar) (n = 7/group). (E) The total circulating NP-specific (NP27 binding) IgM (left) and IgG (center) and high affinity NP-specific (NP2 binding) IgG (right) at day 14 post-immunization of mice receiving vehicle control chow (white bars) or 0.9375mg/kg Idelalisib containing chow (grey bars) (dashed line represents pre-immunization anti-NP serum levels). (Idel= Idelalisib. BG= blood glucose. Bars in D & E represent mean ± SEM. Log-rank (Mantel-Cox) test was used to calculate statistics in B. One-Way ANOVA was used to calculate statistics in D & E. *=p<0.05, **= p<0.01,***=p<0.005, ****=p<0.0001).

NOD mice on low-dose Idelalisib treatment remain immunocompetent

As mentioned previously, B cell depletion therapies are somewhat efficacious in T1D, but are not without safety concerns. Removal of an arm of the adaptive immune system can leave patients susceptible to infection and prevent proper response to immunization. We therefore sought to determine if low dose Idelalisib treatment would spare responsiveness to immunization. Non-diabetic female NOD mice were placed on vehicle control or 0.9375mg/kg Idelalisib containing chow for 4 weeks, immunized with NP4Ova + Alum, and their antibody response assessed (Figure 1C). 14 days post-immunization, the anti-NP IgM response in the spleen was not different between the vehicle control and 0.9375mg/kg Idelalisib treated cohorts. Further, the number of total anti-NP IgM antibody secreting cells (ASCs) per spleen was similar, as were the numbers of high-affinity IgM anti-NP ASCs/spleen (Figure 1D, left panel). We also observed no difference between the vehicle control and 0.9375mg/kg Idelalisib treated cohorts in either total IgG anti-NP or high affinity IgG anti-NP ASCs/spleen (Figure 1D, right panel). The levels of IgM anti-NP and IgG anti-NP found in the periphery were also comparable (Figure 1E) (For pre-immune anti-NP serum levels, see Supplemental Figure 2). There was no difference in B cell numbers in the spleens of animals receiving vehicle control and 0.9375mg/kg Idelalisib containing chow (data not shown). These data show low dose Idelalisib treatment does not affect the ability to respond to immunization, as evidenced by similar levels of class switching and affinity maturation between the treated and untreated groups. Thus, animals receiving doses of Idelalisib sufficient to slow progression of disease and prolong survival in VH125.NOD mice remain immunocompetent, alleviating potential adverse outcomes inherently associated with B cell depletion therapies.

Autoreactive PTEN^−/+ × SHIP-1^−/+B cells re-establish anergy when treated with Idelalisib

We next sought to confirm the specificity of the Idelalisib effect for autoimmunity driven by dysregulation of the PI3K pathway. We and others have previously shown that regulation of the PI3K pathway by the inositol phosphatases PTEN and SHIP-1 is required for maintenance of B cell anergy (27). B cells in T1D, AITD and SLE patients express reduced PTEN and SHIP-1, presumably due to an increase in the miRNAs that regulate them, e.g. mir-7 and mir-155 (23). Studies in animal models utilizing B cell targeted conditional deletion of either of these molecules is sufficient to drive autoreactive B cells out of anergy leading to rapid proliferation and differentiation into antibody secreting cells (27). Removal of a single allele of both PTEN and SHIP-1 is also sufficient to allow for loss of anergy because both degrade PI(3,4,5)P3. To best approximate physiologic conditions, we utilized B cell targeted conditional deletion (huCD20cre^tam) of one allele of PTEN (PTEN^flox/+) and one allele of SHIP-1 (SHIP-1^flox/+), coupled with a YFP-reporter to determine cre-activity, and crossed onto an anti-DNA (Ars/A1) transgenic background that renders B cells anergic (detailed in methods) (50).

Anergic B cells were adoptively transferred into C57BL/6 recipients as shown diagrammatically in Figure 2A. 7 days post tamoxifen treatment anergy was lost (27) and Idelalisib treatment was begun. 14 days post adoptive transfer, mice on 0.9375mg/kg, 3.75mg/kg and the clinically prescribed dose of 30mg/kg of Idelalisib containing chow had significantly decreased serum autoantibody relative to untreated controls (Figure 2B, quantified in Figure 2C). Furthermore, the number of autoreactive ASCs/spleen was significantly reduced in cohorts receiving 0.9375mg/kg and 3.75mg/kg Idelalisib containing chow, with undetected ASCs in cohorts receiving 30mg/kg Idelalisib containing chow (Figure 2D). The reduction in peripheral autoantibody, as well as ASCs/spleen was not due to a differential recovery of transferred cells among treatment groups, as we found no significant difference in total Ars/A1 idiotype+ YFP+ B cells in the spleens of animals at day 14 post transfer regardless of treatment (Figure 2F). Adoptively transferred PTEN^−/+ × SHIP-1^−/+ B cells from cohorts receiving tamoxifen and 0.9375mg/kg, 3.75mg/kg and 30mg/kg Idelalisib containing chow underwent significantly decreased proliferation (Figure 2E top panel, quantified Figure 2G), and plasmablast differentiation as measured by CD138 positivity (Figure 2E bottom panel, quantified Figure 2H) relative to mice receiving vehicle control chow. Thus 0.9375mg/kg Idelalisib, as well as higher doses, is sufficient to constrain an autoreactive B cell response driven by haploinsufficiency of the inositol phosphatases that regulate the PI3K pathway.

Figure 2. Autoreactive PTEN^−/+ × SHIP-1^−/+ B cells re-establish anergy when treated with Idelalisib.

(A) A schematic representation of the experimental protocol. (B) WT Ars/A1 or PTEN^fl/wt×SHIP-1^fl/wt Ars/A1-derived IgM^a anti-Ars antibody detected in serum 14 days post tamoxifen treatment of mice receiving vehicle control chow (open circles), 0.9375mg/kg (closed squares), 3.75mg/kg (closed triangles), 30mg/kg (closed circles) and WT Ars/A1 receiving 0mg/kg (open triangles) Idelalisib containing chow. (C) Quantification of relative response of PTEN^fl/wt×SHIP-1^fl/wt Ars/A1-derived IgM^a anti-Ars antibody detected in the serum 14 days post tamoxifen treatment. (D) Quantification of relative response of PTEN^fl/wt×SHIP-1^fl/wt Ars/A1-derived IgM^a anti-Ars ASCs/spleen 14 days post tamoxifen treatment. (E) Proliferation (top row) and plasmablast differentiation (bottom row) of splenic Ars/A1 Idiotype+ YFP+ B cells of mice on vehicle control chow (open black line, top row) or indicated doses of Idelalisib (shaded line, top row) 14 days post tamoxifen treatment. Enumeration of (F) total recovered transferred PTEN^fl/wt×SHIP-1^fl/wt cells, (G) the unproliferated Ars/A1 Idiotype+ YFP+ population and (H) plasmablasts in the spleens of recipient mice 14 days post tamoxifen treatment (For E, F, G & H gated on: B220+ Ars/A1 Id+ YFP+). (Idel:= Idelalisib. n = 8/group. Data shown are representative of at least three replicated experiments. Bars in C, D, F, G & H represent mean ± SEM. One-Way ANOVA and Tukey’s multiple comparisons test was used to calculate statistics in C, D, F, G & H. *=p<0.05, **= p<0.01, ***=p<0.005, ****=p<0.0001, ND=undetectable).

Autoreactive SHP-1^−/− B cells do not maintain anergy when treated with low dose Idelalisib

SHP-1 is a regulatory SH2-domain containing tyrosine phosphatase which mediates the function of inhibitory receptors such as CD22, PD1 and FCγRIIB, and is necessary for maintenance of B cell tolerance (27, 53, 62). Allelic variants of SHP-1 have been shown to increase risk of developing SLE (24), with studies indicating a subset of SLE patients having reduced SHP-1 protein in their B cells (63). Additionally, reductions in SHP-1 mRNA and protein have been observed in peripheral blood B cells of MS patients (64). Studies in viable motheaten mice have revealed that a mutation in a splice site in Ptpn6, the gene that encodes SHP-1, resulting in an 80–90% reduction in enzymatic activity, leads to severe B cell immunodeficiency and autoantibody production (65). Our laboratory has shown B cell-targeted conditional deletion of SHP-1 from anergic B cells in vivo leads to proliferation and autoantibody production (27). SHP-1 is required to maintain B cell anergy, acting through a pathway distinct from SHIP-1 and PTEN (27).

To test the specificity of Idelalisib effects on non-PI3K pathway dysregulation-mediated autoimmunity we used the adoptive transfer system described in Figure 3A in conjunction with SHP-1^flox/flox B cells. Idelalisib containing chow is administered at day 7 post transfer, upon removal of SHP-1 protein from transferred cells. Unlike its enforcement of anergy caused by PI3K pathway dysregulation, low and intermediate dose Idelalisib treatment had no effect on loss of anergy caused by SHP-1 induced deficiency. However, autoimmunity was blocked by high dose Idelalisib (Figure 3B, quantified in Figure 3C, Figure 3D). Although we saw a trend of reduced recovery of adoptively transferred SHP-1^−/− B cells from the spleens of animals receiving Idelalisib containing chow, this was not significant (Figure 3F). In animals receiving vehicle, 0.9375mg/kg or 3.75mg/kg Idelalisib containing chow, SHP-1^−/− B cells proliferated (Figure 3E top panel, quantified Figure 3G) and differentiated (Figure 3E bottom panel, quantified Figure 3H) comparably. Only in animals receiving 30mg/kg Idelalisib-containing chow did SHP-1^−/− B cells undergo decreased proliferation and differentiation (Figure 3E, Figure 3G and Figure 3H). As illustrated in the visual abstract, these findings lead to the conclusion that the particular risk allele-mimetic conditions at play determine the ability of partial p110δ inhibition to enforce tolerance. Specifically, p110δ inhibition compensates for defects in PI3K pathway regulation, but not defects in regulation by the tyrosine phosphatase SHP-1.

Figure 3. Autoreactive SHP-1^−/− B cells do not maintain anergy when treated with low dose Idelalisib.

(A) A schematic representation of the experimental protocol. (B) WT Ars/A1 or SHP-1^fl/fl Ars/A1-derived IgM^a anti-Ars antibody detected in serum 14 days post tamoxifen treatment of mice receiving vehicle control chow (open circles), 0.9375mg/kg (closed squares), 3.75mg/kg (closed triangles), 30mg/kg (closed circles) or WT Ars/A1 receiving 0mg/kg (open triangles) Idelalisib containing chow. (C) Quantification of relative response of SHP-1^fl/fl Ars/A1-derived IgM^a anti-Ars antibody detected in the serum 14 days post tamoxifen treatment. (D) Quantification of relative response of SHP-1^fl/fl Ars/A1-derived IgM^a anti-Ars ASCs/spleen 14 days post tamoxifen treatment. (E) Proliferation (top row) and plasmablast differentiation (bottom row) of splenic Ars/A1 Idiotype+ CD45.1+ B cells of mice on vehicle control chow (open black line, top row) or indicated doses of Idelalisib (shaded line, top row). Enumeration of (F) total recovered transferred SHP-1^fl/fl cells, (G) the unproliferated Ars/A1 Idiotype+ CD45.1+ population and (H) plasmablasts in the spleens of recipient mice 14 days post tamoxifen treatment (For E, F, G & H gated on: B220+ Ars/A1 Id+ CD45.1+). (Idel:= Idelalisib. n = 8/group. Data shown are representative of at least three replicated experiments. Bars in C, D, F, G & H represent mean ± SEM. One-Way ANOVA and Tukey’s multiple comparisons test was used to calculate statistics in C, D, F, G & H. *=p<0.05, **= p<0.01, ***=p<0.005, ****=p<0.0001, ND=undetectable).

p110δ inhibition suppresses BCR-mediated calcium flux and reduces phosphorylation of its downstream signaling intermediaries

In naïve B cells, antigen receptor stimulation leads to phosphorylation of the two conserved tyrosines in the ITAMs of CD79a/b leading to recruitment of Lyn and Syk to the receptor complex. Lyn phosphorylates CD19, allowing its interaction with Lyn and PI3K and subsequent activation of p110δ. p110δ converts PI(4,5)P2 to PI(3,4,5)P3, generating docking sites for PH-domain containing downstream BCR effectors such as PLCγ, AKT and BTK (21). Multiple parallel pathways emanate from this signalosome, ultimately leading to cell activation, differentiation, proliferation and migration.

To determine whether p110δ inhibition blocks proximal BCR signaling events, we stimulated splenic B cells from C57BL/6 mice with polyclonal F(ab’)₂ anti-Ig heavy and light chain antibodies with simultaneous addition of 0nM, 15nM, 60nM and 490nM Idelalisib. It is noteworthy that these approximate dietary doses of 0mg/kg, 0.9375mg/kg, 3.75mg/kg and 30mg/kg of Idelalisib, respectively, used in vivo, although, due to complex pharmacodynamics, we do not know the precise concentration of Idelalisib in B cells in vivo. All doses of Idelalisib tested suppressed calcium mobilization (Figure 4A, quantified in Figure 4B). Similarly, phosphorylation of AKT, BTK and PLCγ are significantly reduced following exposure to all doses of inhibitor (Figure 4C). It is interesting that although we can achieve a complete abrogation of calcium mobilization with Idelalisib, we observe only modest, albeit significant, decreases in phosphorylation of PLCγ. We investigated this phenomenon further to reveal a direct correlation between the strength of BCR stimulus used and the dose of Idelalisib required to suppress this response (Supplemental Figure 3). These results may seem counterintuitive because of the dependence of calcium responses on PLCγ. However, previous studies indicate that PLCγ phosphorylation is not affected by SHIP-1 activation following FcγRIIB coaggregation with the BCR (unpublished data, Cambier). Additionally, previous work demonstrated that PLCγ interacts directly with the Lyn SRC family kinase during BCR signaling (66). This suggests that PLCγ activation and hydrolysis of PI(4,5)P2 may be a two-step process involving phosphorylation at the BCR receptosome followed by PI(3,4,5)P3 dependent translocation to substrate rich sites in the plasma membrane. In this scenario PLCγ phosphorylation would not by PI3K dependent. These findings lead to the conclusion that low doses of a p110δ inhibitor that enforce anergy while sparing the antibody response has an inhibitory effect on early events in BCR signaling that are predicted to be dependent on PI3K activation.

Dose-dependent Idelalisib inhibition of antibody responses

As shown in Figure 1, low dose Idelalisib does not inhibit antibody responses in NOD mice. To investigate this further we examined the effect of a range of Idelalisib doses on in vivo B cell responses to immunization. We adoptively transferred MD4 B cells loaded with dilution dye into C57BL/6 recipients, and, after allowing the cells to rest for 48 hours, placed recipient mice on various doses of Idelalisib containing chow. 24 hours later, we immunized with HEL conjugated to SRBC, and analyzed the B cell response 5 days later (Figure 5A). MD4 B cells from mice receiving vehicle control chow and 0.9375mg/kg Idelalisib containing chow mounted similar IgM anti-HEL antibody responses, and generated comparable HEL-specific ASCs/spleen (Figure 5D, Figure 5E, and Figure 5F). MD4 B cells in mice receiving 3.75mg/kg and 30mg/kg Idelalisib containing chow prior to immunization mounted significantly reduced IgM anti-HEL antibody responses, and generated a reduced number of ASCs/spleen in comparison to MD4 B cells from vehicle control cohorts (Figure 5D, Figure 5E, and Figure 5F). This dose-dependent reduction in HEL-specific antibody is further reflected in the recovery of MD4 B cells in the spleens of mice from the various cohorts. Mice receiving 3.75mg/kg and 30mg/kg Idelalisib containing chow had significant decreases in recoverable transferred cells, while recovered cells proliferated less than in controls (Figure 5B, Figure 5G, quantified in Figure 5C). These data allowed clearer definition of doses that enforce anergy of autoreactive B cells while sparing B cell responses to exogenous antigen.

Figure 5. Dose-dependent Idelalisib inhibition of antibody responses.

(A) A schematic representation of the experimental protocol. (B) Enumeration of total recovered transferred MD4 B cells and (C) quantification of the unproliferated population of recovered MD4+ B cells in the spleen of recipient mice 5 days post-immunization. (D) MD4-derived IgM^a anti-HEL antibody detected in serum 5 days post immunization of mice receiving vehicle control chow (open circles), 0.9375mg/kg (closed squares), 3.75mg/kg (closed triangles), 30mg/kg (closed circles), and unimmunized mice receiving 0mg/kg (open triangles) Idelalisib containing chow. (E) Quantification of relative response of MD4-derived IgM^a anti-HEL antibody detected in serum and (F) MD4-derived IgM^a anti-HEL ASCs/ spleen 5 days post immunization. (G) Proliferation of splenic MD4+ B cells 5 days post-immunization of mice receiving vehicle control chow (unfilled black line) or indicated doses of Idelalisib (shaded grey line). (Idel:= Idelalisib. -imm= unimmunized. n = 8/group. Data shown are representative of at least three replicated experiments. B, C & G gated on: B220+ HEL binding+. Bars in B, C, E & F represent mean ± SEM. One-Way ANOVA and Tukey’s multiple comparisons test was used to calculate statistics. *=p<0.05, **= p<0.01, ***=p<0.005, ****=p<0.0001).

Low dose p110δ inhibition does not inhibit T cell responses in vitro or in vivo

T cells are essential components of antibody responses to most proteinaceous antigens, including autoantigens. T cells utilize the p110δ isoform, but the role of low dose inhibition of this isoform on T cell function has not been studied. Studies of p110δ knockout mice or functionally inactive p110δ mice have yielded conflicting results with respect to the requirement of this isoform for T cell responses (35, 36, 40, 41, 67–69). Autoantibody responses caused here by compromise of Ars/A1 anti-chromatin B cells is T cell dependent. Upon transfer into TCRα^−/− recipients these B cells fail to proliferate, differentiate and secrete autoantibody (Getahun and Cambier, unpublished data). This raises the possibility that Idelalisib is mediating its effect by inhibiting T cell function. It is noteworthy, however, that if this were the case, the inhibitor should have been equally effective in inhibiting autoimmunity caused by B cell targeted PTEN^−/+ × SHIP-1^−/+ and SHP-1^−/− conditions. Nonetheless, we set out to determine the consequence of low dose p110δ inhibition on T cell responses in vitro and in vivo.

Since we see a therapeutic effect of the inhibitor on VH125.NOD disease progression and survival, reduction in autoantibody responses in PTEN^−/+× SHIP-1^−/+cells, yet comparable autoantibody responses by SHP-1^−/− and MD4 B cells in mice receiving 0.9375mg/kg Idelalisib containing chow, we chose to focus our analysis on effects of this dose on T cell function. 15nM Idelalisib (comparable to 0.9375mg/kg in vivo diet dose), failed to inhibit calcium mobilization of CD4 T cells stimulated by TCR aggregation with biotin-anti-CD3 and avidin (Figure 6A, quantified in Figure 6B). To analyze in vivo CD4 and CD8 T cell responses, we adoptively transferred OT-II CD4 T cells or OT-I CD8 T cells into congenically mismatched recipients and immunized with OVA+P:IC as represented diagrammatically in Figure 6C and Figure 6G, respectively. Five days post-immunization with OVA+P:IC, mice that received vehicle control chow and mice that received 0.9375mg/kg Idelalisib containing chow proliferated [Figure 6E (OT-II)/6I (OT-I) left panel, quantified Figure 6E (OT-II)/6I (OT-I) right panel], and upregulated the activation marker CD44 to similar levels [Figure 6F (OT-II)/6J (OT-I) left panel, quantified Figure 6F (OT-II)/6J (OT-I) right panel]. The transferred OT-II and OT-I cells that proliferated most underwent the greatest upregulation of CD44 [Figure 6D (OT-II) and Figure 6H (OT-I)]. Doses of Idelalisib that enforce anergy of autoreactive B cells, delay disease progression and prolong survival in VH125.NOD mice do not affect CD4 or CD8 T cell responses in vitro or in vivo.

Figure 6. Low dose p110δ inhibition does not inhibit CD4+ or CD8+ T cell responses.

(A) Calcium flux of CD4+ T cells stained with anti-CD3 biotin with simultaneous addition of 0nM (black line) or 15nM (grey line) Idelalisib, and crosslinked with avidin. (B) Quantification of area under the curve of A. (C) A schematic representation of the experimental protocol. (D) Representative flow cytometric plots of proliferation (CFSE) and upregulation of CD44 following immunization. (E) Representative histogram (left) and quantification (right) of proliferation (CFSE) of recovered OT-II T cells from the spleens of recipient mice. (F) Representative histogram (left) and quantification (right) of upregulation of CD44 of recovered OT-II T cells from the spleens of recipient mice. (G) A schematic representation of the experimental protocol. (H) Representative flow cytometric plots of proliferation (CFSE) and upregulation of CD44 following immunization. (I) Representative histogram (left) and quantification (right) of proliferation (CFSE) of recovered OT-I T cells from the spleens of recipient mice. (J) Representative histogram (left) and quantification (right) of upregulation of CD44 of recovered OT-I T cells from the spleens of recipient mice. (Idel:= Idelalisib. imm+/− = immunized/unimmunized. n = 5/group. Data shown are representative of at least two replicated experiments. Bars in B, E, F, I & J represent mean ± SEM. Students T test was used to calculate statistics in B. One-Way ANOVA and Tukey’s multiple comparisons test was used to calculate statistics in E, F, I & J. *=p<0.05, **= p<0.01, ***=p<0.005, ****=p<0.0001).

Discussion

Here we report that autoimmunity caused by failed regulation of PI(3,4,5)P3 levels in B cells can be prevented by treatment with low doses of PI3K p110δ inhibitor, doses that neither inhibit autoimmunity caused by altered expression of the tyrosine phosphatase SHP-1 nor block antibody responses to exogenous antigen. Further we show that in VH125.NOD mice, which express reduced PTEN, low dose p110δ inhibitor therapy delays progression from the hyperglycemic state to diabetes, while sparing the ability to mount antibody responses following immunization. Results speak to the likely success of “precision” approaches to therapy in which effects of specific risk alleles are mitigated by both qualitative and quantitative targeting of the offending gene(s).

Personalized or precision medicine aims to subset, and subsequently treat, patients based on the underlying cause of disease, i.e. genetic polymorphisms, rather than grouping patients based on symptom similarity. This approach would harness genetic and expression information to stratify patients as predictable responders or non-responders to a particular therapy based on the possession of specific risk-conferring alleles. It is estimated that only 10–20% of GWAS hits are attributable to coding-region germline mutations, with the remainder driven by differential expression of proteins governed by non-coding-region mutations (70). Thus heritable differences in promotors and enhancers can, by affecting protein levels, affect risk of disease. It appears that, by affecting protein levels, differences in miRNA expression can also affect risk of disease. Increased expression of mir-7 and associated reduced expression of its target PTEN is associated with lupus (22). Altered expression of mir-155 and its target SHIP-1 has also been implicated in SLE (71, 72). We have recently reported decreased expression of PTEN along with increased regulator miRNAs in B cells from new-onset type 1 diabetics. Finally, PTEN expression is reduced in B cells from NOD mice. PTEN and SHIP-1 are critical regulators of the PI3K pathway, which is important in signaling by a number of immune system receptors, most notably antigen and activating receptors for IgG immunoglobulins. Anergic autoreactive B cells are characterized by increased expression of PTEN relative to naïve B cells (19) and, as discussed below, PTEN is required to maintain anergy.

Our laboratory and others have shown that regulation of the PI3K pathway by the inositol phosphatases PTEN and SHIP-1 is crucial for maintenance of B cell tolerance. Browne et al showed that PTEN is upregulated on anergic B cells in the MD4.ML5 HEL-anti-HEL mouse, and is required to maintain anergy in that model. We subsequently showed that an adoptive transfer model that acutely induced deletion of PTEN or SHIP-1 in anergic anti-DNA B cells leads to rapid cell activation, proliferation and differentiation into autoantibody secreting cells. Similar effects are seen upon acutely induced deletion of SHP-1 (27). Acute induction of haploinsufficiency of either of these genes in anergic B cells has no adverse effects, however haploinsufficiency of both PTEN and SHIP-1 leads to autoimmunity suggesting that these inositol phosphatases function in the same regulatory pathway. This is not surprising given the fact that they attack the same substrate, PI(3,4,5)P3. While acutely induced deletion of the tyrosine phosphatase SHP-1 leads to loss of anergy, induced haploinsufficiency does not. Most importantly, haploinsufficiency of both SHP-1 and SHIP-1 does not lead to autoimmunity suggesting that they function in distinct pathways, both of which are critical for maintenance of anergy. Given this result, it is not surprising that compensatory inhibition of PI3K blocks autoimmunity caused by defective PI3K regulation, but not by defective regulation of tyrosine phosphorylation by SHP-1. This result demonstrates the principle that autoimmunity can be treated by specifically targeting the predisposing genetic defect.

PI3K exists as a heterodimer, comprised of a regulatory and catalytic subunit. The catalytic subunits p110α and p110β are ubiquitously expressed, whereas p110δ and p110γ are restricted to the lymphoid compartment. p110δ knockout and catalytically inactive mice have demonstrated p110δ being the dominant isoform utilized within B cells, with redundant p110 isoforms capable of compensating in T cells for the lack of p110δ (41). Thus it can be expected that Idelalisib, a p110δ isoform-specific inhibitor with 110–453 fold more selectivity over other class I isoforms, should mediate its biologic effects by blocking PI3K p110δ activation in B cells, as indicated by its effectiveness when autoimmunity is driven by a B cell specific defect, and lack of inhibition of T cell function.

It is curious that Idelalisib blocks development of autoimmunity under dosing conditions far below those needed to block growth of B cell tumors and antibody responses to exogenous immunogens. This presumably reflects the fact that autoimmunity can result from only partial dysregulation of PI(3,4,5)P3 levels, and therefore be compensated by partial inhibition of PI3K.

Therapeutic effects of Idelalisib in the NOD mouse model would not have been anticipated prior to our observation of NOD B cells expressing reduced levels of PTEN (Smith and Cambier, in press). Genetic analyses of NOD mice has revealed more than 30 Idd susceptibility loci, with the NOD-derived H-2^g7 MHC as a necessary, but not sufficient, component of disease. While many dominant Idd loci have been described, the causal genes are technically difficult to identify as even small intervals contain a large number of candidate genes. Further, disease risk is likely determined by concerted effects of multiple Idd loci. Interestingly, PTEN and SHIP-1 do not fall into identified Idd loci, nor do the miRNAs that regulate their expression, mir-7 and mir-155, respectively. Thus we hypothesized this inherent inability to regulate the PI3K pathway in the NOD mouse would render our p110δ inhibitor effective in the VH125.NOD mouse. Results suggest that Idd loci may contain a previously unrecognized regulator of PTEN expression, as the known regulators of PTEN do not map to identified loci.

Future therapeutic approaches for T1D must have as their goal prevention of development of life-long dependence on insulin. More specifically, intervention must begin prior to establishment of insulin dependence and prevent progression to insulin dependence. With this in mind, studies described here were designed to test the ability of low dose PI3Kδ inhibitor administration to prevent progression from the modestly hyperglycemic state to disease of such severity that insulin therapy is required, i.e., prior to irreversible beta cell destruction. Results are consistent with utility of initiation of therapy when hyperglycemia and other markers indicate that clinical disease is imminent.

Traditional medicine has operated under the premise that established therapies will work for most patients, but perhaps not all, with a given disease. Personalized medicine intends to target disease while simultaneously reducing or alleviating collateral damage and risk. The future goal of medical professionals involved in treatment of autoimmune diseases should be induction of tolerance/enforcement of anergy and restoration of “normal” immune function. This will be achieved by harnessing genetic and expression information to predict patient response rate to a particular therapy. The studies performed here provide evidence that enforcement of the anergic state in a risk allele-dependent manner while sparing protective immunity in response to immunization can be achieved.

Key Points.

1. Low dose p110δ inhibitor therapy prevents PTEN but not SHP-1 driven autoimmunity.

2. Effective p110δ inhibitor doses spare immune responses to exogenous immunogens.

3. Low dose p110δ inhibitor therapy delays T1D progression in VH125.NOD mice.