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. Author manuscript; available in PMC: 2021 Oct 15.
Published in final edited form as: J Immunol. 2020 Sep 4;205(8):2100–2108. doi: 10.4049/jimmunol.2000584

PIK3IP1 promotes extrafollicular class switching in T-dependent immune responses

Kristina Ottens *, Jalyn Schneider *, Lawrence P Kane †, Anne B Satterthwaite *,‡
PMCID: PMC7541772  NIHMSID: NIHMS1620482  PMID: 32887751

Abstract

PI3 kinase (PI3K) plays multiple roles throughout the life a B cell. As such, its signaling is tightly regulated. The importance of this is illustrated by the fact that both loss and gain of function mutations in PI3K can cause immunodeficiency in humans. PIK3IP1, also known as TrIP, is a transmembrane protein that has been shown to inhibit PI3K in T cells. Results from the ImmGen consortium indicate that PIK3IP1 expression fluctuates throughout B cell development in a manner inversely correlated with PI3K activity; however, its role in B cells is poorly understood. Here we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal immunoglobulin levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and secondary responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response, since germinal center formation and affinity maturation were normal and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with a role early in the response, PIK3IP1 was downregulated at late time points after B cell activation, in a manner dependent on PI3K. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong crosslinking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.

Introduction

The ability of the immune system to respond to foreign antigens by producing antibodies is critical to combat a wide variety of infections and is the basis for many vaccines. During humoral immune responses, B cells are activated in either a T-independent or T-dependent manner, depending on the nature of the antigen (1). In T-dependent responses, B cells receive cognate help from T cells and produce antibodies in two waves. The first is derived from the extrafollicular response. These antibodies can be either IgM or IgG and are of relatively low affinity (1). The second wave of antibodies are produced by germinal center-derived plasma cells, which have undergone somatic mutation and affinity maturation. These antibodies are thus of higher affinity and are more likely to be class switched (1). Memory B cells can also be generated during both the extrafollicular and the germinal center stages of the response (2–7). Both phases are critical for effective immunity. Extrafollicular antibodies provide a rapid response within days of infection, and low affinity, early memory cells are thought to allow for efficient future responses against pathogen variants (2–7). Germinal center-derived plasma cells and memory cells provide longer term, high affinity responses with a wider variety of effector functions (2–7).

During T-dependent responses, B cells receive signals through both the B cell antigen receptor (BCR) and CD40 (8). Both of these receptors signal through PI3 kinase (PI3K) (9–12). The product of PI3K, PIP3, binds to the pleckstrin homology domains of several critical B cell signaling molecules. This results in their localization to the plasma membrane and their subsequent activation (reviewed in (13)). PI3K is required for the initial proliferation of B cells upon antigen encounter (10, 11, 14–16), with the subsequent fate of the B cell shaped by the strength of PI3K signaling. Class switching is limited by strong PI3K signaling and enhanced when PI3K pathway activity is weak (17, 18). The strength of PI3K signaling also controls developmental fate decisions regulating the distribution of B cells among peripheral B cell subsets – follicular (FO), marginal zone (MZ), and B1a B cells (10, 11, 14–16, 18, 19).

Consistent with its critical role in B cells, signaling downstream of PI3K is tightly regulated, mainly by PTEN and SHIP-1. These inositol phosphatases dephosphorylate PIP3, the product of PI3K; PTEN to PI(4,5)P2 and SHIP-1 to PI(3,4)P2 (13). They each have numerous, non-redundant functions in B cell development and activation (18–20), highlighting the importance of maintaining an appropriate balance of PI3K signal strength. Indeed, either too little or too much signaling via the PI3K pathway leads to immunodeficiency in both mice and humans (21, 22).

Much less is understood about mechanisms that act at the level of PI3K itself to limit its activity in B cells. PIK3IP1, also known as TrIP, is a transmembrane protein that interacts with PI3K p85/p110 heterodimers (23). PIK3IP1 downregulates PI3K activity in several non-immune cell types (24–29) and in T cells (30–33). PIK3IP1-deficient T cells exhibit increased pS6/mTOR activation downstream of PI3K (31, 32), produce elevated levels of IFNγ (31, 32), and have enhanced anti-tumor activity (33). The extracellular domain of PIK3IP1 promotes its function in T cells, although a ligand has not been identified (31–33). PIK3IP1 levels are low in several types of malignancy and strategies that upregulate it help sensitize tumors to killing by PI3K inhibitors (25, 34–44), suggesting that understanding control of PIK3IP1 expression may have therapeutic value. One such tumor is of the B lineage, suggesting that PIK3IP1 may act in B cells (36, 37), although nothing is known about its role in normal B cell responses.

Here, we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal immunoglobulin levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and memory responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response, since germinal center formation and affinity maturation were normal and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with it acting early in the response, PIK3IP1 was downregulated at late time points after B cell activation in manner dependent on PI3K signaling. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong crosslinking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.

Materials and Methods

Mice

PIK3IP1f/f mice on the C57BL/6 background (32) were crossed to CD19-cre mice (45) to delete PIK3IP1 specifically in B cells. Foxo3−/− (46) and control mice used in Figure 1c were on the FVB background. Mice were age matched and littermate controls were used whenever possible. All animal experiments were approved by the UT Southwestern Institutional Animal Care and Use Committee.

Flow cytometry

Bone marrow cells, splenocytes, and peritoneal wash cells were depleted of red blood cells and stained with various combinations of the following antibodies. Bone marrow: anti-CD43 FITC (BD Biosciences), anti-IgM PE (BD Biosciences), anti-B220 PerCP-Cy5.5 (Tonbo Biosciences), and anti-CD93 APC (Invitrogen). Spleen: anti-CD21 FITC (BD Biosciences), anti-CD23 PE (BD Biosciences), anti-CD95 PE (BD Biosciences), anti-IgM PerCP-Cy5.5 (BD Biosciences), B220 PerCP-Cy5.5, anti-B220 APC (Tonbo Biosciences), or anti-GL7 APC (BD Biosciences). Peritoneal wash: anti-CD11b FITC (BD Biosciences), anti-CD5 PE (Tonbo Biosciences), anti-IgM PerCP-Cy5.5, anti-B220 APC. Cultured B cells were stained with anti-CD138 PE (BD Biosciences), anti-B220 PerCP-Cy5.5, and anti-IgG1 biotin (BD Biosciences) plus streptavidin APC (Tonbo Biosciences). Samples were run on a FACS Calibur (BD) and analyzed with Flowjo (Treestar).

B cell purification

Splenocytes were depleted of red blood cells and B cells purified by negative selection using anti-CD43 beads and LD columns (Miltenyi Biotech) according to the manufacturer’s instructions.

Real time PCR

Purified splenic B cells were harvested immediately or stimulated with media alone (RPMI 1640 + 10 % FBS + L-glut + pen/strep + β-ME) or 10 ug/ml anti-IgM F(ab)’2 fragments (Jackson Immunoresearch) for 1, 6, or 17 hours. In some experiments cells were pretreated for 15 minutes with vehicle (DMSO) or 10 uM Ly294002 (Sigma-Aldrich) prior to stimulation with anti-IgM. Total RNA prepared using an RNeasy Mini kit (Qiagen), and cDNA subsequently generated with a High Capacity cDNA Reverse Transcription kit (Thermofisher). PIK3IP1 levels were measured by real time quantitative PCR using Taqman reagents (Thermofisher) for PIK3IP1 and the internal control GAPDH and a Biorad CFX96 Real-Time System. Results were normalized to GAPDH using the delta-Ct method.

Western blots

Purified splenic B cells were stimulated at 37oC for varying times from 1 minute to 1 hour with media alone (RPMI 1640 + 10 % FBS + L-glut + pen/strep + β-ME), 10 ug/ml anti-IgM F(ab)’2 fragments (Jackson Immunoresearch), 10 ug/ml anti-CD40 (IC10, Invitrogen), 10 ug/ml anti-IgM F(ab)’2 fragments plus 10 ug/ml anti-CD40, or 0.1, 0.3, or 1 ug/ml CD40L (R&D Systems). Cells were lysed in 2x Laemmli sample buffer (Biorad) and equal cell equivalents subjected to SDS page using a 4–15% gradient gel (Biorad). Gels were transferred to nitrocellulose (GE Healthcare) and blocked in 5% milk. Blots were probed overnight at 4oC with rabbit monoclonal antibodies against pAkt S473 (Cell Signaling Technology), pS6 (Cell Signaling Technology), and β-actin (Cell Signaling Technology) and subsequently for 2 hrs at room temperature with goat anti-rabbit HRP (Biorad). Blots were washed three times in TBST after each antibody incubation. Bands were detected with Clarity ECL reagent (Biorad) and imaged and quantified with a Chemidoc Imaging system (Biorad) and Image Lab software (Biorad).

In vitro class switching

Purified splenic B cells were incubated for 72 hours at 37oC in media alone (RPMI 1640 + 10 % FBS + L-glut + pen/strep + β-ME), 5 ug/ml LPS (Sigma) + 50 ng/ml IL-4 (R&D Systems), or 10 ug/ml anti-CD40 (IC10, Invitrogen) + 50 ng/ml IL-4 (R&D Systems). Cells were then stained with antibodies against B220, CD138, and IgG1 and analyzed by flow cytometry (see above).

Immunizations

To study T-independent responses, mice were prebled, immunized with 100ug/mL NP-Ficoll (Biosearch Technologies) in PBS, and bled 7 and 14 days later. For T-dependent responses, mice were prebled, immunized with 500 ug/mL NP-KLH (Biosearch Technologies) in alum (ThermoFisher Scientific), and bled 7, 14, and 28 days later. After the 28 day bleed, mice were boosted with 1mg/mL NP-KLH (Biosearch Technologies) in PBS and bled one week later. For germinal center studies, mice were immunized with 2.5×108 sheep red blood cells (SRBCs) (Innovative Research) or PBS and germinal centers measured by flow cytometry 5 days later (see above).

ELISAs

Total Ig: Serum from unimmunized mice was analyzed for total IgM, IgG1, IgG2c, IgG3, and IgA levels using the SBA Clonotyping System (Southern Biotech) according to the manufacturer’s instructions. anti-NP Ig: 1:100, 1:400, or 1:1600 dilutions of serum in phosphate buffered saline (PBS) + 0.1% BSA + 0.05% Tween-20 were incubated on Corning® Costar® 96-well flat bottomed plates (Thomas Scientific Holdings LLC) that were previously coated with 25 ug/ml NP>25-BSA or NP8-BSA (Biosearch Technlogies). After washing with PBS + 0.1%BSA + 0.05% Tween-20, wells were incubated with 2 ug/ml alkaline phosphatase labeled anti-IgM, IgG3, or IgG (Southern Biotech). Plates were again washed with PBS + 0.1% BSA + 0.05% Tween-20 and incubated with alkaline phosphatase substrate (Biorad). In all cases, OD405 was measured using an ELx808 ELISA reader (BioTek Instruments, Inc).

Results

Results from the ImmGen Consortium (www.immgen.org) indicate that PIK3IP1 is expressed in the B lineage in an interesting pattern (Figure 1a). In the bone marrow, PIK3IP1 is expressed in a manner inversely correlated with PI3K activity: low in large pre-B cells in which PI3K signaling promotes proliferation and suppresses Rag expression (47, 48), and higher in small pre-B cells in which low PI3K activity allows for cell cycle arrest and light chain rearrangement (47, 48). In the periphery, PIK3IP1 is expressed in most B cell subsets but is particularly low in germinal center B cells (dark blue bar, Fig 1a).

Figure 1: PIK3IP1 expression in B cells.

Figure 1:

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A) PIK3IP1 expression in B cells from www.immgen.org. B-E) PIK3IP1 levels were measured by real time PCR and normalized to GAPDH by the delta-Ct method. (B) To determine whether PIK3IP1 expression changes during the process of B cell activation, purified splenic B cells from wild type mice were stimulated with 10 ug/ml anti-IgM F(ab)’2 fragments for 1 hr, 6 hrs, or overnight (17 hrs). To define the role of PI3K in PIK3IP1 expression, cells were pretreated with vehicle (DMSO) or the PI3K inhibitor LY294002 prior to an overnight (17 hr) stimulation with anti-IgM. The level of PIK3IP1 in stimulated cells relative to unstimulated cells is shown for each condition. Data represent mean +/− SEM, n = 3–8. * p < 0.05, ** p < 0.01 by one way ANOVA. (C) Purified splenic B cells from wild type or Foxo3−/− mice were harvested immediately (open bars) or stimulated with media alone (gray bars) or anti-IgM F(ab)’2 fragments (black bars) for 17 hrs. The level of PIK3IP1 relative to GAPDH is shown. Data represent mean +/− SD, n = 3. *p < 0.05, ns = not significant by unpaired Student’s t-test. (D) Purified splenic B cells were treated with DMSO vehicle (DMSO) or LY294002 for 17 hours. The level of PIK3IP1 in LY294002 treated cells relative to DMSO treated cells is shown. n = 3. ***p < 0.001 by unpaired Student’s t-test. E) PIK3IP1 levels were measured by real time PCR in purified B cells from CD19-cre (open bar) or CD19-cre.PIK3IP1f/f (black bar) mice. The level of PIK3IP1 relative to GAPDH is shown. Data represent mean +/− SEM, n = 5–6. **p<0.01 by unpaired Student’s t-test.

We sought to determine whether PIK3IP1 expression is controlled by PI3K activity in peripheral B cells. We purified wild type splenic B cells, stimulated them with anti-IgM for various times, and measured PIK3IP1 expression by real time PCR. BCR crosslinking for one hour had minimal effect, but PIK3IP1 was downregulated dramatically after 17 hours (Figure 1b). Six hours of treatment resulted in an intermediate response, with PIK3IP1 still present but at reduced levels (Figure 1b). PIK3IP1 downregulation was prevented by LY294002, a PI3K inhibitor (Figure 1b). The transcription factor Foxo3 has been reported to promote PIK3IP1 expression in other cell types (26) and is downregulated by PI3K signaling upon BCR engagement (49). PIK3IP1 expression was normal in freshly isolated Foxo3−/− B cells. However, upon culture in media alone there was a small but significant increase in PIK3IP1 levels in wild type cells that did not occur in the absence of Foxo3 (Figure 1c). This suggests that a low level of PI3K activity present in ex vivo cells is lost during culture without activating stimuli, allowing for an increase in Foxo3-mediated transcription of PIK3IP1. Consistent with this, treatment of wild type cells with LY294002 in the absence of BCR stimulation, which should increase Foxo3 activity, resulted in a 30–40% increase in PIK3IP1 levels compared to DMSO treatment (Figure 1d). BCR stimulation downregulated PIK3IP1 normally in Foxo3−/− B cells, consistent with the known loss of Foxo3 activity and expression in response to this stimulus (49) (Figure 1c). Thus, PIK3IP1 expression is regulated by PI3K signaling in B cells.

To determine the role of PIK3IP1 in B cell development and function, we generated mice with B cell-specific PIK3IP1 deficiency by crossing PIK3IP1f/f mice (32) with CD19-cre mice. CD19-cre deletes efficiently and specifically in B cells, with no cre expression or activity observed in T cells, myeloid cells, or splenic IgM- or B220- cells (45, 50–53). Despite efficient loss of PIK3IP1 expression in CD19-cre.PIK3IP1f/f B cells (Figure 1e), B cell development occurred normally in these mice. B cell subpopulations were present at normal numbers in the bone marrow, spleen, and peritoneal cavity (Table I, Supplemental Figure 1a–c). Steady state levels of all antibody isotypes were also unaffected by the absence of PIK3IP1 (Figure 2a). Thus, PIK3IP1 is dispensable for steady state B cell development and B cell homeostasis.

Table I:

Normal B cell development in CD19-cre.PIK3IP1f/f mice

Population CD19-cre CD19-cre.PIK3IP1f/f
Bone marrow (x 106) (n = 4)
Pro and Pre B (B220+IgM−) 2.9 +/− 1.2 3.3 +/− 1.4
Immature B (B220+IgM+CD93+) 1.2 +/− 0.84 1.1 +/− 0.43
Mature B (B220hiIgM+CD93−) 1.8 +/− 0.91 1.8 +/− 1.0
Spleen (x 107) (n = 9)
Total splenocytes 11.1 +/− 2.7 9.2 +/− 2.1
Newly formed (B220+CD23-CD21−) 0.4 +/− 0.21 0.33 +/− 0.13
Follicular (B220+CD23+CD21+) 3.73 +/− 0.92 3.07 +/− 0.88
Marginal zone (B220+CD23lo/-CD21hi) 0.16 +/− 0.072 0.17 +/− 0.94
Peritoneal cavity (% of lymphocytes) (n = 3)
B-2 (B220+CD5−) 25.4 +/− 8.5 27.3 +/− 8.4
B-1 (B220+CD5+) 10 +/− 2.3 9.14 +/− 1.8
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Total numbers (bone marrow, spleen) or frequency (peritoneal cavity) of cells in each of the indicated populations are indicated.

Figure 2: Normal basal Ig levels and T-independent responses in CD19-cre.PIK3IP1f/f mice.

Figure 2:

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A) Total Ig levels of the indicated isotypes are shown for CD19-cre (open bars) and CD19-cre.PIK3IP1f/f (black bars) mice. Data represent mean +/− SD, n = 6. There was no significant difference between CD19-cre and CD19-cre.PIK3IP1f/f mice. B) CD19-cre (open bars) and CD19-cre.PIK3IP1f/f (black bars) mice were immunized with NP-Ficoll and bled at day 0 (prior to immunization), 7, and 14. Anti-NP IgM and IgG3 was measured by ELISA using a 1:100 dilution of serum. Data represent mean +/− SEM, n = 3. There was no significant difference between CD19-cre and CD19-cre.PIK3IP1f/f mice.

PI3K signaling is known to limit class switching during humoral immune responses. To determine whether this effect is enhanced in the absence of PIK3IP1, we first immunized CD19-cre and CD19-cre.PIK3IP1f/f mice with the T-independent antigen NP-Ficoll. Both IgM and IgG3 responses were normal (Figure 2b), indicating that PIK3IP1 is not required for T-independent responses. We next tested responses to the T-dependent antigen NP-KLH. Primary IgM responses were unaffected (Figure 3a). However, there was a significant delay in the production of anti-NP IgG (Figure 3a), with levels in CD19-Cre.PIK3IP1f/f mice not reaching those of CD19-Cre controls until day 14 post-immunization. To measure secondary responses, we boosted mice at day 28 after initial immunization and collected serum seven days later. Mice with PIK3IP1-deficient B cells failed to demonstrate a significant increase in anti-NP antibodies in response to this boost (Figure 3b). Thus, PIK3IP1 acts in B cells to promote early class switching and memory during T-dependent humoral responses.

Figure 3: Delayed class switching and impaired memory responses to T-dependent antigens in CD19-cre.PIK3IP1f/f mice.

Figure 3:

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A) CD19-cre (open bars) and CD19-cre.PIK3IP1f/f (black bars) mice were immunized with NP-KLH in alum and bled at day 0 (prior to immunization), 7, and 14. Anti-NP IgM and IgG was measured by ELISA using a 1:400 dilution of serum. Data represent mean +/− SEM, n = 5–6. * p < 0.05, ** p < 0.01, ns = not significant by one way ANOVA. B) The mice in (A) were bled at day 28 post immunization, boosted with NP-KLH in PBS, and bled 7 days later. Anti-NP IgM and IgG was measured by ELISA using the indicated dilutions of serum. Each line connects a pre and post boost sample from an individual mouse. ** p < 0.01, by paired Student’s t-test. ns = not significant.

IgG can be produced by antibody secreting cells generated during either the early extrafollicular response or the later germinal center response. The reduction in early, but not late, IgG in CD19-cre.PIK3IP1f/f mice and the low level of PIK3IP1 expression in germinal center B cells suggests that PIK3IP1 is acting to promote class switching at the extrafollicular stage rather than in germinal centers. Consistent with this model, early germinal center formation was unimpaired, as immunization with SRBCs induced an equivalent frequency of germinal center B cells in CD19-Cre (5.4% +/− 2.7, n = 4) and CD19-Cre.PIK3IP1f/fl (5.4% +/− 1.6, n = 4) mice at day 5 (Figure 4a). Germinal centers were also functional; affinity maturation was normal in CD19-cre.PIK3IP1f/f mice, as high affinity IgG was normal at day 14 (Figure 4b). There was also not a general defect in the ability of PIK3IP1-deficient B cells to undergo class switching as measured by IgG1 upregulation in vitro (Figure 4c, Supplemental Figure 1d).

Figure 4: Normal germinal center responses and in vitro class switching in CD19-cre.PIK3IP1f/f mice.

Figure 4:

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A) CD19-cre and CD19-cre.PIK3IP1f/f mice were immunized with SRBCs or a PBS control and splenocytes analyzed by flow cytometry 5 days later. Germinal center B cells are defined as B220+CD95+GL7+. Representative flow cytometry plots are gated on B220+ cells. B) A 1:400 dilution of sera from CD19-cre (open bars) and CD19-cre.PIK3IP1f/f (black bars) mice immunized with NP-KLH in alum was subjected to ELISA analysis using plates coated with NP-BSA8 to measure high affinity antibodies. Data represent mean +/− SEM, n = 6. There was no significant difference between CD19-cre and CD19-cre.PIK3IP1f/f mice. C) Purified splenic B cells were stimulated for 72 hours in LPS + IL-4 or anti-CD40 + IL-4. The frequency of IgG1+ cells was measured by flow cytometry. Data represent mean +/− SD, n = 6 for LPS and 3 for anti-CD40. There was no significant difference between CD19-cre (open bars) and CD19-cre.PIK3IP1f/f (black bars) mice.

During a T-dependent immune response B cells receive signals via both the BCR (from antigen) and CD40 (from T cells). As such, standard in vitro class switching assays do not necessarily reflect the physiological scenario. We thus tested the ability of PIK3IP1 to modulate responses to the BCR and CD40. Two important signaling events that depend on PI3K signaling downstream of these receptors are the phosphorylation of Akt (9, 10, 13, 54, 55) and the phosphorylation of S6 (56–58). B cells expressing a constitutively active form of the p110δ subunit of PI3K demonstrate increased phosphorylated S6 (pS6) in both mice and humans (59–61). Furthermore, in T cells anti-CD3/anti-CD28 induced pS6 depends on PI3K (32) and is inhibited by PIK3IP1 (31, 32). Surprisingly, loss of PIK3IP1 did not result in increased Akt phosphorylation in response to BCR crosslinking or anti-CD40 alone (Figure 5a,b). However, PIK3IP1 deficient B cells demonstrated increased pS6 relative to control cells when stimulated with both anti-IgM and anti-CD40 (Figure 5c). In vivo, B cells encounter CD40L in a trimeric form which crosslinks CD40 more efficiently than anti-CD40. We found that treatment with CD40L also resulted in increased pS6 in the absence of PIK3IP1 relative to control cells (Figure 5d). Thus, PIK3IP1 limits PI3K pathway activity in B cells under conditions reflective of those encountered by B cells during B/T cognate interactions. This explains the reduced production of IgG early in T-dependent responses in vivo, since excessive PI3K signaling limits class switching (17).

Figure 5: Increased pS6 phosphorylation in PIK3IP1 deficient B cells stimulated with anti-IgM + anti-CD40 or CD40L.

Figure 5:

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A, B) Purified splenic B cells from CD19-cre (+/+) and CD19-cre.PIK3IP1f/f (f/f) mice were stimulated with (A) 10 ug/ml anti-IgM or (B) 10 ug/ml anti-CD40 for the indicated times. Whole cell lysates were subjected to Western blot for anti-pAkt S473 and total Akt or β-actin loading controls. Results are representative of 3 independent experiments. C, D) Purified splenic B cells from CD19-cre and CD19-cre.PIK3IP1f/f mice were stimulated with (C) 10 ug/ml anti-IgM plus 10 ug/ml anti-CD40 for the indicated times or (D) the indicated dose of CD40L for 15 min. Whole cell lysates were subjected to Western blot for anti-pS6 and β-actin loading control. Results are representative of 3 independent experiments. Quantitation of each blot is shown in the graph below (open bars = CD19-cre, black bars = CD19-cre.PIK3IP1f/f).

Discussion

Here we have defined a role for PIK3IP1 in B cell functional responses. Our data support a model in which PIK3IP1 normally promotes class switching during the early extrafollicular phase of T-dependent responses by limiting PI3K signaling in response to antigen plus CD40 engagement. In contrast, germinal center formation, affinity maturation, and the late phase of class switching are unaffected in the absence of B cell expressed PIK3IP1, likely because of the low level of PIK3IP1 expression in germinal center B cells. Interestingly, PTEN, another negative regulator of PI3K signaling, is elevated in germinal center B cells (62). This suggests that different regulators of PI3K contribute to different phases of the humoral immune response.

Future studies of signaling in vivo during a T-dependent immune response would provide further support for this model. This could be achieved using an NP-specific BCR transgene (63) to increase the frequency of responding cells in both control and B cell specific PIK3IP1 knockout mice. Activation of PI3K pathway components could then be measured flow cytometry or immunofluorescence in antigen specific extrafollicular and germinal center B cells. Manipulations that eliminate germinal center responses but preserve T-dependent extrafollicular responses, such as deletion of Bcl6 in B cells (6), would also be useful to further define the role of PIK3IP1 in signaling and functional responses in vivo.

It has recently been shown that B cell activation by B cell receptor crosslinking results in activation induced cell death if a second signal such as CD40 engagement is not received within approximately nine hours of initial antigen encounter (64). Thus, there appears to be a limited time window during which B cells can receive cognate help from T cells for an effective immune response (64). Our results suggest that there may be a similar window for efficient CD40-induced class switching during the extrafollicular response. PIK3IP1 promotes class switching but is downregulated at late time points after BCR engagement. Thus, if B cells do not engage T cell help early enough after seeing antigen, their ability to undergo class switching would be decreased due to reduced PI3KIP1 expression.

Cognate T cell help might also promote PIK3IP1 function. PIK3IP1 is a cell surface molecule, and its extracellular domain is required for its function in T cells (31–33). A ligand for PIK3IP1 has been postulated to be expressed on T cells, and the PIK3IP1 extracellular domain can oligomerize (31–33). Thus, it is possible that the ability of PIK3IP1 to promote T-dependent class switching is enhanced by engagement of a ligand (PIK3IP1 itself or another molecule) on T cells during initial cognate interactions. The DCIR2+ subpopulation of dendritic cells are another potential source of a ligand for PIK3IP1. When antigen is targeted to these dendritic cells, they interact with B cells to drive a T-dependent, extrafollicular, class switched response in the absence of germinal center formation (65). This is similar to the response that is promoted by PIK3IP1 expression in B cells. Further studies of the interaction between PIK3IP1 in B cells and its potential ligands on other cell types will shed light on how the context in which B cells are activated shapes their subsequent fate.

Extrafollicular, T-dependent antibody and memory responses are important in clinically relevant situations. Extrafollicular responses contribute to the production of autoantibodies in the MRL.lpr mouse model of lupus (66–70) and in systemic lupus erythematosus patients (70–73). The bacteria Salmonella typhimirium elicits a predominantly T-dependent, extrafollicular antibody response, with germinal centers forming only very late after infection (74–77). Germinal center-independent memory B cells that arise early in T-dependent responses are also of great importance. These lower affinity memory cells likely allow for a secondary response to a broad range of pathogen variants, while germinal center derived high affinity memory cells are more tailored towards responding to reinfection with the original challenge (4, 6, 7, 78). Early IgM memory cells are known to be important in several mouse models of infectious disease, including infection with E. muris, a tick-borne intracellular bacteria (79, 80), and malaria (81, 82). Mice with specific defects in extrafollicular responses, such as those lacking PIK3IP1 in B cells described here, may serve as a useful model to study these diseases.

Here we add PIK3IP1 to the list of negative regulators of PI3K signaling that have unique functions in B cells, further emphasizing the need to keep PI3K activity “just right” a la Goldilocks and the Three Bears (21, 22). While PIK3IP1 interacts with the PI3K p85/p110 heterodimer to limit is ability to signal (23), PTEN and SHIP-1 dephosphorylate the product of PI3K, PIP3, and thus act further downstream (13). Thus, there may be situations in which excess PIP3 produced in the absence of PIK3IP1 is dephosphorylated by PTEN and/or SHIP-1. As such, additional roles for PIK3IP1 in B cells beyond those illustrated here may be revealed in the absence of PTEN or SHIP-1. These could have particular relevance in autoimmune disease or B cell malignancy, which PTEN and SHIP-1 are known to limit (83, 84).

Supplementary Material

1

Key points.

PIK3IP1 is expressed in B cells and is downregulated late after activation.

Lack of PIK3IP1 impairs early class switching during T-dependent responses.

PIK3IP1 is not required for germinal center responses or T-independent responses.

Acknowledgments

This work was supported by NIH Grant AI126051. A.B.S. is a Southwestern Medical Foundation Scholar in Biomedical Research and holds the Peggy Chavellier Professorship in Arthritis Research and Treatment.

Abbreviations

KLH

keyhole limpet hemocyanin

NP

4-Hydroxy-3-nitrophenylacetyl

PIK3IP1

Phosphoinositide-3-Kinase Interacting Protein 1

PIP3

Phosphatidylinositol (3,4,5)-trisphosphate

PTEN

Phosphatase and tensin homolog

References

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