PI3Kδ hyper activation promotes the development of B cells ...6. Refractory Respiratory...

$: PI3Kδ hyper activation promotes the development of B cells ...6. Refractory Respiratory Inflammation Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, Stevenage,$

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PI3Kδ hyper‐activation promotes the development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody‐independent manner Anne‐Katrien Stark1,2*, Anita Chandra1,2,3,4*, Krishnendu Chakraborty1,3, Rafeah Alam1, Valentina Carbonaro1, Jonathan Clark5, Srividya Sriskantharajah6, Glyn Bradley7, Alex G. Richter8,9, Edward Banham‐Hall1,3,4, Menna R. Clatworthy10, Sergey Nejentsev3, J. Nicole Hamblin6, Edith M. Hessel6, Alison M. Condliffe11, Klaus Okkenhaug1,2,#

1. Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge CB21 3AT, UK.

2. Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.

3. Department of Medicine, University of Cambridge, CB2 OQQ, UK. 4. Cambridge University Hospitals NHS Trust, Hills Road, Cambridge, CB2 0QQ 5. Biological Chemistry Laboratory, Babraham Institute, Cambridge CB21 3AT, UK. 6. Refractory Respiratory Inflammation Discovery Performance Unit, Respiratory

Therapy Area, GlaxoSmithKline, Stevenage, SG1 2NY, UK. 7. Computational Biology and Statistics, Target Sciences, GlaxoSmithKline, Stevenage,

SG1 2NY, UK. 8. Department of Immunology, Queen Elizabeth Hospital, Birmingham, UK. 9. Institute of Immunology and Immunotherapy, University of Birmingham, UK. 10. Molecular Immunity Unit, University of Cambridge Department of Medicine, MRC

Laboratory of Molecular Biology, CB2 OQQ, UK. 11. Department of Department of Infection, Immunity and Cardiovascular Diseases,

University of Sheffield, Sheffield S10 2RX, UK.

* Joint first authors. # Correspondence: [email protected]

.CC-BY-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted March 18, 2018. . https://doi.org/10.1101/280651doi: bioRxiv preprint

https://doi.org/10.1101/280651

http://creativecommons.org/licenses/by-nd/4.0/


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Abstract Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death 1 world‐wide. Antibody‐mediated immune responses can offer protection against repeated 2 exposure to S. pneumoniae, yet vaccines only offer partial protection. Patients with 3 Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated 4 a conditional knockin mouse model of this disease and identified a CD19+B220‐ B cell 5 subset that is induced by PI3Kδ signaling, is resident in the lungs, and which promotes 6 increased susceptibility to S. pneumoniae during the early phase of infection via an 7 antibody‐independent mechanism. We show that an inhaled PI3Kδ inhibitor improves 8 survival rates following S. pneumoniae infection in wild‐type mice and in mice with 9 activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the 10 severity of S. pneumoniae infection, representing a novel therapeutic target. 11

Introduction 12

Streptococcus pneumoniae is an invasive extracellular bacterial pathogen and is a leading 13 cause of morbidity and mortality. Although S. pneumoniae can cause disease in 14 immunocompetent adults, it commonly colonizes the upper airways without causing 15 disease. The World Health Organization has estimated that there are 14.5 million episodes 16 of severe pneumococcal disease and that 1.6 million people die of pneumococcal disease 17 every year1. Despite the implementation of global vaccination programs, S. pneumoniae 18 infection remains a major disease burden2‐4. 19 20 Invasive S. pneumoniae infection is a major cause of lower airway infections (pneumonia), 21 sepsis and meningitis. Healthy people at the extremes of age are more susceptible to 22 pneumococcal disease, as are people with chronic obstructive pulmonary disease (COPD), 23 however those at greatest risk are patients with splenic dysfunction or immune deficiency. 24 This increased susceptibility results at least in part from the lack of protective antibodies 25 against conserved protein antigens or against polysaccharides that form part of the 26 pneumococcal capsule5. Indeed, the protective role of antibodies in pneumococcal disease 27 is most obvious in individuals with congenital (primary) immunodeficiencies (PIDs). This was 28 first recognized in a patient with X‐linked agammaglobulinemia (XLA), a syndrome 29 subsequently shown to be caused by a block in B cell development due to loss‐of‐function 30 mutations in BTK6‐8. These patients remain highly susceptible to S. pneumoniae into 31 adulthood, but can be effectively treated by the administration of immunoglobulins from 32 healthy donors. 33 34 We and others have recently described cohorts of immune deficient patients with activating 35 mutations in PIK3CD, the gene encoding the p110δ catalytic subunit of phosphoinositide 3‐36 kinase δ (PI3Kδ)9‐11. PI3Kδ is a lipid kinase that catalyzes the phosphorylation of the 37 phosphatidylinositol‐(4,5)‐bisphosphate lipid to produce phosphatidylinositol‐(3,4,5)‐38 trisphosphate (PIP3). PI3Kδ is expressed in cells of the immune system and regulates many 39 aspects of immune cell signaling, particularly in lymphocytes12, 13. Activated 40 phosphoinositide 3‐kinase δ syndrome (APDS) is a combined immunodeficiency affecting T 41 and B cells. APDS patients suffer from recurrent sinopulmonary infections, with S. 42 pneumoniae being the most commonly isolated pathogen14. 85% of APDS patients have 43 been diagnosed with pneumonia15. APDS patients are also more likely to develop structural 44 lung damage (bronchiectasis) than patients with other PIDs14. The mechanism underpinning 45


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the increased susceptibility to pneumococcal infection in APDS is unclear12. Although APDS 46 patients often lack IgG2, the protection afforded by immunoglobulin replacement therapy is 47 not as robust as that observed in patients with pure antibody deficiencies, suggesting that 48 antibody‐independent PI3Kδ‐driven mechanisms may be involved14. The monogenic nature 49 of APDS allows us to dissect mechanisms of susceptibility to S. pneumoniae infection on 50 cellular and molecular levels, and to determine whether PI3Kδ inhibitors may help reduce 51 the susceptibility to S. pneumoniae. If so, PI3Kδ inhibitors, that are in development for the 52 treatment of inflammatory and autoimmune diseases, might also have wider applications to 53 reduce the pathological consequences of S. pneumoniae infection16. In this study, we have 54 explored mechanisms by which PI3Kδ hyperactivation drives susceptibility to S. pneumoniae 55 infection. We found that the administration of the PI3Kδ‐selective inhibitor nemiralisib 56 (GSK‐22696557)17, 18 reduced the severity of pneumococcal disease in wild‐type mice. To 57 investigate this further, we generated a p110δE1020K mouse model that accurately 58 recapitulates the genetics and immunological phenotype of APDS, and displays increased 59 susceptibility to S. pneumoniae infection. We show that this susceptibility segregates with 60 enhanced PI3Kδ signaling in B cells, which exacerbate S. pneumoniae infection at early time 61 points before the adaptive immune response comes into play. Of note, we have identified a 62 previously unappreciated population of CD19+B220‐ IL‐10‐secreting cells that was present in 63 wild‐type mice but expanded 10‐20 fold in p110δE1020K mice. We demonstrate that 64 nemiralisib reduces the frequency of IL‐10‐producing B cells in the lung and improves 65 survival of p110δE1020K mice. Similarly, a higher proportion of transitional B cells from APDS 66 patients produced IL‐10 and this was reduced by nemiralisib. This study provides new 67 insights into the pathogenesis of the early stages of invasive S. pneumoniae disease and 68 offers the potential of future therapeutic strategy to alleviate the severity of this disease in 69 susceptible patients. 70


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Results 71

Inhaled nemiralisib alleviates the severity of S. pneumoniae infection in mice 72 Given that APDS patients are more susceptible to S. pneumoniae, we sought to determine 73 whether nemiralisib, an inhaled PI3Kδ inhibitor which is in development for the treatment 74 and prevention of COPD excacerbations17, 18, would alter susceptibility to airway infections. 75 We treated mice with nemiralisib and then infected them intranasally with S. pneumoniae. 76 Nemiralisib‐treated mice showed prolonged survival compared to mice given vehicle control 77 (Fig 1). This protection was only effective if the drug was administered before and during 78 infection (Fig 1). By contrast, nemiralisib administration 8h or 24h post infection had no 79 impact on survival of the mice. These data suggest that PI3Kδ modulates the immune 80 response during early S. pneumoniae infection, either by inhibiting protective immunity, or 81 by promoting an adverse response. 82 83

Mouse model of APDS: hyperactive PI3Kδ signaling and altered B and T cell 84

development in p110δE1020K mice 85 We generated a conditional knock‐in mouse harboring mutation E1020K in the Pik3cd gene 86 that is equivalent to the most common APDS‐causing mutation E1021K in humans 87 (Supplementary Fig 1). These mice were subsequently crossed with different Cre‐expressing 88 lines to either generate germline mice where p110δE1020K is expressed in all cells 89 (p110δE1020K‐GL) or selectively in B cells using Mb1Cre (p110δE1020K‐B), in T cells using Cd4Cre 90 (p110δE1020K‐T) or myeloid cells using Lyz2Cre (p110δE1020K‐M). We studied p110δE1020K mice in 91 comparison with wild‐type and p110δD910A mice that have catalytically inactive p110δ19. 92 93 Initially, we tested if p110δE1020K mice have increased PI3Kδ activity and display the 94 characteristic immunological phenotype of APDS. Biochemical analyses of B cells and T cells 95 from p110δE1020K‐GL mice confirmed that the kinase is hyperactive (Fig 2). Measurements of 96 PIP3 in T cells showed that p110δE1020K is about 6 times as active as the wild‐type kinase 97 following stimulation with anti‐CD3 and anti‐CD28, but with no evidence for increased basal 98 activity (Fig 2A). In B cells, p110δE1020K led to increased basal PIP3 levels, but it was further 99 increased only about 2‐fold compared to wild‐type mice after stimulation with anti‐IgM (Fig 100 2B). This pattern resembles results found in patients with APDS10. In wild‐type and 101 p110δE1020K‐GL cells, the PI3Kδ‐selective inhibitor nemiralisib reduced PIP3 to the background 102 level observed in p110δD910A cells, which, as expected, were insensitive to nemiralisib (Fig 103 2A, B). 104 105 PIP3 binds to the protein kinase AKT, supporting its phosphorylation on Thr308 and 106 subsequent activation. Western blotting of purified p110δE1020K‐GL T cells showed increased 107 AKT phosphorylation following stimulation with anti‐CD3 and anti‐CD28 antibodies 108 compared to wild‐type cells, whereas AKT phosphorylation in p110δD910A T cells was below 109 the limit of detection (Fig 2C). In B cells, both basal and anti‐IgM‐induced phosphorylation of 110 AKT were elevated in p110δE1020K‐GL cells, while strongly diminished in p110δD910A B cells (Fig 111 2D). The phosphorylation of ERK and the AKT effector proteins, FOXO and S6, were similarly 112 affected. All phosphorylation events in wild‐type and p110δE1020K‐GL cells were reduced to 113 the levels observed in p110δD910A cells by inhibition with nemiralisib. As expected, p110δ 114 protein expression was not affected by the E1020K or D910A mutations (Figs 2C, D). 115 116


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Figure 1: Prophylactic, but not therapeutic treatment with the inhaled PI3Kδ inhibitor nemiralisibmitigates disease severity following S. pneumoniae infection in wild-type mice.Wild-type mice were treated twice daily with the inhaled PI3Kδ inhibitor nemiralisib for the duration of thestudy: when treatment was started 24h prior to infection with S. pneumoniae, survival rates were improved.When started 8h or 24h post-infection, the treatment had no effect on survival outcome. (-24h: data from 5independent experiments combined n=60; +8h/+24h: data from 3 independent experiments combinedn=36).

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Figure 2: PI3Kδ hyper-activation leads to increased PIP3 and pAKT levels that can be reduced using aselective PI3Kδ inhibitorA: PIP3 levels in purified T cells from wild-type, p110δE1020K and p110δD910A mice, unstimulated or stimulatedwith anti-CD3 and anti-CD28 in the presence or absence of the selective PI3Kδ inhibitor, nemiralisib (n=2-3).B: PIP3 levels in purified B cells from wild-type, p110δE1020K and p110δD910A mice unstimulated, or stimulatedwith anti-IgM in the presence or absence of nemiralisib (n=2-6). C: Western blots of purified T cells from wild-type, p110δE1020K and p110δD910A mice, unstimulated or stimulated with anti-CD3 and anti-CD28 in thepresence or absence of nemiralisib. D: Western blots of purified B cells from wild-type, p110δE1020K andp110δD910A mice, unstimulated or stimulated with anti-IgM in the presence or absence of nemiralisib.(Representative of 2 independent experiments).

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Germline p110δE1020K‐GL mice had near normal numbers of myeloid cells in the bone marrow 117 and spleen. In the bone marrow we observed a significant B cell lymphopenia that was 118 associated with a block in B cell development between Pro‐B and Pre‐B cells and did not 119 extend to the spleen. Although these mice had normal numbers of splenic B cells, there was 120 an increased proportion of marginal zone B and B1 cells with an altered distribution of 121 transitional B cells (Supplementary Fig. 2). The thymus of p110δE1020K‐GL mice was normal 122 except for a mild reduction in single positive CD8+ T cells. In the spleens and lymph nodes 123 there were increased proportions of activated/memory T cells identified by high CD44 124 expression and low CD62L expression, and increased numbers of Foxp3+ T regulatory cells 125 (Supplementary Fig 3). These T cell and B cell phenotypes were recapitulated in p110δE1020K‐T 126 and p110δE1020K‐B mice, respectively (Supplementary Figs 4 and 5.). The reciprocal effects of 127 the inhibitory D910A and activating E1020K mutations on specific cell subsets demonstrate 128 the pivotal role of PIP3 during lymphocyte development and highlight the importance of a 129 tight control of PI3Kδ activity. 130 131 Analysis of serum immunoglobulins showed that p110δE1020K‐GL mice had elevated levels of 132 IgG1 and IgG2b and a trend towards increased levels of IgG2c, IgA, and IgE isotypes 133 compared to wild‐type mice. There was also a trend to hyper IgM in p110δE1020K‐GL mice as 134 is frequently observed in APDS patients 10, 11, 14, whereas p110δD910A mice were antibody 135 deficient (Supplementary Fig 6). The level of serum IgG3, which has been shown to be 136 protective against S. pneumoniae19, was comparable in p110δE1020K‐GL and wild‐type mice 137 (Supplementary Fig 6). 138 139

Susceptibility to S. pneumoniae is caused by PI3Kδ hyper‐activation in B cells 140 Given that the immunological phenotype of p110δE1020K‐GL mice strongly resembled that of 141 APDS, we sought to determine whether these mice recapitulate the increased susceptibility 142 to S. pneumoniae observed in APDS patients. We infected p110δE1020K‐GL, p110δD910A, and 143 p110δWT mice with S. pneumoniae (TIGR4 serotype 4) and followed their survival for 10 days 144 (Fig 3A). Interestingly, despite being antibody‐deficient, p110δD910A mice did not show 145 increased susceptibility to S. pneumoniae. By contrast, p110δE1020K‐GL mice showed 146 accelerated disease onset and increased mortality (Fig 3A). 147 148 In order to determine the cell type responsible for increased susceptibility to S. 149 pneumoniae, we next infected the lineage‐restricted p110δE1020K‐B, p110δE1020K‐T and 150 p110δE1020K‐M mice. Myeloid expression of p110δE1020K had no effect on the course of S. 151 pneumoniae infection (Fig 3B), whereas expression of p110δE1020K in T cells was protective 152 (Fig 3C). Only the p110δE1020K‐B mice replicated the increased susceptibility of the 153 p110δE1020K‐GL mice to S. pneumoniae (Fig 3D). Furthermore, transfer of p110δE1020K‐B bone 154 marrow into irradiated RAG2‐/‐ recipients also conferred increased susceptibility to 155 infection, which was only partially rescued by co‐transferring wild‐type and p110δE1020K‐B 156 bone marrow at a 1:1 ratio (Fig 3E). These results indicate that B cells drive the increased 157 susceptibility to S. pneumoniae infection in p110δE1020K‐GL mice in an immune‐dominant 158 manner. 159 160 Natural antibodies against phosphorylcholine (PC) can offer protection against infection 161 with encapsulated bacteria, including S. pneumoniae 20, 21. We found that p110δD910A mice 162 lacked anti‐PC antibodies, presumably because of the absence of B1 and MZ B cells which 163


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Figure 3: PI3Kδ hyper-activation leads to increased susceptibility to S. pneumoniae infectionA: Germline p110δE1020K-GL mice show accelerated disease development and significantly increased mortalitycompared to control mice in response S. pneumoniae infection, while kinase dead p110δD910A mice do notrespond differently to wild-type mice. B-D: The p110δE1020K mutation was introduced conditionally into myeloidcells, T cells and B cells by crossing onto Lyz2cre, Cd4cre and Mb1cre lines respectively. The myeloid conditionalmutation did not affect survival following S. pneumoniae infection in p110δE1020K-M mice (B), while T cellconditional p110δ hyper-activation lead to improved survival in p110δE1020K-T mice (C). However, introducing thep110δE1020K mutation specifically in B cells (p110δE1020K-B mice) replicated the increased susceptibility to S.pneumoniae seen in p110δE1020K-GL mice (D). E: Transfer of p110δE1020K-B bone marrow into irradiated RAG2-/-

recipients also conferred increased susceptibility to S. pneumoniae infection compared to recipients receivingwild-type bone marrow, and this phenotype was not fully rescued in a 50% BM chimera. F-G: Naïve PI3KδE1020K

mice produce normal levels of anti-phosphorylcholine IgM and significantly higher levels of anti-PC IgG, whilePI3KδD910A mice produce no natural antibody. (Data from 2 independent experiments combined. A: n=24; B:n=22; C: n=20; D: n=20; E: n=20; F-G: WT n=11; E1020K n=8 D901A n=9).

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are the major source of natural antibodies19, 22‐24. By contrast, anti‐PC antibodies in the 164 serum from p110δE1020K mice were similar (IgM) or elevated (IgG)(Fig 3 F , G) compared to 165 wild‐type mice. Therefore, susceptibility to S. pneumoniae in p110δE1020K mice cannot be 166 explained by a failure to produce natural antibodies against conserved bacterial epitopes. 167 168

B cells drive early pathology during S. pneumoniae infection in an antibody‐169

independent manner but are required to prevent chronic infection 170 In order to further investigate antibody‐mediated protection in the context of PI3Kδ hyper‐171 activation, we immunized mice with Pneumovax, a 23‐valent polysaccharide vaccine25. 172 Following infection with S. pneumoniae, wild‐type mice were completely protected by this 173 vaccination protocol, while p110δE1020K‐GL mice were only partially protected, to a level 174 similar to that in non‐immunized wild‐type mice. By contrast, p110δD910A mice did not 175 benefit from vaccination (Fig 4A, B). Interestingly, p110δE1020K‐GL mice and wild‐type mice 176 produced a similar antibody response, in contrast to p110δD910A mice that showed no 177 response (Fig 4C). These data indicate that Pneumovax vaccination clearly protects against 178 S. pneumoniae, as shown in immunized wild‐type mice. Despite this protection p110δE1020K 179 mice remained significantly more susceptible to S. pneumoniae infection. Together, these 180 data suggest that B cells can affect susceptibility to S. pneumoniae by a mechanism that is at 181 least in part antibody‐independent. 182 183 To determine more definitively whether B cells can be pathogenic in the context of S. 184 pneumoniae infection, we infected wild‐type and Ighmtm1 (µMT) mice which lack mature B 185 cells26. Strikingly, Ighmtm1 mice showed reduced susceptibility to S. pneumoniae infection, 186 delaying disease onset from ~2 days in wild‐type mice to ~5 days in Ighmtm1 mice (Fig 5). 187 Although survival in Ighmtm1 mice was increased up to 30 days post infection compared to 188 wild‐type mice, CFU counts from the lungs of mice surviving to this time‐point indicates 189 that, despite appearing clinically healthy, 41% (7/17) of Ighmtm1 mice failed to clear the 190 infection compared to 100% clearance in wild‐type mice (Fig 5). Taken together, these 191 results suggest that during early time‐points in the local infected environment B cells can be 192 pathogenic, while at later stages they are required to prevent chronic infection. 193 194 Increased susceptibility to S. pneumoniae could either be due to uncontrolled bacterial 195 proliferation or be caused by an aberrant immune response to the pathogen. The bacterial 196 titers from lungs and spleens of wild‐type, p110δD910A and p110δE1020K‐GL mice were similar 197 at 24h post infection, suggesting that the different susceptibilities did not correlate with 198 different abilities to control bacterial outgrowth during the early phase of infection 199 (Supplementary Fig 7A). We observed a trend towards increased levels of TNFα, IL‐6, IL‐1β 200 and IL‐1α in the lung tissue of p110δE1020K‐GL mice at 24h post infection (Supplementary Fig 201 7B). Consistent with this, the levels of TNFα, IL‐6 and IL‐1β were reduced in the lungs of 202 wild‐type mice treated with nemiralisib prior to S. pneumoniae infection (Supplementary Fig 203 7C), indicating that, while PI3Kδ signaling affect the amount of pro‐inflammatory cytokines 204 produced in response to infection, the increase in response to PI3Kδ hyper‐activation is 205 modest. 206 207


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GL mice against infection (A), while p110δD910A are not protected (B). C: p110δE1020K-GL mice produce normallevels of total IgG in response to Pneumovax immunisation (anti-pneumococcal serotype 4 shown) whilep110δD910A mice do not respond to vaccine. (A: results from 3 independent studies combined, n=30; B: resultsfrom 2 independent studies combined, n=19; C: results from 2 independent studies combined: WT n=22;E1020K n=8; D910A n=6)

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Figure 5: B cells could drive increased pathology in response to S. pneumoniae infection, independentof bacterial clearance.Ighmtm1 (µMT) mice show delayed disease progression and improved overall survival up to 30 days post S. pneumoniae infection, however 41% (7/17) surviving Ighmtm1 mice failed to clear bacteria from the lung tissue at 30 days post infection compared to 100% clearance in surviving wild-type mice. (Data from 1 study, n=24)

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CD19+ B220‐ B cells produce high levels of IL‐10 in response to S. pneumoniae 208

infection in comparison with conventional CD19+ B220+ B cells 209 We hypothesized that increased susceptibility to S. pneumoniae infection can be mediated 210 by a specific subpopulation of B cells. Therefore, we studied the B cell compartment in 211 various tissues and found an atypical population of CD19+B220‐ B cells that was rare in wild‐212 type mice, but significantly increased in p110δE1020K‐GL mice and was absent in p110δD910A 213 mice (Fig 6A). In the spleen and bone marrow, there was a 10‐ and 5‐fold increase, 214 respectively, in the numbers of CD19+B220‐ B cells in p110δE1020K‐GL mice compared to wild‐215 type mice (Fig 6A). Infection with S. pneumoniae did not induce further expansion of 216 CD19+B220‐ B cells in the lungs or other tissues examined 24h post‐infection (Fig 6B). 217 218 To ascertain if these cells were also present in the lungs and to distinguish resident from 219 circulating cells, we labelled circulating leukocytes in wild‐type and p110δE1020K‐GL mice by 220 intravenous injection of biotin‐conjugated anti‐CD45. We then stained the lung homogenate 221 with fluorochrome‐conjugated anti‐mouse CD45 and streptavidin, and used flow cytometry 222 to distinguish tissue resident leukocytes from those present in the lung capillaries. We found 223 a significant increase in the proportion and number of tissue‐resident, but not circulating, B 224 cells in p110δE1020K‐GL mice compared to wild‐type mice. There was an increase in both 225 CD19+B220+ and CD19+B220– cells among the tissue‐resident B cells which demonstrates 226 that both populations can take residence in the lung (Supplementary Fig 8). 227 228 Given that B cells from p110δE1020K‐GL mice drive susceptibility to S. pneumoniae in an 229 antibody‐independent manner, we sought to look at other properties of B cells. We found a 230 trend towards lower IL‐10 protein levels and a significant 10‐fold decrease in IL‐10 mRNA 231 expression at 24h post infection in whole lung homogenates from wild‐type mice treated 232 with nemiralisib (Supplementary Fig 7C, D), indicating that PI3Kδ signaling can regulate IL‐10 233 levels in the lung post infection. IL‐10 is an important immune‐regulatory cytokine known to 234 affect the course of S. pneumoniae infection27, 28 and therefore increased IL‐10 production 235 could explain the B cell‐dependent but antibody‐independent effects seen in p110δE1020K‐GL 236 mice. To investigate this further, we crossed the p110δE1020K‐GL and p110δD910A mice with a 237 highly sensitive IL‐10 reporter (Il10ITIB) mouse29. 238 239 At 24h post S. pneumoniae infection, the frequencies of the IL‐10‐producing B cells were 240 significantly increased in the lungs of p110δE1020K‐GL Il10ITIB mice compared to in wild‐type 241 Il10ITIB mice (Supplementary Fig 7E). In addition, IL‐10‐producing B cells were barely 242 detected in lungs from p110δD910A mice (Supplementary Fig 7E). Frequencies of the IL‐10‐243 producing CD11b+ myeloid, T and NK cells in lungs were variable, but not consistently 244 increased in p110δE1020K‐GL mice (Supplementary Fig 7E). Further analysis of the CD19+ B cell 245 subset isolated from lungs and spleens at 24h post S. pneumoniae infection showed that the 246 proportion of IL‐10‐producing cells was increased among the B220‐ B cell subset as 247 compared to the B220+ B cell subset in p110δE1020K‐GL mice (5% B220+ vs 68% B220‐) and 248 wild‐type mice (2% B220+ vs 55% B220‐) (Fig 6C). Furthermore, the proportion and absolute 249 number of IL‐10‐producing CD19+B220‐ cells and CD19+B220+ cells was increased in 250 p110δE1020K‐GL mice compared to wild‐type mice, while such IL‐10‐producing cells were 251 virtually absent in p110δD910A mice (Fig 6C). This indicates that CD19+B220‐ cells are the 252 predominant population of B cells that produce IL‐10 in a PI3Kδ‐dependent manner. 253 254


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Figure 6: p110δE1020K mice have an expanded population of IL-10 producing CD19+B220- B cellsA: Naïve p110δE1020K mice show a significant increase in the proportion and number of B220- B cells in bone marrow and spleen. While thispopulation is detectable in wild-type mice, it is absent from p110δD910A mice. Representative pseudocolor plots show mean cell proportions in naïvemice B: S. pneumoniae (S.p) infection does not lead to significant expansion or recruitment of B220- B cells at 24h post infection in bone marrow,blood, spleen or lung tissue. Representative pseudocolor plots show mean cell proportions in naïve mice. C: At 24h post S. pneumoniae infection,IL-10 production is increased in p110δE1020K B cells from both lung and spleen, and reduced in p110δD910A B cells compared to wild-type mice.Furthermore, B220- B cells produce higher levels of IL-10 in response to S. pneumoniae infection compared to B220+ B cells in wild-type andp110δE1020K mice. Representative pseudocolor plots show mean cell proportions in S. pneumoniae infected mice (A: combined data from 2independent experiments, WT n=10; E1020K n=12; D910A n=7, B: representative data from 2 independent experiments, n=6, C: representativedata from 2 independent experiments n=6).

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Previously, B cells that produce IL‐10 have been termed B regulatory cells (Bregs)30. We 255 sought to compare the phenotype of CD19+B220‐ IL‐10‐producing B cells with conventional 256 IL‐10‐producing CD19+B220+ Bregs described previously (Fig 7). Following in vitro 257 stimulation for 5h and analysis of cell surface markers and intracellular IL‐10 we found that 258 there were clear phenotypic differences between conventional B220+ Breg cells and the 259 B220‐ B cells we describe here, including the differential expression of CD43 and IgM (Fig 7). 260 The lack of B220 expression and low IgM expression differentiate the CD19+B220‐ IL‐10‐261 producing B cells from conventional B1 cells31. 262 263

Nemiralisib ameliorates the susceptibility to S. pneumoniae in p110δE1020K mice 264 Given that increased PI3Kδ activity in mice leads to increased mortality after S. pneumoniae 265 infection we explored if nemiralisib would provide protection. Treatment of p110δE1020K‐GL 266 mice with inhaled nemiralisib 24h prior to infection led to a 20% increase in survival. While 267 nemiralisib treatment did not affect the numbers of CD19+B220‐ B cells, the proportion of IL‐268 10 producing CD19+B220‐ B cells in the lungs were reduced in comparison to non‐treated 269 mice (Fig 8A). This was in keeping with our prior observations that nemiralisib treatment 270 reduced IL‐10 mRNA levels in the lung tissue of S. pneumoniae infected wild‐type mice 271 (Supplementary Fig 7D), suggesting a role for IL‐10‐producing CD19+B220‐ B cells in the 272 PI3Kδ‐dependent susceptibility to infection. 273 274

Nemiralisib treatment suppresses IL‐10 production in B cells of APDS patients 275 Cohort studies have shown that 75% of patients with APDS have elevated circulating 276 transitional B cells (CD19+IgM++CD38++)14. Other studies have shown that such transitional B 277 cells can produce high levels of IL‐1032, 33. We obtained blood samples from patients with 278 APDS and healthy controls and confirmed that all of these APDS patients had elevated 279 proportions of transitional B cells (Fig 8B). We isolated PBMCs from two of these APDS 280 patients and three healthy controls and stimulated them for 72h with anti‐CD3 and IL‐2 32. 281 After stimulation we found more IL‐10‐producing B cells and more IL‐10‐producing 282 transitional B cells in PBMCs of APDS patients than in PBMCs of healthy controls, while 283 treatment with nemiralisib effectively suppressed IL‐10 production in these cells (Fig 8C). 284 Therefore, in keeping with reduction of CD19+B220‐IL‐10 producing B cells in the mice, 285 nemiralisib can also reduce IL‐10 producing human transitional B cells. 286


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Figure 7: IL-10 producing B220- B cell subset is a novel B cell subset, expanded in PI3KδE1020K mice.In order to compare surface marker expression from B220- and B220+ IL-10 producing B cells, splenocytesfrom Il10ITIB mice (WT and E1020K) were stimulated with LPS/PdBu/Ionomycin/Brefeldin A for 5 hours andthen stained for cell surface markers as shown. A: After gating on IL-10 producing B cells, CD43++B220- cellswere compared to CD43-B220+ cells. Histograms highlighting the differential surface marker expression asmeasured by median fluorescence intensity (MFI) between these populations are shown. B: Comparison ofsurface marker MFI show that CD19+ B220- IL-10+ B cells express: CD43++CD5int/+ CD23-CD21-CD1dlo/int

IgM+/-IgDlo/-PDL1-CD138- as opposed to conventional Bregs expressing: CD19hi B220hi IL-10+ CD43-

CD5VarCD23-CD21++CD1dhi IgMhi IgDVar/lo PDL1+CD138int. (Data from 1 experiment, n=5)

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Figure 8: Nemiralisib treatment reduces the proportion of IL-10 producing B cells in mice and humans.A: Prophylactic treatment with nemiralisib improved survival in p110δE1020K-GL mice and was associated with asignificant reduction in the proportion of IL-10 producing B220- B cells in the lung at 24h post infection. (A: Combineddata from two independent studies, survival n=24; lung tissue analysis n=12) B: Blood from patients with APDS (n=6)and healthy controls (n=6) was obtained and the B cell phenotype was determined by flow cytometry. Transitional Bcells are identified as CD19+IgD+CD27-CD24+CD38+. Representative contour plots with outliers show the meanproportions of cells. C: Freshly isolated PBMCs were unstimulated or stimulated with plate bound anti-CD3 and anti-IL-2 for 72h in the presence or absence of nemiralisib (n=2-3). The proportion of IL-10 producing cells among the totalB cell and transitional B cell populations was determined by flow cytometry. Representative contour plots with outliersshow mean proportions of IL-10 producing cells. (Combined data from 2 independent experiments).

CD19

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Discussion 287

Our results show that enhanced PI3Kδ signaling leads to the increased susceptibility to S. 288 pneumoniae infection through a B cell‐dependent, but antibody‐independent mechanism. 289 We found an expansion of an aberrant B cell population which could be equivalent to the 290 elevated transitional B cell population found in APDS patients. These cell populations may 291 have a detrimental effect during the early phase of S. pneumoniae infection. 292 293

B cells drive increased susceptibility to S. pneumoniae infection in p110δE1020K mice 294 In this study, we show that B cells can increase the susceptibility to S. pneumoniae infection 295 during the first few hours after exposure. Mice were more susceptible to S. pneumoniae 296 infection when the hyperactive p110δE1020K mutation was expressed in B cells. By contrast, 297 mice expressing the p110δE1020K mutation only in T cells were protected. The basis for the 298 protection remains unknown and is the focus of ongoing study. The p110δE1020K mice have 299 increased numbers of TFH and Treg cells and the conventional T cells have a more activated 300 phenotype. A recent study has documented a population of lung‐resident innate Th17 cells 301 that confers protection against re‐challenge with S. pneumoniae34. Whether such a subset is 302 affected by PI3Kδ activity and can also protect against immediate challenge is not known. 303 However, we did not detect increased proportions of Th17 cells after infection. Regardless 304 of the mechanism, the protection offered by T cells was overcome by the adverse effects of 305 B cells in p110δE1020K‐GL mice. 306 307 We describe a subset of B cells which lack the common B cell marker B220, and whose 308 abundance is correlated with the susceptibility to S. pneumoniae infection. Moreover, we 309 show that the development of this CD19+B220‐ B cell subset is highly dependent on the level 310 of activity of PI3Kδ. Susceptibility to S. pneumoniae infection was not altered in p110δD910A 311 mice as compared to wild‐type mice. Kinase‐dead p110δD910A mice lack natural and anti‐312 capsular antibodies which may make them more susceptible, however, their lack of 313 CD19+B220‐ B cells may counterbalance this antibody deficiency, such that overall the 314 mutation has little net effect on S. pneumoniae susceptibility. 315 316

PI3Kδ signaling controls IL‐10 production in B cells 317 A large proportion of the CD19+B220‐ B cells produced IL‐10. Although increased IL‐10 could 318 potentially reduce the production of inflammatory cytokines, we found similar or increased 319 levels of TNFα, IL‐6 and IL‐1 in the lungs of p110δE1020K mice and no difference in CFU counts 320 at 24h post infection. 321 322 One of the defining characteristics of APDS patients is an increased proportion of 323 CD24+CD38+ B cells defined as transitional B cells 10‐12, 14, 15. Intriguingly, production of IL‐10 324 is a characteristic of these B cells as well. High IL‐10 levels in response to secondary S. 325 pneumoniae infection following influenza A is associated with increased lethality when 326 compared to a primary S. pneumoniae infection, and importantly, the outcome of a 327 secondary S. pneumoniae infection is improved by IL‐10 neutralisation27, 28. This indicates 328 that, while IL‐10 is required for normal immune regulation and resolution of inflammation35, 329 excess IL‐10 during the early stages of S. pneumoniae infection could have an acute 330 detrimental effect. This fits with our observation that nemiralisib treatment improved the 331 outcome of mice infected with S. pneumoniae and reduced IL‐10 mRNA levels in the lung. 332


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Further studies are required to investigate the time and location specific effects of B cell 333 dependent IL‐10 production, keeping in mind that B cells have the potential to produce a 334

number of different cytokines (such as IL‐6, TNF‐, and IL‐35) that could affect S. 335 pneumoniae susceptibility36. 336 337 We postulate that the transitional B cells which are expanded in APDS patients may not just 338 be precursors to more mature B cells, but also include cells that are functionally equivalent 339 to the CD19+B220‐ cells that we have identified in p110δE1020K mice and thus may also 340 contribute to the increased susceptibility to S. pneumoniae infection and hence to the high 341 incidence of bronchiectasis characteristic of this disease15. In this context, it is interesting 342 that CD19+B220‐ B cells had previously been described as immature progenitors in the bone 343 marrow37, rather than a functional PI3Kδ dependent subset found in peripheral tissues as 344 we describe here. Although the CD19+B220‐ B cells we describe resemble B1 cells by some 345 criteria, such as the expression of CD5 and CD43, their lower IgM expression, lack of B220 346 and distribution pattern suggest that they are related but distinct to conventional B1 and 347 B10 cells30, 31. A similar subset has previously been described as being dependent on CD19 348 and increased in the absence of PTEN expression and PTEN haploinsuffiency in humans can 349 lead to an APDS‐like syndrome38‐40. 350 351 Our results do not question the important role that antibodies play in the protection against 352 S. pneumoniae and other encapsulated bacteria. Indeed, in wild‐type mice, immunization 353 with Pneumovax offered complete protection. Rather, our results highlight a hitherto 354 underappreciated subset of B cells that increase the pathological response shortly after 355 infection. It remains to be determined whether this is a feature of the B cells being lung‐356 resident (and hence among the first cells of the immune system to encounter the invading 357 pathogens) and by which mechanisms B cells either condition lung epithelia to become 358 more susceptible to invasion of S. pneumoniae, reduce the clearance of S. pneumoniae by 359 phagocytes and/or increase the collateral adverse effects of the early innate immune 360 response to this pathogen. This is in keeping with the increasing realization that B cells are 361 important sources of different cytokines and can modulate immune responses 362 independently of their capacity to present antigen and produce antibodies36. 363 364

Therapeutic implications 365 Intriguingly, the inhaled inhibitor nemiralisib offered protection in not just in p110δE1020K but 366 also in wild‐type mice infected with S. pneumoniae, when administered prior to infection. 367 This result suggests that preventive inhibition of PI3Kδ can be beneficial not only just in the 368 context of APDS, but also in other conditions associated with PI3Kδ hyperactivation, such as 369 COPD41. 370 371 The results presented herein are relevant to our understanding and the potential treatment 372 of patients with APDS. Clinical trials are currently ongoing using both inhaled and systemic 373 PI3Kδ inhibitors (NCT02593539, NCT02435173 and NCT02859727). Systemic use of PI3Kδ 374 inhibitor has shown promise in reduction of lymphoproliferation and immune cell 375 aberrations in patients with APDS, however, no comments were made regarding recurrent 376 infections or respiratory manifestations42. Systemic use of PI3Kδ inhibitors in malignancies 377 have been associated with serious side effects, including colitis43. It would therefore be of 378 importance to determine whether inhaled PI3Kδ inhibitors could alleviate the respiratory 379


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manifestations of APDS while reducing the risk of the adverse effects associated with 380 systemic inhibition. Moreover, the results may also be relevant to other conditions, such as 381 COPD17, where PI3Kδ may be activated by non‐genetic mechanisms. Overall, our findings 382 suggest that in subjects with increased PI3Kδ activity prophylactic administration of PI3Kδ 383 inhibitors may help alleviate the course of S. pneumoniae infection. 384


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Materials and Methods 385

Mouse strains and gene targeting 386 The p110δE1020K mice were generated by Ozgene, Australia using homologous 387 recombination in ES cells. A duplicate sequence corresponding to the last coding exon in 388 Pik3cd and carrying the E1020K mutation was flanked by loxP sites and inserted 3’ to the 389 wild‐type sequence. Upon Cre‐mediated recombination, the wild‐type sequence is replaced 390 by the mutant E1020K sequence. p110δD910A, Cd4cre, Mb1cre, Lyz2cre, Ighmtm1 (µMT) and 391 Il10ITIB mice have been described previously19, 26, 29, 44‐46. Throughout the study, the 392 p110δE1020K allele was heterozygous whereas the p110δD910A alleles were homozygous. 393 Genotyping was performed by Transnetyx (Cordova, TN). 394 395

S. pneumoniae stock preparation 396 Streptococcus pneumoniae (TIGR4, serotype 4 (provided by Professor Jeremy Brown, 397 University College, London)) was grown to mid‐log phase (OD500 = 0.5‐0.7) in Todd‐Hewitt 398 broth (Oxoid) supplemented with 0.5% yeast extract (Oxoid) at 37°C, 5% CO2. The bacteria 399 were collected by centrifugation, and resuspended in PBS/20% glycerol (Sigma‐Aldrich) prior 400 to freezing in liquid N2 and storage at ‐80 °C. Stocks were assessed for viable CFU counts and 401 homogeneity by plating out serial dilutions of three frozen samples on blood agar plates (LB 402 agar, supplemented with 5% defibrinated sheep blood (Oxoid)) after incubation for 24h at 403 37 °C, 5% CO2. S. pneumoniae colonies were confirmed by the presence of an α‐hemolytic 404 zone and sensitivity to optochin (Sigma‐Aldrich). Virulent stocks were maintained by 405 performing an in vivo passage every 6‐12 months. 406 407

S. pneumoniae infections 408 Frozen stocks were thawed and washed twice by centrifugation in sterile PBS before 409 resuspending at 4x107 CFU/mL in PBS. The suspension was kept on ice at all times, and used 410 for infection within 2h of thawing (no loss of viability was observed under these conditions). 411 Mice were lightly anaesthetized by inhalation of 3% isoflurane and maintained with 2% 412 isoflurane. Mice (males and females aged 8‐12 weeks) were infected intranasally with 50µL 413 S. pneumoniae suspension containing 2x106 CFU. Animals were observed to confirm 414 inhalation of the dose and full recovery from anesthesia. The infection dose was routinely 415 confirmed by plating out serial dilutions of the inoculum on blood agar plates, as described 416 above. 417 418

Survival studies 419 Mice were infected with S. pneumoniae as described above. Pre‐infection body weights 420 were recorded and animals were weighed daily post infection. Animals were monitored 421 three times a day for a period of 10 days post infection. Disease progression was assessed 422 by assigning clinical scores without knowledge of the individual genotypes: 0: Healthy; 1: 423 mild clinical signs; 2: Up to 2 moderate clinical signs; 3: up to 3 moderate signs. Animals 424 were culled when they showed >25% bodyweight loss or reach score 3. The most frequently 425 observed clinical signs were piloerection, hunched posture, tremor, and labored breathing. 426 At the study end‐point, animals were culled and terminal blood samples collected. 427 428


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Bone Marrow transfer 429 A single cell suspension of p110δE1020K‐B and wild‐type (C57Bl/6.SJL) donor bone marrow was 430 prepared in sterile HBSS (Sigma‐Aldrich) as described below (Isolation of immune cells from 431 mouse tissues). Cells from two sex‐matched donors were pooled. RAG2‐/‐ recipient mice 432 were supplied with 4mg/mL Neomycin (Sigma‐Aldrich) in drinking water before sub‐lethal 433 irradiation (one dose of 500Rads over 63 seconds), and received 3x106 donor cells by 434 intravenous (tail vein) injection. Neomycin treatment was maintained for 4 weeks post 435 transfer, and after 8 weeks reconstitution was confirmed by analyzing tail bleeds. 436 437

Vaccination 438 For vaccine studies, mice were immunized with Pneumovax II (pneumococcal 439 polysaccharide vaccine, serotypes: 1‐5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B. 17F, 18C, 440 19F, 19A, 20, 22F, 23F and 33F; Sanofi Pasteur MSD). One 0.5mL dose containing 50µg/mL 441 of each serotype was diluted 1:12.5 in sterile PBS to 4µg/mL. Mice were given one 100µL 442 (0.4µg) dose by intraperitoneal injection, 14 days before infection with S. pneumoniae. 443 Blood samples were taken from the tail vein prior to vaccination and 14 days after 444 vaccination into serum collection tubes (BD microtainer, SST) samples were centrifuged at 445 15000xg for 5 min and the serum stored at ‐20°C until analysis. 446 447

Nemiralisib treatment 448 The PI3Kδ inhibitor nemiralisib (GSK2269557) was supplied by GSK, Respiratory Refractory 449 Inflammation DPU, UK. The nemiralisib suspension was prepared on the day of use in 0.2% 450 Tween80, and administered intranasally to mice twice daily at 0.2mg/kg in a total volume of 451 50µL, as described above (S. pneumoniae infections). Unless otherwise specified, the first 452 dose was given 24h before infection, and treatment was maintained for the duration of the 453 study. 454 455

Isolation of immune cells from mouse tissues 456 Mice were euthanized by CO2 inhalation and cervical dislocation. Lungs were perfused with 457 10mL cold PBS through the right ventricle, and collected into cold PBS. Lungs were 458 homogenized using a GentleMACS tissue dissociator and mouse lung dissociation enzyme kit 459 from Miltenyi according to the manufacter’s instructions. The homogenate was transferred 460 to 15mL tubes (BD Falcon) and washed by centrifugation (500xg) in 10mL cold PBS. The 461 pellet was resuspended in 3mL 37.5% isotonic Percoll (Sigma) at room temperature and 462 centrifuged at 650xg for 20min with low acceleration and no brake. The supernatant 463 including tissue debris was removed, and the cell pellet was washed and resuspended in 464 cold PBS. Where CFU counts were required, the right lung was processed as described 465 above and the left lung was processed for CFU counts as described below. Bone marrow 466 cells were collected by flushing cold PBS through femurs and tibias collected, filtered 467 through 40µm cell strainers and washed by centrifugation in 5mL PBS. Spleens, thymus and 468 peripheral lymph nodes were homogenized in PBS by pushing the tissue through 40µm cell 469 strainers (BD) using a syringe plunge. The cell suspension was then transferred to a 15ml 470 Tube (BD falcon) and washed once in 5ml cold PBS. For blood, bone marrow and spleen, red 471 blood cells (RBC) were lysed using hypotonic ammonium chloride RBC lysis buffer (Sigma). 472 Lysing was quenched with cold PBS and the cells were collected by centrifugation. Single 473 cell suspensions were processed for flow cytometry as described below. 474 475


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S. pneumoniae CFU counts 476 Lungs were homogenized in 1ml PBS using a Bullet Blender using 3x 3mm steel beads: speed 477 8, 3min (Next Advance, USA). Spleens were homogenized as described above in 2mL PBS. 478 Serial dilutions (10‐fold) were performed for spleen and lung homogenates, and samples 479 were plated out on blood agar as described above. Plates were incubated for 24h at 37°C 480 and S. pneumoniae colonies were counted. 481 482

In vitro stimulation of mouse B cells 483 Splenocytes were isolated from naïve wild‐type, p110δE1020K‐GL and p110δD910A mice that had 484 been crossed with the Il10ITIB reporter line as described above and the total cell count was 485 obtained using a CASY counter. The cells were resuspended in complete RPMI (RPMI plus 486 5% (v/v) FCS, 50 μM β‐mercaptoethanol and 100 μg/ml penicillin and streptomycin) and 487 plated at 5x106 cells in 100µL per well in a 96‐well U bottom plate. The cells were stimulated 488 with 10 ng/ml LPS, 50ng/ml PdBu (Sigma, USA), 0.25 μg/ml Ionomycin (Sigma, USA) and 1 489 μl/ml of Brefeldin A (eBioscience) for 5h at 37 °C. The cells were then processed for IL‐10 490 detection and flow cytometry as described below. 491 492

Detection of IL‐10 in Il10ITIB reporter mice: 493 Single cell suspensions from S. pneumoniae infected mice or in vitro stimulated cells were 494 prepared as described above. The cells were incubated with 3.3µM CCF4‐AM, a 495 Fluorescence Resonance Energy Transfer substrate for β‐lactamase (LiveBLAzer kit, 496 Invitrogen), and 3.6mM probenecid (Sigma) in complete RPMI for 90min at 29°C, as 497 previously described 29, then placed on ice and collected by centrifugation. Cells were then 498 processed for flow cytometry as described below. 499 500

Flow cytometry 501 Antibodies used for flow cytometry are listed in Table 1. Single cell suspensions were 502 stained using an antibody master mix in PBS/0.5%BSA for 40min at 4°C. Cells were washed 503 and fixed in 4% paraformaldehyde (Biolegend) for 10min at room temperature before 504 washing 2x in PBS/0.5%BSA. For intracellular detection of IL‐10 in human samples, the cells 505 were fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biocsiences, USA) following 506 surface staining. For FoxP3 staining, the eBioscence FoxP3/Transcription Factor staining 507 Buffer Set was used according to the manufacturer’s instructions. Non‐fluorescent counting 508 beads (AccuCount Blank Particles 5.3µm; Spherotech, USA) were added to quantify absolute 509 cells numbers. Samples were kept at 4°C until analysis (BD Fortessa5). Analysis was carried 510 out using FlowJo (Treestar) analysis software. 511 512

In vitro stimulation of human B cells 513 Freshly isolated PBMCs from patients and control individuals were stimulated with 0.5μg/ml 514 purified plate bound anti CD3 monoclonal antibody (clone‐OKT3) (Invitrogen) and 20ng/ml 515 recombinant human IL2 in presence or absence of, the p110δ inhibitor, nemiralisib (10nM) 516 for 72h. This stimulation led to the activation of CD40L (CD154) on CD4 T cells. The 517 interaction between CD40L on CD4 T cells with CD40 on B cells resulted in the production of 518 IL‐10 in regulatory B cells. For a final stimulation 50ng/ml PdBu (sigma, USA), 0.25 μg/ml 519 ionomycin (Sigma, USA) and 1μl/ml of Brefeldin A (eBioscience) were also added for the last 520 4h. Cells were washed, surface stained, fixed and permeabilized with Cytofix/Cytoperm 521 buffer (BD Biocsiences, USA) for the intracellular detection of IL‐10 as described above. 522


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523

Natural Antibody ELISA 524 Anti‐phosphorylcholine IgM and IgG levels were assessed in serum from naïve mice. Blood 525 samples were collected by cardiac puncture and serum collected as described above. NUNC 526 Maxisorp ELISA plates were coated overnight at 4°C with 20µg/mL phosphorylcholine‐BSA 527 (BioSearch) in sodium bicarbonate coating buffer (pH9) (Biolegend) (100µL/well). Plates 528 were washed 4x with PBS/0.2% Tween20 followed by blocking for 1h at room temperature 529 in PBS/1%BSA. Serum samples were diluted 1:10 in PBS/1% BSA and pooled WT serum was 530 diluted from 1:5 to 1:400 to confirm the linear range of the assay. After removing the 531 blocking solution, samples were added at 100µL/well, and the plates were incubated 532 overnight at 4°C. The plates were then washed 4x in PBS/0.2%Tween20. Polyclonal HRP 533 conjugated goat‐anti‐mouse IgM (abcam ab97023) or IgG (abcam ab97230) was diluted 534 1:5000 in PBS/1% BSA, added at 100µL per well and the plates were incubated for 2h at 535 room temperature. The plates were washed 6x with PBS/0.2%Tween20, TMB substrate 536 (Biolegend) was added at 100µL per well and incubated for 5‐10 min at room temperature. 537 The reaction was stopped by adding 50µL 2N H2SO4 solution. Absorbance was read at 538 450nm (Fluostar omega, BMG Labtech). 539 540

Tissue resident cell analysis (lung) 541 Mice received an intravenous (tail vein) injection of 3µg anti‐mouse CD45 conjugated to 542 biotin (Biolegend, clone 30‐F11) in 100µL PBS. 4min after injection animals were killed and 543 the lungs were collected into cold PBS without prior perfusion. Lungs were then finely 544 minced using a scalpel blade and the pushed through a 100µm cell strainer (BD) the 545 homogenate was collected in a 15mL tube (BD falcon) and washed in 10ml cold PBS. The 546 pellet was resuspended in 3mL 37.5% isotonic Percoll (Sigma) at room temperature and 547 processed as described above. The cells were stained for flow cytometry as described 548 above, including streptavidin APC to identify anti‐CD45‐Biotin labelled circulating cells. 549 550

Cytokine analysis 551 Cytokine levels were measured in the supernatant from lung homogenates prepared for 552 CFU analysis. The homogenates were centrifuged for 1min at 10000xg to remove tissue 553 debris. Samples collected from wild‐type mice treated with nemiralisib were analyzed using 554 Meso Scale Discovery mouse Th1/Th2 9‐plex ultrasensitive kits, according to manufacturer’s 555 instructions. Samples collected from genetically modified mice were analyzed using the flow 556 cytometry based Legendplex mouse inflammation 13‐plex panel (Biolegend). 557 558

Gene Array 559 Wild‐type mice were treated with nemiralisib and infected with S. pneumoniae as described 560 above. At 24h post infection, whole lungs were collected and snap frozen in liquid nitrogen. 561 Samples were randomized and analyzed (Affymetrix Genechip Mouse genome 430 2.0 562 Array) by Expression Analysis, Quintiles Global Laboratories. Data analysis was performed by 563 Computational Biology and Statistics, Target Sciences, GlaxoSmithKline, Stevenage. Data 564 was normalised using the robust multiarray average method47 and quality checked in 565 R/Bioconductor48 using the affy package49. A linear model was fitted to the RMA 566 normalised data for each probset and differiential expression analysis was conducted in the 567 ArrayStudio software (Omicsoft Corporation). P‐values were false discovery rate corrected 568 by the method of Benjamini and Hochberg50. Probes with an absolute fold change >1.5 and 569


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16

an adjusted p‐value <0.05 were called significant. The data been deposited in the National 570 Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible 571 through GEO series accession number GSE109941. 572 573

Luminex antibody analysis 574 Measurement of IgG recognizing pneumococcal polysaccharide of 13 serotypes was 575 performed as described previously 51. The assay was modified in order to measure murine 576 IgG as follows: 50µl of 1:10 diluted mouse sera was added to each well containing the 577 pneumococcal polysaccharides coupled to microspheres and the secondary antibody used 578 was 50µl of 10µg/ml goat F(ab’)2 anti‐mouse IgG (H+L)‐ R‐phycoerythrin (Leinco 579 Technologies, Inc). R‐PE fluorescence levels were recorded for each serotype and median 580 fluorescence intensities were compared for the different genotypes. 581 582 PIP3 quantification 583 CD4 and CD8 cells were isolated from mouse splenocytes using immunomagnetic negative 584 selection by biotinylated cocktail of antibodies and Streptavidin dynabeads (Invitrogen). T 585 cells were stimulated with anti‐CD3 (1mg/ml), (145‐2c11, Biolegend) anti‐ CD28 (2µg/ml), 586 (35.51, Biolegend) antibodies followed by crosslinking with anti‐ Armenian hamster IgG (10 587 µg/ml, Jackson ImmunoResearch labs). Cells were stimulated for 1 min at 37°C. B cells were 588 similarly isolated from mouse splenocytes and stimulated for 1 min at 37 °C with anti‐ IgM 589 2µg/ml (AffinPure F(ab’)2 Fragment goat anti‐mouse IgM from Jackson Immuno‐Research 590 labs). 591 592 After stimulation of the cells, we terminated the reactions by addition of 750µl kill solution 593 (CHCl3:MeOH:1M HCL (10:20:1) and immediately froze the samples on dry ice. PIP3 levels 594 were quantified by mass spectrometry as previously described52. 595 596

Western blot 597 Purified T and B cells were isolated from murine spleens and stimulated as described above 598 (PIP3 measurement). For performing western blots the stimulation period was 5 min. 599 Isolated T cells were stimulated with anti ‐CD3 1µg/ml and anti ‐CD28 (2 µg/ml) with or 600 without 10 Nm nemiralisb; or B cells with anti‐ IgM 4µg/ml (AffinPure F(ab’)2 Fragment goat 601 anti‐mouse IgM from Jackson ImmunosResearch labs). 602 Stimulated T and B cells were lysed with ice‐cold lysis buffer (50mM HEPES, 150mM NaCl, 10mM 603 NaF, 10mM Indoacetamide, 1% IGEPAL and proteinase inhibitors (Complete Ultra tablets, Roche)) for 604 15‐20 min. Lysates were centrifuged at 15,000g for 10 min at 4 °C and supernatants were 605 mixed with NuPage LDS sample buffer (life technologies). Samples were heated at 70°C and 606 resolved on 4‐12% NuPage bis‐tris gel (Invitrogen), transferred to PVDF membranes and 607 probed with the following antibodies: pAKT (T308, Cell Signaling, 1 in 1000 dilution); total 608 AKT1 (2H10, Cell signaling, 1 in 2000 dilution); p110δ (Sc7176, Santa Cruz Biotechnology, 1 609 in 200 dilution); pS6 (S235/236, Cell Signaling, 1 in 500 dilution); pFoxo1/3a (T24/T32, Cell 610 Signaling, 1 in 1000 dilution); pErk (p44/42, Cell Signaling, 1 in 200 dilution); βActin 611 (Sc47778, Santa Cruz Biotechnology, 1 in 2000 dilution). 612

Statistics 613

Data analysis was performed in Graphpad Prism. Where two groups were compared we 614 used student’s t‐test with Welch’s correction. Where three or more groups were compared 615


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17

we used 1‐way ANOVA with Tukey’s multiple comparisons test. Survival data was analyzed 616 using the Gehan‐Breslow‐Wilcoxon test. Statistical significance is indicated by asterisk as 617 follows: p > 0.05 *; p ≤ 0.05 **; p ≤ 0.01 ***; p ≤ 0.001 **** 618

Ethics 619

Animal experiments were performed according to the Animals (Scientific Procedures) Act 620 1986, licence PPL 70/7661. 621 Informed consent was obtained from patients and healthy controls. This study conformed to 622 the Declaration of Helsinki and according local ethical review document 12/WA/0148. 623

Acknowledgements 624

We thank Rahul Roychoudhuri, Bart Vanhaesebroeck and Martin Turner for their invaluable 625 advice on the draft manuscript. We also thank Hicham Bouabe and Jürgen Heesemann (LMU 626 Munich) for providing the Il10ITIB mice and for advice. We thank Ramkumar Venigalla for 627 advice on B cell phenotyping and Alice Denton for advice on the detection of tissue resident 628 cells in the lung. Rainer Döffinger provided advice on the use of Luminex for the 629 quantification of antibodies against pneumococcal serotypes; Keith Burling helped quantify 630 mouse serum immunoglobulins. We gratefully acknowledge the support from the Babraham 631 Institute Biological Services Unit, Biological Chemistry, Mass Spectroscopy and Flow 632 Cytometry Facilities. Funding for the project was from the Medical Research Council 633 MR/M012328/2 (AS, AMC, SN, KO), Wellcome Trust 103413/Z/13/Z and 206618/Z/17/Z 634 (AC), 095691/Z/11/Z (KO), 095198/Z/10/Z (SN) Biotechnology and Biological Sciences 635 Research Council BBS/E/B/000 ‐C0407, ‐C0409, ‐C0427 and ‐C0428 (KO). EBH is currently 636 employed by Cambridge University Hospitals NHS Foundation Trust but is seconded to 637 spend 50% of his time on GSK clinical trial research. He receives no other benefits or 638 compensation from GSK. EBH was funded by the Wellcome Trust Translational Medicine and 639 Therapeutics (TMAT) PhD program. SN is also supported by the National Institute for Health 640 Research (NIHR) Cambridge Biomedical Research Centre. MRC is supported by the National 641 Institute of Health Research (NIHR) Cambridge Biomedical Research Centre and the NIHR 642 Blood and Transplant Research Unit and by a Medical Research Council New Investigator 643 Research Grant (MR/N024907/1) and an Arthritis Research UK Cure Challenge Research 644 Grant (21777). SS, GB, EBH, JNH and EMH are employed by GSK. 645


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18

Table 1: Antibodies 646 647

anti‐mouse Clone Supplier

CCR2 475301 R&D Systems

CD11b M1/70 Biolegend

CD11c N418 Biolegend

CD138 281‐2 Biolegend

CD19 ID3 BD Biosciences

CD1d K253 Biolegend

CD21 7E9 Biolegend

CD23 B3B4 Biolegend

CD25 PC61 Biolegend

CD28 37.51 Biolegend

CD3 2C11 Biolegend

CD4 GK1.5 Biolegend

CD43 S7 BD Biosciences

CD44 IM7 Biolegend

CD45 30‐F11 Biolegend

CD45R(B220) RA3‐6B2 BD Biosciences

CD5 53‐7‐3 eBioscence (ThermoFisher)

CD62L MEL‐14 Biolegend

CD8a 53‐6.7 Biolegend

FoxP3 FJK‐16s eBioscence (ThermoFisher)

IgD 11‐26c.2a Biolegend

IgM II/41 eBioscence (ThermoFisher)

IL‐7R A7R34 Biolegend

KLRG1 2F1 Biolegend

Ly6C HK1.4 Biolegend

Ly6G 1A8 Biolegend

Ly6G/Ly6C(Gr1) RB6‐8C5 Biolegend

NK1.1 PK136 Biolegend

Siglec‐F E50‐2440 BD Biosciences

TCRβ H57‐597 Biolegend

γδTCR UC7‐13D5 Biolegend

anti‐human Clone Supplier

CD19 SJ25C1 BD Biosciences

CD24 ML5 BD Biosciences

CD38 HIT2 BD Biosciences

IL‐10 JES3‐9D7 Miltenyi Biotec

648


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19

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21. Mi, Q.S. et al. Highly reduced protection against Streptococcus pneumoniae after 694 deletion of a single heavy chain gene in mouse. Proc Natl Acad Sci U S A 97, 6031‐695 6036 (2000). 696

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24. Clayton, E. et al. A crucial role for the p110delta subunit of phosphatidylinositol 3‐702 kinase in B cell development and activation. J Exp Med 196, 753‐763 (2002). 703

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27. van der Sluijs, K.F. et al. IL‐10 is an important mediator of the enhanced susceptibility 709 to pneumococcal pneumonia after influenza infection. J Immunol 172, 7603‐7609 710 (2004). 711

28. Barthelemy, A. et al. Influenza A virus‐induced release of interleukin‐10 inhibits the 712 anti‐microbial activities of invariant natural killer T cells during invasive 713 pneumococcal superinfection. Mucosal Immunol 10, 460‐469 (2017). 714

29. Bouabe, H., Liu, Y., Moser, M., Bosl, M.R. & Heesemann, J. Novel highly sensitive IL‐715 10‐beta‐lactamase reporter mouse reveals cells of the innate immune system as a 716 substantial source of IL‐10 in vivo. J Immunol 187, 3165‐3176 (2011). 717

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31. Hardy, R.R. & Hayakawa, K. B cell development pathways. Annu Rev Immunol 19, 720 595‐621 (2001). 721

32. Vlkova, M. et al. Regulatory B cells in CVID patients fail to suppress multifunctional 722 IFN‐gamma+ TNF‐alpha+ CD4+ T cells differentiation. Clin Immunol 160, 292‐300 723 (2015). 724

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39. Browning, M.J., Chandra, A., Carbonaro, V., Okkenhaug, K. & Barwell, J. Cowden's 741 syndrome with immunodeficiency. J Med Genet 52, 856‐859 (2015). 742

40. Tsujita, Y. et al. Phosphatase and tensin homolog (PTEN) mutation can cause 743 activated phosphatidylinositol 3‐kinase delta syndrome‐like immunodeficiency. J 744 Allergy Clin Immunol 138, 1672‐1680 e1610 (2016). 745

41. To, Y. et al. Targeting phosphoinositide‐3‐kinase‐delta with theophylline reverses 746 corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit 747 Care Med 182, 897‐904 (2010). 748

42. Rao, V.K. et al. Effective "activated PI3Kdelta syndrome"‐targeted therapy with the 749 PI3Kdelta inhibitor leniolisib. Blood 130, 2307‐2316 (2017). 750

43. Coutre, S.E. et al. Management of adverse events associated with idelalisib 751 treatment: expert panel opinion. Leuk Lymphoma 0, 1‐8 (2015). 752

44. Lee, P.P. et al. A critical role for Dnmt1 and DNA methylation in T cell development, 753 function, and survival. Immunity 15, 763‐774 (2001). 754

45. Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1‐cre mice. 755 Proc Natl Acad Sci U S A 103, 13789‐13794 (2006). 756

46. Clausen, B.E., Burkhardt, C., Reith, W., Renkawitz, R. & Forster, I. Conditional gene 757 targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 8, 758 265‐277 (1999). 759

47. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density 760 oligonucleotide array probe level data. Biostatistics 4, 249‐264 (2003). 761

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51. Willcocks, L.C. et al. The effect of sirolimus therapy on vaccine responses in 769 transplant recipients. Am J Transplant 7, 2006‐2011 (2007). 770

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773 774


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Exon_21 Loxp

Exon 22

TAG(stop)

PolyA

FRTPolyA

FRTloxp

K1020E1020

Exon 22SASA

TAG(STOP)

PGK Neo

Exon_21 Loxp

Exon_22

TAG-stop

PolyA

FRTloxp

K1020E1020

SA SA

TAG (STOP)3' UTR

Exon 22

Exon_21 loxp

K1020

SA

TAG (STOP)

3' UTR

Exon 22

Frt recombination

Loxp recombination

P110δE1020K-GL

P110δE1020K-T

P110δE1020K-B

P110δE1020K-M

P110δE1020K-Flox

Gene-targeted ES cells

Founders crossed with FlpE transgenic

Conditional KI crossed with Cre transgenic

Exon 21

E1020

SA

TAG(STOP)

3' UTR

Exon 22

Endogenous locus

3' UTR

Supplementary Figure 1: Gene targeting strategy for generating conditional p110δE1020K miceThe p110δE1020K mice were generated by OzGene using homologous recombination in ES cells. Aduplicate sequence corresponding to the last coding exon in Pik3cd was flanked by loxP sites andinserted 3’ to the original sequence. The original sequence encoding E1020 was mutated to K1020.Upon Cre-mediated recombination, the wild-type sequence is replaced by the mutant E1020Ksequence. In this study, we used Tnfrsf4cre to delete in the germline, Cd4cre to delete in T cells, Mb1cre

to delete in B cells and Lyz2Cre to delete in myeloid cells.


https://doi.org/10.1101/280651


Gat

ed o

n B

220+

CD

19+

Gat

ed o

n B

220+

CD

93+

IgM

CD23

Spleen

p110δWT p110δE1020K p110δD910A

Liv

e ce

lls

B220

CD

19

0.3 57 4.0 35 0.01 47

CD

21

CD23

581

10

0.06

94

4

9

57

26

40 28

156

20 53

126

52 11

1112

Supplementary figure 2: Phenotype of B cells in p110δE1020K-GL mice.A: B cell subsets in the spleen and bone marrow from wild-type, p110δE1020K-GL and p110δD910A mice were analyzed by flow cytometry andrepresentative pseudocolor plots with the mean cell proportion are shown. B: In the spleen, total CD19+B220+ B cells were increased inp110δE1020K-GL mice, due to increased numbers of B1, marginal zone (MZ) and T1 transitional cells while follicular B cell numbers were normal.These populations were reduced in p110δD910A mice. Analysis of the bone marrow showed normal pro-B cell numbers in p110δE1020K-GL mice with areduced number of pre-B cells, immature, transitional and mature B cells. Populations of cells are described as follows: Splenic B cells: Total Bcells CD19+B220+, B1 cells CD19+B220+CD23-CD21-, Follicular B cells CD19+B220+CD23+CD21+, Marginal zone B cells CD19+B220+CD23-CD21+,Transitional T1 B cells B220+CD93+IgM+CD23-, Transitional T2 B cells B220+CD93+IgM+CD23+, Transitional T3 B cells B220+CD93+IgM-CD23-;Bone marrow B cells - Immature B cells CD19+B220loIgM+, Mature B cells CD19+B220hiIgM+, Pro-B cells B220+IgM-CD19+CD25-, Pre-B cellsB220+IgMCD19+CD25+, Transitional B cells B220+IgD+. (Combined data from 2 independent experiments, n=8-12).

A

B220

CD

19Ig

MC

D25

IgD

B220

B220

CD19

25 1817

6

54

22

38

43 35

35

2 6

3 45 7 4 5

Bone marrow

Liv

e ce

llsL

ive

cells

Liv

e ce

llsG

ated

on

B

220+

IgM

-


WT

E10

20

K

D91

0A

0.0

5.0×106

1.0×107

1.5×107

2.0×107

CD

19+B

220+

cel

ls/B

M p<0.0001

p<0.0016

WT

E10

20K

D91

0A

0

1×106

2×106

3×106

4×106

Imm

atur

e B

cel

ls/ B

M p<0.0001p<0.0001

p<0.0001

WT

E10

20K

D91

0A

0

2×106

4×106

6×106

Mat

ure

B c

ells

/ BM

p<0.0001

p<0.00384

WT

E10

20K

D9

10A

01×106

2×106

3×106

4×106

5×106

Pro

B c

ells

/ BM

WT

E1

020K

D91

0A

01×106

2×106

3×106

4×106

5×106

Pre

B B

cel

ls/ B

M

p<0.0001

p=0.0001

WT

E10

20K

D91

0A

01×106

2×106

3×106

4×106

5×106

Tran

sitio

nal B

cel

ls /B

M p=0.001p<0.0001

Bone marrow

B Spleen

WT

E1

02

0K

D9

10

A

0

1 107

2 107

3 107

4 107

CD

19+B

220

+ c

ells

/Spl

een

p<0.0001 p<0.0001

WT

E1

02

0K

D9

10

A

0

2 106

4 106

6 106

8 106

1 107

B1

cells

/ sp

leen

p<0.0001p<0.0001

p<0.0004

WT

E1

02

0K

D9

10

A

0

1 106

2 106

3 106

4 106

Mar

gina

l zon

e B

cel

ls /

sple

en

p<0.0001p<0.0001

p<0.0001

WT

E1

02

0K

D9

10

A

0

5 106

1 107

1.5 107

2 107

2.5 107

Fol

licul

ar B

cel

ls/ s

plee

n

p<0.0001

p<0.0001

WT

E1

02

0K

D9

10

A

0

2 106

4 106

6 106

8 106

Tra

nsiti

onal

B c

ells

- T

1/ S

plee

n

p<0.0001p<0.0013

p<0.0001

WT

E1

02

0K

D9

10

A

0

1 106

2 106

3 106

Tra

nsiti

onal

B c

ells

- T

2/ S

plee

n

p<0.0007

p<0.0001

WT

E1

02

0K

D9

10

A

0

5 105

1 106

1.5 106

2 106

Tra

nsiti

onal

B c

ells

- T

3/ S

plee

n

p<0.0001

p<0.0001


https://doi.org/10.1101/280651



CD62L

CD

44

Spleen Spleen

Lymph nodes

CD62L

CD

44



Lymph nodes

Wild

type

E10

20K

D91

0A

0

1 106

2 106

3 106

4 106

CD

4+ C

D25

+ F

oxp3

+

/ spl

een

p<0.0173 p<0.0016

Lymph nodes


Wild

type

E10

20K

D91

0A

0

5 105

1 106

1.5 106

2 106

CD

4+C

D25

+F

oxp3

+

/ th

ymus

p<0.0001

p<0.0001

Spleen Thymus

Foxp3

CD

25

Wild

type

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400000

600000

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pher

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Lymph nodesSpleen

Thymus

A

B

C

Supplementary figure 3: Phenotype of T cells in p110δE1020K-GL mice.T cell populations in the thymus, spleen and lymph nodes of wild-type, p110δE1020K-GL andp110δD910A mice were analyzed by flow cytometry. Representative pseudocolor or contour plotswith outliers show mean cell proportions. A: Thymic T cell numbers in p110δE1020K mice werenormal apart from a reduction in single positive CD8+ cells. B: The number of activated CD4+

and CD8+ T cells (CD44hiCD62lo) as well as memory CD8+ T cells (CD44hiCD62Lhi) in lymphnodes and spleen were increased in p110δE1020K mice. C: The number of CD4+CD25+Foxp3+

regulatory T cells was increased in the lymph nodes and spleen of p110δE1020K mice.(Representative data from 2 independent experiments, n=3-8)

CD8

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CD

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T cells

CD

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T cells

CD

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T cells

0

1 107

2 107

3 107

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p<0.002

Wild

typ

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1.5 107

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CD

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Wild

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Wild

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0.9312.5

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4.242.63

Thymus Thymus


https://doi.org/10.1101/280651


Spleen

Bone marrow

A

WT

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Live

cel

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********

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5.0×100

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% M

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nal z

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ells ***

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7×101

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ular

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cells

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live

cells

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ntr

ol

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020

K

D91

0A

0.0

5.0×100

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rans

ition

al B

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Supplementary figure 4: Phenotype of B cells in p110δE1020K-B mice.B cell subsets in the spleen and bone marrow from wild-type, p110δE1020K-B and p110δD910A mice were analyzedby flow cytometry, and was shown to recapitulate the phenotype of p110δE1020K-GL mice. A: In the spleen, theproportion of B1, marginal zone (MZ) and T1 transitional cells were increased, while the proportion of follicular Bcells were reduced. B: Analysis of the bone marrow showed increased proportions of pro-B cells in p110δE1020K-B

mice with reduced proportions of pre-B cells and immature B cells, and a trend towards reduced proportions oftransitional and mature B cells. Populations of cells are described as follows: Splenic B cells - Total B cellsCD19+B220+, B1 cells CD19+B220+CD23-CD21-, Follicular B cells CD19+B220+CD23+CD21+, Marginal zone Bcells CD19+B220+CD23-CD21+, Transitional T1 B cells B220+CD93+IgM+CD23-, Transitional T2 B cellsB220+CD93+IgM+CD23+, Transitional T3 B cells B220+CD93+IgM-CD23-; Bone marrow B cells - Immature B cellsCD19+B220loIgM+, Mature B cells CD19+B220hiIgM+, Pro-B cells B220+IgM-CD19+CD25-, Pre-B cellsB220+IgMCD19+CD25+, Transitional B cells B220+IgD+. (Combined data from 2 independent experiments, n=6-10).

B


https://doi.org/10.1101/280651


Lymph nodes

Spleen

Thymus

Supplementary figure 5: Phenotype of T cells in p110δE1020K-T

mice.T cell populations in the thymus, spleen and lymph nodes in wild-type, p110δE1020K-T and p110δD910A were analyzed by flowcytometry. A: Thymic T cell proportions in p110δE1020K-T mice werenormal B: In the spleen, the proportions of activated CD4+ andCD8+ T cells (CD44hiCD62lo) were increased, and there was atrend towards increased proportions of memory CD8+ T cells(CD44hiCD62Lhi) in p110δE1020K-T mice. In the lymph nodes ofp110δE1020K-T mice, the proportions of activated CD4+ T cells(CD44hiCD62lo) were increased with a trend towards increasedproportions of activated CD8+ T cells, as well as increasedproportions of memory CD8+ T cells (CD44hiCD62Lhi). C: Theproportion of CD4+CD25+Foxp3+ regulatory T cells were increasedin the lymph nodes and spleen of p110δE1020K-T mice. (Combineddata from 3 independent experiments, n=7-12).

B

C

Lymph nodesSpleen Thymus

A

Wild

typ

e

E10

20K

D91

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5

10

15

% v

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e C

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mu

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* ****

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typ

e

E10

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D91

0A

0

1

2

3

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% v

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+ T

cel

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707580859095

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f D

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****

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40

50

60

70

80

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20

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62L

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***

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E10

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% C

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* ***

0

10

20

30

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50

Wild

typ

e

E10

20K

D91

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5

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20

% C

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/spl

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* *

0

5

10

15

20

25

**

***

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E10

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0A

0

50

100

150

% C

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CD

62L

hi C

D44

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E10

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30

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110

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E10

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110

% C

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Wild

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5

10

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% C

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f C

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T c

ells

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hym

us


https://doi.org/10.1101/280651


Supplementary figure 6: Serum immunoglobulins in p110δE1020K-GL mice.Analysis of serum immunoglobulins from naïve mice (age 8-12 weeks) showed significantly increasedlevels of IgG1 and IgG2b, and a trend towards increased levels IgG2c, IgM, IgA, and IgE in p110δE1020K-GL

mice, while IgG3 levels were similar compared to wild-type mice. p110δD910A mice were antibody deficientfor all isotypes analyzed. (n= 6-7).

WT

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um Ig

Mg/

mL

WT

E10

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0.1

1

10

100

Ser

um I

gEg/

mL **


https://doi.org/10.1101/280651


WT

E1

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A: B:

WT

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A

IL-6

[pg/

mL]

0

100

200

300

400

WT

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A

0

50

100

150

200

250

IL-1

[pg/

mL]

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400

600

800

TNF

[pg/

mL]

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IFN

[pg/

mL]

WT

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[pg/

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Supplementary Figure 7: PI3Kδ signaling regulates B cell specific IL-10 production in the lung following S. pneumoniae infection.A: 24h post S. pneumoniae infection, lung and spleen CFU counts were similar in wild-type, p110δE1020K-GL and p110δD910A mice. B: 24hpost infection, cytokine levels in the lung homogenate showed a trend towards increased TNFα, IL-6 and IL-1 in p110δE1020K mice. C: 24hprophylactic treatment with nemiralisib (nem) led to a significant reduction in TNFα, IL-6 and IL-1β, and a trend towards reduced IFNγ andIL-10, in the lungs of wild-type mice compared to vehicle control (veh) treated animals at 24h post infection. D: Volcano plot (statisticalsignificance against fold change) of the gene expression changes in response to nemiralisib treatment showed reduced levels of pro-inflammatory cytokines as well as IL-10 at 24h post infection compared to vehicle control treatment. All genes analysed are shown (greydots) with the cytokines of interest labelled and coloured; green for those with a negative fold change, red for positive fold change. E:Analysis of immune cell subsets in Il10ITIB reporter mice at 24h post infection showed that the proportion of IL-10 producing B cells issignificantly increased in p110δE1020K-GL mice and reduced in p110δD190A mice compared to wild-type mice, with similar trends in T cells andmyeloid cells not reaching significance. (A: results from 2 independent studies combined, n=14; B: representative data from 3 independentstudies n=8; C: Combined data from 4 independent experiments n=25; D: data from 1 study, n=6; E: representative data from 2independent studies n=6).

C:

ve

h

ne

m

0

500

1000

1500

IFN

pg/

mL

p=0.0715

ve

h

ne

m

0

5000

10000

15000

IL-1

pg/

mL p=0.008

0

20000

40000

60000

80000 p=0.0026

0

100

200

300

400

500 p=0.0918

0

20000

40000

60000

80000 p=0.0933

0

1000

2000

3000

4000

5000 p=0.0062W

T

E1

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10

A%IL

-10+

(C

D11

b+Ly

6G+)

0

20

40

60

80

WT

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0A%

IL-1

0+ (T

CR

+C

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)

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0

5

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15

%IL

-10+

(C

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b+Ly

6C+)

0

10

20

30

40

50 p<0.0001 p<0.0001

WT

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02

0K

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IL-1

0+ (T

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+C

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)

D:

-log1

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e)

E:

log2FoldChange-4 -2 0 2

0

5

10

15


https://doi.org/10.1101/280651


01020304050

7580859095

100105

p=0.0001

0

5

10

15 p<0.0001

0

1 105

2 105

3 105

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02 1044 1046 1048 1041 105

0

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1.5 104 p=0.03

02468

WT

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+

(CD

45+)

WT

E1

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%B

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(CD

19+, C

D45

+)

WT

E1

02

0K

0

10

20

30

%B

220-

(CD

19+,

CD

45+)

WT

E1

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CD

45+

cells

/Lun

g

WT

E1

02

0K

CD

19+

cells

/Lun

g

01 103

2 103

3 103

4 103p=0.0117

02 1024 1026 1028 1021 103 p=0.0044

Supplementary Figure 8: Tissue resident B cell numbers are increased in the lungs of naïve p110δE1020K-GL miceBiotin-conjugated anti-CD45 was injected intravenously to distinguish between circulating (CC) and tissue resident (TRC) cells in the lungs ofnaïve mice. p110δE1020K-GL mice showed an increase in the proportion and number of tissue resident leukocytes (CD45+TRC), but notcirculating leukocytes. There was also an increase in the proportion and number of total CD19+ B cells among the tissue resident cellpopulation. Among circulating leukocytes (CD45+IV), total CD19+ B cell numbers were similar in p110δWT and p110δE1020K mice, andp110δE1020K mice had increased numbers and proportions of CD19+B220- B cells. (Representative data from 2 independent experimentsn=4).

CD

45 (

Circ

ulat

ing)

CD45 (Tissue Resident)

CD

11b

GR1 Gated on CD45(Circulating)

Wild-type E1020K

Gated on SiglecF-

17%

1%

27%

3%

59% 63%49% 60%

6% 24%

94%

1%

42%

18%

83%

11%

63%

20%

CD

11b

GR1 Gated on CD45(Tissue Resident)

TC

Rβ

CD19 Gated on CD11b-GR1-

72% 63%

B22

0

CD19 Gated on CD19+

TC

Rβ

CD19 Gated on CD11b-GR1-

B22

0

CD19 Gated on CD19+

Circulating Cells (CC) Tissue Resident cells (TRC)

Wild-type E1020K Wild-type E1020K

Tot

al le

ukoc

ytes

Tot

al B

ce

llsC

D19

+B

220+

CD

19+B

220-

Tot

al le

ukoc

ytes

Tot

al B

ce

llsC

D19

+B

220+

CD

19+B

220-


https://doi.org/10.1101/280651


PI3Kδ hyper activation promotes the development of B cells ...6. Refractory Respiratory...

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Transcript of PI3Kδ hyper activation promotes the development of B cells ...6. Refractory Respiratory...