Pi3kγ in b cells promotes antibody responses and generation of antibody-secreting cells

ABSTRACT The differentiation of naive and memory B cells into antibody-secreting cells (ASCs) is a key feature of adaptive immunity. The requirement for phosphoinositide 3-kinase-delta

(PI3Kδ) to support B cell biology has been investigated intensively; however, specific functions of the related phosphoinositide 3-kinase-gamma (PI3Kγ) complex in B lineage cells have not.

In the present study, we report that PI3Kγ promotes robust antibody responses induced by T cell-dependent antigens. The inborn error of immunity caused by human deficiency in PI3Kγ results

in broad humoral defects, prompting our investigation of roles for this kinase in antibody responses. Using mouse immunization models, we found that PI3Kγ functions cell intrinsically within

activated B cells in a kinase activity-dependent manner to transduce signals required for the transcriptional program supporting differentiation of ASCs. Furthermore, ASC fate choice

coincides with upregulation of _PIK3CG_ expression and is impaired in the context of PI3Kγ disruption in naive B cells on in vitro CD40-/cytokine-driven activation, in memory B cells on

toll-like receptor activation, or in human tonsillar organoids. Taken together, our study uncovers a fundamental role for PI3Kγ in supporting humoral immunity by integrating signals

instructing commitment to the ASC fate. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through

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support SIMILAR CONTENT BEING VIEWED BY OTHERS RIF1 INTEGRATES DNA REPAIR AND TRANSCRIPTIONAL REQUIREMENTS DURING THE ESTABLISHMENT OF HUMORAL IMMUNE RESPONSES Article Open access 17 January

2025 CONTROL OF GERMINAL CENTER B CELL SURVIVAL AND IGE PRODUCTION BY AN ADAPTOR MOLECULE CONTAINING PH AND SH2 DOMAINS, APS/SH2B2 Article Open access 01 August 2024 A P38Α-BLIMP1

SIGNALLING PATHWAY IS ESSENTIAL FOR PLASMA CELL DIFFERENTIATION Article Open access 28 November 2022 DATA AVAILABILITY Bulk RNA-seq and scRNA-seq and BCR-seq data are available via the Gene

Expression Omnibus (GEO) and were analyzed using mm10 reference genome. The bulk and scRNA-seq data used in the present study are available through the GEO database under accession nos.

GSE269303 and GSE269300, respectively. Source data are provided with this paper. CODE AVAILABILITY Customized scripts in R v.4.3.1 for scBCR analysis were deposited under

bitbucket.org/kleinstein/projects (Lanahan2023 folder). REFERENCES * Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M. & Corcoran, L. M. The generation of antibody-secreting plasma cells.

_Nat. Rev. Immunol._ 15, 160–171 (2015). CAS  PubMed  Google Scholar  * Brandtzaeg, P. et al. Immunobiology and immunopathology of human gut mucosa: humoral immunity and intraepithelial

lymphocytes. _Gastroenterology_ 97, 1562–1584 (1989). CAS  PubMed  Google Scholar  * van der Heijden, P. J., Stok, W. & Bianchi, A. T. Contribution of immunoglobulin-secreting cells in

the murine small intestine to the total ‘background’ immunoglobulin production. _Immunology_ 62, 551–555 (1987). PubMed  PubMed Central  Google Scholar  * Gaudette, B. T. et al. Resting

innate-like B cells leverage sustained Notch2/mTORC1 signaling to achieve rapid and mitosis-independent plasma cell differentiation. _J. Clin. Invest._ 131_,_ e151975 (2021). *

Skrzypczynska, K. M., Zhu, J. W. & Weiss, A. Positive regulation of Lyn kinase by CD148 is required for B cell receptor signaling in B1 but not B2 B cells. _Immunity_ 45, 1232–1244

(2016). * Gaudette, B. T., Jones, D. D., Bortnick, A., Argon, Y. & Allman, D. mTORC1 coordinates an immediate unfolded protein response-related transcriptome in activated B cells

preceding antibody secretion. _Nat. Commun._ 11, 723 (2020). CAS  PubMed  PubMed Central  Google Scholar  * Tumang, J. R., Frances, R., Yeo, S. G. & Rothstein, T. L. Spontaneously

Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors. _J. Immunol._ 174, 3173–3177 (2005). CAS  PubMed  Google Scholar  *

Martin, F., Oliver, A. M. & Kearney, J. F. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. _Immunity_ 14, 617–629 (2001).

CAS  PubMed  Google Scholar  * Klein, U. et al. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. _Nat. Immunol._ 7, 773–782 (2006). CAS  PubMed

  Google Scholar  * Shaffer, A. L. et al. Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program. _Immunity_ 17, 51–62 (2002). CAS 

PubMed  Google Scholar  * Carotta, S. et al. The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation. _J. Exp. Med._ 211, 2169–2181 (2014). PubMed  PubMed

Central  Google Scholar  * Scharer, C. D. et al. Antibody-secreting cell destiny emerges during the initial stages of B-cell activation. _Nat. Commun._ 11, 3989 (2020). CAS  PubMed  PubMed

Central  Google Scholar  * Jones, D. D. et al. mTOR has distinct functions in generating versus sustaining humoral immunity. _J. Clin. Invest._ 126, 4250–4261 (2016). PubMed  PubMed Central

  Google Scholar  * Luo, W. et al. IL-21R signal reprogramming cooperates with CD40 and BCR signals to select and differentiate germinal center B cells. _Sci. Immunol._ 8, eadd1823 (2023).

CAS  PubMed  PubMed Central  Google Scholar  * Luo, W., Weisel, F. & Shlomchik, M. J. B cell receptor and CD40 signaling are rewired for synergistic induction of the c-Myc transcription

factor in germinal center B cells. _Immunity_ 48, 313–326.e315 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Clayton, E. et al. A crucial role for the p110delta subunit of

phosphatidylinositol 3-kinase in B cell development and activation. _J. Exp. Med._ 196, 753–763 (2002). CAS  PubMed  PubMed Central  Google Scholar  * Conley, M. E. et al. Agammaglobulinemia

and absent B lineage cells in a patient lacking the p85alpha subunit of PI3K. _J. Exp. Med._ 209, 463–470 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Coulter, T. I. et al.

Clinical spectrum and features of activated phosphoinositide 3-kinase delta syndrome: A large patient cohort study. _J. Allergy Clin. Immunol._ 139, 597–606.e594 (2017). CAS  PubMed  PubMed

Central  Google Scholar  * Bader, A. G., Kang, S., Zhao, L. & Vogt, P. K. Oncogenic PI3K deregulates transcription and translation. _Nat. Rev. Cancer_ 5, 921–929 (2005). CAS  PubMed 

Google Scholar  * Vanhaesebroeck, B., Stephens, L. & Hawkins, P. PI3K signalling: the path to discovery and understanding. _Nat. Rev. Mol. Cell Biol._ 13, 195–203 (2012). CAS  PubMed 

Google Scholar  * Burke, J. E. & Williams, R. L. Synergy in activating class I PI3Ks. _Trends Biochem. Sci._ 40, 88–100 (2015). CAS  PubMed  Google Scholar  * Rommel, C., Camps, M. &

Ji, H. PI3Kδ and PI3Kγ: partners in crime in inflammation in rheumatoid arthritis and beyond? _Nat. Rev. Immunol._ 7, 191–201 (2007). CAS  PubMed  Google Scholar  * Hirsch, E. et al.

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. _Science_ 287, 1049–1053 (2000). CAS  PubMed  Google Scholar  * Sasaki, T. et al. Function of PI3Kgamma in

thymocyte development, T cell activation, and neutrophil migration. _Science_ 287, 1040–1046 (2000). * Beer-Hammer, S. et al. The catalytic PI3K isoforms p110gamma and p110delta contribute

to B cell development and maintenance, transformation, and proliferation. _J. Leukoc. Biol._ 87, 1083–1095 (2010). CAS  PubMed  Google Scholar  * Reif, K. et al. Cutting edge: differential

roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing. _J. Immunol._ 173, 2236–2240 (2004). * Takeda, A. J. et al. Human PI3Kγ deficiency and its

microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology. _Nat. Commun._ 10, 4364 (2019). PubMed  PubMed Central  Google Scholar  * Suire, S. et al. p84, a new

Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma. _Curr. Biol._ 15, 566–570 (2005). CAS  PubMed  Google Scholar  * Stephens, L. R. et al. The G beta

gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. _Cell_ 89, 105–114 (1997). CAS  PubMed  Google Scholar  * Allen, D., Simon, T., Sablitzky, F., Rajewsky, K.

& Cumano, A. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. _EMBO J._ 7, 1995–2001 (1988). CAS  PubMed  PubMed Central  Google Scholar  *

Elsner, R. A. & Shlomchik, M. J. Germinal center and extrafollicular B cell responses in vaccination, immunity, and autoimmunity. _Immunity_ 53, 1136–1150 (2020). CAS  PubMed  PubMed

Central  Google Scholar  * Casola, S. et al. Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting. _Proc. Natl Acad. Sci.

USA_ 103, 7396–7401 (2006). CAS  PubMed  PubMed Central  Google Scholar  * Breasson, L. et al. PI3Kγ activity in leukocytes promotes adipose tissue inflammation and early-onset insulin

resistance during obesity. _Sci. Signal_. 10, eaaf2969 (2017). * Nojima, T. et al. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. _Nat.

Commun._ 2, 465 (2011). PubMed  Google Scholar  * Monaco, G. et al. RNA-seq signatures normalized by mRNA abundance allow absolute deconvolution of human immune cell types. _Cell Rep._ 26,

1627–1640 e1627 (2019). CAS  PubMed  PubMed Central  Google Scholar  * Lanahan, S. M., Wymann, M. P. & Lucas, C. L. The role of PI3Kγ in the immune system: new insights and translational

implications. _Nat. Rev. Immunol._ 22, 687–700 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Heng, T. S., Painter, M. W. & Immunological Genome Project The Immunological Genome

Project: networks of gene expression in immune cells. _Nat. Immunol._ 9, 1091–1094 (2008). CAS  PubMed  Google Scholar  * Pinto, D. et al. A functional BCR in human IgA and IgM plasma

cells. _Blood_ 121, 4110–4114 (2013). CAS  PubMed  Google Scholar  * Wagar, L. E. et al. Modeling human adaptive immune responses with tonsil organoids. _Nat. Med._ 27, 125–135 (2021). CAS 

PubMed  PubMed Central  Google Scholar  * Le Coz, C. et al. Human T follicular helper clones seed the germinal center-resident regulatory pool. _Sci. Immunol._ 8, eade8162 (2023). PubMed 

PubMed Central  Google Scholar  * Bekeredjian-Ding, I. & Jego, G. Toll-like receptors—sentries in the B-cell response. _Immunology_ 128, 311–323 (2009). CAS  PubMed  PubMed Central 

Google Scholar  * Gong, G. Q. et al. A small-molecule PI3Kα activator for cardioprotection and neuroregeneration. _Nature_ 618, 159–168 (2023). CAS  PubMed  PubMed Central  Google Scholar  *

Donahue, A. C. & Fruman, D. A. Distinct signaling mechanisms activate the target of rapamycin in response to different B-cell stimuli. _Eur. J. Immunol._ 37, 2923–2936 (2007). CAS 

PubMed  Google Scholar  * Thian, M. et al. Germline biallelic PIK3CG mutations in a multifaceted immunodeficiency with immune dysregulation. _Haematologica_ 105, e448–e492 (2020). PubMed

Central  Google Scholar  * Camps, M. et al. Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. _Nat. Med._ 11, 936–943 (2005). CAS 

PubMed  Google Scholar  * Barber, D. F. et al. PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus. _Nat. Med._ 11, 933–935 (2005). CAS 

PubMed  Google Scholar  * Del Prete, A. et al. Defective dendritic cell migration and activation of adaptive immunity in PI3Kγ-deficient mice. _EMBO J._ 23, 3505–3515 (2004). PubMed  PubMed

Central  Google Scholar  * Nobs, S. P. et al. PI3Kγ is critical for dendritic cell-mediated CD8+ T cell priming and viral clearance during influenza virus infection. _PLoS Pathog._ 12,

e1005508 (2016). PubMed  PubMed Central  Google Scholar  * Laidlaw, B. J. et al. The Eph-related tyrosine kinase ligand Ephrin-B1 marks germinal center and memory precursor B cells. _J. Exp.

Med._ 214, 639–649 (2017). CAS  PubMed  PubMed Central  Google Scholar  * Rossbacher, J. & Shlomchik, M. J. The B cell receptor itself can activate complement to provide the complement

receptor 1/2 ligand required to enhance B cell immune responses in vivo. _J. Exp. Med._ 198, 591–602 (2003). CAS  PubMed  PubMed Central  Google Scholar  * Luo, W. et al. SREBP signaling is

essential for effective B cell responses. _Nat. Immunol._ 24, 337–348 (2023). CAS  PubMed  Google Scholar  * Study to Evaluate the Efficacy/Safety of IPI-549 in Combination With Nivolumab in

Patients With Advanced Urothelial Carcinoma (MARIO-275)_. NIH_ https://clinicaltrials.gov/ct2/show/NCT03980041 (2019). * Evaluation of IPI-549 Combined With Front-line Treatments in Pts.

With Triple-Negative Breast Cancer or Renal Cell Carcinoma (MARIO-3)_. NIH_ https://clinicaltrials.gov/ct2/show/NCT03961698 (2019). * Evans, C. A. et al. Discovery of a selective

phosphoinositide-3-kinase (PI3K)-γ inhibitor (IPI-549) as an immuno-oncology clinical candidate. _ACS Med. Chem. Lett._ 7, 862–867 (2016). CAS  PubMed  PubMed Central  Google Scholar  *

Haniuda, K. & Kitamura, D. Induced germinal center B cell culture system. _Bio Protoc._ 9, e3163 (2019). PubMed  PubMed Central  Google Scholar  * Pahl, M. C. et al. Implicating effector

genes at COVID-19 GWAS loci using promoter-focused Capture-C in disease-relevant immune cell types. _Genome Biol._ 23, 125 (2022). CAS  PubMed  PubMed Central  Google Scholar  * Ramaswamy,

A. et al. Immune dysregulation and autoreactivity correlate with disease severity in SARS-CoV-2-associated multisystem inflammatory syndrome in children. _Immunity_ 54, 1083–1095.e1087

(2021). CAS  PubMed  PubMed Central  Google Scholar  * Barmada, A. et al. Cytokinopathy with aberrant cytotoxic lymphocytes and profibrotic myeloid response in SARS-CoV-2 mRNA

vaccine-associated myocarditis. _Sci. Immunol._ 8, eadh3455 (2023). CAS  PubMed  PubMed Central  Google Scholar  * Lopez, R., Regier, J., Cole, M. B., Jordan, M. I. & Yosef, N. Deep

generative modeling for single-cell transcriptomics. _Nat. Methods_ 15, 1053–1058 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY:

large-scale single-cell gene expression data analysis. _Genome Biol._ 19, 15 (2018). PubMed  PubMed Central  Google Scholar  * Ye, J., Ma, N., Madden, T. L. & Ostell, J. M. IgBLAST: an

immunoglobulin variable domain sequence analysis tool. _Nucleic Acids Res._ 41, W34–40 (2013). PubMed  PubMed Central  Google Scholar  * Lefranc, M. P. et al. IMGT, the international

ImMunoGeneTics information system. _Nucleic Acids Res._ 37, D1006–1012 (2009). CAS  PubMed  Google Scholar  * Alsoussi, W. B. et al. A potently neutralizing antibody protects mice against

SARS-CoV-2 infection. _J. Immunol._ 205, 915–922 (2020). CAS  PubMed  Google Scholar  * Gupta, N. T. et al. Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire

sequencing data. _Bioinformatics_ 31, 3356–3358 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Nouri, N. & Kleinstein, S. H. A spectral clustering-based method for identifying

clones from high-throughput B cell repertoire sequencing data. _Bioinformatics_ 34, i341–i349 (2018). CAS  PubMed  PubMed Central  Google Scholar  * Stern, J. N. et al. B cells populating

the multiple sclerosis brain mature in the draining cervical lymph nodes. _Sci. Transl. Med._ 6, 248ra107 (2014). PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS

We thank the patients and their families for participation in research and all clinical care staff for their contributions. We thank L. Wirth for technical assistance, J.-M. Carpier for

critical feedback and training, E. Fagerberg and J. Attanasio for contributions to RNA-seq experimental design and A. Rice for his contributions. We acknowledge D. Kitamura, Tokyo University

of Science, Organization for Research Advancement, Research Institute for Biomedical Sciences for the 40LB feeder cell line used in Extended Data Fig. 3. C.L.L. received funding for this

work from the Colton Center for Autoimmunity at Yale, NIH/NIAID (grant no. R21AI144315) and Yale University. M.P.W. was funded by the Swiss National Science Foundation (SNF; grant no.

310030_189065). N.R. is funded by the NIH, NIAID (grant no. AI146026 to N.R.), the Chan Zuckerberg Initiative Pediatric Human Cell Atlas and the Jeffrey Modell Foundation. BioRender.com was

used for schematics in Extended Data Fig. 2h and Extended Data Fig. 6. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Immunobiology, Yale University School of Medicine, New

Haven, CT, USA Stephen M. Lanahan, Lucas Yang, Kate M. Jones, Zhihong Qi, Anjali Ramaswamy, Anis Barmada, Dinesh Babu Uthaya Kumar, Lan Xu, Peiying Shan, Steven H. Kleinstein & Carrie L.

Lucas * Division of Immunology and Allergy, Children’s Hospital of Philadelphia, Philadelphia, PA, USA Emylette Cruz Cabrera & Neil Romberg * Immunology Graduate Group, Perelman School

of Medicine, Philadelphia, PA, USA Lauren Y. Cominsky * Department of Pathology, Yale University School of Medicine, New Haven, CT, USA Gisela Gabernet & Steven H. Kleinstein *

Department of Biomedicine, University of Basel, Basel, Switzerland Matthias P. Wymann * Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Steven H.

Kleinstein * Primary Immunodeficiency Clinic, Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD, USA V. Koneti Rao * Division of Allergy and Immunology, Department

of Pediatrics, Nationwide Children’s Hospital, Columbus, OH, USA Peter Mustillo * Department of Pediatrics, Perelman School of Medicine, Philadelphia, PA, USA Neil Romberg * Department of

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CONTRIBUTIONS S.M.L. was involved in idea generation, performed experiments, analyzed data and wrote the manuscript. L.Y. and K.M.J. performed experiments, analyzed data and wrote methods

for their work. Z.Q., D.B.U.K., L.X. and P.S. conducted experiments and analyses. E.C.C., L.Y.C. and N.R. conducted and analyzed the tonsillar organoid experiments. A.R., A.B., G.G. and

S.H.K. performed analysis of single-cell transcriptome and BCR-seq. K.R., P.M. and R.A. provided patient care and analyses relating to patient A.1’s samples. M.P.W. provided KO and floxed

mice and input. C.L.L. supervised overall research and data analysis, was involved in idea generation and wrote and edited the manuscript. All authors discussed and reviewed the manuscript.

CORRESPONDING AUTHOR Correspondence to Carrie L. Lucas. ETHICS DECLARATIONS COMPETING INTERESTS C.L.L. reports an advisory/consulting role for Pharming Healthcare Inc. and unrelated funding

support from Ono Pharma. S.H.K. receives consulting fees from Peraton. R.S.A. discloses support from ClinGen, _Journal of Immunology_ and Clinical Laboratory Standards Institute. The

remaining authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Immunology_ thanks Sidonia Fagarasan and the other, anonymous, reviewer(s) for their

contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the

editorial team. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA

EXTENDED DATA FIG. 1 PI3KΓ IS REQUIRED FOR T-DEPENDENT ANTIBODY RESPONSES. (A-B) Serum IgM (A) and IgA (B) levels in Patient A.1. (C-E) Mice were immunized (as in Fig. 1b, c) and

antigen-specific IgM (day 5) (C), primary (non-boosted) IgG1 (day 12) (D), or IgG2c (E) was measured via high conjugation ratio NP-BSA ELISA. (F) Mice were immunized (as in Fig. 1d, e) and

IgG2b was measured via NP-BSA ELISA. Data are from 2-3 independent experiments and are presented as box-and-whisker plots showing median, min, and max. Data points represent different

biological samples. Statistical analysis was performed using non-parametric two-tailed unpaired T-tests. (c) n = 12 for control, n = 12 for KO. (d) n = 15 for control, n = 14 for KO. (e) n =

 12 for control, n = 11 for KO. (f) n = 5 for control, n = 6 for KO. Source data EXTENDED DATA FIG. 2 PI3KΓ IS REQUIRED FOR GC B CELLS TO ADOPT ASC GENE SIGNATURES. (A) Stacked bar plots

showing proportions of cell subsets within Naïve or GC B cells used for CSR/SHM analysis. (B) Percentage of high-affinity W38L mutations from the total number of sequences with IGHV1-72 gene

and lambda light chains. Statistical analysis was Wilcoxon Rank Sum (C-D) Quantification of marginal zone B cells (C) and follicular B cells (D) from the spleens of indicated mice assessed

by flow cytometry. Data are representative of 2 independent experiments (n = 5 for each condition). (E) Volcano plot showing gene expression from RNA sequencing in sorted naïve B cells (IgD+

B220+) from WT versus PI3Kγ KO mice 28 days after SRBC i.p. immunization. Data are from one experiment. (F) Volcano plot of differentially expressed genes from bulk RNA sequencing of sorted

GC B cells at day 28 post immunization, as described for Fig. 2e. (G) Expression of ‘Activated B cell’ UPR program genes in sorted splenic GC B cells from _Pik3cg_ heterozygous or KO mice

28 days after SRBC immunization. Transcriptional program is defined by Gaudette, et. al 2020. Data are from one experiment (n = 3 for het, n = 4 for KO). (H) Diagram of germinal center B

cells that are beginning to adopt expression of antibody secreting cell surface markers (CD138). Diagram was created with BioRender.com. (C-D) Each dot represents different biological

samples and data are presented as box-and-whisker plots showing median, min, and max. Statistical analysis was performed using non-parametric two-tailed unpaired T-tests. Source data

EXTENDED DATA FIG. 3 B CELLS LACKING PI3KΓ ACTIVATE NORMALLY AND ARE CAPABLE OF FORMING GERMINAL CENTERS AND NORMAL IGA RESPONSES. (A) Littermate control or conditional _Pik3cg_ KO mice were

immunized and boosted with NP-OVA precipitated in alum. Antigen-specific IgG2c (day 28) was measured via NP-BSA ELISA, and arbitrary units were defined using pooled immunized sera as a

standard. (B) Immunofluorescence imaging of spleens from indicated mice 28 days after SRBC immunization. Data are representative of two independent repeats (n = 6 for each genotype). (C-H)

Murine naïve B cells were stimulated using TI stimulation with TLR agonists (CpG or LPS) or TD stimulation with anti-CD40, anti-IgM F(ab’)2 and assessed for proliferation using cell trace

violet (gating on live cells after 4 days) and activation by staining for CD69 or CD86 (gating on live cells after 2 days). Data (n = 3 for each condition) are representative of two

independent experiments. (I-N) Quantification of B cell subsets from Peyer’s patches of mice assessed by flow cytometry. Data are representative of three independent experiments (n = 13 for

control and 14 for KO). (O) Quantification of fecal IgA in control versus B cell-specific PI3Kγ KO mice (n = 6 per group). (P) Quantification of surface IgG-expressing memory and GC B cells,

normalized to control animals, in control versus B cell-specific PI3Kγ KO mice (n = 3 for control and 5 for KO). (Q-R) _In-vitro_ generation of GC B cells using 40LB feeder cells, as in

Nojima et al. 2011. Data are from two independent experiments (n = 5 for each condition). Data are presented as a bar graph (median ± SD) or box-and-whisker plots showing median, min, and

max. Each dot represents different biological samples and statistical analysis was performed using non-parametric two-tailed unpaired T-tests, except for panels q-r which uses non-parametric

paired T-test. Source data EXTENDED DATA FIG. 4 FUNCTIONAL PI3KΓ IS REQUIRED FOR OPTIMAL ASC DIFFERENTIATION _IN VITRO_ AND _IN VIVO_ DESPITE BEING DISPENSABLE FOR INNATE-LIKE B CELL IGM

RESPONSES. (A) Expression of PI3K genes in human/mouse immune cells derived from Immgen3. (B) Expression of _Pik3cg_ in mouse B cells comparing splenic follicular and germinal center B cells

to innate-like marginal zone and B1a B cells. (C-D) Mice were immunized i.p. with TI antigens and serum was taken on day 5 to measure antigen-specific IgM via ELISA. (E-F) Antigen-specific

ASCs were measured from splenocytes via ELISPOT in WT mice treated as described for Fig. 4e, f. (G-H) Assessment of _in-vitro_ differentiation of naïve mouse B cells into ASCs (anti-CD40,

IL-4, IL-5 gating on CD138+ after 4 days) in utilizing B cells from Blimp1-YFP mice treated _in vitro_ with DMSO vehicle or the PI3Kγ inhibitor IPI-549. Data are from 2-4 independent

experiments. Data presented as box-and-whisker plots show median, min, and max. (c) n = 11 for controls, n = 12 for KOs. (d) n = 12 for both groups. (e) n = 15 for both groups. (f) n = 13

for both. (g-h) n = 6 for each group. Each dot represents different biological samples and statistical analysis was performed using non-parametric two-tailed unpaired T-tests, with the

exception of panel H which used non-parametric paired T-test. Source data EXTENDED DATA FIG. 5 PI3KΓ IS REQUIRED FOR ROBUST HUMAN ASC DIFFERENTIATION. (A-H) Cells from human tonsillar

organoid model were assessed via flow cytometry (n = 5). Graphs depict percentage of each subset or percentage of live cells in each subset. (I) Assessment of _in-vitro_ differentiation of

primary healthy human peripheral blood pan-B cells into IRF4+ cells with 100 nM dose of PI3Kγi IPI-549 versus DMSO (shown as a proportion) after 5 days in the presence of DMSO control or

PI3Kα activator (UCL-TRO-1938). (J-K) Overnight IPI-549 treatment of isolated primary human CD138+ ASCs from peripheral blood. ELISPOTs and quantification are from 2 independent experiments

(n = 6). Each dot represents different biological samples and data are presented as mean ± SD. Statistical analysis was performed using non-parametric two-tailed unpaired T-tests and paired

T-test for (i). Source data EXTENDED DATA FIG. 6 MODEL SUMMARIZING THE FUNCTION OF PI3KΓ IN ANTIBODY RESPONSES. B cell-intrinsic PI3Kγ supports the transition to the ASC transcriptional

program and ASC differentiation in activated B cells. Diagram was created with BioRender.com. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Patient A.1 antibody titers elicited by

indicated vaccines. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE

DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source

data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. RIGHTS

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lanahan, S.M., Yang, L., Jones, K.M. _et al._ PI3Kγ in B cells promotes antibody responses and generation of antibody-secreting cells. _Nat

Immunol_ 25, 1422–1431 (2024). https://doi.org/10.1038/s41590-024-01890-1 Download citation * Received: 06 July 2023 * Accepted: 07 June 2024 * Published: 03 July 2024 * Issue Date: August

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