B Cell−Intrinsic mTORC1 Promotes Germinal Center−Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell This information is current as Generation in Humoral Immunity of September 26, 2021. Ariel L. Raybuck, Sung Hoon Cho, Jingxin Li, Meredith C. Rogers, Keunwook Lee, Christopher L. Williams, Mark Shlomchik, James W. Thomas, Jin Chen, John V. Williams

and Mark R. Boothby Downloaded from J Immunol published online 12 March 2018 http://www.jimmunol.org/content/early/2018/03/11/jimmun ol.1701321 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 12, 2018, doi:10.4049/jimmunol.1701321 The Journal of Immunology

B Cell–Intrinsic mTORC1 Promotes Germinal Center–Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell Generation in Humoral Immunity

Ariel L. Raybuck,* Sung Hoon Cho,* Jingxin Li,* Meredith C. Rogers,†,‡,1 Keunwook Lee,*,2 Christopher L. Williams,*,3 Mark Shlomchik,‡ James W. Thomas,x Jin Chen,x,{,‖,# John V. Williams,†,‡,1 and Mark R. Boothby*,{,‖

B lymphocytes migrate among varied microenvironmental niches during diversification, selection, and conversion to memory or

Ab-secreting plasma cells. Aspects of the nutrient milieu differ within these lymphoid microenvironments and can influence Downloaded from signaling molecules such as the mechanistic target of rapamycin (mTOR). However, much remains to be elucidated as to the B cell–intrinsic functions of nutrient-sensing signal transducers that modulate B cell differentiation or Ab affinity. We now show that the amino acid–sensing mTOR complex 1 (mTORC1) is vital for induction of Bcl6—a key transcriptional regulator of the germinal center (GC) fate—in activated B lymphocytes. Accordingly, disruption of mTORC1 after B cell development and activation led to reduced populations of Ag-specific memory B cells as well as plasma cells and GC B cells. In addition, induction

of the germ line transcript that guides activation-induced deaminase in selection of the IgG1 H chain region during class switching http://www.jimmunol.org/ required mTORC1. Expression of the somatic mutator activation-induced deaminase was reduced by a lack of mTORC1 in B cells, whereas point mutation frequencies in Ag-specific GC-phenotype B cells were only halved. These effects culminated in a B cell–intrinsic defect that impacted an antiviral Ab response and drastically impaired generation of high-affinity IgG1. Collec- tively, these data establish that mTORC1 governs critical B cell–intrinsic mechanisms essential for establishment of GC differ- entiation and effective Ab production. The Journal of Immunology, 2018, 200: 000–000.

cell receptor–initiated signals in mature lymphocytes are i.e., Ab class-switch recombination and the point mutations that vital for Ag-activated B cell proliferation and clonal diversify and heighten affinity in the memory and Ab repertoires by guest on September 26, 2021 B expansion (1, 2). After several days and divisions, these (3, 4, 8–10). B cell activation and costimulation in the GCs and signals can also yield functionally important class-switch recom- extrafollicular milieux occur in the context of a variegated inter- bination as well as somatic hypermutation (SHM) coupled to the stitial environment (11–13). How sensors of environmental cues iterative selection of B cells with higher affinity Ag-specific BCR influence B lymphocyte–intrinsic mechanisms of differentiation in germinal center (GC) reactions (3–6). Humoral immunity then and function that determine Ab response properties through so- stems from the differentiation of B lymphocytes into Ab-secreting matic mutation is incompletely understood. plasmablasts and plasma cells (1, 4, 7). Activation-induced de- Ag-specific BCR-derived signals (2, 14) are modulated by aminase (AID), which is most highly induced in GCs, is essential CD19 along with a menu of additional B cell surface recep- for both classes of genome revision crucial in humoral immunity, tors (namely, CD40; IL-4R and IL-21R; TLR4 and 7) whose

*Department of Pathology, Microbiology, and Immunology, Vanderbilt University This work was supported by National Institutes of Health Grants AI113292 and Medical Center, Nashville, TN 37232; †Division of Infectious Diseases, Department HL106812 (to M.R.B.) and AI085062 (to J.V.W.) for the experiments. Additional of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 27232; support was provided by National Institutes of Health Grant T32 HL069765 ‡Department of Immunology, University of Pittsburgh Medical Center, Pittsburgh, (to C.L.W.), Korean Ministry of Science Grant NRF-2014M3A9D5A01073841 PA 15261; xDivision of Rheumatology and Immunology, Department of Medicine, (to K.L.), and National Institutes of Health Shared Instrumentation Grant Vanderbilt University Medical Center, Nashville, TN 27232; {Medical and Research 1S10OD018015, as well as scholarships through Cancer Center Support Grant Services, Department of Veterans Affairs, Tennessee Valley Healthcare System, CA068485 and Diabetes Research Center Grant DK0205930 to help defray costs Nashville, TN 37212; ‖Program in Cancer Biology, Vanderbilt University School of the Vanderbilt Cores. of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232; and #De- Address correspondence and reprint requests to Dr. Mark R. Boothby, Department of partment of Cell and Developmental Biology, Vanderbilt University School of Med- Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, icine, Vanderbilt University Medical Center, Nashville, TN 37232 A-5301 M. C. N., 1161 21st Avenue South, Nashville, TN 37232. E-mail address: 1Current address: Children’s Hospital and Department of Pediatrics, University of [email protected] Pittsburgh Medical Center, Pittsburgh, PA. The online version of this article contains supplemental material. 2Current address: Department of Biomedical Science, Hallym University, Chuncheon, Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AID, activation- Korea. induced deaminase; ASC, Ab-secreting cell; 2DG, 2-deoxyglucose; DZ, dark zone; 3Current address: Institute for Cellular Therapeutics, University of Louisville, Louisville, FSC, forward-scattered (light) channel; GC, germinal center; GLT, germ line tran- KY. script; HA, hemagglutinin; hu, human; IgHa, Ig CH allotype-disparate; IRF, IFN response factor; LZ, light zone; mTOR, mechanistic target of rapamycin; mTORC1, ORCIDs: 0000-0002-6463-4032 (S.H.C.); 0000-0003-1798-6294 (M.C.R.); 0000- mTOR complex 1; SHM, somatic hypermutation; SSC, side-scattered (light) channel; 0002-2098-6458 (J.W.T.); 0000-0002-5557-2079 (J.C.); 0000-0001-8377- T , T follicular helper; WT, wild type. 5175 (J.V.W.). FH Received for publication September 18, 2017. Accepted for publication February 14, Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 2018.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701321 2 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY engagement and activity are crucial for the acquisition of a GC unanswered questions as to which functions of mTORC1 are in- B cell program (15–19). In the GC reaction, activated B cells trinsic to B lineage cells in vivo. Notable among these are: 1) what receive cognate help from T cells that specialize into a T follic- molecular defects of mTORC1-deficient B cells could explain a ular helper (TFH) phenotype (3, 4). This reaction can involve GC impact, 2) whether lower Aicda gene expression is accom- iterative rounds of proliferation and AID-dependent somatic mu- panied by defects of SHM, 3) if affinity maturation is altered, and tation in the dark zone (DZ), followed by restimulation and se- 4) to what extent the mTORC1 function within B cells influences lection in a TFH-enriched light zone (LZ) (5, 20–22). Diversification their capacity to generate memory. of the BCR on lymphoblasts by somatic mutation is one, but not the To investigate B cell–intrinsic actions of mTORC1, we used only, factor promoting high-affinity Ab (8, 23). The GC ultimately multiple Cre-driven approaches to cause loss-of-function after generates Ag-specific memory cells, plasmablasts, and long-lived establishment of normal B cell populations. In vivo and in vitro plasma cells that constitutively secrete Ab (3, 4). The B lineage also analyses with these systems revealed requirements for mTORC1 generates plasma cells and some memory through extrafollicular in promoting progression to GC and memory stages of the B responses, either TLR driven or aided by T cells (24–30). The lineage. Depletion of the core mTORC1 protein Raptor not only memory B cell population that arises from extrafollicular processes reduced AID expression but also limited the induction of germ is dominated by unswitched IgM+ cells (28, 30). Along each of line transcripts (GLT) that direct Ab class switching, leading to a these cellular pathways, B cells encounter and depend on different B cell–intrinsic block in switched Ab in models of immunization arrays and densities of receptor ligands, suggesting that differences or virus infection. These effects were associated with loss of in signal transduction determine the relative efficiency of each BCL6 induction, weakened B cell support of a TFH population, outcome. and reductions in somatic point mutations. Strikingly, we found Downloaded from Many receptors that regulate B lineage fates increase the activity that quantitative decreases in high-affinity Ab deriving from of PI3K, a set of lipid kinases essential for development, survival, mTORC1-deficient B cells were more profound than effects on and function of B cells (31). Phosphatidyl-inositol 3, 4, 5-tri- SHM. Collectively, the findings indicate that presence of phosphate generated from PIP2 by this enzyme recruits pleckstrin mTORC1, a sensor of nutrient supply, critically impacts the homology domain-containing proteins and thereby activates qualities of the primary humoral response.

downstream pathways (31). Activity of the mammalian mecha- http://www.jimmunol.org/ nistic target of rapamycin (mTOR), a serine-threonine kinase, is Materials and Methods stimulated by PI3K (32, 33). mTOR complex 1 (mTORC1) ac- Reagents tivity also is regulated by sensing levels of nutrients such as mAbs (purified, biotinylated, or fluorophore -conjugated) were from BD glucose or certain amino acids (34). Thus, mTORC1 function is of Pharmingen (San Jose, CA) or Tonbo Biosciences (San Diego, CA) unless particular interest as it integrates information about metabolite otherwise specified. Recombinant BAFF was purchased from Adipogen (San Diego, CA); IL-4, CellTrace Violet, BrdU, and CFSE (CFDA-SE) for levels with conventional signal transduction by surface receptors. proliferation assays, and TRIzol reagent for nucleic acid isolation were from Rapamycin bound to an endogenous cellular protein acutely but Thermo Fisher Scientific (Carlsbad, CA); LPS, tamoxifen, and 4-hydroxy- incompletely inhibits the multiprotein mTORC1 by an allosteric tamoxifen from Sigma-Aldrich (St. Louis, MO); NP-BSA and NP-PSA for mechanism dependent on the structural component Raptor (35). ELISA and ELISpot, NP-KLH for immunizations, and NP-O-succinimide by guest on September 26, 2021 Of note, however, rapamycin cripples a second complex— (NP-Osu for NP-APC conjugation) from Biosearch Technologies (Novato, CA). SRBC were obtained from Remel (San Diego, CA) via Thermo mTORC2—in lymphoid lineage and dendritic cells (36–39). Apart Fisher Scientific and used within 2 wk. Fluorescent APC protein Prozyme from PI3K, the activating inputs of the two respective mTOR (Hayward, CA) was used in conjugation reactions with NP-Osu. Rapa- complexes differ, as do their functional targets (35). Previous work mycin and oligomycin were from EMD Millipore (Billerica, MA) and has indicated that disruption of mTORC2 in B cells by genetic CellTak for Seahorse assays from Corning (Corning, NY). approaches cripples B cell development, survival, and Ab re- Mice and B cell transfer models sponses (32, 40). Accordingly, genetic approaches are essential for All mice, C57BL/6J, B6-mMT, CD45.1 congenic, Ig CH allotype-disparate more fully understanding the molecular mechanisms related to (IgHa), B6-Rptorf/f Cre [human (hu) CD20-CreERT2 (50); Rosa26- mTOR. CreERT2 (37); Cg1-Cre (51)], were housed in ventilated microisolators Early work in this area showed that rapamycin blocked human under specified pathogen-free conditions in a Vanderbilt University mouse B cell proliferation and Ab production in vitro (41). This finding facility. A balanced mix of males and females was used starting at 6–8 wk may correlate with in vitro data indicating that mTORC1 inacti- of age and following approved mouse protocols, generally taking litter- mates that were housed together except for some CreERT2 Rptorf/f, which vation attenuated switching to IgG1 for anti-CD40–activated were bred separately from the CreERT2 Rptor+/+ mice used to control for B cells (42). Experiments in vitro and in mice, including deletion effects of inducible Cre. In vivo analyses have found this wild-type (WT) of mTOR with CD19-Cre, indicate that mTOR promotes Ab control yields results no different from Creneg Rptorf/f controls. Other than T2 T2 production and marginally increases high-affinity Ab (43–45), huCD20-CreER (50) and Rosa26-CreER (37), breeding stock for each line was from the Jackson Laboratory (Bar Harbor, ME). For influenza albeit with differences among the results. Intriguingly, systemic infection experiments, transfer recipient mice were moved to an ABSL-2 rapamycin administration during a primary response to immuni- facility under a separate protocol (J.V.W.) during periods of a potential zation with influenza A virus dramatically decreased GC and GC infectious nature, and handled only by personnel with appropriate training B cell frequencies yet led to better heterosubtypic protection in and the approval of Institutional Biosafety. Healthy donor and recipient cross-strain challenges 4 wk after immunization (46). This cross- mice of the appropriate genotype were selected randomly for experiments + without size or gender preference. For adoptive transfer experiments to protection required CD4 T cells as well as B cells, and the anti- measure primary immune response, donor (Rptor+/+, Rosa26-CreERT2+; influenza Ab specificities generated in rapamycin-treated mice Rptorf/f, Rosa26-CreERT2+) lymph node and spleen cell suspensions were appeared to differ from those of controls (46). With regard to depleted of T cells using biotinylated anti-Thy1.2 Ab and streptavidin- mTOR activity, mTORC1 and 2 are each inhibited after hours of conjugated microbeads in the BD iMag system. Recipient mice then re- ceived i.v. transfers of 5–10 3 106 cells in sterile saline. rapamycin treatment and each has crucial functions in CD4+ T cells that include follicular help to Ab production (37, 38, 47– Infections and immunizations, ELISA, and ELISpot 49), and cell-intrinsic functions in dendritic cells as well (39). For influenza and other adoptive transfer experiments, Rosa26-CreERT2 These studies of rapamycin and of mTOR, each of which im- mice of all three genotypes at the Rptor locus (Rptorf/f, Rptorf/+, and +/+ pairs T cell differentiation into functional TFH cells, raise crucial Rptor ) received a sequence of three tamoxifen injections every other The Journal of Immunology 3 day as described (32), i.e., 3 mg per injection, dissolved in safflower oil, and proliferation were carried out as described above. For proliferation and were harvested 1 wk later. To measure the donor-specific humoral assays cells were labeled after purification with either CFSE or CellTrace response stemming from transferred B cells in immune competent recip- Violet (as indicated) and then cultured for 4 or 5 d (respectively) in the ients, purified donor B cells were transferred i.v. into B6-IgHa allotype presence of BAFF, or BAFF, LPS, and IL4. Radiotracer measurements of mice followed by immunization with NP20-OVA as described previously glycolysis were conducted as described previously (37). (32). Alternatively, donor-derived GC-phenotype and NP-specific GC- phenotype B cells were enumerated after i.v. transfers of B cells purified Flow cytometry from tamoxifen-injected mice into B6-CD45.1 recipients followed by Flow cytometric phenotyping of in vivo immune responses was con- m immunization with NP-KLH [NP16-KLH; 100 g i.p.) precipitated in alum ducted 5–10 d after booster immunization as indicated. For detection of adjuvant (Imject; Thermo Fisher Scientific). To analyze the characteristics 6 NP-specific GC responses, spleen or lymph node cells (2 3 10 )were of a serological response to influenza virus immunization and later in- stained with GL7, anti-CD95, -CD38, -B220, -IgM, NP-APC, and a fectious challenge, purified B cells from donor mice were transferred i.v. dump mixture containing monoclonal Abs for IgD, 7-AAD, CD11b, m into MT recipients that were then immunized by i.p. injection of the CD11c, F4/80, and Gr1. The flow cytometric gating strategy for analysis A/PR8/34 (H1N1) vaccine strain of influenza virus. After interval, sera of NP-binding, memory B cell, and GC B cell populations is shown in were collected from mice by phlebotomy (1 and 3 wk after immunization), Supplemental Fig. 1F. For intracellular staining of BCL6 and IFN re- mice were then challenged by intranasal administration of influenza A/X- sponse factor (IRF) 4, cultured cells were stained for viability (7-AAD) 31 (H3N2). Mice were monitored (with daily weights and clinical as- and B220, followed by fixation and permeabilization (4˚C for 30 min) sessment to allow for humane endpoint termination if indicated) for 7 d using commercial transcription factor buffer (Tonbo), washing with postinfection and then harvested (organs and terminal bleed for sera). For T2 f/f permeabilization buffer (Tonbo), and then staining (1 h at 4˚C) with other immunization experiments, huCD20-CreER mice (Rptor and 6 +/+ anti–BCL6-APC (eBioscience) (5 mlper10 cells) or anti-IRF4 (2 ml Rptor ) 6 controls received serial tamoxifen injections as described for per 10 cells) (Thermo Fisher Scientific). Cells were then washed and adoptive transfers. Then 7d after the third such injection, mice were im- IRF4-stained cells were incubated(20minat4˚C)withAlexa647 munized with NP -KLH as described above for the adoptive transfers into Downloaded from 16 chicken anti-rabbit IgG second Ab, and washed with permeabilization B6-CD45.1 recipients. buffer. For splenocytes recovered after immunization, samples were depleted 7 Measurement of humoral responses of Thy1.2 as in the preceding section, stained (10 cells per sample) with IgD, CD19, GL7, and CD95 as well as 7-AAD and B220, and then processed ELISA was performed as described previously (11, 32). Sera from for direct and indirect immune fluorescent staining of BCL6 and IRF4 as for influenza-challenged mice were assayed using total viral lysate prepared the cultured cells. All samples were then resuspended in wash buffer (2% from stocks of each strain (A/PR8/34; A/ X-31) or purified recombinant FBS in PBS) followed by flow cytometry. For these and other flow cyto- strain-specific H1 and H3 hemagglutinin (HA) proteins (Sino Biological, metric analyses, fluorescence emission data on cell suspensions were col- http://www.jimmunol.org/ Beijing). Relative levels of all- and high-affinity anti-NP Abs were mea- lected on BD LSR or FACSCanto flow cytometers driven by BD FACS Diva sured by ELISA capturing the hapten-binding Ab on plate-immobilized software, then processed using FlowJo software (FlowJo, formerly Tree Star, NP20-BSA or NP2-porcine serum albumin, respectively; anti-KLH Ab Ashland, OR). were measured by ELISA with plate-bound KLH. Allotype- or isotype- specific Abs were then detected using HRP-conjugated or biotinylated Abs Seahorse assays (Southern Biotechnology Associates, Birmingham, AL). Ab-secreting cells Purified B cells (conditionally Raptor-depleted and WT) from lymphoid analyzed by ELISpot were quantitated using the ImmunoSpot Analyzer organs were plated (2 3 106 cells per ml in 12-well clusters) and then (Cellular Technology, Shaker Heights, OH). Ag-specific GC and memory cultured (2 d) with BAFF alone, or LPS, BAFF, and IL-4. Viable cells B cell populations were analyzed by flow cytometry using APC-conjugated recovered from these cultures were seeded at 150,000 cells per well on NP and an FL-3 dump channel consisting of 7-aminoactinomycin D (7- manufacturer-supplied 96-well plates after precoating wells with CellTak by guest on September 26, 2021 AAD) (Thermo Fisher Scientific, previously Invitrogen) and PerCP- (Corning) at 2.5 mg/ml because optimization pilots showed this configu- Cy5.5–conjugated IgD (BioLegend, San Diego, CA), F4/80, Gr1, CD11b, ration to yield comparable adhesion along with more vigorous B lym- and CD11c (Tonbo Bio). phoblasts as compared with 22.4 mg/ml recommended by Seahorse, whereas poly-D-lysine did not yield suitable adhesion. Measurements of Immunoblots extracellular pH were then made over time on an XFe96 metabolic flux Relative protein or phosphopeptide abundance in whole cell extracts was analyzer (Agilent Technologies, Santa Clara, CA) and converted to cal- determined by immunoblotting as described (32, 37). In brief, denatured, culated or estimated rates of oxygen consumption, extracellular acidifi- reduced proteins separated by SDS-PAGE with a stacking-resolving gel cation, glycolysis, glycolytic flux, and glycolytic capacity using Microsoft system were transferred to polyvinylidene difluoride membranes by elec- Excel (Mac OS X) with formulae from Seahorse protocols. trophoresis. For improved transfer of Raptor, gels were first incubated Somatic hypermutation analyses (5 min at 22˚C) in Trypsin-EDTA (Life Technologies-BRL Life Tech- nologies division of Thermo Fisher Scientific). Membranes were probed WT (Rosa26- Cre ERT2+, Rptor+/+)orRptor knockout (Rosa26-CreER T2+, with primary antisera at supplier-recommended dilutions, after blocking by Rptor f/f -.D/D) B cells were transferred into mMT recipients (5 3 106 preincubation with 5% milk, and rinsed with 0.1% Tween in TBS. After cells per recipient). The mice were immunized with NP-KLH in alum as rinsing, membranes were incubated with goat anti-rabbit-specific, or for actin above, and harvested 10 d later. Viable [Ghost dye-excluding cells in the quantitation donkey anti-goat, secondary anti-IgG Ab conjugated to viable lymphoid forward-scattered (light) channel (FSC) 3 side-scattered Alexa680 nm fluorophore (Invitrogen Molecular Probes of Thermo Fisher (light) channel (SSC) gate] NP-specific GC B cells as well as naive (GL72 Scientific), rinsed, and bands visualized and quantified by scanning with an IgD+) B cells were flow sorted and lysed for cellular DNA. [This viable Odyssey Imaging system (Li-Cor, Lincoln, NE). For measurement of cell gate is devoid of early apoptotic cells (i.e., annexin-V+ or cleaved phosphorylation of S6, anti-phosphopeptide antisera (Cell Signaling Tech- caspase-3+ events).] Nested PCR was performed as previously described nologies, Danvers, MA) directed to the S235/S236 phosphorylated epitopes (43) and gel-purified products were subcloned into TA vector (Promega, (a target of both p70 S6K and p90 RSK) were used. Other primary Abs for Madison, WI) for sequencing. Sequences were aligned and scored for immunoblotting were from the same supplier [anti-S6 (5G10), anti-phospho mutations using MEGA software. S6K(T389), anti-S6K] or Thermo Fisher Scientific for anti-cyclophilin B. Quantitation of mRNA by quantitative PCR B cell cultures and assays of metabolism mRNA was isolated from flow-sorted GC phenotype B cells (dumpneg Splenocytes were enriched for B cells by Thy1.1 or Thy1.2 depletion B220+ GL7+), memory B cells (dumpneg B220+ GL7neg), and naive (dependent on mouse strain used) using biotinylated anti-Thy1.1 or -Thy1.2 (dumpneg B220+ IgD+) B cells using TRIzol reagent and then purified mAb and the BD iMag system. For class-switch cultures, cells were cultured following the standard manufacturer’s protocol. B cell cultures grown to at a density of 0.5 3 106 cells per ml in 2 ml cultures on 12-well plates with measure mRNA were harvested into TRIzol, from which RNA was purified BAFF, BAFF and LPS, or BAFF, LPS, and IL-4. Unless otherwise spec- using the manufacturer’s protocol. RNA concentrations were measured ified, cells were cultured in IMDM supplemented with 10% FBS. Cultures using a NanoDrop spectrophotometer and then used to synthesize to measure the effect of 2-deoxyglucose (2DG) received agent (1.5 mM) cDNA using the Promega AMV Reverse Transcriptase kit. All mRNA on day 0 only. Cells were cultured for 2–5 d, followed by flow cytometry, quantifications were normalized to hypoxanthine phosphoribosyltransfer- and ELISA measurements of relative Ab concentrations in culture super- ase. Primer pair sequences are tabulated and further details freely available natants. Cultures for measurements of class-switch, AID-GFP expression, upon request. 4 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY

Statistical methods and tests of significance The primary analyses were conducted on pooled data points from independent samples and replicates (minimum two, generally three, biologically and temporally independent replicate experiments for all data, with multiple independent samples in the case of two biological replicates), using an un- paired two-tailed Student t test with posttest validation of its suitability. Two- way ANOVAwith Bonferroni correction for multiple comparisons was used for statistical analysis across ELISA titration curves to compare WT to each mutant sample set. When this determination indicated rejection of the null hypothesis (i.e., p , 0.05), two-tailed comparisons of WT versus mutant were performed at single dilution values as above (typically, Student t test). Data are displayed as mean (6SEM), i.e., center values were means as average. Results were considered statistically significant when the p value for the null hypothesis of a comparison was ,0.05. Most p values are shown in the figure panels, with some in the figure legends or in Results. All resulted from statistical tests of null hypotheses with posthoc testing to assure ap- propriateness. Because the extent or direction of difference between samples was unknown, and regulations mandate reducing the number of animals used to the lowest feasible level, no statistical methods were used to deter- mine prespecified sample sizes and mice of both genders were selected in balanced numbers without bias. The experiments were not randomized and

the investigators were not blinded during the experiments. Downloaded from Tabulated details. A tabulation of names, sources, and stock numbers for Ab reagents and other such details are freely available on request.

Results Raptor is essential for a class-switched but not IgM response to hapten http://www.jimmunol.org/ We first used transfer models with a chemically activated Cre and conditional loss-of-function for Raptor to investigate how mTORC1 affects the capacity for B cells to yield Ab responses. When Raptor-deficient B cells were transferred into immune- FIGURE 1. B cell–autonomous mTORC1 regulates the class switch and competent allotype-disparate recipients followed by immuniza- production of Abs in response to NP-KLH immunization. (A) Equal tion (Fig. 1A), the switched IgG1 anti-hapten response derived numbers of B cells from tamoxifen-treated WT (Rptor +/+) or Raptor- T2+ from donor (IgHb) B cells was completely eliminated, whereas deficient (Rptor fl/fl) CreER mice were transferred into CD45.1 (Fig. 2F, a Ag-specific IgMb concentrations were the same as those derived 2G) or IgH -[a allotype]–disparate recipients that were then immunized B C following the schedule in the schematic. ( and ) Relative Ab concen- by guest on September 26, 2021 from WT control B cells (Fig. 1B). Internal control on immuni- trations (B), and prevalence of splenocytes that secrete Ag-specific Ab of zation quality was provided by analyses of the recipient (a allo- the indicated isotype after harvesting immunized IgHa mice that received type) Abs, which were no lower in recipients of Raptor-null IgHb B cells (WT or Raptor-depleted, closed and open symbols, respec- B cells than controls. Similar data were obtained by measuring tively) (C). (B) The mean (6 SEM) data from three independent replicate donor-allotype Ab secreting cells (ASCs) (Fig. 1C). Control experiments of Ig allotype–disparate ELISAs for all-affinity IgM/G1a re- analyses performed with B cells purified after tamoxifen injections cipient Ab and IgM/G1b donor Ab responses. (C) Mean (6 SEM) data showed that the chemically induced Rosa26-Cre–mediated dele- from allotype-specific measurements of ASC by ELISPOT in the same tion was efficient. Little residual Raptor protein was detected, and three independent experiments (nine mice for each genotype of donor B D phosphorylation of a protein downstream from mTORC1 was cells). ( ) Efficient mTORC1 inactivation via deletion of the conditional reduced substantially (Fig. 1D). Because the donor-derived B cells Rptor allele. Shown are immunoblots performed using the indicated Ab and extracts of B cells purified from mice of the indicated genotypes (one are a small minority of Ag-specific and activated B cells in these experiment representative of two to three replicates with biologically in- experiments, these data indicate that a switched response can have dependent samples). a strong B cell–autonomous dependence on mTORC1. Parallel work indicated that homozygous Rptor depletion led to a failure to elicit sustainable IgM (44). However, our results show that B cells yielded evidence of a reduction in the number of B cells recovered can yield a normal primary IgM response despite essentially a 4–5 wk after the onset of Raptor depletion (Fig. 2A, 2B, complete loss of mTORC1 function after Raptor depletion. Supplemental Fig. 1A–C). Within the population of B cells, the frequency of GL7+ Fas+ (GC B phenotype) cells was reproducibly Reduced BCL6 expression and Ag-specific GC cells reduced, albeit only to a modest extent (flow plots; Fig. 2C), after Prior work with mTOR, rapamycin, and Rptor loss-of-function has immunization of tamoxifen-injected huCD20-CreERT2 mice that indicated that mTORC1 promotes GC reactions (43–49) but the were Rptor f/f, leading to a reduction to ∼two thirds of control cellular or molecular mechanisms for this impairment are not numbers (Fig. 2C). However, the frequency of NP-binding cells known. We tested how disruption of mTORC1 in B cells affected among B lymphocytes was more substantially affected by loss of proliferation, the populations of B cells, and their expression of Raptor than was the overall population (Fig. 2D). Cumulatively, key transcriptional mediators of the GC B cell program after the numbers of Ag-specific GC-phenotype B cells were reduced to immunization. To do so, we used both in situ Rptor gene dis- ∼0.43 those of control mice when naive and activated B lineage ruption in transgenic models that allow B lineage–restricted cells were deficient for mTORC1 (Fig. 2D). Activation of the B tamoxifen activation of Cre-mediated Rptor gene inactivation lineage–restricted huCD20-driven CreERT2 led to sustained, al- (32, 52) (Fig. 2) and adoptive transfers into immunocompetent, though not complete, loss of Raptor in the B cell population allotype-disparate mice. In situ deletion in B cells driven by present after immunization (Fig. 2E). Comparison of the data from chemical activation of Cre using the huCD20-CreERT2 transgene longer-term loss-of-function (Fig. 2E) with the more immediate The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. mTORC1 promotes production of class-switched Abs by B cell–intrinsic mechanisms regulating Ag-specific GC B cells. (A) Schematic of experimental design used for (B–E). (B–D) Splenocytes harvested 1 wk after the booster immunization were counted and analyzed by flow cytometry. (B)B cell counts in spleens at harvest after immunization and boost of huCD20-CreERT2 mice (Rptor +/+ or D/D). Each point, calculated from the enumeration of splenocytes and the fraction of B220+ cells (Supplemental Fig. 1A), represents an individual mouse (nine mice of each genotype, distributed evenly across four independent replicate experiments) and the horizontal bars represent means. (C and D) Preferential impact of intrinsic mTORC1 on Ag-specific GC B cells. (C) One representative result (with inset numbers representing percentages in the indicated GC-phenotype gate) as well as quantitation of the mean (6 SEM) frequencies (italicized) of such B cells in the nine independent mice of each genotype in the four biological replicate experiments as in (B). (D) NP- binding B cells in the gate for viable, IgDneg, myeloid lineageneg cells that were GL7+ (GC phenotype), with a representative result from one experiment shown along with mean (6 SEM) quantitation as in (C). (E) Recovered cells remained Raptor-depleted. CD19+ B cells were microbead-purified from spleens of tamoxifen-injected, immunized mice (huCD20-CreERT2+ Rptor+/+, and huCD20-CreERT2+ Rptor fl/fl), activated with LPS, cultured (2 d), and then analyzed by immunoblotting to measure Raptor levels and phosphorylation of S6 [anti-pS6(S235/236)] as well as actin, shown as fluorescence units quantitated on the Odyssey IR imager. (F and G) Purified WT and Raptor-depleted B cells (as in Fig. 1A) were transferred into CD45.1 recipients that were then immunized with NP-KLH, boosted 3 wk later, and harvested 1 wk after the boost. (F) Representative flow plots from within the viable cell, donor (CD45.2+) gate, with inset numbers representing the percentage GC-phenotype (dumpneg GL7+ B220+) B cells from spleens of CD45.1 recipient mice (upper panel pair) and the Ag-specific (NP-APC+) events in the GL7+ population (lower panel pair), which are then quantified in (G) with circles rep- resenting recipient mouse B cell phenotyping (CD45.1+) and squares representing the donor (CD45.2+) population (eight mice per donor cell genotype, in balanced distributions among three independent replicate experiments). Representative flow data for donor CD45.2 staining specificity are in Supplemental Fig. 1E.

B cell isolation (Fig. 1D) suggests that the selective outgrowth of program for GC B cells, beyond loss of surface IgD and induction B cells in which Rptor deletion failed was, at most, modest. These of the GL7 epitope. To investigate this issue, we purified GL7+ 2 results were complemented by independent experiments in which IgD CD38int viable (7-AADneg events in the lymphoid FSC 3 mTORC1-depleted B cells competed against a majority of normal SSC gate) B cells by flow sorting after immunization of the B cells (Fig. 2F, 2G, Supplemental Fig. 1D, 1E). In this compet- tamoxifen-injected, huCD20-CreERT2+ mice. Quantitation of itive setting, almost no NP-binding GL7+ B cells were observed mRNA in these purified mTORC1-depleted cells revealed sub- after transfers of B cells from WT or Raptor-depleted B cells stantial or dramatic reductions in Irf4 and Bcl6 gene expression, (CD45.2) into CD45.1 recipients followed by immunization. respectively, in the viable GC-phenotype B cells (Fig. 3A). Notwithstanding reduced proliferation of mTORC1-deficient Consistent with this, Aicda mRNA also was lower (Fig. 3A). B cells in vitro [(49) and data in this study], these findings indi- The GC makes major contributions to B cell memory (3, 4, 25, cated that there is an intrinsic impairment of the differentiation 29). Of note, Irf4 and Aicda mRNA levels also were lower in 6 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY

Raptor-depleted memory-phenotype B cells (Fig. 3B). In the de- velopment of follicular helper characteristics among CD4 T cells, mTORC1 improves translational efficiency of Bcl6 but not Irf4 mRNA (52). Consistent with this report, BCL6 protein was far lower in the Rptor-inactivated GC B subsets by intracellular staining after immunization as compared with WT controls (Fig. 3C). Analyses of T cells demonstrated links among trans- porters, uptake of leucine (via LAT1) and glutamine (via ASCT2), and differentiation (53, 54). When we tested if mTORC1 promotes gene expression for Slc7a5 and Slc1a5 (encoding LAT1 and ASCT2, respectively) in GC B cells, we found striking decreases in transporter induction (Fig. 3D). Consistent with this finding, mTORC1-deficient B lymphoblasts in vitro expressed less of these transporters (Supplemental Fig. 2A). Induction of the CD98 H chain, which pairs with LAT1 and ASCT2 along with other transport proteins, was also reduced (Fig. 3E). Together, these findings indicated that the capacity to express genes crucial for GC B cell growth and function is impaired in Raptor-depleted B cells. Rapamycin treatment during and after immunization almost Downloaded from completely eliminated mouse GCs (46), but rapamycin impacts both mTORC2 and mTORC1 and impacts all cells. Moreover, TFH cells depend on both mTORC1 and mTORC2. GCs also were absent after disruption of mTORC2 via Rictor gene inactivation (32) or in mutant mice whose mTOR is functionally hypomorphic

(43, 45). Therefore, we tested how B cell–intrinsic mTORC1 http://www.jimmunol.org/ deficiency impacts GC anatomy. Immunofluorescent microscopy of spleens harvested after immunization of tamoxifen-injected huCD20-CreERT2+ mice (i.e., WT or Rptor D/D B cells) showed that an IgD-negative zone was organized within IgD+ IgM+ fol- + FIGURE 3. Bcl6 and Irf4 gene expression along with neutral amino acid licles surrounded by CD35 cores (Supplemental Fig. 2B). These transporters require mTORC1 in the GC-phenotype B cell. (A and B)Rel- structures were present in essentially normal numbers [means (6 ative mRNA levels were quantified by quantitative real-time PCR using SEM) of 43 (6 5.1) for WT and 38 (6 5.7) for Rptor D/D, p . 0.1], cDNA of naive, GC-, and memory-phenotype B cells (huCD20-CreERT2+, whereas GC structures with GL7+ centers depended on mTORC1

+/+ by guest on September 26, 2021 either Rptor or D/D) purified from the splenocyte suspensions by flow [mean values were 40 (6 5.0) for WT versus 10 (6 2.4) when sorting B cells according to IgD, GL7, CD38, and viability indices (FSC, , + B cells were Rptor D/D, p 0.01] (Supplemental Fig. 2B). These SSC, and 7-AAD dye). Relative mRNA is normalized to flow-purified IgD anatomic data indicate that GC were reduced, but suggest that GL7neg naive B cells, whose relative mRNA level is defined as 1 for each initial elements of a GC might form, possibly from a reduced amplification. Shown are mean (6 SEM) measurements of independent samples in two biological replicate experiments, each with three WT number of B cells in which some mTORC1 remained. Notwith- (CreERT2+, Rptor+/+) and knockout (CreERT2+, Rptorf/f). (A) Reduced standing this, across the population, mTORC1 mediated at least a + mRNA encoding AID (Aicda)aswellasBCL6(Bcl6) and IRF4 (Irf4), 10-fold increase of BCL6 in GL7 B cells. When Raptor is de- transcription factors essential for GC B cell differentiation, in Raptor-de- pleted at the onset of B cell activation, the yield of Ag-specific pleted GL7+ Bcells.(B) Reduced mRNA encoding IRF4 in Raptor-depleted memory-phenotype B cells also was reduced (Fig. 3F) to an extent 2 2 memory-phenotype (IgD GL7 CD38hi) B cells. (C) Induction of BCL6 in that exceeds any effect on B cell numbers overall. IgD2 GL7+ B cells required mTORC1. Mice (huCD20-CreERT2+ Rptor +/+, and huCD20-CreERT2+ Rptor fl/fl) were injected with tamoxifen and initially Regulation of Ig1 GLT by mTORC1 and glycolysis in B cells immunized with NP-KLH in alum as in Fig. 2A, boosted with the same There were major defects of the class-switched anti-viral Ab re- amount of NP-KLH in alum 1 wk later, harvested 1 wk thereafter, processed sponse in rapamycin-treated mice, and anti-HA IgM increased due 3 6 as described in the Materials and Methods,followedbycollectionof2 10 to rapamycin in this model (46), and reduced AID levels have been viable, dumpneg events on the flow cytometer. Shown to the left are repre- implicated as a mechanism for these findings (43). In contrast, Ag- sentative flow profiles for intracellular staining of BCL6 in the GL7+ CD95+ gate or IgD+ control gate, as indicated, with B cells that were WT (+/+)or specific IgM production failed within days of hapten-carrier im- Raptor-depleted (D/D), flanked by a bar graph with the mean (6 SEM) mean munization (44). We found in vitro switching to IgG1 was 3-fold fluorescence intensity for BCL6 in the replicate samples (two independent higher when B cells expressed Raptor (Fig. 4A). In addition to an experiments, each with two mice of each genotype). To the right are the effect on Aicda mRNA, however, the induction of Ig1 GLT, which analogous data for intracellular IRF4 in splenic B cells of the same immu- is essential for directing recombination to the switch region for nized mice. (D) mTORC1 dependence of large, neutral amino acid trans- IgG1 (55), was drastically reduced (Fig. 4B). Induction of Aicda porter gene expression in GC B cells. (E) Reduced cell surface CD98hc on gene expression and functional class-switching require time and Raptor-depleted GC-phenotype B cells. Representative and aggregate [itali- cell divisions (6). Even when flow cytometric gating restricted 6 + neg cized mean ( SEM)] flow cytometry results for IgD GL7 B cells of the measurements to B lymphoblasts that had divided several times, immunized mice. For the reduced CD98hc on mTORC1-depleted B cells the fractions of mTORC1-deficient B cells with switched Ab class relative to controls p , 0.01. (F and G) Formation of Ag-specific memory B cells requires mTORC1. (F) Representative flow phenotyping results for (Supplemental Fig. 2C) and their levels of AID expression marked spleens from immunized and boosted huCD20-CreERT2 mice analyzed in by fluorescence of an AID-GFP translational fusion were reduced Fig. 2A–E. Inset and italicized (mean 6 SEM) numbers are as in Fig. 2C. (G) (Fig. 4C, Supplemental Fig. 2D). Of note, AID protein expression Absolute numbers of memory-phenotype and NP-binding memory B cells in was maintained among B cells that divided several times (Fig. the spleens of huCD20-CreERT2 mice, derived from phenotyping in (F). 4C), although the frequency of cells that become GFP-positive The Journal of Immunology 7

Thus, although there still is no in vitro system that accurately recapitulates the GC reaction, data in this model were congruent with those we obtained from in vivo GC-phenotype activated B cells. We conclude that mTORC1 promotes the induction of transcription factors essential for GC B cells, as well as gene targets crucial for Ig class switching. We then explored if there is a connection between mTOR regulation of cellular metabolism and the observed effects of Raptor depletion on class switching. Glycolysis in T cells is promoted by mTORC1 (47, 48). Flux of radiolabel from glucose into water via glycolytic generation of pyruvate was halved when equal numbers of WT and Rptor D/D B cells that had been acti- vated and grown in vitro were assayed (Fig. 4E). These data were supported by an analogous decrease in extracellular acidification rates (Supplemental Fig. 3A). To decrease glycolytic flux exper- imentally, in a manner independent of the known impact of such a reduction on TFH cells, we tested the effect of 2DG on switching by purified B cells and found that it substantially reduced the

frequency of IgG1+ IL-4–treated B lymphoblasts (Fig. 4F) without Downloaded from undermining viability (Supplemental Fig. 3B). Inhibition of gly- colytic flux prevented induction of the Ig1 GLT (Fig. 4G), a proliferation-independent event. The frequency of B cells that became AID-GFP+ also decreased (Supplemental Fig. 3C), as did the activation-induced increases in BCL6 and IRF4 (Supplemental

Fig. 3D), although the magnitudes of these effects were less http://www.jimmunol.org/ striking than those stemming from lack of mTORC1 or the impact of glycolysis inhibition on Ig1. We conclude that mTORC1 de- fects reduce glycolysis, and interference with glycolytic flux as a primary intervention attenuates Ab class switching via a failure to

FIGURE 4. Glycolysis promotes IgCH germ line Ig1 transcription and induce GLT. However, additional mechanisms that regulate levels CSR. (A) Flow cytometric measurements of Ab class switching to IgG1 of AID likely collaborate in the overall reduction. after WT and Raptor-depleted B cells were activated (LPS) and cultured B cell–intrinsic raptor function promotes SHM and with BAFF and IL-4. Shown are the flow plots of samples in one repre- sentative experiment. Italicized mean (6 SEM) values in the three inde- high-affinity Ab by guest on September 26, 2021 pendent replicates [four (WT) and five (Rptor D/D) samples] and inset How B cell–intrinsic mTORC1 influences SHM rates or affinity + + numbers denote the fractions of IgG1 cells in the viable B220 gate for maturation in physiological conditions is unclear. Using the B the particular experiment. ( ) Decreased Aicda and germ line Ig1 tran- tamoxifen-triggered huCD20-CreERT2 system to effect Raptor scripts, measured by quantitative real-time PCR in mTORC1-deficient and depletion in B cells prior to immunization led to lower concen- control B cells 2 d after activation and culture as in (A). (n = 3 independent trations of Ab, particularly of switched isotype (Fig. 5A). Strik- replicate experiments). (C) Decreased AID in Raptor-depleted B lympho- blasts. WT and Raptor-deleted B cells encoding a AID-GFP BAC transgene ingly, the impact on high-affinity Ab (stably bound to low-valency were stained with CellTrace Violet before activation and culture as in (A), NP in the ELISA) was as much as 20-fold greater than that on the followed by flow cytometric analysis. Left panels: A representative division all-affinity Ab (Fig. 5A), so that the NP2/NP20 ratio averaged history (dye partitioning); right: frequencies of GFP+ B cells in the gates for 0.053 control. This result contrasts with a prior finding that B two and four divisions of one representative experiment, with italicized mean lineage–specific inactivation of the gene encoding the kinase (6 SEM) values for the samples in four independent replicate experiments. mTOR resulted in a modest reduction of higher-affinity anti-NP (D) Bcl6 and Irf4 mRNA levels in the samples of (C), measured by quan- Ab relative to all anti-NP, i.e., to ∼0.93 control levels (43). The E titative RT-PCR. ( ) Reduced glycolysis in mTORC1-deficient B lympho- impact of mTORC1 also was evident in measurements of ASC A blasts. Purified B cells, activated and grown as in ( ), were assayed for flux frequencies (Fig. 5B). The decreases in high-affinity Ab, together of 3H from tritiated glucose into water via glycolytic generation of pyruvate. with the reduced AID, prompted us to explore underlying mech- Mean (6 SEM) results from four different samples, distributed equally in two biologically independent experiments. (F)Asin(A) except 2DG was added as anism(s). Affinity maturation involves both the rate of imposing indicated, with data from six independent experiments graphed as individual mutations and a mechanistically distinct process of selecting for values (black or gray circles) along with each mean (horizontal line). (G) coding sequence changes that affect BCR affinity for Ag. Ac- Glycolytic flux supports induction of Ig1 GLT. Results of quantitative real- cordingly, we quantified somatic mutations in noncoding (and time PCR using cDNA made from RNA of B cells cultured in BAFF alone hence nonselected) DNA of the Ig H chains of NP-binding B cells. (resting B cell control) or with IL-4 after LPS activation; each sample was This analysis revealed less frequent noncoding somatic mutations normalized to the BAFF control culture. Shown are individual values from in NP-binding Raptor-depleted B cells (Fig. 5C). These data show F four independent experiments and mean results, displayed as in ( ). that the reduction in mTORC1 limited both AID and the rate of mutation. However, the quantified decrease in mutation rate was reduced. Irf4 and Bcl6 mRNA levels were substantially lower (∼0.53 WT rate) was an order of magnitude less than the loss of in Raptor-deficient B cells after activation (Fig. 4D, Supplemental high-affinity IgG1 anti-NP (∼0.053 WT concentrations). These Fig. 2E), as observed in the freshly sorted GC B cells after im- differences suggest that selective pressure on the B cells is higher munization (Fig. 3A). These defects culminated in a reduction in whereas the mutation rate is lower, so that the combined alterations accumulation of class-switched Ab in vitro (Supplemental Fig. 2F) lead to an Ab response of dramatically lower affinity. Survival and + proportionally similar to the impaired generation of IgG1 B cells. affinity maturation depend on competition for a limiting pool of TFH 8 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY

FIGURE 5. B cell–intrinsic mTORC1 promotes somatic mutation of Ig VH DNA and dramatically enhances affinity maturation. (A) All-affinity (anti-NP20) and high-affinity (anti-NP2) IgM and IgG1 in sera of immunized and boosted, tamoxifen-injected huCD20-CreERT2 mice that were either Rptor+/+ or Rptorf/f (as in Fig. 2A). Shown are the mean (6 SEM) values across serial 4-fold dilutions for the independent mice (n = 8, Cre+ WT; n = 7 Cre+ Rptor D/D), in three separate experiments. (Initial dilutions:

NP20, 1:400; NP2, 1:100.) (B) Mean (6 SEM) frequencies of ASCs quantified as all-affinity (anti-NP20) and high- affinity (anti-NP2) IgM- and IgG1-secreting cells in spleens of the immunized mice of Fig. 2 and (A). (C) B cells from tamoxifen-treated CreERT2+ mice (WT and Rptor D/D) were transferred into mMT recipients that were immunized Downloaded from with NP-KLH. After flow purification of viable NP-bind- ing IgDneg GL7+ B cells, rates of somatic mutation in a noncoding interval of VH186-2 of genomic DNA were scored as in (43). (D) Raptor-deficient B cells fail to sup- port the TFH population. Splenocytes from immunized and boosted mice as in Fig. 2A–E and (A and B) were analyzed by flow cytometry to determine the fraction and number of http://www.jimmunol.org/ + + + TFH (CXCR5 PD-1 ) cells among activated (CD44 ) CD4+ T cells. Graphs display the values measured for each individual mouse, along with means (horizontal bars). by guest on September 26, 2021

cells, and the capacity to sustain a TFH phenotype in CD4 T cells whereas the IgM anti-NP response was normal (Fig. 6B). As requires sustained interactions with GC B cells (4, 5). Thus, it was expected, B cell numbers were normal (Fig. 6C) because only a striking that the B lineage defects after huCD20-CreERT2–driven low fraction of the B cell population would be activated by Rptor gene disruption caused a profound decrease in activated immunization and turn on transcription of the Cg1-Cre allele. CXCR5+ CD4+ T cells (Fig. 5D). These data are consistent with a Nonetheless, the frequencies of GC-phenotype and of NP- model in which mTORC1-deficient B cells provide insufficient binding B cells among them were reproducibly reduced + support for stabilization of a TFH program among cognate CD4 (Fig. 6D). The prevalence of TFH-phenotype T cells also was T cells with which they interact. lower after Cg1-Cre–driven Raptor depletion in vivo (Fig. 6E), Because results in these and earlier studies might in part be due consistent with both the results of experiments with huCD20- to limitations of the model systems, we used variant approaches. CreERT2 and the characteristics of each means of deleting the First, a hapten facilitates detection and semiquantitation of af- targeted Rptor gene segment. Because the Cg1-Cre transgene finity maturation in the overall Ab population, but the nature of acts only after activation and proliferation of B cells, these data the Ag potentially could influence results. We found here that indicate that 1) B cell–intrinsic mTORC1 is required even mTORC1 depletion had a similar effect on concentrations of after initial B cell activation, and 2) the results are not a function class-switched anti-KLH and anti-NP Ab after immunizations of of either the adoptive transfer system or the immune effects of T2 the huCD20-CreER mice: IgG1 anti-KLH was ∼30-fold higher tamoxifen. in sera of controls than in the mice whose B cells were Raptor- deficient, whereas IgM only decreased to ∼0.53 control levels B cell–intrinsic mTORC1 inhibits heterosubtypic Ab (Fig. 6A). Second, a potential concern with any work in which generation after influenza immunization the signaling pathway is eliminated first is that the loss-of- To evaluate B cell–intrinsic function(s) of mTORC1 in immune function might yield an abnormal population of cells prior to memory using an immunization model incapable of generating immunization, or that results might be due to a defect arising at new B cells, we used chemical activation of Cre-mediated Rptor the time of initial B cell activation. Moreover, tamoxifen has gene deletion followed by transfers of the resultant B cells into strong effects on humoral immunity (56, 57). Thus, it was pos- B cell–deficient mice (Fig. 7A). Recipient mice were then im- sible that either this strong nuclear hormone receptor ligand or munized by inoculation with one strain of influenza (A/PR8/34; the transfer model would somehow not be representative of H1N1) at a nonpermissive site, followed 6 wk later by intranasal physiological requirements. However, with Cg1-Cre mice that inoculation with a recombinant influenza (FLU) strain of heter- were Rptor +/+ or f/f, all-affinity IgG1 was ∼1/64 of the control ologous surface type (A/ X-31; H3N2). Based on reports of concentrations and no high-affinity anti-NP IgG1 was detectable rapamycin-resistant mTORC1 (39), we tested heterozygous (D/+) The Journal of Immunology 9

FIGURE 6. Raptor depletion causes re- duced switching and affinity maturation in hapten- or tamoxifen-independent systems. (A) Relative concentrations of anti-KLH Ab were measured by ELISA using sera from the eight mice immunized with NP-KLH for analyses of Fig. 5A. Shown are mean (6 SEM) ELISA results across serial 4-fold dilutions (1:50–1:12,800). After screening statistical significance by ANOVA, the null hypothesis was tested pairwise and achieve the indicated p values for IgG1 in samples at the dilutions 1:80, 1:200, and 1:800. (B–E)Cg1-Cre knock-in mice (Rptor +/+ or f/f, six of each genotype distributed equally in two independent experiments) were im- munized as in Fig. 2A. Shown are sero- Downloaded from logical analyses (B) as well as B cell numbers (C), flow staining to measure the frequencies of B cells of GC phenotype (D), or (E)TFH cells as in Fig. 5D. Shown in (B) are ELISA determinations of circulating anti-NP Ab (all affinity, left panels; high

affinity, right panels) across serial 10-fold http://www.jimmunol.org/ dilutions (1:10–1:108), with each point representing mean (6 SEM) data for the individual samples (six WT, six mutant). After screening the overall curves by ANOVA, statistical significance was tested pairwise and yielded the indicated p values for samples at the dilutions 1:10 through 1:104.(D and E) Shown are flow data from a representative mouse of each genotype in one experiment, with inset numbers and by guest on September 26, 2021 italicized means (6 SEM) as in Fig. 2C.

B cells as well as homozygous loss-of-function. Measurements of the immunogen subtype, H1. Strikingly, Rptor haplo-insufficiency the Abs were performed both with virus lysates and with reduced but did not eliminate Ag-specific IgG1 whereas IgG2c recombinant strain-specific HA proteins, which are surface dis- anti-H1 was almost completely eliminated. After a period for played and a major target of protective Abs (58). The post- formation of memory, inoculation with a different influenza sub- challenge response as measured with virus lysates demonstrated type at a site permissive for replication rapidly stimulated anti-HA that Raptor depletion caused modest increases in IgM anti- responses in which the IgM anti-H1 levels derived from Raptor- influenza concentrations postinfection to elicit Ab responses at a depleted B cells were higher than for the WT controls (Fig. 7C, memory phase after immunization (Fig. 7B). Consistent with left panel of upper row). Although little IgG1 or IgG2c anti-H1 Ab systemic rapamycin effects, major reductions in the anti-viral was detected (Fig 7C), anti-H3 reactivities were elicited by in- IgG1 and IgG2c were observed in mice whose B cells were tranasal X-31 inoculation. Switched and IgM responses were ro- mTORC1 deficient (Fig. 7B). Type-specific Ab responses were bust for controls, whereas Rptor D/D B cells yielded only IgM measured at three stages: 1) the primary response (3 wk), 2) a production (Fig. 7C, lower row of panels). However, the second- prechallenge estimate of more durable Ab concentrations, and 3) ary virus challenge elicited substantial anti-H3 IgG1 from haplo- postchallenge at a time point (1 wk postinoculation) chosen to bias insufficient Rptor D/+ B cells akin to the primary anti-H1 Ab the measurement of class-switched Ab toward those derived from response (Fig. 7C, middle panels). The strength of haplo-insufficiency recall responses of memory B cells. The primary IgM response to in recall humoral immunity was intriguing in that, at most, a the immunizing H1 strain (PR8) was robust and not reduced by modest effect was noted if Raptor was present during a primary mTORC1 inactivation (Fig. 7C, upper row panels). Ab directed immunization and only then was one allele inactivated (44). The against the X-31 HA (H3) were virtually undetectable in the pri- straightforward basis for such findings would be that the integrity mary response derived from WT B cells (Fig. 7C, lower row of mTORC1 is more critical during B cell activation and the es- panels). However, Raptor-depleted B cells yielded a low but re- tablishment of memory than once a memory B cell population is producible and statistically significant increase in IgM that bound in place. However, extensive direct comparisons will be needed to HA of the X-31 challenge strain (Fig. 7D). Complete loss of test this model. Notwithstanding this, we conclude that graded Raptor eliminated the primary IgG1 and IgG2c responses against reductions in B cell–intrinsic mTORC1 progressively eliminated 10 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY

FIGURE 7. mTORC1 level programs the spectrum of anti-influenza Ab in primary and recall responses. (A) Schema of adoptive transfer, immunization, and respiratory challenge experiments to test B cell– intrinsic function of mTORC1 in humoral responses to influenza virus. Donor B cells were harvested from Rosa26-CreERT2 mice with WT or flox alleles of Rptor after tamoxifen injections as in Fig. 1A. (B) Mean (6 SEM) ELISA results on sera 1 wk after PR8-immu- nized mice were challenged with intranasal X-31 strain influenza virus, using lysates of X-31 virions to capture anti-FLU Ab in sera from mice whose B cells were WT, haplo-insufficient for Rptor, or homozygous for the deleted Rptor allele. Isotype-specific ELISA results after serial 2-fold dilutions starting at 1:50 serum di- lution. For sera of WT versus Rptor-depleted B cell recipients, p # 0.001 by ANOVA calculated for each of the three isotypes as specified in the Materials and Downloaded from Methods, followed by pairwise comparisons at single dilutions. (C)Mean(6 SEM) results of ELISA per- formed with recombinant HA proteins to capture anti-H1 (PR8) and anti-H3 (X-31) Ab of the indicated isotypes, using sera (1:100 dilutions) from the mice in (B) after identification of serum dilutions in the linear range of the assay. Data capture the sera from individual recip- http://www.jimmunol.org/ ient mice for each genotype of donor B cells (eight Rptor +/+, nine Rptor D/+, and nine Rptor D/D, each distributed evenly among three independent experi- ments). (D) Titrations for cross-reactive IgM anti-H3 in the primary response to immunization with A/PR8, separating panels comparing D/+ (left) and D/D (right) to the same WT controls for clarity. Significance testing was performed using two-way ANOVAwith Bonferroni correction for multiple comparisons, followed by test- by guest on September 26, 2021 ing the null hypothesis for pairwise comparisons at 1:25 dilutions.

the Ig class switch while reprogramming the repertoire of B cell humoral responses after vaccination, during and after an infection, responses. Consequently, the primary response yielded a modest or in disease due to autoantibodies. Previous work has provided amount of heterosubtypic IgM Ab against the HA of the X-31 evidence that mTOR, mTORC1, and mTORC2 promote humoral challenge strain (H3) and an increase in the IgM anti-H3 upon immunity. Several recent papers underscored T cell–intrinsic specific challenge. functions of mTORC1 impact TFH phenotype and functions (47– Using this nonlethal challenge inoculum, we also explored how 49). Other findings suggest that both mTORC2 (32) and mTORC1 much protection against illness resulted from the cross-reactive exert cell-intrinsic functions in B lineage ontogeny and function (X-31, or H3-binding) IgM response to immunization with A/PR8 (42–44, 59). However, crucial unanswered questions remain about influenza. At most, subtle differences were observed in the lung the mechanisms for these findings, and the interplay between pathology, amount of weight loss, and the number of infiltrating cells mTORC1 and metabolism in the B cell contribution to Ab regu- recovered from lungs 1 wk after intranasal challenge (Supplemental lation. We have shown in this study that following depletion of Fig. 4A–C). Modest differences suggestive of some protective effect Raptor, an essential component of mTORC1, in B cells, inclusion were not statistically significant, and the protection was at most very of activated B cells in GC is drastically reduced when the analyses modest. Activated T cells were in the inflamed lungs in similar involve physiological frequencies of Ag-specific cells in the naive numbers (Supplemental Fig. 4D–G). Consistent with evidence in repertoire and immunization. Furthermore, mTORC1 was crucial the protein immunization systems (Figs. 1–6), however, lack of for normal induction of the GC-determining transcription factor mTORC1 reduced the populations of GC and memory-phenotype BCL6. AID expression was increased when this signaling pathway B cells in spleens of the mMT recipients (Supplemental Fig. 4H, 4I). was active, accompanied by increased rates of somatic mutations. We conclude that a B cell–intrinsic defect of mTORC1 liberated Thus, one critical function of mTORC1 in B cells is to support production of heterospecific anti-HA Ab, but that the resultant hu- acquisition of the full functionality of lymphocytes whose acti- moral memory was insufficient for any substantial protective effect. vation sufficed for loss of IgD and gain of GL7 expression. Ulti- mately, perturbations of mTORC1 that were restricted to B cells Discussion sufficed to alter the nature of antiviral Ab specificities. Moreover, Elucidation of extrinsic and cell-intrinsic regulators of Ab gen- our studies connect mTORC1 effects on class switching to in- eration and qualities is central to the potential to manipulate creased glycolysis in B cells. Thus, in a setting independent from The Journal of Immunology 11

TFH contributions, mTORC1 promoted glycolytic flux of TLR- mTORC2 could fit with observed increases in switched Ab after activated B cells. Strikingly, reduced glycolytic flux limited the using catalytic site inhibitors of mTOR (42). Closer to mTORC1, capacity to undergo Ab class switching, express AID, and induce a inactivation of Tsc1 via Cd19-Cre prior to full B cell maturation GLT crucial for IgH switch region targeting by AID. Collectively, caused sustained increases in mTORC1 activity and develop- these findings are consistent with a capacity of the metabolic mental defects of B cells along with a mild defect of CSR and Ab programming—and perhaps microenvironments deficient in responses (65). More recently, although the effect on develop- nutrients—to impact the qualities of humoral immunity. mental progression was not reported, Tsc1 inactivation via mb1- Our data reveal that at physiological precursor frequencies and Cre also increased mTORC1 activity (as expected) and under- with Ag-carrier immunization akin to vaccination, mTORC1 in mined affinity maturation, whereas a separate gain-of-function B cells strongly promotes affinity maturation and the primary mutant in RRaga confined B cells to the DZ but did not alter humoral response as well as recall Ab production. The underlying VH somatic mutation rates (63). Subject to a caveat from the likely frequency of somatic mutation was halved among the reduced pool abnormalities of B cell development, these findings suggest that of NP-binding GC-phenotype B cells when Raptor was depleted. excess mTORC1 undermines CSR and affinity selection but not Although absolutes rarely apply in immunity, the somatic muta- SHM per se. Taken together, sustained increases in mTORC1 may tions of Abs are a fundamental property of the GLT reaction (3, 4, reduce rates of somatic mutation but our data indicate that de- 60). Accordingly, it is intriguing to consider how this reduction creases in mTORC1 activity undermine SHM in a physiological may relate to the dynamics of GC reactions. CXCR4-dependent repertoire in a primary response to immunization. iterative shuttling of B cells between the GC dark and LZs is a Ultimately, an important aspect of dissecting these mechanisms prominent feature of these dynamics (3, 4, 20–22), and although is to assess how they affect immunization, infection, or autoim- Downloaded from Aicda mRNA levels are on average at most 2-fold different be- mune processes. Rapamycin in vivo can limit autoantibody pro- tween DZ and LZ B cells (61, 62), it is thought that mutations are duction in mouse models, as can a combination of 2DG and imposed in the proliferating B cells of the DZ. Inactivation of metformin (44, 66, 67). These rapamycin effects involve de- CXCR4 gene expression in B cells disrupted shuttling and trapped creased plasma cell generation and function (42, 44). In addition B cells in the DZ. Quantitation of SHM in Cxcr4-deficient GC- 2DG dramatically reduces the efficiency of plasma cell differen-

phenotype B cells after influenza infection, i.e., in the absence of tiation (J. Li, A.L. Raybuck, and M.R. Boothby, unpublished http://www.jimmunol.org/ iterative LZ/DZ shuttling, about a 50% reduction (22). This effect observations). In this regard, 2DG reduced IgG1 in media after size matches what we have determined for mTORC1-deficient anti-IgM–activated B cells were provided help from mixtures of B cells. Intriguingly, work in parallel to our studies found that follicular T cells (TFH and follicular regulatory cells) ex vivo (68). rapamycin inhibited the movement of transferred B1-8 B cells Our findings suggest that this includes a B cell–specific effect from light to the DZs of GC in mice immunized prior to the involving switching. One of the more striking findings has been transfer (63), but the effects of this intervention on mTORC2, that rapamycin treatment during a primary immunization led to somatic mutation, and affinity of the resulting Ab are not clear. It dramatic cross-protection against lethal challenge by an HA- is tempting to relate the elegant findings in this transfer system to disparate influenza virus notwithstanding the virtual elimination the results of B cell–restricted Raptor depletion. One caveat, of GCs (46). CD4+ cells as well as B cells needed to be present for by guest on September 26, 2021 however, is that with a physiological Ag-specific B cell repertoire this protective effect, dendritic cell function also is rapamycin- prior to immunization, depletion of B cell mTORC1 via Cg1-Cre sensitive (39), and recent findings have highlighted a require- expression undermined B cell support for the TFH population. ment for mTORC1 in TFH cell development and function (47–49). Moreover, Raptor-deficient B cells yielded a drastically reduced Evidence in this study, generated with genetic approaches, shows number of Ag-specific GC phenotype B cells, and GC-like that the block to forming class-switched Abs against HA gener- structures with few B cells. Thus, SHM was reduced in Raptor- ated by loss of mTORC1 is B cell intrinsic. Moreover, although and CXCR4-deficient B cells to a quantitatively similar degree. the use of transfers into B cell–deficient recipients means that However, understanding the relative contributions of shuttling there would be an element of homeostatic proliferation, the results versus impaired GC functionality due to mTORC1-deficiency of provide support for a licensing of the emergence of IgM clones Ag-specific B cells under physiological conditions awaits further specific for epitopes on an HA-subtype differing from the im- studies. Our data indicate that mTORC1 integrity promotes higher munogen and definitively map this to the B cell. Indeed, our ev- concentrations of high-affinity Ab to a greater extent than the idence indicates that even a partial restriction of mTORC1 activity effect on SHM. This disparity points to more stringent selection, restricted to B cells sufficed to increase cross-protective anti-HA which appears to differ from the finding that a ubiquitously IgM. However, the durability of the humoral memory and extent expressed hypomorphic mTOR led to a very modest affinity re- of protection against disease are not clear, and probably need to duction quantitatively similar to a decrease in JH4 SHM to ∼0.6 3 invoke rapamycin’s effects on mTORC2 as well as mTORC1 in WT frequency (43). follicular T cells and others. Indeed, IgM memory B cells persist Our data from immunization experiments in vivo confirm and for extended periods yet the cross-reactive anti-HA Ab concen- extend earlier evidence that loss of Raptor decreased switching to trations had declined within a month, so it may be that a sub- IgG1 in vitro (42). However, earlier work had indicated that hy- stantial fraction of the response was extrafollicular or derived peractivity of PI3K suppressed CSR as well as Aicda gene ex- from shorter-lived plasma cells (26). In any event, CD4 T cells pression (64). PI3K actions in immunity in vivo may mostly be were required for the protective effect on survival upon influenza attributable to mTORC1 and mTORC2 because disease resulting challenge in the rapamycin-treated immunization model, as were from constitutive PI3K activity operates primarily along rapamycin- B cells (46). Our work indicates that a haplo-insufficient B cell– sensitive pathways (33). Thus, increased PI3K had, on the sur- autonomous, mTORC1-specific mechanism suffices to reduce face, the same effect as a block of mTORC1 activity. Although switching and liberate the emergence of heterosubtypic anti-HA mTORC2-depleted B cells appeared to switch normally in vitro IgM. However, these Ab did not improve the clinical outcome. (32), some of this apparent paradox might be due to mTORC2 or These findings suggest that the protective effect required rapa- other signaling resulting from sustained increases in phosphatidyl- mycin action on the CD4 T cells, and perhaps also dendritic cells, inositol 3, 4, 5-triphosphate. For instance, a suppressive role of during and after immunization. 12 B CELL–INTRINSIC mTORC1 PROMOTES BCL6, IRF4, SHM, AND AFFINITY

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