An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry

Accepted Manuscript An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry

Nicholas A. Zwang, Balaji B. Ganesh, Kim T. Cardenas, Anita S. Chong, Patricia W. Finn, David L. Perkins PII: DOI: Reference:

S0022-1759(18)30171-6 doi:10.1016/j.jim.2018.10.002 JIM 12527

To appear in:

Journal of Immunological Methods

Received date: Revised date: Accepted date:

7 May 2018 10 July 2018 3 October 2018

Please cite this article as: Nicholas A. Zwang, Balaji B. Ganesh, Kim T. Cardenas, Anita S. Chong, Patricia W. Finn, David L. Perkins , An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry. Jim (2018), doi:10.1016/ j.jim.2018.10.002

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ACCEPTED MANUSCRIPT TITLE. An optimized protocol to quantify signaling in human transitional B cells by phospho flow cytometry. AUTHOR NAMES AND AFFILIATIONS. Nicholas A. Zwang MDa, Balaji B. Ganesh PhDb, Kim T. Cardenas PhDc , Anita S. Chong PhDd, Patricia W. Finn MDe, David L. Perkins MD PhDa a

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Division of Nephrology. Department of Medicine, the University of Illinois at Chicago. 820 South Wood Street (MC 793). Chicago, IL 60612 b Director, Flow Cytometry Core. The University of Illinois at Chicago. Medical Science Building, 835 South Wolcott Avenue (E-25C). Chicago IL 60612 c Technical Application Scientist, BioLegend. 9727 Pacific Heights Blvd. San Diego, CA 92121 d Department of Surgery. Section of Transplantation Surgery, the University of Chicago. 5841 South Maryland Avenue (SBRI J547/MC 5026). Chicago, IL 60637 e Department of Medicine, the University of Illinois at Chicago. 840 South Wood Street Suite 1020N (MC 787). Chicago, IL 60612

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CORRESPONDING AUTHOR. Nicholas A. Zwang MD ([email protected])

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ABSTRACT

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Background and purpose Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets.

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Results TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38-), and late memory (CD24-CD38-) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation.

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Conclusions We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies. KEY WORDS. Immunobiology. Flow cytometry. Signaling. B cells.

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ABBREVIATIONS EM Early memory Erk Extracellular signal regulated kinase LM late memory MAPK mitogen-activated protein kinase NF-κB nuclear factor-kappa B PMA phorbol 12-myristate 13-acetate PMT photomultiplier tube TrBs Transitional B cells

ACCEPTED MANUSCRIPT 1. INTRODUCTION

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Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations.[1, 2] This technique is particularly useful when traditional immunoblotting is unfeasible due to cell scarcity in patient samples.[3] Unlike traditional immunoblotting, phospho flow cytometry allows for quantification of signaling among multiple subsets within the same sample. This technique, however, typically requires harsh conditions for cell fixation (paraformaldehyde) and permeabilization (methanol), which can denature surface antigens or antibody-conjugated fluorochromes.[4] Less harsh, saponin-based permeabilization may be suitable for labeling of some phosphorylated targets.[5] Methanol-based permeabilization, however, is more universally effective for analysis of intracellular signaling.[6]

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Assessment of signaling by phospho flow cytometry generally involves labeling of surface epitopes following permeabilization.[7-9] Unfortunately, methanol-based permeabilization degrades many cell surface epitopes essential for immune subset identification. One such epitope is CD19. For this reason, many protocols rely on CD20 rather than CD19 to identify B cells flow cytometrically.[10-12] CD20 expression, however, may be variable in early B cell development[13] and lost in late B cell maturation[14], thus CD19 is preferable as a pan-B cell marker. One approach to preserve flow cytometric identification of markers such as CD19 is to label cells prior to stimulation[15, 16] or even after stimulation but before fixation.[17] Conceivably, however, antibodies used for labeling could be stimulatory or inhibitory thus labeling after fixation would be preferable. Another approach is to sort or enrich cells prior to stimulation.[18, 19] This approach has several disadvantages, including additional cost, manipulation that could alter signaling, and inability to compare signaling from different subpopulations from one sample. Finally, sequential labeling has been recommended for epitopes degraded by methanol.[20] This latter approach has not been employed widely. Potential concerns include disruption of antibody-antigen binding by methanol and degradation of fluorochrome conjugates.

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Technical limitations have been a barrier for quantification of signaling in certain unique B cell subsets. Recent studies have elucidated the importance of a unique population of B cells with suppressive function in kidney transplantation.[21-24] Such “regulatory” B cells with a transitional phenotype (TrBs) suppress autoimmunity in mouse models partly through elaboration of anti-inflammatory cytokines such as interleukin 10 (IL-10).[25, 26] An unanticipated but consistent marker of kidney allograft tolerance in clinical studies is an enhanced B cell gene signature, prompting a hypothesis that B cells, and specifically regulatory B cells, play a critical role modulating graft survival.[27-29]. Though low in overall frequency, deficient numbers of TrBs correlates with antibody-mediated rejection.[16, 30-32] Abnormalities of TrBs also have been found in autoimmune diseases such as lupus,[33] anti-neutrophil cytoplasmic antibody (ANCA) vasculitis,[34] graft-versus-host disease (GVHD),[35, 36] and even aging.[17] B cell depletion therapies like rituximab may paradoxically promote rejection by depleting this regulatory cell population.[37-40] Finally, kinetics of TrB repopulation may have important prognostic implications for response to depleting immunotherapy.[41] High numbers of TrBs in rejection-free patients may reflect either a skewing towards a TrB phenotype or a failure to differentiate into mature B cell subsets as a result of clinical immunosuppression. In this vein, TrBs do not appear to be an ontologically distinct cell population but rather a developmental step in B cell differentiation.[42, 43] Activated TrBs not only suppress alloreactivity [25] but can mature to naïve B cells, whereupon antigen encounter promotes differentiation to memory B cells and plasmablasts.[43]. It is possible that cell-intrinsic differences among B cell subsets generate different effects from the same stimuli. For example,

ACCEPTED MANUSCRIPT B cell receptor (BCR)-mediated antigen stimulation in TrBs may induce apoptosis and clonal deletion[44], whereas in mature B cells, BCR-signaling induces activation, clonal expansion and differentiation into memory B cells and plasma cells.[45] Therefore the effects of BCR signaling in both TrB and mature alloreactive B cells would be undesirable in transplantation (or, conversely, desirable in clinical oncology).

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The mechanisms by which human TrBs are activated to exert regulatory activity, differentiate away from this phenotype, or to undergo BCR-mediated apoptosis, are not defined. However, T cell help via CD40/CD40 ligand (CD40L) interactions may promote different signaling and cellular consequences in TrB compared to mature B cell subsets.[18] For example, disrupted TrB STAT3-mediated activation following exposure to T cell-derived stimuli has been implicated in lupus[33] and GVHD.[36] Interestingly, experimentally generated murine tumor-derived Bregs demonstrating constitutive STAT3 activity fail to suppress breast cancer metastasis.[46] Clearer understanding of the signaling mechanisms responsible for TrB activation and maturation could yield key insights to diagnose and treat not only rejection in transplantation but many immunemediated diseases.

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Here we have optimized a protocol to quantify phospho-signaling in human TrBs by flow cytometry. TrBs were defined flow cytometrically as CD19 +CD24hiCD38hi [33, 47-49] and distinguished from mature B cell subsets based on CD24 and CD38 expression. With this approach, TrBs were identified flow cytometrically among a mixed population of peripheral blood mononuclear cells (PBMCs), allowing comparison with mature B cell subsets from the same sample. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to fixation and permeabilization buffers, photomultiplier tube (PMT) voltages (particularly for the surface marker CD38), and compensation. The optimized protocol reliably distinguished TrBs from naïve, early memory, and late memory CD19 + B cells. Finally, normal human TrBs showed diminished phosphorylation of several key signaling molecules following nonspecific stimulation in vitro.

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2. MATERIALS AND METHODS

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Peripheral blood mononuclear cells De-identified normal human donor buffy coat preparations were obtained with Institutional Review Board approval (2016-1163) from LifeSource. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient and frozen as aliquots (in fetal bovine serum containing 10% dimethyl sulfoxide) at -196oC. For experiments, PBMCs were rapidly thawed into pre-warmed Phosphate Buffered Saline without calcium or magnesium; there was recovery of 99% viability (Supplementary Figure 1). Cells were counted using a Bio-Rad TC20 automated cell counter and used at 4x105 cells/test. Antibodies Fluorochrome-conjugated antibodies to surface epitopes and clones are listed as follows and were tittered to 4x105 cells/test: CD3 (UCHT1) Brilliant Violet 510; CD19 (HIB19) Brilliant Violet 605; CD24 (ML5) Brilliant Violet 421; and CD38 (HIT2) FITC or PE-Cy7. Additional clones tested are listed as follows: CD3 (OKT3), CD19 (SJ25C1), and CD38 (HB-7, REA671). We also tested IgD (both goat polyclonal and IA6-2) and CD20 (2H7) but found inadequate separation of CD20+ from CD20- lymphocytes. Fluorochrome-conjugated antibodies to intracellular phosphorylated epitopes and clones are listed as follows and used undiluted: ERK 1/2 pT202/pY204 (6B8B69) AlexaFluor 488 (also tested PE and PerCP-Cy5.5); NF-κB p65 pS529 (B33B4WP) PE; p38 MAPK pT180/pY182

ACCEPTED MANUSCRIPT (36/p38) APC (also tested PerCP-Cy5.5); and Akt pS473 (M89-61) AlexaFluor 647. Other fluorochrome-conjugated antibodies tested included: pan-tyrosine (PY20) APC and FITC; S6 pS235/pS236 (cupk43k) PE and APC; Akt pS473 (SDRNR) APC; and STAT3 pY705 (13A3-1) APC.

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Stimulation Thawed PBMCs were aliquoted into round-bottom polystyrene tubes and rested at 37oC in 100 µL RPMI media containing 10% fetal bovine under 4-5% CO2 for 30 minutes prior to stimulation. Samples were then stimulated in a 37oC water bath with an equal volume of the same media containing phorbol 12-myristate 13-acetate (final concentration 5 ng/mL) and ionomycin (final concentration 0.5 μmol/L).

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Fixation, labeling, and permeabilization Following stimulation, samples were immediately fixed with an equal volume of pre-warmed Fixation Buffer (BioLegend, 420801) to halt further signaling. Samples were centrifuged at 350 x g and washed twice in FACS buffer (0.5% bovine serum albumin in phosphate buffered saline without calcium or magnesium). Surface epitope labeling was performed in approximately 100 µL FACS buffer in the dark at room temperature for 60 minutes. They were then washed twice. For permeabilization, samples were mixed by pipetting up and down prior to the addition of 400 µL pre-chilled TruePhos™ Perm Buffer (BioLegend, 425401). Samples were vortexed and then incubated at -20oC for 60 minutes. Thereafter, samples were centrifuged at 1,000 x g and washed twice in FACS buffer. Labeling with phospho-specific antibodies was performed in approximately 100 µL FACS buffer in the dark at room temperature for 60 minutes. Finally, samples were washed twice in FACS buffer prior to flow cytometric analysis.

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Flow cytometry Data were collected on a 4-color (488 nm blue, 638 nm red, 405 nm violet, and 561 nm yellowgreen) LSR Fortessa (BD, USA). Final PMT voltages were as follows: (485V FITC, 425V PE, 575V PerCP-Cy5.5, 595V PE/Cy7, 545V APC, 447V Brilliant Violet 421, 425V Brilliant Violet 510, and 430V Brilliant Violet 605.

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Data analysis and compensation controls Flow cytometric data were analyzed using FlowJo (TreeStar, Ashland, OR). The same compensation controls were consistently applied to all quantitative analyses of signaling. Compensation controls for surface markers were singly-labeled cells: CD38 (PE/Cy7 channel) or CD3 (Brilliant Violet 421, Brilliant Violet 510, and Brilliant Violet 605 channels). For phosphospecific markers, compensation controls were OneComp compensation beads (Thermo Fisher, USA) for the FITC, APC, and PerCP-Cy5.5 channels. The compensation control for the PE channel was singly labeled cells (CD3) due to off-scale brightness of labeled beads. To account for autofluorescence, unlabeled cells were used as negative controls in setting cytometer PMT voltages. The final compensation matrix was consistently applied to all signaling analyses. Cells were gated for lymphocytes and singlets. Numerical data were analyzed with Microsoft Excel. Statistical analyses were performed using paired one-tailed Student’s t-Tests. Statistical significance for p<0.05 was noted. Graphs were created with Prism GraphPad (La Jolla, CA). 3. RESULTS 3.1 Surface epitope identification is preserved after paraformaldehyde-based fixation but not methanol-based permeabilization Initially we aimed to avoid labeling live cells prior to stimulation and fixation for concern that labeling itself could activate or inhibit intracellular signaling, and tried to label desired surface

ACCEPTED MANUSCRIPT epitopes following methanol-based permeabilization. Unfortunately, preservation of epitope labeling after exposure to methanol was highly variable.

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Essential markers to identify different B cell subsets included CD3, CD19, CD24, and CD38. We also tested a goat polyclonal IgD antibody as described by others.[12] IgD labeling by a goat polyclonal antibody was preserved after fixation but not permeabilization; labeling with a mouse monoclonal antibody was lost after fixation (Supplementary Figure 2). Ultimately, we chose to focus on CD24 and CD38 to identify B cell subsets as this schema is more commonly employed. Additionally, commercially available fluorochrome conjugates to goat anti-human IgD are FITC, PE, and APC (or equivalent Alexa Fluor fluorochromes), which we reserved for phospho-specific antibodies. We tested different antibody clones, as epitope labeling may be preserved by some but not other clones. We found that CD3 (not shown), CD19, CD24, and CD38 antibody labeling was poorly preserved when labeling was performed after permeabilization (Figure 1). Epitope labeling was well preserved when performed after fixation but before permeabilization. Labeling of CD38 in particular was best performed with a bright fluorochrome such as PE/Cy7 to distinguish CD38-, CD38+, and CD38hi labeling. Even at a high PMT voltage, the fluorochrome FITC was not bright enough for optimal labeling of CD38. To our surprise, CD19 and CD24 labeling appeared enhanced when performed after fixation compared with labeling of live cells and subsequent fixation. Further investigation revealed that paraformaldehyde-based fixation disrupted nonspecific monocyte binding of these antibodies, therefore increasing available antibody for specific lymphocyte labeling (Supplementary Figure 3). This difference importantly affected labeling since antibodies were used at tittered rather than saturating concentrations.

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FIGURE 1. Surface epitope identification is preserved after paraformaldehyde -based fixation but not methanol-based permeabilization. Key B cell markers included CD19, CD24, and CD38. Normal human peripheral blood mononuclear cells (PBMCs) were labeled live then subject to paraformaldehyde-based fixation (red); fixed then labeled (blue) or fixed, subject to methanol-based permeabilization, and then labeled (orange). Comparison was made with fixed, unlabeled cells (gray). Top panel. CD38 was best identified when labeled with a PE/Cy7conjugated antibody (single-antibody labeling). The same clone and antibody concentration conjugated to FITC yielded less distinct labeling, even at a high photomultiplier tube (PMT) voltage. CD38 identification was preserved when labeling was performed after fixation (indicated with a star) but not after permeabilization. Bottom panel. CD19 (singly-labeled) and CD24 (colabeling after fixation with CD19 conjugated to APC, for which there was no compensation to or from Brilliant Violet 421) identification were preserved with labeling after fixation but not permeabilization. Identification of CD19 and CD24 actually appeared enhanced with labeling after fixation. Subsequent investigation (Supplementary Figure 2) revealed monocyte binding of these antibodies in un-fixed (but not fixed) cells. Since all antibodies used were titrated to labeling of fixed cells, this phenomenon resulted in less antibody to bind PBCMs when labeling was performed in live cells prior to fixation. FIGURE 1. Surface epitope identification is preserved after paraformaldehyde -based fixation but not methanol-based permeabilization. Key B cell markers included CD19, CD24, and CD38. Normal human peripheral blood mononuclear cells (PBMCs) were labeled live then subject to paraformaldehyde-based fixation (red); fixed then labeled (blue) or fixed, subject to methanol-based permeabilization, and then labeled (orange). Comparison was made with fixed, unlabeled cells (gray). Top panel. CD38 was best identified when labeled with a PE/Cy7conjugated antibody (single-antibody labeling). The same clone and antibody concentration

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conjugated to FITC yielded less distinct labeling, even at a high photomultiplier tube (PMT) voltage. CD38 identification was preserved when labeling was performed after fixation (indicated with a star) but not after permeabilization. Bottom panel. CD19 (singly-labeled) and CD24 (co-labeling after fixation with CD19 conjugated to APC, for which there was no compensation to or from Brilliant Violet 421) identification were preserved with labeling after fixation but not permeabilization. Identification of CD19 and CD24 actually appeared enhanced with labeling after fixation. Subsequent investigation (Supplementary Figure 2) revealed monocyte binding of these antibodies in un-fixed (but not fixed) cells. Since all antibodies used were titrated to labeling of fixed cells, this phenomenon resulted in less antibody to bind PBCMs when labeling was performed in live cells prior to fixation.

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3.2 Methanol-based permeabilization yields an acceptable degree of fluorochrome degradation and affects spillover relevant for compensation. Following the determination that labeling of desired surface epitopes could be preserved when performed after fixation but before permeabilization, we next assessed the extent to which fluorochrome conjugates themselves were degraded by a methanol-based permeabilization buffer. We reserved FITC, PE, and APC for labeling phosphorylation of intracellular targets (i.e., labeling following permeabilization). Figure 2A demonstrates an acceptable degree of fluorochrome degradation after PE/Cy7 and Brilliant Violets 421, 510, and 605 were exposed to the methanol-based permeabilization buffer. PerCP-Cy5.5 was completely degraded by this buffer (data not shown). Importantly, permeabilization buffer-induced degradation affected fluorochrome signal spillover relevant to compensation. This is illustrated in Figure 2B. For the Brilliant Violet dyes, diminution of fluorochrome intensity corresponded with diminution of spillover into other channels. For PE/Cy7, however, exposure to methanol-based permeabilization actually increased spillover into the PE channel. There was no appreciable effect of permeabilization buffer-induced degradation on tandem breakdown.

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FIGURE 2. Methanol-based permeabilization yields an acceptable degree of fluorochrome degradation and affects spillover relevant for compensation A) Methanol-based permeabilization yields an acceptable degree of fluorochrome degradation. Singly-labeled compensation beads were labeled with antibodies used for B cell subset identification (gray). A duplicate set was subject to methanol-based permeabilization (red). There was an acceptable degree of signal intensity diminution. B) Methanol-based permeabilization increases PE/Cy7 spillover into PE. As in 2A, compensation singly-labeled beads were labeled with antibodies used for B cell subset identification (gray). A duplicate set was subject to methanol-based permeabilization (red). Labeling for the indicated fluorochrome was potted against the channel into which spillover was highest. For Brilliant Violet 421, 510, and 605, there was less spillover into Briliant Violet 510, Brilliant Violet 605, and PE, respectively, with diminution of signal intensity. In contrast, spillover of PE/Cy7 into PE actually increased. There was no appreciable increase in tandem breakdown of PE/Cy7 after exposure to methanol-based permeabilization.

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FIGURE 2. Methanol-based permeabilization yields an acceptable degree of fluorochrome degradation and affects spillover relevant for compensation A) Methanol-based permeabilization yields an acceptable degree of fluorochrome degradation. Singly-labeled compensation beads were labeled with antibodies used for B cell subset identification (gray). A duplicate set was subject to methanol-based permeabilization (red). There was an acceptable degree of signal intensity diminution. B) Methanol-based permeabilization increases PE/Cy7 spillover into PE. As in 2A, compensation singly-labeled beads were labeled with antibodies used for B cell subset identification (gray). A duplicate set was subject to methanol-based permeabilization (red). Labeling for the indicated fluorochrome was potted against the channel into which spillover was highest. For Brilliant Violet 421, 510, and 605, there was less spillover into Briliant Violet 510, Brilliant Violet 605, and PE, respectively, with diminution of signal intensity. In contrast, spillover of PE/Cy7 into PE actually increased. There was no appreciable increase in tandem breakdown of PE/Cy7 after exposure to methanol-based permeabilization.

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3.3 Optimal labeling of CD38 requires a bright fluorochrome with adequately high PMT voltage. Optimal separation of CD38-, CD38+, and CD38hi labeling required both a bright fluorochrome (PE/Cy7) and high PMT voltage. The latter is illustrated in Figure 3A. Optimal PE/Cy7 PMT voltage was at least 575 volts when PE/Cy7 labeling is performed without exposure to methanol-based permeabilization buffer. Slightly higher PMT voltages (around 600 volts) were required to accommodate buffer-induced signal degradation. To achieve optimal separation of CD38hi, CD38+, and CD38- labeling, it would be tempting to increase the PMT voltage of the PE/Cy7 channel up to 650 volts. Higher PE/Cy7 PMT voltage, however, caused greater spillover of APC and Brilliant Violet 605 into the PE/Cy7 channel. The degree of spillover is quantified in Figure 3B. We paid special attention to compensation from APC (conjugated to phospho-specific antibodies) and Brilliant Violet 605 (conjugated to CD19, of which there is some variability of expression among B cells). Excessive spillover could artefactually increase the CD38hi population or phosphorylation among these subsets. Careful optimization of PMT

ACCEPTED MANUSCRIPT voltages for PE/Cy7-conjugated CD38 labeling was critical to balance CD38hi, CD38+, and CD38- separation on the one hand and spillover from other fluorochromes on the other hand.

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FIGURE 3. Optimal labeling of CD38 requires a bright fluorochrome with adequately high PMT voltage. A) Optimal identification of CD38 requires high photomultiplier tube (PMT) voltages. Normal human PBMCs were fixed and then labeled with CD38 conjugated to PE/Cy7. Labeled cells from a single sample were analyzed at progressively higher PMT voltages. Optimal identification of CD38hi cells occurred at higher voltages. B) Relative photomultiplier tube (PMT) voltages of PE/Cy7 compared to APC and Brilliant Violet 605, markedly affect compensation values. Normal human PBMCs were singly labeled with CD3 conjugated to APC or Brilliant Violet 605. The same samples analyzed at increasing PE/Cy7 PMT voltages yielded increased spillover into PE/Cy7. Degree of spillover of CD3 or Brilliant Violet 605 into the PE/Cy7 channel is quantified (color-coded) for each PE/Cy7 PMT voltage in the upper right hand corner. An upper bound for the PE/Cy7 PMT was set by the relative difference in PMT voltages between PE/Cy7 and APC and Brilliant Violet 605.

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FIGURE 3. Optimal labeling of CD38 requires a bright fluorochrome with adequately high PMT voltage. A) Optimal identification of CD38 requires high photomultiplier tube (PMT) voltages. Normal human PBMCs were fixed and then labeled with CD38 conjugated to PE/Cy7. Labeled cells from a single sample were analyzed at progressively higher PMT voltages. Optimal identification of CD38hi cells occurred at higher voltages. B) Relative photomultiplier tube (PMT) voltages of PE/Cy7 compared to APC and Brilliant Violet 605, markedly affect compensation values. Normal human PBMCs were singly labeled with CD3 conjugated to APC or Brilliant Violet 605. The same samples analyzed at increasing PE/Cy7 PMT voltages yielded increased spillover into PE/Cy7. Degree of spillover of CD3 or Brilliant Violet 605 into the PE/Cy7 channel is quantified (color-coded) for each PE/Cy7 PMT voltage in the upper right hand corner. An upper bound for the PE/Cy7 PMT was set by the relative difference in PMT voltages between PE/Cy7 and APC and Brilliant Violet 605.

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3.4 Quantification of PE labeling requires optimization of PE/Cy7 signal intensity and PE PMT voltages. PE was one of the three key fluorochromes used to quantify intracellular phosphorylation. Not only is PE brightness useful for such quantitation, but many commercially available phospho antibodies are conjugated to PE (in addition to FITC and APC). An important consideration with use of PE in our system was that brighter PE/Cy7 staining caused greater spillover into the PE channel (Figure 4A). This was a key parameter to optimize for compensation between PE/Cy7 and PE. Use of brightly labeled CD3 + cells resulted in overcompensation into the PE channel; use of dimly labeled CD38 hi lymphocytes with dilute antibody concentrations resulted in under-compensation. Since PE was used to detect quantitative changes in intracellular phosphorylation, compensation had to be as precise as possible. Optimal compensation therefore required use of cells labeled for CD38 at the same antibody concentration used for stimulation experiments and then exposed to methanol-based permeabilization buffer. Additional optimization of PE compensation further required adjustment of the PE voltage. Figure 4B illustrates that higher PE voltages caused greater spillover of the signal from the tandem dyes PE/Cy7 and Brilliant Violet 605 into the PE channel. On the one hand, brighter PE labeling was desirable for quantitative detection of PE-labeled phosphorylated targets. On the other hand, overly bright staining could artefactually increase the PE labeling of CD38hi cells. Therefore, we chose a lower PE channel PMT voltage for our experiments. As shown below, there was actually diminished NF-κB (PE-conjugated) phospho signaling in CD38hi TrB cells in our system; this finding excludes the possibility that PE/Cy7 into PE was undercompensated. Finally, the fluorochrome Brilliant Violet 605 was used for CD19 labeling, which is dichotomous rather than continuous. Therefore, any spillover of Brilliant Violet 605 into PE equally affected B cell subsets. FIGURE 4. Quantification of PE labeling requires optimization of PE/Cy7 signal intensity and PE PMT voltages. A) Higher PE/Cy7 signal intensity increases spillover into PE. Normal donor PBMCs were singly labeled after fixation with CD3 or CD38 conjugated to PE/Cy7 at two dilutions. PE/Cy7 signal intensity was highest in CD3 + cells and lowest in CD38hi cells labled with CD38 at the lower dilution. Spillover into PE was highest in the brightest CD3 + cells (blue) and lowest in the CD38hi cells labeled with a lower dilution of antibody (gray). B) Relative PMT voltage of PE affects spillover of tandem dyes into PE Normal human PBMCs were singly labeled with CD3 conjugated to PE/Cy7 or Brilliant Violet 605, both tandem conjugates. The same samples analyzed at increasing PE PMT voltages yielded increased spillover into PE. Thus, an upper bound for the PE PMT (used in these studies for phospho antibody labeling)

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was set by the relative difference in PMT voltages between PE and PE/Cy7 and Brilliant Violet 605.

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FIGURE 4. Quantification of PE labeling requires optimization of PE/Cy7 signal intensity and PE PMT voltages. A) Higher PE/Cy7 signal intensity increases spillover into PE. Normal donor PBMCs were singly labeled after fixation with CD3 or CD38 conjugated to PE/Cy7 at two dilutions. PE/Cy7 signal intensity was highest in CD3 + cells and lowest in CD38hi cells labled with CD38 at the lower dilution. Spillover into PE was highest in the brightest CD3 + cells (blue) and lowest in the CD38hi cells labeled with a lower dilution of antibody (gray). B) Relative PMT voltage of PE affects spillover of tandem dyes into PE Normal human PBMCs were singly labeled with CD3 conjugated to PE/Cy7 or Brilliant Violet 605, both tandem conjugates. The same samples analyzed at increasing PE PMT voltages yielded increased spillover into PE. Thus, an upper bound for the PE PMT (used in these studies for phospho antibody labeling) was set by the relative difference in PMT voltages between PE and PE/Cy7 and Brilliant Violet 605. 3.5 Optimization of the above parameters allows for distinct identification of B cell subsets. Having optimized each of the parameters discussed above, we could accurately distinguish B cells (CD19+) from T cells (CD3+) and B cell subsets as show in Figure 5: Early memory (CD24hiCD38-), late memory (CD24-CD38-), naïve (CD24+CD38+), and transitional B cells (CD24hiCD38hi). Fluorescence minus one controls, separately overlaid on the full surface marker panel, were used to help delineate the gating strategy. Gating was also consistent with relevant literature. As described, use of the bright fluorochrome PE/Cy7 with optimal PMT

ACCEPTED MANUSCRIPT voltages was necessary for separation of CD38-, CD38+, and CD38hi labeling. The fluorochrome FITC, even at a high PMT voltage, was inadequate to distinguish CD38 -, CD38+, and CD38hi labeling. Labeling of additional normal donors is shown in Supplementary Figure 4.

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Compensation values were favorable (Supplementary Figure 5), with limited spillover to or from PE/Cy7, PE, and APC in particular. Compensation controls for surface markers were singly labeled cells as described in the Methods section. While autofluorescence and brightness differed between compensation beads and cells (Supplementary Figure 6A), choice of beads or cells as compensation controls had limited effects upon the final compensation matrix (Supplementary Figure 6B). These findings are consistent with findings in current flow cytometry literature.[50-52]

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FIGURE 5. Optimized labeling and gating schematic to distinguish transitional B cells (TrBs) from other B cell subsets. Surface epitope labeling, photomultipler tube (PMT) voltages, and compensation were adjusted as above. Normal donor PBMCs were fixed; labeled for CD3 (Brilliant Violet 510), CD19 (Brilliant Violet 605), CD24 (Brilliant Violet 421), and CD38 (FITC at a high PMT voltage or PE/Cy7); and subject to methanol-based permeabilization. Gating of B cell subsets is shown for the full surface panel (red). Fluorescence minus one (FMO) controls for CD24 and CD38 are overlaid (black and blue, respectively) on the same surface marker labeling (bottom panel) to help delineate gating. Even at a high PMT voltage, FITC conjugated CD38 labeling was sub-optimal to distinguish CD38hi from CD38+. Abbreviations: EM, early memory B cells; FMO, fluorescence minus one; LM, late memory B cells; TrB, transitional B cells.

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FIGURE 5. Optimized labeling and gating schematic to distinguish transitional B cells (TrBs) from other B cell subsets. Surface epitope labeling, photomultipler tube (PMT) voltages, and compensation were adjusted as above. Normal donor PBMCs were fixed; labeled for CD3 (Brilliant Violet 510), CD19 (Brilliant Violet 605), CD24 (Brilliant Violet 421), and CD38 (FITC at a high PMT voltage or PE/Cy7); and subject to methanol-based permeabilization. Gating of B cell subsets is shown for the full surface panel (red). Fluorescence minus one (FMO) controls for CD24 and CD38 are overlaid (black and blue, respectively) on the same surface marker labeling (bottom panel) to help delineate gating. Even at a high PMT voltage, FITC conjugated CD38 labeling was sub-optimal to distinguish CD38hi from CD38+. Abbreviations: EM, early memory B cells; FMO, fluorescence minus one; LM, late memory B cells; TrB, transitional B cells.

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3.6 There is diminished phospho signaling in transitional B cells upon phorbol 12myristate 13-acetate (PMA) and ionomycin stimulation. Finally, the kinetics of phosphorylation among several key signaling molecules for lymphocyte survival and activation were quantified. These targets were: Extracellular signal regulated kinase (ERK), nuclear factorkappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Akt. Phospho flow cytometry distinguished phosphorylation in each of these molecues as illustrated in Figure 6A. For ERK, median fluorescence intensities (MFIs) were analyzed because even brief stimulation yielded phosphorylation in nearly 100% of all subsets analyzed. For all other signaling molecules, the percentage of phosphorylated cells was analyzed. There was high basal phosphorylation of MAPK, and stimulation further increased phosphroylation. Gating of phosphorylation by percentage (for each target) was held consistent across experiments. Stimulation did not affect surface epitope labeling (Supplementary Figure 7). Kinetics of phosphorylation among subsets were similar among B cell subsets, but the extent of phosphorylation was diminished especially in TrBs (illustrated in Figure 6B). There was, however, considerable inter-individual variability in the pecentage of phosphorylated molecules at different time points. Therefore, quantitative phosphorylation was normalized for each of three donors. For each individual and at each time point, phospho target normalization was performed to the most highly phosphorylated B cell subset (Figure 6C). That is, for a given time point, the most highly phosphorylated subset (by MFI for ERK and percentage for all other targets) was normalized as 1. Target phosphorylation of other subsets was was normalized to this maximum. Normalization in this way revealed diminished signaling among TrBs for each target analyzed. Preliminary data and review of the literature suggested the hypothesis that TrB signaling would be diminished compared to more mature B cell subsets. Therefore, statistical analysis was performed as a one-tailed paired T test. FIGURE 6. Diminished phospho signaling in transitional B cells upon phorbol 12myristate 13-acetate (PMA) and ionomycin stimulation. Normal human PBMCs were thawed from frozen and rested at 37oC under 5% CO2 for 30 minutes in RPMI medium containing 10% fetal bovine serum and L-glutamine. Samples were subsequently stimulated with phorbol 12myristate 13-acetate (PMA) and ionomycin for kinetic studies. At indicated times, cells were fixed with paraformaldehyde-based buffer. They were subsequently labeled for surface epitopes, permeabilized with methanol-based buffer, and finally labeled for intracellular phosphorylated targets. These targets were: ERK (pT202/pY204, AlexaFluor 488), NF-kB p65 (pS529, PE), p38 MAPK (pT180/pY182, APC), and Akt (pS473, AlexaFluor 647). The first three were analyzed from the same sample, and the last was analyzed from a duplicate sample. A) Representative labeling of unstimulated (gray) or stimulated (red) late memory (CD24 -CD38-) B

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cells. Percentage of phosphorylated cells is indicated for each target except for ERK, where median fluorescence intensity (MFI) was analyzed. This was because most stilulated cells were phosphorylated at ERK, but MFI varied with duration of stimulation. B) Representative kinetics of phosphorylation at ERK, NF-kB, p38 MAPK, and Akt among B cell subsets. C) Normalized data from three separate donors (n=3). At each time point for each donor, phosphorylation was normalized to the most highly phosphorylated subset (i.e., normalized percentage = 1 for the most highly phosphorylated subset). Error bars indicate standard error of the mean. A one-tailed paired T test (given our hypothesis of and preliminary data suggesting reduced signaling in TrBs) was used to compare TrBs to other B cell subsets. Statistical significance of p < 0.05 is indicated in comparison of TrBs to naïve (#), early memory (*), and late memory (^) B c ells. Abbreviations: EM, early memory B cells; ERK, extracellular signal regulated kinase; LM, late memory B cells; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B; TrB, transitional B cells.

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FIGURE 6. Diminished phospho signaling in transitional B cells upon phorbol 12-myristate 13acetate (PMA) and ionomycin stimulation. Normal human PBMCs were thawed from frozen and rested at 37oC under 5% CO2 for 30 minutes in RPMI medium containing 10% fetal bovine serum and L-glutamine. Samples were subsequently stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for kinetic studies. At indicated times, cells were fixed with paraformaldehyde-based buffer. They were subsequently labeled for surface epitopes, permeabilized with methanol-based buffer, and finally labeled for intracellular phosphorylated targets. These targets were: ERK (pT202/pY204, AlexaFluor 488), NF-kB p65 (pS529, PE), p38 MAPK (pT180/pY182, APC), and Akt (pS473, AlexaFluor 647). The first three were analyzed from the same sample, and the last was analyzed from a duplicate sample. A) Representative labeling of unstimulated (gray) or stimulated (red) late memory (CD24-CD38-) B cells. Percentage of phosphorylated cells is indicated for each target except for ERK, where median fluorescence intensity (MFI) was analyzed. This was because m ost stilulated cells were phosphorylated at ERK, but MFI varied with duration of stimulation. B) Representative kinetics of phosphorylation at ERK, NF-kB, p38 MAPK, and Akt among B cell subsets. C) Normalized data from three separate donors (n=3). At each time point for each donor, phosphorylation was normalized to the most highly phosphorylated subset (i.e., normalized percentage = 1 for the most highly phosphorylated subset). Error bars indicate standard error of the mean. A one-tailed paired T test (given our hypothesis of and preliminary data suggesting reduced signaling in TrBs) was used to compare TrBs to other B cell subsets. Statistical significance of p < 0.05 is indicated in comparison of TrBs to naïve (#), early memory (*), and late memory (^) B cells. Abbreviations: EM, early memory B cells; ERK, extracellular signal regulated kinase; LM, late memory B cells; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B; TrB, transitional B cells. 4. DISCUSSION

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B cells can have regulatory properties (Bregs), however, unlike human regulatory T cellsthere is no single defining marker such as Foxp3 for Bregs.[21] While T-cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) has emerged as a murine regulatory B cell (Breg) marker,[53] this has been less useful as a human TrB or Breg marker.[54] TrBs have also been implicated as having regulatory activity; in this study TrBs were defined by surface marker expression (e.g., CD24hiCD38hi). As an alternative to CD24 expression, some groups have defined TrBs as IgD+CD38hi. [41, 55, 56] This schema yields six rather than four B cell subsets, complicating analysis somewhat. Thus, the analysis of TrB functionality and activation demands precise flow cytometry, and our approach allows for substitution of IgD for CD24 as IgD labeling was better preserved than anti-CD24, with labeling by a goat polyclonal antibody after fixation. Ultimately, TrBs are functionally defined by their capacity to produce IL-10 upon stimulation in vitro.[57] Such stimulation, of course, would obscure the earlier quantitative signaling events. Therefore, IL-10 is not a useful marker for defining TrB subsets flow in conjunction with phospho flow cytometry experiments. Among the parameters optimized, CD38 labeling required the most careful attention. Separation of CD38-, CD38+, and CD38hi populations required a bright antibody-conjugated fluorochrome such as PE/Cy7 collected at a sufficiently high PMT voltage. Fortunately, PE/Cy7 epitope labeling was preserved following exposure to a paraformaldehyde-based fixative; PE/Cy7 itself showed an acceptable degree of degradation (but not tandem breakdown) following exposure to a methanol-based permeabilization agent. Bright CD38 labeling affected spillover of PE/Cy7 into the PE channel and both APC and Brilliant Violet 605 into the PE/Cy7 channel. Our optimized protocol minimized compensation requirements, allowing us to quantitatively assess signaling

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Transitional B cells (TrBs) have recently been identified to have important roles as suppressor cells in transplantation, autoimmunity, and lately in oncology.[58] Current techniques to study intracellular signaling have limited progress in understanding the mechanisms responsible for TrB signaling and activation. Careful optimization of phospho flow cytometry should enable analysis of normal TrB signaling, dysfunction in disease, and response to B cell-targeted therapies. In recent years, numerous small molecules and antibodies have been developed to target B cells in autoimmunity and cancer.[59-63] There may even be a role for in vitro TrB expansion with subsequent therapeutic infusion,[64] similar to Treg expansion protocols.[65] Understanding the effects of pharmacologic manipulation on TrB function is critical to safe and effective use of these treatments. Phospho flow cytometry also has great potential for drug discovery and immune monitoring after treatment.[66, 67] An effective and standardized protocol to quantify B cell subset signaling upon stimulation would be a key tool toward this end. Our optimized phospho flow cytometry protocol can serve as a basis for quantifying B cell subset signaling in health and disease. Finally, the approach can be a model for analysis of signaling in other rare immune cells. 5. CONCLUSIONS

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Here we have optimized a protocol to quantify phospho-signaling in human TrBs by flow cytometry. We were reliably able to distinguish TrBs from other B cell subsets: naïve, early memory, and late memory. Following nonspecific stimulation with PMA and ionomycin, MAPK/Erk and Akt signaling was quantitatively diminished in TrBs compared to mature B cell subsets. This protocol can serve as a basis to assess B cell signaling in health and disease.

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INFORMED CONSENT De-identified normal human donor buffy coat preparations were obtained with Institutional Review Board approval (2016-1163) from LifeSource. Studies were determined not to meet the definition of human subject research as defined by 45 CFR 46.102(f)

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DECLARATION OF INTEREST The authors have no conflicts of interest to disclose.

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FUNDING Institutional support from the The University of Illinois at Chicago Department of Medicine. ACKNOWLEDGEMENTS The authors would like to thank Suresh Ramasamy, PhD for his assistance as a Research Specialist in the University of Illinois at Chicago Flow Cytometry Core.

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Highlights  Phospho flow cytometry is a powerful technique to analyze signaling  Technical limitations are a barrier to quantify B cell subset signaling  B cell surface marker labeling was best after fixation but before permeabilization  CD38 labeling required careful PMT voltage and compensation optimization  Diminished signaling in human CD24hiCD38hi TrBs after stimulation