Human germinal center B cells differ from naïve and memory B cells in CD40 expression and CD40L-induced signaling response

Abstract

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing germinal center reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 minutes following CD40L activation. Prior to CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naive and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 minutes following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing germinal center reactions.

INTRODUCTION

The germinal center (GC) is the main site for affinity maturation and differentiation of B cells into memory B cells or Ig-producing plasma cells (1). During the GC reaction, B cells with high-affinity B-cell receptors are selectively expanded by capture of antigen and subsequent presentation of antigen-derived peptides to T cells. Duration of the interaction with T follicular helper cells shapes B-cell fate through CD40L-CD40 signaling interactions (2). Signals from CD40 are necessary for effective B-cell responses to T cell-dependent antigens, specifically during initiation of a GC reaction and development of memory B cells (3,4). Non-malignant GC reactions are disrupted in B-cell follicular lymphoma (FL) (5), and abnormal CD40 signaling via NFκB p65 in the malignant B cells is associated with overall survival in FL (6). A better understanding of the signaling mechanisms required for effective GC reactions could provide insight into modulating humoral immune responses to improve therapies for autoimmunity and cancer.

CD40 is a member of the TNF receptor family and is a co-stimulatory receptor on B cells and other antigen-presenting cells. Upon binding of CD40L to CD40, TNF receptor-associated (TRAF) proteins are recruited to the cytoplasmic domain of CD40 and subsequently activate signaling pathways including the nuclear factor κB (NFκB) pathway, phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinases (MAPK) (7). Activation of NFκB, both via canonical and non-canonical pathways, is initiated by activation of inhibitor of κB (IκB) kinase (IKK) complexes. Dimers of NFκB subunits, such as the abundant heterodimer of RELA/p65 and NFKB1/p50, are sequestered in the cytoplasm by IκBα (8). Upon activation of the canonical NFκB pathway, TNFR-associated factor (TRAF)-mediated receptor signaling triggers IκBα degradation via activation of IKK, and releases NFκB dimers that translocate to the nucleus and activate transcription. The NFκB subunits play different roles during the GC reaction as c-REL is needed for establishing and maintaining the GC, whereas p65 is crucial for terminal plasma cell differentiation (9). Phosphorylation and other post-translational modifications of NFκB subunits can further fine-tune transcriptional activity (10).

Despite the important role of CD40 activation and signaling for B-cell function and fate during the germinal center reaction, the expression of CD40 and its signaling capacity in human GC B cells is not fully understood. We previously developed phospho-flow cytometry assays to characterize signaling kinetics in healthy peripheral blood B cells and lymphoma B cells (11,12). More recently, this approach showed that human GC B cells are hypersensitive to redox signaling (13) and revealed clinically significant differences in B-cell receptor (BCR) and CD40L signaling responses (6) and basal signaling (14) in lymphoma B cells. Here, phospho-flow was used to characterize CD40L signaling kinetics across stages of healthy, mature B cells from human tonsils. Germinal center B cells were distinguished from naïve and memory B cells in the same tissue samples by both the starting state of the signaling network, including surface expression of CD40, and CD40L-induced phospho-protein response kinetics.

MATERIALS AND METHODS

Human samples

Tonsils were obtained from patients undergoing tonsillectomy with written informed consent in accordance with the Declaration of Helsinki and the Regional Committees for Medical and Health Research Ethics, Region Eastern Norway (REK#2010/1147a). The tonsils were processed to single-cell suspension by mincing and then stored in liquid nitrogen, as in prior studies of tonsillar tissue (5,13). The cells were thawed and rested in RPMI w/10% FCS at 37 °C for 15 min before further processing.

Reagents

All antibodies used are listed in Ting Information Table S1. Pacific Blue and Alexa 750 dyes used for barcoding (6) were from Molecular Probes. rhCD40L was from Peprotech (0.05 μg/ml) or Enzo Life Siences (0.25 μg/ml). CD40L from Enzo is fused to a FLAG® tag and was pre-incubated for 30 min with an Enhancer for ligands (anti-FLAG antibody; 1 μg/ml) to enhance the biological activity, according to manufacturer’s recommendations. CD40L from Enzo was used at a saturating concentration which potently induces proliferation and plasma cell differentiation in vitro when combined with IL-21 (Supporting Information Fig. S1). CD40L from Peprotech gave similar signaling responses without crosslinking (Supporting Information Fig. S2). CD40L from Enzo was used for Figure 2 and 4, CD40L from Peprotech was used for Figure 3.

Figure 2. Reduced CD40-mediated NFκB p65 phosphorylation in GC B cells despite increased surface CD40 expression.

NFκB p65 phosphorylation (p-p65) at serine 529 following 15 min CD40L stimulation was measured by mass cytometry. Populations were gated on biaxial plots; GC B cells as CD3⁻CD19⁺CD20⁺⁺CD38⁺ and non-GC B cells as CD3⁻CD19⁺CD38⁻. The non-GC population was further gated to enrich for IgD⁺ naïve B cells (possibly containing unswitched memory B cells) and IgD⁻ memory B cells. A) Histograms of p-p65 from one representative tonsil (T19). B-C) Relative induction of p-p65 by CD40L (B) and surface CD40 expression in unstimulated cells (C) was measured simultaneously. Mean ± SD. * p < 0.05; repeated measurement one-way ANOVA and Tukey’s multiple comparison test.

Figure 4. CD40L-induced IκBα degradation is not proportional to level of CD40L binding.

CD40L-induced signaling (IκBα and p-p65-S529) in human tonsillar cells was measured by fluorescence flow cytometry as a function of ligand uptake (bound CD40L). Cells were stimulated with CD40L and a secondary antibody was used to detect bound CD40L after permeabilization of the cells. Non-GC (naïve and memory) B cells were gated as CD20⁺CD38⁻ and GC B cells as CD20^hiCD38⁺. (A) The cells were further gated into 7 populations based on the level of bound CD40L. (B-C) Signaling is shown as arcsinh ratio relative to the lowest level of bound CD40L in non-GC B cells. D) Signaling responses is shown as arcsinh ratio relative to the unstimulated condition for each cell type and for each level of CD40L. (C-D) Mean values ± SEM, n = 4 individual tonsils. * p < 0.05 between GC and naïve/memory in a two-sided paired t-test.

Figure 3. CD40L signaling responses distinguish GC B cells from naïve and memory B cells in human tonsils.

CD40L-induced signaling (A: total IκBα, B: p-p65-S529, C: p-S6-Ser235/236) in human tonsillar cells was measured by fluorescence flow cytometry. Values represent MFI relative to unstimulated naïve B cells in one representative tonsil (A-C) or mean values ± SEM (D-F), n = 3–4 individual tonsils. After debarcoding, GC B cells were gated as CD20^hiCD38⁺, non-GC (CD20⁺CD38⁻) were split into CD27⁻IgD⁺ naïve and CD27⁺IgD⁻ memory B cells. * p < 0.05 between GC and naïve, # p < 0.05 between GC and memory, § p < 0.05 between naïve and memory, Repeated measurement one-way ANOVA and Tukey’s multiple comparison test.

Phenotyping by mass cytometry

Live cells were stained with antibodies for surface targets on ice for 30 min, then fixed with paraformaldehyde (PFA; final concentration 1.6%) at room temperature for 5 min, permeabilized with ice-cold methanol (final concentration > 90%) and stored at −80 °C. At the day of acquisition, cells were rehydrated by washing with PBS and then with PBS with 1% BSA, stained with antibodies for intracellular targets for 15–30 min at room temperature, incubated with iridium intercalator (Fluidigm), washed at least twice by centrifugation in PBS, resuspended in water and run on a CyTOF1 (Fluidigm). For phenotyping by fluorescence flow cytometry, live cells stained on ice for 30 min before acquisition on a FACS Canto (BD).

Activation of signaling

Cells were transferred to wells or tubes at a final concentration of 10 million cells per ml and rested for an additional 20 min before stimulation with CD40L. Signaling was stopped by fixation with PFA and cells were permeabilized with methanol and stored at −80 °C.

In Figure 4, an anti-mouse antibody to detect the Enhancer for ligands (anti-FLAG mouse antibody) was used to detect bound CD40L. The staining with the anti-mouse antibody was performed after permeabilization and barcoding, before staining with the other antibodies. In the unstimulated condition, cells were fixed, then incubated with CD40L+enhancer for 10 minutes, followed by permeabilization and staining with the anti-mouse antibody to measure the basal level of CD40 without inducing signaling.

Phospho-specific flow cytometry

For mass cytometry, staining for surface targets was performed after PFA fix and before permeabilization (the anti-IgD antibody lost some sensitivity when used on fixed cells). At the day of acquisition, cells were rehydrated, stained with intracellular antibodies, incubated with intercalator and run on a CyTOF1 as for phenotyping.

When using fluorescence cytometry, barcoding was used by encoding all the samples from one tonsil in a 4×2 barcoding grid (4 levels of Pacific Blue (0.3, 0.8, 4 and 25 ng/ml) and 2 levels of Alexa 750 (5 and 15 ng/ml)). At the day of acquisition, cells were rehydrated by washing twice in PBS and incubated with barcoding dyes for 30 min in the dark, before washing with PBS with 1% BSA and combining samples into one tube (6). Antibody staining was performed at room temperature for 15–30 min before analyzing cells on a BD FACSCanto or LSR II.

Computational analysis

Expression values from flourescence and mass cytometry data are shown on the log-like arcsinh scale, calculated by arcsinh(MFI/cofactor), and differences in protein expression were also quantified on this scale, as previously described (6). The scale-specific cofactor represents the noise or background in the data and standard cofactors of 15 (mass cytometry) and 150–300 (fluorescence cytometry) were used. A cofactor of 15 is necessary for data collected on a CyTOF 1.0 instrument as it is less sensitive for lower intensity signals than the modern CyTOF 2.0 and Helios CyTOF 3.0 instruments where a cofactor of 5 is sufficient.

viSNE was run in Cytobank (15), on the markers indicated in Supporting Information Table S1, following established methods (16). In Figure 1, a computational light-chain channel was created by selecting the highest value of either Igκ or Igλ for each cell event, hence reporting the light chain level for each B cell regardless of isotype, as previously described (5). Marker enrichment modeling (MEM) was run in R using the published MEM package (17,18). Slicing according to level of bound CD40L was done by making 7 parallel gates in a plot displaying B cells. Gates were fixed across all samples in each experiment (2 tonsils and 3 conditions) and were placed to ensure that each gate represented a distinct level and to ensure sufficient cells for subsequent analysis. This strategy was chosen to compare equivalent levels of bound CD40L between conditions and resulted in gates that did not contain the same number of cells (Supporting Information Table S2 and S3).

Figure 1. MEM identifies CD40 as a positively enriched marker in GC B cells.

Single cells from three human tonsils were analyzed by mass cytometry. All live lymphocytes were computationally analyzed by viSNE and GC, naïve and memory B cells were identified in the viSNE maps as shown in Supporting Information Fig. S3. A) Marker enrichment modeling (MEM) scores were calculated for each population (using the other two populations as reference population) and the scores were clustered by hierarchical clustering. All markers used for gating and Ig heavy chains were left out of the MEM analysis. B) Mean MEM scores for each population across tonsils were calculated. Only mean MEM scores ≥ 2 are listed. C) Representative expression intensity of CD40 in viSNE map for tonsil 25. D) Median CD40 intensities measured by mass cytometry. Mean ± SEM, n = 3. * p < 0.05, Repeated measurement one-way ANOVA and Tukey’s multiple comparison test. E) Comparing a histogram view of CD40 expression measured by mass cytometry (same data as in C) and fluorescence flow cytometry (representative of three tonsils).

Statistics

Two-tailed t-test for paired samples or one-way ANOVA was applied to determine the level of statistical significance. Data were considered statistically significant when p < 0.05. In Figure 4, linear regression was performed in GraphPad Prism to investigate the relationship between signaling and level of bound CD40L.

RESULTS

MEM identified CD40 as a positively enriched surface protein in GC B cells

A previously published dataset of human tonsils phenotyped by mass cytometry (5) was analyzed to investigate the expression of CD40 and other surface proteins in GC B cells compared to naïve and memory B cells (Supporting Information Fig. S3). Single cells from three human tonsils were stained by a 27-marker antibody panel which included markers to identify B and T cells (CD3, CD19), B-cell subsets (CD20, CD38, IgD, CD27) and other B-cell relevant markers such as BCR-complex components and chemokine receptors (Supporting information Table S1). Some markers gave weak staining in the three B-cell populations (CD27, IgG, CD79B and CD86), but the specificity was validated by comparing the appropriate cell types for enrichment of the markers in question. B-cell populations were gated on viSNE maps (19) (Supporting Information Fig. S3), and hierarchical clustering was performed on median marker intensities for each population from the three tonsils, identifying GC, naïve and memory B cells as three separate clusters (Supporting Information Fig. S3C).

Marker Enrichment Modeling (MEM), a newly developed algorithm to objectively report quantitative features of a cell population (17,18), was next used to calculate MEM scores for each of the three populations GC, naïve and memory B cells. Markers used for gating the B-cell populations and immunoglobulin (Ig) heavy chains were not included in the analysis in order to focus the analysis on other phenotypic differences. The reference population for MEM was selected as “all other cells” in order to identify differential enrichment between the three populations (see Supplementary Figure 1 from MEM manuscript for more information on reference population selection (17)). The B-cell populations were subjected to hierarchical clustering based on the MEM scores (Fig. 1A). The GC populations demonstrated highest similarity and clustered together, whereas the naïve and memory populations were more diverse and had few features that were specifically enriched on the population as a whole (Fig. 1A, B).

MEM identified CD40 as the highest positively enriched marker on GC B cells with a mean MEM score of +4 (Fig. 1B). In addition, GC B cells were distinguished by low CCR6, CD44 and Ig light chain (computationally combined Igκ/Igλ) expression, and high expression of HLA-DR. In addition, GC B cells had a small positive enrichment for CD86 (mean MEM score of 0.8) and negative enrichment for CD79B (mean MEM score of −0.6), whereas naïve B cells were positively enriched for CD22 (mean MEM score of 2). Ig light chain expression varied most across the B-cell populations, being highly enriched in naïve B cells (mean MEM score +7) and negatively enriched in GC B cells (mean MEM score −8). The MEM scores for CXCR4 and CXCR5 varied within the populations, and the other markers, CD43, CD62L, CD36, CCR4 and CD24, had weak or no expression across the populations.

The viSNE map showed that CD40 expression varied in all three populations (Fig. 1C), but the median expression was significantly higher in the GC population (Fig. 1D). The differences in CD40 expression between GC and naïve/memory B cells observed, and the heterogeneity within the populations, were further validated by traditional fluorescence flow cytometry (Fig. 1E).

CD40-mediated NFκB p65 phosphorylation was lower despite high CD40 expression in GC B cells

The increased expression of CD40 on GC B cells could imply that GC B cells exhibit stronger CD40 signaling responses than non-GC B cells. To investigate this hypothesis, single-cell suspensions from human tonsils were stimulated with CD40L and phosphorylation of NFκB p65 at serine 529 (p-p65) was measured by mass cytometry. Upon CD40 activation, lower levels of p-p65 were observed in GC B cells, as compared to naïve B cells (Fig. 2A,B). This diminished p-p65 signaling response contrasted with the increased surface CD40 expression measured on GC B cells in the same experiments (Fig. 2C). CD40L-induced signaling was also lower in memory B cells compared to naïve B cells, despite similar surface CD40 levels. This suggested that CD40 receptor level alone does not dictate signaling strength in B cells.

Higher basal IκBα level and increased CD40L-induced IκBα degradation in GC B cells

Kinetics of CD40L stimulation were next used to characterize the duration of the GC B cell signaling response, as compared to naïve and memory B cells (Fig. 3). For these experiments we chose to use fluorescence flow cytometry, to get optimal separation of populations. The p-p65 response following CD40L stimulation was dampened in GC B cells and significantly lower signaling responses were observed at 7, 10 and 15 minutes post stimulation (Fig. 3B,E). Despite differences in total p-p65, comparable signaling kinetics were observed; p-p65 levels peaked at 15 minutes following CD40L in all three B-cell subsets. To investigate if the levels of basal signaling was causing the difference in signaling response, the signaling for all three populations was normalized against the same control (unstimulated naïve cells). However, no significant p65 phosphorylation was observed in any of the three B-cell populations prior to stimulation.

CD40L increased phosphorylation of PI3K/AKT pathway effector S6 less in GC B cells than in naïve and memory B cells (Fig. 3C,F). This lowered response might be due to the relatively higher basal starting level of p-S6 observed in GC B cells prior CD40L stimulation (Fig. 3C,F). GC B cells ultimately achieved the highest observed levels of p-S6 at 30–60 minutes following CD40L (Fig. 3C,F).

The IκBα repressor of NFκB p65 was heterogeneously expressed in the B-cell subsets, but GC B cells had higher expression levels than naïve and memory B cells (Fig. 3A,D). CD40L stimulation induced rapid IκBα destruction in all three B cell types, and IκBα reached and maintained comparably low absolute levels after 30 min of CD40L stimulation (Fig. 3A,D).

CD40L-induced p-p65, but not degradation of IκBα, was proportional to the level of bound CD40L

Next, the level of CD40L binding and levels of signaling response was compared between GC and non-GC B cells with equivalent surface CD40 expression. This was achieved by detecting bound rhCD40L by a PE-conjugated secondary antibody against the mouse anti-FLAG antibody (Enhancer for ligands) used to crosslink rhCD40L. On a population level, binding of CD40L correlated with surface expression of CD40 (Supporting Information Fig. S4). Signaling readouts, p-p65 and IκBα, were then quantified as a function of CD40L binding by gating on thin slices of bound CD40L (Fig. 4A).

To measure the basal levels of the signaling readouts relative to distinct levels of CD40 expression, CD40L was added to the cells after fixation (i.e. no in vitro activation of cells). The basal IκBα levels were significantly higher in GC compared to non-GC (naïve/memory) B cells across most levels of CD40L (Fig. 4C). The basal p-p65 levels differed less but were higher in non-GC B cells (Fig. 4C). Furthermore, the relationship between level of bound CD40L and basal level of p-p65 and IκBα was investigated by linear regression. For non-GC B cells there was a positive relationship between increasing CD40L levels and higher basal levels of p-p65 and IκBα (Table 1, Supporting Information Fig. S5, r²=0.68 and 0.54; p<0.0001). A similar but weaker relationship was also observed for GC B cells (Table 1, Supporting Information Fig. S5). Cell size was not a confounding factor in these measurements as no relationship was found between FSC and CD40L levels (Supporting Information Fig. S6).

Table 1.

Linear regression to model the relationship between signaling and level of bound CD40L¹

IkBa		Non-GC		GC
		r2	p-value	r2	p-value
	Basal (unstim)	0.6806	<0.0001	0.1869	0.0216
	10 min	0.02249	0.4462	0.08672	0.1282
	30 min	0.03558	0.3364	0.003960	0.7504
p-p65		Non-GC		GC
		r2	p-value	r2	p-value
	Basal (unstim)	0.5441	<0.0001	0.1663	0.0312
	10 min	0.1739	0.0273	0.4040	0.0003
	30 min	0.2587	0.0057	0.4635	<0.0001

¹This table reports the r² and p-value of a linear regression of signaling readout (p-p65 or IκBa) and level of bound CD40L (data from Fig. 4). A scatter plot was made where each tonsil was plotted as separate data points (Supporting Information Fig. S5). The p-value indicates if the slope of the regression line is significantly different from zero.

Next, CD40L-induced p-p65 and IκBα degradation was plotted against level of bound CD40L (Fig. 4D). By normalizing all three B cell populations to its own unstimulated control, we here highlight the induced changes in p-p65 and IκBa, not taking the starting level into account. GC responded with less p-p65 than non-GC B cells at the 10 minutes time point, also when comparing equivalent levels of bound CD40L, and the total CD40-induced degradation of IkBa was higher in GC B cells at the 30 minutes time point, as seen in Figure 3A,D (Fig. 4D). Cells with more bound CD40L displayed greater CD40L-induced p-p65, and linear regression showed that this trend was significant for both GC and non-GC B cells (Table 1; p<0.05, Supporting Information Fig. S3). In contrast, equivalent CD40L-induced degradation of IκBα occurred across low, medium, and high levels of CD40L input (Table 1; Supporting Information Fig. S3). This further supported the idea that CD40 levels alone do not control CD40L-induced signaling activity. Together, these results reveal distinct starting states and signaling responses of the classical NFκB pathway in GC cells. In the starting signaling network state, GC B cells have two opposing methods of regulating NFκB p65 signaling: 1) high expression of CD40 and 2) high expression of IκBα. In GC B cells, IκBα degradation was triggered in an all-or-nothing manner that did not depend on number of bound CD40L molecules. NFκB p65 phosphorylation responses differed and were attenuated in GC B cells. This diminished NFκB p65 phosphorylation could be offset by higher CD40 expression. Thus, although CD40L stimulation induced robust signaling responses in both GC and non-GC B cells, different intracellular contexts tuned the responses. These results revealed that CD40 engagement induced robust signaling responses in GC and naive/memory B cells through degradation of IKBα and suggested that cell-intrinsic differences in GC B cells fine-tune NFκB p65 transcriptional activity by tightly regulating phospho-protein signaling.

DISCUSSION

CD40 plays a crucial role in T-cell dependent humoral immune responses as seen by lack of germinal centers and virtual absence of class switched B cells in patients with hyper-IgM syndrome caused by mutated CD40L (20,21). Similarly, CD40 or CD40L knock-out mice are unable to produce germinal centers or IgA, IgE and IgG antibodies in response to T-cell dependent antigens (3,22). Whether CD40-signaling is regulated intrinsically in B cells by regulation of receptor level or by ligand availability has not been determined in humans. Here, we employed phospho-specific flow cytometry and a tailored gating strategy to map signaling responses to distinct levels of bound CD40L. Measurement of IκBα, the main regulator of canonical NFκB activation, revealed higher average basal levels of IκBα expression in GC B cells, as compared to naïve and memory B cells. Despite this difference in starting state, CD40L-induced degradation of IκBα was triggered easily in all B cells and did not depend on the level of bound CD40L. This contrasted with CD40L-induced NFκB p65 phosphorylation, which was dependent on the level of bound CD40L and dampened in GC B cells compared to naïve and memory B cells. Phosphorylation of NFκB p65 at S529 depends on release from IκBα (23) and is thought to fine-tune the transcriptional activity of NFκB (10,24). These experiments cannot determine whether the diminished induction of NFκB p65 phosphorylation seen from 7 to 15 minutes following CD40L was mediated by IκBα. If the active CD40L signal is fading by 7 minutes, the remaining higher IκBα levels may be sufficient to restrain NFκB. High IκBα levels in GC B cells may also serve as a check on activation of the NFκB transcriptional program by signals other than CD40.

The high basal level of IκBα in GC B cells might indicate lower in vivo NFκB activity, and would be consistent with previous studies showing that GC B cells from human tonsils do not express a CD40-induced gene expression signature and that NFκB p65 is retained in the cytoplasm in most GC B cells (25,26). In contrast, the presence of a CD40 gene expression signature was observed in naïve and memory B cells from tonsils (25). The regulation of IκBα is complex, but the high basal IκBα expression observed in GC B cells likely results from a prior signal encountered earlier in the GC reaction, such as BCR engagement or feedback from a prior CD40 signal (27). Prior studies showed that CD40 signaling is activated during the initiation of GC and during the selection process in the light zone (3,4). Indeed, Basso et al. observed that a small subset of GC B cells in the light zone displayed nuclear localization of NFκB, demonstrating active NFκB signaling (25). Several studies suggest that access to stimulation from T follicular helper cells is the limiting factor in selection of B cells in the germinal center (28–30), as these cells are the main source of CD40L and mainly localized in the light zone (1,31). Furthermore, co-engagement of CD40 and BCR is needed for phosphorylation of S6 and induction of cMyc in GC B cells, leading to positive selection in the germinal center (32).

We have previously developed phospho-specific flow cytometry as a tool to dissect signaling events in healthy and malignant B cells (6,33). Here, key experiments were performed by both mass and fluorescence cytometry, providing similar results. The signaling strength of CD40L-induced p-p65 (S529) in malignant B-cells has been shown to vary considerably between B-cell lymphoma patients (14,34), and had clinical significance in both follicular lymphoma (6) and small lymphocytic leukemia/marginal zone lymphoma (35). In these studies, reduced CD40 signaling correlated with poor survival. Our findings here show that induction of p-p65 depended on cell intrinsic factors beyond the level of CD40 expression alone. The contrasting regulation of IκBα and p-NFκB (p65) in response to the same signal, CD40L engagement, show the importance of measuring multiple nodes in a signaling network to fully understand cell signaling dynamics. Phospho-flow provided the ability to track this signaling network over time in rare populations of primary human cells. Furthermore, individual cells within a population could be used to model mathematical relationships between bound ligands and levels of signaling effectors. Thus, the single cell profiling approach revealed novel differences in B cell signaling network regulation that had been overlooked in previous studies of B-cell populations. Notably, human naïve, GC, and memory B cells studied here were all capable of responding to CD40L stimulation. Our findings suggest that early signals in the GC reaction fine-tune subsequent responses of GC B cells. These results provide a detailed map of CD40 signaling kinetics in human B cells undergoing germinal center reactions that can serve as a reference for comparisons with human diseases, including allergy, autoimmunity, and cancer.