Interleukin-2-secreting T helper cells promote extra-follicular B cell maturation via intrinsic regulation of a B cell mTOR-AKT-Blimp-1 axis

Abstract

During antigen-driven responses, B cells can differentiate at extra-follicular (EF) sites or initiate germinal centers (GCs) in processes that involve interactions with T cells. Here, we examined the roles of interleukin (IL)-2 secreted by T helper (Th) cells during cognate interactions with activated B cells. IL-2 boosted the expansion of EF plasma cells and the secretion of low-mutated immunoglobulin G (IgG). Conversely, genetically disrupting IL-2 expression by CD4⁺ T cells, or IL-2 receptor (CD25) expression by B cells, promoted B cell entry into the GC and high-affinity antibody secretion. Mechanistically, IL-2 induced early mTOR activity, expression of the transcriptional regulator IRF4, and metabolic changes in B cells required to form Blimp-1-expressing plasma cells. Thus, T cell help via IL-2 regulates an mTOR-AKT-Blimp-1 axis in activated B cells, providing insight into the mechanisms that determine EF versus GC fates and positioning IL-2 as an early switch controlling plasma cell versus GC B cell commitment.

Keywords: CD4 T cells; IL-2 cytokine; T follicular helper cells; Tfh cells; antibodies; mTOR; metabolism; plasma cells; vaccine.

Figure 1.. In vivo IL-2 administration enhances EF B_PC differentiation and antibody secretion upon eOD-GT5 immunization

(A) Adoptive transfer of CD45.2⁺ Smarta T cells and CD45.2⁺ VRC01^gHL B naïve cells into CD45.1⁺ B6 recipients prior to intra-peritoneal (i.p.) immunization with 20 µg alum-precipitated eODGT5_gp6160mer. Mice were treated (day 1 to 3) with recIL-2 or vehicle (PBS). Box cartoon showing GT5⁺⁺ and VRC01^gHL B cells detection with tetramers. (B) Total B_GC frequency on day 3.5 and day 7. (C) B_PB/B_PC gating strategy on day 3.5. (D) Frequency of endogenous EF B_PB on day 3.5. (E) Analysis of eOD-GT5⁺⁺ B-endo. (F) Quantitation of EF B_PB/_PC VRC01^gHL on day 3.5. Data from n=2 experiments. (G) Adoptive transfer of CD45.1⁺ Smarta T cells and VRC01^gHL uGFP B naïve cells (number of cells transferred: 1 x10³ (squares in graphs) or 1 x10⁵ (circles in graphs)) into CD45.2⁺ B6 recipients prior to immunization as in (A) and recIL-2 treatment (from day 3 to 6). (H) PC to GC ratio of total B cells on day 7 (Experiments #1–4). (I) Day 7 analysis of eOD-GT5⁺⁺ B cells recognizing non-CD4bs (KO⁺ probe) and CD4bs (KO⁻ probe) and their differentiation into GC- or PC-like cells (Experiments #5 and #6). (J) Flow plots and quantitation of B_GC (GL7⁺CD138⁻), B_PC (GL7⁻CD138⁺ or B220⁻CD138⁺) and B_PB (B220^+/lo CD138⁺) VRC01^gHL GFP⁺B220^+/− B cells. (K) VRC01 ^gHL B_PC and B_GC correlation (L) VRC01^gHL B_PC to B_GC ratio. Data in J-L are from day 7 (Experiments #1–4). Each dot represents an individual mouse. P values were determined by two-tailed Student’s t test. See also Figure S1 and S2.

Figure 2.. IL-2 treatment enhances the formation of low mutated plasma cells with increased antibody-secretion capacity

(A) ELISpot enumeration of splenic IgG-secreting ASC (day 7) (Experiments #1–3). (B) ELISA sera tested against eOD-GT8 and HxB2^N276D probes. Each dot represents an individual mouse. Lines in graphs are mean values. P values were determined by a two-tailed Student’s t test. (C) Adoptive cell transfer and VRC01^gHL B cell population sorted and BCR analyses steps. (D) Frequency of observed heavy chain (HC) aa mutations per residue position at day 12 on VRC01^gHL B_PC. CDRs are highlighted in grey. Data from n= 2 experiments. Mixed-linear effect statistics (two-way ANOVA) were applied. (E) Representative pies showing the fraction of germline (GL) and accumulated mutations. Ratio of non-GL VRC01-VH reads is shown on the left graph with applied unpaired t-test. See also Figure S1 and S2.

Figure 3.. IL-2⁺ Smarta cells regulate VRC01^gHL cell GC or PC differentiation

(A) Adoptive cell transfer of CRISPR/Cas9-modified gCd8a or gIl2 Smarta cells and VRC01^gHL uGFP naïve B cells into CD45.2⁺ B6 recipient mice prior to immunization with 20 µg of eODGT5_gp6160mer immunogen. (B) BCL6, CXCR5, and PD-1 expression on gRNA Smarta cells (CD4⁺Ametrine⁺). (C) Quantitation of total Smarta and Smarta Tfh cells from mice receiving gCD8a Smarta (controls, n=26) and gIl2 Smarta (KO, n=24). (D) Day 7 splenocytes re-stimulated with gp-66 or PMA/ionomycin. FACS plots show IL-2 and CD40L. (E) Quantitation of IL-2 upon stimulation of gCd8a or gIl2 Smarta. (F) FACS plots showing gCd8a Smarta Tfh (CXCR5⁺PSGL-1⁻) and non-Tfh (CXCR5⁻PSGL-1⁺) subsets and expression of IL-2 and CD40L. (G) Relative quantitation of IL-2 secreting cells from each indicated population as in (F). Data from n=3 experiments. (H) FACS plots showing the quantitation of VRC01^gHL cells (gated as viable, CD3⁻CD19⁺ GFP⁺B220^+/− B cells) and their differentiation into B_GC (GL7⁺CD138⁻) or B_PC (GL7⁻CD138⁺, B220⁻CD138⁺ and B220⁻IRF-4⁺) scored on day 7. (I) Quantitation of VRC01^gHL B cell populations. Each dot represents an individual mouse. Lines in graphs represent the mean value. Data from n=3 or 4 experiments. P values were determined by a two-tailed Student’s t-test. See also Figure S3 and S4.

Figure 4.. IL-2 is required to promote in vitro terminal plasma cell differentiation and acts intrinsically on CD25 expressing B cells

(A) Diagram showing the B cell culture system. (B) Representative FACS plots showing IRF4, PAX5, B220 and CD138 expression on day 4. (C) Relative quantitation of each indicated subset and stimulus tested. (D) Representative FACS plots showing the expression of B220, CD138, Blimp-1 YFP (YFP-reporter mice), and CD25 on day 4. (E) Quantitation of differentiated B cells into CD138^posB220^neg, CD138^negB220^neg. Blimp-1^posCD20^neg and Blimp-1^posCD25^pos cells. Lines in graphs are mean values. Data from n=13 experiments. (F) Diagram showing the gRNA VRC01^gHLxCas9^EGFP culture system. (G) Representative FACS plots showing gCd8a (control) and gIl2ra (CD25 deficient) B cells differentiated into B220⁻CD138⁺Blimp-1 YFP⁺ plasma cells and (H) their quantitation on day 2 post in vitro stimulation. Data from n=20 experiments. (I) ELISA quantitation of secreted IgM and IgG from supernatants collected on day 2 and day 4. Data from n=3 experiments. (J) ELISpot quantitation of IgM and IgG ASC at day 4. Data from n=5 experiments. Each dot represents an individual mouse. Error bars represent the SEM. P values were determined by a two-tailed Student’s t or paired ANOVA test for dosing experiments. See also Figure S4.

Figure 5.. In vivo IL-2 signals intrinsically control B cell maturation and their EF B_PC versus B_GC differentiation

(A) Adoptive cell transfer of 5 x10⁴ Smarta T cells CD45.1⁺ and transduced VRC01^gHL x Cas9^EGFP B cells (0.5–2 x 10⁶/mouse) into wild type B6 CD45.2⁺ recipient mice. Mice were immunized with 20 µg of eODGT5_gp6160mer in alum and spleens analyzed at 8–10 days. Sera tested in ELISA at 3 months post-immunization. (B) FACS plots showing VRC01^gHL x Cas9^EGFP transduced B cells (Cas9⁺Ametrine⁺) and their differentiation into B_PC (B220⁻GL-7⁻CD138⁺) or B_GC (GL-7⁺CD138⁻). (C) Frequency of splenic gCd8a and gIl2ra VRC01^gHL x Cas9^EGFP . (D) Quantitation of B_PC VRC01^gHL cells as CD138⁺GL7⁻ and CD138⁺B220⁻ cells, day 8–10. (E) Quantitation of B_GC VRC01^gHL cells as CD138⁻GL7⁺. (F) Ratio of PC/GC of Ametrine-positive VRC01^gHL. (G) FACS plots showing GL-7/IgG1-positive cells. (H) Analysis of membrane-bound IgG1 (mbIgG1) VRC01^gHL B cells from gCd8a, gIl2ra B cells and gAicda VRC01^gHLxCas9^EGFP B cells. Data from n=5 experiments. Line and error bars represent the mean. (I) ELISA test for eOD-GT8 IgG and (J) their A.U.C. quantitation. (K) eOD-GT8- and (L) HxB2^N276D-specific IgG from sera collected on day 90 post-immunization and (M) A.U.C for HxB2^N276D IgG. Each dot represents an individual mouse. P values were determined by a two-tailed Student’s t test. See also Figure S5 and S6.

Figure 6.. IL-2 controls the plasma cell gene regulatory network by inducing high expression of IRF4 and Blimp-1, in a Bach2 independent manner

(A) FACS plots and quantitation of B220^negIRF4^pos/hi PC. Data are from n=4 or 5 experiments. (B) IRF4 expression on day 4 proliferated CTV^lo gRNA VRC01^gHL B cells. Data from n=3 pooled experiments. (C) Dot plots B_PB (B220⁺CD138⁺) and B_PC (B220⁻CD138⁺) and IRF4-expressing cells on day 4 and (D-F) their relative quantitation. (E, G) Ratio of B_PB/B_PC. Data from n=4 experiments. (H) Quantitation and B_PB/B_PC ratio upon stimulation of gCd8a and BACH2 deficient (gBach2) VRC01^gHL cells on day 2. Data from n=7 experiments. (I) Simple model of IL-2 regulation of B cells differentiation. P values were determined by a two-tailed Student’s t-test or paired ANOVA test for dosing experiments. See also Figure S5 and S7.

Figure 7.. IL-2 acts via the mTOR-AKT-Blimp-1 axis to control the B cell metabolic state

(A) CD98 and CD71 expression on day 4. (B) Quantitation of CD98⁺CD71⁺ B cells on day 2 and (C) day 4. (D) Analysis of B_PC on day 4. Data from n=5 experiments. (E) CD98 and CD71 expression on gCD8a and gPrdm1 VRC01^gHL YFP-reporter cells. Data from n=4 experiments. (F) FACS plots showing the expression of MitoTracker Green (MTG), MitoTracker Red CMXRos (CMXRos), and their MFIs compared to unstained control (day 4 of culture). (G) Mito^low cells on day 4. (H) MitoSOX-positive B cells on day 4. Data from n=5 experiments. Error bars represent SEM. (I) TMRM staining and quantitation. (J) Calculated membrane potential. Data from n=3 experiments. (K) CD98 and CD71 expression on endogenous B, non-PC (day 3.5) and their quantitation. (L) CD98 and CD71 expression on VRC01^gHL B, non-PC (day 3.5) and their quantitation. (M) Overlay of CD98 and pS6 and quantitation on day 3.5. (N) CD98, CD71, pS6, and Ly6C expression on B cells upon in vivo immunization. Quantitation of CD98, CD71, pS6, and Ly6C expressing B cells on day 3.5 (O) and day 7 (P). Data from n=2 experiments on day 3.5 and n=2 experiments on day 7 (Experiments #5 and #6). P values were determined by an unpaired Student t-test. See also Figure S8 and S9.