Second signals rescue B cells from activation-induced mitochondrial dysfunction and death

Abstract

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response–activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.

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Fig. 1: Similar metabolic changes occur immediately following B cell stimulation through either the BCR or TLR9.
Fig. 2: Glycolytic capacity and maximal mitochondrial respiration are correlated with B cell survival.
Fig. 3: Increases in mitochondrial mass in response to activation via the BCR and/or TLR9.
Fig. 4: B cells stimulated only via their BCRs in vitro show mitochondrial dysfunction.
Fig. 5: B cells stimulated with antigen alone in vivo show mitochondrial dysfunction.
Fig. 6: Antigen-induced mitochondrial dysfunction in B cells correlates with the strength and duration of the BCR stimulation.
Fig. 7: T cell help prevents antigen-induced mitochondrial dysfunction in B cells.
Fig. 8: Antigen-induced mitochondrial dysfunction results from increases in intracellular calcium.

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Acknowledgements

We thank S. Bolland (National Institutes of Health) for TLR9-deficient mice; I. Gery (National Institutes of Health) for 3A9mice; O. Voss (National Institutes of Health) for the NIH3T3 mouse fibroblast cell line; P. Allen (Washington University) for the 3A9 mouse T cell hybridoma line; R. Kissinger for preparing the illustration in Supplementary Fig. 2; and P.W. Sheehan, T. Leto, J. Brzostowski and J. Manzella-Lapeira for assistance and advice in various experiments. Supported by the National Institutes of Health Intramural Research Program, National Institute of Allergy and Infectious Diseases and National Heart, Lung, Blood Institute.

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M.A. wrote the manuscript; M.A., J.T., A.S.R.,P.M., B.A., B.P.T., H.S., M.P., M.S. and D.W.D. carried out experiments; M.A., J.T., A.S.R., P.M., B.A., B.P.T., H.S., J.K., E.D. and J.S. analyzed data; M.A., J.T., A.S.R., P.M., B.P.T. and S.K.P. designed the experiments; M.A. and S.K.P. conceived of the project; M.N.S. and S.K.P. edited the manuscript; and S.K.P. secured funding;

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Correspondence to Munir Akkaya or Susan K. Pierce.

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Integrated supplementary information

Supplementary Figure 1 Early changes in B cell metabolism following B cell stimulation do not require cellular remodeling.

a-j) Purified mouse B cells were stimulated with 1 μM CpG and/or 5 μg/ml anti-IgM or left unstimulated (media only). The flow cytometry plots (a,c) and MFI graphs (b,d) of TMRM alone, TMRM + Oligomycin, TMRM + FCCP at 1 h (a,b) and 4 h (c,d) post stimulation; flow cytometry plots (e,f) and MFI graphs (g) of GLUT 1 and 3 at 4 h post stimulation; change in MFI of 2NBDG (added to the culture at 10 μM) between 0–2 h (h) post stimulation and representative flow cytometry plot showing the 2NBDG levels at 2 h (i) and expression levels of TOM20 at 4 h (j) post stimulation are shown. Data represents three independent experiments each done with triplicates. Bars indicate the mean of the triplicates and error bars represent the standard deviation. (P > 0.05 = n.s.) (one sided two-way ANOVA).

Supplementary Figure 2

Schematic illustration, depicting the metabolic functions of genes that are transcriptionally regulated in response to B cell activation through TLR9 and /or BCR.

Supplementary Figure 3 The roles of glycolysis and oxidative phosphorylation in B cell functionality and survival.

a) Representative flow cytometry plots for the experiment outlined in Fig. 1i. b-c) Purified mouse splenic B cells were stained with e450 proliferation dye and then cultured in growth media alone or media supplemented with 1 μM CpG and/or 5 μg/ml anti-IgM. Flow cytometry plots (b) and bar graphs (c) demonstrating the total proliferating cells and cells that have proliferated at least two times at 24 h and 48 h post stimulation are shown. Bars and error bars indicate mean and standard deviation respectively. Data is representative of three independent experiments. d) Graphs representing the changes in the fold expression of CD69 in the experiment outlined in Fig. 1j are shown. (P > 0.05 = n.s.; P ≤ 0.0001 = ****) (One-way ANOVA with Tukey’s adjustment).

Supplementary Figure 4 Long term cellular and metabolic changes following B cell stimulation.

a,b) Unstimulated B cells and B cells stimulated with anti-IgM (5 μg/ml) were harvested at 0 (unstimulated only) 3, 8 or 24 h post stimulation and the percentages of live, early apoptotic, late apoptotic and necrotic cells were determined by staining the cells with both 7AAD and VAD (FAM-FLICA). Representative flow cytometry plots (a) and quantification of each population in triplicates for each time point (b). Bars and error bars represent mean and standard deviation respectively. Data are representative of two independent experiments. c) Representative flow cytometry plots showing the GLUT 1 expression 24 h post stimulation of WT and TLR9 KO B cells in the experiment outlined in Fig. 2i. d) Representative flow cytometry plot showing the 2NBDG staining at 24 h (120 min after addition of 2NBDG) in the experiment outlined in Fig. 2j e) Representative flow cytometry plots for the experiment outlined in Fig. 3a

Supplementary Figure 5 BCR activation induced mitochondrial changes in B cells.

a) B cells were purified from WT mice and cultured in growth media alone or media supplemented with 1 μM CpG and/or 5 μg/ml anti-IgM for 24 h. Cells were then harvested and stained with Live/DEAD stain and MitoTracker Green. Stained cells were immobilized in chambers coated with Poly-L-lysine and imaged under confocal microscope. Representative images showing the MitoTracker Green staining in viable B cells for each stimulation condition are shown. b-e) Representative flow cytometry plots for experiments outlined in Fig. 4c. (b), Fig. 4d (c), Fig. 4i (d) and Fig. 4k (e) are shown.

Supplementary Figure 6 Outline of the adoptive transfer strategy.

a) Depiction of the experimental design described in Fig. 5. Purified B cells from the spleens of WT (CD45.1) and MD4 (CD45.2) mice were mixed 1:1, stained with e450 and adoptively transferred into WT (CD45.2) recipient mice (4.5 × 106 cells per mouse). Mice were injected i.v. 24 h post transfer with 200 μl PBS alone, PBS containing HEL (100 μg HEL/mouse), PBS containing CpG (100 μg CpG) or PBS containing HEL and CpG, 24 h later mice were euthanized and splenocytes were harvested. b) Adoptively transferred B cells from WT and MD4 mice were identified in the spleens of the recipient mouse using the gating strategy shown. Gating of singlets and live cells carried out prior to this step are not shown.

Supplementary Figure 7 BCR stimulation mediated changes in mitochondrial function are linked to the intracellular Calcium levels.

a) Representative flow cytometry plot for the experiment outlined in Fig. 6a. b) Histogram overlay demonstrating the expression levels of recombinant proteins consisting of rCD4 attached to WT or mutant HEL or DEL proteins on the surfaces of NIH3T3 cells as detected by anti rCD4 antibody. c-f) Representative flow cytometry plots for the experiment outlined in Fig. 8 h-k respectively.

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Akkaya, M., Traba, J., Roesler, A.S. et al. Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nat Immunol 19, 871–884 (2018). https://doi.org/10.1038/s41590-018-0156-5

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