Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis

Yazicioglu, Yavuz F.; Marin, Eros; Sandhu, Ciaran; Galiani, Silvia; Raza, Iwan G. A.; Ali, Mohammad; Kronsteiner, Barbara; Compeer, Ewoud B.; Attar, Moustafa; Dunachie, Susanna J.; Dustin, Michael L.; Clarke, Alexander J.

doi:10.1038/s41590-023-01484-3

Download PDF

Article
Open access
Published: 24 April 2023

Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis

Nature Immunology volume 24, pages 991–1006 (2023)Cite this article

11k Accesses
9 Citations
47 Altmetric
Metrics details

Subjects

Abstract

Germinal center (GC) B cells undergo proliferation at very high rates in a hypoxic microenvironment but the cellular processes driving this are incompletely understood. Here we show that the mitochondria of GC B cells are highly dynamic, with significantly upregulated transcription and translation rates associated with the activity of transcription factor A, mitochondrial (TFAM). TFAM, while also necessary for normal B cell development, is required for entry of activated GC precursor B cells into the germinal center reaction; deletion of Tfam significantly impairs GC formation, function and output. Loss of TFAM in B cells compromises the actin cytoskeleton and impairs cellular motility of GC B cells in response to chemokine signaling, leading to their spatial disorganization. We show that B cell lymphoma substantially increases mitochondrial translation and that deletion of Tfam in B cells is protective against the development of lymphoma in a c-Myc transgenic mouse model. Finally, we show that pharmacological inhibition of mitochondrial transcription and translation inhibits growth of GC-derived human lymphoma cells and induces similar defects in the actin cytoskeleton.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy

Article Open access 10 April 2024

Jack D. Chan, Christina M. Scheffler, … Phillip K. Darcy

MEF2C regulates NK cell effector functions through control of lipid metabolism

Article 08 April 2024

Joey H. Li, Adalia Zhou, … Timothy E. O’Sullivan

Dictionary of immune responses to cytokines at single-cell resolution

Article Open access 06 December 2023

Ang Cui, Teddy Huang, … Nir Hacohen

Main

The germinal center (GC) reaction is a highly spatially organized process in secondary lymphoid tissue essential for humoral immunity¹. B cells responding to antigen captured by follicular dendritic cells introduce random mutations in their immunoglobulin genes in a process known as somatic hypermutation (SHM), which occurs in the anatomically defined dark zone, and are then selected through competitive interaction with follicular helper T cells (T_FH) in the light zone. GC B cells cycle between these zones, leading to antibody affinity maturation and eventually formation of memory B or plasma cells. During SHM, mutations are introduced in immunoglobulin gene loci through the action of activation-induced cytidine deaminase (AID encoded by AICDA). This can give rise to oncogenic mutations, for example, the translocation of MYC with the IGH or IGL loci². GC B cells are the origin of most diffuse large B cell lymphomas (DLBCL), the most common non-Hodgkin lymphoma.

The metabolic processes that support GC B cell homeostasis are incompletely understood. GC B cells are highly proliferative, with division times as short as 4–6 h, and reside within a hypoxic microenvironment^3,4,5. Typically, rapidly proliferating immune cells mainly use aerobic glycolysis, but GC B cells rely on fatty acid oxidation (FAO) and oxidative phosphorylation, although to what extent is somewhat controversial^{4,5,6,7,8,9,10}. This metabolic phenotype is carried over into a substantial proportion of DLBCLs, which are also oxidative phosphorylation-dependent¹¹. In vitro, mitochondria have been shown to be regulators of B cell signaling through redox-related mechanisms, although whether these studies can be generalized to the hypoxic microenvironment of the GC found in vivo is uncertain^12,13,14.

Regulation of mitochondrial transcription and translation is important in other immune cell types, including cytotoxic CD8⁺ T cells, where it controls cell killing independent of the effects on metabolism, and cytokine production in CD4⁺ T cells^15,16,17. A key regulator of mitochondrial transcription and translation is transcription factor A, mitochondrial (TFAM), a DNA-binding high mobility box group protein that aids in the packaging of the mitochondrial genome into nucleoids, analogous to the role of histone proteins, and also controls transcription and translation of mitochondrial DNA, serving as a regulator of mitochondrial biogenesis^18,19. In T cells, TFAM controls immunosenescence by restraining the production of inflammatory cytokines²⁰; in fibroblasts and myeloid cells, it acts to regulate an antiviral immune state²¹. Recent work has shown that the mitochondrial DNA helicase TWINKLE is required for GC formation and plasma cell differentiation²². However, the dynamics, function and regulation of mitochondria in GC B cell biology remain incompletely understood.

We show that GC B cell mitochondria are highly dynamic organelles, undergoing profound structural changes as they transition through the GC reaction. We find that TFAM is dynamically regulated in B cells and is required for their development and transcriptional and spatial entry into the GC reaction by modulating cellular motility. We also demonstrate, using genetically modified mouse models, that TFAM is essential for the development of lymphoma and that pharmacological inhibition of mitochondrial transcription and translation in human lymphoma cells is a potential treatment target for human disease.

Results

GC B cells undergo active mitochondrial remodeling

To determine mitochondrial density and structure in GC B cells, we first immunized mitochondrial reporter mice expressing green fluorescent protein (GFP) and mCherry (mito-QC)²³ with sheep red blood cells (SRBCs) and examined spleen by confocal microscopy and flow cytometry. The mitochondrial GFP signal was strongly localized to GCs and significantly higher than the surrounding B cell follicle (Fig. 1a,b), which we confirmed by flow cytometry (Fig. 1c). We found that the mitochondrial GFP signal was highest in the gray zone, followed by the light zone and then the dark zone (Fig. 1c). We identified highly distinct mitochondrial morphology between naive and GC B cells, with small, fragmented mitochondria in naive B cells and large fused mitochondria in GC B cells (Fig. 1d and Extended Data Fig. 1a). The mito-QC reporter mouse also allows detection of autophagy of mitochondria (mitophagy), as mCherry is resistant to quenching in the acid environment of the autolysosome. Using multispectral imaging flow cytometry, we screened GC B cells for the presence of GFP⁻mCherry⁺ punctae. Although autophagy has been reported in GC B cells²⁴, we did not identify evidence of active mitophagy (Extended Data Fig. 1b–d). We hypothesized that for B cells entering the GC reaction to acquire the high mitochondrial mass we observed, activation of mitochondrial transcription and translation would be required. Using Aicda-Cre × Rosa26^STOPtdTomato reporter mice to label GC B cells, we found markedly increased levels of cytochrome c oxidase subunit 1 (COXI), part of the electron transport chain (ETC) complex IV, in GC B cells (Fig. 1e,f).

To examine mitochondrial transcription, we next labeled B cells in vivo or ex vivo with 5-ethynyl uridine (5-EU), which is incorporated into actively synthesized RNA. We detected colocalization between 5-EU and COXI, with 5-EU incorporation highly prominent in GC B cells and most often seen in areas of high COXI signal (Fig. 1g).

To understand the regulation of this high rate of mitochondrial protein transcription and translation, we examined the expression and distribution of TFAM. We found TFAM protein organized into punctate structures representing mitochondrial nucleoids, which colocalized with 5-EU (Fig. 1g). These were more numerous and larger in GC B cells (Fig. 1h) and had a more elliptical morphology, which is associated with elevated transcriptional activity (Extended Data Fig. 1e)²⁵. We confirmed the increase in nucleoid number using stimulated emission depletion (STED) super-resolution microscopy (Fig. 1i). Mitochondria are therefore highly dynamic in GC B cells, with prominent transcriptional activity associated with increased TFAM-nucleoid content.

Tfam is essential during B cell development and differentiation

TFAM has a role in the prevention of long-term inflammatory immunosenescence in T cells but is dispensable for their development^20,26. Whether TFAM is important for B cell development or function is unknown, as are the dynamics of ETC component expression in B cell developmental trajectories. To address these questions, we first conditionally deleted Tfam in B cells using Cd79a-Cre (hereafter B-Tfam), which is active from the early pro-B cell stage²⁷. TFAM was efficiently deleted in mature splenic B cells (Extended Data Fig. 2a,b). B-Tfam mice appeared healthy, with no clinical signs of immunosenescence or overt autoimmunity. However, B-Tfam mice had a profound reduction in the peripheral B cell compartment (Fig. 2a and Extended Data Fig. 2c–e). Analysis of B cell development in the bone marrow indicated a failure of progression from the pro- to the pre-B cell stage (Fig. 2b,c and Extended Data Fig. 2f). We noted that heterozygous B-Tfam mice had normal B cell development and an intact peripheral B cell compartment (Extended Data Fig. 2g,h).

To understand the expression of ETC proteins during B cell development, and the effect of Tfam deletion, we quantified a panel of ETC proteins by high-dimensional spectral flow cytometry in B wild-type (B-WT) and B-Tfam bone marrow and spleen (Fig. 2d,e). We found that in B-WT bone marrow, there was a progressive increase in the expression of most ETC proteins from the pre-pro-B stage (Hardy fraction A²⁸), peaking at the earliest pre-B subset (fraction C′) and then substantially falling in the later stages (fractions D–F). Interestingly, TFAM expression peaked early (fraction A), suggesting that it might initiate a mitochondrial transcription program leading to upregulation of ETC proteins. The maximum expression of ETC proteins corresponded to the developmental block seen in B-Tfam mice (pro- to pre-B cell stage). B-Tfam mice deleted TFAM from fraction A onwards in the bone marrow and TFAM remained deleted in splenic B cell subsets; accordingly, mitochondrially encoded COXI was downregulated. The nuclear-encoded ETC components COXIV, cytochrome C, ATP5A1 and succinate dehydrogenase A (SDHA) were increased.

In the periphery, ETC component expression was similar across transitional and follicular B cells but increased in marginal zone B cells (Fig. 2e). TFAM deletion led to a much more marked upregulation of nuclear-encoded ETC proteins. This was evident in a mismatch in the ratio of proteins encoded by mitochondrial or nuclear genomes in the same ETC complex, that is, COXI and COXIV of complex IV. Extracellular flux analysis of unstimulated peripheral B cells showed no difference in oxygen consumption rate (OCR) but a significant increase in extracellular acidification rate in B-Tfam mice (Extended Data Fig. 2i).

Also increased in B-Tfam B cells were the mitochondrial unfolded protein response (UPR^mt)-associated proteins heat shock protein 60 (HSP60) and mitochondrial 70-kDa heat shock protein (mtHSP70) (Fig. 2d,e). These results demonstrate that ETC components are dynamically regulated across B cell developmental trajectories and that TFAM is required for the expression of mitochondrially encoded proteins.

We next examined the ability of B-Tfam mice to generate GCs. Examination of immunized B-Tfam spleens revealed smaller and fewer B cell follicles and an almost complete absence of GL-7⁺ GCs compared to B-WT mice (Fig. 2f,g). Tfam heterozygosity in B cells also led to a significantly reduced GC response after SRBC immunization (Extended Data Fig. 2j). These results collectively suggest that TFAM is essential for normal B cell differentiation and that in situations of low-energy demand, partial respiratory compensation is possible despite substantially reduced translation of mitochondrial proteins.

GC B cells require TFAM

Having established a role for TFAM in B cell development and ETC complex balance, we next focused on its function in the GC reaction. To specifically delete Tfam in GC B cells, we generated Aicda-Cre × Tfam^l^oxP × Rosa26^STOPtdTomato × Blimp1-mVenus mice (hereafter Aicda-Tfam), which allow simultaneous identification of cells that have expressed Aicda (activated, GC, memory B and plasma cells) and/or currently express Blimp1 (encoded by Prdm1) (by detecting the fluorescent reporter proteins tdTomato and mVenus, respectively) (Fig. 3a). Aicda-Tfam mice were immunized with SRBCs and analyzed on day 12. Immunofluorescence staining of Aicda-Tfam spleen sections demonstrated small GCs in reduced numbers compared with Aicda-WT controls (Fig. 3b). Clusters of plasma cells in the splenic cords were much smaller in Aicda-Tfam mice and Blimp1-mVenus⁺tdTomato⁺ cells were poorly represented, suggesting impairment of GC output (Fig. 3c). The proportion of CD138⁺tdTomato⁺ long-lived plasma cells in bone marrow was also significantly lower (Extended Data Fig. 3a). We found that the generation of CD19⁺IgD^loCD38⁻GL-7⁺ (and correspondingly CD19⁺IgD^loCD38⁻tdTomato⁺) GC B cells was substantially reduced in Aicda-Tfam mice as measured by flow cytometry (Fig. 3d,e). We also confirmed a reduction in Blimp1-mVenus⁺tdTomato⁺ post-GC plasma cells (Fig. 3f).

Next, we examined antigen-specific GC formation by immunizing mice with (4-hydroxy-3-nitrophenyl) acetyl-chicken gamma globulin (NP-CGG). The proportion and absolute numbers of NP-binding GC B cells were significantly reduced in Aicda-Tfam mice, as were those of plasma cells measured in situ; the plasma cell to GC B cell ratio was correspondingly decreased (Fig. 3g and Extended Data Fig. 3b–d). Affinity maturation was also compromised, with decreased binding of IgG1 antibodies to NP_1–4 compared with NP_>20 (Fig. 3h and Extended Data Fig. 3e,f). However, there was no difference in IgM anti-NP antibodies, which is in keeping with the extrafollicular origin of this response, produced by plasmablasts that have not expressed Aicda (Extended Data Fig. 3c,g).

To understand if loss of Tfam compromised B cell memory, we immunized Aicda-Tfam and Aicda-WT mice with NP-CGG in alum and then boosted them on day 30 with NP-CGG in PBS. On day 70 there were substantially fewer NP-binding tdTomato⁺CD38⁺IgG1⁺ memory B cells (Fig. 3i,j) and on day 49 a reduction in IgG1 anti-NP antibodies reactive against NP_1–4 and NP_>20 (Extended Data Fig. 3h). The number of plasma cells in the bone marrow was also significantly reduced on day 70 (Fig. 3k).

Mitochondria play a central role in the regulation of apoptosis, but surprisingly the apoptosis rate detected by means of active caspase 3 staining in Aicda-Tfam GC B cells was comparable with that of Aicda-WT controls (Extended Data Fig. 3i). In situ TUNEL also demonstrated unaltered apoptosis (Extended Data Fig. 3j). There were no significant differences in cell cycle dynamics between Aicda-Tfam and Aicda-WT mice (Extended Data Fig. 3k,l). We also confirmed that Tfam deletion did not affect proliferation or cell viability in vitro (Extended Data Fig. 3m–p).

These data demonstrate that loss of Tfam markedly compromises GC B cell differentiation and output but without detectable effects on proliferation and survival in those cells already committed to the GC fate.

TFAM controls transcriptional entry into the GC program

To examine the effects of Tfam deletion on the transcriptional program of GC B cells, we performed combined single-cell gene expression profiling and V(D)J sequencing. We immunized Aicda-Tfam and Aicda-WT mice with NP-CGG and sorted tdTomato⁺ cells on day 14 (Fig. 4a). This population includes any B cells that have expressed Aicda and so will include pre-GC, GC, memory and plasma cells. We identified nine shared clusters, broadly separated into B cells from GC, CD38⁺ non-GC and plasma cell populations (Fig. 4b,c). Cluster 0 (C0), which expressed markers of immaturity suggestive of an activated precursor (AP) state (Ighd, Ccr6, Gpr183, Cd38, Sell and low levels of Bcl6), was significantly expanded in Aicda-Tfam compared with Aicda-WT mice (Fig. 4d).

Examination of differential gene expression in C0 revealed, as expected, broad dysregulation of mitochondrial gene expression in Aicda-Tfam mice, in keeping with the function of TFAM as a regulator of mitochondrial transcription (Fig. 4e). Pathway analysis²⁹ demonstrated substantial downregulation of translation initiation and elongation gene sets (Fig. 4f). Gene components of the activator protein 1 (AP-1) signaling pathway Jun, Junb, Jund and Fos were all significantly upregulated (Fig. 4g). The AP-1 pathway is broadly upregulated by cellular stress signaling, reactive oxygen species and in response to environmental cues^30,31.

Analysis of the main GC cluster (C1) demonstrated as before dysregulation of mitochondrial and ribosomal gene transcription, with upregulation of Jun, Fos and Junb (Fig. 4h,i). Notably increased in Aicda-Tfam cells was Rgs1 (regulator of G-protein signaling-1), a GTPase-activating protein that has an important role in the negative regulation of cell movement in response to chemokines (for example, CXCL12)^32,33 (Fig. 4j). Downregulated were Coro1a (coronin-1), which encodes an actin-binding protein required for cell migration³⁴, and Arpc3 (actin-related protein 2/3 complex subunit 3) (Fig. 4h), which mediates branched actin polymerization and actin foci formation³⁵.

We next examined B cell clonality by evaluating V(D)J sequences. Because we sorted all tdTomato⁺ B cells, the overall clonal diversity of all samples was high (Fig. 4k). As expected in the anti-NP immune response, V_H1-72 usage was dominant (Fig. 4l). However, there was more diversity in Aicda-Tfam mice, with fewer larger clones, suggesting that there was less ability for the evolution of dominant clones.

There was significantly less SHM in Aicda-Tfam B cells (Extended Data Fig. 4a), including in the Ighv1-72 gene in GC B cells (Extended Data Fig. 4b,c). The W33L mutation in CDR1 and substitution of K59 of the V_H1-72 heavy chain confer increased affinity for NP^36,37. There were significantly lower W33L and K59 substitution rates in the Aicda-Tfam GC B cell cluster, in keeping with our observation that high-affinity NP binding was reduced (Fig. 4m). There was no W33L mutation and negligible K59 substitution in the AP cluster (C0) of either Aicda-WT or Aicda-Tfam mice, reflecting their pre-GC state (Extended Data Fig. 4d).

Therefore, transcriptional analysis revealed evidence of failure of Aicda-Tfam B cells to transition from an AP to a committed GC B cell phenotype.

TFAM is required for GC B cell commitment

Given the relative accumulation in immunized Aicda-Tfam mice of AP B cells with high expression of Sell (selectin L), Ccr6 and a GC transcriptional profile suggestive of altered cell trafficking and cytoskeleton dynamics, we hypothesized that Tfam is required for activated B cells to enter the GC and remain appropriately spatially positioned.

We first confirmed the proportional expansion of APs, defined as tdTomato⁺CD38⁺IgD⁺, in Aicda-Tfam mice after NP-CGG immunization (Fig. 5a). Despite a significant numerical reduction in GC B cells, the AP population was maintained (Extended Data Fig. 5a). This was also seen to a more striking extent in B-Tfam mice immunized with SRBC, with APs defined as IgD⁺GL-7^int (Extended Data Fig. 5b). We found that in Aicda-WT mice, a lower proportion of AP B cells bound NP compared to GC B cells, and did so with lower affinity, in keeping with their lack of a W33L mutation and low levels of SHM (Extended Data Fig. 5c).

**Fig. 5: TFAM is required for GC B cell commitment.**

We then examined the splenic sections of immunized Aicda-Tfam mice under high magnification. We found a highly disorganized GC architecture, with poor GC B cell compartmentalization; within the follicle, there were relatively many more tdTomato⁺IgD⁺ B cells in Aicda-Tfam mice (Fig. 5b). The level of B cell lymphoma 6 (BCL6) protein, a master transcriptional regulator of GC commitment and entry, was lower in Aicda-Tfam GC B cells (Extended Data Fig. 5d).

We next asked whether it was possible to overcome the failure of AP B cells to enter the GC by adoptively transferring preactivated Tfam^−/− AP-like B cells into primed congenically marked mice. We used the induced GC B cell (iGB) culture system³⁸ and TAT-Cre to delete Tfam in Tfam^loxP × Rosa26^STOPtdTomato B cells, or with WT congenically marked CD45.1/2 control B cells. This experimental design allows competitive transfer of activated B cells to take place (Fig. 5c). The resulting Tfam^−/− iGB cells effectively deleted TFAM on day 4 of culture and this resulted in decreased expression of mitochondrially encoded ETC proteins and upregulation of nuclear-encoded proteins, as seen in splenic B cells from B-Tfam mice (Extended Data Fig. 5e). Loss of Tfam did not affect cell expansion over 4 days of culture (Extended Data Fig. 5f). However, after 5 days, Tfam^−/− iGB cells were at a substantial competitive disadvantage in GC participation after adoptive transfer (Extended Data Fig. 5g and Fig. 5d–f).

We reasoned that the defects we observed on deletion of Tfam might be due to abnormalities in T_FH-B cell interaction, or a defective T_FH pool. Although there was no numerical defect in the T_FH compartment (Fig. 5g,h), and antigen presentation was intact (Extended Data Fig. 5h), we noted impairment of T_FH-induced in vitro GC B cell differentiation (Fig. 5i,j and Extended Data Fig. 5i).

To determine whether the defect we saw in Aicda-Tfam mice was B cell-intrinsic, we generated mixed competitive bone marrow chimeras, in which T_FH generation would be intact. Aicda-Tfam GC B cells were outcompeted by WT cells compared with Aicda-WT controls in the spleen and Peyer’s patches (Extended Data Fig. 5j). Overall these data suggest that TFAM promotes the entry or maintenance of activated B cells into the GC and that this function is principally cell-intrinsic.

TFAM regulates mitochondrial translation in activated B cells

We next examined the expression of TFAM and mitochondrial ETC components after immunization. There was dynamic expression of TFAM, which peaked at the AP stage and then subsided after GC entry, and a progressive increase in COXI, which was maximal in GC B cells from Aicda-WT mice (Fig. 6a,b). Other ETC proteins were also highly expressed in GC B cells, including those encoded in nuclear and mitochondrial DNA. Deletion of Tfam led to a program of alteration in mitochondrial ETC protein expression in AP and GC B cells in B-Tfam mice (Fig. 6c), much like that observed during B cell development.

To directly confirm the activity of mitochondrial translation, we then measured incorporation of the amino acid analog O-propargyl-puromycin (OPP) in isolated ex vivo mitochondria from Aicda-WT and Aicda-Tfam B cells by flow cytometry (Fig. 6d,e). The proportion of red fluorescent protein (RFP)⁺ mitochondria (originating from tdTomato⁺ AP/GC B cells) (Extended Data Fig. 6a) was substantially lower in Aicda-Tfam mice, reflecting their defective GC formation (Fig. 6e). We detected significantly more OPP incorporation in RFP⁺ mitochondria than those of non-AP or GC B cell origin (RFP⁻) (Fig. 6f). Translation was decreased in RFP⁺ mitochondria from Aicda-Tfam B cells compared to Aicda-WT cells (Fig. 6g); using Tfam^−/− iGB cells, treatment with the mitochondrial translation inhibitor and oxazolidinone antibiotic chloramphenicol (CHL)³⁹ did not lead to additional suppression, confirming the defective translation in mitochondria (Extended Data Fig. 6b).

We then examined the metabolic consequences of Tfam deletion on APs and GC B cells, quantifying cytoplasmic protein translation rate by the incorporation of OPP as a proxy for metabolic capacity, as recently reported⁴⁰. GC B cells had significantly higher basal OPP incorporation than AP B cells, reflecting their high levels of metabolic activity (Extended Data Fig. 6c). Loss of Tfam led to an increase in glucose dependence and a marked reduction in FAO/amino acid oxidation (AAO) capacity in AP B cells, seen to a lesser extent in GC B cells (Fig. 6h and Extended Data Fig. 6c,d). These results were mirrored by Seahorse extracellular flux analysis in activated B cells and iGBs (Fig. 6i and Extended Data Fig. 6e,f).

Therefore, loss of TFAM compromised GC B cell mitochondrial translation and impaired metabolic homeostasis.

Tfam deletion disrupts B cell mobility

We found that Aicda-Tfam GCs were poorly compartmentalized, with smaller dark zones (Fig. 7a) and a disrupted dark zone to light zone ratio measured using flow cytometry (Fig. 7b).

Positioning of B cells in the GC is controlled by the chemokines CXCL12 and CXCL13, which promote migration to the dark zone and light zone, respectively⁴¹. As our preceding data indicated that AP B cells need TFAM to enter the GC, and given that the transcriptional profile of Aicda-Tfam GC B cells was suggestive of cytoskeletal and mobility defects, we hypothesized that TFAM was required for proper cellular positioning in GCs.

We examined the cellular actin network of TFAM-deficient B cells and found a significant increase in filamentous actin (F-actin) in Aicda-Tfam GC B cells (Fig. 7c,d), which was also evident in B-Tfam B cells (Extended Data Fig. 7a,b). Rearrangement of the actin cytoskeleton is critical for B cell migration⁴²; to understand if Tfam deletion compromised GC B cell motility, we performed a transwell migration assay to determine chemotaxis in response to the chemokines CXCL12 and CXCL13. We found that Aicda-Tfam GC B cells migrated poorly, with significantly reduced chemotaxis compared to Aicda-WT cells (Fig. 7e).

B cell receptor (BCR) and chemokine-driven cytoplasmic calcium mobilization critically regulates F-actin organization in B cells⁴³. As mitochondria are an important reservoir of intracellular calcium⁴⁴, we asked whether Tfam deletion led to dysregulated intracellular calcium levels. After CXCL12 stimulation, B cells from B-Tfam mice failed to sustain peak cytoplasmic calcium levels (Fig. 7f), which we also observed after BCR stimulation with anti-IgM (Fig. 7g). Interestingly, we also detected a significant upregulation in the levels of the mitochondrial calcium uniporter (MCU), suggesting elevated mitochondrial calcium uptake capacity in B-Tfam B cells, whereas CD3⁺ T cells showed comparable MCU expression (Fig. 7h and Extended Data Fig. 7c).

Mitochondrial reactive oxygen species (mtROS) were also substantially increased in B-Tfam naive and APs (Fig. 7i and Extended Data Fig. 7d). ROS activate the AP-1 signaling pathway³¹ and this observation was therefore consistent with the transcriptional profile of Aicda-Tfam AP B cells.

Given these observations, we next tested whether either suppression of mtROS with the scavenger mitoTEMPO⁴⁵ or inhibition of MCU function with the ruthenium compound Ru265 (ref. ⁴⁶) improved cell motility in B-Tfam B cells in response to CXCL12. MCU inhibition did not improve transwell migration but strikingly mitoTEMPO largely rescued the defect in B-Tfam cells (Fig. 7j).

Our data therefore collectively suggest that TFAM is required for proper cellular motility to enable entry into and movement within the GC, and that this effect is associated with regulation of ETC function and mtROS generation.

Tfam deletion in B cells prevents lymphoma

Having demonstrated an essential role for TFAM in B cell development and entry into the GC, we next asked whether it was required for the development of lymphoma. One of the most common mutations giving rise to DLBCL is translocation of MYC to immunoglobulin gene loci, leading to its unregulated expression. c-Myc is a key transcription factor regulating cell cycle and growth, cellular metabolism and mitochondrial biogenesis⁴⁷. We reasoned that deletion of Tfam would counter the oncogenic effects of c-Myc overexpression. We employed the well-established Eμ-Myc transgenic mouse model of lymphoma, in which Myc is expressed under the control of the Igh enhancer⁴⁸. Eμ-Myc mice develop lymphoma with high penetrance from a median age of 11 weeks. To understand what effects overexpression of Myc had on mitochondrial translation, we transferred established lymphoma cells from Eμ-Myc mice into WT CD45.1 congenic hosts to allow us to compare B cells from the same environment (Fig. 8a). We found very markedly higher expression of COXI in transferred lymphoma cells compared with WT B cells, with significantly upregulated TFAM (Fig. 8b,c).

We next generated Cd79a-Cre × Tfam^loxP × Eμ-Myc mice (B-Tfam-Myc). B-Tfam-Myc mice were completely protected from the development of lymphoma during the observation period of 30 weeks, compared with Eμ-Myc controls, which had a median survival of 12.5 weeks (Fig. 8d).

Finally, to establish whether inhibition of mitochondrial transcription and translation might be a therapeutic target in human lymphoma, we treated Daudi B cell lymphoma cells (originally arising from Burkitt lymphoma) with IMT1, a specific inhibitor of mitochondrial RNA polymerase (POLRMT), which functions along with TFAM to initiate mitochondrial RNA transcription⁴⁹. We found that treatment with IMT1 led to a progressive reduction of COXI levels, with increasing COXI:succinate dehydrogenase B (SDHB) mismatch (Fig. 8e,f). Imaging of Daudi cells treated with IMT1 revealed mitochondrial enlargement and confirmed the loss of COXI (Fig. 8g). IMT1 reduced cell growth, inhibited cell cycle progression, increased mtROS levels and recapitulated the F-actin dysregulation we observed with Tfam deletion (Fig. 8h–k and Extended Data Fig. 8a). To inhibit mitochondrial translation, we used CHL. CHL reduced COXI expression and increased expression of F-actin, and the UPR^mt protein LON peptidase 1 (LONP1) in Daudi cells (Fig. 8l,m and Extended Data Fig. 8b). Cell growth was substantially reduced (Fig. 8n).

These results define high rates of mitochondrial translation enabled by Tfam expression as an essential requirement for the development of B cell lymphoma and show the therapeutic potential of POLRMT and mitochondrial translation inhibition in human disease.

Discussion

In this study, we show that on entry to the GC, B cells dramatically remodel their mitochondria, increasing their mass and radically altering their morphology. As part of this transition, mitochondrial translation is highly active and we demonstrate that the nuclear-encoded mitochondrial transcriptional and translational regulator TFAM is not only required for B cell development but also for their entry into the GC program, proper spatial anchoring and for the subsequent development of lymphoma.

GC B cells have a highly distinct metabolism, predominantly relying on oxidative phosphorylation despite their very rapid rate of proliferation in a hypoxic microenvironment^6,8. For rapidly dividing cells to maintain high mitochondrial mass in the face of dilution to their daughter cells, a high rate of mitochondrial protein translation and division must be maintained. We were surprised to find that TFAM is required for entry into the GC program itself; when deleted, there is a proportional accumulation of B cells with an AP phenotype, which have expressed Aicda but maintain markers of immaturity. Recently, the TCA metabolite ɑ-ketoglutarate was shown to be upregulated by interleukin-4 (IL-4) in B cells; this leads to epigenetic alteration of the Bcl6 locus⁹. It is therefore possible that TFAM is required for an initial burst of mitochondrial biogenesis to facilitate GC program entry.

Deletion of TFAM substantially altered the balance of ETC protein expression, with loss of mitochondrially encoded subunits and compensatory upregulation of nuclear-encoded proteins. This was reflected in metabolic disturbance, with impaired oxidative phosphorylation after activation, upregulation of glycolysis and, importantly, mtROS generation, which was seen even in unstimulated cells. Temporal control of Tfam deletion either very early in B cell development using Cd79a-Cre, or after activation with Aicda-Cre or in vitro with TAT-Cre did not lead to unexpected or major phenotypic differences but it is possible that adaptations might occur in a dynamic manner.

An increase in F-actin was consistent across genetic or pharmacological interference with mitochondrial transcription and translation. Dynamic actin cytoskeletal modification is essential for normal cell movement and its probable disruption through ROS accumulation, reversible with the addition of a ROS scavenger, probably contributes to the positioning and motility defects we observed⁵⁰. This adds weight to the idea that mitochondria are intimately linked to cytoskeletal function and that this role may operate independently of ATP generation⁵¹.

These defects collectively have the potential to compromise cellular interaction, in particular that with T_FH cells required to enter the GC program, and for normal dynamics once within. We observed that Tfam^−/− B cells failed to differentiate normally into GC B cells in an ex vivo T_FH coculture system and yet were unaffected when CD40 ligation was artificially provided in the iGB culture system. The capacity of Tfam^−/− iGB cells to present antigen to cognate T cells was normal, however, as were T_FH cell numbers within GCs after immunization. How Tfam deletion in B cells may affect T cell interaction is therefore deserving of future study. We have used the iGB system developed by Nojima et al.³⁸ to precisely control deletion of Tfam before adoptive transfer; while this is an important and widely used tool to generate GC B cell precursors in vitro, which can then participate in the GC reaction in vivo, uncertainties remain about their fidelity to true APs and this would also benefit from future study.

Although we have directly established the importance of TFAM as a regulator of B cell development and activation, the factors driving the counterintuitive switch to oxidative phosphorylation in GC B cells is to be uncovered, as do the signaling mechanisms controlling the differences in mitochondria we observed between GC microenvironments. Disruption of mitochondrial integrity also induces a phenotype associated with immune aging; to what extent the mechanisms we describe might hold true in the diminished humoral immune response seen with age is another area deserving of further exploration²⁰.

We show that deletion of Tfam is sufficient to completely prevent the development of Myc-driven lymphoma. Although loss of Tfam at an early developmental stage leads to B cell lymphopenia, and thus the pool of B cells that may become malignant is reduced, the high penetrance of the model contrasted with the complete protection against lymphoma suggests that this is insufficient to explain the phenotype we observed. How TFAM acts to support lymphomagenesis requires further study but may be associated with its promotion of mitochondrial translation⁵². Our observation that inhibition of mitochondrial transcription and translation reduces growth of lymphoma cells suggest that this should be prioritized as a therapeutic target.

Methods

Mice

B6.Cg-Tfam^tm1.1Ncdl/J (strain 026123), B6.C(Cg)-Cd79a^tm1(Cre)Reth/EhobJ (strain 020505), B6.129P2-Aicda^tm1(Cre)Mnz/J (strain 007770), B6;129S6-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J (strain 007905), B6.Cg-Tg(TcraTcrb)425Cbn/J (strain 004194) and B6.Cg-Tg(IghMyc)22Bri/J (strain 002728) were purchased from The Jackson Laboratory. Tg(Prdm1-Venus)1^Sait [Blimp1-mVenus] (strain 3805969) was a kind gift from M. Saitou (Kyoto University). Gt(ROSA)26Sor^{tm1(CAG-mCherry/GFP)Ganl} (mito-QC) was a kind gift from I. Ganley (University of Dundee). B6.SJL.CD45.1 mice were provided by the central breeding facility of the University of Oxford. Male and female mice between the ages of 6 and 15 weeks were used. Mice were bred and maintained under specific pathogen-free conditions at the Kennedy Institute of Rheumatology, University of Oxford. Mice underwent regular checks to ensure they did not have any pathogenic microorganisms. They were housed in cages that had individual ventilation and were provided with items to stimulate their environment. The temperature was kept between 20 and 24 °C, with a humidity level of 45–65%. They were exposed to a 12-h cycle of light and darkness (7:00 to 19:00), with a 30-min period of dawn and dusk. All procedures and experiments were performed in accordance with the UK Scientific Procedures Act (1986) under a project license authorized by the UK Home Office (PPL no. PP1971784).

Immunization

For SRBC immunization, 1 ml sterile SRBCs (catalog no. 12977755, Thermo Fisher Scientific) were washed twice with 10 ml ice-cold PBS and reconstituted in 3 ml PBS and administered as 0.2-ml injections intraperitoneally. In some experiments, an enhanced SRBC immunization method was followed to maximize GC B cell yield by immunizing mice with 0.1 ml SRBC on day 0 followed by a 0.2-ml second injection on day 5 (ref. ⁵³). For protein antigen immunizations, 50 μg NP_(30–39)-CGG (catalog no. N-5055D-5, Biosearch Tech) was mixed with alum (Thermo Fisher Scientific) or PBS for boost immunization at a 1:1 ratio and rotated at 20 °C (room temperature) for 30 min before intraperitoneal injection. For NP-CGG- and SRBC-based immunizations, day 14 and day 12 were used as read-out time points, respectively, unless specified otherwise.

Flow cytometry and imaging flow cytometry

Flow cytometry was performed as described previously⁵⁴. Briefly, collected spleens were injected with ice-cold PBS and mashed through a 70-μm strainer (Falcon) or crushed between the rough ends of microscope slides. For Peyer’s patch and lymph node dissociation, a 40-μm strainer and 70-µm strainer (VWR) were used, respectively. RBCs were depleted by incubating splenocytes with ACK Lysis Buffer (Gibco) for 3–4 min at 20 °C. Single-cell suspensions were incubated with Fixable Viability Dye eFluor 780 (eBioscience) in PBS (30 min on ice), followed by Fc Block (5 min) and surface antibodies (30 min on ice) in FACS buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA). For intracellular staining, cells were fixed at 20 °C with 4% freshly prepared paraformaldehyde (PFA) (Cell Signaling Technology) for 15 min and permeabilized with 90% freezer-cold methanol (10 min on ice with frequent vortexing) unless specified otherwise. Phalloidin-based F-actin (catalog no. A30107, Thermo Fisher Scientific) staining was performed using the BD Perm/Wash reagent (catalog no. 554723) after 4% PFA fixation. For in vivo cell cycle analysis, 5-ethynyl-2′-deoxyuridine (EdU) (1 mg, catalog no. A10044, Thermo Fisher Scientific) was injected intraperitoneally and mice were sacrificed after 2.5 h. Cells were stained for surface markers, fixed and permeabilized then labeled using Click chemistry according to the manufacturer’s instructions (Click-iT Plus EdU Flow cytometry kit, catalog no. C10634, Thermo Fisher Scientific). FxCycle Violet (catalog no. F10347, Thermo Fisher Scientific) reagent was used for cell cycle characterization. For mitochondrial superoxide Deep Red (mtSOX, Dojindo) uptake indicating mtROS, after viability dye and surface staining, cells were resuspended in warm complete Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with mtSOX (10 μM) and incubated for 30 min at 37 °C. Cells were washed twice before flow cytometry acquisition. Flow cytometry was performed on a BD Fortessa X-20 or LSR II instrument or using a Cytek Aurora (5-laser) spectral flow cytometer. For imaging cytometry, single-cell suspensions were prepared from spleens of mito-QC mice, incubated with LIVE/DEAD dye, stained for surface markers and then fixed with 4% PFA. Washed cells were then resuspended in 50 μl FACS buffer and run on an Amnis ImageStream^X Mark II Imaging Flow Cytometer and analyzed with the Amnis IDEAS and FCS Express software (v.7.12). Flow cytometry data was analyzed using FlowJo (FlowJo LLC).

Cell sorting

Naive B cells were isolated using the Pan B cell Isolation II Kit, anti-CD43, and/or anti-CD23 Microbeads (all Miltenyi Biotec) according to the manufacturer’s instructions. Purity validated by flow cytometry was more than 90%. Isolation of the dark zone, light zone and gray zone subsets of GC B cells and T_FH cells (CD19⁻CD4⁺CXCR5⁺ICOS⁺GITR⁻) was performed via fluorescence-activated cell sorting (FACS).

For some experiments, untouched GC B cells were isolated using a magnetic bead-based protocol as described by Cato et al.⁵⁵. Briefly, spleens were collected from SRBC-immunized mice and single-cell suspensions were prepared in ice-cold magnetic-activated cell sorting (MACS) isolation buffer (PBS with 0.5% BSA + 2 mM EDTA) followed by ACK RBC lysis (Gibco) for 4 min at 20 °C with occasional mixing every 30 s. After washing, cells were labeled with anti-CD43 microbeads (Miltenyi Biotec) and biotinylated antibodies against CD38 and CD11c (clones 90 and N418, respectively; both eBioscience), followed by incubation with anti-biotin microbeads, and were subsequently run through a MACS LS column (Miltenyi Biotec). Purity was confirmed by flow cytometry and immunocytochemistry (ICC) and exceeded 95%.

When sorting for single-cell RNA sequencing (scRNA-seq), spleens were crushed using the rough ends of microscope slides to maximize cell yield. Subsequently, non-B cells were depleted using the Pan B cell Isolation II Kit (Miltenyi Biotec). Enriched cells were then incubated with viability dye, anti-CD16/32 (Fc Block) and surface flow antibodies, including markers for an exclusion (dump) channel (anti-CD3, anti-Gr1 and anti-CD11c); live Dump⁻tdTomato⁺ cells were sorted by BD FACSAria.

Detection of ETC and UPR^mt proteins with spectral flow cytometry

A complete list of antibodies is shown in the antibody table (see the spectral flow cytometry antibody panel section). All antibodies targeting intracellular mitochondrial proteins were directly conjugated and of mouse origin, with the exception of rabbit anti-Tfam monoclonal antibody (Abcam). Goat anti-rabbit AF405 Plus secondary antibody (Thermo Fisher Scientific) was used for detection. All antibodies used in the panel had been either validated for flow cytometry by the vendor or in the literature. Mouse anti-mitochondrial complex 1 antibody was conjugated in house using a Lightning-link PE-Cy7 Antibody Labeling kit (catalog no. 762-0005, Novus Biologicals). Cells were labeled with Zombie NIR viability dye (BioLegend) and Fc Block in PBS for 30 min on ice in 96-well V-bottom plates (Sarstedt). After washing, surface staining was performed in Brilliant stain buffer (catalog no. 563794, BD Biosciences) and FACS buffer (at a 1:1 ratio) for 30 min. Cells were then fixed in 4% freshly-made PFA at 20 °C for 15 min and permeabilized in freezer-cold methanol for 10 min with occasional vortexing. Anti-Tfam primary staining was performed in 50 μl FACS buffer supplemented with 2% goat serum for 45 min at 20 °C. After two washes, goat anti-rabbit secondary antibody and the remaining directly conjugated antibodies for ETC and UPR^mt proteins were added for 30 min at 20 °C. The cells were subsequently washed in FACS buffer and acquired on a Cytek Aurora (5-laser) spectral flow cytometer. Exploratory pilot experiments were performed to determine the most suitable single-stained references (beads or cells) for individual marker–fluorochrome combinations. If cells were used as reference controls, they were obtained from matching organs (spleen or bone marrow). Reference controls were processed similarly to fully stained samples, with parallel fixation, permeabilization and washing steps. Acquired samples were unmixed using SpectroFlo and analyzed with FlowJo software. The ‘Autofluorescence (AF) as a fluorescent tag’ option was enabled during unmixing to minimize AF interference. In rare cases, minor adjustments were applied to unmixing on the SpectroFlo software. Geometric mean fluorescence intensity (gMFI) values for ETC and UPR^mt proteins were calculated using FlowJo.

Single-mitochondrion translation assay

The technique was performed as described previously but with modifications⁵⁶. Total B cells were isolated from the spleens of Aicda-WT and Aicda-Tfam mice immunized with NP-CGG using magnetic beads (Pan B cell Isolation Kit II). Experiments were performed in batches so that one Aicda-WT and one Aicda-Tfam mouse spleen were processed in each replicate. Cells were pulsed with OPP (20 μM, catalog no. NU-931-5, Jena Bioscience) in complete RPMI 1640 supplemented with the cytoplasmic translation inhibitor harringtonine (1 μg ml⁻¹, catalog no. ab141941-5mg, Abcam) for 45 min at 37 °C. After a wash with complete medium, cells were resuspended in ice-cold mitochondria isolation buffer (1×, 320 mM sucrose, 2 mM EGTA,10 mM Tris-HCl, at pH 7.2 in water, prepared as 2× stock and stored in aliquots at −20 °C). Cells were then homogenized with a Dounce homogenizer with a 2-ml reservoir capacity (catalog no. ab110171, Abcam), using ten strokes with a type B pretzel. The homogenizer was rinsed with distilled water before each sample was processed to avoid cross-contamination. Homogenates underwent differential centrifugation at 4 °C, with intact cells and isolated nuclei first pelleted at 1,000g for 8 min. The supernatant containing the mitochondria was then transferred into new microcentrifuge tubes and centrifuged at 17,000g for 15 min. Enriched mitochondria, which appeared as brown-colored pellets, were fixed in 1% PFA in 0.5 ml PBS on ice for 15 min, followed by a wash with PBS. Permeabilization and subsequent antibody staining was performed in 1× BD Perm/Wash buffer (diluted in MilliQ-filtered water) at 20 °C. Preadsorbed primary rabbit anti-RFP (1:250, catalog no. 600-401-379, Rockland) antibody labeling was performed at 20 °C for 30 min. After a single wash, the Click reaction was performed using the Click-iT Plus OPP AF647 Protein Synthesis Assay Kit (catalog no. C10458, Thermo Fisher Scientific) at 20 °C for 30 min. After a wash, mitochondria were resuspended in 1× BD Perm/Wash buffer containing AF488-conjugated anti-COXIV (1:100, catalog no. 66110-1-Ig, Proteintech) and PE-conjugated donkey anti-rabbit IgG (1:200, catalog no. 406421, BioLegend). After washing with Perm/Wash buffer, mitochondria were resuspended in 250 μl filtered (0.2 μm) PBS and acquired using a BD Fortessa X-20 flow cytometer. The threshold for SSC-A (log-scale) was set to the minimum value (20,000) to allow acquisition of subcellular particles. Submicron Particle Size Reference Beads (catalog no. F13839, Thermo Fisher Scientific) were also used to identify mitochondria. For analysis, mitochondria were identified based on the COXIV and SSC-A properties; RFP⁺ (mitochondria with AP-GC origin) and RFP⁻ (mitochondria from naive B cells) were gated based on the anti-RFP antibody signal. The gMFI for the OPP-AF647 fluorescence was calculated for the RFP⁺ and RFP⁻ mitochondrial subsets and their ratio was used as a translation index and pooled after batch correction. Mitochondria from reporter-free cells, and those untreated with OPP, served as negative controls. In the validation experiments, 300 μg ml⁻¹ CHL or ethanol vehicle was used to inhibit mitochondrial translation along with the cytoplasmic translation inhibitor harringtonine; mitochondria were detected by COXIV-AF488 (1:100, catalog no. 66110-1-Ig, Proteintech) and SDHA-AF594 antibodies (1:250, catalog no. ab170172, Abcam) as described above.

Immunohistochemistry (IHC)

Collected spleens were immediately transferred to Antigenfix (Diapath) solution in 1.5 ml and fixed overnight at 4 °C. The next day, spleens were washed in PBS followed by overnight incubation in 30% sucrose (in PBS) at 4 °C for cryoprotection. On the following day, spleens were snap-frozen in 100% methanol on dry ice and stored at −80 °C until cryosectioning at 8–12-μm thickness. Slides were then rehydrated in PBS at 20 °C, then permeabilized and blocked in PBS containing 0.1% Tween-20, 10% goat serum and 10% rat serum at 20 °C for 2 h. For panels requiring intracellular detection, Tween-20 was replaced by 0.5% Triton X-100. All primary antibody staining was performed overnight at 4 °C in PBS supplemented with 2% goat serum and 0.1% Triton X-100 (intracellular) or Tween-20 (surface); secondary staining was performed at 20 °C for 2 h the next day in PBS 0.05% Tween-20. TUNEL imaging was performed using the Click-iT Plus TUNEL In Situ Imaging Far Red kit according to the manufacturer’s protocol (catalog no. C10619, Thermo Fisher Scientific). Slides were mounted with Fluoromount G (catalog no. 0100-01, Southern Biotech) and imaged with a ZEISS LSM 980 equipped with an Airyscan 2 module. See the relevant image analysis section in Supplementary Methods.

ICC

Isolated cells were transferred onto 18-mm coverslips coated with 0.01% (weight/volume) poly-l-lysine (catalog no. P8920-100ML, 10× stock, Merck) and incubated for 10 min at 37 °C to ensure cell attachment. Cells were then fixed in prewarmed 1× PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 4 mM MgSO₄·7H₂0, pH 6.8) with 4% PFA for 10 min at 37 °C, followed by permeabilization and blocking in 0.2% Triton X-100 with 10% goat serum for 30 min. Primary antibody labeling was performed overnight at 4 °C; secondary antibody staining was performed for 45 min at 20 °C (see antibody table). For the mitochondrial transcription assay based on 5-EU incorporation, isolated untouched naive B cells and GC B cells were resuspended in complete RPMI 1640 supplemented with 1 mM 5-EU (catalog no. C10330, Thermo Fisher Scientific) and transferred to 18-mm coverslips coated with poly-l-lysine. After incubation for 45 min, cells were briefly washed and fixed in warm 4% PFA diluted in PHEM buffer. After permeabilization and blocking for 30 min, incorporated 5-EU was detected by the Click-iT RNA AF594 Imaging Kit (catalog no. C10330, Thermo Fisher Scientific). Intracellular antibody labeling was performed after the Click reaction to minimize the interference of Click reagents with fluorochromes. For in vivo measurement of mitochondrial RNA synthesis, 2 mg 5-EU (Thermo Fisher Scientific) was injected intraperitoneally on day 12 after SRBC immunization and similar preparation and labeling steps described for the ex vivo 5-EU assay were followed. DAPI (catalog no. D8417-1MG, Sigma-Aldrich) staining was performed at 1 μM at 20 °C for 5 min, followed by a brief wash and mounting in Fluoromount G. For MitoTracker staining, cells were labeled with MitoTracker Red CMX ROS (150 nM, catalog no. M7512, Thermo Fisher Scientific). Slides were imaged with a ZEISS LSM 980 equipped with an Airyscan 2 module. See the relevant image analysis section in Supplementary Methods.

iGB culture system

The iGB culture system was described previously by Nojima et al.³⁸. Briefly, the 3T3 fibroblast cell line of BALB/c origin stably expressing CD40 ligand and B cell activating factor (BAFF) (40LB cell line), was cultured and maintained in high-glucose DMEM with GlutaMAX (catalog no. 31966021, Thermo Fisher Scientific) medium supplemented with 10% FCS and 50 U ml⁻¹ penicillin/streptomycin (catalog no. 15070063, Thermo Fisher Scientific) in T75 tissue culture flasks (catalog no. 658175, Sarstedt). Once cells were confluent, they were detached using trypsin/EDTA (catalog no. 25200056, Gibco) treatment, washed and collected in 15-ml tubes in 5 ml medium and irradiated (80 Gy). After irradiation, cells were washed, counted and seeded at 3 × 10⁶ per dish (100 mm, catalog no. G664160, Greiner) or 0.5 × 10⁶ per well (6-well plate, catalog no. 353046, Falcon). Fibroblast attachment and stretching were allowed overnight at 37 °C and 5% CO_2. The next day, naive B cells were isolated using anti-CD43 microbeads and treated with TAT-Cre (approximately 1.5 μM or 66.7 U ml⁻¹, catalog no. SCR508, Merck) for 45 min as described in Supplementary Methods. After three washes, cells were counted and cultured on an irradiated 40LB layer at 5 × 10⁵ (100-mm dish) and 5 × 10⁴ (per well, 6-well plate) for 4–6 days.

iGB adoptive transfer

iGB cells were generated as above. On day 4, fibroblasts and in vitro-differentiated plasmablasts (generally less than 10% frequency) were removed using biotinylated anti-H-2Kd (catalog no. 116604, BioLegend) and anti-CD138 (catalog no. 142511, BioLegend) followed by anti-biotin microbeads (Miltenyi Biotec); negative selection was performed using LS columns (Miltenyi Biotec). In some experiments, FACS was used to purify tdTomato⁺ iGB cells. For competitive experiments, purified WT iGBs (CD45.1/2⁺) were mixed 1:1 (ratio confirmed by flow cytometry before transfer) with CD45.2⁺ tdTomato⁺ Tfam^−/− iGBs and injected intravenously (6 × 10⁶ total cells in competitive setting or 3 × 10⁶ cells in noncompetitive setting) into CD45.1⁺ or CD45.2⁺ congenic hosts that were immunized with SRBC according to the enhanced protocol to maximize the recruitment of transferred iGB cells into the GC. On day 6 after iGB adoptive transfer, spleens were collected and analyzed by flow cytometry and confocal imaging to assess GC entry.

Transwell migration assay

A total of 5 × 10⁵ enriched total B cells isolated from SRBC-immunized Aicda-Tfam and Aicda-WT mice were placed in a 6.5-mm transwell chamber with 5-μm pore size (catalog no. 3421, Corning) and incubated at 37 °C for 2 h in the presence of murine CXCL12 (200 ng ml⁻¹, BioLegend) or CXCL13 (1 μg ml⁻¹, BioLegend) in complete RPMI 1640. The relative chemotaxis/migration index was calculated as follows: percentage of GC B cells (CD38⁻GL-7⁺tdTomato⁺) in migrated live total cells divided by the percentage of GC B cells in total input cells . A total of 2 × 10⁵ purified total B cells from B-Tfam and B-WT mice were placed in a 6.5-mm transwell chamber with 5-μm pore size and incubated for 5 h in the presence or absence of murine CXCL12 (100 ng ml⁻¹, BioLegend) with or without mitoTempo (100 μM, Merck) and Ru265 (30 μM, Merck). After 5 h, cells were incubated with LIVE/DEAD and anti-B220 AF488 antibody and resuspended in 100 μl in 96-well V-bottom plates and acquired on a Cytek Aurora flow cytometer at high-flow setting with a stopping volume of 60 μl. Technical duplicates were also included. Specific migration (%) was calculated according to this formula: 100 × (number of B220⁺ cells migrated in response to CXCL12 − number of B220⁺ cells migrated in the absence of CXCL12)/number of input B cells.

Lymphoma cell culture system

Daudi cells were cultured in RPMI 1640 medium (pH 7–7.4) supplemented with 10% FCS, 1× GlutaMAX (Gibco), 10 mM HEPES (Gibco) and 50 U ml⁻¹ penicillin/streptomycin and maintained at 37 °C in a humidified incubator with 5% CO₂. IMT1 (as a 1 mM stock solution in dimethylsulfoxide (DMSO), catalog no. HY-134539, MedChem Express) was used at 0.1 μM, 1 μM and 10 μM concentrations for a 0–120 h time window. CHL (catalog no. C0378-5G, Merck) was used at 10 μg ml⁻¹ and 25 μg ml⁻¹ concentrations (prepared in 100% ethanol fresh for each culture experiment) for a 0–120 h time window. Cell numbers were determined by manual counting using Trypan blue dye for dead cell exclusion at each time point.

scRNA-seq analysis

Approximately 17,000 cells per sample were loaded onto the 10X Genomics Chromium Controller (Chip K). Gene expression and BCR sequencing libraries were prepared using the 10X Genomics Single Cell 5′ Reagent Kits v2 (Dual Index) according to the manufacturer’s user guide (CG000330 Rev B). The final libraries were diluted to approximately 10 nM for storage. The 10-nM library was denatured and further diluted before loading on the NovaSeq 6000 sequencing platform (v.1.5 chemistry, 28/98 bp paired-end, Illumina) at the Oxford Genomics Centre.

Filtered output matrices from Cellranger v.6.0.1 were loaded in Seurat v.4.1.0. Cells with more than 5% mitochondrial reads and fewer than 200 genes were removed from the analysis. Data were normalized and transformed with SCTransform, with regression of cell cycle phase and mitochondrial reads, and integrated with the FindIntegrationAnchors and IntegrateData functions. The RunUMAP, FindNeighbors and FindClusters functions were used to cluster cells. Marker genes between clusters were identified using the FindAllMarkers function. Two small contaminant clusters (less than 1% of cells) were identified based on the expression of non-B cell genes and were removed from subsequent analyses. Clusters were identified by expression of canonical markers. Cluster proportions were calculated using DittoSeq. For visualization of uniform manifold approximation and projection (UMAP), equal number of cells from each experimental condition were displayed by random downsampling. Differential gene expression between conditions was calculated using the FindMarkers function with the ‘t-test’ parameter. Pathway analysis was performed using the R package single-cell pathway analysis (SCPA). Pseudobulk differential gene expression between individual biological replicates was performed using EdgeR after count aggregation across cells using Scuttle.

Filtered contig outputs generated by Cellranger v.6.0.1 from cells processed in the Seurat workflow above were combined, filtered and visualized using scRepertoire v.1.4. For quantification of mutational load, the Immcantation pipeline was used. Germline segment assignment was performed with Change-O; the SHM count was calculated using SHazaM.

Statistical analysis

The statistical tests used are indicated in the respective figure legends, with error bars indicating the mean ± s.e.m. P ≤ 0.05 was deemed to indicate significance. Analyses were performed with Prism 9 (GraphPad Software) or R v.4.1. No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications⁵⁷. The distribution of data was determined using normality testing to determine appropriate statistical methodology; otherwise, it was assumed to be normally distributed. For the in vivo experiments, we matched the sex and age of the mice in the experimental batches; however, other modes of randomization were not performed. Data collection and analysis were not performed blind to the conditions of the experiments in most of the experiments. Mice with complete absence of GCs and lacking alum spots after immunization were considered as failed intraperitoneal immunization and therefore excluded from the analysis. In selected experiments, batch correction was performed with the R package Batchma.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The scRNA-seq data have been deposited with the Gene Expression Omnibus under accession no. GSE208021. Source data are provided with this paper.

Code availability

The code used to analyze the scRNA-seq data is available upon reasonable request and can be found at: https://github.com/alexclarke7/Yazicioglu_et_al.

References

Young, C. & Brink, R. The unique biology of germinal center B cells. Immunity 54, 1652–1664 (2021).
Article CAS PubMed Google Scholar
Ott, G., Rosenwald, A. & Campo, E. Understanding MYC-driven aggressive B-cell lymphomas: pathogenesis and classification. Blood 122, 3884–3891 (2013).
Article CAS PubMed Google Scholar
Victora, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143, 592–605 (2010).
Article CAS PubMed PubMed Central Google Scholar
Abbott, R. K. et al. Germinal center hypoxia potentiates immunoglobulin class switch recombination. J. Immunol. 197, 4014–4020 (2016).
Article CAS PubMed Google Scholar
Cho, S. H. et al. Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system. Nature 537, 234–238 (2016).
Article CAS PubMed PubMed Central Google Scholar
Weisel, F. J. et al. Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis. Nat. Immunol. 21, 331–342 (2020).
Article CAS PubMed PubMed Central Google Scholar
Boothby, M. R. et al. Over-generalizing about GC (hypoxia): pitfalls of limiting breadth of experimental systems and analyses in framing informatics conclusions. Front. Immunol. 12, 664249 (2021).
Article CAS PubMed PubMed Central Google Scholar
Chen, D. et al. Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturation. Nat. Immunol. 22, 904–913 (2021).
Article CAS PubMed Google Scholar
Haniuda, K., Fukao, S. & Kitamura, D. Metabolic reprogramming induces germinal center B cell differentiation through Bcl6 locus remodeling. Cell Rep. 33, 108333 (2020).
Article CAS PubMed Google Scholar
Luo, W. et al. SREBP signaling is essential for effective B cell responses. Nat. Immunol. 24, 337–348 (2023).
Article CAS PubMed Google Scholar
Caro, P. et al. Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma. Cancer Cell 22, 547–560 (2012).
Article CAS PubMed PubMed Central Google Scholar
Tsui, C. et al. Protein kinase C-β dictates B cell fate by regulating mitochondrial remodeling, metabolic reprogramming, and heme biosynthesis. Immunity 48, 1144–1159 (2018).
Article CAS PubMed PubMed Central Google Scholar
Akkaya, M. et al. Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nat. Immunol. 19, 871–884 (2018).
Article CAS PubMed PubMed Central Google Scholar
Jang, K.-J. et al. Mitochondrial function provides instructive signals for activation-induced B-cell fates. Nat. Commun. 6, 6750 (2015).
Article CAS PubMed Google Scholar
Lisci, M. et al. Mitochondrial translation is required for sustained killing by cytotoxic T cells. Science 374, eabe9977 (2021).
Article CAS PubMed Google Scholar
Almeida, L. et al. Ribosome-targeting antibiotics impair T cell effector function and ameliorate autoimmunity by blocking mitochondrial protein synthesis. Immunity 54, 68–83 (2021).
Article CAS PubMed PubMed Central Google Scholar
O’Sullivan, D. et al. Fever supports CD8⁺ effector T cell responses by promoting mitochondrial translation. Proc. Natl Acad. Sci. USA 118, e2023752118 (2021).
Article PubMed PubMed Central Google Scholar
Kaufman, B. A. et al. The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures. Mol. Biol. Cell 18, 3225–3236 (2007).
Article CAS PubMed PubMed Central Google Scholar
Hillen, H. S., Temiakov, D. & Cramer, P. Structural basis of mitochondrial transcription. Nat. Struct. Mol. Biol. 25, 754–765 (2018).
Article CAS PubMed PubMed Central Google Scholar
Desdín-Micó, G. et al. T cells with dysfunctional mitochondria induce multimorbidity and premature senescence. Science 368, 1371–1376 (2020).
Article PubMed Google Scholar
West, A. P. et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520, 553–557 (2015).
Article PubMed PubMed Central Google Scholar
Urbanczyk, S. et al. Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle. Cell Rep. 39, 110912 (2022).
Article CAS PubMed Google Scholar
McWilliams, T. G. et al. mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J. Cell Biol. 214, 333–345 (2016).
Article CAS PubMed PubMed Central Google Scholar
Martinez-Martin, N. et al. A switch from canonical to noncanonical autophagy shapes B cell responses. Science 355, 641–647 (2017).
Article CAS PubMed PubMed Central Google Scholar
Brüser, C., Keller-Findeisen, J. & Jakobs, S. The TFAM-to-mtDNA ratio defines inner-cellular nucleoid populations with distinct activity levels. Cell Rep. 37, 110000 (2021).
Article PubMed Google Scholar
Baixauli, F. et al. Mitochondrial respiration controls lysosomal function during inflammatory T Cell responses. Cell Metab. 22, 485–498 (2015).
Article CAS PubMed PubMed Central Google Scholar
Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl Acad. Sci. USA 103, 13789–13794 (2006).
Article CAS PubMed PubMed Central Google Scholar
Hardy, R. R. & Hayakawa, K. B cell development pathways. Annu. Rev. Immunol. 19, 595–621 (2001).
Article CAS PubMed Google Scholar
Bibby, J. A. et al. Systematic single-cell pathway analysis to characterize early T cell activation. Cell Rep. 41, 111697 (2022).
Article CAS PubMed Google Scholar
Shaulian, E. & Karin, M. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, E131–E136 (2002).
Article CAS PubMed Google Scholar
Suzuki, Y. J., Forman, H. J. & Sevanian, A. Oxidants as stimulators of signal transduction. Free Radic. Biol. Med. 22, 269–285 (1997).
Article CAS PubMed Google Scholar
Ansel, K. M., Harris, R. B. S. & Cyster, J. G. CXCL13 is required for B1 cell homing, natural antibody production, and body cavity immunity. Immunity 16, 67–76 (2002).
Article CAS PubMed Google Scholar
Han, S.-B. et al. Rgs1 and Gnai2 regulate the entrance of B lymphocytes into lymph nodes and B cell motility within lymph node follicles. Immunity 22, 343–354 (2005).
Article CAS PubMed Google Scholar
Föger, N., Rangell, L., Danilenko, D. M. & Chan, A. C. Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis. Science 313, 839–842 (2006).
Article PubMed Google Scholar
Bolger-Munro, M. et al. Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation. eLife 8, e44574 (2019).
Article CAS PubMed PubMed Central Google Scholar
Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. & Cumano, A. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7, 1995–2001 (1988).
Article CAS PubMed PubMed Central Google Scholar
Weiser, A. A. et al. Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations. Int. Immunol. 23, 345–356 (2011).
Article CAS PubMed Google Scholar
Nojima, T. et al. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. Nat. Commun. 2, 465 (2011).
Article PubMed Google Scholar
McKee, E. E., Ferguson, M., Bentley, A. T. & Marks, T. A. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob. Agents Chemother. 50, 2042–2049 (2006).
Article CAS PubMed PubMed Central Google Scholar
Argüello, R. J. et al. SCENITH: a flow cytometry-based method to functionally profile energy metabolism with single-cell resolution. Cell Metab. 32, 1063–1075 (2020).
Article PubMed PubMed Central Google Scholar
Allen, C. D. C. et al. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat. Immunol. 5, 943–952 (2004).
Article CAS PubMed Google Scholar
Tolar, P. Cytoskeletal control of B cell responses to antigens. Nat. Rev. Immunol. 17, 621–634 (2017).
Article CAS PubMed Google Scholar
Maus, M. et al. B cell receptor-induced Ca²⁺ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes. J. Leukoc. Biol. 93, 537–547 (2013).
Article CAS PubMed Google Scholar
Williams, G. S. B., Boyman, L., Chikando, A. C., Khairallah, R. J. & Lederer, W. J. Mitochondrial calcium uptake. Proc. Natl Acad. Sci. USA 110, 10479–10486 (2013).
Article CAS PubMed PubMed Central Google Scholar
Trnka, J., Blaikie, F. H., Smith, R. A. J. & Murphy, M. P. A mitochondria-targeted nitroxide is reduced to its hydroxylamine by ubiquinol in mitochondria. Free Radic. Biol. Med. 44, 1406–1419 (2008).
Article CAS PubMed Google Scholar
Woods, J. J. et al. A selective and cell-permeable mitochondrial calcium uniporter (MCU) inhibitor preserves mitochondrial bioenergetics after hypoxia/reoxygenation injury. ACS Cent. Sci. 5, 153–166 (2019).
Article CAS PubMed PubMed Central Google Scholar
Li, F. et al. Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis. Mol. Cell. Biol. 25, 6225–6234 (2005).
Article CAS PubMed PubMed Central Google Scholar
Harris, A. W. et al. The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells. J. Exp. Med. 167, 353–371 (1988).
Article CAS PubMed Google Scholar
Bonekamp, N. A. et al. Small-molecule inhibitors of human mitochondrial DNA transcription. Nature 588, 712–716 (2020).
Article CAS PubMed Google Scholar
Klemke, M. et al. Oxidation of cofilin mediates T cell hyporesponsiveness under oxidative stress conditions. Immunity 29, 404–413 (2008).
Article CAS PubMed Google Scholar
Campello, S. et al. Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J. Exp. Med. 203, 2879–2886 (2006).
Article CAS PubMed PubMed Central Google Scholar
D’Andrea, A. et al. The mitochondrial translation machinery as a therapeutic target in Myc-driven lymphomas. Oncotarget 7, 72415–72430 (2016).
Article PubMed PubMed Central Google Scholar
Dominguez-Sola, D. et al. The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry. Nat. Immunol. 13, 1083–1091 (2012).
Article CAS PubMed PubMed Central Google Scholar
Yazicioglu, Y. F., Aksoylar, H. I., Pal, R., Patsoukis, N. & Boussiotis, V. A. Unraveling key players of humoral immunity: advanced and optimized lymphocyte isolation protocol from murine Peyer’s patches. J. Vis. Exp. https://doi.org/10.3791/58490 (2018).
Cato, M. H., Yau, I. W. & Rickert, R. C. Magnetic-based purification of untouched mouse germinal center B cells for ex vivo manipulation and biochemical analysis. Nat. Protoc. 6, 953–960 (2011).
Article CAS PubMed PubMed Central Google Scholar
Delaunay, S. et al. Mitochondrial RNA modifications shape metabolic plasticity in metastasis. Nature 607, 593–603 (2022).
Article CAS PubMed PubMed Central Google Scholar
Clarke, A. J., Riffelmacher, T., Braas, D., Cornall, R. J. & Simon, A. K. B1a B cells require autophagy for metabolic homeostasis and self-renewal. J. Exp. Med. 215, 399–413 (2018).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank J. Webber for flow sorting, T. Arnon and P. Magill (University of Oxford) for providing the mice, and L. Dustin (University of Oxford) for providing the Daudi cells. We thank D. Kitamura (Tokyo University of Science) for providing the 40LB cell line. We also thank K. Morten (University of Oxford) for helpful suggestions. We thank L. Uhl and G. Pirgova for their assistance and helpful guidance. We also thank the Kennedy Institute BSU staff for their support. Funding for this work was provided by the Wellcome Trust (no. 211072/Z/18/Z) and Cancer Research UK/Versus Arthritis (no. C70663/A29547) to A.J.C., the Kennedy Trust for Rheumatology Research to Y.F.Y. and M.L.D., and the US National Institutes of Health (no. HL118979) to M.L.D. S.J.D. is funded by an National Institute for Health and Care Research (NIHR) Global Research Professorship (no. NIHR300791). For the purpose of open access, the author has applied a CC BY public copyrightlicense to any Author Accepted Manuscript version arising from this submission. Flow cytometry and microscopy facilities were supported by the Kennedy Trust for Rheumatology Research through the Cell Dynamics Platform. We thank the Wolfson Imaging Centre Oxford for providing microscope facility support and the Don Mason flow cytometry facility and staff (R. Hedley and V. Tsioligka) of the Sir William Dunn School of Pathology, University of Oxford. The computational aspects of this research were supported by the Wellcome Trust Core Award grant no. 203141/Z/16/Z and the NIHR Oxford Biomedical Research Centre. The views expressed are those of the authors and not necessarily those of the NHS, NIHR or the Department of Health. The image of a laboratory mouse used was created by Gwilz and distributed under an CC BY-SA 4.0 license.

Author information

Authors and Affiliations

Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK
Yavuz F. Yazicioglu, Eros Marin, Ciaran Sandhu, Ewoud B. Compeer, Moustafa Attar, Michael L. Dustin & Alexander J. Clarke
Medical Sciences Division, University of Oxford, Oxford, UK
Ciaran Sandhu & Iwan G. A. Raza
Medical Research Centre Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
Silvia Galiani
Nuffield Department of Medicine Centre For Global Health Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK
Mohammad Ali, Barbara Kronsteiner & Susanna J. Dunachie
Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand
Mohammad Ali, Barbara Kronsteiner & Susanna J. Dunachie
National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Oxford, UK
Susanna J. Dunachie

Authors

Yavuz F. Yazicioglu
View author publications
You can also search for this author in PubMed Google Scholar
Eros Marin
View author publications
You can also search for this author in PubMed Google Scholar
Ciaran Sandhu
View author publications
You can also search for this author in PubMed Google Scholar
Silvia Galiani
View author publications
You can also search for this author in PubMed Google Scholar
Iwan G. A. Raza
View author publications
You can also search for this author in PubMed Google Scholar
Mohammad Ali
View author publications
You can also search for this author in PubMed Google Scholar
Barbara Kronsteiner
View author publications
You can also search for this author in PubMed Google Scholar
Ewoud B. Compeer
View author publications
You can also search for this author in PubMed Google Scholar
Moustafa Attar
View author publications
You can also search for this author in PubMed Google Scholar
Susanna J. Dunachie
View author publications
You can also search for this author in PubMed Google Scholar
Michael L. Dustin
View author publications
You can also search for this author in PubMed Google Scholar
Alexander J. Clarke
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Y.F.Y. and A.J.C. conceived and designed the study. Y.F.Y. performed most of the experiments. E.M., E.B.C., S.G., C.S., M. Ali, B.K. and M. Attar performed the experiments. Y.F.Y. and A.J.C. wrote the paper. Y.F.Y. performed the image analysis and A.J.C. and I.G.A.R. analyzed the single-cell data. M.L.D. and S.J.D. provided advice and guidance. A.J.C. supervised the study.

Corresponding author

Correspondence to Alexander J. Clarke.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: L. A. Dempsey in collaboration with Nature Immunology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 GC B cells undergo active mitochondrial remodeling.

(a) Flow sorting strategy for DZ, LZ, and GZ from MACS-enriched GC B cells isolated from SRBC-immunized (enhanced protocol, day 12) Mito-QC mice. (b) Representative ImageStream image galleries of splenic CD19⁻ non-B cells and CD19⁺ B cells defined as undergoing mitophagy. Arrows indicate mitophagic foci of lysosomal-associated membrane protein 1 (LAMP1⁺) MitoQC-mCherry without MitoQC-GFP colocalization. (c) Representative ImageStream image galleries of splenic GC B cells (CD19⁺CD38⁻GL-7⁺). (d) Proportions of mitophagy⁺ population in CD38⁻GL-7⁺ GC B cells and non-GC B cells. Mitophagy was defined as in b. Representative of two independent experiments with n = 4 mice. (e) Quantification of average major radius and aspect ratio (major radius/second radius) of mitochondrial nucleoids based on 3D fitted ellipsoid volume model in naïve (n = 20 cells for major radius and n = 22 cells for aspect ratio quantification) and GC B cells (n = 24 cells in both panels). Representative of two independent experiments. Statistical significance was calculated by two-tailed Mann Whitney U (d) and unpaired two-tailed t-test with Welch’s correction (e). Data are presented as the mean ± s.e.m.

Source Data

Extended Data Fig. 2 Tfam is essential during B cell development and differentiation.

(a) 3D Airyscan confocal images of B cells from unimmunized B-WT and B-Tfam mice, stained for TFAM and with MitoTracker CMX ROS and DAPI. Scale bar, 3µm. Representative of three independent experiments. (b) Representative histogram of TFAM staining by intracellular flow cytometry in splenic CD19⁺ B cells from unimmunized B-WT and B-Tfam mice. Representative of three independent experiments. (c) Representative images of spleens from B-WT and B-Tfam littermate mice. (d) Flow cytometry gating strategy for splenic follicular (B220⁺CD23⁺CD21^int) and marginal zone B cells (B220⁺CD23⁻CD21⁺) and representative plots for CD19⁺CD93⁺ transitional B subsets (T1,T2, and T3) from B-WT and B-Tfam mice (quantified in Fig. 2a). (e) Proportional comparison of splenic follicular and marginal zone B cells from B-WT and B-Tfam mice (n = 5 per group). Representative of three independent experiments. (f) Cell counts of bone marrow B cell subsets from B-Tfam and B-WT mice (n = 4 per group) according to Hardy classification (Fr A-F). Representative of two independent experiments. (g) Representative flow cytometry plots of bone marrow B cell precursors in B-WT (n = 3) and B-Tfam heterozygous (Cd79a-Cre × Tfam^loxP/+) mice (n = 4). Representative of two independent experiments. (h) Proportional comparison of B220⁺ B cells in spleen, Peyer’s patches, and precursor bone marrow B cells from B-WT (n = 3) and B-Tfam heterozygous mice (n = 4). Representative of two independent experiments. (i) OCR and ECAR measurements of unstimulated naïve B cells from B-Tfam and B-WT mice and quantification of basal OCR and ECAR values (n = 3 mice per group), representative of two independent experiments. (j) Comparison of CD38⁻GL-7⁺ GC B cell proportions in spleens of SRBC-immunized B-Tfam Het (n = 4) and B-WT (n = 3) mice. Results representative of two independent experiments. Statistical significance was calculated by unpaired two-tailed t-test (e,f, i,j) or two-way ANOVA with Šidák’s multiple comparison test (h). Data are presented as the mean ± s.e.m.

Source Data

Extended Data Fig. 3 GC B cells require TFAM.

(a) tdTomato⁺CD138⁺ plasma cell percentages within Dump¯ bone marrow cells from Aicda-WT (n = 6) and Aicda-Tfam (n = 5) mice at day 12 post SRBC-immunization. (b) Proportional comparison of NP-PE or NP-APC-binding GC B cells in NP-CGG-immunized Aicda-WT (n = 9) and Aicda-Tfam (n = 8) mice at day 14. (c) Plasma cell clusters in splenic red pulp following NP-CGG immunization. Scale bar, 50μm. Counts of tdTomato⁺Blimp1-mVenus⁺CD138⁺ post-GC plasma cells and tdTomato⁻Blimp1-mVenus⁺CD138⁺ plasmablasts. Results pooled from n = 3 non-serial sections per mouse (n = 2 mice per genotype). (d) Ratio of GC B cells (IRF4⁻ CD38⁻ tdTomato⁺) to post-GC plasma cells (IRF4⁺tdTomato⁺) in Aicda-Tfam (n = 6) and Aicda-WT mice (n = 5). (e-h). ELISA quantifications and dilution curves of IgG1 or IgM anti-NP antibodies (NP_1-4-BSA and NP_>20-BSA respectively) in sera from Aicda-Tfam (n = 5) and Aicda-WT mice (n = 6) at day 14 (e-g) or day 49 (h) (n = 6 per genotype) following NP-CGG immunization. (i) Active caspase 3⁺ apoptotic GC B cell percentages in Aicda-Tfam and Aicda-WT mice (n = 9 per group). (j) In situ TUNEL assay on Aicda-WT and Aicda-Tfam spleens following SRBC immunization. Scale bar, 50μm. (k) Representative flow cytometry plots and quantification of M and G2 cell cycle stages in GC B cells from Aicda-WT (n = 4) and Aicda-Tfam mice (n = 5). (l) Representative flow cytometry plots and quantification of EdU⁺ GC B cells at S phase from Aicda-WT (n = 7) and Aicda-Tfam mice (n = 8). (m-p) Naïve B cells from Rosa26^STOPtdTomato-WT and Rosa26^STOPtdTomato-Tfam^loxP mice (n = 2) were TAT-Cre treated and in vitro-stimulated with anti-IgM + anti-CD40 + IL-4 for four days. Representative flow cytometry plots of tdTomato (m), TFAM (n), and CTV (o), and viability (p). Statistical significance was calculated by unpaired two-tailed t-test (b,d,e,h,i,k,l), two-tailed Mann Whitney U test (a), two-way ANOVA with Šidák’s multiple comparison test (c). Data are presented as the mean ± s.e.m. Data representative of ≥2 independent experiments in all cases.

Source Data

Extended Data Fig. 4 TFAM regulates B cell clonality.

(a) Quantification of somatic hypermutation by Igh mutation count for indicated immunoglobulin isotype across all sequenced B cells in which isotype call could be made. Data are as described in Fig. 4a (IgG1 = 53 cells, IgG2b = 116 cells, IgG3 = 50 cells, IgM = 1038 cells, pooled from n = 3 Aicda-WT and n = 3 Aicda-Tfam mice). (b) Quantification of overall mutation rate for Ighv1-72 gene segment (n = 76 cells in Aicda-WT, n = 89 in Aicda-Tfam, pooled from n = 3 Aicda-WT and n = 3 Aicda-Tfam mice). (c) Amino acid substitution rate across Ighv1-72 in GC B cell cluster for Aicda-WT and Aicda-Tfam mice (n = 76 cells in Aicda-WT, n = 89 in Aicda-Tfam, pooled from n = 3 Aicda-WT and n = 3 Aicda-Tfam mice). (d) Amino acid substitution rate across Ighv1-72 in AP B cell cluster for Aicda-WT and Aicda-Tfam mice (n = 24 cells in Aicda-WT, n = 154 in Aicda-Tfam, pooled from n = 3 Aicda-WT and n = 3 Aicda-Tfam mice). Statistical significance was calculated by two-tailed t-test with correction for multiple comparison by the Benjamini-Hochberg method(a), or two-tailed unpaired t-test (b). In a,b the box and whisker plots depicts the minimum and maximum values no greater than ±1.5 × the IQR, the lower and upper quartiles, and the median.

Extended Data Fig. 5 TFAM is required for GC B cell commitment.

(a) Counts of AP and GC B cells from NP-CGG-immunized Aicda-WT (n = 4) and Aicda-Tfam mice (n = 10). (b) Flow cytometry plot and quantification of AP and GC B cell subsets in B-WT (n = 3) and B-Tfam (n = 4) mice immunized with SRBC (enhanced protocol). (c) NP-binding rates of naïve B cells, APs, and GC B cells from Aicda-WT (n = 6) to high (NP_hi) and low (NP_lo) NP-APC conjugates. (d) Quantification of BCL6 expression (gMFI) in GC B cells from Aicda-WT (n = 4) and Aicda-Tfam mice (n = 6). (e) Heatmap of row Z-scores for gMFI of indicated mitochondrial proteins, measured by flow cytometry in TAT-Cre treated Tfam^−/− or WT iGB cells (n = 3 mice per group). (f) Live cell counts of WT and Tfam^−/− iGB cells at day 4. Results representative of two independent experiments with n = 6 technical replicates from three mice. (g) Pre-transfer tdTomato⁺Tfam^−/− and tdTomato⁻CD45.1/2⁺ WT iGB cell ratio in competitive iGB transfer experiment. (h) GFP⁺ activated OTII-Tg CD4⁺ T cells were mixed with tdTomato⁺ WT or Tfam^−/− iGBs pulsed with OVA 323-339 peptide. Percentage of GFP⁺ tdTomato⁺ doublets indicating T-B conjugates was quantified. Three technical replicates of pooled n = 2 mice shown. Representative of two independent experiments with n = 3 mice per group in total. (i) Gating and flow sort strategy for MACS-enriched CD4⁺ICOS⁺CXCR5⁺GITR⁻ T_FH cells. (j) Quantification of CD45.2⁺ GC B cells from spleens and Peyer’s patches of Aicda-WT and Aicda-Tfam (n = 5) 50:50 competitive bone marrow chimeras at day 7 following SRBC immunization, normalized to CD45.1 WT GC B cell proportions. Statistical significance was calculated by unpaired two-tailed t-test (a,b,d,j), two-tailed Mann Whitney U test (f) or two-way ANOVA with Šidák’s multiple comparison test (c,h). Data are presented as the mean ± s.e.m. Data representative of ≥2 independent experiments in all cases.

Source Data

Extended Data Fig. 6 TFAM regulates mitochondrial translation in activated B cells.

(a) Airyscan in situ confocal image and signal intensity chart of GC B cells expressing tdTomato depicting the diffusion of RFP into TOMM20⁺ mitochondria. Scale bar, 5µm. Representative of four independent experiments. (b) Mitochondrial OPP incorporation assay performed on WT and Tfam^−/− iGB cells at day 6. Flow cytometry gating strategy of mitochondria as COX IV⁺ SDHA⁺ particles. OPP-AF647 signal with harringtonine alone (baseline)(H, 1μg/ml) and chloramphenicol (CHL, 300μg/ml) or vehicle (ethanol) treatments depicted in flow cytometry histogram plots. Representative of two independent experiments. (c) Flow cytometry histogram plots depicting OPP incorporation in splenic IgD⁺GL-7^int AP and IgD⁻CD38⁻GL-7⁺ GC B cells from B-WT and B-Tfam mice in response to metabolic inhibitors (oligomycin and/or 2-DG), shifts in OPP-AF647 signal indicates metabolic properties. Representative of three independent experiments. (d) Quantification of mitochondrial dependence and glycolytic capacity of cells based on OPP incorporation in splenic IgD⁺GL-7^int AP (n = 3 mice per group) and IgD⁻GL-7⁺CD38⁻ GC B cells from B-WT (n = 7 mice) and B-Tfam mice (n = 5), treated ex vivo with metabolic inhibitors (oligomycin and/or 2-DG). Data pooled from two independent experiments. (e) ECAR measurements (MitoStress test) of B-Tfam (n = 4) and B-WT (n = 3 mice) B cells stimulated overnight with anti-CD40 + IL-4. Data pooled from two independent experiments. (f) OCR and ECAR measurements (MitoStress test) of 2×10⁵ iGB cells (day 5, after overnight rest in IL-4) from TAT-Cre treated WT (Tfam^+/+) and Tfam^loxp (Tfam^−/−) B cells. Results representative of two independent experiments. Statistical significance was calculated by two-way ANOVA with Šidák’s multiple comparison test (d) or unpaired two-tailed t-test (e). Data are presented as the mean ± s.e.m.

Source Data.

Extended Data Fig. 7 Tfam deletion disrupts B cell mobility.

(a) 3D Airyscan confocal images of F-actin phalloidin-stained total B cells from unimmunized B-WT and B-Tfam mice. Scale bar, 3 μm. Representative of two independent experiments. (b) Representative flow cytometry histogram of F-actin phalloidin fluorescence of IgD⁺ B cells from unimmunized B-WT and B-Tfam mice. Representative of two independent experiments. (c) Representative flow cytometry histogram of MCU fluorescence of CD3⁺ T cells from unimmunized B-WT and B-Tfam mice. Representative of three independent experiments. (d) Representative flow cytometry histogram and quantification of mtROS Deep Red fluorescence in IgD⁺ GL-7^int AP cells from immunized B-WT (n = 4) and B-Tfam mice (n = 5). Data representative of two independent experiments. Statistical significance was calculated by unpaired two-tailed t-test (d). Data are presented as the mean ± s.e.m.

Source Data

Extended Data Fig. 8 Tfam deletion in B cells prevents lymphoma.

(a) Flow cytometry-based cell cycle stage characterization (G1, S, G2-M) in Daudi cells at 120h following IMT1 treatment. Quantification of Daudi cells in S phase, representative of two independent experiments with n = 3 technical replicates. (b) Representative confocal images of Daudi cells treated with IMT1 (1μM) and CHL (25μg/ml) for 5 days. Quantification of UPR^mt associated protease LONP1 normalized to mitochondrial mass (TOMM20 signal). Scale bar, 2 μm. Each symbol represents a cell. Vehicle (n = 8 cells), IMT1 (n = 7 cells) and CHL (n = 9 cells). Statistical significance was calculated by ordinary one-way ANOVA with Dunnet’s multiple comparisons test (a,b). Data are presented as the mean ± s.e.m.

Source Data

Supplementary information

Supplementary Information

Supplementary Methods.

Reporting Summary

Peer Review File

Supplementary Table 1

Table of antibodies used.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Yazicioglu, Y.F., Marin, E., Sandhu, C. et al. Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis. Nat Immunol 24, 991–1006 (2023). https://doi.org/10.1038/s41590-023-01484-3

Download citation

Received: 04 July 2022
Accepted: 09 March 2023
Published: 24 April 2023
Issue Date: June 2023
DOI: https://doi.org/10.1038/s41590-023-01484-3

This article is cited by

Germinal centers FAMished without TFAM
- Julia Jellusova
Nature Immunology (2023)

Subjects

Abstract

Similar content being viewed by others

Main

Results

GC B cells undergo active mitochondrial remodeling

Tfam is essential during B cell development and differentiation

GC B cells require TFAM

TFAM controls transcriptional entry into the GC program

TFAM is required for GC B cell commitment

TFAM regulates mitochondrial translation in activated B cells

Tfam deletion disrupts B cell mobility

Tfam deletion in B cells prevents lymphoma

Discussion

Methods

Mice

Immunization

Flow cytometry and imaging flow cytometry

Cell sorting

Detection of ETC and UPRmt proteins with spectral flow cytometry

Single-mitochondrion translation assay

Immunohistochemistry (IHC)

ICC

iGB culture system

iGB adoptive transfer

Transwell migration assay

Lymphoma cell culture system

scRNA-seq analysis

Statistical analysis

Reporting summary

Data availability

Code availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Extended data

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Search

Quick links

Detection of ETC and UPR^mt proteins with spectral flow cytometry