ABSTRACT To gain new insight into the role of B-cell autophagy, we generated two novel mouse models deficient for the autophagy-related gene (_Atg_)_5_, one from the outset pro-B cell stage

(_Atg5__f/−_ Mb1 cre) and the other in mature B cells only (_Atg5__f/−_ CD21 cre). We show that autophagy is dispensable for pro- to pre-B cell transition, but necessary at a basal level to

maintain normal numbers of peripheral B cells. It appears non-essential for B-cell activation under B-cell receptor stimulation but required for their survival after lipopolysaccharide

stimulation that drives plasmablast differentiation and for specific IgM production after immunization. Results obtained using Atg5_f/−_ CD21 cre × C57BL/6_lpr/lpr_ autoimmune-prone mice

AN AGED IMMUNE RESPONSE Article Open access 22 March 2024 ABERRANT METTL1-MEDIATED TRNA M7G MODIFICATION ALTERS B-CELL RESPONSES IN SYSTEMIC AUTOIMMUNITY IN HUMANS AND MICE Article Open

access 05 December 2024 DHX33 PROMOTES B-CELL GROWTH AND PROLIFERATION BY CONTROLLING ACTIVATION-INDUCED RRNA UPREGULATION Article 11 January 2023 MAIN Macroautophagy is a catabolic process

allowing the degradation of cytoplasmic material in double membrane vesicles, ultimately fusing with lysosomes. Macroautophagy, initially implicated in the generation of nutrients under

metabolic stress, is known to have multiple roles, in different physiologic compartments, such as in vacuole trafficking, cell signalling, and cell death. Macroautophagy is deeply involved

in the regulation of immunity.1 It has been shown that autophagy can regulate inflammation related to inflammasome activation and to type I interferon secretion. Moreover, it contributes to

antigen presentation by both major histocompatibility complex (MHC) class I and class II molecules.2 Macroautophagy is also tightly linked to lymphocyte activation and survival. It has

central roles in T-cell basal homeostasis, survival, and polarization.3 It is also involved in the regulation of T-cell signalling by downregulating the NF-_κ_B pathway4 and apoptosis

processes through the procaspases 3 and 8 degradation.5 Macroautophagy has additionally been described to regulate B-cell lineage, in particular during B-cell development. Thus, it has been

shown that B cells generated from fetal liver chimaeras, with a complete deletion of the essential autophagy-related gene (_Atg_)_5,_ exhibited a block at the pro- to pre-B stage

transition.6, 7 However, as the genetic deletion is systemic and occurs very early during development, the question remains over whether the developmental blockade could be due to defects

resulting from early haematopoietic development. Indeed, macroautophagy has been shown to be fundamental to haematopoietic stem cell survival and renewal.8 Moreover, conditional deletion of

_Atg5_ under the control of CD19 promoter expressed from the pre-B stage does not lead to major developmental breaks, except a decrease in B-1a B-cell population.6 The contrast with results

obtained with chimaeric mice could be due to the partial deletion induced by cre expression under the control of CD19 promoter.9 At this stage, however, it remains unclear whether

macroautophagy is really needed in the first steps of B-cell development. Studies based on mouse models deleted for autophagy genes at an early stage of development led to fundamental

findings regarding the importance of macroautophagy in B cell-related immune responses. Two independent studies, based on T cell-dependent and -independent model antigens or on infectious

agents,10, 11 concluded that macroautophagy is necessary _in vivo_ for humoral immune responses by regulating plasma cell differentiation and survival. The former study11 showed that

macroautophagy is involved in regulating endoplasmic reticulum (ER) load along with plasma cell differentiation, thus limiting ER stress and contributes to long-lived plasma cell survival. A

role for macroautophagy in B-cell early activation was excluded, especially in germinal centre (GC) formation. Two more recent studies, one based on a mouse model of influenza infection and

the other on model antigens used as immunogens, demonstrated that in addition to its effect on plasma cell survival, macroautophagy is necessary for the survival of B-cell memory

compartment.12, 13 These findings therefore also implicate macroautophagy in humoral autoimmunity. Studying the involvement of autophagy in autoimmunity is justified by its central

regulatory impact in inflammation and its role on antigen presentation and on lymphocyte activation and survival.14, 15 Our team and others described a deregulation of macroautophagy in T

cells from both lupus-prone mice and patients suffering from systemic lupus erythematosus (SLE).16, 17, 18 This deregulation could contribute to autoreactive T-cell survival and can be put

in line with the deregulation of both macroautophagy and chaperone-mediated autophagy (CMA) in B cells that has been recently described to occur in lupus.19, 20 To date, however, these data

remain correlative and no study has been published describing an _in vivo_ model, prone to systemic autoimmunity, with specific autophagy deletion in B cells. Here, we describe the

generation of two new mouse models of conditional ATG5 deletion, one under the control of a promoter active early during B-cell development (Mb1 cre) and the other active in mature B cells

only (CD21 cre). They were designed and constructed to clarify the role of macroautophagy in B-cell development and homeostasis. Comparison of the two models should allow us to define

whether identified defects are linked to developmental issues or deregulation of homeostasis. In addition, _Atg5__f/−_ CD21 cre autophagy-deficient mice were crossed with autoimmune-prone

animals to further examine the role of macroautophagy _in vivo_ on long-term humoral autoimmunity and study some lupus-related pathophysiological features. RESULTS BASAL LEVELS OF AUTOPHAGY

ARE NECESSARY FOR B-CELL MAINTENANCE We generated two new mouse models deficient for ATG5 specifically in B cells. The first one, with cre recombinase expression under the control of Mb1

promoter, was deleted early during B-cell development while the second one, under the control of CD21 promoter, was designed to be deleted in mature B cells only. Both mouse models were born

at the predicted Mendelian ratio and showed no particular morphologic phenotype. _Atg5__f/−_ CD21 cre (CD21 cre) mice exhibited no deletion of ATG5 in splenic T cells and in these cells,

conversion of LC3-I to LC3-II occurred normally (Figure 1a). However, as expected, B cells from CD21 cre mice showed a specific deletion of ATG5 compared with B6 mice (wild type, WT) and

littermate _Atg5__f/+_ CD21 cre mice at both transcriptional and protein levels (Figures 1b and d). A poor conversion of LC3 was observed in _Atg5__f/−_ CD21 cre mice validating the impeding

of the autophagy process, even under stimulation by anti-IgM. As previously reported by others,19 no increase in autophagic activity was found after BCR stimulation in WT and littermate

mice. We also checked whether autophagy was decreased in B cells from _Atg5__f/−_ Mb1 cre (Mb1 cre) mice. We saw a very efficient deletion of ATG5 in splenic B cells from _Atg5__f/−_ Mb1 cre

mice, correlating with a total impairment of LC3-II generation (Figures 1c and d). _Atg5_ mRNA expression was reproducibly lower than for CD21 cre B cells. Although B cells from CD21 cre

mice exhibited a reproducible LC3-II knockdown (Figure 1d), LC3-II was no longer observed in Mb1 cre B cells, arguing for a specific deletion of autophagy that is stronger in this model than

in CD21 cre mice. Although ATG5 deletion is observed in littermates relative to both CD21 cre and Mb1 cre mice compared with WT C57BL/6 mice, autophagy levels (deduced from LC3-II staining)

were comparable and no significant difference was observed in terms of autophagic flux intensity (Supplementary Figure S1). To more specifically evaluate the potential autophagy-independent

effects of decreased ATG5 expression on phenotypes of both littermates, we then conducted a series of experiments using CD21 cre and Mb1 cre mice separately compared with WT mice. First, we

quantified the different subpopulations of B cells in the spleen of these two defective models. Although both types of littermate mice exhibited levels of B cells comparable to wild-type

mice, both Mb1 cre and CD21 cre mice showed a diminished number of B cells in the spleen (Figures 2a and b). A decrease in follicular B-cell number and T1 B cells was also observed in both

CD21 cre and Mb1 cre mice, as compared with WT mice. However, the homeostatic defect of follicular (FO) B cells in CD21 cre was less severe than in Mb1 cre mice, arguing for a more efficient

autophagy impairment in the latter deletion strategy, as described above in Figure 1. Mb1 cre T2 B cells were also diminished compared with the corresponding littermates. However, marginal

zone B cells were not seen to be affected by autophagy deletion in both deletion models. These results led us to propose that _Atg5_ deletion in B cells induces a defect in peripheral

homeostasis, the latter being more pronounced in the context of Mb1 cre-mediated deletion. Next, we assessed the repartition of B-cell populations from the peritoneum. A decrease in both

B-1a and B-2 B-cell proportions was observed in Mb1 cre mice while, in contrast, a decrease in peritoneal B-2 B cells only was visualized in CD21 cre mice (Figures 2f and g). This finding

led us to conclude that early during development, autophagy is necessary for the generation of B-1a B cells. The loss of B-2 cells in both mice might be due to sensitivity to cell death of

these Ab-secreting cells in the absence of autophagy, or to a defective replenishment from circulating cells. Altogether, these results show that autophagy is central during development for

B-1a B-cell generation. Basal levels of autophagy are necessary to maintain a normal number of B cells in the periphery. B-2 B cells of the peritoneum are particularly sensitive to the

decrease in autophagic activity. BASAL LEVELS OF AUTOPHAGY ARE NOT NECESSARY FOR B-2 B-CELL DEVELOPMENT The survival defects of peripheral B cells found in Mb1 cre mice could be linked to a

developmental issue as described in earlier studies using bone marrow fetal liver chimaeras.6 This does not seem to be only attributable to developmental issues as some defects were also

found in CD21 cre mice. We checked the number of precursor cells of the B-cell lineage in the bone marrow of Mb1 cre and CD21 cre mice, and compared the data with WT and littermate mice

(Figures 3a and b). As expected, no anomaly in B-cell number or in subset distribution was detected in CD21 cre mice when autophagy deletion occurs later during development (Figures 3c and

f). In sharp contrast with previous studies,6 we did not find any significant decrease in the number of B-cell precursors in Mb1 cre mice. Previous data suggested a developmental block at

the transition between the pro- and pre-B cell stages.6 In this study, normal proportions of pro- and immature B cells were detected in the bone marrow of early ATG5-deleted mice (Figures 3c

and d). Distinction of pre-B and immature B cells from pro-B cells by CD43 staining did not reveal any decrease in the proportion of pre-B cells in Mb1 cre mice, but instead showed an

increase in this population (Figures 3e and f). Thus, autophagy seems dispensable for the pro- to pre-B cell differentiation. However, as previously described by others6 using CD19 cre mice,

we observed a decrease in the proportion of mature B cells, corresponding to Fraction F defined from Hardy nomenclature (Figures 3c and f).21 As the overall number of B cells does not vary

between Mb1 cre and their littermates, the increased proportion of pre-B cells in these mice could reflect a spatial compensation consecutive to the loss of mature B cells. As these latter

cells are described as recirculating cells coming from the periphery, they may not necessarily constitute a final developmental stage occurring in the bone marrow. They have also been

considered as cells secreting Abs against blood-borne pathogens.22 The acute deletion of autophagy in Mb1 cre mice might thus lead to sensitivity to cell death of this B-cell subtype,

because of their long-lived nature or of their Ab secretion properties. Taken together, these results show that autophagy is dispensable for the transition between pro- and pre-B cell stage.

However, basal levels of autophagy are necessary to maintain a normal number of mature recirculating B cells in the bone marrow. AUTOPHAGY IS DISPENSABLE FOR B-CELL ACTIVATION IN RESPONSE

TO BCR STIMULATION We then assessed in our two models the potential relevance of autophagy activation in B-cell response to BCR stimulation. We found no difference in terms of B-cell

proliferation in Mb1 cre and CD21 cre mice stimulated by anti-IgM Abs with or without anti-CD40 stimulation (Figures 4a and b) nor in B-cell survival (Figures 4c and d), compared with their

respective littermates. These results are in line with previous reports showing that short-term B-cell stimulation by antigen does not require autophagic activity. In contrast, under

lipopolysaccharide (LPS) stimulation, both Mb1 cre and CD21 cre mice showed a significantly reduced survival. LPS is known to drive differentiation of mouse B cells into plasmablasts. These

findings highlight a preferential role of autophagy in Ab-secreting cell survival rather than in initial B-cell activation. Incidentally, no exclusive annexin-V staining in CD138-negative

population was found in these conditions, suggesting that B cells upregulate this plasma cell marker normally, but then fail to survive in the absence of autophagy (Figure 5a). Pengo _et

al._11 previously described a defect in ER homeostasis, during plasma cell differentiation in the absence of autophagy. Here, we show further that both CD21 cre and Mb1 cre B cells exhibit

an increased mitochondrial content after LPS stimulation as shown by mitotracker staining compared with littermates (Figures 5b and c). Furthermore, mitotracker deep red staining experiments

revealed the presence in both deficient models of an increased cell population containing damaged mitochondrial content. We thus propose that autophagy participates in early plasmablast

survival by limiting the potential damages related to increased and/or damaged mitochondrial content. We then examined short-term humoral responses against the T cell-dependent model antigen

OVA (Figure 6a). No difference was observed in the total levels of IgM and IgG in Mb1 cre and CD21 cre mice compared with controls (Figure 6b). Likewise, the anti-OVA IgG secretion in CD21

cre mice and control mice was similar (Figure 6c). This observation could be explained by the fact that in our immunization conditions, long-lived IgG secreting plasma cell differentiation

is normal in the absence of autophagy in B cells, and that survival defects might impact IgG secretion at a longer term. However, a significant drop in anti-OVA IgM secretion was observed in

both deficient mice (Figure 6c), which could reflect decreased survival of short-lived plasma cells in the absence of autophagy. Our results do not fully recapitulate the conclusions stated

in the work of Cenci’s group,11 where both IgM and IgG secretions were altered in the absence of autophagy. This could be explained by the difference in the immunization protocol. We thus

applied a protocol closer to the one used by Pengo and immunized Mb1 cre mice and their littermates only once, without boost. Although a slight decrease in both IgG and IgM levels was

observed, the differences did not reach significance (Supplementary Figure S2). We cannot totally exclude that other factors linked to immunization, such as the adjuvant for example, may

have influenced the data. The use of Freund’s adjuvant for repetitively immunizing mice might effectively amplify the deleterious effect of autophagy deletion in short-lived plasmablasts.

However, the results we generated in our experimental settings strongly suggest that although antigen-specific responses are altered, continuous differentiation of naïve B cells into plasma

cells allows uninterrupted secretion of Ig, as shown by normal levels of total Ig. Overall, these results demonstrate that in the absence of autophagy in B cells, activation driven by BCR

stimulation is largely normal. However, early specific humoral response is compromised, probably due to a defect of short-lived plasma cells, as illustrated by the high mortality rates after

stimulating B cells deficient for autophagy by LPS. AUTOPHAGY IN B CELLS IS INTEGRAL TO THE LONG-TERM MAINTENANCE OF AUTOIMMUNE RESPONSES Both macroautophagy and CMA have recently been

shown to be deregulated in B cells from lupus-prone mice and patients with lupus.19, 20 To complete our analysis, we thus asked the question about whether deficiency in _Atg5_ could

influence autoimmune responses. Owing to the defect in peripheral B-cell homeostasis observed in Mb1 cre mice, these experiments were performed using CD21 cre mice exhibiting a milder

phenotype, to be crossed with B6_lpr/lpr_ mice. The B6_lpr/lpr_ strain develops a mild autoimmunity characterized by the secretion of autoAbs directed against nuclear antigens and Ig and

complement deposition in kidneys.23 Crossed mice were born at the expected Mendelian ratio and did not exhibit differential mortality rates before the age of observation. Compared with CD21

cre _Atg5__f/+ lpr/lpr_ littermate mice (littermates_lpr/lpr_), there was no significant difference in CD21 cre _Atg5__f/−lpr/lpr_ mice (CD21 cre_lpr/lpr_) when total IgM or specific

anti-dsDNA IgM levels were measured (Figure 7a). In sharp contrast, however, CD21 cre_lpr/lpr_ mice exhibit a markedly reduced hypergammaglobulinemia and secreted much less anti-dsDNA IgG

(Figure 7a). Several sets of experiments were then designed to investigate whether this reduction could be due to a decreased B-cell survival in peripheral lymphoid organs. No change in

splenic B-cell proportions was observed in CD21 cre_lpr/lpr_ mice compared with controls (Figure 7b) and no difference was detected in GC area relative to the surface of the spleen section

scanned by confocal microscopy (Supplementary Figure S3). These results indicate that in our model of autoimmunity, autophagy does not have any significant role in the early steps of B-cell

activation. A slight reduction of splenic plasma cells was noted, but it did not reach statistical significance, consistent with the fact that IgM autoAb levels remain unaffected. These

findings may result from a greater replenishment of new short-lived plasma cells in the _lpr_ model, compensating their loss in the absence of autophagy. In contrast to the above

observations, a reproducible drop in CD138+B220_−_ plasma cell counts was found in the bone marrow of CD21 cre_lpr/lpr_ mice, compared with controls (Figures 6c and d). For some samples,

intracellular IRF4 costaining was performed to ensure that the cell population previously identified corresponds to plasma cells. These results fully agree with the above-described

diminished levels of IgG secreted by long-lived CD138+B220_−_ plasma cells, which reside in bone marrow niches. Finally, we investigated the pathophysiological relevance of the decreased

autoAb secretion levels observed in CD21cre_lpr/lpr_ mice. In agreement with the above-described results, immunofluorescence imaging of renal glomeruli clearly showed that IgG deposit

staining was weaker in CD21 cre_lpr/lpr_ mice compared with littermate controls (Figure 8). Altogether, these results indicate that in a mild spontaneous model of lupus, autophagy is a

central component of the long-term maintenance of autoAb production by plasma cells and Ab deposits in target organs, while B-cell proportion remains unaffected. DISCUSSION The main purpose

of this study was to provide new insight into the role of autophagy in the development of B cells. It was previously assumed that autophagy was important for the transition from pro- to

pre-B cells.6 These results were obtained using fetal liver chimaeras with ATG5-deficient cells, in which autophagy was deleted at the early stages of embryonic life, and thus before the

initial haematopoietic stem cell development took place. Owing to the major role of autophagy in the survival of haematopoietic stem cells and in lymphocyte precursors,8 it thus remained

possible that accumulation of defects in the absence of autophagy in B-cell precursors led to the observed defects on pre-B cell survival. Indeed no such defects were observed using CD19,

expressed from the pre-B cell stage, to drive cre expression.6 It could be argued further that CD19 leads to incomplete ATG5 deletion at this early developmental stage. We thus created a new

mouse expressing cre recombinase under the control of the strong Mb1 promoter known to drive very efficient deletion at the pro-B cell stage. In the mice generated in this study, contrary

to previous observations, we did not find any developmental blockade at the pro/pre-B cell transition. We thus concluded that autophagy is dispensable for B-cell development in the bone

marrow. However, we found a reproducible decrease in mature recirculating B cells in the bone marrow. These cells occupying a specific niche in this central lymphoid organ, are described as

long-lived cells, secreting Abs against blood-borne pathogens.22 As CD21 cre mice exhibit no such loss, we cannot totally rule out a developmental issue in B-cell precursors leading to

selective defects in this population. It is also possible that the suboptimal deletion of ATG5 in CD21 cre mice is responsible for their normal survival contrary to Mb1 cre mice where the

deletion was total. Thus, basal levels of autophagy are particularly necessary for the survival of mature B cells in the bone marrow. In line with previous reports, we found a strong

decrease in B-1a B cells in the peritoneum. Again this loss may be relative to developmental issue, as Mb1 cre mice exhibit this defect but CD21 cre mice did not. Overall, these data support

the idea that early precursors of B-1a B cells critically depend on autophagy to generate viable cells at long term. Interestingly, we also found a diminished survival of B-2 B cells in the

peritoneum of both CD21 cre and Mb1 cre mice. This could be explained by the secretory activity of these cells,24, 25 or by their nature of mature recirculating cells. In the latter case

and in contrast to what occurs in lymphoid organs, a slower replacement rate of dying cells by new B-cell emigrants issued from the bone marrow could account for these observations. The

survival of naive B-2 cells in lymphoid organs was found to be compromised in both CD21 cre and Mb1 cre mice, in a stronger way for the latter deletion strategy. This difference could be

linked to the very efficient deletion of ATG5 in Mb1 cre mice, whereas some basal autophagy remained in B cells from CD21 cre mice. It seems that, contrary to what was previously assumed,

basal levels of autophagy are indeed required for the survival of B cells in the periphery. This observation may be related to an impairment of homeostatic maintenance, as proliferation and

survival upon BCR signalling were normal in both Mb1 cre and CD21 cre mice. Interestingly, in our study, the only effective stimulus leading to survival defect was the one generated by LPS,

and this was observed in both our deletion models. LPS is known to drive the differentiation of mouse B cells into plasmablasts. Thus, the selective survival defect could also be linked to

the role autophagy plays in the survival of Ab-secreting cells. A defective IgM production against the T cell-dependent antigen OVA was observed in both Mb1 cre and CD21 cre mice. In

contrast, IgG production against OVA appeared to be normal in the absence of autophagy. A possible explanation for this outcome would be the defective survival of short-lived plasma cells in

secondary lymphoid organs in the absence of autophagy while long-lived plasma cells could transiently resist the absence of autophagy. In this setting, defective IgG production could be

observed on the long term only. It is also possible that selection processes allow autophagy-competent cells only to survive, as it has been described in mice with CD19 cre-mediated ATG5

deletion.11 This latter argument remains however uncertain, knowing the very efficient ATG5 deletion observed in our models, especially in Mb1 cre mouse B cells. To study the long-term

impact of autophagy deletion in B cells, we used the spontaneous systemic autoimmunity model driven by the _lpr_ mutation on a B6 genetic background.26 This mutation on _Fas_ gene leads to

secretion of autoAbs directed against nuclear antigens, which can deposit in several organs, especially in the kidneys of affected animals.23 The pathology developed over time, although

relatively mild in comparison with MRL/lpr mice, for example, mimicking some lupus features. _Atg5__f/−_ CD21 cre × B6_lpr/pr_ autophagy-deficient autoimmune mice showed no survival failures

in B cells, supporting the minimal role of autophagy in B-cell survival, even in an autoimmune context. In contrast, and in good agreement with data reported in other settings,11, 12, 13 we

found a decrease in plasma cell proportion in the bone marrow reflecting selective defects in long-lived cells. This drop in long-lived Ab-secreting cells may be responsible for the

reduction of hypergammaglobulinemia and anti-dsDNA Abs observed in our autophagy-defective mice. Note that we cannot formally exclude a possible contribution of the reduced memory B-cell

compartment in _lpr_ mice deficient for autophagy.12, 13 Interestingly, we also found less IgG deposits in the kidneys of CD21 cre_lpr/lpr_ mice. Considering the above-described findings, we

propose that this feature is mainly due to autophagy impairment in plasma cells, and not in initial B-cell activation, as no major defect in GC formation was noticed. In summary, using

three novel mouse models defective for autophagy process, we report here that autophagy is dispensable for B-2 B-cell development contrary to B-1a B cells and that basal levels of autophagy

are required in B cells to maintain their normal number in lymphoid organs. We confirm in a new setting that autophagy is a major player of long-lived B cells and Ab-secreting cell survival.

We describe further that mice exhibiting signs of lupus autoimmunity and in which autophagy was diminished, show significantly less biological (lower anti-dsDNA IgG Ab levels) and clinical

(less IgG deposits in their kidneys) lupus features, without having their whole B-cell compartment adversely affected. Therefore, autophagy modulation appears to be a good therapeutic option

to limit autoAb-linked inflammation in systemic autoimmunity. MATERIALS AND METHODS MICE _Atg5__f/f_ mice, with a flox sequence flanking exon 3 of _Atg5_ gene backcrossed on a C57BL/6 (B6)

background have been described27 were a kind gift from Prof. N Mizushima. We also used mice with systemic deletion of exon 3 (_Atg5__+/−_) to generate Atg5_f/−_mice. _Atg5__f/f_ mice were

crossed with _Atg5__+/−_ CD21 cre28 or _Atg5__+/−_Mb1 cre9 mice for B-cell lineage-specific deletion. In all experiments, B6 _Atg5__f/__−_ CD21 cre or B6 _Atg5__f/−_ Mb1 cre mice with B-cell

specific deletion of _Atg5_ were compared with littermates (B6 _Atg5__f/+_CD21cre mice and _Atg5__f/+_ Mb1cre mice). In some experiments, B6 _Atg5__f/−_ CD21cre mice were crossed with B6

mice bearing the _lpr_ mutation affecting _Fas_ gene, driving a spontaneous systemic autoimmunity. In this setting, lupus-prone B6_lpr/lpr_ _Atg5__f/−_ CD21 cre (3 females, 1 male) were

compared with age-matched B6_lpr/lpr_ _Atg5__f/+_ littermates (4 females, 1 male). Mice were genotyped for the _Atg5_ alleles (WT, with constitutive or induced deletion) with the primers A

(exons 3–1), 5′-GAATATGAAGGCACACCCCTGAAATG-3′; B (short 2), 5′-GTACTGCATAATGGTTTAACTCTTGC-3′; C (check2), 5′-ACAACGTCGAGCACAGCTGCGCAAGG-3′; D (5L2), 5′-CAGGGAATGGTGTCTCCCAC-3′ using PCR (94 

°C (4 min); 30 cycles of 94 °C (30 s), 60 °C (30 s), 72 °C (1 min); 72 °C (5 min)). The cre transgene expressed under the control of CD21 promoter was detected with primers cre-F

5′-ACGACCAAGTGACAGCAATG-3′; cre-R 5′-CTCGACCAGTTTAGTTACCC-3′ and for the cre expressed under Mb1 promoter with primers, cre-F 5′-ACCTCTGATGAAGTCAGGAAGAAC-3′; cre-R

5′-GGAGATGTCCTTCACTCTGATTCT-3′ using PCR (94 °C (4 min); 25 cycles of 94 °C (30 s), 60 °C (30 s), 72 °C (1 min); 72 °C (5 min)). The _lpr_ allele was identified by PCR using a sense primer,

5′-AGGTTACAAAAGGTCACCC-3′, in intron 2 and two antisense primers, 5′-GATACGAAGATCCTTTCCTGTG-3′ and 5′-CAAACGCAGTCAAATCTG CTC-3′ using PCR (94 °C (4 min); 30 cycles of 94 °C (45 s), 55 °C (1 

min), 72 °C (1 min); 72 °C (5 min)). We genotyped mice with the REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO, USA). All mice were bred and maintained in accordance with

guidelines of the local Institutional Animal Care and Use Committee (CREMEAS). FLOW CYTOMETRY The majority of antibodies (Abs) used for flow-cytometry analyses were purchased from BD

Biosciences (Le Pont-De-Claix, France): allophycocyanin (APC)-Cyanin 7-labelled anti-mouse TCR-_β_ (clone H57-597, 553139), phycoerythrin (PE)-labelled anti-mouse CD69 (clone H1.2F3,

553236), fluorescein isothiocyanate (FITC), APC or peridinin chlorophyll (PerCP) cyanine 5.5-labelled anti-mouse B220 (clone R13-6B2, 553087, 553092, and 552771), FITC-labelled anti-mouse

CD3ɛ (clone 145.2C11, 553061), FITC-labelled anti-mouse CD21/CD35 (clone 7G6, 553818); PE-labelled anti-mouse CD23 (clone B3B4, 553139), APC-labelled anti-mouse CD5 (clone 53-7-3, 550035),

APC-labelled anti-mouse CD43 (clone S7, 560663), APC-labelled anti-mouse IgD (clone 11-26 c.2a, 560868), APC-labelled anti-mouse CD19 (clone 1D3, 550992), and APC-labelled anti-mouse CD138

(clone 281-2, 558626). FITC-conjugated polyclonal anti-mouse IgM was purchased from Jackson Immunoresearch (Newmarket, UK; 115-095-020) and PE-labelled anti-IRF4 from Biolegend (San Diego,

CA, USA; clone 4.3E4, 646404). Cells were incubated with fluorochrome-conjugated Abs and with unlabelled rat anti-mouse CD16/CD32 monoclonal Ab (mAb, clone 2.4G2, 553142) to block Fc

receptors, for 15 min at 4 °C in phosphate-buffered saline (PBS) pH 7.4 containing 2% (v/v) fetal calf serum (FCS). Intracellular staining was performed after surface marker labelling using

the FoxP3 detection kit purchased from eBiosciences (San Diego, CA, USA; 00-5523-00). Mitochondria content and membrane potential were measured by incubating cells 30 min at 37 °C in

complete medium in the presence of mitotracker green and mitotracker deep red (Fisher Scientific, Pittsburgh, PA, USA; M-7514 and M22426) at the recommended concentrations. Data were

collected on a Gallios flow cytometer (Beckman Coulter, Fullerton, CA, USA) and analysed using FlowJo software (FlowJo LLC, Ashland, OR, USA). CELL CULTURE AND ISOLATION Splenic B cells were

collected from B6 _Atg5__f/−_ CD21 cre, B6 _Atg5__f/−_ Mb1 cre, B6_lpr/lpr_ _Atg5__f/−_ CD21 cre mice or from littermates and immediately cultured at 37 °C, 5% CO2 in complete RPMI-1640

medium (Lonza BioWhittaker, Levallois, France) containing 10% FCS, 10 _μ_g/ml gentamycin (Lonza BioWhittaker), 10 mM HEPES (Lonza, Basel, Switzerland) and 0.05 mM _β_-mercaptoethanol (Lonza)

at a concentration of 5 × 106 cells/ml. Splenic B cells were purified by negative selection. Briefly, spleen cell suspensions were depleted from monocytes, granulocytes, T cells, and NK

cells using the Pan B cell isolation Kit II (130-090-862; Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. Resulting TCR-β-/B220+ mouse B-cell

preparations were >95% pure as determined by flow cytometry. Cells were stimulated with goat F(ab)’2 anti-IgM (Jackson Immunoresearch; 115-006-020) with or without anti-CD40 Ab (5 

_μ_g/ml, BD Biosciences, clone HM40-3, 553721) or LPS alone (5 _μ_g/ml, Sigma, Saint-Quentin-en-Yvelines, France). Alternatively, T cells were sorted from spleen cell suspension with the

Dynabeads untouched mouse T cell isolation kit (ThermoFisher Scientific, Illkirch, France; 11413D) and were stimulated by anti-CD3ɛ Ab (clone 145.2C11, 553057) purchased from BD Biosciences.

For the analysis of proliferation, cells were stained with 0.5 _μ_ M carboxyflurescein isothiocyanate succinimidyl ester diacetate (CFSE, Sigma-Aldrich) before the stimulation. Cell death

was assessed by Annexin-V-FITC/7-AAD double staining (BD Biosciences, respectively, 559925 and 556419). After acquisition by flow cytometry, proliferation and cell death results were

analysed with FlowJo Software. IMMUNOBLOTS The Abs used for western immunoblotting were specific for ACTB (Santa Cruz Biotechnology, Heidelberg, Germany, clone C4, sc-47778), LC3 (MBL,

Nagoya, Japan, clone 51-11, ref M115-3), and ATG5 (Polyclonal, Novus, Littleton, CO, USA). In some experiments, lysosomal protease inhibitors E64d and pepstatin A (Sigma-Aldrich; P5318 and

E8640) were added at 5 _μ_g/ml each. When indicated, cells were treated with goat F(ab)’2 anti-IgM (5 _μ_g/ml, Jackson Immunoresearch; 115-006-020). To evaluate the autophagosomal membrane

load, whole-cell proteins were extracted from cultured cells using Laemmli buffer (125 mM Tris-HCl, pH 6.8; 2% (w/v) sodium dodecyl sulphate (SDS); 10% (v/v) glycerol; 5% (v/v)

_β_-mercaptoethanol). Cell lysates were separated using 4–20% gradient gels (Bio-Rad, Hercules, CA, USA) and proteins transferred onto a polyvinylidene difluoride membrane. Membranes were

blocked with PBS containing 0.1% (v/v) Tween-20 (PBS-T) and 3% (w/v) non-fat dry milk for 1 h and then incubated overnight at 4 °C with 1 _μ_g/ml anti-LC3 Ab in PBS-T containing 1% non-fat

dry milk, or for 1 h at room temperature with 1 _μ_g/ml anti-ATG5 Ab in PBS-T containing 1% non-fat dry milk. After washing with PBS-T, membranes were incubated for 30 min at room

temperature with goat anti-mouse IgG Ab (Southern Biotech, Birmingham, AL, USA; 1030-05) conjugated to horseradish peroxidase (HRP). Signal was detected using enhanced chemiluminescence

detection reagents (Immobilon Western, Merck Millipore, Darmstadt, Germany; WBKLS0500). When indicated, LC3-II and ATG5 staining was normalized by densitometry to ACTB staining using the

ImageJ Software (National Institute for Health, Washington, DC, USA). REAL-TIME PCR Total RNA was isolated from 5 × 106 purified B cells using the RNeasy Mini Kit (Qiagen, Courtabeuf,

France, ref 74103) according to the manufacturer’s instructions. After treatment by DNAse (Qiagen, ref 79254) to remove residual genomic DNA, mRNA was retro-transcribed with the Maxima

first-strand synthesis kit for cDNA (ThermoFisher, Illkirch, France). Fifteen nanograms of cDNA was used for real-time PCR (RT-PCR) on StepOne apparatus (ThermoFisher). Briefly, _Atg5_ and

_Gapdh_ cDNA was amplified using Taqman Gene Expression Assays provided by ThermoFisher (ref Mm00504340_m1 and Mm99999915_g1). Amplicons and probe were designed to span two exons, limiting

the risk of amplifying residual genomic DNA. Relative _Atg5_ mRNA quantifications were made by defining _ΔC_T (_C_T Gapdh−_C_T _Atg5_ where _C_T is 'Cycle Threshold') and _ΔΔC_T

(_Δ_CT sample−ΔCT of one C57BL/6 mouse sample used for each plate) using StepOne software (ThermoFisher). Results shown represent 2_−__ΔΔ_CT values where one same control sample is used on

each plate and arbitrarily equal to 1. IMMUNIZATION Eight- to twelve-week-old mice were injected intraperitoneally (i.p.) at days 0, 10, and 20 with the antigen suspension, and bled on days

5, 15, and 25 after the first immunization. Mice received 100 _μ_g ovalbumin (OVA, Sigma) in complete Freund’s adjuvant (CFA, Sigma) for the first injection and 100 _μ_g OVA in incomplete

Freund’s adjuvant (IFA, Sigma) for the second and third injections. Alternatively, mice were injected only once with 100 _μ_g OVA in CFA and were bled at days 7 and 14 to follow the anti-OVA

response. ANTIBODY DETECTION BY ELISA IgG or IgM titres were measured in serum from immunized or lupus-prone mice. To measure anti-OVA specific Abs, 96-well ELISA Maxisorp plates (NUNC,

Fisher Scientific) were coated with OVA (10 _μ_g/ml, Sigma) in 50 mM sodium carbonate buffer (pH 9.6). The wells were blocked with PBS-T 0.1%, 5% (w/v) milk for 1 h at 37 °C and incubated

with diluted anti-sera for 1 h at 37 °C. HRP-conjugated anti-mouse isotype-specific Abs (Jackson Immunoresearch) were used to reveal bound Abs. Absorbance was measured at 450 nm, after

revelation with tetramethylbenzidine to the wells, and stop of the reaction by 1m HCl. The same protocol was used for detection of anti-double stranded (ds)DNA Abs except that ELISA plates

were coated with 100 ng/ml non-sheared dsDNA (from calf thymus, Sigma-Aldrich) in 25 mM citrate buffer, pH 5. Titres correspond to the inverse of the last dilution giving an absorbance equal

to 0.2. IMMUNOHISTOFLUORESCENCE Spleen/kidneys were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA; 4583) and snap frozen with liquid nitrogen. To visualize IgG

deposits in the kidney, tissue sections were then stained with goat anti-mouse IgG-FITC (Southern Biotech; 1030-02) and DAPI. To detect GC in the spleen, tissue sections were stained with

peanut agglutinin (PNA, Vector, Burlingame, CA, USA; FL-1071), IgD-APC, and DAPI. Images were acquired with a spinning disk confocal microscope (Zeiss, Oberkochen, Germany) with an A-Plan

20x/0.8 Zeiss × 20 objective. Images were analysed with the ImageJ Software. The quantification of IgG deposits in the kidney was performed as follows: glomeruli were identified via typical

structures shaped by DAPI staining. A zone comprising 20 glomeruli was delimited for each kidney section. Fluorescence intensity given by the anti-IgG staining was measured in each

glomerulus, and background intensity of the delimited zone was subtracted. The quantification for GC in spleen section was performed by delimiting zones positive for PNA staining, measuring

the area of the zone, and by normalizing it to the total area of the scanned section. STATISTICAL ANALYSES The data were analysed with the Prism software (GraphPad, San Diego, CA, USA) using

two-tailed unpaired _t_-tests or Mann–Whitney _U-_test. Error bars represent standard deviations (SD). ABBREVIATIONS * Abs: antibodies * ACTB: _β_-actin * _Atg_ : autophagy-related genes *

BCR: B-cell receptor * CFA/IFA: complete/incomplete Freund adjuvant * CFSE: carboxyfluorescein succinimidyl ester * CMA: chaperone-mediated autophagy * dsDNA: double-stranded DNA * ER:

endoplasmic reticulum * FO: follicular * GC: germinal centre * IRF4: interferon-responding factor 4 * LC3: light-chain 3 standing for _MAP1LC3B_, microtubule-associated protein light-chain

3B * LPS: lipopolysaccharide * MHC: major histocompatibility complex * MZ: marginal zone * OVA: ovalbumin * PNA: peanut agglutinin * SLE: systemic lupus erythematosus * T1 and T2:

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tail. _J Exp Med_ 2001; 194: 455–469. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Prof. Noboru Mizushima for the gift of Atg5_f/f_

mice. This work was funded by the French Centre National de la Recherche Scientifique, the Laboratory of Excellence Medalis (ANR-10-LABX-0034) and the EquipEx program I2MC (ANR-11-EQPX-022),

Initiative of Excellence (IdEx), Strasbourg University, and the Fondation Arthritis Courtin. JA was a recipient of pre-doctoral fellowship from the Ministère de la Recherche et de

l’enseignement supérieur and from Association de Recherche Contre le Cancer; DM was a recipient of pre-doctoral fellowship from the Fond National de Recherche of Luxembourg; FA was a

recipient of pre-doctoral fellowship from the Ministère de la Recherche et de l’enseignement supérieur. AUTHOR CONTRIBUTIONS JA, JDF and FG performed and designed experiments. DM and FA

performed experiments. SM and FG wrote the article. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * CNRS, Immunopathology and Therapeutic Chemistry/Laboratory of Excellence MEDALIS, Institut

de Biologie Moléculaire et Cellulaire, Strasbourg, France J Arnold, D Murera, F Arbogast, J-D Fauny, S Muller & F Gros * University of Strasbourg Institute for Advanced Study,

Strasbourg, France S Muller * University of Strasbourg, Strasbourg, France F Gros Authors * J Arnold View author publications You can also search for this author inPubMed Google Scholar * D

Murera View author publications You can also search for this author inPubMed Google Scholar * F Arbogast View author publications You can also search for this author inPubMed Google Scholar

* J-D Fauny View author publications You can also search for this author inPubMed Google Scholar * S Muller View author publications You can also search for this author inPubMed Google

Scholar * F Gros View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to F Gros. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by M Piacentini Supplementary Information accompanies this paper on Cell Death and Differentiation website

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THIS ARTICLE CITE THIS ARTICLE Arnold, J., Murera, D., Arbogast, F. _et al._ Autophagy is dispensable for B-cell development but essential for humoral autoimmune responses. _Cell Death

Differ_ 23, 853–864 (2016). https://doi.org/10.1038/cdd.2015.149 Download citation * Received: 24 April 2015 * Revised: 04 October 2015 * Accepted: 12 October 2015 * Published: 20 November

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