ATG5 контролирует выражение CD80 в клетках B и фигурах, соединяющих CD4+T реакции клеток.

Introduction

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS). The genetic association with HLA class II haplotypes and the dependence on myelin-reactive CD4 + T cells in experimental adoptive transfer models of demyelinating disease, such as experimental autoimmune encephalomyelitis (EAE), indicate an important role for CD4 + T cells in the pathogenesis of MS [ 1 ]. However, experimental and clinical evidence has accumulated, specifically over the past 15 years, that strongly implicates B cells in driving disease development and progression. Precise pathogenic functions of B cells in MS remain incompletely understood but are believed to include B- and T-cell interactions through antigen presentation and production of proinflammatory cytokines [ 2 , 3 ].

B-cell functions are not featured well in classical EAE models in which accumulation of autoreactive T cells within the CNS and clinical disease development depend on the presence of ITGAX/CD11c + antigen-presenting cells (APCs) interacting with myelin-specific CD4 + T cells [ 4 ]. We recently demonstrated that this effector phase, induced by adoptive transfer of primed MOG (myelin oligodendrocyte glycoprotein)-specific CD4 + T cells into naïve mice, requires ATG (autophagy related) protein-dependent phagocytosis in dendritic cells (DCs) [ 5 ]. Autophagy plays a differential role in B-cell subsets, being essential for the efficient development and self-renewal of B-1 B cells, that respond to T-independent antigens, but not to B-2 B cells, that respond to T-dependent antigens [ 6 , 7 ]. Although there is evidence that autophagy is dispensable for B-cell activation following BCR stimulation [ 8 ], B cells lacking ATG7, another component of the MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) conjugation system, are impaired in their ability to develop memory responses upon influenza infection [ 9 ].

Here, we hypothesized that ATG5-dependent antigen processing in B cells, similar to our findings in DCs, enhances MHC class II presentation to cognate CD4 + T cells. We further hypothesized that targeted deletion of Atg5 in B cells protects from EAE development. To address this hypothesis, we capitalized on an adjuvant-free co-transfer EAE model, in which MOG-specific B cells provide the critical stimulus for MOG-specific T-cell activation and disease development [ 10 ].

Results

Deleting Atg5 in activated B cells accelerates EAE development

To address whether ATG5 in B cells modulates EAE susceptibility and progression, we generated conditional knockout mice (C57BL/6) for disruption of Atg5 in MOG-specific B cells by crossing IgH MOG -CD19-Cre mice with Atg5 flox/flox mice for at least five generations, designated B MOG - atg5 −/− mice. IgH MOG mice carry the heavy chain from the MOG-specific antibody 8.18c5 as a knock-in [ 11 ] with 90% of B cells recognizing MOG via their BCR, as determined by MOG-protein-tetramer staining [ 12 ]. Notably, IgH MOG mice show no signs of spontaneous EAE, however, when crossed with 2D2 mice, which carry MOG-specific TCR transgenic CD4 + T cells, about 60% of animals spontaneously develop severe EAE, indicating that antigen-specific B and T cells cooperate to induce autoimmunity [ 13 , 14 ]. IgH MOG -CD19-Cre −/−
Atg5 flox/flox mice, designated B MOG - Atg5 +/+ mice, were used as littermate controls Figure 1(a) .
Figure 1. Lack of Atg5 in activated B-cells accelerates EAE development. (A) breeding strategy for atg5 deletion in MOG-specific B cells. IgH MOG -CD19-Cre mice were crossed with Atg5 flox/flox mice to generate B MOG - Atg5 +/+ and B MOG - atg5 −/− experimental mice. Created with BioRender. (B) experimental strategy for an adjuvant-free B cell dependent EAE induction. B cells were isolated from B MOG - Atg5 +/+ and B MOG - atg5 −/− mice and cultivated in the iGB culture system for activation and expansion. After differentiation, iGB cells were loaded with MOG 1–125 for 3 h and 1.5 × 10 7 cells were adoptively transferred together with 5 × 10 6 unmanipulated 2D2 CD4 + T cells into rag1 -KO recipients. Created with BioRender. (C and D) their differentiation to germinal center B cells was confirmed by expression of FAS, GL7, CD38 and PNA in CD19 + CD138 − gated cells. (E) expansion of B MOG - Atg5 +/+ and B MOG - atg5 −/− after iGB culture. (F) clinical data are shown as mean ± SEM for 3 mice ( Atg5 +/+ ) or 7 mice per group ( atg5 −/− ) pooled from 2 independent experiments. Two-way-ANOVA was applied for statistical analysis and differences are expressed as * p  < 0.05 or ** p  < 0.01. (g) % incidence of EAE is shown as SE.

Lack of Atg5 in activated B-cells accelerates EAE development. (A) breeding strategy for atg5 deletion in MOG-specific B cells. IgH MOG -CD19-Cre mice were crossed with Atg5 flox/flox mice to generate B MOG - Atg5 +/+ and B MOG - atg5 −/− experimental mice. Created with BioRender. (B) experimental strategy for an adjuvant-free B cell dependent EAE induction. B cells were isolated from B MOG - Atg5 +/+ and B MOG - atg5 −/− mice and cultivated in the iGB culture system for activation and expansion. After differentiation, iGB cells were loaded with MOG 1–125 for 3 h and 1.5 × 10 7 cells were adoptively transferred together with 5 × 10 6 unmanipulated 2D2 CD4 + T cells into rag1 -KO recipients. Created with BioRender. (C and D) their differentiation to germinal center B cells was confirmed by expression of FAS, GL7, CD38 and PNA in CD19 + CD138 − gated cells. (E) expansion of B MOG - Atg5 +/+ and B MOG - atg5 −/− after iGB culture. (F) clinical data are shown as mean ± SEM for 3 mice ( Atg5 +/+ ) or 7 mice per group ( atg5 −/− ) pooled from 2 independent experiments. Two-way-ANOVA was applied for statistical analysis and differences are expressed as * p  < 0.05 or ** p  < 0.01. (g) % incidence of EAE is shown as SE.

We capitalized on a recently developed adjuvant-free B- and T-cell cotransfer EAE model [ 10 ]. The model employs an induced germinal center B-cell (iGB) culture system [ 15 ] to activate, expand, and manipulate B cells. Briefly, sorted primary B cells are cultured on fibroblastic feeder cells expressing CD40LG/CD40L and TNFSF13B/BAFF (40LB cells), which leads to extensive B-cell proliferation and expansion [ 15 ]. B cells acquire a GC phenotype characterized by the upregulation of FAS and GL7 [ 15 ]. To induce EAE, iGB cells are loaded with MOG protein and then transferred together with unmanipulated MOG-specific 2D2 CD4 + T cells into rag1 -KO mice that lack endogenous B and T cells [ 10 ] Figure 1(b) .

We first tested whether absence of ATG5 in IgH MOG B cells is associated with fundamental differences in their ability to differentiate into GC B cells. To this end, we sorted splenic B cells from B MOG - atg5 −/− and B MOG - Atg5 +/+ mice and cultured them on 40LB cells with IL4 as previously described [ 15 ]. After 4 days, the cells were collected and their phenotype was assessed by flow cytometry. All CD19 + SDC1/CD138 − cells exhibited a cell-surface phenotype typical of GC B cells: FAS + GL7 + CD38 low and PNA + , and no differences in the expansion rate or expression of GC B-cell-related markers were observed between B MOG - atg5 −/− and B MOG - Atg5 +/+ cells Figure 1(c-e ).

To assess whether lack of ATG5 in B cells restrains cognate T-cell responses and protects from EAE development such as observed in DCs [ 5 ], iGB cells derived from B MOG - atg5 −/− and B MOG - Atg5 +/+ mice were loaded with MOG protein (aa 1–125) and transferred together with 2D2 CD4 + T cells into rag1 -KO mice. Surprisingly, animals that received atg5 knockout iGB cells showed an accelerated EAE development with the first signs of EAE becoming evident approximately 1 week earlier than those mice that received Atg5 -sufficient iGB cells Figure 1(f) . Disease susceptibility was similar in both groups investigated Figure 1(g) . Thus, contrary to our hypothesis, the lack of Atg5 expression in iGB cells did not protect against the development of CD4 + T-cell-mediated EAE, but rather promoted it.

Atg5 deficiency in iGB cells compromises CD80 internalization

In order to unravel the mechanisms that could lead to accelerated EAE development induced by iGB cells derived from B MOG - atg5 −/− mice together with 2D2 T cells, we first tracked the ability of atg5 −/− and Atg5 +/+ MOG-specific iGB cells for cognate antigen uptake. To this end, MOG 1–125 protein was conjugated with the pH-sensitive pHrodo dye and loaded onto iGB cells. Atg5 +/+ and atg5 −/− MOG-specific iGB cells did not differ in intracellular uptake and processing of MOG antigen Figure 2(a ).
Figure 2. Atg5 deficiency in iGB cells stabilizes CD80 surface expression. (A) Atg5 +/+ or atg5 −/− iGB cells were incubated during 30, 60, 120 or 180 min with MOG 1–125 previously labelled with pHrodo red dye. MFI of pHrodo is show and data is representative of 2 experiments. (B) surface expression of MHC and co-stimulatory molecules are shown for Atg5 +/+ or atg5 −/− iGB cells. Pooled data from 3 experiments are shown. MFI values were normalized to the mean of the control group ( Atg5 +/+ ), which was set to 100. Dots represent individual mice. Unpaired t-test was applied. Difference is referred as ** p  < 0.01. (C) CD80 gene expression was measured by RT-PCR. (D) CD80 recovery over time was analyzed using a flow cytometry-based recycling assay. Data shown is representative of 3 experiments. (E) immunofluorescence staining for CD80. Nuclear DNA was counterstained with DAPI. The presented data summarize 2 independent experiments. (F) quantification of vesicular CD80 in Atg5 +/+ or atg5 −/− iGB cells. (G) mean vesicle area of CD80-positive vesicles.

Atg5 deficiency in iGB cells stabilizes CD80 surface expression. (A) Atg5 +/+ or atg5 −/− iGB cells were incubated during 30, 60, 120 or 180 min with MOG 1–125 previously labelled with pHrodo red dye. MFI of pHrodo is show and data is representative of 2 experiments. (B) surface expression of MHC and co-stimulatory molecules are shown for Atg5 +/+ or atg5 −/− iGB cells. Pooled data from 3 experiments are shown. MFI values were normalized to the mean of the control group ( Atg5 +/+ ), which was set to 100. Dots represent individual mice. Unpaired t-test was applied. Difference is referred as ** p  < 0.01. (C) CD80 gene expression was measured by RT-PCR. (D) CD80 recovery over time was analyzed using a flow cytometry-based recycling assay. Data shown is representative of 3 experiments. (E) immunofluorescence staining for CD80. Nuclear DNA was counterstained with DAPI. The presented data summarize 2 independent experiments. (F) quantification of vesicular CD80 in Atg5 +/+ or atg5 −/− iGB cells. (G) mean vesicle area of CD80-positive vesicles.

We next profiled expression of MHC class I and class II along with costimulatory molecules, that could directly affect the T-cell activation. We found that atg5 −/− iGB cells displayed higher CD80 surface expression than their Atg5 flox/flox littermate-derived controls. In contrast, surface expression of MHC class I, class II, other costimulatory molecules including CD40, CD83, CD86, PDCD1LG2/CD273, CD274, ICOSL/CD275 was unchanged, indicating that deficiency in the core machinery of autophagy increases CD80 expression on iGB cells but does not affect other surface molecules involved in antigen presentation Figure 2(b) .

The autophagy machinery supports several cellular pathways that transport material of both endogenous or exogenous origin to the endolysosomal compartments. Pathways that facilitate antigen processing leading to MHC class II presentation include macroautophagy and the non-canonical autophagy pathway (LC3)-associated phagocytosis (LAP) where LC3 is conjugated to phagosome membranes using a portion of the canonical autophagy machinery. Activation of membrane-bound CYBB/NOX2, essential for the execution of LAP, and the consecutive production of superoxide leads to recruitment and binding of LC3 to the outer membrane of the LAPosome [ 16 ].

To investigate whether conditional deletion of Cybb in B cells affects CD80 surface expression, we generated conditional knockout mice (C57BL/6) for disruption of Cybb , in MOG-specific B cells by crossing IgH MOG -CD19-Cre with Cybb flox /flox mice for at least five generations, designated B MOG - cybb −/− mice. B cells derived from B MOG - cybb −/− mice were compared to those derived form B MOG - Cybb +/+ littermates.

Absence of CYBB/NOX2 in IgH MOG B cells was neither associated with differences in the ability to differentiate into GC B cells nor with surface expression levels of CD80 (Fig. S1), indicating that the increased surface CD80 expression on iGB cells observed in B MOG - atg5 −/− mice is not dependent on CYBB/NOX2 expression and LAP function.

To better define the mechanism mediating increased CD80 expression in absence of ATG5, we first ruled out a difference in CD80 gene expression, by quantifying CD80 transcript levels in Atg5 +/+ and atg5 −/− iGB cells Figure 2(c ). We next tested whether absence of ATG5 leads to elevated trafficking of CD80 molecules to the cell surface, possibly due to enhanced vesicular transport [ 17 , 18 ]. To assess CD80 recycling rates, we measured the newly resurfaced CD80 molecules after the cell surface CD80 had been blocked with unconjugated anti-CD80 antibodies. We did not see any difference in the rate of de novo expression of CD80 molecules on the cell surface Figure 2(d) . Next, we investigated whether the absence of ATG5 attenuates CD80 internalization. To this end, we analyzed vesicular CD80 levels in Atg5 +/+ and atg5 −/− iGB cells using an immunofluorescence-based CD80 internalization assay. Indeed, we observed a substantial reduction in vesicular CD80 pools in atg5 −/− iGB cells as demonstrated in Figure 2(e-f) . All together, these results suggest that the higher surface expression of CD80 molecules on atg5 −/− iGB cells is not related to enhanced gene transcription or elevated trafficking of de novo formed molecules but associated with compromised internalization.

Lack of Atg5 in iGB cells enhances cognate T-cell stimulation through CD80-costimulation

To investigate whether increased CD80 levels on atg5 −/− iGB cells affect their CD4 + T-cell stimulatory capacity, we co-cultured Atg5 +/+ and atg5 −/− MOG-specific iGB cells with MOG 1–125 protein with 2D2 cells and assessed T-cell proliferation. We observed higher MOG-specific CD4 + T-cell proliferative responses with atg5 −/− compared to Atg5 +/+ iGB cells Figure 3(a) . Increased proliferation was also observed after loading atg5 −/− iGB cells with MOG 35–55 peptide, indicating that the enhanced T-cell stimulatory potential was not dependent on intracellular antigen uptake and processing Figure 3(a,b) . Antibody-mediated blockade of CD80 resulted in a dose-dependent reduction in T-cell proliferation Figure 3(c,d) and decreased IFNG/IFN-γ and CSF2/GM-CSF production Figure 3(e,f ). T-cell activation did not result in IL17 production Figure 3(g) in line with previous reports that 2D2 CD4 + T cells primarily produce IFNG [ 10 ]. Notably, the enhanced T-cell stimulatory capacity of atg5 −/− iGB cells was lost after antibody-mediated blockade of CD80, altogether demonstrating that genetic ablation of Atg5 in MOG-specific iGB cells enhances cognate T-cell activation through CD80 co-stimulation.
Figure 3. Atg5 -deficient iGB cells enhance cognate T-cell responses through CD80-costimulation. (A) 2.5 × 10 4 CD4 + T cells labelled with CTV were cocultured with 5 × 10 3
Atg5 +/+ or atg5 −/− iGB cells for 72 h. MOG 1–125 and MOG 35–55 were used to stimulate T cell proliferation with and without antigen processing by iGB cells respectively. PBS was used as negative control. OVA 323–339 was used as peptide control. (B) CD4 + T cell proliferation after 72 h of coculture with Atg5 +/+ or atg5 −/− iGB cells in the presence of MOG 35–55 with addition of CD80 blocking antibody (5 µg/ml). (C) Representative histograms of CTV dilution within CD4 + T cells cultured with Atg5 +/+ or atg5 −/− iGB cells in the presence of MOG 35–55  + anti-CD80 at different concentrations (0.01, 0.1, 1, or 5 µg/ml). (D) histograms corresponds to CTV dilution within CD4 + T cells cultured with Atg5 +/+ or atg5 −/− iGB cells in the presence of PBS, OVA 323–339 , MOG 35–55 or MOG 35–55  + anti-CD80 (5 µg/ml) showed in (B). (E) IFNG, (F) CSF2 and (G) IL17 levels measured by ELISA in the supernatants of cocultures showed in (B). All data are shown as mean ± SEM and are representative of two experiments. Dots represent individual mice. An unpaired t-test was applied. Differences are reported as * p  < 0.05; ** p  < 0.01; or *** p  < 0.001.

Atg5 -deficient iGB cells enhance cognate T-cell responses through CD80-costimulation. (A) 2.5 × 10 4 CD4 + T cells labelled with CTV were cocultured with 5 × 10 3
Atg5 +/+ or atg5 −/− iGB cells for 72 h. MOG 1–125 and MOG 35–55 were used to stimulate T cell proliferation with and without antigen processing by iGB cells respectively. PBS was used as negative control. OVA 323–339 was used as peptide control. (B) CD4 + T cell proliferation after 72 h of coculture with Atg5 +/+ or atg5 −/− iGB cells in the presence of MOG 35–55 with addition of CD80 blocking antibody (5 µg/ml). (C) Representative histograms of CTV dilution within CD4 + T cells cultured with Atg5 +/+ or atg5 −/− iGB cells in the presence of MOG 35–55  + anti-CD80 at different concentrations (0.01, 0.1, 1, or 5 µg/ml). (D) histograms corresponds to CTV dilution within CD4 + T cells cultured with Atg5 +/+ or atg5 −/− iGB cells in the presence of PBS, OVA 323–339 , MOG 35–55 or MOG 35–55  + anti-CD80 (5 µg/ml) showed in (B). (E) IFNG, (F) CSF2 and (G) IL17 levels measured by ELISA in the supernatants of cocultures showed in (B). All data are shown as mean ± SEM and are representative of two experiments. Dots represent individual mice. An unpaired t-test was applied. Differences are reported as * p  < 0.05; ** p  < 0.01; or *** p  < 0.001.

Discussion

Our study identifies an unexpected role of ATG5 in iGB cells as its genetic depletion results in enhanced T-cell stimulatory capacity through stabilized surface expression of CD80 and accelerated CD4 + T-cell-mediated autoimmune disease development. These data corroborate a critical function for CD80 co-stimulation for disease development in 2D2×IgH MOG mice as CD80 expression on B cells is reported to be specifically enhanced in sick mice [ 13 , 14 ] and since IgH MOG B cells are perfectly capable to present MOG and induce activation of MOG-specific 2D2 T cells in vitro [ 13 , 14 ]. Furthermore, our findings highlight non-redundant roles of ATG5 in B cells versus professional APCs such as DCs in EAE pathology.

CNS-infiltrating, myelin-specific CD4 + T cells require phagocytosis of intact myelin by CD11c + APCs and its processing to induce demyelination and clinical disease development [ 19 , 20 ]. ATG-dependent phagocytosis in professional APCs support downstream CD4 + T-cell responses by promoting sustained MHC class II antigen presentation [ 21 , 22 ] and absence of ATG5 and ATG7 in DCs abrogates myelin presentation to CD4 + T cells and protects from EAE development [ 5 , 23 ]. Processing of intact myelin by ITGAX/CD11c + -professional APCs is however preserved in rag1 -deficient recipient mice used in our study as these cells were not genetically modified. Consequently, rag1 -deficient recipient mice exposed to atg5 −/− iGB/2D2 cells were as susceptible to EAE development as those exposed to Atg5 +/+ iGB/2D2 cells. The difference observed was an accelerated disease development in mice receiving a tg5 −/− iGB cells, consistent with a greater initial T-cell stimulatory capacity of these cells due to enhanced CD80 expression.

Loi et al. previously described a role for lipidated LC3B in recruiting the internalization machinery to MHC class I molecules in DCs [ 24 ]. Genetic ablation of Atg5 and Atg7 results in decreased endocytosis and degradation of MHC class I surface molecules, associated with elevated anti-viral CD8 + T-cell responses in vitro and in vivo [ 24 ]. Notably, loss of Atg5 also affects CD80 expression in the aforementioned study as its cell surface expression is higher in some atg5 −/− DC populations (mainly CD8 + splenic DCs), while surface expression of other investigated molecules including MHC class II, CD86, ITGB1/CD29, CD44, and TFRC (transferrin receptor), remains unchanged, indicating that CD80 expression and internalization in DCs is regulated in a similar fashion as MHC class I molecules. We previously observed that loss of ATG5 in DCs additionally impairs CD1d internalization, leading to increased expression of stimulatory CD1d-glycolipid complexes and enhanced iNKT-cell activation [ 25 ]. We cannot exclude the possibility that the internalization is ATG5 dependent but not necessarily macroautophagy dependent or dependent on additional essential macroautophagy proteins such as ATG7, as our observations are based on genetic deletion of Atg5 . While we did not formally compare MOG-specific CD4 + T-cell expansion upon transfer, a potential limitation of our study, we clearly observed that in vitro proliferation of these cells depends on ATG5 and CD80 expression by cognate iGB cells. All together, these data support the concept that the core autophagic machinery in DCs couples to T lymphocyte activation through regulating expression levels of MHC class I and costimulatory molecules.

MHC class I internalization in DCs can be mediated by both clathrin-dependent and -independent pathways. The macroautophagy machinery has been previously suggested to support clathrin-dependent endocytosis. Components of the AP2 complex have been found to interact with lipidated LC3B for Alzheimer precursor protein internalization and degradation [ 26 ]. In line with the identification of AP2 as an LC3-interacting protein, loss of Atg5 impairs trafficking of both AP2 and CD1d molecules into endosomal compartments [ 25 ]. However, AP2 components were not significantly reduced in immunoprecipitates of classical MHC class I molecules derived from atg5 -deficient cells compared with floxed littermates [ 24 ]. Mechanisms regulating CD80 internalization in both DCs and B cells are poorly understood but might involve different pathways and mechanisms as described for classical and nonclassical MHC class I molecules in DCs.

In conclusion, our findings indicate that the autophagic machinery regulates cognate B- and T-cell interactions through stabilizing CD80 expression. These data broaden the concept of Atg-mediated antigen presentation and indicate that the core autophagy machinery does not only enhance antigen presentation, as described previously for MHC class II-restricted presentation of peptide antigens in DCs, but can also limit and tune presentation of antigens by B cells and their capacity to activate CD4 + T cells.

Materials and methods

Animals

To knock out Atg5 specifically in B cells, we applied a Cre-loxP recombination system in which IgH MOG -CD19-Cre ± mice were crossed with Atg5 flox/flox mice. After at least five generations we obtained IgH MOG -CD19-Cre −/− - Atg5 flox/flox referred as control mice or B MOG - Atg5 +/+ and IgH MOG -CD19-Cre ± - atg5 flox/flox referred to as B MOG - atg5 −/− . rag1 -KO mice (B6.129S7-Rag1tm1Mom/J) and 2D2 mice (C57BL/6-Tg [Tcra2D2Tcrb2D2]1Kuch/J) were purchased from The Jackson Laboratory (002216; 006912). All mice were housed and bred in the University of Münster animal facilities in individually ventilated cages on a 12 h light/dark cycle with food and water available ad libitum according to institutional guidelines, as well as German animal laws. All animal protocols were approved by and conducted following the State Agency for Nature, Environment and Consumer Protection North Rhine-Westphalia, Germany (81–2 April 2021.A416).

Genotyping

DNA was extracted from tail or ear biopsies of IgH MOG -CD19-Cre- Atg5 flox/flox and the genotype was determined by PCR using the following primers. IgH MOG Primer 1: GCA TCC TGA TAC CTG ACG CA (MOG forward), Primer 2: GGA GAC TGT GAG AGT GGT GCC T (MOG reverse), Primer 3: TCT CTT CTA TGC TTT CTT TGT CCC (wild type forward) and Primer 4: GTC ATT GTT CCA CAT TCT GTT CC (wild type reverse). CD19-Cre Primer 1: GAC TCC TCA CCT GTA TCT TCT GA (Common), Primer 2: AGA GCT CCT CTT TAC CTC CAC A (wild type reverse) and Primer 3: CAG GTT CTT GCG AAC CTC AT (mutant reverse). Atg5 flox/flox Primer 1: GAA TAT GAA GGC ACA CCC CTG AAA TG (exon3–1), Primer 2: GTA CTG CAT AAT GGT TTA ACT CTT GC (short2), Primer 3: ACA ACG TCG AGC ACA GCT GCG CAA GG (check2) and Primer 4: CAG GGA ATG GTG TCT CCC AC.

iGB cell culture

40LB cells (BALB/c3T3 fibroblasts expressing CD40LG and TNFSF13B/BAFF) were kindly provided by Annely Peters (Ludwig-Maximilians-Universität München, Germany) and routinely cultured in 40LB medium – Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific; 31966021) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, A5256701), 1% penicillin-streptomycin (Thermo Fisher Scientific 115,448,876), 2 mm L-glutamine (Thermo Fisher Scientific 11,534,546), 100 µM nonessential amino acids (Thermo Fisher Scientific 11,140,035), 1 mm sodium pyruvate (Thermo Fisher Scientific 11,360,039), and 5.72 µM β-mercaptoethanol (Thermo Fisher Scientific 31,350,010) – and kept subconfluent (37°C, 10% CO 2 ). One day before iGB culture, 40LB cells were seeded at 2 × 10 5 cells/cell culture dish and allowed to adhere overnight. Next, 1.5 × 10 6 B cells were purified by using B cell isolation kit mouse (Miltenyi Biotec, 130-090-862) from B MOG - Atg5 +/+ and B MOG - atg5 −/− and cultured on 40LB cells with 1 ng/ml recombinant mouse IL4 (BioLegend 574,306) for 4 days in iGB medium – Roswell Park Memorial Institute 1640 medium (RPMI 1640; Thermo Fisher Scientific; 11530586) containing 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 2 mm L-glutamine, 100 µM non-essential amino acids, 1 mm sodium pyruvate, 1 M HEPES (Thermo Fisher Scientific 11,560,496), pH 7.2, and 5.72 µM β-mercaptoethanol at 37°C, 5% CO 2 . Both 40LB medium and iGB medium were prepared as described previously [ 10 ]. The iGB cells were then collected by incubation with pre-warmed PBS (Sigma Aldrich, D8537) containing 0.5% BSA (Sigma, A4503) and 2 mm EDTA (Sigma, ED2SS). The differentiation of naive B cells into iGB cells was analyzed by flow cytometry using the expression of GL7, FAS, CD38, and PNA (Merck, L7381) ligands on CD19 + CD138 − gated cells.

Quantitative PCR

Both iGB cells, Atg5 +/+ and atg5 −/− , were transferred to microcentrifuge tubes, and RNA isolation was performed using the Quick RNA Microprep Kit (Zymo Research, R1050) according to the manufacturer’s instructions. The RNA was stored on ice, quantified using a Nanodrop machine, and finally stored at −80°C until cDNA conversion. To convert RNA to cDNA, we employed the GoScript Reverse Transcription System (Promega, A5000). For CD80 quantification, we perform a TaqMan Gene expression assay (Thermo Fisher 4,351,370) using the Cd80 gene symbol (Thermo Fisher 4,331,182). Quantifications were performed in triplicates for the genes of interest and the housekeeping gene ( Rn18s ). Real-time PCR was performed on the StepOne software v2.1 using the advanced setup to measure the ∆∆CT values.

B cell-dependent adoptive transfer EAE

iGB cells were collected and washed by centrifugation at 300 × g for 10 min at RT. Then, iGB cells were loaded with MOG protein (codon-optimized rodent MOG 1–125 ), produced according to the protocol previously described [ 27 ], by incubating them at a concentration of 1 × 10 7 cells/ml in the presence of MOG 1–125 (10 µg/ml) for 3 h at 37°C. Then, the cells were washed and filtered through a 70-µm filter, washed two more times, and resuspended in prewarmed PBS. Then, 1.5 × 10 7 iGB cells/mouse were injected i.p. into rag1 -KO recipients. MOG-specific CD4 + T cells were isolated from spleens of transgenic 2D2 mice by positive selection using CD4 (L3T4) MicroBeads (Miltenyi Biotec, 130-117-043) according to the manufacturer’s instructions. After isolation, CD4 + 2D2 T cells were resuspended in PBS, and 5 × 10 6 cells/mouse were injected i.p. into rag1 -KO recipients. Animals were monitored daily for clinical signs of EAE according to the following scoring criteria: 0, no signs of EAE; 0,5 tail tip paralysis; 1 tail paralysis; 1.5 tail paralysis and hind leg weakness; 2 unilateral partial hind leg paralysis; 2.5 bilateral partial hind leg paralysis; 3 complete bilateral hind leg paralysis; 3.5 complete bilateral hind leg paralysis and partial foreleg paralysis; 4 tetraplegic and 5 moribund.

PHrodo assay

MOG protein (MOG 1–125 ) was labeled with the pH-sensitive dye pHrodo Red, succinimidyl ester (Thermo Fisher, P36600 ) before addition to iGB cells at different time points as follows: the pHrodo dye was dissolved in 1 ml dimethyl sulfoxide (DMSO) and used at a 1:100 ratio per milligram of protein. MOG 1–125 was resuspended in PBS (pH 8) and incubated with pHrodo for 1 h at room temperature in the dark. Following conjugation, unreacted dye was removed using an Amicon® Ultra centrifugal filter (Millipore, UFC5010). The labeled protein solution was transferred to the filter unit and centrifuged at 4,000 × g for 10 min. The retained MOG 1–125 -pHrodo conjugate was washed three times with PBS (pH 7.4) to ensure complete removal of excess dye. The purified conjugate was resuspended in PBS (pH 7.4) and used immediately. After incubation at 37°C with 5% CO₂ for the indicated time spans, iGB cells were stained for flow cytometry analysis.

Flow cytometry

Single-cell suspensions were transferred to a 96-well plate and prepared for extracellular marker staining. Each sample underwent a 10-min incubation at 4°C with anti-FCGR3/CD16-FCGR2B/CD32 (BioLegend 101,302) to avoid unspecific binding of antibodies to Fc receptors. Following this, samples were washed once with PBS and then stained in the dark for 20 min at 4°C using a freshly prepared master mix of antibodies along with Zombie Aqua Fixable Viability Kit (BioLegend 423,102) and FITC-conjugated peanut agglutinin (PNA; Vector Laboratories; FL-1071). After antibody incubation, samples were washed with PBS at 400 × g and 4°C for 5 min and resuspended in FACS buffer (PBS 1% BSA). All samples were measured on CytoFlex S flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA), with subsequent analysis conducted using FlowJo_v10.9.0 software (Tree Star Inc).

Antibodies for flow cytometry

CD19 (BD Biosciences 552,854); SDC1/CD138 (BioLegend 142,508); FAS (BioLegend 152,604); GL7 (BioLegend 144,608 or 144,608); CD38 (BioLegend 562,770); MHC-I (BioLegend 116,512); MHC-II (BioLegend 107,652); CD40 (BioLegend 124,612); CD80 (BioLegend 104,706); CD83 (Invitrogen 12,083,182); CD86 (BioLegend 105,122); PDCD1LG2/CD273 (BD Bioscience 740,430); CD274 (BioLegend 124,321); ICOSLG/CD275 (BioLegend 107,405); CD3 (BioLegend 100,222); CD4 (BioLegend 116,022).

Recycling assay

iGB cells of Atg5 +/+ - and atg5 −/− -derived splenocytes were incubated with protein synthesis inhibitor cycloheximide (Sigma-Aldrich 01,810–1 G) used at 1 µg/ml in RPMI 1640 (Thermo Fischer Scientific 11,530,586) 10% FBS (60 min at 37°C, 5% CO 2 ). After extensive washing with PBS, cells were either incubated with unconjugated anti-CD80 antibody (Thermo Fisher Scientific, 14-0801-82) for 30 min (pre-blocked) or left untreated (unblocked) at 4°C. After a washing step, cells were divided into the following groups (1 × 10 5 /group): 30 min on ice (pre-blocked), 30 min on ice (unblocked), 30, 20, 10, and 5 min at 37°C, 5% CO 2 . After respective incubation times, cells were washed with PBS and incubated with an anti-CD80 antibody (Thermo Fischer Scientific 104,706) using the same clone as for pre-blocking, anti-CD19, and Zombie Aqua Fixable Viability Kit (BioLegend 423,102) for 20 min on ice in the dark. Samples were fixed with 3% PFA in PBS and acquired with the CytoFlex S system. All data were analyzed using FlowJo software (Tree Star Inc.). Experiments were performed in triplicate, and the values were expressed as the level of CD80 present on the cell surface after each incubation time. Data were normalized to the level of CD80 present on the cell surface after 30 min of incubation at 4°C (time 0) so that the surface level of CD80 at time 0 was 100.

In vitro iGB- and T-cell co-culture

Atg5 +/+ and atg5 −/− iGB cells were transferred to 96-well U-bottom plates (Sarstedt, 83.3925). CD4 + T cells were isolated from the spleens of 2D2 mice by positive selection using the CD4 + L3T4 T cell Isolation Kit (Miltenyi Biotec, 130-117-043). CD4 + T cells were labeled with Amicon® Violet (CTV; Thermo Fisher; C34557 ) according to the manufacturer’s instructions. Briefly, up to 1 × 10 7 cells were resuspended in 1 ml PBS and incubated with 5 µM CTV for 20 min at RT protected from light. Then, 5 ml iGB medium was added to stop the staining reaction and the cells were incubated for 5 min at RT. After subsequent centrifugation, the cells were resuspended in iGB medium and the cell number was determined. iGB cell subsets and 2D2 CD4 + T cells were co-cultured in a 1:5 ratio, respectively (5 × 10 3 iGB + 2.5 × 10 4 CD4 + ) in the presence of 50 µg/ml MOG 1–125 , MOG 35–55 (Peptides&Elephants, EP02030) or OVA 323–339 (Peptides&Elephants, EP04609) for 72 h at 37°C, 5% CO 2 . Then, CD4 + T-cell proliferation was assessed by flow cytometry and supernatant was stored at −80°C for cytokine production measurement.

Enzyme-linked immunosorbent assay

IFNG quantification in the supernatants of iGB- and T-cell co-culture was assessed by ELISA using commercially available ELISA MAX Standard Set Mouse IFNG (BioLegend 430,801). CSF2 quantification was assessed by using commercially available ELISA MAX Standard Set Mouse GM-CSF (BioLegend 432,201). IL17 quantification was assessed by using commercially available ELISA MAX Deluxe Set Mouse IL-17A/F (BioLegend 436,204). Samples were stored at −80°C until the assay was performed. Results are expressed as pg/ml.

Immunofluorescence

Both iGB cells, Atg5 +/+ and atg5 −/− , were collected, and resuspended in fresh iGB medium and incubated with the Fc-blocker purified anti-mouse FCGR3-FCGR2B (BioLegend 101,302) for 20 min at 4°C, followed by labeling for CD80 (Thermo Fisher, PA5–79001) for 30 min at 4°C. Next, they were incubated at 37°C, 5% CO 2 for 1 h to allow internalization, washed with PBS, and resuspended in iGB medium. A volume of 100 µL of the cell suspension was loaded into cytospin chambers and centrifuged at 600 × g for 5 min using the Rotofix 32 A centrifuge (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), equipped with a cytospin adapter. Glass microscope slides (Epredia, J1800AMNZ) were used to collect the deposited cells that were then fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were washed with PBS, permeabilized with 0.1% Triton X-100 (Sigma Aldrich, X100), and saturated with 1% FBS for 1 h. The binding of the primary antibody was detected with Alexa Fluor 488 donkey anti-rabbit IgG (BioLegend, A21206), and cell nuclei were stained with DAPI. Fluorescence was analyzed with a Zeiss ELYRA 7 Structured Illumination Microscope using the Apotome Mode (automated reconstruction) and a Plan-Apochromat 63×/1.4 oil objective. Image analysis and particle quantification were performed using CellProfiler (version 4.2.6) with Cellpose extension. In CellProfiler, cells were segmented using the Cellpose module with the DAPI and 488-channel signals. Particles per cell were identified using the IdentifyPrimaryObjects module with automatic thresholding set to Otsu, and relating the particles to the segmented cells. The particle data, including counts and morphological metrics, were exported for further analysis [ 28 , 29 ].

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 10. Appropriate statistical tests were selected as indicated in the figure legends. Differences between the two groups were determined using an Unpaired two-tailed Student’s t-test using Welch´s correction, when necessary. Differences between EAE incidence curves were evaluated using the Mixed-effects analysis test with adjusted p values for multiple comparisons using the Holm – Sidak method, when necessary.

Supplementary Material

ATG5_B_cell_Autophagy_SUPPLEMENTAL_MATERIAL_250506 R4.docx