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. 2015 Mar;144(3):461–471. doi: 10.1111/imm.12392

A role for mitochondria in antigen processing and presentation

Laura C Bonifaz 1, Mariana P Cervantes-Silva 1,2, Elizabeth Ontiveros-Dotor 1,2, Edgar O López-Villegas 3, F Javier Sánchez-García 2,✉
PMCID: PMC4557683  PMID: 25251370

Abstract

Immune synapse formation is critical for T-lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen-presenting cells (APCs) and T lymphocytes is a two-way signalling process. However, the role of mitochondria in APCs during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing and -presentation process. Here we show that hen egg white lysozyme (HEL) -loaded B lymphocytes, as a type of APC, undergo a small but significant mitochondrial depolarization by 1–2 hr following antigen exposure, suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca2+ uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analysed. Oligomycin treatment reduced the amount of specific MHC–peptide complexes but not total MHC II on the cell membrane of B lymphocytes, which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes, which endogenously express HEL and by B lymphocytes loaded with the HEL48–62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taken together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APC mitochondria were found to re-organize towards the APC–T immune synapse.

Keywords: antigen presentation, antigen processing, immune synapse, mitochondria

Introduction

Activation of conventional αβ T lymphocytes in a primary immune response requires that specific antigenic peptides associate with MHC molecules to be presented by antigen-presenting cells (APCs).1 APCs include, among other cell types, dendritic cells, macrophages and B lymphocytes.2–4 Upon APC–T-lymphocyte interaction, a number of APC cell membrane molecules interact with molecular partners on the T-lymphocyte cell membrane, so providing the necessary signals for T-cell activation.5 The site of contact between APCs and T lymphocytes has been referred to as the immune synapse6,7 and their molecular characterization has provided important clues on their function.8,9

More recently, it has become clear that in addition to cell membrane molecules, mitochondria also re-organize towards the T-cell immune synapse,10 contributing to the T-cell activation process, by regulating cytoplasmic calcium concentration or providing local energy.10–12

Natural killer cells form an immune synapse when targeted to a tumour cell.13 In this case, mitochondria from both natural killer cells and tumour cells polarize towards the immune synapse, indicating that the mobilization of mitochondria may have a role in both cell types, at the time of interaction.14

Studies on the role of mitochondria in the T-cell immune synapse have been carried out mainly by using anti-CD3/CD28 microbeads10,15 and therefore, the role of mitochondria in APCs has somehow been neglected.

Antigen-presenting cells endocytose whole microorganisms or soluble proteins and then process them in the phagolysosome compartment into antigenic peptides, which are then bound to MHC class II molecules, being the optimal peptide length of approximately 18–20 amino acids.16 The MHC class II–peptide complexes are then exported to the cell membrane where they have a half-life of 10–150 hr.1,17,18 Binding of the specific T-cell receptor to this MHC II–peptide complex on APCs, along with other molecular partners at the immune synapse, ensures T-cell activation.

By using a model of antigen-processing and presentation based on in vitro HEL-loading of LK35.2 B lymphocytes or an LK35.2 derivative cell line (LKKDEL) that endogenously express HEL [both of which present the 48–62 peptide of hen egg white lysozyme (HEL48–62) to 3A9 or C10 T lymphocytes whose T-cell receptor is specific for HEL48–62 peptide associated with the I-Ak MHC II haplotype],19,20 combined with antigen uptake and antigen processing assays, based on the fluorescence of ovalbumin (OVA)-Alexa fluor 488 or that of processed DQ-OVA, respectively, we show here that mitochondria from HEL-loaded B-lymphocytes undergo a subtle and transient but significant depolarization. This suggests an increase in the metabolic demand of B lymphocytes as antigen processing takes place. Disruption of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase,21 diminished the amount of MHC II–peptide on the cell membrane of HEL-loaded LK35.2 B lymphocytes, but not that of total MHC II. This correlated with the diminished ability of oligomycin-treated and HEL-loaded B lymphocytes to activate HEL48–62-specific T lymphocytes. In addition, oligomycin also reduced antigen presentation of endogenously expressed HEL, as well as that of cells pulsed directly with HEL48–62 peptide.

By separately analysing antigen uptake and antigen processing it is shown that this effect is due to antigen processing but not to antigen uptake disruption. Likewise, inhibition of the mitochondrial calcium uniporter (MCU; mitochondrial calcium uptake) with Ruthenium red22 disrupts antigen processing but not antigen uptake. We also provide evidence that HEL-loaded B-lymphocyte mitochondria re-organize towards the immune synapse. Taken together, our results suggest that mitochondria play an active role in antigen processing and presentation.

Materials and methods

Cells, antibodies and antigen-loading of B cells

The mouse B-cell hybridoma LK35.2 (H-2k,d) (American Type Culture Collection, Manassas, VA)23 and the LK35.2 derivative cell line LKKDEL20 that endogenously express HEL were used as a source of antigen-presenting B cells. 3A9 and C10 T lymphocytes that harbour a T-cell receptor that specifically recognizes the MHC II molecule in complex with the HEL48–62 peptide, was used to assess antigen presentation, and as the cell partner for B–T immune synapse formation. Both cell lines were cultured in RPMI-1640 (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc.), 100 μg/ml streptomycin and 100 U/ml penicillin (Sigma, St. Louis, MO) (complete medium). C4H3 monoclonal antibody, which recognizes the MHC II–HEL48–62 peptide, was kindly provided by Dr Ronald Germain through Dr José Moreno, and H11632 monoclonal antibody, which recognizes total MHC II, was kindly provided by Dr José Moreno. For HEL-loading of B cells, 5 × 105 LK35.2 cells in 1 ml of RPMI-1640 were cultured in 12-well plates for 12 hr in the presence of HEL (Sigma) at a final concentration of 250 μg/ml.

Mitochondrial membrane potential

LK35.2 B lymphocytes cultured in complete RPMI-1640 medium were plated in 12-well culture plates at a density of 5 × 105 cells/ml and incubated overnight at 37° in a 5% CO2 atmosphere. Cells were then treated with 250 μg/ml of HEL for 15, 30 min, and 1, 2, and 4 hr, or left untreated. Cells were loaded with 100 nm tetra-methyl rhodamine methyl ester (TMRME) (Thermo Fisher Scientific Inc.), for 30 min at 37° and then washed with PBS. Mitochondrial membrane potential (Δψm) was assessed by flow cytometry, as described previously.24 Mean fluorescence intensity in the FL-2 channel, indicative of Δψm, was recorded. To confirm that the observed fluorescence was really due to Δψm, a respiratory chain uncoupler (carbonyl cyanide 3-chlorophenylhydrazone, CCCP) (Sigma) was added to the cell suspension. A drop in fluorescence was indicative of mitochondrial depolarization.

Mitochondrial calcium

LK35.2 B lymphocytes (5 × 105) in calcium-free PBS in 12-well culture plates were labelled with 10 μm Rhod-2/AM (Thermo Fisher Scientific Inc.), a mitochondrial calcium indicator, for 45 min as described previously,10 cells were washed with PBS and supplemented with fresh RPMI-1640 medium and 2 mm CaCl2 (Sigma), cells were then pulsed with 250 μg/ml of HEL for 15 min, 30 min and 2 hr, or left untreated, and incubated at 37° in a 5% CO2 atmosphere. Cells were scraped and transferred into FACS tubes. As a lipopolysaccharide (LPS) contamination control, we carried out an exact replicate of the procedure just described, in the presence of the LPS inhibitor polymyxin B (Sigma) at a final concentration of 10 μg/ml, as described previously.25 Mitochondrial calcium was assessed by flow cytometry. Cell activation with 100 ng/ml of Ionomycin (Sigma) was used as a positive control for mitochondrial calcium uptake.

Antigen uptake assays

LK35.2 B lymphocytes (5 × 105 cells) were cultured overnight in complete RPMI-1640 medium at 37° in a 5% CO2 atmosphere, in 12-well culture plates. Cells were then washed with PBS and 1 ml of fresh RPMI-1640 medium was added to each well, cells were either left untreated or supplemented with oligomycin (10 μg/ml) (Sigma) or Ruthenium red (5 μm) (Sigma), cells were incubated for 30 min at 37° and then OVA-Alexa fluor 488 (25 μg/ml) (Thermo Fisher Scientific Inc.) was added and cells were further incubated. At the indicated time-points (15, 30, 60 and 90 min) cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, scraped out and transferred into FACS tubes (BD Biosciences, San Jose, CA) for FACS analysis (BD Biosciences). Data analysis was performed with cell quest software (BD Biosciences). Mean fluorescence intensity is indicative of OVA uptake.

Analysis of MHC–peptide complexes and total MHC class II on the cell membrane of B lymphocytes

LK35.2 B lymphocytes cultured in 75-cm2 flasks were washed with PBS, and suspended in complete RPMI-1640 medium at a density of 5 × 105 cells/ml; 1 ml of the cell suspension was added to each well in a 12-well culture plate and incubated at 37° in a 5% CO2 atmosphere for about 8 hr. Cells were then loaded with 250 μg/ml of HEL and further incubated for 12 hr. For ATP synthase inhibition, oligomycin (2 μg/ml or 10 μg/ml) (Sigma) was added 20 min before the addition of HEL and left in the culture medium for the entire incubation period. After incubation, LK35.2 B lymphocytes were scraped from the tissue culture plates, washed with ice cold PBS, suspended in 100 μl of 0·01% BSA, 0·01% Na2N PBS (staining solution) containing 2·5 μg/ml biotin-labelled C4H3 monoclonal antibody (anti-MHC II–HEL peptide) or 2·5 μg/ml biotin-labelled H11632 monoclonal antibody (anti-total MHC II), cells were incubated at 4° for 30 min and then washed with PBS and suspended in 100 μl of staining solution containing 1 μg/ml of streptavidin-Alexa fluor 488 (Thermo Fisher Scientific Inc.). Cells were again incubated at 4° for 30 min and then washed with PBS and fixed with 4% paraformaldehyde-PBS. Expression of MHC II–peptide on the cell membrane was analysed by flow cytometry (FACScalibur; BD Biosciences) and cellquest software (BD Biosciences).

Antigen presentation assays

LK35.2 or LKKDEL B lymphocytes, as a source of APCs, were plated in 12-well culture plates and then left untreated or loaded with 250 μg/ml HEL, or pre-treated with 2 μg/ml oligomycin for 20 min and then loaded with 250 μg/ml HEL or with 1 μm HEL48–62 peptide. Cells were then incubated for 12 hr at 37° in a 5% CO2 atmosphere. Afterwards, cells were washed with ice-cold PBS and fixed with 0·75% paraformaldehyde-PBS. After 15 min cells were washed with PBS and co-cultured with C10 T lymphocytes (1 : 2 ratio). Cells were then incubated for 24 hr. Cell supernatants were collected and assayed for interleukin-2 (IL-2) content as a way to evaluate APC-mediated T-cell activation. The relative amount of IL-2 in the supernatants was evaluated by their proliferative effect on the IL-2-dependent CTLL-2 cell line.26 Sixteen hours before harvesting, 0·5 μCi of [3H]thymidine was added to each CTLL-2-containing well. Cells were harvested with a semi-automatic cell harvester (Tomtec, Hamden, CT), and DNA synthesis was determined in a scintillation counter (Wallac, Waltham, MA). Results are expressed as counts per minute (cpm).

Antigen-processing assays

LK35.2 lymphocytes (2 × 105 cells/well) grown in Lab-Tek chambers (Thermo Fisher Scientific Inc.) were pulsed with DQ-OVA (Thermo Fisher Scientific Inc.) at a final concentration of 1 mg/ml, for 15 min at 37° and then washed three times with ice-cold PBS, in a similar way as previously described.27 Fresh complete RPMI-1640 medium was added and oligomycin (10 μg/ml) or Ruthenium red (5 μm) was added when indicated, a set of cells were washed and fixed with 100 μl of Perm/Fix (BD Biosciences), another set of cells were incubated at 37° for 1 hr, and then washed with PBS and fixed with Perm/Fix. After fixing, cells were covered with DAPI-containing Vectashield (Vector Laboratories, Burlingame, CA) and analysed using an LSM Pascal confocal microscope (Zeiss, Oberkochen, Germany), using a 63 × oil immersion objective; raw data were analysed with the LSM5 image browser (Zeiss).

Mitochondrial distribution

LK35.2 B lymphocytes and 3A9 T lymphocytes were cultured as mentioned in the previous section. LK35.2 B lymphocytes (1 × 107) were plated into 10 × 100 mm Petri dishes and cultured overnight at 37° in 5% CO2 atmosphere. 3A9 T lymphoctyes (1 × 107) were added on top of the LK35.2 B lymphocyte monolayer. Cell-to-cell interaction was allowed to proceed for 15–30 min, after which cells were carefully washed with 0·1 m phosphate buffer solution at pH 7·0 (PBS) and fixed with 3% potassium permanganate/PBS (J.T. Baker, Center Valley, PA) for 2 hr at 4°. After fixing, cells were carefully scraped and spun down in a 15-ml Falcon tube and then in an Eppendorf tube, where cells were washed several times with PBS. Cells where dehydrated with ethanol, embedded in EPON 812 (Electron Microscopy Sciences, Hatfield, PA) and cured in an oven at 60° for 24 hr. Ultrathin sections (70 nm) were obtained, contrasted for 5 min each in 4% uranyl acetate and Reynold’s lead citrate solutions, and observed with a Jeol JEM1010 electron transmission microscope, operated at 60 kV. Electron microscopy images were analysed with the imagej software (National Institutes of Health, Bethesda, MD).

Cell viability

LKB35.2 B lymphocytes untreated or exposed to HEL (250 μg/ml) and oligomycin (10 μg/ml) for 12 hr were stained with Hoescht 33258 (Thermo Fisher Scientific Inc.) and analysed by flow cytometry to assess cell viability. Viable cells stain positive for Hoescht whereas dead cells are not stained.

Results

HEL loading induces mitochondrial depolarization in B lymphocytes

Mitochondrial membrane potential (Δψm) has long been referred to as a key indicator of the energy status of a cell,28 and it has been shown, for instance, that stimuli such as LPS in macrophages depolarize mitochondria in a matter of a few hours, and this has been interpreted as a cell response aimed at fulfilling new metabolic demands that may even lead to apoptosis.29 We wondered whether antigen-uptake by B lymphocytes depolarizes mitochondria in an analogous way. Figure1(a,b) shows that upon HEL loading, Δψm in LK35.2 remains unchanged after 15 and 30 min. However, a subtle but significant mitochondrial depolarization is observed between 1 and 2 hr after B-lymphocyte exposure to HEL (250 μg/ml). By 4 hr Δψm seems to return to base levels, as no significant difference in Δψm at this time, compared with unstimulated cells, was observed. Treatment with the mitochondrial uncoupler CCCP was used as a positive control for Δψm dissipation. Figure1(a) shows that indeed, CCCP treatment caused maximal mitochondrial depolarization, so validating Δψm data.

Figure 1.

Figure 1

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Mitochondrial membrane potential (Δψm) during antigen-uptake by B lymphocytes. LK35.2 B cells (5 × 105 cells/ml) in 12-well culture plates were treated with 250 μg/ml of hen egg white lysozyme (HEL) for 15 and 30 min, and 1, 2 and 4 hr, or left untreated. Cells were loaded with the Δψm indicator TMRME and the TMRME mean fluorescence intensity, indicative of Δψm was assessed by flow cytometry. (a) Representative dot-plot depicting cell size [forward scatter (FSC)] versus mean fluorescence intensity of TMRM (Δψm) at different times after HEL-loading, (b) integrated results from duplicates from three independent experiments. The uncoupler CCCP was used as a positive control for mitochondrial depolarization. (c) Mitochondrial Ca2+ uptake at different time-points after HEL loading, as assessed by Rhod-2/AM and flow cytometry. The Ca2+ ionophore Ionomycin was used as a positive control for Ca2+ uptake. Analysis of variance, and Turkey′s multiple comparison test, were used for statistical analyses.

As mitochondrial calcium uptake is critically important for cell function and an association between mitochondrial calcium and Δψm has been observed (ref. 32), mitochondrial calcium concentration following HEL (250 μg/ml) loading was assessed on LK35.2 B lymphocytes. Figure1(c,d) shows that HEL-loading tends to increase mitochondrial calcium concentration at 15 min, 30 min and 2 hr post HEL-loading, although results were not statistically significant. Exact replicates of mitochondrial calcium uptake experiments following HEL-loading were simultaneously performed in the presence of 10 μg/ml polymyxin B. Results were similar to those performed in its absence, so ruling out the possibility that the observed effects were the result of LPS contamination of the HEL preparation. LK35.2 B lymphocytes were stimulated with ionomycin as a positive control of mitochondrial calcium uptake. As shown, mitochondrial calcium concentration (Rhod-2/AM signal) in ionomycin-treated cells significantly increased, so validating the mitochondrial Ca2+ uptake assays. The timing of mitochondrial depolarization suggests that this is due to antigen processing rather than to antigen uptake.

Inhibition of ATP synthase and of MCU has no effect on antigen uptake by B lymphocytes

To assess the hypothesis that inhibition of ATP synthase disrupts antigen processing but not antigen uptake, we compared the mean fluorescence intensity of OVA-Alexa fluor 488-pulsed cells immediately after pulsing (time 0 min), versus OVA-Alexa fluor 488 pulsed cells that were incubated for 15, 30, 60 and 90 min at 37°. Exact replicates were performed in cells cultured in medium alone, in the presence of the ATP synthase inhibitor oligomycin, or in the presence of the MCU inhibitor Ruthenium red. Figure2 shows that the mean fluorescence intensity due to OVA-Alexa fluor 488 has a significant increase from 15 to 90 min in all three culture conditions. However, no differences were observed when comparing the three conditions at the same time-point, suggesting that neither ATP synthase inhibition (oligomycin) nor MCU inhibition (Ruthenium red) has any effect on antigen (OVA-Alexa fluor 488) uptake.

Figure 2.

Figure 2

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Effect of ATP synthase inhibition and of mitochondrial calcium uniporter inhibition on the ability of B lymphocytes for antigen uptake. LK35.2 B cells were left untreated or supplemented with 10 μg/ml of oligomycin (ATP synthase inhibitor) or with 5 μm of Ruthenium red (mitochondrial calcium uniporter inhibitor), cells were incubated for 30 min at 37° and then pulsed with 25 μg/ml of ovalbumin (OVA) -Alexa fluor 488 and further incubated at 37°. After 15, 30, 60 and 90 min post-pulsing, cells were washed with PBS, fixed with 4% paraformaldehyde and analysed by flow cytometry. (a) Representative dot-plot depicting LK35.2 cell size (forward scatter) versus fluorescence intensity due to OVA-Alexa Fluor 488 uptake. (b) Mean fluorescence intensity (mean ± SD), indicative of OVA-Alexa Fluor 488 uptake. Results are from three independent experiments. Raw data were analysed by non-parametric analysis of variance.

Inhibition of ATP synthase decreases the relative amount of MHC II–peptide complexes on antigen-presenting B lymphocytes

The finding that antigen loading induces a subtle but significant mitochondrial depolarization on LK35.2 B lymphocytes and that ATP synthase inhibition does not affect antigen uptake suggests that it is antigen processing that imposes a metabolic demand on antigen-presenting cells. Therefore, we wanted to address if pharmacological inhibition of ATP synthase (oligomycin treatment) after HEL-loading would have an effect on antigen processing evaluated by the amount of MHC II–peptide complexes expressed on the LK35.2 B-lymphocyte cell membrane. Figure3(a,b) shows that inhibition of ATP synthase significantly reduces B-lymphocyte cell membrane expression of I-Ak MHC II bound to the HEL48–62 peptide, as assessed by flow cytometry.

Figure 3.

Figure 3

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Effect of ATP synthase inhibition on MHC II expression by antigen-presenting B lymphocytes. LK35.2 B lymphocytes (5 × 105 cells/ml) in 12-well culture plates were loaded with 250 μg/ml of hen egg white lysozyme (HEL) for 12 hr at 37° to assess the cell membrane expression of MHC II–peptide, by staining with the MHCII–peptide-specific C4H3 monoclonal antibody, followed by an Alexa fluor 488-labelled secondary antibody. (a) Representative FACS histogram depicting MHC II expression in LK35.2 B lymphocytes incubated with no HEL, with 250 μg/ml of HEL, or with 250 μg/ml of HEL plus 2 μg/ml of oligomycin (ATP synthase inhibitor). (b) Integrated results for MHC II expression from three independent experiments (by duplicate). Results are expressed as fold increase, taking non-stained cells as the reference. (c) Total MHC II expression in LK35.2 B lymphocytes loaded with 250 μg/ml of HEL for 12 hr at 37°. Cells were stained with total MHC II-specific H11632 monoclonal antibody. The effect of oligomycin and Ruthenium red was assessed. No statistically significant differences were observed. Polymyxin B was also used to analyse if the observed effects were due to contaminant lipopolysaccharide in the HEL preparation. Analysis of variance and Turkey′s multiple comparison test were used for statistical analyses.

In addition, total MHC molecules were assessed on HEL-loaded LK35.2 B lymphocytes in the presence or in the absence of oligomycin. Figure3(c) shows no statistically significant differences in total MHC expression among the treatments, which suggests that ATP synthase inhibition disrupts antigen processing rather than the transport of MHC class II to the cell membrane.

Inhibition of ATP synthase renders antigen-presenting B lymphocytes less efficient to activate T-lymphocyte proliferation

After finding that mitochondrial ATP synthesis inhibition decreases the extent of cell membrane expression of MHC II–peptide in LK35.2 B lymphocytes, we wanted to investigate whether inhibition of ATP synthase of LK35.2 B lymphocytes also has some effect on their ability for antigen presentation and therefore on T-lymphocyte activation. Figure4 shows the results of antigen presentation. As indicated in the Materials and methods, this assay is based on the proliferative response of the IL-2-dependent CTLL-2 cell line, when cultured with the supernatants from 24 hr co-cultures of HEL-loaded LK35.2 cells with HEL-specific C10 T lymphocytes. So there is a direct correlation between CTLL-2 proliferation, the amount of IL-2 in the supernatants, and the ability of LK35.2 B cells to activate C10 T lymphocytes. Unloaded LK35.2 B cells did not induce T-cell activation whereas HEL-loaded LK35.2 B cells significantly induced C10 T-cell activation, inhibition of ATP synthase previous to HEL-uptake, antigen processing and MHC II–peptide export to the cell membrane significantly reduced the ability of LK35.2 B lymphocytes to present the HEL48–62 peptide to C10 T lymphocytes. As shown, when LK35.2 cells are pulsed directly with the HEL48–62 peptide instead of complete HEL, oligomycin treatment also reduces antigen presentation. Likewise when the LK35.2 cell derivative cell line (LKKDEL) that endogenously expresses HEL20 was used, oligomycin treatment reduced antigen presentation to the HEL48–62-specific C10 T lymphocytes. It is worth noting that exogenous antigen presentation was completely inhibited by oligomycin whereas antigen presentation of endogenously expressed HEL and HEL48–62 peptide was only partially inhibited.

Figure 4.

Figure 4

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Effect of ATP synthase inhibition on the ability of hen egg white lysozyme (HEL) -loaded antigen-presenting B cells to induce T-cell activation. LK35.2 B cells or LKKDEL (HEL endogenous) were treated for 20 min with oligomycin (10 μg/ml) or left untreated and then either pulsed with HEL (250 μg/ml) or HEL peptide (1 μm) or left untreated for 12 hr. Cell lines were then washed, fixed with paraformaldehyde and co-cultured with the C10 T-cell hybridoma (at a 1 : 2 ratio) for 24 hr. Interleukin-2 (IL-2) -dependent CTLL-2 cells (10 000 cells/well) were cultured with 100 μl of co-culture supernatants for 24 hr and pulsed with [3H]thymidine (0·5 μCi/well) for the last 12 hr of culture. (a) CTLL-2 proliferation was measured by [3H]thymidine incorporation. Data shown are from duplicate values from three independent experiments. Mann–Whitney U-test was used for statistical analysis. ***P < 0·0001, **P < 0·005. (b) Cell viability was assessed by Hoescht staining in LK35.2 B cells that were treated with HEL plus oligomycin, HEL alone or left untreated. Viable cells stain positive for Hoescht. Depicted is a representative results out of two independent experiments.

Cell viability of untreated LK35.2, cells pulsed with 250 μg/ml of HEL for 16 hr and cells pulsed with 250 μg/ml of HEL for 16 hr in the presence of 10 μg/ml of oligomycin was determined. Figure4(b) shows that cell viability in all three conditions was > 95%, ruling out the possibility that oligomycin-dependent impairment of antigen presentation was due to cell death.

Inhibition of ATP synthase or MCU inhibits antigen processing by B lymphocytes

To confirm that mitochondrial function is relevant for antigen processing, we used a well-established fluorescence-based method to assess antigen processing,27 comprising the use of a self-quenching conjugate of OVA (DQ-OVA) (Thermo Fisher Scientific Inc.) which, upon proteolysis, yields highly fluorescent peptides that can be quantified, we compared the mean fluorescence intensity of DQ-OVA-pulsed cells immediately after pulsing (time 0), versus DQ-OVA pulsed cells that were incubated for 1 hr at 37°, so allowing antigen processing to take place. These assays were carried out in medium alone, in the presence of the ATP synthase inhibitor oligomycin, or in the presence of the MCU inhibitor Ruthenium red, to assess if mitochondrial ATP synthesis and mitochondrial Ca2+ uptake are required for OVA processing. DQ-OVA peptide-associated fluorescence was observed and quantified by confocal microscopy. Figure5(a) (confocal microscopy images) shows that in the absence of oligomycin, fluorescence was higher at 60 min than at 0 min and therefore that antigen processing had taken place. In contrast, when cells were cultured for 60 min in the presence of 10 μg/ml of oligomycin, or in the presence of 5 μm of Ruthenium red, no difference in fluorescence (as indicative of antigen processing) was observed, compared with cells at 0 min and Fig.5(b) shows the fold change in fluorescence (median ± SD) for each culture condition, indicating the involvement of both ATP synthesis and mitochondrial Ca2+ uptake in antigen processing.

Figure 5.

Figure 5

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Effect of ATP synthase inhibition and of mitochondrial calcium uniporter inhibition on the ability of B lymphocytes for antigen processing. LK35.2 B cells were pulsed for 20 min with 1 mg/ml of DQ-ovalbumin (OVA), which upon proteolytic degradation exhibits bright green fluorescence. Cells were either fixed immediately after being pulsed (0 min) or incubated for 60 min at 37° in medium alone with 10 μg/ml oligomycin (ATP synthase inhibitor) or with 5 μm Ruthenium red (mitochondrial calcium uniporter inhibitor), after which cells were washed, fixed and covered with DAPI-containing Vectashield. (a) Green fluorescence, as indicative of DQ-OVA processing, was analysed by confocal microscopy (representative result of three independent experiments and 15 microscopic fields). (b) Mean fluorescence intensity (MFI) was normalized and given a value of 1 to the MFI obtained at time 0 min and then calculating the fold change in MFI at 60 min after DQ-OVA processing. Results show the mean ± SD of 15 microscopic fields from three independent experiments for each treatment. Statistical significance was analysed by non-parametric analysis of variance.

Mitochondria re-organize towards the immune synapse in antigen-presenting B lymphocytes

Mitochondrial function is also dependent on their location within the cell and therefore we wanted to analyse the mitochondrial position in HEL-loaded APC versus unloaded LK35.2 lymphocytes upon contact with specific T lymphocytes. Figure6(a) shows the distribution of mitochondria in HEL-loaded LK35.2 B lymphocytes (+HEL) and in untreated LK35.2 cells (–HEL) upon contact with 3A9 T lymphocytes, by transmission electron microscopy. The relative amount of mitochondria per area was assessed by imagej analysis. Figure6(b) shows that there are more mitochondria by area in HEL-loaded than in untreated LK35.2 cells upon contact with specific T lymphocytes, which suggests a reorganization of APC mitochondria to the immune synapse.

Figure 6.

Figure 6

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Mitochondrial distribution in antigen-presenting B lymphocytes. LK35.2 B cells (1 × 107) in Petri dishes were left untreated or pulsed with 250 μg/ml of hen egg white lysozyme (HEL) for 12 hr at 37° and then washed and layered over with 3A9 T lymphocytes (1 × 107). Cell-to-cell interaction was allowed to proceed for 15–30 min, after which cells were carefully washed with PBS and fixed. After fixing, cells were carefully scraped, spun down, and prepared for electron microscopy analysis as indicated in Materials and methods. (a) Representative images of B–T conjugates in HEL-loaded LK35.2 B cells (+HEL) and unloaded LK35.2 B cells (−HEL), depicting a cell–cell contact, a magnification, and the way image-j analysis was performed, i.e. an area was defined, the mitochondria were highlighted (red) and the % of the area in red with respect to the total defined area was calculated. (b) Relative amount of mitochondria (expressed as the percentage of mitochondria in a defined area) in LK35.2 cells at the immune synapse when they were loaded with HEL (+HEL) or not (−HEL). Images are representative of multiple fields from three independent experiments. image-j analyses were carried out from 30 images for each condition. Statistical significance was analysed by non-parametric analysis of variance.

Discussion

Here we provide some evidence that mitochondrial function may play a role in antigen processing and presentation by using HEL48–62 peptide-presenting B lymphocytes (LK35.2 B cells) and HEL48–62-specific 3A9 and C10 T lymphocytes, as well as an LK35.2 cell derivative (LKKDEL) that endogenously expresses HEL, as model systems for antigen presentation.19,20 First, we observed that HEL loading of LK35.2 cells induced a subtle and transitory but statistically significant mitochondrial depolarization (Fig.1a,b), which suggested an increase in the metabolic demands of these cells. It has previously been shown that antigen processing takes place between 30 and 60 min after loading,27 and that peptide-loaded MHC II molecules start to be expressed on the APC cell membrane by 4 hr after antigen uptake30 so, the length of our Δψm determinations covered all this process.

Next, we evaluated mitochondrial Ca2+ concentration [Ca2+]m, because mitochondrial function is largely dependent on this ion, and both Δψm and [Ca2+]m are indicative of mitochondrial energy status.28,31,32 In addition, the small reduction in Δψm observed would indicate that not all mitochondria are being affected by HEL-loading but rather that calcium microdomains and only a subset of mitochondria are at play, as has been demonstrated for T-lymphocyte immune synapses.33 For this, we used the [Ca2+]m indicator Rhod-2/AM combined with flow cytometry. HEL loading did not induce a statistically significant mitochondrial uptake at the times tested (Fig.1c,d). However, further experiments demonstrated that inhibition of the MCU, and therefore of mitochondrial Ca2+ uptake, had significant effects on antigen processing.

Antigen uptake by APC begins within minutes of exposure to the antigen and is followed by antigen processing and antigen presentation. Therefore we wanted to separately analyse the effect that inhibition of ATP synthase or inhibition of MCU have on antigen uptake, antigen processing and antigen presentation.

Antigen uptake was analysed by loading LK35.2 cells with OVA-Alexa fluor 488 and flow cytometry. It was observed that antigen uptake persisted up to the last time tested (90 min) in otherwise untreated cells and that this was not disrupted by oligomycin or Ruthenium red treatments and so, in this system, inhibition of ATP synthase or inhibition of MCU had no effect on antigen uptake (Fig.2).

Once on the cell membrane, the MHC II–peptide complexes have a half-life of about 150 hr for immunodominant peptides.18 So, in order to analyse the expression of MHC II–peptide and total MHC II, LK35.2 cells were loaded with HEL for 12 hr, after this time the amount of MHC II–peptide on the cell membrane increased about twofold, compared with the negative control (unloaded LK35.2 B cells), as assessed by the mean fluorescence intensity resulting from the cell labelling with the MHC II-HEL48–62-specific monoclonal antibody.

Inhibition of ATP synthase by oligomycin treatment 20 min before HEL-uptake and the remaining 12 hr of culture significantly decreased the expression of MHC II–peptide, suggesting that antigen processing and presentation require mitochondrially generated ATP. Expression of MHC II–peptide was not completely lost and so mitochondrial ATP synthesis is not the only requirement for MHC II–peptide export to the cell membrane (Fig.3a–b). However, total MHC II cell membrane expression after 12 hr of HEL loading was not significantly affected by oligomycin or Ruthenium red treatment (Fig.3c). Polymyxin B pre-treatment was carried out to indirectly analyse a possible contamination of HEL with LPS. Our results ruled out this possibility. The oligomycin-dependent decrease in MHC II–peptide expression (Fig.3a,b) was in keeping with the finding that HEL-loaded, and oligomycin-treated cells were less efficient to activate HEL48–62-specific T lymphocytes (antigen presentation) than HEL-loaded B lymphocytes, as shown in Fig.4(a). Antigen presentation of LK35.2 B lymphocytes loaded with HEL48–62 peptide and the antigen presentation of endogenously expressed HEL by LKKDEL cells was also inhibited by oligomycin, although to a lesser extent than that of exogenous HEL. Hence, it seems that antigen presentation of exogenous antigen is more sensitive to ATP synthase inhibition than that of endogenous antigen or direct peptide engagement on cell membrane MHC II molecules.

To further analyse the role of functioning mitochondria in antigen processing, we used DQ-OVA, a self-quenching conjugate that, upon proteolysis, renders fluorescent DQ-OVA-derived peptides that can be quantified by fluorescence-based techniques.27 Figure5(a) shows confocal microscopy images of DQ-OVA processing. In otherwise untreated LK35.2 cells, fluorescence, indicative of antigen processing, is evident after 60 min. However, at this time-point, LK35.2 cells that were treated with oligomycin or Ruthenium red at time 0 (i.e. 15 min after loading with DQ-OVA) presented a less intense fluorescence. Figure5(b) shows the fold change in DQ-OVA fluorescence intensity for the three culture conditions. Taken together these findings indicate that antigen processing requires functional mitochondria, i.e. mitochondria with functioning ATP synthase and with the capacity for MCU-dependent calcium uptake.

Finally, when HEL-loaded LK35.2 B lymphocytes were co-cultured with the HEL48–62-specific 3A9 T lymphocytes, a mobilization of B-lymphocyte mitochondria towards the immune synapse was observed. This would suggest that local mitochondrial energy is required for antigen presentation. However, the antigen presentation assays were performed with HEL-loaded and paraformaldehyde-fixed LK35.2 B lymphocytes, i.e. metabolically inactive cells. Therefore we suggest that APC mitochondria may have a role in immune synapses other than to provide local energy for antigen presentation.

In T lymphocytes, immune synapse formation is critical for activation, and mitochondria have a role in this process, by localizing close to the immune synapse,10,34 and mitochondrial metabolism is a critical component of T-cell activation.35 Immune synapse is a two-way interaction, as it has been shown that APCs also receive activation signals from T lymphocytes.36 This is to the best of our knowledge the first evidence that inhibition of mitochondrial ATP synthase or inhibition of MCU disrupts antigen processing and antigen presentation.

Increased ATP levels at the immune synapse are thought to be required for energy consuming signalling and also for the centripetal flux of T-cell receptor to the cSMAC, which is important for the termination of T-cell receptor signals by internalization and degradation.37 Accordingly, it has been proposed that mitochondria strengthen but also terminate immune synapse signalling.38 Local mitochondria in APCs immune synapses may serve analogous purposes, and this remains to be analysed.

Acknowledgments

We thank the two anonymous reviewers who kindly contributed to improve this work; Dr José Moreno for kindly providing LK35.2, LKKDEL, 3A9, and C10 cell lines; and Dr Eneida Campos (postgraduate Immunology programme core laboratory) for technical assistance with confocal microscopy. This work was financed in part by CONACYT (CB-2010-157018), SIP (2013-1132) and CONACYT (CB-2010-158340) grants. LCB is an SNI fellow, EOLV and FJSG are COFAA/EDI and SNI fellows, MPCS was supported by a CONACYT studentship.

Disclosures

The authors declare no conflict of interests.

References

  1. Trombetta ES, Mellman I. Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol. 2005;23:975–1028. doi: 10.1146/annurev.immunol.22.012703.104538. [DOI] [PubMed] [Google Scholar]
  2. Unanue ER. Antigen presenting function of the macrophage. Annu Rev Immunol. 1984;2:395–428. doi: 10.1146/annurev.iy.02.040184.002143. [DOI] [PubMed] [Google Scholar]
  3. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245. doi: 10.1038/32588. [DOI] [PubMed] [Google Scholar]
  4. Vascotto F, Le Roux D, Lankar D, Faure-André G, Vargas P, Guermonprez P, Lennon-Duménil AM. Antigen presentation by B lymphocytes: how receptor signaling directs membrane trafficking. Curr Opin Immunol. 2007;19:93–8. doi: 10.1016/j.coi.2006.11.011. [DOI] [PubMed] [Google Scholar]
  5. Kuhns MS, Davis MM. TCR signaling emerges from the sum of many parts. Front Immunol. 2012;3:159. doi: 10.3389/fimmu.2012.00159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dustin ML, Long EO. Cytotoxic immunological synapses. Immunol Rev. 2010;235:24–34. doi: 10.1111/j.0105-2896.2010.00904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML. The immunological synapse: a molecular machine controlling T cell activation. Science. 1999;285:221–7. doi: 10.1126/science.285.5425.221. [DOI] [PubMed] [Google Scholar]
  8. Davis DM, Dustin ML. What is the importance of the immunological synapse? Trends Immunol. 2004;25:323–7. doi: 10.1016/j.it.2004.03.007. [DOI] [PubMed] [Google Scholar]
  9. Singleton KL, Gosh M, Dandekar RD, et al. Itk controls the spatiotemporal organization of T cell activation. Sci Signal. 2011;4:ra66. doi: 10.1126/scisignal.2001821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Quintana A, Schwindling C, Wenning AS, Becherer U, Rettig J, Schwarz EC, Hoth M. T cell activation requires mitochondrial translocation to the immunological synapse. Proc Natl Acad Sci U S A. 2007;104:14418–23. doi: 10.1073/pnas.0703126104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Quintana A, Hoth M. Mitochondrial dynamics and their impact on T cell function. Cell Calcium. 2012;52:57–63. doi: 10.1016/j.ceca.2012.02.005. [DOI] [PubMed] [Google Scholar]
  12. Schwindling C, Quintana A, Krause E, Hoth M. Mitochondria positioning controls local calcium influx in T cells. J Immunol. 2010;184:184–90. doi: 10.4049/jimmunol.0902872. [DOI] [PubMed] [Google Scholar]
  13. Dustin ML, Shaw AS. Costimulation: building an immunological synapse. Science. 1999;29:649–50. doi: 10.1126/science.283.5402.649. [DOI] [PubMed] [Google Scholar]
  14. Abarca-Rojano E, Muñiz-Hernández S, Moreno-Altamirano MMB, Mondragón-Flores R, Enriquez-Rincón F, Sánchez-García FJ. Re-organization of mitochondria at the NK cell immune synapse. Immunol Lett. 2009;122:18–25. doi: 10.1016/j.imlet.2008.10.008. [DOI] [PubMed] [Google Scholar]
  15. Christie DA, Kirchhof MG, Vardhana S, Dustin ML, Madrenas J. Mitochondrial and plasma membrane pools of stomatin-like protein 2 coalesce at the immunological synapse during T cell activation. PLoS ONE. 2012;7:e37144. doi: 10.1371/journal.pone.0037144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. O’Brien C, Flower DR, Feighery C. Peptide length significantly influences in vitro affinity for MHC class II molecules. Immunome Res. 2008;4:6. doi: 10.1186/1745-7580-4-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Vyas JM, Van der Veen AG, Hidde L, Ploegh HL. The known unknowns of antigen processing and presentation. Nat Rev Immunol. 2008;8:607–18. doi: 10.1038/nri2368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lazarski CA, Chaves FA, Jenks SA, Wu S, Richards KA, Weaver JM, Sant AJ. The kinetic stability of MHC class II:peptide complexes is a key parameter that dictates immunodominance. Immunity. 2005;23:29–40. doi: 10.1016/j.immuni.2005.05.009. [DOI] [PubMed] [Google Scholar]
  19. Allen PM, Unanue ER. Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridomas. J Immunol. 1984;132:1077–9. [PubMed] [Google Scholar]
  20. Bonifaz LC, Arzate S, Moreno J. Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments. Eur J Immunol. 1999;29:119–31. doi: 10.1002/(SICI)1521-4141(199901)29:01<119::AID-IMMU119>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  21. Linnett PE, Beechey RB. Inhibitors of the ATP synthetase system. Methods Enzymol. 1979;55:472–518. doi: 10.1016/0076-6879(79)55061-7. [DOI] [PubMed] [Google Scholar]
  22. Bae JH, park J-W, Kwon TK. Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochem Biophys Res Commun. 2003;303:1073–9. doi: 10.1016/s0006-291x(03)00479-0. [DOI] [PubMed] [Google Scholar]
  23. Kappler J, White J, Wegmann D, Mustain E, Marrack P. Antigen presentation by Ia B cell hybridomas to H-2-restricted T cell hybridomas. Proc Natl Acad Sci U S A. 1982;79:3604. doi: 10.1073/pnas.79.11.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Floryk D, Houstek J. Tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements of mitochondrial membrane potential in cells treated with digitonin. Biosci Rep. 1999;19:27–34. doi: 10.1023/a:1020193906974. [DOI] [PubMed] [Google Scholar]
  25. Cardoso LS, Araujo MI, Goés AM, Pacífico LG, Oliveira RR, Oliveira SC. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microb Cell Fact. 2007;6:1–6. doi: 10.1186/1475-2859-6-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Graham DB, Akilesh HM, Gmyrek GB, et al. ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling. Blood. 2010;116:3208–18. doi: 10.1182/blood-2009-10-250415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Daro E, Pulendran B, Brasel K, et al. Polyethylene glycol-modified GM-CSF expands CD11bhighCD11chigh but not CD11blowCd11chigh murine dendritic cells in vivo: a comparative analysis with Flt3 ligand. J Immunol. 2000;165:49–58. doi: 10.4049/jimmunol.165.1.49. [DOI] [PubMed] [Google Scholar]
  28. Beltran B, Mathur A, Buchen MR, Erusalimsky JD, Moncada S. The effect of nitric oxide on cell respiration: a key to understanding its role in cell survival or death. Proc Natl Acad Sci U S A. 2000;97:14602–7. doi: 10.1073/pnas.97.26.14602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kuwabara T, Imajoh-Ohmi S. LPS-induced apoptosis is dependent upon mitochondrial dysfunction. Apoptosis. 2004;9:467–74. doi: 10.1023/B:APPT.0000031453.90821.6a. [DOI] [PubMed] [Google Scholar]
  30. Nashar TO, Drake JR. The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation. J Immunol. 2005;174:1306–16. doi: 10.4049/jimmunol.174.3.1306. [DOI] [PubMed] [Google Scholar]
  31. Liesa M, Shirihai OS. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab. 2013;17:491–506. doi: 10.1016/j.cmet.2013.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium ATP, and ROS: a mitochondrial love–hate triangle. Am J Physiol Cell Physiol. 2014;287:C817–33. doi: 10.1152/ajpcell.00139.2004. [DOI] [PubMed] [Google Scholar]
  33. Quintana A, Pasche M, Junker C, et al. Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation. EMBO J. 2011;30:3895–912. doi: 10.1038/emboj.2011.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Baixauli F, Martın-Cofreces NB, Morlino G, Carrasco YR, Calabia-Linares C, Veiga E, Serrador JM, Sanchez-Madrid F. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signaling at the immune synapse. EMBO J. 2011;30:1238–50. doi: 10.1038/emboj.2011.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sena LA, Li S, Jairaman A, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013;38:225–36. doi: 10.1016/j.immuni.2012.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Duchez S, Rodrigues M, Bertrand F, Valitutti S. Reciprocal polarization of T and B cells at the immunological synapse. J Immunol. 2011;187:4571–80. doi: 10.4049/jimmunol.1100600. [DOI] [PubMed] [Google Scholar]
  37. Lee KH, Dinner AR, Tu C, et al. The immunological synapse balances T cell receptor signaling and degradation. Science. 2003;302:1218–22. doi: 10.1126/science.1086507. [DOI] [PubMed] [Google Scholar]
  38. Junker C, Hoth M. Immune synapses: mitochondrial morphology matters. EMBO J. 2011;30:1187–9. doi: 10.1038/emboj.2011.72. [DOI] [PMC free article] [PubMed] [Google Scholar]

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