Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

Abstract

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

Fig. 1. Ubl3 regulates surface expression of MHC II and CD86 in MutuDCs.

a Schematic of CRISPR/Cas9 genetic screen. MutuDCs were lentivirally transduced with the GeCKOv2 library A and puromycin-resistant cells were stained for surface MHC II. The 5% highest MHC II expressing cells were sorted by flow cytometry, genomic DNA extracted, and amplified. gRNAs were quantified by Illumina NextSeq sequencing and analyzed with edgeR. Volcano plots show detected gRNAs enriched in sorted cells with increased MHC II expression compared to the unsorted library. P values were assessed using a negative binomial generalized linear model with a two-sided likelihood ratio test, with adjustment for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method. Guide RNAs with an absolute log2 fold change >1 and FDR of <0.01 were considered significantly enriched (orange dots), with genes hit by at least two gRNAs highlighted (large red circles). b Surface expression of MHC II, CD86, or MHC I were analyzed by flow cytometry for MutuDCs lacking B2m (pool), Marchf1, or Ubl3 (single-cell clones), compared with control cells expressing non-targeting hBim gRNA. Top: representative histograms, with gray solid histograms: control MutuDCs expressing non-targeting hBim gRNA, black lined histograms: CRISPR/Cas9 modified MutuDC lines as indicated. Bottom: quantification of flow cytometry analysis from three experiments. Bars indicate mean+SD of geometric mean fluorescence intensity (gMFI), relative to untransfected cells. Statistical analysis was performed using a one-way ANOVA and Bonferroni’s multiple comparisons test, comparing each sample to hBim controls, p values shown above bars, ns (not significant) p > 0.05.

Fig. 2. UBL3 does not control MHC II via altering synthesis, peptide loading, or UBL3 modification of MHC II.

a Analysis of Ii degradation and MHC II peptide loading in control MutuDCs expressing non-targeting hBim gRNA or ΔUbl3 MutuDCs. Cells were metabolically labeled for 30 min, washed, and cultured in a growth medium for the indicated time points before lysis. MHC II molecules were immunoprecipitated with JV1 rabbit antisera, or normal rabbit serum (NRS), and protein G-Sepharose. Samples were loaded before (NB) or after boiling (B) at 95 °C (b), subjected to denaturing SDS-PAGE, transferred onto PVDF membranes, and exposed to a storage phosphor screen. Sizes corresponding to free MHC II α and β, full-length invariant chain (Ii), the Ii spliced variant Ii_p41, the degradation intermediate Ii_p10, and SDS-stable complexes αβIi_p10, and αβ-peptide (pep), are indicated on the right. Data from one experiment. b ΔUbl3 MutuDCs or ΔUbl3 MutuDCs expressing Myc-UBL3 were lysed and post-nuclear supernatants (PNS) were subjected to immunoprecipiation (IP) with anti-Myc-Tag agarose (clone 9E10) or anti-MHC II antibody (M5/114) crosslinked to protein G-sepharose. Per lane, samples equivalent to 5 × 10⁶ cells (Myc IP), 2.5 × 10⁶ cells (MHC II IP), or 400,000 cells (PNS) were analyzed by non-denaturing SDS-PAGE and western blotting using antibodies against UBL3 (ab113820), MHC II β chain (JV2), Myc (71D10), and ubiquitin (P4D1). Shown are representative blots and quantification of relative ubiquitination, with bars displaying mean + 95% confidence intervals for four experiments.

Fig. 3. UBL3 regulation of MHC II and CD86 requires membrane anchoring.

a–d Clonal ΔUbl3 MutuDCs were retrovirally transduced with Myc-tagged wild type UBL3 (“+Myc-UBL3_WT”), or UBL3_C114S mutant (“+Myc-UBL3_C114S”). a Post-nuclear supernatants of 2.5 × 10⁵ cells were analyzed by non-denaturing SDS-PAGE and western blotting using anti-actin (20-33) anti-Myc (71D10) and anti-Ubl3 (ab113820) antibodies. Representative blot is shown for two independent experiments with similar results. b Proximity ligation assay (PLA). ΔUbl3 MutuDC expressing Myc-UBL3_WT or Myc-UBL3_C114S were stained with plasma membrane (PM) CytoPainter. After washing, fixing, and permeabilization, cells were stained with Hoechst 33342, rabbit anti-MHC II (JV2) antiserum, and mouse anti-Myc mAb (9E10) and subjected to PLA. Scale bar = 10 µm. PLA spots indicate spatial proximity between MHC II and UBL3 and were enumerated within plasma membrane boundaries (violin plots). One experiment, P value as indicated, unpaired Welch’s t test (two-sided). c Immunofluorescence microscopy. Cells were fixed, permeabilized, and stained with biotinylated rat anti-MHC II mAb (M5/114), mouse anti-Myc mAb (9E10), streptavidin Alexa Fluor 647, donkey anti-mouse Alexa Fluor 594, and 0.5 μg/ml DAPI. Scale bar = 10 µm. Representative image shown for two independent experiments with similar results. d Flow cytometry analysis of MHC II and CD86. Left: Dot plots show MHC II (x axis) and GFP (y axis). Center: histograms show corresponding expression levels of MHC II or CD86, with gray solid histogram: control MutuDCs expressing non-targeting hBim gRNA; black line: ΔUbl3 MutuDCs, red line: ΔUbl3 MutuDCs expressing Myc-UBL3_WT, blue line: ΔUbl3 MutuDCs expressing Myc-UBL3_C114S. Right: quantification of gMFI from three experiments, normalized to highest gMFI of each experiment, with bars mean ± SD and symbols. ****P < 0.0001, one-way ANOVA with Bonferroni’s test.

Fig. 4. UBL3 regulates MHC II and CD86.

a Ubl3^−/− mice. b Spleen cDC surface MHC II and CD86 by flow cytometry. Representative histograms and graphs showing gMFI relative to highest signal, bars mean ± SD, symbols  individual mice (n = 9, three independent experiments). One-way ANOVA with Bonferroni’s test. c Quantitative real-time PCR of H2-Ab1 and Marchf1 mRNA in spleen cDCs. Bars mean ± SD relative to WT, n = 2 samples, two experiments. d Spleen cDCs labeled with FIP-conjugated mAb for MHC II, CD86, or MHC I were incubated for 30 mins at 37°C. Fluorescence was quenched (+Q) and internalization was calculated as described in Methods. CD86 internalization not shown for cDC2 (low expression). Data representative of two independent experiments with similar results (MHC II) or pooled from two (CD86) or three (MHC I) experiments performed in triplicate, mean ± SD, one-way ANOVA with Bonferroni’s test. e MHC II was immunoprecipitated (IP) from spleen cDCs using anti-MHC II antibody, analyzed by non-denaturing SDS-PAGE, and immunoblotted (IB) for ubiquitin and MHC II β-chain. Left: representative blot. Right: MHC II ubiquitination relative to WT, bars mean + 95% confidence interval for three biological replicates. f–i Flow cytometry of surface MHC II and CD86 of f professional antigen-presenting cells (thymus: n = 14, four experiments (MHC II), n = 5, one experiment (CD86); blood: n = 7 for WT, 10 for Ubl3^−/−, 6 for Marchf1^−/−, two experiments; peritoneal cavity: n = 5 for WT, 4 for Ubl3^−/−, one experiment), g spleen myeloid cells (n = 5 for WT, 4 for Ubl3^−/−, one experiment), h TEC (n = 11 for WT, 12 for Ubl3^−/−, 12 for Marchf8^−/−, four experiments), and i lung epithelial cells (n = 5, one experiment). Graphs show gMFI relative to highest gMFI, bars mean ± SD, symbols represent individual mice. One-way ANOVA with Bonferroni’s test, unpaired t test (two-sided) with Holm–Sidak correction. SPM small peritoneal macrophages, LPM large peritoneal macrophages, Mφ macrophages, mo monocytes, AECII type II lung alveolar epithelial cells, BEC bronchial epithelial cells, EC lung endothelial cells. p values above bars, with ****p < 0.0001, ns (not significant) p > 0.05.

Fig. 5. UBL3 is required for normal thymic T_reg development.

Quantification of a Foxp3⁺CD25⁺ T_reg or b Foxp3⁻CD25⁺GITR⁺ T_reg precursor frequencies in thymus. Representative plots are shown on the left. Graphs on the right show pooled data from a n = 12 mice examined over four experiments and b n = 9 mice examined over three experiments, with each symbol representing an individual mouse and bars designating mean ± SD. P values shown above bars, one-way ANOVA with Bonferroni’s multiple comparisons test.

Fig. 6. Lack of UBL3 impairs DC-targeted antigen presentation in vivo.

a Left: spleen cDC1 expression of CLEC9A from wild type (WT) or Ubl3^−/− mice by flow cytometry, n = 11 mice examined over three experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). Right: Spleen cDCs from WT or Ubl3^−/− mice were labeled with FIP-conjugated anti-CLEC9A mAb. After 30 mins at 37°C, quencher (Q) was added and percentage internalization was calculated as described in Methods. Histograms show representative CLEC9A-FIP signal. Right: quantification of internalization from one experiment performed in triplicate, with bars mean ± SD, ns (not significant) p > 0.05, unpaired t test (two-sided). b In vivo antigen presentation assay. Purified CellTrace Violet (CTV)-labeled OT-I and OT-II cells were adoptively transferred into WT and Ubl3^−/− mice. 24 hours later, mice were injected with 0.2 µg anti-CLEC9A mAb-targeted OVA and spleens harvested after 64 hours. Antigen presentation capacity was assessed by flow cytometric analysis of CTV-dilution by OT-I and OT-II proliferation. Data from n = 9 (OT-I) and n = 8 for WT, 7 for Ubl3^−/− (OT-II) mice, two experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). c Quantification of spleen cDC1 and cDC2 from WT or Ubl3^−/− mice. Data from n = 9 mice, three independent experiments, bars mean ± SD, symbols representing individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). d Left: C3 expression on spleen cDCs from WT, Ubl3^−/− or Marchf1^−/− mice by flow cytometry. Data from n = 5 from one experiment, bars mean ± SD, symbols represent individual mice. Two-way ANOVA with Bonferroni’s test. Right: Quantification of trogocytic B cells gated on B220⁺CD19⁺ cells in Nycodenz-enriched splenocytes from WT, Ubl3^−/− or Marchf1^−/− mice. Representative dot plots and data from one experiment with n = 5, bars mean ± SD, and symbols representing individual mice. Significant p values shown above bars, with ****p < 0.0001, ns (not significant) p > 0.05, one-way ANOVA with Bonferroni’s test.

Fig. 7. Lack of UBL3 alters cDC function and phenotype.

a Ex vivo antigen presentation assay. WT and Ubl3^−/− mice were injected with 1 µg of anti-CLEC9A-OVA mAb. Indicated numbers of spleen cDC1 and cDC2 were purified and co-cultured with OT-I or OT-II cells, and divided OT-I or OT-II cells enumerated by flow cytometry. Data with n = 3, representative of two experiments, mean ± SD, ****p < 0.0001, two-way ANOVA with Bonferroni’s test. b Flow cytometry analysis of relative cell surface marker expression of spleen cDC1 or cDC2 isolated from WT and Ubl3^−/− mice, with n = 8 mice, two independent experiments, symbols represent individual mice, bars mean ± SD, ****p < 0.0001, unpaired t test (two-sided) with Holm–Sidak adjustment. c OVA-Cy5 uptake assay. Purified spleen cDC1 or cDC2 from WT or Ubl3^−/− mice were incubated with 50 µg/ml OVA-Cy5 for the indicated times, and washed before flow cytometry analysis. Graphs show mean ± SD, with data from one experiment performed in triplicate, two-way ANOVA with Bonferroni’s test. d Proteolysis assay. Purified spleen cDC1 or cDC2 from WT or Ubl3^−/− mice were pulsed with DQ-OVA for 15 min, washed twice and DQ-OVA signal was measured by flow cytometry at different chase time points. Graphs show mean ± SD, with data pooled from two independent experiments performed in triplicate, two-way ANOVA with Bonferroni’s test. e Quantification of cathepsin (cat) activity in spleen cDCs. Top: representative gel indicating active cathepsin X/B/S/L, and actin immunoblot. Bottom: relative protease activity normalized to actin, with bars showing mean ± SEM of one experiment with three biological replicates. Four spleens were pooled for each biological replicate, ns not significant, unpaired t test (two-sided). f Cytokine expression of purified Ubl3^−/− and wild-type spleen cDC1 and cDC2 stimulated with CpG, IFN-γ, and GM-CSF. Bars display mean ± SD of cells stimulated in duplicate, using purified cDCs pooled from eight mice, representative of two independent experiments.

Fig. 8. UBL3 function is conserved in humans.

Human CD14⁺ monocytes were infected with non-targeting control shRNA or shRNA against UBL3 (sh1 UBL3 or sh2 UBL3) and analyzed after five days in culture. Top left: Cell lysates were probed with antibodies against UBL3 (LS-C661402) and actin, and quantification of gene silencing was determined by UBL3 signal intensity. Immunoblot depicts a scenario of approximately 95% UBL3 knockdown. Data from n = 6 donor samples examined in a single experiment, with symbols connected with a line representing individual donors. Top right: Surface MHC II, CD86, and MHC I on monocyte-derived DCs (CD16⁻ CD1a⁺) and monocyte-derived macrophages (CD16⁺ CD1a⁻) were analyzed by flow cytometry. Histograms are representative of analysis from one donor. Bottom: Graphs showing data from n = 6 (DCs) or n = 4 (macrophages) donor samples examined in a single experiment, with symbols connected with a line representing individual donors, p values shown above points, ns not significant, repeated measures one-way ANOVA.