Fig 1.
Reduced MHC I surface expression in MARCH1-deficient MuTu DC line.
MHC II and MHC I expression was examined by flow cytometry for isghBim and isgMarch1 MuTu DCs. The graph displays the fold-change in MFI for MHC II or MHC I expressed by isghMarch1 cells relative to isghBim cells. Each symbol represents an independent experiment. MFI, mean fluorescence intensity. Control is fluorescence minus one (FMO).
Fig 2.
Reduced MHC I surface expression in MARCH1-deficient primary haemopoietic cells.
(A) MHC II and MHC I surface expression was examined by flow cytometry for blood B220+ B cells. Graph displays an individual experiment where each symbol is data obtained from single mouse. Data is representative of 5 independent experiments with a total of 17 wild type mice and 13 March1-/- mice. Statistical analysis was performed using an unpaired Student’s t test. (B) MHC II and MHC I surface expression was examined by flow cytometry for blood B220+ B cells. Graphs display an individual experiment where each symbol is data obtained from a single mouse. Data is representative of 2 independent experiments with a total of 6 March1+/+, 5 March1-/+ and 6 March1-/- mice. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (C) MHC II and MHC I surface expression was examined by flow cytometry for splenic CD11c+ DCs. Data is displayed from 5 independent experiments, with each of the 5 experiments comprising 1 wild type sample (4–8 wild type spleens pooled) and 1 March1-/- sample (4–8 March1-/- spleens pooled). Statistical analysis was performed using a ratio paired t-test. (D) MHC I H-2Kb and H-2Db surface expression was examined by flow cytometry for blood B220+ B cells. Graphs display an individual experiment where each symbol is data obtained from a single mouse. Data is representative of 3 independent experiments with a total of 9 wild type mice and 6 March1-/- mice. Statistical analysis was performed using an unpaired Student’s t test (A, C and D). Control is fluorescence minus one (FMO). **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant. MFI, mean fluorescence intensity.
Fig 3.
MARCH1 does not alter MHC I ubiquitination or surface internalization.
(A) Wild type and March1-/- spleen DCs were isolated and whole cell lysis performed (input). MHC II or MHC I was immunoprecipitated (IP) and immunoblots probed with antibodies specific for MHC II, MHC I and ubiquitin. The high MW band in the immunoblots probed for ubiquitin is non-specific. (B) Internalization of surface MHC II and MHC I was measured for wild type and March1-/- spleen DCs. Cells were labeled with FIP-conjugated anti-MHC II or anti-MHC I and after 30 min of culture at 37°C, cells were exposed to quencher (Q). Percentage internalization was calculated as described in Materials and Methods. Data are pooled from 4 independent experiments performed in triplicate. Graph represents the mean internalization ± SEM. Data were analyzed using unpaired Student’s t test. **P < 0.01. ns, not significant. (C) Immunofluorescence microscopy of isghBim and isgMarch1 MuTu DCs. Cells were fixed and permeabilized. Cells were stained with rabbit anti-MHC I polyclonal antiserum (generated to the cytosolic tail of MHC I) donkey anti-rabbit Alexa Fluor 647 and 0.5 μg/ml DAPI. Images were collected from 55 isghBim and 45 isgMarch1 MuTu DCs.
Fig 4.
Reduced MHC I surface expression in MARCH1-deficient cells requires MHC II expression.
(A) MHC I surface expression was examined by flow cytometry for blood CD4+ T cells. Graphs display an individual experiment where each symbol is data obtained from a single mouse. Data is representative of 4 independent experiments with a total of 12 wild type mice and 10 March1-/- mice. Statistical analysis was performed using an unpaired Student’s t test. (B) MHC II, CD86 and MHC I surface expression was examined by flow cytometry for blood B220+ B cells. Data is pooled from 5 independent experiments with a total of 22 March1+/+ I-Aα+/+, 4 March1-/- I-Aα+/+, 5 March1-/- I-Aα-/- and 11 March1+/+ I-Aα-/- mice. The MFI of MHC II, CD86 and MHC I for cells isolated from March1-/- I-Aα+/+, March1-/- I-Aα-/-, March1+/+ I-Aα-/- was expressed as a fold-change relative to the mean MFI for wild type (March1+/+ I-Aα+/+) cells. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001,**** P < 0.0001, ns, not significant. MFI, mean fluorescence intensity. (A, B) Control is fluorescence minus one (FMO).
Fig 5.
Reduced MHC I surface expression in MHC II K225Rki/ki cells.
(A) MHC II, CD86 and MHC I expression was examined by flow cytometry for MuTu DCs overexpressing, or not, MHCII K225R. Data is from 1 experiment. (B) MHC II and MHC I surface expression was examined by flow cytometry for splenic CD11c+ DCs. Data is displayed from 8 independent experiments, with each of the 8 experiments comprising of 1 wild type sample (4–8 wild type spleens pooled) and 1 MHC II K225Rki/ki sample (4–8 MHC II K225Rki/ki spleens pooled). Statistical analysis was carried out using a ratio paired t-test. (C) MHC II and MHC I expression on wild type and MHC II K225Rki/ki B cells. Graphs display an individual experiment where each symbol is data obtained from a single mouse. Data is representative of 6 independent experiments with a total of 18 wild type and 18 MHC II K225Rki/ki mice. (D) MHC I H-2Kb and H-2Db expression on blood-circulating B cells. Graphs display an individual experiment where each symbol is data obtained from a single mouse. Data is representative of 3 independent experiments with a total of 9 wild type and 9 MHC II K225Rki/ki mice. (B-D) Statistical analysis was performed using unpaired Student’s t tests, *** P < 0.001**** P < 0.0001. MFI, mean fluorescence intensity. (A-D) Control is fluorescence minus one (FMO).
Fig 6.
Altered MHC II ubiquitination reduces the ability of dendritic cells to present antigen via MHC I.
(A) Purified wild type or MHC II K225Rki/ki spleen CD11c+ DCs were pulsed with SIINFEKL peptide. SIINFEKL-pulsed DCs were incubated with CellTrace Violet-labeled OT-I T cells for 3 days. The number of dividing CD8+ TCRVα2+ OT-I T cells was determined by flow cytometry. (B) Purified spleen CD8+ CD11c+ cDC1 wild type and MHC II K225Rki/ki were incubated with CellTrace Violet-labeled OT-I T cells. DCs were pulsed with OVA-coated splenocytes. The number of dividing CD8+ TCRVα2+ OT-I T cells was determined by flow cytometry following three days of cell culture. (A, B) Graphs display mean ± SEM. Data is representative of 2 independent experiments, performed in triplicate. *P < 0.05, **P < 0.01.