asci  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: A panel of MR1 mutants differentially translocates to the cell surface in response to stabilizing ligand . A , MR1-GFP expression was induced in clonal cell lines derived from the polyclonal parent cell line described in with 2 μg/ml of doxycycline (dox, dark colors) or not ( light gray ) and either treated with 100 μM of 6-FP (filled histograms) or an equal volume of solvent control 0.01 M NaOH (empty histograms) overnight. Data are representative of three independent experiments. The same unstained BEAS-2B WT control is included in each graph for reference. Calibration beads were included in each experiment. B , MR1-GFP expression was measured over time after removal of 2 μg/ml dox. Data are pooled from four independent experiments resulting in three or four data points for each time point with each cell line normalized to its starting level in each experiment and shown as mean with standard deviation (SD). Best fit parameters for straight lines were determined by least-squares regression. The fit of one curve fit to all the data sets was compared with the fit of individual curves fit to each data set using the extra sum-of-squares F test. For full test results see . C , whole cell lysates from cell lines induced with 2 μg/ml dox overnight were analyzed for expression of MR1-GFP and loading control Vinculin (VINC) by WB. Primary antibodies were from different species and detected in parallel with species-specific secondary antibodies conjugated to distinct IRDyes, and both channels exported as grayscale. Molecular weight markers are indicated in kDa on the left . Data are representative of three independent experiments. See for remaining blots. D , the region immediately around the sgRNA target site was analyzed by next-generation sequencing and mutations resulting in intact reading frames are summarized. See for complete sequence analysis. aa, amino acid; GeoMFI, geometric mean fluorescence intensity; M, marker; MR1, MHC class I-related protein 1; RF, reading frame; sgRNA, single-guide RNA; WT, wild type.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Expressing, Derivative Assay, Solvent, Control, Standard Deviation, Molecular Weight, Next-Generation Sequencing, Sequencing, Fluorescence, Marker

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1 mutants have a defect in presenting exogenous ligands but present ligands derived from intracellular infection and endosomal processing . MR1-GFP expression was induced with adjusted dox concentrations (see and text) overnight, and cells were used as APCs in IFNγ ELISpots. A , B , D , cells were preincubated with indicated dilutions of M . smegmatis ( M . smeg ) supernatant ( A ), deazalumazine ( B ), or 5-A-RU prodrug ( D ) for at least 1 hour before addition of MAIT cell clone D481-C7. C , APCs in C were infected with auxotroph Mtb (Aux Mtb) overnight and diluted as indicated. MR1-GFP expression at the time of the ELISpot was measured by flow cytometry and is shown to the right of each plot. Each dot represents a single technical replicate from one independent experiment. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration, and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test and statistically significant differences are indicated. 5-A-RU, 5-amino-6-D-ribitylaminouracil; aa, amino acid; APC, antigen presenting cell; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Derivative Assay, Infection, Expressing, Enzyme-linked Immunospot, Flow Cytometry, Concentration Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Mutated MR1 does not accumulate in vesicular compartments at baseline . A , expression of MR1-GFP was induced in clonal cell lines with twice the adjusted dox concentrations (1 μg/ml, 2 μg/ml, and 4 μg/ml, respectively—see and text), and cells were incubated with 100 μM of 6-FP or an equal volume of the solvent control 0.01 M NaOH overnight. Images are representative of three independent experiments. Scale bar indicates 10 μm. B–D , clonal cell lines D4 and D6 were induced to express MR1-GFP with 1 μg/ml and 2 μg/ml of dox, respectively, and treated with CellLight BacMam 2.0 for late endosomes. B , representative images. Scale bar indicates 20 μm. C , enumeration of MR1-GFP + vesicles per cell. D , colocalization of MR1-GFP + vesicles and Rab7a + vesicles. Data in C and D represent 24 cells from 24 images for each cell line, pooled from three independent experiments. Experimental groups in C were compared by two-tailed, unpaired t test. 6-FP, 6-formylpterin; MR1, MHC class I-related protein 1.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Expressing, Incubation, Solvent, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Mutated MR1 differentially interacts with calnexin, B2M, HLA-Ia, TPP1, OLFML2A, and SQSTM1/p62 . Expression of MR1-GFP was induced in clonal cell lines D4 and D6 with 10 μg/ml of dox overnight before incubation with 20 μg/ml dox and 100 μM 6-FP overnight. A , B , C , MR1-GFP was immunoprecipitated, and bound proteins were analyzed by mass spectrometry in DDA mode ( A ) or DIA mode ( B + C ). A , average size-adjusted intensity of proteins identified in both D4 and D6 normalized to MR1-GFP intensity in each sample. The MR1-GFP construct, B2M, and two isoforms of CALX (Uniprot IDs P27824 and P27824-2) are highlighted. Data in A are pooled from three independent experiments. Data in B and C are pooled from three independent experiments with three or four technical replicates each. Each dot in C represents one mass spectrometry injection for proteins with p adjusted ≤ 0.01 and |log2 fold-change| ≥ 1. For statistical analysis, see mass spectrometry Experimental Procedures section. For full lists of identified proteins and peptides see and . D , the lysates of the three experiments shown pooled in B and C were analyzed by WB. CANX and B2M were analyzed on one gel with the membrane cut horizontally before primary antibody incubation. Both the red and the green channel were exported in grayscale to allow visualization of the molecular weight marker. MR1-GFP and SQSTM1/p62 were analyzed on another gel with primary antibodies from different species. Species-specific secondary antibodies conjugated to distinct IRDyes were used, and each channel was exported individually in greyscale. aa, amino acid; B2M, β2-microglobulin; DDA, data-dependent acquisition; DIA, data independent acquisition; expt, experiment; HLA-Ia, human leukocyte antigen class Ia; LFQ, label-free quantification; M, marker; MR1, MHC class I-related protein 1; MW, molecular weight; OLFML2A, olfactomedin-like protein 2A; SQSTM1, sequestosome 1; TPP1, tripeptidyl-peptidase 1.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Expressing, Incubation, Immunoprecipitation, Mass Spectrometry, Construct, Injection, Membrane, Molecular Weight, Marker, Data-dependent acquisition, Data-independent acquisition, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: SQSTM1/p62 is not required for MR1-mediated antigen presentation . A–D , clonal cell lines D4 and D6 ( A + B ) or BEAS-2B WT cells ( C + D ) were transfected with missense (mis, filled symbols) siRNA or siRNA targeting SQSTM1/p62 (KD, empty symbols) after inducing MR1-GFP expression with adjusted dox concentrations (see , A + B only) and used as APCs in IFNγ ELISpots with the indicated antigens. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right in A . Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups in A and B were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. Comparisons in C and D were performed with two-tailed, paired t tests. SQSTM1/p62 KD was confirmed by WB except for one of the independent experiments in B as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux, auxotroph; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units; SQSTM1, sequestosome 1.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Immunopeptidomics, Transfection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Two Tailed Test, Fluorescence, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1 in mutant cell line D6 binds B2M with lower apparent affinity. A , clonal cell lines D4 and D6 were transiently transfected to overexpress B2M (full symbols) or mock transfected (empty symbols) before use as APCs in an ELISpot. Data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. B2M overexpression was confirmed by WB as shown in . B , MR1-GFP was immunoprecipitated from clonal cell lines D4 and D6 induced to express MR1-GFP with 0.5 or 8 μg/ml dox, respectively, overnight followed by incubation with the same concentrations of dox plus 100 μM 6-FP overnight. Immunoprecipitated samples were incubated at 37 °C for the indicated time periods and MR1 bound to the beads, B2M bound to the beads, and B2M in the supernatant (unbound) were measured by WB. A representative blot is shown on the left and pooled data from three experiments, normalized to MR1 for each sample, and t = 0 for each cell line is shown on the right as mean with SD. Primary antibodies were detected in parallel with species-specific secondary antibodies conjugated to IRDye800, and both channels exported as grayscale to visualize the molecular weight markers. The 800 channel was exported individually for MR1-GFP. See for the remaining blots. C , clonal cell lines D4 and D6 were induced to express MR1-GFP with 4 and 8 μg/ml dox, respectively, overnight followed by incubation with the same concentrations of dox with 100 μM 6-FP overnight to bring MR1 to the cell surface before incubation with brefeldin A (BFA) for the indicated amounts of time. MR1 surface levels ( left ) and total MR1 expression ( right ) were measured by flow cytometry. Best fit parameters for one-phase exponential decay curves were determined by least-squares regression in B and C . The fit of one curve fit to both data sets was compared with the fit of individual curves fit to each data set using the extra sum-of-squares F test. For full test results, see . 5-A-RU, 5-amino-6-D-ribitylaminouracil; 6-FP, 6-formylpterin; A.U., arbitrary units; aa, amino acid; APC, antigen presenting cell; B2M, β2-microglobulin; IFNγ, interferon-γ; IP, immunoprecipitation; M, marker; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Mutagenesis, Transfection, Enzyme-linked Immunospot, Concentration Assay, Over Expression, Immunoprecipitation, Incubation, Molecular Weight, Expressing, Flow Cytometry, Marker

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Knock down of B2M differentially affects exogenous and endosomal antigen presentation pathways . Clonal cell lines D4 and D6 were transfected with missense (mis, filled symbols ) siRNA or siRNA targeting B2M (KD, empty symbols ) before induction with adjusted dox concentrations (see ) and used as APCs in IFNγ ELISpots with the indicated antigens ( A ) or infected with auxotroph Mtb ( B ) and then used as APCs in IFNγ ELISpots. Dox concentrations during Aux Mtb infection were 0.4 and 0.8 μg/ml, respectively. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and shown to the right . Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. B2M KD was confirmed by WB as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux Mtb, Mycobacterium tuberculosis auxotroph; Aux, auxotroph; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Knockdown, Immunopeptidomics, Transfection, Infection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: The effect of B2M knock down depends on MR1 expression levels . A , B , BEAS-2 B WT cells ( A ) or BEAS-2B MR1 KO (KO) and BEAS-2B MR1 overexpressing (OE) cells ( B ) were transfected with missense (mis, filled symbols) siRNA or siRNA targeting B2M (KD, empty symbols) and used as APCs in IFNγ ELISpots with the indicated antigens. C , B2M was knocked down in the clonal cell line D4 as in A and B , and MR1-GFP expression was induced with the indicated concentrations of dox before use as APCs in IFNγ ELISpots with the indicated antigens. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right. Each dot represents a single technical replicate from one of three independent experiments. ELISpot data in all panels are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations ( A + B ), or the highest and lowest dox concentrations ( C ) are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test for more than two groups or a two-tailed, paired t test for two groups and statistically significant differences are indicated. Comparisons of all experimental groups for the flow cytometry analysis in C are shown in . B2M KD was confirmed by WB as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Knockdown, Expressing, Transfection, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Two Tailed Test, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: B2M is required for endosomal antigen presentation in the presence of high levels of MR1 . Clonal cell lines D4 and D6 cells were electroporated with RNPs containing scrambled nontarget control sgRNAs (NT) or three pooled sgRNAs targeting B2M (KO). Single clones were induced to express MR1-GFP with adjusted dox concentrations (see ) and used as APCs in IFNγ ELISpots with the indicated antigens ( A ) or infected with auxotroph Mtb ( B ) and then used as APCs in IFNγ ELISpots. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right. Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test and statistically significant differences are indicated. B2M KO was confirmed by WB, PCR, and qRT-PCR as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux, auxotroph; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KO, knock out; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; RNP, ribonucleoprotein; SFU, spot forming units; sgRNA, single-guide RNA.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Immunopeptidomics, Control, Clone Assay, Infection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Quantitative RT-PCR, Fluorescence, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1-mediated presentation of exogenous antigen is limited by binding to B2M, whereas presentation of antigen derived from intracellular infection is limited by the availability of MR1 itself . Cellular mechanisms for MR1-dependent presentation of free antigen that originates in the extracellular environment ( left ) differ from those for pathogen-derived antigen generated within the endosomal compartment ( right ). A reduced ability to bind B2M due to either AB loop mutation (baseline in D6) or KD of B2M reduces ER-based presentation of exogenous ligand whereas endosomal presentation in the context of intracellular infection remains functional as long as MR1 protein levels are not limiting. Complete deficiency for B2M abrogates all MR1-mediated antigen presentation. B2M, β2-microglobulin; ER, endoplasmic reticulum; KD, knock down; KO, knock out; MR1, MHC class I-related protein 1; Mtb , Mycobacterium tuberculosis ; OE, overexpressing; WT, wild type.

Article Snippet: PCR products were then cloned into our pCI AscI MR1 res expression vector which features an internal ribosomal entry site GFP (Addgene #214752) ( ) and transformed into NEBalpha competent bacteria (NEB).

Techniques: Binding Assay, Derivative Assay, Infection, Generated, Mutagenesis, Functional Assay, Immunopeptidomics, Knockdown, Knock-Out