Figure 3
From: Alternative splicing of MR1 regulates antigen presentation to MAIT cells
MR1B inhibits T cell activation by MR1A. Wild type Beas2B cells were transduced with a lentivirus targeting MR1 using CRISPR/Cas9 gene editing to generate a Beas2B MR1 knockout cell line (Beas2B:MR1_KO). (A) Surface expression of MR1A assessed using flow cytometry of antibody staining to MR1A (α-MR1, 26.5) following exposure of wt Beas2B and Beas2B:MR1_KO cells to 6FP overnight. (B) wt Beas2B and Beas2B:MR1_KO cells were treated with either M.smegmatis supernatant (left), pronase treated Mtb cell wall (middle), or CFP102-9 and (right) utilized to stimulate either a MR1-restricted T cell clone (left), an HLA-B45 restricted T cell clone (middle), or a HLA-E restricted T cell clone (right). IFN-γ production is measured by ELISpot and reported as IFN-γ spot forming units/5,000 T cells (IFN-γ SFU). Error bars represent mean and standard deviation of the mean from duplicate wells. Data are representative of > 3 independent experiments. (C) A549:MR1_KO and Beas2B:MR1_KO cells were transfected with plasmids encoding either MR1AGFP, MR1BRFP or MR1CRFP, or a pCI empty vector. Cells were infected overnight with M. smegmatis at a multiplicity of infection (MOI) of 3 and utilized as antigen presenting cells to stimulate MAIT production of IFN-γ in an ELISpot as described in (B). (z) Beas2B:MR1_KO cells were transfected as described and MR1BRFP or MR1CRFP expression was measured by detection of total RFP by flow cytometry. (E) A549:MR1_KO or Beas2B:MR1_KO cells were cotransfected with plasmids encoding MR1AGFP along with either the pCI empty vector, MR1BRFP, or MR1CRFP. (Left, Middle) Cells were infected for 1 h with M. smegmatis and used as in (B). MOI is indicated on x-axis. Data are pooled from at least 3 independent experiments with duplicate wells and error bars represent mean and error from n = 6 replicates. (Right) Beas2B:MR1_KO cells were infected with BCG overnight at MOI of 15, and used to stimulate IFN-γ production by MAIT cells. Number of infected APCs used are indicated on x-axis. Data are pooled from at least 2 independent experiments with duplicate wells, and error bars represent mean and error of n = 4 replicates from 2 independent experiments. (F) Beas2B overexpressing MR1AGFP were transiently transfected with either the pCI empty vector or pCI_MR1BRFP. Flow cytometric analysis was performed to quantify total MR1A expression, as measured by detection of GFP, following transfection. Data are representative of > 2 independent experiments.
