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. 2021 Sep 2;10:e70055. doi: 10.7554/eLife.70055

Figure 3. Downregulation of protein synthesis in tissue-resident intestinal intraepithelial T lymphocytes (T-IEL).

(a) Box plots showing the estimated total cytoplasmic (left) and mitochondrial (right) ribosomal protein copies for lymph node (LN) TCRβ CD8αβ T cells and all T-IEL subsets. The sums of all copy numbers are available in Figure 3—source data 1. (b) Box plots showing the estimated summed total protein copies for the protein subunits that are exclusive to RNA polymerases I, II, and III, respectively, for LN TCRβ CD8αβ T cells and all T-IEL subsets. The sum of all copy numbers are available in Figure 3—source data 1. (c) Bar plots showing the estimated protein copy numbers of the amino acid transporters, SLC7A5 and SLC38A2, for wild-type (WT) LN TCRβ CD8αβ and all three subsets of T-IEL. (d) Bar plots showing the estimated protein copy numbers of arginase-2 (ARG2; left) and alanine aminotransferase (glutamic-pyruvic transaminase [GPT]; right) for WT LN TCRβ CD8αβ and all three subsets of T-IEL. (e) Bar plots showing the estimated protein copy numbers of PKR-like endoplasmic reticulum kinase (PERK) for WT LN TCRβ CD8αβ and all three T-IEL subsets. (f) Bar plots showing the O-propargyl puromycin (OPP) incorporation (n = 3 biological replicates) in ex vivo WT LN TCRβ CD8αβ and T-IEL. As a negative control, OPP incorporation was inhibited by cycloheximide (CHX) pre-treatment. OPP incorporation was assessed by flow cytometry 15 min after administration. Bar graph represents the geometric MFI of the OPP-AlexaFluor 647 in each T cell subsets normalised to the geometric MFI of the CHX pre-treated T cells. The p-values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparisons. For all box plots, the bottom and top hinges represent the first and third quartiles. The top whisker extends from the hinge to the largest value no further than 1.5× IQR from the hinge; the bottom whisker extends from the hinge to the smallest value at most 1.5× IQR of the hinge. All bar plots show the mean and SEM. Symbols on the bars represent the biological replicates. The proteomics data displayed on the plots include WT TCRαβ CD8αβ T cells (n = 6 biological replicates), TCRαβ CD8αβ T-IEL and TCRαβ CD8αα T-IEL (n = 3 biological replicates), and TCRγδ CD8αα T-IEL (n = 4 biological replicates). The p-values for individual proteins (c,d,e) were calculated in R with LIMMA, where ** = p < 0.001 and fold change greater than or equal to the median+1 standard deviation, *** = p < 0.0001 and fold change greater than or equal to the median±1.5 standard deviations, and in (a,b) in R with Welch’s t-test.

Figure 3—source data 1. Sum of median copy numbers for the cytoplasmic and mitochondrial ribosomes.

Figure 3.

Figure 3—figure supplement 1. Selective regulation of RNA polymerases and the urea cycle in T-IEL.

Figure 3—figure supplement 1.

(a) Box plots showing the median Log2 fold change ((tissue-resident intestinal intraepithelial T lymphocytes/lymph node [T-IEL/LN]) CD8 T cells) for RNA polymerase 1, 2, and 3 complexes. Each grey dot represents one of the polymerase subunits. The p-values were calculated using one-sample t-tests, where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant. (b) Schematic representation of the urea cycle. Coloured heatmap squares represent protein expression Log2 fold change (T-IEL/LN CD8 T cells) in, from left to right, T-IEL TCRβ CD8αβ, T-IEL TCRβ CD8αα, and T-IEL TCRγδ CD8αα. Proteins expressed only by T-IEL are highlighted by red squares, representing estimated protein copy numbers (mean from at least three biological replicates). For protein names, see Supplementary file 4.