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. 2021 Dec 31;10:e74272. doi: 10.7554/eLife.74272

Figure 5. eIF3 binding to the TCRA and TCRB mRNA 3’-UTR elements is required for a rapid burst in TCR translation and robust activation of primary human T cells.

(A) Western blots of TCRA protein levels in T cells as a function of time after different modes of activation. HSP90 was used as a loading control. (B) Western blots measuring TCRA protein levels as a function of time after anti-CD3/anti-CD28 activation. Cell lines used are labeled on the left: TCRA ΔPAR, TCRB ΔPAR, and SC (scrambled gRNA). HSP90 was used as a loading control. Schematics of TCRA and TCRB mRNAs with and without eIF3 PAR-CLIP sites are shown above. SC control cells have the WT 3’-UTRs for TCRA and TCRB mRNAs. (C) The number of T cells with one or more TCR clusters measured by anti-TCRA/anti-TCRB protein staining and epifluorescence microscopy as a function of time after anti-CD3/anti-CD28 activation. A total of 100 cells from each donor were imaged for TCRA ΔPAR (n = 2 donors, stained with anti-TCRA antibody), TCRB ΔPAR (n = 2 donors, stained with anti-TCRB antibody), and SC cell lines (n = 2 donors, each stained separately with anti-TCRA and anti-TCRB antibodies). Values are mean ± standard deviation. (D) Flow cytometric analysis measuring T cell activation markers CD69 (early activation marker) and CD25 (mid-activation marker), quantifying the mean percent of CD4+ T cells that are CD69+ CD25+. (E) Flow cytometric analysis of CD8+ T cells, quantifying the mean percent of CD8+ T cells that are CD69+ CD25+. Cells sorted as shown in Figure 5—figure supplement 2. (F) Quantification of IL2 secreted from SC, TCRA ΔPAR, TCRB ΔPAR, and ΔTCR cell populations at different time points after stimulation with anti-CD3/anti-CD28 antibodies, as determined by ELISA. (G) Quantification IFNγ secreted from the cells in F, as determined by ELISA. In panels D–G, all experiments were carried out in triplicate (three separate wells per condition), with mean and standard deviations shown. Representative results from one donor are shown.

Figure 5.

Figure 5—figure supplement 1. Generation and analysis of TCRA ΔPAR and TCRB ΔPAR primary human T cells.

Figure 5—figure supplement 1.

(A) CRISPR/Cas9 RNP-mediated genome editing at the TCRA and TCRB genomic loci in primary human T cells. Location of gRNA sites targeting the TCRA 3’-UTR, to generate TCRA ΔPAR cells is highlighted in red. In TCRA, gRNA1 and gRNA2 target hg38 genomic locations chr14:22,551,700 and chr14:22,552,073, respectively. Location of gRNA sites targeting the TCRB 3’-UTR, to generate TCRB ΔPAR cells is highlighted in blue. In TCRB, gRNA1 and gRNA2 target hg38 genomic locations chr7:142,802,561 and chr7:142,802,695, respectively. (B) Top gel: Analysis of TCRA 3’-UTR PAR-CLIP site deletion efficiency. Total genomic DNA extracted from WT, TCRA ΔPAR, TCRB ΔPAR (here serving as a negative control), scrambled gRNA nucleofected (S) and non-nucleofected (N) cells was analyzed by PCR to measure the editing efficiency. TCRA ΔPAR cells produced a 1283 bp PCR product compared to 1676 bp in S, N or TCRB ΔPAR cells (See panel A). Bottom gel: Analysis of TCRB 3’-UTR PAR-CLIP site deletion efficiency. Total genomic DNA extracted from WT, TCRA ΔPAR (here serving as a negative control), TCRB ΔPAR, scrambled gRNA nucleofected (S) and non-nucleofected (N) cells were analyzed by PCR to measure the editing efficiency. TCRB ΔPAR cells produced a 1022 bp PCR product compared to 1190 bp in S, N or TCRA ΔPAR cells (See panel A). The percentage of alleles with PAR-CLIP site deletion, quantified by imageJ. (C) Representative immunofluorescence images used to count TCR clusters in WT, TCRA ΔPAR and TCRB ΔPAR primary human T cells. The white arrows indicate the cells that were scored as having TCR clusters. (D) Percentage of cells expressing TCR on the cell surface of activated T cells measured by flow cytometric analysis. TCR expressing cells in the cell populations tested: N, Non-nucleofected cells; SC, Scrambled gRNA; 1gA, TCRA gRNA 1; 2gA, TCRA gRNA 2; 1 + 2 gA, TCRA gRNA 1 + 2 (i.e. TCRA ΔPAR); 1 gB, TCRB gRNA 1; 2 gB, TCRB gRNA 2; 1 + 2 gB, TCRB gRNA 1 + 2 (i.e. TCRB ΔPAR); ΔTCR, TCR gRNA targeting the CDS of TCRA. (n = 3 replicates from three separate wells, with mean and standard deviation shown.) (E) Representative density plots showing the percentage of TCR on the cell surface in activated T cells. The cell lines shown are: SC, TCRA ΔPAR, TCRB ΔPAR, ΔTCR (negative control). In panels D and E, representative results from one of two donors are shown. (F) Gating strategy for flow cytometric analysis of CD69 and CD25 levels. Primary human T cells were gated to isolate lymphocytes, followed by isolation of single cells, then live cells. Next, cells were gated to separate CD4+ and CD8+ cells expressing T cell activation markers CD69 and CD25. The gates for CD69+ and CD25+ cells were set based on CD69 and CD25 levels in the ΔTCR T cells. Shown is the workflow of the FACS gating, with an example of T cells activated with anti-CD3/anti-CD28 antibodies for 8 hr.
Figure 5—figure supplement 2. Effects of TCRA ΔPAR and TCRB ΔPAR mutations on different steps of CD8+ T cell activation.

Figure 5—figure supplement 2.

Representative density plots showing the percentage of CD8+ T cells expressing CD69 (early activation marker), CD25 (mid-activation marker) or CD69 and CD25 after activation with anti-CD3/anti-CD28 antibodies at different time points, analyzed by flow cytometry. The plots represent one of the two donors. The cell lines shown are SC, TCRA ΔPAR, TCRB ΔPAR, and ΔTCR (negative control). The gating for CD69+ and CD25+ cells was set based on CD69 and CD25 levels in ΔTCR T cells.
Figure 5—figure supplement 3. Effects of TCRA ΔPAR and TCRB ΔPAR mutations on different steps of CD4+ T cell activation.

Figure 5—figure supplement 3.

Representative density plots showing the percentage of CD4+ T cells expressing CD69 (early activation marker), CD25 (mid-activation marker) or CD69 and CD25 after activation with anti-CD3/anti-CD28 antibodies at different time points, analyzed by flow cytometry. The plots represent one of the two donors. The cell lines shown are SC, TCRA ΔPAR, TCRB ΔPAR, and ΔTCR (negative control). The gating for CD69+ and CD25+ cells was set based on CD69 and CD25 levels in ΔTCR T cells.
Figure 5—figure supplement 4. Effects of TCRA ΔPAR and TCRB ΔPAR mutations on different steps of T cell activation.

Figure 5—figure supplement 4.

(A and B) Flow cytometric analysis quantifying the mean percent of A, CD8+ T and B, CD4+ T cells that are only CD69+. (C and D) Flow cytometric analysis quantifying the mean percent of C, CD8+ T and D, CD4+ T cells that are CD69+ CD25+. (E and F) Quantification of E, secreted IL-2 and F, secreted IFNγ, for all cell populations tested, as determined by ELISA. The cell populations tested: N, Non-nucleofected cells; SC, Scrambled gRNA; 1gA, TCRA gRNA 1; 2gA, TCRA gRNA 2; 1 + 2 gA, TCRA gRNA 1 + 2 (i.e. TCRA ΔPAR); 1 gB, TCRB gRNA 1; 2 gB, TCRB gRNA 2; 1 + 2 gB, TCRB gRNA 1 + 2 (i.e. TCRB ΔPAR); ΔTCR gRNA targeting the CDS of TCRA. All experiments were carried out in triplicate (three separate wells per condition), with mean and standard deviations shown. Representative results from one donor are shown.
Figure 5—figure supplement 5. eIF3 binding to the TCRA and TCRB mRNA 3’-UTR elements in Jurkat cells.

Figure 5—figure supplement 5.

(A) Western blots of TCRA protein levels as a function of time after anti-CD3/anti-CD28 activation. Cell lines used: WT Jurkat cells, TCRA ΔPAR and TCRB ΔPAR, Jurkat cells in which the eIF3 PAR-CLIP sites in the 3’-UTRs of the TCRA and TCRB mRNAs, respectively, have been deleted. Schematics of TCRA and TCRB mRNAs with and without eIF3 PAR-CLIP sites are shown above. HSP90 was used as a loading control. Western blots are representative of two biological replicates. (B) TCRA and TCRB mRNA levels in WT Jurkat cells, or Jurkat cells with the TCRA eIF3 3’-UTR PAR-CLIP site deleted, as determined by qRT-PCR at different time points after anti-CD3/anti-CD28 activation. (C) TCRA and TCRB mRNA levels in WT Jurkat cells, or Jurkat cells with the TCRB eIF3 3’-UTR PAR-CLIP site deleted, as determined by qRT-PCR at different time points after anti-CD3/anti-CD28 activation. (D) Schematic of immunoprecipitation of eIF3 using an anti-EIF3B antibody, followed by qRT-PCR to quantify the amount of nanoluciferase mRNA bound to eIF3. (E) Immunoprecipitation of eIF3 as shown in D to quantify the amount of TCRA or TCRB mRNA bound to eIF3 in Jurkat cells after activation with anti-CD3/anti-CD28 antibodies for 5 hours (left) and 8 hr (right). The percent mRNA bound to anti-EIF3B beads is calculated relative to total mRNA isolated from the cells. In panels B–E, all experiments were carried out in triplicate (three separate wells per condition), with mean and standard deviations shown. Representative results from one of two biological replicates are shown.