Figure 5.
Figure 5. Identification of a novel recurrent, gain-of-function RLTPR mutation (p.Q575E) in CTCL. (A) Schematic of the recurrent RLTPR (p.Q575E) amino acid alteration. CBR, capping protein binding region; HD, helical domain; PH, pleckstrin homology; Pro, proline-rich region. (B) Confirmation of the RLTPR mutation by Sanger sequencing. Chromatogram of Sanger sequencing of genomic DNA from CTCL tumor cells (upper) and matched germline controls (ie, monocytes; lower). (C) Protein structure of the RLTPR homolog, CARMIL, showing the location of the p.Q575E amino acid alteration. CARMIL harbors 38% identity more than 670 amino acids when aligned to RLTPR, and p.Q575 is conserved between the 2 proteins. (PDB ID: 4K17).36 (D) Expression of RLTPR in Jurkat cells nucleofected with siCTRL or siRLTPR. Immunoblot analysis with RLTPR antibody, and β-actin as the loading control. (E) Downregulation of RLTPR inhibits TCR signaling in Jurkat cells. IL-2 mRNA expression in Jurkat cells nucleofected with siCTRL or siRLTPR in the presence of pharmacological TCR mimics (PMA/ionomycin) and a CD28 ligand, CD86. Data are shown as mean ± standard error from 4 independent biological replicates. P value was determined by 2-sided paired ratio t test (*P < .05; **P < .01). (F) Expression of RLTPR in isolated CD4+ T cells nucleofected with siCTRL or siRLTPR. (G) Downregulation of RLTPR inhibits TCR signaling in CD4+ T cells. IL-2 mRNA expression in CD4+ T cells nucleofected with siCTRL or siRTLPR in the presence of anti-CD3/anti-CD28 activation beads. Data are shown as mean ± standard error from 7 independent biological replicates from peripheral blood mononuclear cells of 2 different healthy donors. P value was determined by 2-sided paired ratio t test (***P < .001). (H) The RLTPR mutation significantly increases TCR signaling. Jurkat cells were transduced with RLTPR WT or RLTPR p.Q575E. These cells were treated with vehicle control or PMA (50 ng/mL)/ionomycin (300 ng/mL) and CD86 at the indicated concentrations. Data represent as mean ± standard error from 6 independent experiments. P value was determined by 2-sided paired ratio t test. *P < .05; ***P < .001.

Identification of a novel recurrent, gain-of-function RLTPR mutation (p.Q575E) in CTCL. (A) Schematic of the recurrent RLTPR (p.Q575E) amino acid alteration. CBR, capping protein binding region; HD, helical domain; PH, pleckstrin homology; Pro, proline-rich region. (B) Confirmation of the RLTPR mutation by Sanger sequencing. Chromatogram of Sanger sequencing of genomic DNA from CTCL tumor cells (upper) and matched germline controls (ie, monocytes; lower). (C) Protein structure of the RLTPR homolog, CARMIL, showing the location of the p.Q575E amino acid alteration. CARMIL harbors 38% identity more than 670 amino acids when aligned to RLTPR, and p.Q575 is conserved between the 2 proteins. (PDB ID: 4K17).36  (D) Expression of RLTPR in Jurkat cells nucleofected with siCTRL or siRLTPR. Immunoblot analysis with RLTPR antibody, and β-actin as the loading control. (E) Downregulation of RLTPR inhibits TCR signaling in Jurkat cells. IL-2 mRNA expression in Jurkat cells nucleofected with siCTRL or siRLTPR in the presence of pharmacological TCR mimics (PMA/ionomycin) and a CD28 ligand, CD86. Data are shown as mean ± standard error from 4 independent biological replicates. P value was determined by 2-sided paired ratio t test (*P < .05; **P < .01). (F) Expression of RLTPR in isolated CD4+ T cells nucleofected with siCTRL or siRLTPR. (G) Downregulation of RLTPR inhibits TCR signaling in CD4+ T cells. IL-2 mRNA expression in CD4+ T cells nucleofected with siCTRL or siRTLPR in the presence of anti-CD3/anti-CD28 activation beads. Data are shown as mean ± standard error from 7 independent biological replicates from peripheral blood mononuclear cells of 2 different healthy donors. P value was determined by 2-sided paired ratio t test (***P < .001). (H) The RLTPR mutation significantly increases TCR signaling. Jurkat cells were transduced with RLTPR WT or RLTPR p.Q575E. These cells were treated with vehicle control or PMA (50 ng/mL)/ionomycin (300 ng/mL) and CD86 at the indicated concentrations. Data represent as mean ± standard error from 6 independent experiments. P value was determined by 2-sided paired ratio t test. *P < .05; ***P < .001.

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