Figure 2.
FCRL3 expression is sufficient to modulate T cell activation. (A) Schematic representation of the experimental setup for repetitive T cell stimulation. Memory CD8+ T cells isolated from peripheral blood were activated on plate-bound anti-CD3 antibody with or without costimulation with an anti-CD28 antibody or restimulation. (B and C) Surface expression of FCRL3 measured 5 days after activation with anti-CD3/CD28 or with two consecutive stimulations (day 0 and day 3) with anti-CD3 antibody. One representative donor (B) and N = 8 independent experiments (C) are shown. Each dot represents one donor. Mean; paired t test, two-tailed. *P = 0.0147. (D) Schematic representation of the FCRL3 locus, with the exons (4 and 7) targeted by the selected sgRNAs. Only the exons targeted by the sgRNAs are shown. (E) Experimental workflow for FCRL3 CRISPR-KO in FCRL3+ CD8+ T cells. (F–H) FCRL3 (F), CD25 (G), and CD137 (H) expression on CD8+ FCRL3+ cells, transfected with RNPs containing sgRNAs targeting the FCRL3 gene, or control. Surface expression was monitored 10 days after reactivation with anti-CD3. N = 6–8 independent experiments. Mean; paired t test, two-tailed. From left to right: (F) ***P = 0.001, (G) *P = 0.0145, (H) *P = 0.0171. (I) Experimental workflow for the ectopic expression of FCRL3 in CD8+ T cells, by lentiviral transduction. After activation with plate-bound anti-CD3/CD28 and transduction, transduced cells are selected by puromycin treatment, followed by recovery and surface staining for FCRL3 and AIMs. (J) Surface expression of FCRL3 in memory CD8+ T cells transduced with FCRL3-encoding lentivirus or empty lentivirus as a control. One representative experiment of N = 4. (K) Surface CD25 expression in FCRL3-transduced memory CD8+ T cells, 6 and 10 days after the initial activation. The left panel shows the result for one representative donors, while the bar graphs represent the results for N = 4 independent donors at day 10. The gMFI and percentage of CD25+ cells are both shown. Mean ± SD; paired t test, two-tailed. *P = 0.0306, **P = 0.0076. Data underlying this figure can be found in Data S4.

FCRL3 expression is sufficient to modulate T cell activation. (A) Schematic representation of the experimental setup for repetitive T cell stimulation. Memory CD8+ T cells isolated from peripheral blood were activated on plate-bound anti-CD3 antibody with or without costimulation with an anti-CD28 antibody or restimulation. (B and C) Surface expression of FCRL3 measured 5 days after activation with anti-CD3/CD28 or with two consecutive stimulations (day 0 and day 3) with anti-CD3 antibody. One representative donor (B) and N = 8 independent experiments (C) are shown. Each dot represents one donor. Mean; paired t test, two-tailed. *P = 0.0147. (D) Schematic representation of the FCRL3 locus, with the exons (4 and 7) targeted by the selected sgRNAs. Only the exons targeted by the sgRNAs are shown. (E) Experimental workflow for FCRL3 CRISPR-KO in FCRL3+ CD8+ T cells. (F–H) FCRL3 (F), CD25 (G), and CD137 (H) expression on CD8+ FCRL3+ cells, transfected with RNPs containing sgRNAs targeting the FCRL3 gene, or control. Surface expression was monitored 10 days after reactivation with anti-CD3. N = 6–8 independent experiments. Mean; paired t test, two-tailed. From left to right: (F) ***P = 0.001, (G) *P = 0.0145, (H) *P = 0.0171. (I) Experimental workflow for the ectopic expression of FCRL3 in CD8+ T cells, by lentiviral transduction. After activation with plate-bound anti-CD3/CD28 and transduction, transduced cells are selected by puromycin treatment, followed by recovery and surface staining for FCRL3 and AIMs. (J) Surface expression of FCRL3 in memory CD8+ T cells transduced with FCRL3-encoding lentivirus or empty lentivirus as a control. One representative experiment of N = 4. (K) Surface CD25 expression in FCRL3-transduced memory CD8+ T cells, 6 and 10 days after the initial activation. The left panel shows the result for one representative donors, while the bar graphs represent the results for N = 4 independent donors at day 10. The gMFI and percentage of CD25+ cells are both shown. Mean ± SD; paired t test, two-tailed. *P = 0.0306, **P = 0.0076. Data underlying this figure can be found in Data S4.

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