Characterization of the CD8 ⁺ FCRL3 ⁺ T cell subset. (A) Differentially expressed genes in FCRL3⁺ versus FCRL3⁻ memory CD8⁺ T cells sorted from the peripheral blood of N = 5 healthy donors and analyzed by RNA-seq (FDR ≤ 0.05 and log₂FC ≥|0.5|). (B) RNA-seq tracks for selected transcripts enriched in FCRL3⁺ or FCRL3⁻ cells. (C) Differentially expressed proteins in sorted CD8⁺ FCRL3⁺ versus CD8⁺ FCRL3⁻ memory T cells, measured by shotgun mass spectrometry (log₂FC ≥ |0.5|; FDR [Benjamini and Hochberg multiple t test correction] ≤0.05; N = 7 independent donors). (D) Heatmap showing the differential expression of selected proteins from C. (E and F) TOX (E) and EOMES (F) intracellular staining in sorted FCRL3⁺ and FCRL3⁻ memory T cells. One representative experiment and the results from N = 8 (TOX) or N = 6 (EOMES) independent donors are shown; each dot represents one donor. Paired t test, two-tailed. (E) ****P < 0.0001, (F) *P = 0.0352. (G) Granzyme B (left) and perforin (right) expression in sorted CD8⁺ FCRL3⁺ and CD8⁺ FCRL3⁻ memory T cells as determined by intracellular staining. The histograms show the results from different donors (N = 7 for granzyme and N = 3 for perforin). Each dot represents one donor. Mean ± SD; paired t test, two-tailed. From left to right: **P = 0.0018, *P = 0.0182. (H) Cytotoxicity of CD8⁺ FCRL3⁺ and FCRL3⁻ memory T cells. CD8⁺ FCRL3⁺ and FCRL3⁻ memory T cells were sorted and cocultured with the JeKo B cell line preincubated with the bispecific antibody blinatumomab. JeKo cell viability was then monitored with Live/Dead staining at the indicated time points. The FACS plots on the left are representative of the 8-h time point. The graph on the right shows the percentage of live JeKo cells normalized at time 0 h. N = 4–9 biological replicates (independent donors), mean ± SD, paired t test. From left to right: *P = 0.0260, *P = 0.0185. (I) Degranulation assay in sorted FCRL3⁺ and FCRL3⁻ memory CD8⁺ T cells. T cells were stimulated for 5 h with PMA and ionomycin, and surface CD107A (LAMP-1) expression was measured. One representative experiment and the results from N = 4 independent experiments are shown. Each dot represents one donor. Paired t test, two-tailed. *P = 0.0408. FC, fold change. Data underlying this figure can be found in Data S4.