Figure 3. Chemotherapy induced increase in SLAMF6⁺PD-1⁺ CD8⁺ T_PEX is not restricted to tumor antigen-specific CD8⁺ T cells. (A) Experiment timeline. BALB/c recipients received CL4xThy1.1 splenocytes 1-day prior to AB1-HA tumor inoculation. Mice were treated with two doses of either PBS or GEM when tumors reached 30–40 mm² in size. Tumors (TUM) and tumor draining lymph nodes (DLN) were harvested for flow cytometry 3 days after the second dose of chemotherapy (+6). (B) Dot plots showing the percentage of Thy1.1⁺ or Thy1.1⁻ SLAMF6⁺PD-1⁺ T_PEX out of CD8⁺ T cells in PBS and GEM treated DLNs (top) and TUM (bottom). (C) Representative flow cytometry plots of SLAMF6 and PD-1 expression on CD8⁺Thy1.1⁺ and CD8⁺Thy1.1⁻ T cells in DLNs (top) and TUM (bottom). (D) Dot plots presenting the frequency of TIGIT, TIM-3 and LAG-3 expression on CD8⁺Thy1.1⁺ (left) and CD8⁺Thy1.1⁻ (right) T cells in DLNs (top) and TUM (bottom). (E) Representative histograms of LAG-3 expression on CD8⁺Thy1.1⁺ (left) and CD8⁺Thy1.1⁻ (right) T_PEX in DLNs and TUM. Data represented as mean±SD. Two-way analysis of variance with Tukey’s multiple comparisons test was used to compare between treatment groups and cell types. Sample sizes n=4 per treatment group. *p<0.05, **p<0.01, ****p≤0.0001. GEM, gemcitabine; LAG-3, lymphocyte activating gene-3; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; SALMF6, SLAM family member 6; TIGIT, T-cell immunoreceptor with immunoglobulin and ITIM domain; TIM-3, T-cell immunoglobulin and mucin-domain containing-3; T_PEX, progenitor exhausted CD8⁺ T cells.