PMC

CD4⁺ T cells isolated from splenocytes of previously healthy or cancer septic animals were analyzed for coinhibitory receptor expression by flow cytometry. A, Representative flow plots of LAG-3, BTLA, 2B4, and PD-1 expression. Frequencies (B) and absolute numbers (C) of LAG-3⁺, BTLA⁺, 2B4⁺, and PD-1⁺ CD4⁺ T cells from previously healthy vs. cancer septic animals. D, Representative flow plots of CD127 expression. MFI of CD127 expression on CD4⁺ T cells from previously healthy vs. cancer septic animals. n = 11/group. ***p<0.001, *p<0.05.

CD4⁺ T cells isolated from splenocytes of previously healthy or cancer septic animals were analyzed by flow cytometry. A, Representative flow plots of CD44 and CD62L expression. Frequencies (B) and absolute numbers (C) of naïve, central memory, and effector memory T cells from previously healthy vs. cancer septic animals. D, Representative flow plots of CD69 and CD25 expression. Frequencies (E) and absolute numbers (F) of CD25⁺ and CD69⁺ CD4⁺ T cells from previously healthy vs. cancer septic animals. n = 11/group. ***p<0.001.

CD4⁺ T cells isolated from splenocytes of previously healthy or cancer septic animals were A) incubated for 30 min ex vivo with fluorescently labeled 2-NBDG as described in Materials and Methods or B) stained intracellularly for Ki-67 as described in Materials and Methods. A, Representative flow plots of 2-NBDG uptake in CD4⁺ T cells cells isolated from previously healthy (blue histograms) vs. cancer septic (red histograms) mice. B, Summary data of frequencies of 2-NBDG⁺ CD4⁺ T cells. (C) Representative flow plots and D) summary data of Ki-67 staining in CD4⁺ T cells populations isolated from previously healthy vs. cancer septic mice. Summary data tabulated from n = 5/group. **p<0.01.

Mice were injected with LLC tumor cells subcutaneously in the inner thigh as described in Materials and Methods. Tumors were allowed to grow for 3 weeks, at which time cancer mice and previously healthy age-matched controls were subjected to CLP (A). 24 h later, splenocytes were harvested and frequencies (B, C) and absolute numbers (D) of CD4+ T cells were assessed by flow cytometry. n = 11/group. ***p<0.001. PH = previously healthy septic. CA = cancer septic.

CD4⁺ T cells isolated from splenocytes of previously healthy or cancer septic animals were restimulated ex vivo with PMA/ionomycin as described in materials and methods and stained intracellularly for IL-2, TNF, and IFN-γ. A, Representative flow plots of IL-2 and TNF expression in unstimulated (top panels) and stimulated (bottom panels) cells isolated from previously healthy (left panels) vs. cancer septic (right panels) mice. B-D, Summary data of frequencies of IL-2⁺ (B), TNF⁺ (C), and IFN-γ⁺ (D) CD4⁺ T cells from previously healthy (PH) vs. cancer (CA) septic animals. n = 11/group. **p<0.01.

Traditional flow cytometry data generated from cells in were manually gated in FlowJo on CD3⁺CD4⁺ lymphocytes and new FCS files were created containing only these gated events. The pre-gated data files were uploaded to Cytobank and SPADE trees were generated using CD44, CD62L, CD69, CD25, and CXCR4 as the clustering channels. A, Representative SPADE trees are shown. Fold Change Groups was used to make comparisons with the data files from previously healthy animals set as the baseline samples. SPADE trees depicted are colored by the parameter “percent total ratio log” or log₁₀ (percent of total sample/average percent of total baseline). The percent of total CD4⁺ cells in each node was compared between previously healthy and cancer septic animals, and phenotypically similar nodes demonstrating significant differences between previously healthy and cancer animals were grouped into clusters for further analysis. B. Frequencies of total CD4⁺ T cells falling into each Cluster within each experimental group are depicted. n = 6/group, representative of two independent experiments with a total of n = 11/group. C, The expression patterns of CD44, CD62L, CD69, and CD25 within each Cluster are depicted.

Traditional flow cytometry data generated from cells in were manually gated in FlowJo on CD3⁺CD4⁺ lymphocytes and new FCS files were created containing only these gated events. The pre-gated data files were uploaded to Cytobank and SPADE trees were generated using CD44, LAG-3, BTLA, 2B4, PD-1, and CD127 as the clustering channels. A, Representative SPADE trees are shown. Fold Change Groups was used to make comparisons with the data files from previously healthy animals set as the baseline samples. SPADE trees depicted are colored by the parameter “percent total ratio log” or log₁₀ (percent of total sample/average percent of total baseline). The percent of total CD4⁺ cells in each node was compared between previously healthy and cancer septic animals, and phenotypically similar nodes demonstrating significant differences between previously healthy and cancer animals were grouped into clusters for further analysis. B. Frequencies of total CD4⁺ T cells falling into each Cluster within each experimental group are depicted. n = 6/group, representative of two independent experiments with a total of n = 11/group. C, The expression patterns of CD44, LAG-3, BTLA, 2B4, PD-1, and CD127 within each Cluster are depicted. D, Summary data of MFI of CD44, LAG-3, BTLA, 2B4, PD-1, and CD127 on the cells in each Cluster for n = 6 mice/group are shown. **p<0.01.

Traditional flow cytometry data generated from cells in were manually gated in FlowJo on CD3⁺CD4⁺ lymphocytes and new FCS files were created containing only these gated events. The pre-gated data files were uploaded to Cytobank and SPADE trees were generated using TNF, IL-2, IFN-γ, BTLA, 2B4, and PD-1 as the clustering channels. A, Representative SPADE trees are shown. Fold Change Groups was used to make comparisons with the data files from previously healthy animals set as the baseline samples. SPADE trees depicted are colored by the parameter “percent total ratio log” or log₁₀ (percent of total sample/average percent of total baseline). The percent of total CD4⁺ cells in each node was compared between previously healthy and cancer septic animals, and phenotypically similar nodes demonstrating significant differences between previously healthy and cancer animals were analyzed further. B. Frequencies of total CD4⁺ T cells falling into each Node within each experimental group are depicted. n = 6/group, representative of two independent experiments with a total of n = 11/group. C, The expression patterns of TNF, IL-2, IFN-γ, BTLA, 2B4, and PD-1 within each Node are depicted. D, Summary data of MFI of TNF, IL-2, IFN-γ, BTLA, 2B4, and PD-1 on the cells in each Node for n = 6 mice/group are shown. The MFIs of cells positive for each parameter (derived from other nodes) are also depicted as positive controls.