CD4⁺ T cell populations in cancer septic animals exhibit increased coinhibitory receptor expression relative to previously healthy septic controls.

<p>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.</p

Increased frequencies and absolute numbers of effector memory CD4⁺ T cells in cancer septic animals relative to previously healthy septic controls.

<p>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.</p

CD4⁺ T cell populations in cancer septic hosts are characterized by the increased prevalence of two distinct populations of cells.

<p>Traditional flow cytometry data generated from cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191065#pone.0191065.g001" target="_blank">Fig 1</a> 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.</p

SPADE analysis identifies three primary clusters that distinguish CD4⁺ T cells from cancer septic mice as compared to previously healthy controls.

<p>Traditional flow cytometry data generated from cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191065#pone.0191065.g001" target="_blank">Fig 1</a> 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.</p

CD4⁺ T cells are increased in cancer septic mice as compared to previously healthy controls.

<p>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.</p

CD4⁺ T cell populations in cancer septic animals exhibit increased TNF secretion relative to previously healthy septic controls.

<p>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.</p

Cancer sepsis impacts CD4⁺ T cell proliferation but not glucose uptake.

<p>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.</p

Effect of impaired intestinal lipid transport on serum lipoproteins.

<p>Lipoprotein distribution measured by fast protein liquid chromatography in control (A) and <i>Mttp-IKO</i> (B) mice. Pooled samples of serum from n = 5–10 mice per genotype were analyzed in sham animals and both before and 24 hr after induction of pneumonia in the experimental groups. Cholesterol was assayed enzymatically and peaks corresponding to fractions 10–16 indicate particles in the low density lipoprotein (LDL) range while fractions 19–26 correspond to high density lipoprotein (HDL). (C) Expression of genes implicated in hepatic cholesterol efflux were analyzed by qRT-PCR on samples of RNA from the indicated groups (n = 4 mice per genotype and treatment) (D) and (E). Expression of hepatic Abca1, apoA1 and apoA4 protein by SDS-PAGE and western blot. Gapdh was used as loading control. Panel D shows representative Western blotting results. Panel E shows densitometric scanning from groups of 4 mice per genotype and treatment. (F) Expression of genes implicated in intestinal lipid metabolism were analyzed by qRT-PCR on samples of small intestinal RNA from the indicated groups (n = 4 mice per genotype and treatment). *p<0.05.</p

Effect of impaired intestinal lipid transport on lung cytokines.

<p>Cytokines were measured in bronchoalveolar lavage (BAL) fluid 24 hr after induction of pneumonia. Although several cytokines were elevated during pneumonia, there were no differences between septic <i>Mttp-IKO</i> and control mice. n = 8–10/group.</p

Effect of impaired intestinal lipid transport on intestinal epithelial apoptosis.

<p>Intestinal epithelial apoptosis was evaluated by active caspase-3 staining (A) and H&E staining (B) in 100 crypts. Control mice subjected to <i>P. aeruginosa</i> pneumonia exhibited increased intestinal apoptosis by both methods. In contrast, <i>Mttp-IKO</i> mice with pneumonia had similar levels of intestinal apoptosis as sham mice.n = 6–7 shams/genotype, n = 16–18 septics/genotype. The gene expression ratios of pro-apoptotic Bax to anti-apoptotic Bcl-2 (C) and Bcl-xL (D) were evaluated. Septic <i>Mttp-IKO</i> mice had significantly decreased ratios compared to septic control mice. n = 11/group.</p