Stressful presentations: mild cold stress in laboratory mice influences phenotype of dendritic cells in naïve and tumor-bearing mice

Abstract

The ability of dendritic cells (DCs) to stimulate and regulate T cells is critical to effective anti-tumor immunity. Therefore, it is important to fully recognize any inherent factors which may influence DC function under experimental conditions, especially in laboratory mice since they are used so heavily to model immune responses. The goals of this report are to 1) briefly summarize previous work revealing how DCs respond to various forms of physiological stress and 2) to present new data highlighting the potential for chronic mild cold stress inherent to mice housed at the required standard ambient temperatures to influence baseline DCs properties in naïve and tumor-bearing mice. As recent data from our group shows that CD8(+) T cell function is significantly altered by chronic mild cold stress and since DC function is crucial for CD8(+) T cell activation, we wondered whether housing temperature may also be influencing DC function. Here we report that there are several significant phenotypical and functional differences among DC subsets in naïve and tumor-bearing mice housed at either standard housing temperature or at a thermoneutral ambient temperature, which significantly reduces the extent of cold stress. The new data presented here strongly suggests that, by itself, the housing temperature of mice can affect fundamental properties and functions of DCs. Therefore differences in basal levels of stress due to housing should be taken into consideration when interpreting experiments designed to evaluate the impact of additional variables, including other stressors on DC function.

Keywords: anti-tumor immunity; cold stress; mouse models of cancer; norepinephrine; thermoregulation.

Figure 1

Splenocytes, tumor size, and body weight are increased when mice are maintained at ST compared to TT. 4T1 tumor-bearing BALB/c mice and age-matched controls were maintained at ST or TT. (A) Splenocytes obtained from control and tumor-bearing mice were counted and (B) tumor weight and (C) volume were measured. Data presented as mean ± SEM; n = 5/group; Student’s t-test; *p < 0.05, **p < 0.01. (D) Change in weight from the start of the experiment was measured. - - - - indicates day of tumor inoculation. Data presented as mean ± SEM; n = 5/group; two-way ANOVA with Bonferroni post-tests; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Splenic myeloid cells are increased in tumor-bearing mice maintained at ST compared to TT. Single cell suspensions of splenocytes from 4T1 tumor-bearing mice and age-matched controls were stained for CD11b and analyzed by flow cytometry. (A) Representative dot plots from each group show the gating strategy used to select CD11b⁺ cells. Percentage of cells are shown above their respective gate. (B) The absolute number of CD11b⁺ cells calculated from the total number of splenocytes counted in each individual mouse. (C) The percentage of CD11b⁺ cells of the total population of live cells as determined by DAPI staining. Data presented as mean ± SEM; n = 5/group; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Tumor-bearing mice maintained at ST have an increased frequency of DCs compared to those at TT. Single cell suspensions of splenocytes from 4T1 tumor-bearing mice and age-matched controls were stained for CD11c and (D–H) B220 and analyzed by flow cytometry. (A) Representative dot plots from each group show the gating strategy used to select CD11c⁺ cells. Percentage of cells are shown above their respective gate. (B) The absolute number of CD11c⁺ cells calculated from the total number of splenocytes counted in each individual mouse. (C) The percentage of CD11c⁺ cells of the total population of live cells as determined by DAPI staining. (D) Representative dot plots from each group show the gating strategy used to select B220⁺CD11c⁺ and B220⁻CD11c⁺ cells. Percentage of cells are shown above their respective gate. (E) The absolute number of B220⁺CD11c⁺ cells calculated from the total number of splenocytes counted in each individual mouse. (F) The percentage of B220⁺CD11c⁺ cells of the total population of live cells as determined by DAPI staining. (G) The absolute number of B220⁻CD11c⁺ cells calculated from the total number of splenocytes counted in each individual mouse. (H) The percentage of B220⁻CD11c⁺ cells of the total population of live cells as determined by DAPI staining. Data presented as mean ± SEM; n = 5/group; Student’s t-test; *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 4

Tumor-bearing mice maintained at ST have proportionally more non-plasmacytoid DCs than those at TT, but these DCs primarily display an immature phenotype. Single cell suspensions of splenocytes from 4T1 tumor-bearing mice and age-matched controls were stained for CD8α, CD4, CD11c, MHCII, CD86 and analyzed by flow cytometry. (B–G) Quantification of data describing CD8α⁺ non-plasmacytoid DCs. (A) Representative dot plots from each group show the gating strategy used to select CD4 and CD8α cells from the non-plasmacytoid parent population shown in Figure 3D. Percentage of cells are shown above their respective gate. (B) The absolute number of CD8α⁺MHC II⁻CD86⁻ B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (C) The percentage of MHC II⁻CD86⁻ cells of the total population of CD8α⁺ non-plasmacytoid cells. (D) The absolute number of CD8α⁺MHC II⁺CD86⁻B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (E) The percentage of MHC II⁺CD86⁻ cells of the total population of CD8α⁺ non-plasmacytoid cells. (F) The absolute number of CD8α⁺MHC II⁺CD86⁺B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (G) The percentage of MHC II⁺CD86⁺ cells of the total population of CD8α⁺ non-plasmacytoid cells. (H–M) Quantification of data describing CD4⁺ non-plasmacytoid DCs. (H) The absolute number of CD4⁺MHC II⁻CD86⁻B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (I) The percentage of MHC II⁻CD86⁻ cells of the total population of CD4⁺ non-plasmacytoid cells. (J) The absolute number of CD4⁺MHC II⁺CD86⁻B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (K) The percentage of MHC II⁺CD86⁻ cells of the total population of CD4⁺ non-plasmacytoid cells. (L) The absolute number of CD4⁺MHC II⁺CD86⁺B220⁻CD11c⁺ cells calculated from the total number of non-plasmacytoid cells. (M) The percentage of MHC II⁺CD86⁺ cells of the total population of CD4⁺ non-plasmacytoid cells. Data presented as mean ± SEM; n = 5/group; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

T cells are activated better by splenocytes from mice at TT than ST. Total splenocytes from tumor-free and tumor-bearing BALB/c mice and lymphocytes from C57BL/6 mice were cultured 1:2. T cell proliferation was measured by ³H-thymidine incorporation. Data presented as mean ± SEM; n = 5/group; Student’s t-test; *p < 0.05, **p < 0.01,***p < 0.001.