Antigen-specific T cell cytokine production in control and cancer mice following intracellular cytokine stimulation.

<p>CD8⁺Thy1.1⁺ T cells in the control and cancer groups were stimulated with SIINFEKL peptide. No statistically significant differences were noted in intracellular production of IFN-γ or IL-2; n = 8–10.</p

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

<div><p>While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with <i>Listeria monocytogenes</i>, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.</p></div

Correlation of coinhibitory receptor expression to antigen-specific cytokine production.

<p>A) The frequency of PD-1, 2B4, and BTLA expression was compared to the frequency of cells producing IFN-γ. Increased inhibitory receptor expression is correlated with reduced cytokine production. n = 8–10 (PD-1 and BTLA) and 4–5 (2B4). B) The frequency of PD-1, 2B4, and BTLA expression was compared to the frequency of cells producing IL-2. Increased inhibitory receptor expression is correlated with increased cytokine production; n = 8–10 (PD-1 and BTLA) and 4–5 (2B4).</p

Effect of cancer and LM-OVA infection on coinhibitory receptor expression.

<p>A) Data presented is following selection of the CD8⁺Thy1.1⁺ T cell population. CD8⁺Thy1.1⁺ T cells in cancer group had increased mean frequencies of BTLA⁺ T cells. They were 8.58±1.07% in control vs. 20.72±5.58% in cancer mice following infection (*p<0.05; n = 8–10) at day 5. Mean frequencies of 2B4 (n = 4–5) and PD-1 (n = 8–10) had a trend toward increased expression in the cancer group, but were not statistically significant. B) In the absence of infection (day 0), the control and cancer groups did not have any significant differences in the expression or co-expression of 2B4, BTLA, and PD-1 on CD4⁺ T cells. At day 5, however, CD4⁺ T cells in the cancer group expressed a higher frequency of BTLA (4.03±0.46 vs. 6.77±0.59) and 2B4 (2.67±0.22 vs. 3.95±0.25). The inhibitory profile was further altered when looking at co-expression. The cancer group had higher expression of PD-1⁺BTLA⁺ (3.76±0.47 vs. 5.65±0.42) and 2B4⁺PD-1⁺ (2.50±0.23 vs. 3.76±0.26) CD4⁺ T cells. Following resolution of infection at day 14, the expression of 2B4 (n = 4–5), 2B4⁺PD-1⁺ (n = 4–5), or PD-1 was no longer different in both groups, however, the frequency of expression CD4⁺ PD-1⁺BTLA⁺ T cells remained elevated in the cancer group (3.99±0.44 vs. 5.56±0.48). **p<0.01; *p<0.05; n = 8–10 at days 0, 5, and 14, unless otherwise stated. C) At baseline (day 0), cancer mice had reduced expression of CD8⁺PD-1⁺ T cells compared to control mice (14.03±0.61 in control vs. 11.91±0.47 in cancer). However, expression of 2B4, BTLA, 2B4⁺PD-1⁺ or PD-1⁺BTLA⁺ was unchanged prior to infection. At day 5 following infection, cancer mice had increased frequencies of expression of CD8⁺BTLA⁺ (8.78±0.95 vs. 13.66±0.64) and CD8⁺PD-1⁺BTLA⁺ (6.55±0.83 vs. 9.22±0.47) T cells. Following resolution of infection at day 14, only the mean frequency of BTLA expression in the cancer group remained elevated (6.72±1.40 vs. 8.76±2.03); **p<0.01; *p<0.05; n = 8–10.</p

Experimental design and bacterial antigen-specific immune responses.

<p>A) C57BL/6 mice were randomized to control vs. cancer groups. The cancer group received a subcutaneous injection of Pan02 cells. Following 3 weeks, all mice were given an IV injection of transgenic (Tg) T cell receptor (TCR) CD8⁺Thy1.1⁺ T cells. After 24 hours, uninfected mice were sacrificed for the day 0 time point, and all other mice were given an intraperitoneal injection of LM-OVA and underwent spleen collections at days 5 and 14 post-infection. B) At days 5 or 14 following infection, there were no significant differences in antigen-specific CD8+T cells expansion between the control and cancer groups. The mean frequencies in control vs. cancer were 6.97±1.49% vs. 6.89±2.49% at day 5, respectively and 5.38±0.73% vs. 4.90±1.19% at day 14, respectively; n = 8–10 at all time points.</p