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

IL-2 (but not IFN-γ or TNF) production by CD4+ T cells is decreased 72h following CLP in alcohol-fed animals.

<p>Representative flow plot and summary data 72h following sepsis, demonstrating a trend toward decreased IL-2 production due to alcohol alone between sham groups which did not reach significance, but there was a significant decrease in IL-2 in alcohol septic animals compared with water septic (16.15±1.7% vs 26.7±1.74%, p = 0.004). There were no differences in the frequencies of IFN-γ or TNF- producing CD4+ T cells between any of the groups. n = 7-12/group.</p

Alcohol delays the kinetics of CD69 expression of naïve CD4+ T cells and prolongs CD69 expression on memory CD4+ T cells.

<p>A) At 24h, CD69 expression increased in the water septic group over water sham (11.6±1% vs 8.6±0.6%, p = 0.01). At 72h, CD69 increased due to sepsis in both water and alcohol-fed groups (H2O sham 10.1±0.9% vs H2O CLP 19.9±1.4%, p = 0.04; EtOH sham 9.4±1% vs EtOH CLP 23.6±1.7%, p = 0.004). B) In naïve CD4s at 24h, the water septic group exhibited increase in CD69 expression (10.2±1.1% vs 5.6±1.0%, p = 0.04), while the alcohol fed group did not. By 72h, both alcohol and water fed groups exhibit CD69 upregulation in sepsis (H2O sham 4.4±0.5% vs H2O CLP 10.9±0.9%, p = 0.04; EtOH sham 4.3±0.4% vs EtOH CLP 13.2±0.5%, p = 0.005). C) In memory CD4s at 24h, sepsis increased CD69 in both water and alcohol-fed groups (H2O sham 18.7±0.8% vs H2O CLP 33.6±2.6%, p = 0.006; EtOH sham 23.5±0.9% vs EtOH CLP 36.4±0.8%, p = 0.03). At 72h, CD69 remained increased in alcohol septic mice only (EtOH sham 27.2±1.7% vs and EtOH sepsis 55.6±4%, p = 0.0003). n = 6-9/group.</p

Serum cytokine suggests Th2 skewing in EtOH-fed septic relative to water-fed septic animals.

<p>A) Chronic alcohol ingestion induced elevated baseline levels of serum IL1β over water-feeding in sham animals (906.5±187.7 vs 1883±444.6, p = 0.01). B) Alcohol ingestion increased baseline serum IL-4 concentration in sham animals (19322±473.2 vs 26584±2516, p = 0.02), with a concurrent strong trend toward increase in septic animals (19629±523 vs 24433±1686, p = 0.05). C) Chronic alcohol ingestion increased baseline serum IL12 concentration in sham animals (1073±421.5 vs 35.9±3.1, p = 0.04). D) Alcohol ingestion increased baseline serum TNF concentration in sham animals (1093±79.9 vs 823.8±34.7, p = 0.005). E) Serum IL-6 was increased in alcohol sepsis over water sepsis (58697±27081 vs 896.7±356, p = 0.02). F) Serum IL-10 concentration was increased is alcohol sepsis over water sepsis (10057±4412 vs 979.7±896.2, p = 0.01). n = 8/group.</p

Alcohol delays O-glycosylation of CD43 on memory CD4+ T cells in sepsis.

<p>A) At 24h, there was no significant increase in CD43 expression in total CD4 cells in either water or alcohol-fed mice due to sepsis. By 72h, both water sepsis and alcohol sepsis groups increased CD43 expression over shams (H2O sham 81.4±7.0 vs H2O CLP 334.5±27.6, p = 0.004; EtOH sham 96.6±3.7 vs EtOH CLP 420.0±65.0, p = 0.04). B) At 24h, there was no significant increase in CD43 expression in naïve CD4 cells. By 72h, both water sepsis and alcohol sepsis groups increased CD43 expression over shams (H2O sham 1.6±0.4% vs H2O CLP 12.9±1.6%, p = 0.01; EtOH sham 1.7±0.1% vs EtOH CLP 20.2±4.8%, p = 0.01). C) At 24h, water septic mice showed significant upregulation of CD43 on memory CD4s (H2O sham 27.8±0.5% vs H2O CLP 55.4±4.2%, p = 0.02), while alcohol septic mice did not. By 72h, CD43 expression is increased in both water and alcohol sepsis groups compared to sham (H2O sham 31.9±0.5% vs H2O CLP 56.7±3.3%, p = 0.03; EtOH sham 29.1±1.4% vs EtOH CLP 50.6±4.5%, p = 0.02).</p

CD8+ T cell frequencies and counts.

<p>A) There were no relevant differences in CD8 frequencies or counts specifically due to sepsis or ethanol alone at 24h. B) At 72h, there was a strong trend toward a decrease in absolute count due to sepsis in both the water and alcohol fed groups. n = 6-9/group.</p

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

Experimental design.

<p>A, 4–6 week old C57BL/6 mice were provided either ethanol or water for 12 weeks followed by sepsis induction via CLP or sham laparotomy. Spleens were harvested at 24 and 72 hours for flow cytometric analysis of lymphocytes. B, Body weights were not different between water-fed and alcohol-fed mice at the end of the 12-week period.</p

Alcohol delays increase in the CD69+CD43+ population in naïve and memory CD4+ T cells.

<p>A) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 24h. B) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 72h. C) By 24h, there was significant increase in CD69+ CD43+ population in naïve CD4s in water sepsis (H2O sham 0.4±0.04% vs H2O CLP 1.6±0.4%, p = 0.03). This population was not increased in alcohol sepsis. By 72h, both water septic and alcohol septic groups showed significant increase in the CD69+ CD43+ population (H2O sham 0.3±0.08% vs H2O CLP2.5±0.4%, p = 0.02; EtOH sham 0.3±0.05% vs EtOH CLP 3.5±0.5%, p = 0.01). D) In memory CD4s, at 24h there was a significant increase in CD69+ CD43+ population in water sepsis (H2O sham 6.2±0.7% vs H2O CLP 23.7±4.2%, p = 0.02) but not alcohol sepsis. By 72h, both water and alcohol septic groups show significant increase in this population above sham controls (H2O sham 11.4±-.2% vs H2O CLP 30.8±1.8%, p = 0.04; EtOH sham 10.3±0.9% vs EtOH CLP 32.9±2.6%, p = 0.005). n = 4-8/group.</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