K. M. Ramonell1, C. Chen1, Z. Liang1, C. M. Coopersmith1, M. L. Ford1 1Emory University School Of Medicine,Department Of Surgery,Atlanta, GA, USA
Introduction: Patients with pre-existing malignancy are nearly ten times more likely to develop sepsis than the general population and mice with pre-existing tumors exhibit worsened survival following cecal ligation and puncture (CLP) as compared to previously healthy controls. Here, we aimed to determine whether alterations in T regulatory (Treg) cell frequency or suppressive capacity play a role in the increased mortality observed in the setting of cancer and sepsis.
Methods: Cancer was induced via subcutaneous injection of 125,000 Lewis lung cancer cells into the right inner thigh of C57BL/6 mice, which were then followed for three weeks for the development of palpable tumors. Mice with cancer and previously healthy mice were then made septic via 2x25g CLP or underwent sham laparotomy. Spleens were harvested 24hr post-sepsis induction and splenocytes were stained with fluorescently conjugated mAbs specific for CD3, CD4, CD8, CD25, FoxP3, TIGIT, and Helios. Tregs were defined as CD3+CD4+CD25+FoxP3+ cells. Data was collected on an LSRII Flow Cytometer and analyzed with One-Way ANOVA testing. For all data points, n=5-10 mice/group.
Results: In cancer septic mice, the frequency of Tregs was significantly increased compared to septic mice without cancer (14.5% vs 8.2%; p=0.02). Absolute counts followed a similar trend (4.7 x 105 vs. 3.2 x 105; p=0.05) but differences were not statistically significant. Additionally, cancer septic mice had the greatest frequency of Helios and FoxP3 double positive Tregs compared to non-cancer septic mice (12.5% vs. 8.0%; p=0.004) and cancer sham mice (12.5% vs. 10.0%; p=0.08). When further analyzing Helios+ Tregs, cancer septic mice exhibited greater frequencies of TIGIT+ cells compared to non-cancer septic mice (14.0% vs 7.1%; p<0.001) and cancer sham mice (11.5% vs. 14.0%; p=0.09). When analyzing non-Treg CD4 T cells, the frequency of Helios+TIGIT+ cells was significantly less compared to Helios+TIGIT+ Tregs (9.0% vs. 0.8%; p<0.001) isolating Helios and TIGIT expression on CD4 T cells to Tregs.
Conclusion: Sepsis in the setting of cancer significantly increases the frequency of total Tregs and Helios+TIGIT+ Tregs. This phenotype has been previously reported to be highly suppressive and could contribute to the exaggerated mortality observed when these two conditions occur together. These findings help elucidate the specific derangements in the immune profile of co-existing cancer and sepsis in order to develop effective therapies for this vulnerable population.