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CD4+ regulatory T cells (Tregs) are acutely activated by traumatic injury, which suggests that they may react to injury with similar kinetics as memory T cells. Here, we used a mouse burn trauma model to screen for memory-like T cell responses to injury by transferring T cells from sham or burn CD45.1 mice into CD45.2 mice and performing secondary injuries in recipient mice. Among all T cell subsets that were measured, only Tregs expanded in response to secondary injury. The expanded Tregs were a CD44high/CD62Llow subpopulation, markers indicative of memory T cells. CyTOF (cytometry by time-of-flight) mass cytometry was used to demonstrate that injury-expanded Tregs expressed higher levels of CD44, CTLA-4, ICOS, GITR, and Helios than Tregs from noninjured mice. Next, we tested whether a similar population of Tregs might react acutely to burn trauma. We observed that Tregs with a phenotype that matched the injury-expanded Tregs were activated by 6 h after injury. To test if Treg activation by trauma requires functional MHC class II, we measured trauma-induced Treg activation in MHC class II gene deficient (MHCII−/−) mice or in mice that were given Fab fragment of anti-MHC class II antibody to block TCR activation. Injury-induced Treg activation occurred in normal mice but only partial activation was detected in MHCII−/− mice or in mice that were given Fab anti-MHCII antibody. These findings demonstrate that trauma activates a memory-like Treg subpopulation and that Treg activation by injury is partially dependent on TCR signaling by an MHC class II dependent mechanism.

BackgroundImmune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%–20% of patients and underpinnings of resistance remain undefined.MethodsStarting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models.ResultsAnti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1− (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions.ConclusionsThese data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

This retrospective study aimed to evaluate the relationship between fecal organic acids and mortality in critically ill patients. The minimum values of fecal acetic acid and propionate in non‐survivors were significantly lower than in survivors. The altered balance of fecal organic acids was associated with mortality in critically ill patients., Aim This study aimed to evaluate the relationship between fecal organic acids and mortality in critically ill patients. Methods This retrospective study included 128 patients who fulfilled the criteria of systemic inflammatory response syndrome and had a serum C‐reactive protein level of greater than 10 mg/dL. Patients were treated in the intensive care unit for more than 2 days. Patients were divided into two groups: survivors and non‐survivors. We measured and compared eight kinds of fecal organic acids between the two groups. We focused on the minimum and maximum value of each fecal organic acid and evaluated prognostic factors by using classification and regression tree (CART) and multivariate logistic regression analyses. Results We included 90 patients as survivors and 38 as non‐survivors. The CART analysis revealed that the dominant factors for mortality were the minimum values of propionate and acetate and the maximum values of lactate and formic acid. In the evaluation of the minimum values of fecal organic acids, propionate was significantly associated with increased mortality (odds ratio, 0.11 [95% confidence interval, 0.024–0.51]; P = 0.005), acetate (0.047 [0.005–0.49]; P = 0.01), and age (1.048 [1.015–1.083]; P = 0.004). In the evaluation of the maximum values, lactate was significantly associated with increased mortality (5.21 [2.024–13.42], P = 0.001) and age (1.050 [1.017–1.084]; P = 0.003). Conclusion An altered balance of fecal organic acids was significantly associated with mortality in critically ill patients.

Prolonged exposure to hyperoxia has deleterious effects on the lung, provoking both inflammation and alveolar injury. The elements of hyperoxic injury, which result in high rates of lethality in experimental models, are thought to include multicellular immune responses. To characterize these alterations in immune cell populations, we performed time-of-flight mass cytometry (CyTOF) analysis of CD45-expressing immune cells in whole lung parenchyma and the bronchoalveolar space of mice, exposed to 48 hours of hyperoxia together with normoxic controls. At the tested time point, hyperoxia exposure resulted in decreased abundance of immunoregulatory populations (regulatory B cells, myeloid regulatory cells) in lung parenchyma and markedly decreased proliferation rates of myeloid regulatory cells, monocytes and alveolar macrophages. Additionally, hyperoxia caused a shift in the phenotype of alveolar macrophages, increasing proportion of cells with elevated CD68, CD44, CD11c, PD-L1, and CD205 expression levels. These changes occurred in the absence of histologically evident alveolar damage and abundance of neutrophils in the parenchyma or alveolar space did not change at these time points. Collectively, these findings demonstrate that pulmonary response to hyperoxia involves marked changes in specific subsets of myeloid and lymphoid populations. These findings have important implications for therapeutic targeting in acute lung injury.

Trauma induces a complex immune response, requiring a systems biology approach to capture multicellular changes. Using mass cytometry by time-of-flight (CyTOF), we evaluated time-dependent changes in peripheral blood in trauma patients to identify changes correlated with infection.Total leukocytes were prepared via red blood cell lysis using peripheral blood samples from trauma patients with an Injury Severity Score greater than 20 at Days 1, 3, and 5 after injury, and from age- and sex-matched uninjured controls. Cells were stained using a 33-marker immunophenotyping CyTOF panel. Statistics were calculated using one-way analysis of variance with multiple comparisons.The CyTOF staining demonstrated changes in many cell subsets. The mean expression intensity of CD86 on monocytes decreased significantly at all time points after injury. When the patients were stratified based on development of infection, there was a trend to decreased CD86 expression on monocytes of those patients that developed subsequent infection. Based on stratification, we identified significantly increased expression of CD39 on NK cells only in patients that developed an infection.This study used a systems biology approach to identify novel changes in circulating immune cell subsets in trauma patients correlating with post-traumatic infection. Decreased expression of CD86, a costimulatory molecule, on monocytes demonstrates that trauma affects the innate system's ability to control T-cell immunity. We also found that CD39 expression on NK cells increased significantly in patients with subsequent infection. CD39 is a protein that generates adenosine, which has immunosuppressive effects on several immune cell types including NK cells. In summary, our results point to pathways that may be central to second-hit infections and further study to delineate these pathways could be key to generating clinical biomarkers or targeted immune therapies for trauma patients.Prognostic study, level II.

The response rate of 15-20% with anti-PD1 in head and neck squamous cell carcinoma (HNSCC) highlights the urgent need for strategies to overcome resistance. Our lab has previously developed a carcinogen-induced immunocompetent murine oral carcinoma (MOC) model to study HNSCC immunobiology. Specifically, MOC1 is an immunogenic cell line that, despite sensitivity to anti-PD-1, exhibits occasional development of escape tumors (MOC1esc). MOC1esc escape tumors display a resistance phenotype similar to those observed in HNSCC patients undergoing anti-PD1 therapy. When independent escape tumors are harvested and re-transplanted into naïve mice, they grow progressively and are resistant to anti-PD1 therapy. Intriguingly, whileMOC1esc is resistant to anti-PD1, it is completely rejected in tumor bearing mice treated with anti-CTLA4. Therefore, the anti-PD1 responsive MOC1 and resistant MOC1esc mouse model is an isogenic system that provides an excellent opportunity to study mechanism(s) in adaptive resistance to anti-PD1 therapy of HNSCC. To gain a comprehensive insight into the tumor microenvironment (TME) as a contributor to adaptive resistance, we analyzed tumor infiltrating lymphocytes (TIL) in naïve MOC1 and MOC1esc tumors using mass cytometry time-of-flight (CyTOF) with a 38-cell marker panel.MOC1esc tumors were highly infiltrated by CD103+ effector/memory regulatory T cells (Tregs) and M2-like tumor associated macrophages (TAMs), while MOC1 tumors have more M1-like TAMs and neutrophils. Furthermore, we observed that both anti-PD1 and anti-CTLA4 treatment dramatically expanded CD8+ T cells and decreased neutrophils in MOC1esc tumors. In responding MOC1esc tumors, anti-CTLA4 treatment resulted in depleted Tregs, decreased M2-like TAMs and neutrophils, as well as a striking increase in M1-like TAMs compared with isotype control treated tumors. In contrast, anti-PD1 treated resistant MOC1esc tumors showed decreased M1-like TAMs, while M2-like TAMs were increased compared with controls. Tregs were not affected by anti-PD1 treatment. Therefore, the comparison between the TME of anti-PD1 treated resistant tumors and anti-CTLA4 treated responding tumors suggeststhat Tregs, neutrophils, and TAMs may contribute to the sensitivity (or resistance) to checkpoint blockade therapy. Ongoing studies will test the functional contribution of these distinct TME components in antitumor immunity including cytokine production, proliferative capacity, and their roles in immunotherapy resistance. In summary, this study identified immune modulators within TME involved in adaptive immunotherapy resistance of HNSCC. Findings from this study have advanced our understanding of HNSCC immunotherapy resistance and will accelerate the discovery of new therapeutic targets and biomarkers for adaptive resistance. Citation Format: Liye Zhou, Yasutaka Nakahori, Fei Guo, Joshua Keegan, Rachel Riley, James A. Lederer, Ravindra Uppaluri. Identification of immunotherapy resistance mechanisms within tumor microenvironment in a mouse model of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1509.

Background The gut is an important target organ of injury during critically ill conditions. Although Gram staining is a common and quick method for identifying bacteria, its clinical application has not been fully evaluated in critically ill conditions. Aims This study’s aims were to identify patterns of Gram-stained fecal flora and compare them to cultured bacterial counts and to investigate the association between the patterns and septic complications in patients with severe systemic inflammatory response syndrome (SIRS). Methods Fifty-two patients with SIRS were included whose Gram-stained fecal flora was classified into three patterns. In a diverse pattern, large numbers of multiple kinds of bacteria completely covered the field. In a single pattern, one specific kind of bacteria or fungi predominantly covered the field. In a depleted pattern, most bacteria were diminished in the field. Results In the analysis of fecal flora, the numbers of total obligate anaerobes in the depleted pattern was significantly lower than those in the diverse pattern and single pattern (p

Background The gut is an important target organ for injury after severe insult, and resolution of feeding intolerance is crucial for critically ill patients. We investigated gut flora and motility to evaluate the impact of gastrointestinal dysmotility on septic complications in patients with severe systemic inflammatory response syndrome (SIRS). Methods Sixty-three ICU patients with severe SIRS were divided into two groups depending on their intestinal condition. Patients with feeding intolerance comprised patients who had feeding intolerance, defined as ≥300 mL reflux from nasal gastric feeding tube in 24 h, and patients without feeding intolerance comprised patients with no feeding intolerance. We compared fecal microflora, incidences of bacteremia, and mortality between these groups. Key Results Analysis of feces showed that patients with feeding intolerance had significantly lower numbers of total obligate anaerobes including Bacteroidaceae and Bifidobacterium, higher numbers of Staphylococcus, lower concentrations of acetic acid and propionic acid, and higher concentrations of succinic acid and lactic acid than those in patients without feeding intolerance (P ≤ 0.05). Patients with feeding intolerance had higher incidences of bacteremia (86%vs 18%) and mortality (64%vs 20%) than did patients without feeding intolerance (P ≤ 0.05). Conclusions & Inferences Gut flora and organic acids were significantly altered in patients with severe SIRS complicated by gastrointestinal dysmotility, which was associated with higher septic mortality in SIRS patients.