Circulating Th17 T cells at treatment onset predict autoimmune toxicity of PI3Kδ inhibitors.

PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity, despite promising efficacy and evidence that toxicity is associated with improved clinical outcomes. Prior phenotypic evaluation by CyTOF has identified increases in activated CD8 T cells with activation of Th17 T cells, as well as decreases in Tregs, particularly in patients with toxicity. Here we sought to further understand the effects of idelalisib and duvelisib in vitro, and demonstrate that both idelalisib and duvelisib can inhibit T cell proliferation as well as Th1 and Treg differentiation in vitro, while promoting Th2 and Th17 differentiation. We further demonstrate directly using intracellular flow cytometry that autoimmune toxicity in patients is associated with higher absolute numbers of CD4 and CD8 T cells with Th17 differentiation in peripheral blood prior to therapy, and that gastrointestinal tissues from patients with active autoimmune complications of PI3Kδ inhibitors show infiltration with Th17+ T cells. These same tissues show depletion of Tregs as compared to CLL patients without toxicity, suggesting that loss of Tregs may be permissive for Th17 activation to lead to autoimmune toxicity. Clinical trials to restore this balance are warranted.

Activated CLL cells regulate IL-17F-producing Th17 cells in miR155-dependent and outcome-specific manners.

Chronic lymphocytic leukemia (CLL) results from expansion of a CD5+ B cell clone that requires interactions with other cell types, including T cells. Moreover, patients with CLL have elevated levels of circulating IL-17A+ and IL-17F+ CD4+ T (Th17) cells, with higher numbers of IL-17A+ Th17 cells correlating with better outcomes. We report that CLL Th17 cells expressed more miR155, a Th17-differentiation regulator, than control Th17 cells, despite naive CD4+ T (Tn) cell basal miR155 levels being similar in both. We also found that CLL cells directly regulated miR155 levels in Tn cells, thereby affecting Th17 differentiation, by documenting that coculturing Tn cells with resting or activated (Bact) CLL cells altered the magnitude and direction of T cell miR155 levels; CLL Bact cells promoted IL-17A+ and IL-17F+ T cell generation by an miR155-dependent mechanism, confirmed by miR155 inhibition; coculture of Tn cells with CLL Bact cells led to a linear correlation between the degree and direction of T cell miR155 expression changes and production of IL-17F but not IL-17A; and Bact cell-mediated changes in Tn cell miR155 expression correlated with outcome, irrespective of IGHV mutation status, a strong prognostic indicator. These results identify a potentially unrecognized CLL Bact cell-dependent mechanism, upregulation of Tn cell miR155 expression and subsequent enhancement of IL-17F+ Th17 generation, that favors better clinical courses.

Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Chronic lymphocytic leukemia-like monoclonal B-cell lymphocytosis exhibits an increased inflammatory signature that is reduced in early-stage chronic lymphocytic leukemia.

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation.

Constitutive Vagus Nerve Activation Modulates Immune Suppression in Sepsis Survivors.

Patients surviving a septic episode exhibit persistent immune impairment and increased mortality due to enhanced vulnerability to infections. In the present study, using the cecal ligation and puncture (CLP) model of polymicrobial sepsis, we addressed the hypothesis that altered vagus nerve activity contributes to immune impairment in sepsis survivors. CLP-surviving mice exhibited less TNFα in serum following administration of LPS, a surrogate for an infectious challenge, than control-operated (control) mice. To evaluate the role of the vagus nerve in the diminished response to LPS, mice were subjected to bilateral subdiaphragmatic vagotomy at 2 weeks post-CLP. CLP-surviving vagotomized mice exhibited increased serum and tissue TNFα levels in response to LPS-challenge compared to CLP-surviving, non-vagotomized mice. Moreover, vagus nerve stimulation in control mice diminished the LPS-induced TNFα responses while having no effect in CLP mice, suggesting constitutive activation of vagus nerve signaling in CLP-survivors. The percentage of splenic CD4+ ChAT-EGFP+ T cells that relay vagus signals to macrophages was increased in CLP-survivors compared to control mice, and vagotomy in CLP-survivors resulted in a reduced percentage of ChAT-EGFP+ cells. Moreover, CD4 knockout CLP-surviving mice exhibited an enhanced LPS-induced TNFα response compared to wild-type mice, supporting a functional role for CD4+ ChAT+ T cells in mediating inhibition of LPS-induced TNFα responses in CLP-survivors. Blockade of the cholinergic anti-inflammatory pathway with methyllcaconitine, an α7 nicotinic acetylcholine receptor antagonist, restored LPS-induced TNFα responses in CLP-survivors. Our study demonstrates that the vagus nerve is constitutively active in CLP-survivors and contributes to the immune impairment.

Identification and characterization of distinct IL-17F expression patterns and signaling pathways in chronic lymphocytic leukemia and normal B lymphocytes.

Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of B lymphocytes. T cell abnormalities are a common feature of CLL and contribute to impaired immune function in these patients. T cells are ineffective in eliminating the leukemic clone and may actually promote tumor growth and survival. Previous work from our laboratory documented elevated circulating levels of IL-17A-producing Th17 cells in CLL patients as compared to healthy age-matched control subjects. These high circulating Th17 levels associated with better prognostic markers and significantly longer overall survival, even among patients whose clones used unmutated IGHVs (U-CLL). Recent studies suggest that Th17 cells are heterogeneous, expressing different profiles of cytokines, and that different subsets of Th17s mediate different biological functions. In the present study, we found significantly higher levels of IL-17F-expressing CD4(+) T cells in CLL versus healthy peripheral blood mononuclear cells following in vitro stimulation in the presence of Th17-promoting cytokines. Furthermore, the differentiation of IL-17F-expressing Th17 cells was significantly enhanced when purified CD4(+) T cells from CLL patients were cultured in the presence of autologous CLL B cells. Lastly, single-cell network profiling revealed that IL-17F triggers NFκB phosphorylation in T and B cells from patients with CLL, but not age-matched healthy controls. Taken together, our data suggest that the phenotype of Th17 cells in CLL patients is distinct from healthy individuals, expressing higher levels of IL-17F, and that B and T cells from CLL patients are particularly responsive to IL-17F, as compared to healthy age-matched control individuals.

Th17 and non-Th17 interleukin-17-expressing cells in chronic lymphocytic leukemia: delineation, distribution, and clinical relevance.

BACKGROUND: The levels and clinical relevance of Th17 cells and other interleukin-17-producing cells have not been analyzed in chronic lymphocytic leukemia. The objective of this study was to quantify blood and tissue levels of Th17 and other interleukin-17-producing cells in patients with this disease and correlate blood levels with clinical outcome. DESIGN AND METHODS: Intracellular interleukin-17A was assessed in blood and splenic mononuclear cells from patients with chronic lymphocytic leukemia and healthy subjects using flow cytometry. Interleukin-17A-producing cells were analyzed in formalin-fixed, paraffin-embedded spleen and lymph node sections using immunohistochemistry and immunofluorescence. RESULTS: The absolute numbers of Th17 cells in peripheral blood mononuclear cells and the percentages of Th17 cells in spleen cell suspensions were higher in patients with chronic lymphocytic leukemia than in healthy subjects; in six out of eight paired chronic lymphocytic leukemia blood and spleen sample comparisons, Th17 cells were enriched in spleen suspensions. Circulating Th17 levels correlated with better prognostic markers and longer overall survival of the patients. Two "non-Th17" interleukin-17-expressing cells were identified in chronic lymphocytic leukemia spleens: proliferating cells of the granulocytic lineage and mature mast cells. Granulocytes and mast cells in normal spleens did not express interleukin-17. Conversely, both chronic lymphocytic leukemia and healthy lymph nodes contained similar numbers of interleukin-17+ mast cells as well as Th17 cells. CONCLUSIONS: Th17 cells are elevated in chronic lymphocytic leukemia patients with better prognostic markers and correlate with longer survival. Furthermore, non-Th17 interleukin-17A-expressing cells exist in chronic lymphocytic leukemia spleens as maturing granulocytes and mature mast cells, suggesting that the microenvironmental milieu in leukemic spleens promotes the recruitment and/or expansion of Th17 and other IL-17-expressing cells. The pathophysiology of Th17 and non-Th17-interleukin-producing cells in chronic lymphocytic leukemia and their distributions and roles in this disease merit further study.

Effects of pneumoperitoneal gases and pressures on transitional cell carcinoma adhesion, growth, apoptosis and necrosis: an in vitro study.

PURPOSE: We studied the effects of insufflation gas and pressure on the adhesion, growth, apoptosis and necrosis of transitional cell carcinoma (TCC) in an in vitro model. MATERIALS AND METHODS: Tumor adhesion and cell growth of AY-27 rat TCC was measured after 3-hour incubation with CO2, N2 and He insufflation at different pressures (0, 10 and 15 mm Hg) in vitro. The effects of these gases on the rate of tumor cell apoptosis and necrosis were compared. RESULTS: In vitro the tumor adhesion rate was lowest with CO2 and highest with N2. Higher gas pressures resulted in decreased adhesion rates for CO2 and He but increased adhesion rates for N2. N2 enhanced tumor cell proliferation at all pressures studied. He and CO2 resulted in an initial increase in cell proliferation in the first 24 hours, followed by a decrease in tumor growth. Extracellular medium turned acidic in CO2 (pH 6.27 to 6.39) but basic in N2 and He (pH 8.39 to 8.84). At all insufflation pressures studied apoptosis and necrosis rates were increased in the first 24 hours, followed by a decrease for CO2 and N2. He resulted in increasing apoptosis and necrosis throughout the study period. CONCLUSIONS: The type of gas and insufflation pressure affects cell adhesion and tumor growth. There was a significant increase in tumor adhesion and proliferation with N2 insufflation compared with CO2 and He at 0 to 15 mm Hg pressures. CO2 demonstrated the greatest decrease in TCC adhesion and proliferation at 15 mm Hg pressure. Apoptosis and necrosis were highest for He compared with the other gases.