Eicosanoids tailor the innate immune response by supporting local inflammation and exhibiting immunomodulatory properties. Prostaglandin (PG) E 2 is the most abundant eicosanoid in the inflammatory milieu due to the robust production elicited by pathogen-associated molecular patterns on cells of the innate immune system. The different functions and cell distribution of E prostanoid receptors explain the difficulty encountered thus far to delineate the actual role of PGE 2 in the immune response. The biosynthesis of eicosanoids includes as the first step the Ca 21 -and kinase-dependent activation of the cytosolic phospholipase A 2 , which releases arachidonic acid from membrane phospholipids, and later events depending on the transcriptional regulation of the enzymes of the cyclooxygenase routes, where PGE 2 is the most relevant product. Acting in an autocrine/ paracrine manner in macrophages, PGE 2 induces a regulatory phenotype including the expression of interleukin (IL)-10, sphingosine kinase 1, and the tumor necrosis factor family molecule LIGHT. PGE 2 also stabilizes the suppressive function of myeloidderived suppressor cells, inhibits the release of IL-12 p70 by macrophages and dendritic cells, and may enhance the production of IL-23. PGE 2 is a central component of the inflammasomedependent induction of the eicosanoid storm that leads to massive loss of intravascular fluid, increases the mortality rate associated with coinfection by Candida ssp. and bacteria, and inhibits fungal phagocytosis. These effects have important consequences for the outcome of infections and the polarization of the immune response into the T helper cell types 2 and 17 and can be a clue to develop pharmacological tools to address infectious, autoimmune, and autoinflammatory diseases.

Backgroundβ-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.Principal FindingsSerum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE2 biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE2. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.ConclusionsThese results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A2 route.

Background: Phagocytosis and Th17 immune response control fungal invasion. Results: ␤-Glucans mobilize nuclear C/EBP homologous protein (CHOP) and increase activating transcription factor 2 (ATF2) binding to the il23a promoter. Conclusion: ␤-Glucans direct proapoptotic CHOP to phagosomal vesicles and induce ATF2-dependent il23a transcription. Significance: The mechanisms underlying the unfolded protein response and the trans-activation of il23a elicited by ␤-glucans are ascertained.

Merino J. Enhanced sensitivity of flow cytometry for routine assessment of minimal residual disease. Haematologica. 2010; 95:691-692. doi:10.3324/haematol.2009 (approximately 20-30, 40-60, and 80-200 © F e r r a t a S t o r t i F o u n d a t i o n Minimal residual disease (MRD) was measured in samples from 5 patients with multiple myeloma (MM), 5 patients with B-cell chronic lymphocytic leukemia (B-CLL), and 4 patients with T-cell lymphoproliferative disorders (T-CLPD) by detecting three different levels of malignant cells

The relevance of the immune system in cancer has long been studied. Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients by flow cytometry. Using this technology, CTLs bound through T cell receptor (TCR) to tumor cells can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. These CTLs display higher percentage of effector cells and marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in patients diagnosed with acute myeloid leukemia.

Introduction Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients. Using this approach, CTLs stably bound through T cell receptor to tumor cells (doublet forming T-cells) can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. Methods Co-cultures between PBMC from AML patients in complete remission and AML tumor cells (PKH-stained) from the same patient were performed to study the percentage of doublet-forming T cells (CD3+PKH+) (T cell bound to a tumor cell). After 15 hours of co-culture, cells were stained and sorted. Secondary co-cultures with autologous tumor cells (used in primary co-culture) were performed to study the cytotoxic activity and cytokine production of T-cells capable or not to form stable joints with the leukemic cells (doublet population vs non-doublet population). Results Doublet-forming T cells from AML patients were identified in a range of 2% to 6% (mean=3.83%, n=5). Immunophenotyping analysis showed differences between doublet-forming T cells (CD3+PKH+) and those T cells which did not form stable and strong interactions with target cells (CD3+PKH-). Doublet T cells displayed a higher percentage of CD8+ T cells and higher percentage of effector CD4+ and CD8+ T cells compared to non-doublet T cells. Next, we explored, among effector CD4+ and CD8+ cells, those with cytotoxic phenotype. As expected, a high percentage of effector CD8+ doublet T cells showed Granzyme B and perforin expression, thus corresponding with a cytotoxic immune-phenotype (n=3, mean 65.51%). Within effector CD4+ doublet T cells, a mean of 9.053 % showed expression of both Granzyme B and perforin corresponding with CD4+ CTL (n=3). Regarding CD57 and CD16 markers, a mean of 18.62% of effector CD4+ doublet T cells were positive for both markers, compared to 65.84% of effector CD8+ doublet T cells (n=3). Further, we performed secondary co-cultures to analyze the CD69 activation marker after 24h of co-culture. A high percentage of CD69+ cells was observed in co-cultures with doublet-forming T cells against target cells as compared to non-doublet T cells (n=3, p=0.0053). Finally, analysis of supernatants of co-culture of doublet T cells and non-doublet T cells with target cells revealed specific secretion of IFNγ and IL-2 (n=3, p=0.0001; p=0.0005, respectively). The cytolytic activity was evaluated comparing the viability of tumor cells cultured alone or with doublet-forming T cells or non-doublet T cells from the same patient. A significant increase of the specific lysis of AML cells was observed when doublet T cells were co-cultured as compared to non-doublet T cells (p=0.0424, n=5). This encouraged us to examine whether we were able to identify doublet-forming T cells from bone marrow of AML patients at diagnosis. Analyses of bone marrow by flow cytometry reveled a small percentage of CD3+CD34+ population corresponding with bone marrow-doublet-forming T cells (n=3, mean=2.9%). Interestingly, bone marrow-doublet-forming T cells show a higher percentage of CD4+ T cells, whereas bone marrow-non-doublet T cells show a higher percentage of CD8+ T cells. Conclusions Our data demonstrate that when T cells from AML patients are co-cultured with tumor cells, a "doublet T cell" population appears. This population consists of T cells capable to bind tumor cells. These CTLs display higher percentage of effector cells and a marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in both bone marrow and peripheral blood from patients diagnosed with acute myeloid leukemia. Figure. Figure. Disclosures Sanchez-Abarca: Virgen del Rocio University Hospital: Patents & Royalties. Ramos:Takeda Oncology: Research Funding.