Lymphoid Subsets Research Articles

Levamisole stimulates proliferation of circulating and intestinal immune cell subsets, gut health and performance in weaned pigs

Valpotić, H., Šperanda, M., Kovšca-Janjatović, A., Ðidara, M., Lacković, G., Božić, F., Habrun, B., Srečec, S., Mataušić-Pišl, M. and Valpotić, I. 2014. Levamisole stimulates proliferation of circulating and intestinal immune cell subsets, gut health and performance in weaned pigs. Can. J. Anim. Sci. 94: 43–53. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly stated that exogenous modulation through the use of substances able to modulate immune functions represents an important prophylactic/therapeutic approach in prevention/treatment of both stress- and F4+ and F18+ enterotoxigenic E. coli (ETEC)-induced infections accompanied weaning. The aim of this study was to evaluate the effectiveness of levamisole (LEVA; 2.5 mg kg−1 BW in 10 mL) applied per os to weaned pigs in proliferation of circulating and intestinal immune cell subsets throughout a period of 5 wk. Changes in proportion or number of peripheral blood and ileal mucosal leukocytes tested were studied either weekly by flow cytometry or at the end of the experiment (day 35) by immunohistology/histomorphometry, respectively. Pigs treated with LEVA had increased proportions of peripheral blood CD45+ lymphoid cells, CD4+ and CD8+ T cells, and CD21+ B cells (P<0.01) between days 14 and 35 following the treatment. Also, LEVA stimulated the proliferation of CD45RA+ naïve lymphoid cells in interfollicular (P<0.001) and follicular areas (P<0.05) of ileal Peyer’s patches at day 35 of the experiment. These pigs had a significantly higher (P<0.05) average body weight (19.7 vs. 17.1 kg) and weight gain at the end of experiment compared with the control pigs (for 15%). We conclude that LEVA stimulated the proliferation of circulating and intestinal lymphoid cell subsets tested and improved performance in weaned pigs, and thus, the drug may nonspecifically enhance their immunity/resistance to F4+ and F18+ ETEC strains.

Splenic Lymphoid Subsets With Less Well-Recognized Phenotypes Mimic Aberrant Antigen Expression

Flow cytometry can assist in the diagnosis of lymphoma by identifying aberrant antigen expression. Recognition of aberrancy requires knowledge of the phenotype of normal lymphoid cells. Lymphoid subsets were characterized in 20 spleens removed for traumatic rupture, using 8-color flow cytometry. Normal variation in splenic lymphoid subsets was highlighted and several well-recognized subsets were identified: CD5+ B cells (20/20 specimens), CD7- T cells (20/20), and CD3 brightγδT cells (16/20). In addition, less well-recognized lymphoid subsets that resemble those described in lymphoma were identified in all specimens: CD5- T cells (4.5 ± 5.1% of T cells), CD2- natural killer (NK) cells (38 ± 7% of NK cells), and CD7dim+ NK cells. Similar populations were identified in 20 control peripheral blood specimens, where they represented a smaller proportion of total lymphoid cells. Familiarity with the phenotype of normal lymphoid subsets can help prevent misinterpreting flow cytometric data. Furthermore, in the context of neoplastic cells, the phenotype may suggest expanded normal subsets rather than aberrant antigen expression.

Embryonic hematopoiesis

Blood cells are continually produced from a pool of progenitors that derive from hematopoietic stem cells (HSCs). In vertebrates, the hematopoietic system develops from two distinct waves or generation of precursors. The first wave occurs in the yolk sac, in mammals or equivalent embryonic structure, and produces nucleated primitive erythrocytes that provide the embryo with the first oxygen transporter and are, therefore, essential for the viability of the embryo. The yolk sac also produces myeloid cells that migrate to the central nervous system and to the skin to form the microglia and skin specific macrophages, the Langerhans cells. The second wave occurs in the dorsal aorta and produces multipotential hematopoietic progenitors. These cells are generated once in the lifetime from mesoderm derivatives closely related to endothelial cells, during a short period of embryonic development. Newly generated cells do not reconstitute the hematopoietic compartment of conventional recipients; therefore, they are designated as immature or pre-HSCs. They undergo maturation into adult HSCs in the aorta or in the fetal liver accompanied by the expression of MHC class I, CD45, CD150, Sca-1 and the absence of CD48. Differentiation of HSCs first occurs in the fetal liver, giving rise to mature blood cells. HSCs also expand in the fetal liver, and in a short time period (four days in the mouse embryo), they increase over 40-fold. HSCs and progenitor cells exit the fetal liver and colonize the spleen, where differentiation to the myeloid lineage and particular lymphoid subsets is favored.

Open questions: a few that need answers in immunology

The cells of the immune system fall into two broad categories - innate and adaptive - with the cells of the innate immune system providing frontline defense by means of invariant receptors to conserved microbial components, and the B and T lymphocytes of the adaptive immune system, with highly variable somatically diversified receptors, providing reinforcement after selective proliferation in response to specific antigen. While this principle still holds, the ‘old’ view of these systems as developmentally separate and functionally distinct has been eroded, most recently and dramatically by the discovery of a whole new group of innate lymphoid cell subsets. Another development has been the increasing emphasis on studying immune responses in vivo in interaction with stromal components that are beginning to be seen as essential contributors to the development of immune responses, rather than just as matrix or scaffold components in lymphoid organs or non-lymphoid peripheral tissues. A major focus now is on immune defense at epithelial barriers and homeostatic coexistence with a healthy microbiota, as it is becoming increasingly clear that intestinal dysbiosis strongly influences immune responses even at distal sites. However, whether dysbiosis is a cause or consequence of dysregulated inflammatory responses remains to be unraveled. I outline below some of the open questions raised by these developments.

In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production.Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts.We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures:No relevant conflicts of interest to declare.

Mass Cytometry Analysis Of Myelofibrosis and Secondary Acute Myeloid Leukemia Reveals Constitutive and Cytokine Induced Signaling Abnormalities With Differential Sensitivities To Ruxolitinib

The classic myeloproliferative neoplasms (MPNs), including primary and secondary myelofibrosis (MF), are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations induce hyperactivated JAK-STAT signaling in cell lines and mouse models. Other mutations have been found recurrently in human MPNs, however, including mutations associated with transformation of chronic MPNs to secondary acute myeloid leukemia (sAML). The aggregate effects of these mutations on in vivo myeloproliferative signaling and disease phenotypes are not yet well understood.While targeted inhibitors of JAK2 have shown activity in MPNs, evidence of a selective effect on the underlying malignant clone has been lacking. These findings suggest that dysregulation of other signaling molecules may be important in MPN pathogenesis, and that therapeutic targeting of these molecules could be beneficial. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed.Mass cytometry is a novel technology that merges flow cytometry with mass spectrometry and enables the simultaneous measurement of 30+ parameters at the single cell level. We utilized this approach to examine bone marrow or peripheral blood samples from four MF patients and five sAML patients, as well as three normal controls. Cells were exposed to five different perturbation conditions ex vivo, including the cytokines thrombopoietin (TPO) and G-CSF, and/or the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 18 surface markers and 16 intracellular signaling effectors and analyzed on a CyTOF mass cytometer.Single cell data was analyzed using traditional gating strategies, as well as with SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets based on shared surface marker expression. These methods grouped cells into distinct cell populations including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict basal and induced activation of each intracellular marker.MF and sAML patient samples exhibited abnormal cytokine-induced signaling that varied in a patient-specific manner. HSPC responses to TPO ranged from hyposensitive to hypersensitive compared with normal. Markedly abnormal basal (unstimulated) signaling in the HSPC compartment was observed in all MF and sAML patients. Each sample exhibited increased total IkBα and/or pCREB, typically accompanied by elevated phosphorylation of one or more additional signaling molecules. Both basal elevation and cytokine hypersensitivity were frequently observed within a MAP kinase signaling axis represented by pERK and downstream targets pCREB and pS6. Conversely, repressed basal STAT1 phosphorylation was observed in all patients. These abnormalities were not exclusive to sAML versus MF, nor specific to patients with or without the JAK2 V617F mutation. Widespread dysregulation of total IkBα, pERK-pCREB-pS6, and pSTAT1 suggest that these signaling effectors may be characteristic of a general myeloproliferative signaling phenotype.Ex vivo ruxolitinib effectively inhibited the majority of observed hypersensitive cytokine-stimulated signaling effects. In contrast, most basal signaling elevations were insensitive to ruxolitinib. The signaling molecules most frequently insensitive to ruxolitinib were those most frequently basally elevated in MF and sAML HSPC, namely pCREB and total IkBα. In contrast, phosphorylation of the JAK2 substrates STAT3 and STAT5 was almost always sensitive to ruxolitinib. These results suggest that targeting of myeloproliferative signaling pathways either downstream or independent of JAK2 activity may be valuable for improved treatment of MF and sAML patients.Ongoing experiments are focused on determining whether constitutive ruxolitinib-insensitive signaling abnormalities can be identified in patients treated with ruxolitinib in vivo, which may underlie the incomplete responses observed clinically. Taken together, these approaches will provide a deeper understanding of altered signaling networks in the context of targeted therapies in MPNs. Disclosures:Oh:Incyte: Consultancy, Research Funding, Speakers Bureau.

Immune Reconstitution In Fanconi Anemia Patients Following Allogeneic Bone Marrow Transplantation

BackgroundFanconi anemia is an autosomal recessive or X-linked genetic disorder characterized by bone marrow (BM) failure/aplasia. Failure of hematopoiesis results in depletion of the BM stem cell reservoir, which leads to severe anemia, neutropenia and thrombocytopenia, frequently requiring therapeutic interventions including hematopoietic stem cell transplantation (HSCT). Successful BMT requires reconstitution of normal immunity. MethodsIn the present study, we performed a detailed analysis of the distribution of peripheral blood subsets of T, B and NK lymphocytes in 23 patients with Fanconi anemia before and after BM transplantation (at days D+30, D+60 and D+100, D+180 D+270 and D+360). In parallel, we also compared the patients groups who received related and unrelated bone marrow, pre and one year after BMT. ResultsAfter transplantation, we found different kinetics of recovery for the distinct major lymphocyte subsets. NK cells were the first to recover, followed by cytotoxic CD8+T-cells and B-cells, and finally also the CD4+ T-helper cells. Early recovery was at the expenses of memory cells which would therefore be potentially derived from the graft whereas, recent thymic emigrant (CD31+ CD45RA+) and naive CD4+ or CD8+T-cells, rose only 6 months after HSCT, in the presence of immunosuppressive graft-vs-host disease (GVHD) prophylactic agents. While only slight differences were observed in the early recovery of cytotoxic CD8+ T-cells among cases receiving a graft from related vs. unrelated donors, cases suffering from GVHD displayed more marked differences vsother patients. ConclusionsOverall, our results support the utility of post-transplant monitoring of peripheral blood lymphoid subsets, for improved monitoring and follow-up of Fanconi anemia patients undergoing BMT. Disclosures:No relevant conflicts of interest to declare.

Critical Role of p38 and GATA3 in Natural Helper Cell Function

Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.

Declined presentation: Influence of granulocytic colony-stimulating factor on peripheral lymphoid subsets in patents with arteriosclerosis obliterans

Declined presentation: Influence of granulocytic colony-stimulating factor on peripheral lymphoid subsets in patents with arteriosclerosis obliterans

SLE Peripheral Blood B Cell, T Cell and Myeloid Cell Transcriptomes Display Unique Profiles and Each Subset Contributes to the Interferon Signature

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19+ B lymphocytes, CD3+CD4+ T lymphocytes and CD33+ myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33+ myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3+CD4+ T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.

TH2, allergy and group 2 innate lymphoid cells

The initiation of type 2 immune responses by the epithelial cell-derived cytokines IL-25, IL-33 and TSLP has been an area of extensive research in the past decade. Such studies have led to the identification of a new innate lymphoid subset that produces the canonical type 2 cytokines IL-5, IL-9 and IL-13 in response to IL-25 and IL-33. These group 2 or type 2 innate lymphoid cells (ILC2 cells) represent a critical source of type 2 cytokines in vivo and serve an important role in orchestrating the type 2 response to helminths and allergens. Further characterization of ILC2 cell biology will enhance the understanding of type 2 responses and may identify new treatments for asthma, allergies and parasitic infections. Interactions between ILC2 cells and the adaptive immune system, as well as examination of potential roles for ILC2 cells in the maintenance of homeostasis, promise to be particularly fruitful areas of future research.

MiR‐155: an ancient regulator of the immune system

MicroRNAs (miRNAs) are a newly recognized class of regulatory genes which repress the expression of protein-coding genes. Numerous studies have uncovered a complex role for miRNAs regulating many aspects of a variety of cellular processes including cell growth, differentiation, and lineage commitment. In the immune system, miR-155 is unique in its ability to shape the transcriptome of activated myeloid and lymphoid cells controlling diverse biological functions ranging from inflammation to immunological memory. Not surprisingly, a tight control of miR-155 expression is required to avoid malignant transformation, as evidenced by miR-155 overexpression in many cancers of B-cell origin. In this review, we discuss the potential of miR-155 as a molecular target for therapeutic intervention and discuss the function of miR-155 in the context of protective immunity. We first look back into the emergence of miR-155 in evolution, which is coincidental with the emergence of the ancestors of the antigen receptors. We then summarize what we have learned about the role of miR-155 in the regulation of lymphoid subsets at the cellular and molecular level in the context of recent progress in this field.

Dual infections with low virulent chicken infectious anaemia virus (lvCIAV) and intermediate infectious bursal disease virus (iIBDV) in young chicks increase lvCIAV in thymus and bursa while decreasing lymphocyte disorders induced by iIBDV

The use of attenuated vaccines or the occurrence of low virulent T-lymphotropic or B-lymphotropic viruses in flocks may alter the immune responses of young chicks in spite of the absence of clinical signs. Infections with a low virulent T-lymphotropic chicken infectious anaemia virus (lvCIAV) followed by infection with an intermediate B-lymphotropic infectious bursal disease virus (iIBDV) were conducted in specific pathogen free chicks. Thirty-six 1-day-old chicks were infected with the lvCIAV strain (CAV-VAC®) and a similar number of chicks were inoculated with phosphate-buffered saline. At 14 days after lvCIAV infection, one group of 18 lvCIAV-infected chicks and one group of 18 uninfected chicks were infected with an iIBDV strain. At 4, 7 and 14 days post infection with iIBDV, six chicks from each group were euthanized and lymphoid organs were collected. Detection of lvCIAV and iIBDV genomes was conducted by polymerase chain reaction and reverse transcriptase-polymerase chain reaction, respectively. Double-labelled lymphoid subsets from the thymus, spleen and bursa were studied by cytofluorometric analysis. The results reveal that previous infection with lvCIAV increases the occurrence of the lvCIAV and iIBDV genome in thymus and/or bursa without the occurrence of clinical signs in dually lvCIAV/iIBDV-infected chicks. However, the decreases of B cells in spleen and bursa and increases of T-cell subsets in bursa observed in chicks infected with iIBDV did not occur in chicks previously infected with lvCIAV. Taken together, these results suggest that previous infection of young chicks with lvCIAV decreases lymphoid disorders induced by iIBDV while subsequent iIBDV infection increases the lvCIAV genome in lymphoid organs.

Single Cell Mass Cytometry of Dysregulated Signaling Networks in Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia

AbstractAbstract ▪703▪This icon denotes a clinically relevant abstractThe classic myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis (PMF) are frequently associated with the JAK2 V617F mutation or other genetic alterations in members of the JAK-STAT axis. These mutations have been shown to cause hyperactivated JAK-STAT signaling in cell lines and mouse models. How accurately these models recapitulate human MPN pathogenesis remains uncertain, as in vivo signaling in MPNs is likely modulated by other genetic changes and regulatory dynamics. In addition, the phenotypic changes that accompany transformation of chronic MPNs to secondary acute myeloid leukemia (sAML) have not been well characterized.While targeted inhibitors of JAK2 have shown activity in MPNs, the incomplete responses observed clinically have called into question the utility of JAK2 as a therapeutic target, suggesting that dysregulation of other signaling pathways may be important in MPN pathogenesis. Therefore, a more complete assessment of JAK-STAT and related signaling pathways in MPNs is needed.Mass cytometry is a novel technology that merges aspects of flow cytometry with mass spectrometry – cells are labeled with antibodies conjugated to elemental isotope reporters and then analyzed on the CyTOF mass cytometer. Each mass channel is distinct, such that no compensation is required, thus circumventing the spectral limitations of fluorescence-based flow cytometry and enabling the simultaneous measurement of 30+ parameters at the single cell level. We have utilized this approach to examine multiple signaling effectors in cell populations throughout hematopoietic differentiation.Our initial experiment included samples from three MPN patients (PV, PMF, post-PV sAML), and one normal donor. Cells were exposed to nine different perturbation conditions ex vivo, including cytokines and the JAK1/2 inhibitor ruxolitinib. Cells were stained with a panel of 17 surface markers and 13 dynamic intracellular signaling effectors and analyzed on the CyTOF. Single cell data was uploaded into SPADE (spanning-tree analysis of density-normalized events), which distills multidimensional data down to interconnected cell subsets and creates 2D tree plots based on shared surface marker expression. These plots identified recognizable cell subsets, including hematopoietic stem/progenitors (HSPCs) and myeloid and lymphoid lineage subsets. Heat maps were constructed to depict the relative induction of each intracellular marker in response to each condition.In the HSPC compartment, several expected responses were observed, particularly in PV. Erythropoietin-mediated activation of STAT3 and thrombopoietin (Tpo)-mediated activation of STAT3/5 were enhanced in PV committed progenitors. On a broader level, PV HSPCs exhibited heightened signaling sensitivities involving several cytokines and downstream effectors. Notably, CREB and S6 phosphorylation were strongly induced by Tpo, G-CSF, and IL-3. Ruxolitinib pre-treatment markedly inhibited signaling mediated by Tpo in PV CD34+ cells, indicating that the HSPC compartment can be effectively targeted by ex vivo JAK1/2 inhibition.In contrast to PV, PMF HSPCs exhibited lesser sensitivity to cytokine stimulation. In several instances, such as IL-3 induction of pSTAT5, the responses were in fact suppressed compared to normal. CD34+ HSPC from the sAML patient generally exhibited subnormal signaling responses. However, widespread hyperactivation following exposure to the phosphatase inhibitor pervanadate (PVO4) was observed in sAML CD34+ cells, suggesting that these signaling pathways were activated in vivo, but that feedback inhibition in response to persistent activity led to downregulation of their ex vivo inducibility.Based on these preliminary findings, we hypothesize that the fundamental chronic phase MPN state is one of heightened cytokine signaling sensitivity, while the advanced phase (especially sAML) state is one of tonic or constitutive downstream signaling activity with persistent feedback inhibition on cytokine signaling pathways. To test this hypothesis, experiments with a larger cohort of MPN samples are currently underway. These studies will provide a comprehensive framework of altered signaling in MPNs and provide deeper insights into the role of targeted therapy for MPNs. Disclosures:No relevant conflicts of interest to declare.

Dissecting Stem Cell Proliferation and Differentiation in Association with the Central Cell Cycle Regulator APC/CCdh1

AbstractAbstract 1236Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation.The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis.To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing.Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures:No relevant conflicts of interest to declare.

Natural Killer T cells – balancing the regulation of tumor immunity

Natural killer T (NKT) cells are one of the minor lymphoid subsets that, similar to γ/δ T cells and NK cells, are emerging as essential factors in the linkage of innate and acquired immunity. This book provides a detailed and comprehensive review of the considerable basic science behind our understanding of the biology of these cells and their role in tumour immunology. The final chapters cover the clinical trials of NKT-activating agents and of adoptive anti-tumour immunotherapy with NKT.

IL-21R EXPRESSION ON CD8+T CELLS PROMOTES CD8+T CELL ACTIVATION IN COXSACKIEVIRUS B3 INDUCED MYOCARDITIS

ObjectivesTo evaluate the role of IL-21 in promoting CD8 T cell mediated cardiac injury in myocarditis.MethodsTo evaluate the role of IL-21 in promoting CD8 T cell mediated cardiac injury in...

Reversal of Paralysis and Reduced Inflammation from Peripheral Administration of β-Amyloid in T H 1 and T H 17 Versions of Experimental Autoimmune Encephalomyelitis

β-Amyloid 42 (Aβ42) and β-amyloid 40 (Aβ40), major components of senile plaque deposits in Alzheimer's disease, are considered neurotoxic and proinflammatory. In multiple sclerosis, Aβ42 is up-regulated in brain lesions and damaged axons. We found, unexpectedly, that treatment with either Aβ42 or Aβ40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aβ42 and Aβ40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive T helper 1 (T(H)1) or T(H)17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aβ42 and Aβ40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major proinflammatory cytokines and chemokines were reduced in the blood after Aβ peptide treatment. Protection conferred by Aβ treatment did not require its delivery to the brain: Adoptive transfer with lymphocytes from donors treated with Aβ42 attenuated EAE in wild-type recipient mice, and Aβ deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aβ treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aβ, there is exacerbated clinical EAE disease progression. Because Aβ42 and Aβ40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.

The bloodline of CD8α(+) dendritic cells.

The immune system is highly coordinated by various cell types. Dendritic cells (DCs) orchestrate immune responses at various stages and bridge innate immunity and adaptive immunity. DCs are a family of cells consisting of various subsets distinguished by surface markers, locations, and transcription factors that govern their development, differentiation, and homeostasis. The complexity of DC subset biology has hindered the understanding of the functional differences among DC subsets. The subset expressing the surface molecule CD8α is of particular interest, due to the efficiency of this DC subset in priming CD8(+) cytotoxic T cells and cross-presenting exogenous antigens to CD8(+) T cells. CD8α(+) DCs maintain tolerance to autologous antigens at steady state, but when activated secrete IL-12, polarizing T helper (Th) 1 responses. Recently, novel DC subsets were found to be present in peripheral tissues and the relationship between CD8α(+) DCs in lymphoid organs and DC subsets in peripheral tissues has been revealed. This review describes the pedigree of CD8α(+) DCs and related subsets, including a history of the discovery of DC subsets and their functional characterization.