Neutrophil Subsets Research Articles

Targeting neutrophilic inflammation in severe neutrophilic asthma: can we target the disease-relevant neutrophil phenotype?

In severe, neutrophilic asthma, neutrophils are thought to have an important role in both the maintenance of the disease and during exacerbations. These patients often display excessive, mucosal airway inflammation with unresolving neutrophilia. Because this variant of asthma is poorly controlled by glucocorticoids, specific pharmacologic targeting of neutrophils seems a plausible therapeutic approach. However, most attempts with this approach have failed in the clinic. We propose that this could be attributed, in part, to an incomplete understanding of the emerging new insights underlying neutrophil homeostasis and life span, neutrophil reverse transmigration, neutrophil phenotypes, and neutrophil transdifferentiation in human health and disease. Of clinical relevance, recent translational studies have started to uncover distinct neutrophil subsets in humans, namely mature and hypersegmented phenotypes that have bimodal immunomodulatory functions during an acute inflammatory response. In this review, we will elaborate on some of the novel insights in neutrophil biology and attempt to translate them into potential consequences for pharmacologic intervention of severe neutrophilic asthma. We speculate that the disease-relevant neutrophil phenotype should be targeted selectively without compromising the immunomodulatory functions essential for homeostasis and pulmonary immunity. However, the identity and exact functional role of distinct neutrophil phenotypes in inflammatory diseases of the human airway remain elusive.

A protective role for splenic neutrophils against Streptococcus pneumoniae infection (INC4P.338)

Abstract The spleen is integral for protection against infections to encapsulated bacteria, such as S.pneumoniae. A recently identified splenic resident neutrophil subset has been proposed to contribute to this protection by modulating the antibody production of marginal zone B cells. To date, little is known regarding the function and behavior of these neutrophils in vivo and whether they actively contribute to protection against infection. Employing intravital microscopy we aimed to characterize splenic neutrophil behavior and function. Under basal conditions, two subsets of neutrophils are observed, a circulating phenotype and a resident phenotype. Infection with S. pneumonia stimulates a robust recruitment of neutrophils to the spleen and an enhanced localization within the marginal zone. This recruitment is accompanied by neutrophil activation typified by a reduction in cellular movement. Neutrophil depletion within the spleen during infection resulted in a reduction in bacterial clearance and a significant decrease in survival. Depletion of marginal zone B cells mimicked these parameters, suggesting both cell types contribute to a common protective mechanism. Our findings suggest that splenic neutrophils play a role in the protection against S. pneumoniae infection. Future studies will determine the contribution of resident vs recruited neutrophils to this protection and the role of these populations in mediating S.pneumoniae specific antibody production.

Pak2 regulates hematopoietic progenitor cell proliferation, survival, and differentiation.

p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.

Regulation of CCN1 (Cyr61) in a porcine model of intestinal ischemia/reperfusion.

Intestinal ischemia is a serious condition that may lead to both local and systemic inflammatory responses. Restoration of blood supply (reperfusion) to ischemic tissues often increases the extent of the tissue injury. Cysteine-rich angiogenic inducer 61 (Cyr61)/CCN1 is an extracellular matrix-associated signaling protein that has diverse functions. CCN1 is highly expressed at sites of inflammation and wound repair, and may modify cell responses. This study aimed to investigate regulation and cellular distribution of CCN1 in intestinal ischemia/reperfusion (I/R) injury in pigs. After intestinal I/R, increased expression of CCN1 was detected by quantitative RT-PCR, Western blot analysis and immunohistochemistry compared with non-ischemic intestine. Immunoflorescence staining revealed that CCN1 was mainly up-regulated in intestinal mucosa after intestinal I/R. Microvillus epithelial cells and vascular endothelial cells were strongly positive for CCN1 in intestinal I/R, while natural killer cells and/or subsets of neutrophils were only modestly positive for CCN1. Furthermore, blood samples taken from the portal and caval veins during ischemia and after reperfusion showed no change of the CCN1 levels, indicating that CCN1 was locally regulated. In conclusion, these observations show, for the first time, that the CCN1 molecule is up-regulated in response to intestinal I/R in a local manner.

Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

AUTOANTIBODY LEVELS INFLUENCES THE NUMBER AND PHENOTYPE OF INFILTRATING NEUTROPHILS IN INFLAMED JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

AUTOANTIBODY LEVELS INFLUENCES THE NUMBER AND PHENOTYPE OF INFILTRATING NEUTROPHILS IN INFLAMED JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

Balancing the innate immune system in tumor development

Cells of the innate immune system have a dual role in cancer development in both tumor initiation and progression. Innate immune cells can, on the one hand, aid malignant transformation and tumor outgrowth and, on the other hand, prevent tumor progression. The innate immune system has the ability to tune the inflammatory response and is a key player in cancer-related inflammation, which can precede the development of malignancy or be induced by oncogenic changes promoting a protumor inflammatory milieu. In this review, we discuss the emerging cellular and molecular mechanisms of the innate immune system and inflammation in tumor initiation and progression, and point to the outstanding questions that remain.

Systemic mastocytosis emerging after azacitidine treatment of refractory anaemia with excess blasts type 2

A 73-year-old man was investigated for a pancytopenia of recent onset. He had no skin lesions, lymphadenopathy or splenomegaly. A full blood count showed a haemoglobin concentration of 94 g/l, neutrophil count of 0 76 9 10/l and platelet count of 40 9 10/l. A bone marrow aspirate showed a hypercelluar marrow with left shifted granulopoiesis and an increased proportion of promyelocytes and myelocytes; a subset of myeloid precursors and mature neutrophils had dysplastic features with hypogranular cytoplasm. There were 17% blast cells, which were intermediate to large in size, with scanty blue agranular cytoplasm and round nuclei that had open reticular chromatin and prominent nucleoli (left); the blast cells were CD34 positive. A diagnosis of refractory anaemia with excess of blasts type 2 (RAEB-2) was made. The patient was treated with azacitidine using a standard protocol for high risk myelodysplastic syndrome (MDS). He responded initially with improved blood counts but clinically became worse. Another bone marrow aspirate performed 5 months later, when neutropenia had resolved, remained hypercellular with a moderate reduction in blast cells (to 9%); new findings were increased mast cells (9%) and eosinophils (18%). Computerized tomography of the abdomen and pelvis demonstrated ascites and splenomegaly that had not been present at diagnosis. Azacitidine treatment was withheld. Another bone marrow apirate 1 month later showed increased abnormal, spindle-shaped mast cells (13%) (right), blasts (3%) and eosinophils (5%). Trephine biopsy demonstrated aggregates of spindle-shaped mast cells in a paratrabecular/multi-focal distribution. Immunohistochemistry showed expression of CD117, tryptase and CD25, while CD34 was negative, confirming these as neoplastic mast cells. Investigation was positive for both the rare KIT D816H mutation, previously reported in a case of systemic mastocytosis (SM) with acute myeloid leukaemia, and KIT K818R, not previously reported in SM. Serum tryptase was elevated to >200 ng/ml. Thus, our patient met the major criteria and all four minor criteria for a diagnosis of SM. CD117, CD25 and tryptase applied to the original biopsy specimen showed no evidence of SM. A diagnosis of systemic mastocytosis with associated clonal haematological non-mast cell disease (SM-AHNMD) was made. The patient deteriorated and died within weeks. SM-AHNMD is the second most common variant of SM, accounting for 40% of all cases of SM. SM-MDS is uncommon and our case is very unusual as SM emerged only after the patient showed a response to azacitidine treatment for high risk MDS. Clonal identity of the two neoplasms was not investigated, and one can only speculate that the pressure of medication could have altered the direction of differentiation of a pluripotent haemopoietic stem cell.

Differential expression of granulopoiesis related genes in neutrophil subsets distinguished by membrane expression of CD177.

ObjectiveDifferential gene expression in CD177+ and CD177− neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV).MethodsNeutrophils were isolated from healthy controls (HC) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177− subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177− subsets in microarray analysis were re-assessed using quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry.ResultsThe proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177− neutrophils resulted in 14 genes with fold change (fc) >3 difference in expression. Interestingly, 10 of these genes have been reported to change significantly in expression during neutrophil maturation, and most of these genes were granule protein (GP) coding genes. mRNA expression levels measured by RT-PCR of a number of these GP, and of PR3 and MPO were higher in the CD177− neutrophil subset in HC, however, particular granule protein amounts were comparable between CD177+ and CD177− neutrophil subsets. AAV patients had higher amounts of CD177+ neutrophils, but contrary to neutrophils from HC expression of GP-genes was increased, possibly due to activation.ConclusionThe neutrophil population can be distinguished by membrane expression of CD177 into subsets that are different in expression of GP mRNA but not in GP protein production. GP gene expression is also elevated in AAV patients, which is not explained by skewed distribution of CD177+ and CD177− subsets but may be associated with neutrophil activation during on-going inflammation.

THU0055 Elevated Cfdna is Associated with Active Lupus Nephritis and May Mainly Derive from Netosis in Systemic Lupus Erythematosus

BackgroundAbnormal formation and insufficient clearance of neutrophil extracellular traps (NETs) has been involved in lupus nephritis (LN). The residual NETs is one potential source of circulating cell-free DNA (cfDNA). However,...

The Ironman Triathlon shifts innate/adaptive immune cell populations and increases systemic inflammation (705.3)

The Ironman Triathlon shifts innate/adaptive immune cell populations and increases systemic inflammation Colleen X. Munoz1, Jenna M. Apicella1, Shlomit Radom‐Aizik2, Carl M. Maresh1, Fadia Haddad2, Dan M Cooper2, Cherryl Nugas2, Frank P Zaldivar2, Douglas J. Casa1, Elaine C. Lee1 1University of Connecticut, Storrs, CT2University of California Irvine, Irvine, CA Exercise and environmental stressors threaten cellular function and integrity. We recently showed that whole body acute stress determines the extent of measurable cellular stress. In this study, we tested the hypothesis that the profile of circulating immune cells, level of systemic inflammation, and chaperone response would reflect the whole body stress of an Ironman. To test this hypothesis, we recruited 40 race participants (29 males, 11 females) and 17 control subjects (9 males, 8 females). Blood was drawn between 2‐3 days before the triathlon (baseline), and during recovery at 1‐, 24‐, and 48‐h after completion. CBC (with differential), multiplex cytometric bead array, and ELISA were used to determine cell counts, inflammatory cytokines, and chaperone HSP70, respectively. Statistical significance was defined by repeated measure ANOVA with Bonferroni‐corrected post hoc tests (p<0.05). Total white blood cell counts increased post‐exercise and returned to normal by 48h post‐race. Neutrophil and monocyte subsets increased post‐exercise and remained elevated even 2 days post‐race, while lymphocyte, eosinophil, and basophil subsets transiently decreased post‐exercise before increasing above normal 24h and 48h later. Inflammatory cytokines IL‐10, IL‐6, and IL‐8 increased >10 fold post‐race and returned to normal by 24h post‐race. HSP70 similarly increased ~2‐fold post‐ race and returned to normal levels by 24h later. The stress of the Ironman event likely shifted the balance of innate/adaptive immune cells, and induced a systemic inflammatory response and chaperone response.Grant Funding Source: Timex, Inc.

The development of giant phagocytes in long-term neutrophil cultures

We tested the hypothesis that in long-term culture conditions, some neutrophils remain viable and participate in debris clearance, and autophagy is involved in their prolonged survival. Neutrophils, classified as professional phagocytes, have the shortest half-life among leukocytes and are constitutively committed to apoptosis. Apoptotic neutrophils are actively removed by Mφ/DCs. However, early and acute inflammatory infiltrates primarily consist of neutrophils. Recently, neutrophils were suggested to facilitate debris clearance at inflammatory sites when the Mφ/DC system is insufficient. Here, purified CD15(+)/CD66b(+)/CD63(+) neutrophils were followed up to 7 days in culture using light, time-lapse, and confocal microscopy. After 3 days in culture, Annexin-V(-)/LC3B(+) large vacuolated cells, engulfing cellular residues, were noted among apoptotic neutrophils and cell debris. Thereafter, these cells were vastly enlarged and exhibited a neutrophilic phenotype (CD15(+)/CD63(+)/MPO(+)/CD66b(+)), phagocytosis, and oxidative burst activity. They also expressed CD68 scavenger receptors and internalized oxLDL. But, unlike in fresh neutrophils or cultured monocytes, oxLDL treatment increased their ROS production. Additionally, these phagocytes contained LC3B-coated vacuoles and LC3B aggregates, indicating the activation of autophagy. An intensive LC3B accumulation was also noted during oxLDL internalization. Importantly, the inhibition of autophagy by 3-MA or BafA1 prevented their development. In conclusion, the internalization of neutrophil remnants may induce activation of autophagic mechanisms in some neutrophil subsets or precursors. This may lead to cell adaptation and survival, resulting in their transformation into long-lived Gφ and potentially suggesting their involvement in inflammatory/anti-inflammatory processes.

Characterization of Neutrophil Subsets in Healthy Human Pregnancies

We have previously shown that in successful pregnancies increased arginase activity is a mechanism that contributes to the suppression of the maternal immune system. We identified the main type of arginase-expressing cells as a population of activated low-density granulocytes (LDGs) in peripheral blood mononuclear cells and in term placentae. In the present study, we analyzed the phenotype of LDGs and compared it to the phenotype of normal density granulocytes (NDGs) in maternal peripheral blood, placental biopsies and cord blood. Our data reveal that only LDGs but no NDGs could be detected in placental biopsies. Phenotypically, NDGs and LDGs from both maternal and cord blood expressed different levels of maturation, activation and degranulation markers. NDGs from the maternal and cord blood were phenotypically similar, while maternal, cord and placental LDGs showed different expression levels of CD66b. LDGs present in cord blood expressed higher levels of arginase compared to maternal and placental LDGs. In summary, our results show that in maternal and cord blood, two phenotypically different populations of neutrophils can be identified, whereas in term placentae, only activated neutrophils are present.

Role of neutrophils in acquired immune responses

Neutrophils play important roles not only in innate immune responses but also in acquired immune responses.As newly defined professional antigen presenting cells(APCs),neutrophils can present antigens to T cells and induce T cell differentiation.Splenic neutrophils have also been shown to support T cell-independent antibody responses by direct activation of marginal zone(MZ)B cells.A subset of human neutrophils with phenotype of CD11cbright/CD62Ldim/CD11bbright/CD16 bright can suppress T cell activation in response to acute inflammation.These new findings about neutrophils can help us to better understand the mechanisms of chronic inflammatory diseases,autoimmune diseases,and cancer,and may also eventually contribute to the discovery of new therapy targets for these diseases.

Neutrophil Aging, Regulated By Microbiota-Derived Signals, Promotes Sickle Cell Vaso-Occlusion

Vaso-occlusion is one of the most common complications in sickle cell disease (SCD). Our previous studies have shown that neutrophils play an important role in promoting sickle cell vaso-occlusion by capturing sickle red blood cells (sRBC) through activated αMβ2 integrin (Mac-1). However, high Mac-1 activity was only observed in a small subset of adherent neutrophils (Nat. Med. 2009;15:384). Recent studies have shown that a CD62Llow CXCR4high subset of neutrophils represents the cells that have truly aged in vivo (Cell. 2013;153:1025). Since aged neutrophils may have experienced activation signals when they survey the whole body, we hypothesized that they are over-active cells that promote sickle cell vaso-occlusion. To test this possibility, we first analyzed the correlation between CD62L expression and Mac-1 activity of adherent neutrophils using multichannel fluorescence intravital microscopy and albumin-coated fluosphere beads that specifically bind activated Mac-1. We found a strong inverse correlation where neutrophils with lower CD62L expression showed greater bead binding capacity (P < 0.001). Mice deficient in P-selectin (Selp-/-), an adhesion molecule required for neutrophil clearance from the circulation, showed a dramatic increase in the percentages of CD62Llow CXCR4high aged neutrophils (wild-type / Selp-/-: 9.0 ± 1.0% / 74.7 ± 2.1%, P < 0.01). When purified neutrophils from Selp-/- mice were transferred into wild-type recipients, these aged neutrophils exhibited significantly higher Mac-1 activity compared to those purified from wild-type animals (wild-type / Selp-/-: 0.26 ± 0.06 / 1.52 ± 0.35 beads per adherent neutrophil, P < 0.05). Since microbial products derived from the microbiota could translocate into the system and modulate the innate immunity (Nat. Rev. Microbiol. 2011;9:233), we hypothesized that microbiota-derived signals could regulate the neutrophil aging process. To test this idea, we depleted the microbiota by treating mice with ampicillin, neomycin, metronidazole and vancomycin for 4 weeks. In these mice, the numbers of CD62Llow CXCR4high aged neutrophils were significantly decreased, and this reduction was reversible by intragastric gavage of Lipopolysaccharide (LPS; Control / Antibiotics-treated (ABX) / ABX + LPS: 87.2 ± 20.3 / 12.9 ± 2.0 / 83.9 ± 60.0 cells / μL blood, P < 0.05 between first two groups). To analyze the kinetics of neutrophil aging in vivo, we transferred blood from CD45.1+ mice into CD45.2+ control or antibiotics-treated mice, and monitored the percentages of CD62Llow CXCR4high population in CD45.1+ donor neutrophils. Strikingly, the aging process of donor neutrophils was significantly slower in antibiotics-treated recipients (Control / ABX: 78.2 ± 3.4% / 45.7 ± 7.7%, 5h after transfer, P < 0.01; 95.5 ± 0.7 / 67.1 ± 7.8%, 9h after transfer, P < 0.05). Since MyD88 mediates the signaling of most toll-like receptors (TLRs), we analyzed the neutrophil aging phenotypes in LysM-Cre/MyD88-flox mice, in which MyD88 is specifically deleted in the myeloid lineage. Similarly, we observed a dramatic decrease in the numbers of aged neutrophils in these mice (wild-type / LysM-Cre/MyD88-flox: 70.1 ± 22.2 / 23.8 ± 3.7 cells / μL blood, P = 0.08), and also a significantly slower aging process when we transferred blood from LysM-Cre/MyD88-flox mice into wild-type recipients (wild-type / LysM-Cre/MyD88-flox: 15.0 ± 3.1% / 5.0 ± 1.0%, 10min after transfer, P=0.09; 49.7 ± 5.2% / 12.0 ± 1.5%, 5h after transfer, P < 0.05; 59.6 ± 2.0% / 19.5 ± 3.2%, 9h after transfer, P < 0.01). To test whether neutrophil aging was relevant in SCD, we depleted the microbiota in a humanized SCD mouse model (“Berkeley” mice). Five weeks after depletion, we observed a dramatic decrease in the numbers of aged neutrophils (Control SCD / ABX SCD: 4392 ± 574 / 819 ± 358 cells/ μL blood, P < 0.01), and a significant improvement of splenomegaly (Control SCD / ABX SCD: 691.7 ± 46.4 / 453.5 ± 28.5 mg, P<0.05). Further, preliminary data indicate that microbiota depletion could protect against vaso-occlusive crisis induced by TNF-α and surgical trauma, leading to prolonged survival. Taken together, these data suggest that aged neutrophils, modulated by microbiota-derived signals, represent an over-active subset of neutrophils that promotes sickle cell vaso-occlusion. Gaining understanding of neutrophil aging may lead to novel ways to control the manifestations of the disease and its complications. Disclosures: No relevant conflicts of interest to declare.

Double-Unit Cord Blood (CB) Transplantation Combined With Haplo-Identical CD34+ Cell-Selected PBSC Results In 100% CB Engraftment With Enhanced Myeloid Recovery

BackgroundDouble-unit CB transplantation (DCBT) has provided high rates of sustained donor engraftment in patients with hematologic malignancies. However, delayed engraftment is frequent with a median neutrophil & platelet recovery of 25 & 48 days, respectively, in adult DCBT recipients at our center. This delay is associated with increased transplant-related mortality (TRM). It is also associated with prolonged hospitalization with a median discharge time of +42 days (range 25-76) in recent adult myeloablative DCBT recipients. MethodsWe investigated the combined transplantation of a 4-6/6 HLA-A,-B antigen, -DRB1 allele matched double-unit CB allograft (infused on day 0) with peripheral blood stem cell derived Miltenyi column selected haplo-identical CD34+ cells (haplo-CD34+, infused on day 0 or +1) to speed myeloid recovery. We used DCB grafts to facilitate comparison with historic/concurrent DCB controls transplanted without haplo-CD34+. ResultsOf 23 protocol eligible patients, 6/23 (26%) underwent DCBT only due to the lack of any suitable haplo-identical donor. Thus, 17 patients [median 39 years (range 16-69), median 78 kg (range 63-133)] were transplanted 9/2012-6/2013 with DCB plus haplo-CD34+ cells for high-risk hematologic malignancies. Diagnoses included 12 acute leukemias & 5 lymphomas. Conditioning was myeloablative with CSA/MMF immune suppression & no ATG. Median infused CB TNC x 107/kg was 2.29 (larger unit, range 1.73-2.95) & 1.82 (smaller unit, range 1.26-2.48). Haplo-identical donors (median 37 years, range 19-71) had a median donor-recipient HLA-match of 5/10 (range 5-7). 15 patients received the targeted infused haplo-CD34+ cell dose of 3 x 106/kg whereas 2 each received haplo-CD34+ cell doses of 1 x 106/kg. The median infused haplo-CD3+ dose was 0.6 x 103/kg (range 0.3-1.6). One patient died on day 14. Of 16 remaining evaluable patients, all (100%) engrafted with a median neutrophil recovery of 13.5 days (range 11-31) in 14 patients who received 3 x 106/kg haplo-CD34+ cells, and 26 and 18 days in the 2 patients who received 1 x 106/kg haplo-CD34+ cells. Platelet recovery ≥ 20 × 109/l has occurred in 12/15 patients (median 27 days, range 18-46) to date. Serial chimerism results demonstrating the contribution of haplo-CD34+ cells & each CB unit to date is shown (Table). While myeloid recovery on day 14 was predominantly haplo-CD34+ cell mediated, one CB unit dominated by day 28 in both neutrophil & T-cell subsets. The median total donor chimerism was 100% the dominant CB unit by day 100. With a median follow-up of survivors of 5 months (range 1-10), to date 9 of 15 evaluable patients have developed grade II-IV aGVHD by day 100 (7 grade II, 1 grade III, 1 grade IV). One patient with refractory leukemia transplanted with disease has relapsed, & 4 have died of TRM (2 organ failure, 1 grade IV aGVHD, 1 CMV infection). Excluding early deaths, of patients who were discharged in the first 100 days (n = 13), the median day of discharge was day +33 (range 21-60). ConclusionsDouble-unit CBT supplemented by haplo-CD34+ cells is safe. The incidence of neutrophil engraftment is high & the speed of neutrophil recovery is enhanced compared with recent DCBT controls. A shorter time of initial hospitalization (9 days) has offset the cost of the addition of haplo-CD34+ cells. It is intriguing that the dominant CB unit can rapidly reject the haplo-identical donor. This may be facilitated by omission of ATG, and the determinants of the speed of haplo-donor rejection are under investigation. Whether the same results could be achieved with a single CB unit plus haplo-CD34+ cells requires investigation. Addition of haplo-CD34+ cells is also an alternative to expansion, although expansion remains an important strategy to augmenting myeloid recovery given some patients do not have any suitable haplo-identical donors. Disclosures:No relevant conflicts of interest to declare.

IFN-γ-Stimulated Neutrophils Suppress Lymphocyte Proliferation through Expression of PD-L1

During systemic inflammation different neutrophil subsets are mobilized to the peripheral blood. These neutrophil subsets can be distinguished from normal circulating neutrophils (CD16bright/CD62Lbright), based on either an immature CD16dim/CD62Lbright or a CD16bright/CD62Ldim phenotype. Interestingly, the latter neutrophil subset is known to suppress lymphocyte proliferation ex vivo, but how neutrophils become suppressive is unknown. We performed transcriptome analysis on the different neutrophil subsets to identify changes in mRNA expression that are relevant for their functions. Neutrophil subsets were isolated by fluorescence-activated cell sorting from blood of healthy volunteers that were administered a single dose of lipopolysaccharide (2 ng/kg i.v.) and the transcriptome was determined by microarray analysis. Interestingly, the CD16bright/CD62Ldim suppressive neutrophils showed an interferon-induced transcriptome profile. More importantly, IFN-γ, but not IFN-α or IFN-β stimulated neutrophils, acquired the capacity to suppress lymphocyte proliferation through the expression of programmed death ligand 1 (PD-L1). These data demonstrate that IFN-γ-induced expression of PD-L1 on neutrophils enables suppression of lymphocyte proliferation. Specific stimulation of neutrophils present at the inflammatory sites might therefore have a pivotal role in regulating lymphocyte-mediated inflammation and autoimmune disease.

The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon in vivo transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue in vivo. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.

Modulation of granulocyte kinetics by GM-CSF/IFN-γ in a human LPS rechallenge model

Inflammation in response to infection or trauma can lead to CARS, which is characterized by leukocyte dysfunction. In this study, we used a human model system for CARS to study the effect of GM-CSF and IFN-γ treatment on this immunoparalyzed state. Healthy human volunteers were treated with GM-CSF (4 μg/kg), IFN-γ (100 μg), or placebo in between two challenges with Escherichia coli LPS/endotoxin (2 ng/kg). Serial leukocyte blood counts were measured. Neutrophil subsets were discriminated using CD16 and CD62L expression. LPS rechallenge resulted in increased mobilization of mature neutrophils, whereas banded neutrophils decreased. GM-CSF and IFN-γ treatment did not restore these changes. GM-CSF treatment did, however, increase the number of CD16(bright)/CD62L(dim) neutrophils that were previously shown be able to suppress T cell proliferation. IFN-γ treatment decreased neutrophilia seen after LPS rechallenge. Our study shows that LPS rechallenge was associated with changes in the distribution of neutrophil subsets, whereas no additional changes in kinetics of other granulocyte populations were observed. GM-CSF and IFN-γ treatment induced a shift in granulocyte composition toward an anti-inflammatory direction by increasing CD16(bright)/CD62L(dim) cells or decreasing neutrophil counts, respectively.

SP0181 Detrimental effects of interferon alpha on angiogenesis

Patients with systemic lupus erythematosus (SLE) have up to a 50-fold increased risk of developing atherosclerotic cardiovascular disease. Traditional CV risk factors seem to be less important predictors of CV...