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We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-C5ar1(fl/fl)) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice. C5ar1(fl/fl).Lyz2Cre(+/−) mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1(fl/fl) mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-β(+) GFP(+) cells, we developed C5ar1(fl/fl).Foxd1Cre(+/−) mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-β(+) pericytes isolated from FA-treated C5ar1(fl/fl).Foxd1Cre(+/−) mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1(fl/fl) mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis. NEW & NOTEWORTHY This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated

The COVID-19 pandemic has brought biosafety to the forefront of many life sciences The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs) In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument ?failure modes? Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in ?failure modes? These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated This article is protected by copyright All rights reserved

The type I IFN (IFN-α) response is crucial for viral clearance during primary viral infections. Plasmacytoid dendritic cells (pDCs) are important early responders during systemic viral infections and, in some cases, are the sole producers of IFN-α. However, their role in IFN-α production during memory responses is unclear. We found that IFN-α production is absent during a murine viral memory response, despite colocalization of virus and pDCs to the splenic marginal zone. The absence of IFN was dependent on circulating Ab and was reversed by the transgenic expression of the activating human FcγRIIA receptor on pDCs. Furthermore, FcγRIIB was required for Sendai virus immune complex uptake by splenic pDCs in vitro, and internalization via FcγRIIb prevented cargo from accessing TLR signaling endosomes. Thus, pDCs bind viral immune complexes via FcγRIIB and prevent IFN-α production in vivo during viral memory responses. This Ab-dependent IFN-α regulation may be an important mechanism by which the potentially deleterious effects of IFN-α are prevented during a secondary infection.

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid-protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcγRγ and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.

A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.

NK-cells undergo a "licensing" process as they develop into fully-functional cells capable of efficiently killing targets. NK-cell differentiation is accompanied by an increased surface expression of inhibitory killer immunoglobulin-like receptor (KIR) molecules, which is positively associated with cytotoxicity against the HLA-deficient K562 cell line. NK-cells are rapidly redeployed between the blood and tissues in response to acute exercise, but it is not known if exercise evokes a preferential trafficking of differentiated NK-cells or impacts NK-cell cytotoxic activity (NKCA) against HLA-expressing target cells. Sixteen healthy cyclists performed three 30-min bouts of cycling exercise at -5%, +5%, and +15% of lactate threshold. Blood samples obtained before, immediately after, and 1h after exercise were used to enumerate NK-cells and their subsets, and determine NKCA and degranulating subsets (CD107+) against cell lines of multiple myeloma (U266 and RPMI-8226), lymphoma (721.221 and 221 AEH), and leukemia (K562) origin by 4 and 10-color flow cytometry, respectively. Exercise evoked a stepwise redeployment of NK-cell subsets in accordance with differentiation status [highly-differentiated (KIR+/NKG2A-) >medium-differentiated (KIR+/NKG2A+)>low-differentiated (KIR-/NKG2A+)] that was consistent across all exercise intensities. NKCA per cell increased ∼1.6-fold against U266 and 221 AEH targets 1h post-exercise and was associated with a decreased proportion of NK-cells expressing the inhibitory receptor CD158b and increased proportion of NK-cells expressing the activating receptor NKG2C, respectively. We conclude that exercise evokes a preferential redeployment of NK-cell subsets with a high differentiation phenotype and augments cytotoxicity against HLA-expressing target cells. Exercise may serve as a simple strategy to enrich the blood compartment of highly cytotoxic NK-cell subsets that can be harvested for clinical use.

Previous studies have identified a novel food allergy driven by IgE antibodies specific for galactose-α-1,3-galactose (alpha-gal), an oligosaccharide found in red meat. While it is known that B cells play an important role in allergy as the producers of IgE antibodies that drive the allergic response, little is known about the phenotype of these B cells. The number of markers used to identify the major human B cell subsets by flow cytometry has been limited to common B cell proteins and thus precludes high dimensional immune phenotyping of B cell subsets, including unique phenotypes present in allergic individuals. We have addressed this problem by using mass cytometry (CyTOF), which enables the simultaneous analysis of up to 40 markers in a single staining panel. Here we analyzed the expression of 23 cell surface markers in PBMCs from 19 alpha-gal-allergic patients and 20 non-allergic controls by CyTOF. Additionally, we combined our CyTOF data with clinical endpoints to identify markers that may correlate with allergic disease. Our data reveals substantial heterogeneity within major B cells subsets on an individual level. Furthermore, our analysis identifies a number of markers that vary significantly in their expression in allergic versus non-allergic B cells and correlate with serum alpha-gal IgE titers. We hypothesize that B cells with this phenotype play an important role in mediating alpha-gal allergy. These findings demonstrate the power of using CyTOF and analytical tools to extract a hierarchy from high dimensional cytometry data in an unsupervised manner to identify known B cell subsets as well as to find novel B cell populations that differ between alpha-gal allergic and non-allergic individuals.

Macrophages are innate immune cells that play key roles in regulation of the immune response and in tissue injury and repair. In response to specific innate immune stimuli, macrophages may exhibit signs of endoplasmic reticulum (ER) stress and progress to apoptosis. Factors that regulate macrophage survival under these conditions are poorly understood. In this study, we identified B cell adapter protein (BCAP), a p85 PI3K-binding adapter protein, in promoting survival in response to the combined challenge of LPS and ER stress. BCAP was unique among nine PI3K adapter proteins in being induced >10-fold in response to LPS. LPS-stimulated macrophages incubated with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor that induces ER stress, underwent caspase-3 activation and apoptosis. Macrophages from BCAP−/− mice exhibited increased apoptosis in response to these stimuli. BCAP-deficient macrophages demonstrated decreased activation of Akt, but not ERK, and, unlike BCAP-deficient B cells, expressed normal amounts of the NF-κB subunits, c-Rel and RelA. Retroviral transduction of BCAP-deficient macrophages with wild-type BCAP, but not a Y4F BCAP mutant defective in binding the SH2 domain of p85 PI3K, reversed the proapoptotic phenotype observed in BCAP-deficient macrophages. We conclude that BCAP is a nonredundant PI3K adapter protein in macrophages that is required for maximal cell survival in response to ER stress. We suggest that as macrophages engage their pathogenic targets, innate immune receptors trigger increased expression of BCAP, which endows them with the capacity to withstand further challenges from ongoing cellular insults, such as ER stress.

The immunosuppressive function of IL10 producing regulatory B cells (Bregs) has been shown in several murine models of inflammation and autoimmune disease. However, there is a paucity of data regarding the existence of an equivalent regulatory B cell subset in healthy individuals and their potential role in the pathogenesis of chronic graft-versus-host disease (cGVHD) remains unknown. Here, we examined the functional regulatory properties of peripheral blood (PB)-derived human B cell subsets from healthy individuals. In addition, we carried out studies to explore their role in cGVHD, using B cells from patients following allogeneic stem cell transplantation (HSCT). We first determined whether human IL-10 producing B cells are enriched within any othe previously described human B cell subsets: CD19+IgM+CD27+ IgM memory, CD19+IgM-CD27+ switched memory, CD19+IgM+CD27- naive, and and transitional CD19+CD24hiCD38hi. Following in vitro stimulation with CD40 ligand, the majority of IL-10 producing B cells were found within the CD24hiCD38hi transitional and CD19+IgM+CD27+B cell subsets. We next assessed the regulatory properties of the PB-derived B cell subsets, by sort-purifying IgM memory (CD19+IgM+CD27+), switched memory (CD19+IgM-CD27+), naïve (CD19+IgM+CD27-) and transitional (CD19+CD24hiCD38hi) B cells from healthy controls, and cultured them 1:1 with autologous magnetic-bead purified CD4+ T cells. CD3/CD28 stimulated CD4+ T cells cultured with either CD19+IgM+CD27- naïve or CD19+IgM-CD27+ switched memory B cells proliferated to the same extent and produced equivalent amounts of IFN-γ to cultures containing CD4+ T cells alone. In contrast, culture of CD4+ T cells with IgM memory and transitional B cells significantly suppressed CD4+ T cell proliferation [median percent proliferating CD4+ T cells 52.5%; (33%-75%)] and 51% (25%-63%)], respectively when compared with CD3/CD28 stimulated CD4+ T cells (positive control) [89.5% (75%-92%], p=0.0001. The inhibitory effect of IgM memory and transitional B cells on CD4+ T cell proliferation was cell dose dependent with the highest suppression observed at a ratio of 1:1. These data suggest that human PB transitional and IgM memory B cells are endowed with regulatory function. We next examined if the in vitro suppressive effect of transitional and IgM memory B cells is mediated by regulatory T cells (Tregs). For this purpose, CD4+ T cells were depleted of CD127lo CD25hi CD4+ T cells by magnetic cell purification. B cell subsets were cultured with CD3/CD28 stimulated CD4+ CD25- T cells at a ratio of 1:1. IgM memory and transitional B cells were able to significantly suppress the proliferation and Th1 cytokine response by CD4+ CD25- T cells compared to cultures containing CD4+ CD25-T cells alone, indicating that the suppressive activity of Bregs is independent of Tregs. To further understand the underlying mechanims though which Bregs exert T-cell suppression, we used antibody blockade experiments and showed that this suppressive effect was mediated partially via the provision of IL-10, but not TGF-ß. Using transwell experiments, we further determined that the suppressive function of Bregs is also partly dependent on direct T cell/B cell contact. We next assessed whether the activity of Breg cells might be altered in patients with cGVHD. B cells from patients with cGVHD were refractory to CD40 stimulation and produced less IL-10 when compared to patients without cGVHD post-SCT and healthy controls, [1.02% (0.22-2.26) vs.1.72% (0.8-5.52) vs. 2.16 (1.3- 5.6), p=0.001]. Likewise, the absolute number of IL-10 producing B cells was significantly lower in cGvHD patients compared to patients without cGVHD and healthy controls (p=0.007), supporting both a qualitative and quantitative defect in IL-10 producing B cells in cGvHD. Our combined studies provide important new data defining the phenotype of B cell populations enriched in regulatory B cells in healthy humans and provide evidence for a defect in the activity of such cells in patients with cGVHD post-SCT. In association with previous reports showing defects in Treg cell activity in GVHD, our results suggest the existence of a broad range of deficiencies in immune regulatory cell function in cGvHD patients. * Both Anushruti Sarvaria and Ahmad K contributed equally. Disclosures: No relevant conflicts of interest to declare.

CD4+ T cells are important in the establishment of long-lived pathogen-specific immunity. However, the mechanisms by which antigen specific CD4 T resist insult by lymphocytotoxic agents and are sustained long-term is not well defined. A recent report described the existence of a subset of long-lived CD8+ memory T cells with stem-like properties (Turtle et al, 2009), including the ability to efflux cellular toxins through the ABC–superfamily multidrug efflux protein ABCB1. We hypothesized that a similar subset of T cells with drug-effluxing properties also exists within the CD4+ T cell compartment. We used multiparameter flow cytometry to measure the capacity of CD4+ T cells from donors to efflux the fluorescent substrate Rh123. We identified a subset of memory CD4+ T cells with rapid drug-effluxing ability, defined as CD161+CD95+CD45RA-CD127hiCD28+CD25int that shared remarkable phenotypic similarity to CD8+drug-effluxing memory T cells. The stem cell marker c-kit was preferentially expressed on Rh123 effluxing CD4+CD161+ T cells, whereas CD57, a marker of terminal differentiation, was exclusively expressed on non-effluxing CD4+CD161+ T cells. Rh123 effluxing CD4+CD161+ T cells also displayed differential expression of CD31, CD38, CD58, CD122 and IL-18RA. Rh123 effluxing CD4+ CD161+ T cells were undetectable in cord blood, but found in adult blood, consistent with the emergence of this subset of memory T cells as a consequence of antigen exposure during childhood and adult life. We reasoned that this subset may be enriched within the viral-specific T cell repertoire. Indeed, CMV-specific CD4+ T cells were found to share the same phonotypic markers as Rh123 effluxing CD4+CD161+ T cells. We purified CMV-specific CD4+ T cells using the interferon gamma capture assay (Miltenyi), and showed that CMV-enriched CD4+T cells preferentially and rapidly efflux Rh123. The high ABCB1-mediated drug efflux capacity of CD4+ CD161+ memory cells also facilitated their in vitro resistance to daunorubicin, which was abrogated by competitive inhibitors of ABCB1. In keeping with the in vitro data, we found a significant increase in the frequencies of CMV-specific CD4+ T cells in the peripheral blood of patients with AML after recovery from remission induction chemotherapy, suggesting that CMV-specific CD4+ T cells can preferentially survive and proliferate following chemotherapy. Since interleukin (IL)-7 and IL-15 drive the proliferation of T cells during lymphopenia to restore homeostasis, we assessed the response of CD4+CD161+ T cells to stimulation with CD3/CD28 +IL7 and IL15. Both effluxing and non-effluxing sort-purified central and effector memory CD4+CD161+ T cells proliferated and upregulated Ki67 in vitro. Whereas CD4+CD161+ T cells were able to differentiate into CD4+CD161- T cells, a subset retained CD161 expression. These data suggest that although CD4+CD161+ T cells share phenotypic similarities with terminally differentiated cells, they are able to fully proliferate, differentiate to CD161-ve cells and self-renew to preserve the pool of memory T cells CD161 is also a hallmark of Th17 cells. We examined the cytokine profile of CD4+CD161+ T cells stimulated with a pool of overlapping MHC class II CMV pp65 peptides. After 6 and 24 hrs of in vitro stimulation we failed to detect significant IL-17 production. Furthermore, by real time qPCR, the Th1 transcription factor Tbet, rather than RORC2 (a Th17 hallmark), was found to be preferentially expressed in CMV enriched CD4+CD161+ T cells, indicating that CMV-specific CD4+CD161+T cells in fact represent a unique subset of Th1 cells, distinct from Th17 cells. Our data delineate novel findings related to a distinct subset of drug-effluxing CD4+CD161+ viral-specific memory T cells. Signaling pathways leading to CD4+CD161+ABCB1+ differentiation, the role of this subset in drug resistance and the presence or absence of “stemness” which may impart this subset with extended longevity are being explored. †Muharrem Muftuoglu and Abdullah Alsuliman contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Multiple myeloma (MM) plasma cells are equipped with an extensive endoplasmic reticulum (ER) for antibody production. Misfolding of newly synthesized immunoglobulins leads to ER stress and cell death. Plasma cells evade this by enhancing protein degradation through the proteasome pathway and the autophagy machinery. Because of this, MM cells are sensitive to proteasome inhibitors (PIs). However, autophagy induced by PIs and by reactive oxygen species (ROS) can increase drug resistance and lead to eventual relapse. We have previously demonstrated in vitro and in vivo sensitivity of MM cells to cord blood-derived natural killer (NK) cells, and have observed that this effect may involve NKG2D and NKP30. In this study we used confocal microscopy, chromium cytotoxicity assays and flow cytometry and we aimed to determine if there is a unique mechanism of NK cell-mediated cytotoxicity on MM cells involving ER stress, ROS and its dependence on NK cell receptors NKG2D and NKp30. We first demonstrated a difference in NK-mediated killing of MM cells vs K562 cells. As expected inhibition of GranzymeB (GrB) and Caspase3 pathway yielded a 13% reduction in NK-cytotoxicity against K562 cells; however, a 12% increase in killing was seen against ARP1 cells (p In summary, we describe a new mechanism of killing of MM cells by NK cells mediated by increased ER stress, decreased autophagy and dependent on NKG2D and NKP30. Furthermore, this cytotoxicity can be transmitted between MM cells. These findings could lead to new cellular therapeutic strategies for MM treatment. Disclosures: Shah: Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Background Natural killer (NK) cells are a key component of innate immunity, with the potential to recognize and kill transformed malignant cells without prior sensitization. A balance between activating and inhibitory signals from cell surface receptors determines NK cell cytotoxicity and cytokine release. Therapeutic approaches to augmenting NK cell function are being explored in various malignancies. Little is known about NK phenotype and function in patients with childhood acute lymphoblastic leukemia (ALL), the most common childhood cancer. Here we describe an inhibitory phenotype and impaired cytolytic function in NK cells from pediatric ALL patients at diagnosis, compared with healthy pediatric controls. Restoring NK function may be a useful therapeutic approach in ALL. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 25 patients with newly diagnosed B-ALL, age 1-16 years, and 7 healthy controls, age 2-13 years, in order to compare NK cell frequency, immunophenotype, and functional activity. NK frequency was assessed by flow cytometric staining for CD56+CD3- cells. NK phenotype was assessed by surface expression of activating receptors NKp30, NKp44, NKp46 and NKG2D and inhibitory receptors KIR2DL1/S1, KIR2DL2/S2, KIR3DL1 and NKG2A. Functional activity was determined by incubation of NKs with target cells, followed by flow cytometric measurement of degranulation (surface CD107a) and cytokine release (intracellular IFNg and TNFa). Targets included the MHC class I deficient K562 cell line and, where available, autologous ALL blasts. Results ALL patients demonstrated significantly lower absolute NK cell counts compared with healthy controls (mean absolute count 168 vs. 406 cells/uL, p = 0.0002). They also exhibited significantly fewer NK cells expressing the activating marker NKp46 (mean absolute count 70 vs. 165, p = 0.016); and a significantly higher percentage of cells expressing the inhibitory marker NKG2A (mean 20.5% vs. 1.95% in controls, p = 0.012) (Fig 1A). In co-culture assays with K562 target cells, ALL patients' NK cells demonstrated inferior degranulation and cytokine release compared to healthy controls (representative data in Fig 1B; mean IFNγ production of 1.2% vs. 4.8%, p = 0.02; mean TNFα production of 1.8% vs. 3.8%, p = 0.06; and mean surface CD107a of 5.4% vs. 15.1%, p = 0.08). ALL samples (n = 3) demonstrated little to no cytokine release when incubated with autologous blasts compared with the response elicited by PMA-ionomycin (representative data in Fig 1C; mean CD107a 0.92% vs. 7.85%, p = 0.04; mean IFNγ 0.26% vs 40.47%, p = 0.10; mean TNFα 0.2% vs 41%, p = 0.008). Conclusion At diagnosis, pediatric ALL patients exhibit a lower frequency of NK cells, an inhibitory phenotype, and decreased cytolytic activity compared to healthy pediatric controls, particularly against autologous leukemic blasts. These results suggest that augmentation of the NK response may be useful therapeutically to improve outcomes in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Colonna, L., G. Catalano, C. Chew, V. D'Agati, J. W. Thomas, F. S. Wong, J. Schmitz, E. S. Masuda, B. Reizis, A. Tarakhovsky, and R. Clynes. Therapeutic targeting of Syk in autoimmune diabetes. J. Immunol . 185: [1532–1543][1]. The original description of the statistical analysis in the Materials

In APCs, the protein tyrosine kinase Syk is required for signaling of several immunoreceptors, including the BCR and FcR. We show that conditional ablation of the syk gene in dendritic cells (DCs) abrogates FcγR-mediated cross priming of diabetogenic T cells in RIP-mOVA mice, a situation phenocopied in wild-type RIP-mOVA mice treated with the selective Syk inhibitor R788. In addition to blocking FcγR-mediated events, R788 also blocked BCR-mediated Ag presentation, thus broadly interrupting the humoral contributions to T cell-driven autoimmunity. Indeed, oral administration of R788 significantly delayed spontaneous diabetes onset in NOD mice and successfully delayed progression of early-established diabetes even when treatment was initiated after the development of glucose intolerance. At the DC level, R788 treatment was associated with reduced insulin-specific CD8 priming and decreased DC numbers. At the B cell level, R788 reduced total B cell numbers and total Ig concentrations. Interestingly, R788 increased the number of IL-10–producing B cells, thus inducing a tolerogenic B cell population with immunomodulatory activity. Taken together, we show by genetic and pharmacologic approaches that Syk in APCs is an attractive target in T cell-mediated autoimmune diseases such as type 1 diabetes.