Role Of CD38 Research Articles

333 HCV-INFECTION IS NOT A MAJOR DETERMINANT OF IMMUNESENESCENCE IN AGING DRUG USERS

increased the in vitro anti-HCV activity of NK cells. A role for CD27 in anti-HCV NK cell activity was furthermore supported by the observation that in HIV(+) patients with acute hepatitis C significantly higher frequencies of CD27+ NK cells were found in patients who were subsequently able to clear HCV infection than in those becoming chronically infected. Discussion: Our data indicate that CD27(+) NK cells display antiHCV activity via robust secretion of IFN-g after binding to CD70. In this way, co-stimulation of NK cells via CD27 may be involved in spontaneous clearance of acute hepatitis C in HIV(+) patients.

Infant B-Cell Signaling after ABO-Incompatible Heart Transplantation (ABOi HTx): Defining the Role of the Inhibitory Molecule CD22

Purpose Immune immaturity allows ABOi HTx to be performed safely in infants and results in B-cell tolerance to donor ABO antigens (Ags). In experimental settings, T-independent (TI) B-cell activation has been shown to be inhibited by binding of the B-cell co-receptor CD22 to CD22 ligands leading to B-cell tolerance. Since immunity to TI Ags, including ABO Ags, are generally reduced in early childhood, we hypothesize a role for CD22, possibly contributing to ABOi HTx tolerance. Previously, we showed that CD22 expression is higher on CD27+IgM+ B-cells in infants when compared to older individuals. In this study we performed functional assays with CD27+IgM+ B-cells to assess the presence of ABO Ag-specific IgM antibody (Ab)-secreting cells (ASC). In addition, we established a phospho-flow assay (PPF) to assess B-cell signaling. Methods and Materials CD27+IgM+ and CD27-IgM+ B-cells were isolated from human splenocytes (n=3 infant, n=6 adult) by cell sorting and stimulated with CpG plus IL-2, IL-10, and IL-15. After 1 week the frequency of ABO Ag-specific ASC was detected by ELISPOT. The PPF was optimized using a Ramos B-cell line and intracellular protein phosphorylation of CD22(pY822) and PLCγ2(pY759) were examined after stimulation with anti-IgM Abs. Results The total number of ASC was lower in infant samples compared to older individuals and ELISPOT analysis revealed that the majority of ABO Ag-specific ASC were derived from CD27+IgM+ B-cells in both groups. Preliminary results by PPF (n=8) showed pY759 signaling beginning at 0.5min and peaking at 4min, with a 7-fold increase in median fluorescence intensity (MFI) of pY759. pY822 signaling began at 2min with a modest peak at 12min, with a 1.6-fold increase in MFl of pY822. Conclusions The overall increased expression of CD22 on infant CD27+IgM+ B-cells, which include ABO Ag-specific ASC precursors, may increase their susceptibility to decreased signaling, leading to inactivation. CD22 may therefore play an inhibitory role in infant immune responses to ABO Ags after ABOi HTx.

Fostamatinib Inhibits BCR Signaling, and Reduces Tumor Cell Activation and Proliferation in Patients with Relapsed Refractory Chronic Lymphocytic Leukemia.

Abstract 2882B-cell receptor (BCR) signaling contributes to the pathogenesis of chronic lymphocytic leukemia (CLL). Spleen tyrosine kinase (SYK) activated directly downstream of the BCR is essential for the induction of proliferation and survival pathways. The SYK inhibitor fostamatinib disrupts BCR signaling and was the first such inhibitor to show significant clinical activity in patients with mature B-cell malignancies. Fostamatinib has been shown to both induce apoptosis in unstimulated CLL cells as well as to inhibit BCR induced anti-apoptotic signals in vitro (Gobessi et al., 2009; Quiroga et al., 2009). Similarly, using the Eμ-TCL1 transgenic mouse model, fostamatinib has been shown to inhibit the growth of malignant B-cells without significant alteration of normal B-cells (Suljagic et al., 2010). In the first phase I/II clinical trial investigating fostamatinib in relapsed B-cell non-Hodgkin’s lymphoma (NHL) and CLL, clinical efficacy was observed in a variety of histologies with the highest response rate in CLL/SLL patients (Friedberg et al., 2010). Eleven CLL/SLL patients enrolled in this trial donated cellular material for correlative studies. Using these primary tumor samples, we evaluated the effects of fostamatinib on CLL cells in vivo after one cycle of treatment.We first validated the on-target effect of fostamatinib by using quantitative RT-PCR to measure expression of validated pathway specific gene signatures. Fostamatinib greatly down-regulated 12/12 evaluated BCR signature genes and significantly reduced the BCR gene signature score (computed as the average expression of the pathway specific genes; p=.002) Effective inhibition of BCR signaling was confirmed by a significant reduction in the phosphorylation of both BTK and ERK; two key BCR signaling molecules located downstream of SYK activation. Interestingly, BCR signaling was inhibited in CLL cells from all patients regardless of response to therapy. We next expanded our analysis to look at NF-κB and MYC gene signatures. We found that 11/11 representative NF-κB signature genes and 5/5 MYC signature genes were also down-regulated resulting in a significant reduction in both gene signature scores (p=.004 and p=.020, respectively). Confirming these results, we also observed a significant reduction in JUNB (p<.001) and MYC (p=.026) at the protein level. Interestingly, the reduction in NF-κB and MYC signature scores was highly correlated with the degree of reduction in BCR signaling suggesting that these pathways are linked. In addition to changes in the gene signatures we also observed a significant reduction in the cellular activation immunephenotype; CD69 and CD86 expression were significantly reduced by fostamatinib (p=.033 and p=.004, respectively). Further, we found that CD38 (an activation marker with prognostic significance) was also reduced on treatment although not to a significant extent. Finally, fostamatinib significantly reduced tumor proliferation as determined by the percentage of CLL cells expressing Ki67 (p=.005). Eight of the 11 patients in this study achieved a clinically significant response; interestingly however, the 3 non-responders demonstrated significantly brighter CD38 expression with an MFI up to 9-times the CD38 MFI in responders. A possible role of CD38 as a biomarker for response should be further explored in patients treated with BCR directed kinase inhibitors.In conclusion, fostamatinib and other inhibitors of BCR-related kinases constitute a major advance in the treatment of CLL. In vitro data with these compounds suggests that interruption of BCR signaling and survival pathways activated in the tissue microenvironments are likely responsible for the observed clinical response as only a moderate direct induction of apoptosis is seen in vitro. Here we demonstrate that inhibition of BCR-mediated signaling by fostamatinib results in a reduction in CLL proliferation and activation in vivo. Together these data provide a blueprint to further study the mechanism of action and resistance mechanisms of not only fostamatinib but also other BCR targeted therapeutics.This work was supported in part by the Intramural Research Program of the National, Heart, Lung and Blood Institute and by the University of Rochester SPORE in lymphoma P50 CA13080503, Rigel and the James P. Wilmot Foundation. Disclosures:Friedberg:Rigel: Research Funding.

Increased percentage of CD8+CD28− T cells correlates with clinical activity in primary Sjögren’s syndrome

The role of CD28− T cell subpopulations in primary Sjögren’s syndrome (pSS) has become controversial. Changes in the number of CD28- T cells have been demonstrated in autoimmune diseases in co-existence with Sjögren’s syndrome. The study aimed to indicate differences in the number of CD4+CD28− and CD8+CD28− T cells in patients with sicca syndrome and suspected pSS. Thirty patients with sicca syndrome at baseline were studied and followed up for 5months. After final diagnosis, comparison was made of the previously recorded lymphocyte subpopulations in patients with pSS and those in other defined subgroups. Notably high percentages of CD8+CD28− T cells were indicated in pSS patients, which correlated with the severity of the sicca symptoms and cutaneous and muscular systemic disease activity. Changes in CD8+CD28− T cell percentages may thus assist in the early differential diagnosis of pSS patients from those with similar clinical symptoms.

Unlike CD4+ T‐cell help, CD28 costimulation is necessary for effective primary CD8+ T‐cell influenza‐specific immunity

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

CD20+ Tumor-Infiltrating Lymphocytes Have an Atypical CD27− Memory Phenotype and Together with CD8+ T Cells Promote Favorable Prognosis in Ovarian Cancer

Tumor-infiltrating lymphocytes (TIL), in particular CD8(+) T cells and CD20(+) B cells, are strongly associated with survival in ovarian cancer and other carcinomas. Although CD8(+) TIL can mediate direct cytolytic activity against tumors, the role of CD20(+) TIL is poorly understood. Here, we investigate the possible contributions of CD20(+) TIL to humoral and cellular tumor immunity. Tumor and serum specimens were obtained from patients with high-grade serous ovarian cancer. CD8(+) and CD20(+) TIL were analyzed by immunohistochemistry and flow cytometry. Immunoglobulin molecules were evaluated by DNA sequencing. Serum autoantibody responses to the tumor antigens p53 and NY-ESO-1 were measured by ELISA. The vast majority of CD20(+) TIL were antigen experienced, as evidenced by class-switching, somatic hypermutation, and oligoclonality, yet they failed to express the canonical memory marker CD27. CD20(+) TIL showed no correlation with serum autoantibodies to p53 or NY-ESO-1. Instead, they colocalized with activated CD8(+) TIL and expressed markers of antigen presentation, including MHC class I, MHC class II, CD40, CD80, and CD86. The presence of both CD20(+) and CD8(+) TIL correlated with increased patient survival compared with CD8(+) TIL alone. In high-grade serous ovarian tumors, CD20(+) TIL have an antigen-experienced but atypical CD27(-) memory B-cell phenotype. They are uncoupled from serum autoantibodies, express markers of antigen-presenting cells, and colocalize with CD8(+) T cells. We propose that the association between CD20(+) TIL and patient survival may reflect a supportive role in cytolytic immune responses.

CD38 deficiency in the tumor microenvironment attenuates glioma progression and modulates features of tumor-associated microglia/macrophages

Gliomas are the most frequent primary tumors of the brain, and for highly malignant gliomas there is no successful treatment. The tumor microenvironment contains large numbers of infiltrating microglia and macrophages (MM). There is increasing evidence that the tumor-associated MM support glioma expansion. CD38 is a multifunctional ectoenzyme that uses nicotinamide adenine dinucleotide as a substrate to generate second messengers. Previously we showed that CD38 deficiency modulates microglial "activation" and impaired recovery from head trauma by a microglia-associated mechanism. In view of the supportive role of MM in glioma progression and the role of CD38 in microglia activation, we hypothesize that deficiency of CD38 in the tumor microenvironment would inhibit glioma progression. Using the syngeneic GL261 model of glioma progression in wild-type and CD38 null mice, we show here that CD38 deficiency significantly attenuates glioma expansion and prolongs the life span of the glioma-bearing mice. The CD38 deficiency effect was associated with increased cell death and decreased metalloproteinase-12 expression in the tumor mass, as well as modulation of the tumor-induced MM properties, as indicated by a reduction in the expression of the MM marker F4/80 and matrix metalloproteinases. Our results thus suggest that CD38 participates in the tumor-supporting action of MM and that targeting CD38 might be a potential therapeutic approach for glioma treatment.

CD45 is a Major Receptor Involved in Galectin‐8 Signaling of Preaparesis in HL‐60 cells

The mechanisms of leukocyte turnover during an inflammatory response and its resolution are poorly understood. We recently showed that upon interactions with Galectin‐8 (Gal‐8), a 34 kilodalton heterodimeric tandem‐repeat galectin expressed by endothelial and epithelial cells, activated leukocytes express phosphatidylserine (PS) on their surface in the absence of apoptosis or DNA fragmentation. This can lead to phagocytosis of the cells in a process termed preaparesis, which fundamentally differs from that induced by factors signaling apoptosis. To identify the surface receptors involved in Gal‐8 signaling we took a proteomic approach to identify Gal‐8‐binding glycoproteins from human HL‐60 cells, a promyelocytic leukemic cell line with many signaling pathways in common to human neutrophils. Our previous studies showed that glycans containing poly‐N‐acetyllactosamine (poly‐N‐LacNAc) on HL‐60 cells were important for signaling. One major glycoprotein identified as a Gal‐8‐binding receptor on HL‐60 cells is CD45, a membrane glycoprotein that bears poly‐N‐LacNAc. We further characterized CD45/Gal‐8 interaction, by Western‐Blot, flow cytometry and confocal microscopy. Inhibition of CD45 cytoplasmic phosphatase activity reduced Gal‐8 induced PS exposure indicating a possible role of CD45 in Gal‐8 signaling preaparesis in human leukocytes.This work was supported by National Institutes of Health Grant HL085607 to R.D.C.

64 High Expression of the B-Cell Inhibitory Molecule CD22 on Memory B-Cells in Early Life May Contribute to Better Transplant Outcomes

Purpose: ABO-incompatible heart transplantation (ABOi HTx) can be performed safely in infants and is generally followed by B-cell tolerance to donor ABO antigens (ags). ABO antibodies are thought to arise in a T-independent (TI) manner. TI B-cell activation can be inhibited by the interaction of CD22, an inhibitory B-cell co-receptor, with sialic acids on cells/tissues, leading to B-cell tolerance. Since immune responses to TI-ags are limited in early childhood, we hypothesize that the role of CD22 may be elevated during infancy compared to later in life. In this study, we determined CD22 expression on B-cell subsets from infancy to adulthood. Methods and Materials: We analyzed splenocytes from organ donors of various age (n 41; age 4 d 74 yr) by flow cytometry to quantify CD22 expression on the surface of CD19 B-cell subsets including CD27IgM IgD CD38high (previously defined as recent bone marrow emigrants), CD27-IgM IgD , CD27-IgM-IgD , CD27-IgM-IgD-, and the memory-like phenotypes CD27 IgMand CD27 IgM . Results: CD22 Median Fluorescence Intensity (MFI) differed amongst various B-cell subsets (p 0.0001; Kruskal Wallis test). Post testing with Dunn’s multiple comparison test showed that CD27-IgM-IgDcells had lower CD22 expression (p 0.001) and CD27 IgM cells had higher expression (p 0.001) compared to other subsets. Furthermore, CD22 on CD27 IgM cells was inversely correlated with age (p 0.001): infant samples had the highest CD22 expression, which decreased with increasing age. Conclusions: Based on previous studies showing the inhibitory role of CD22 on B-cell activation, our findings suggest that higher CD22 expression on the memory subset may cause infant B-cells to be more susceptible to down-regulation of B-cell signaling leading to subsequent inactivation. CD22 may thus play a role in the restricted TI responses to ABO ags after ABOi HTx. Functional assays are underway to determine its role in B-cell signalling during infancy and in regulation.

The Signaling Protein CD38 Is Essential for Early Embryonic Development

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d(+)CD38(+) and CD49d(+)CD38(-) primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d(+)CD38(-) CLL-derived cell line Mec-1. Results indicate that CD49d(+)CD38(+) cells adhered more efficiently onto CD49d-specific substrates than CD49d(+)CD38(-) cells (P < 0.001). Upon adhesion, CD49d(+)CD38(+) cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d(+)CD38(-) cells (P = 0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d(+)CD38(+) cells were more resistant to serum-deprivation-induced (P < 0.001) and spontaneous (P = 0.03) apoptosis than the CD49d(+)CD38(-) counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

Differences in allergen-induced T cell activation between allergic asthma and rhinitis: Role of CD28, ICOS and CTLA-4

BackgroundTh2 cell activation and T regulatory cell (Treg) deficiency are key features of allergy. This applies for asthma and rhinitis. However with a same atopic background, some patients will develop rhinitis and asthma, whereas others will display rhinitis only. Co-receptors are pivotal in determining the type of T cell activation, but their role in allergic asthma and rhinitis has not been explored. Our objective was to assess whether allergen-induced T cell activation differs from allergic rhinitis to allergic rhinitis with asthma, and explore the role of ICOS, CD28 and CTLA-4.MethodsT cell co-receptor and cytokine expressions were assessed by flow cytometry in PBMC from 18 house dust mite (HDM) allergic rhinitics (R), 18 HDM allergic rhinitics and asthmatics (AR), 13 non allergic asthmatics (A) and 20 controls, with or without anti-co-receptors antibodies.ResultsIn asthmatics (A+AR), a constitutive decrease of CTLA-4+ and of CD4+CD25+Foxp3+ cells was found, with an increase of IFN-γ+ cells. In allergic subjects (R + AR), allergen stimulation induced CD28 together with IL-4 and IL-13, and decreased the proportion of CTLA-4+, IL-10+ and CD4+CD25+Foxp3+ cells. Anti-ICOS and anti-CD28 antibodies blocked allergen-induced IL-4 and IL-13. IL-13 production also involved CTLA-4.ConclusionsT cell activation differs between allergic rhinitis and asthma. In asthma, a constitutive, co-receptor independent, Th1 activation and Treg deficiency is found. In allergic rhinitis, an allergen-induced Treg cell deficiency is seen, as well as an ICOS-, CD28- and CTLA-4-dependent Th2 activation. Allergic asthmatics display both characteristics.

Clarifying the Mechanism of Superantigen Toxicity

Superantigens are bacterial proteins that generate a powerful immune response by binding to Major Histocompatibility Complex class II molecules on antigen-presenting cells and T cell receptors on T cells. A recent article reveals that at least one of the superantigens, staphylococcal enterotoxin B (SEB), also binds the co-stimulatory molecule CD28, suggesting that a much larger and potentially more stable complex is formed at the immunological synapse than was previously thought. This revelation greatly clarifies some of the mystery surrounding how and why these toxins are able to elicit such a toxic immune response at extremely low concentrations. These findings also highlight a novel role for CD28 in microbial pathogenicity.

Binding of Superantigen Toxins into the CD28 Homodimer Interface Is Essential for Induction of Cytokine Genes That Mediate Lethal Shock

Author SummaryInduction of protective immunity involves the expression of inflammatory cytokines, proteins that mediate and respond to immune signals. However, excessive cytokine induction can lead to disease, including, at very high levels, lethal toxic shock. Staphylococcal and streptococcal superantigens are a broad family of bacterial protein toxins that induce such a lethal cytokine storm, orders of magnitude higher in intensity than that elicited during normal immune responses. A key participant in every immune response is the costimulatory receptor CD28, which forms a protein dimer to mediate the immune response. Hitherto, CD28 was not known to bind microbial components. Here, we show that superantigens co-opt CD28 as their receptor and that to induce a cytokine storm, superantigens must bind directly into the dimer interface of CD28. The interaction between CD28 and the bacterial superantigen can be blocked with peptides—short protein fragments that mimic the contact domains in the intact superantigen or in CD28. These peptides attenuate inflammatory cytokine gene induction and thus protect animals from lethal toxic shock. Our finding that CD28 is a receptor for the superantigen toxins broadens the scope of microbial pathogen recognition mechanisms and provides a novel approach for designing therapeutics that protect against toxic shock.

Role of CD56 and CD57 uterine natural killer cells and E-cadherin in either successful implantation or recurrent abortion

Role of CD56 and CD57 uterine natural killer cells and E-cadherin in either successful implantation or recurrent abortion

Investigating the role of CD38 and functionally related molecular risk factors in the CLL NOD/SCID xenograft model

We explored the role of CD38 and functionally associated molecular risk factors in a recently described chronic lymphocytic leukemia (CLL) nonobese diabetic/ severe combined immunodeficient xenograft model. Intravenous injection of peripheral blood mononuclear cells from 73 patients with CLL into 244 mice resulted in robust engraftment of leukemic cells into the murine spleens detected 4 wks after transplantation. Leukemic cell engraftment correlated significantly (P < 0.05) with markers reflecting disease activity, e.g., Binet stage and lymphocyte doubling time, and the expression of molecular risk factors including CD38, CD49d, ZAP-70, and IgVH mutational status. Increased engraftment levels of CD38+ as compared to CD38- CLL cells could be attributed, in part, to leukemic cell proliferation as evidenced by combined immunostaining of murine spleen sections for Ki-67 and CD20. In short-term (24 h) homing assays, CD38+ CLL cells migrated more efficiently to the bone marrow of the recipient animals than their CD38- counterparts. Finally, CD38 expression by the leukemic cells was found to be dynamic in that it was regulated not only by elements of the murine microenvironment but also by co-engrafting non-malignant human T cells. This model could be useful for evaluating the biological basis of CLL growth in the context of the hematopoietic microenvironment as well as preclinical testing of novel compounds.

Knocking out of CD38 accelerates development of a lupus-like disease in lpr mice

CD38 participates in lymphocyte ontogeny and function and may be involved in autoimmunity. Absence of CD38 accelerates development of non-obese diabetic (NOD) mice diabetes and anti-CD38 antibodies are good markers of human disease. Little is known regarding systemic autoimmunity. Active SLE patients have higher numbers of CD38(+) T and B cells. CD38 is a candidate gene for the murine Lmb2 lupus locus. We aimed to investigate whether CD38 was involved in lupus development. We developed Cd38(-/-)-Fas(lpr)/Fas(lpr) mice and monitored them for development of a lupus-like disease through measurement of protein excretion in urine, histological assessment of the kidneys, quantification of circulating immunoglobulins and autoantibodies. We have also immunophenotyped 2- and 6-month old Cd38(-/-)-Fas(lpr)/Fas(lpr) mice. We found that absence of CD38 accelerated disease development: female Cd38(-/-)-Fas(lpr)/Fas(lpr) mice presented severe proteinuria, GN, deposition of ICs in the renal medulla and increased amounts of circulating immunoglobulin G (IgG), although anti-dsDNA autoantibodies and RF were not significantly increased at disease onset. We have found that Cd38(-/-)-Fas(lpr)/Fas(lpr) male mice, similarly to other murine models of lupus, were able to control disease. Absence of CD38 in lpr mice altered differentiation of T cells and dendritic cells (DC). Although the role of CD38 in tolerance is still to be elucidated, we provide evidence that it may play an active role in the control of a murine lupus-like disease.

Fibrocyte-like cells recruited to the spleen support innate and adaptive immune responses to acute injury or infection

Bone marrow (BM)-derived fibrocytes are a population of CD45(+) and collagen Type I-expressing cells that migrate to the spleen and to target injured organs, such as skin, lungs, kidneys, and liver. While CD45(+)Col(+) fibrocytes contribute to collagen deposition at the site of injury, the role of CD45(+)Col(+) cells in spleen has not been elucidated. Here, we demonstrate that hepatotoxic injury (CCl(4)), TGF-β1, lipopolysaccharide, or infection with Listeria monocytogenes induce rapid recruitment of CD45(+)Col(+) fibrocyte-like cells to the spleen. These cells have a gene expression pattern that includes antimicrobial factors (myleoperoxidase, cathelicidin, and defensins) and MHC II at higher levels than found on quiescent or activated macrophages. The immune functions of these splenic CD45(+)Col(+) fibrocyte-like cells include entrapment of bacteria into extracellular DNA-based structures containing cathelicidin and presentation of antigens to naïve CD8(+) T cells to induce their proliferation. Stimulation of these splenic fibrocyte-like cells with granulocyte macrophage-colony stimulating factor or macrophage-colony stimulating factor induces downregulation of collagen expression and terminal differentiation into the dendritic cells or macrophage. Thus, splenic CD45(+)Col(+) cells are a population of rapidly mobilized BM-derived fibrocyte-like cells that respond to inflammation or infection to participate in innate and adaptive immune responses.

Abstract 3291: Macrophages and granulocytes promote the malignant glioma phenotype

Abstract Solid tumors, including glioblastoma, contain large populations of both malignant and non-malignant cells. The tumor microenvironment contains a multitude of different cell types and recent evidence supports the role of CD45+ bone marrow cells such as vascular modulating macrophages. Tumor associated macrophages accumulate in hypoxic or necrotic regions of human tumors where they release VEGF. Recent studies have demonstrated that the number of M2-skewed macrophages (CD163+/CD204+) in human tumors correlates with glioma grade, cell proliferation and microvascular density. Myeloid cells including macrophages and granulocytes are thought to promote the malignant phenotype of many solid tumor types but little is known about their influence on glioma biology. To evaluate the impact of macrophages and granulocytes on the malignant phenotype, we used co-culture experiments to determine the influence of these cells on glioma cytokine and angiogenic factor release, glioma cell invasion and proliferation, and resistance to chemotherapy. Mouse macrophages or granulocytes (ATCC) were cultured alone or together with U87 or glioma stem cells using a Boyden chamber to maintain cell separation. Conditioned media and RNA were evaluated for expression of both mouse and human chemokine expression using ELISA and quantitative PCR. Co-culture resulted in a significant increase in macrophage VEGF-A, CXCL1, HGF, VEGF-C and PlGF expression and granulocyte IGF1, FGF, CCL11, VEGF-C and VEGF-D compared to baseline suggesting that myeloid cell-glioma crosstalk may promote VEGF-A-independent angiogenesis in glioma tumors. Co-culture promoted glioma cell expression of PDGF, CCL11, CXCL9, and INF-β. We next evaluated the impact of myeloid cells on glioma invasion. At baseline, U87 demonstrates minimal transwell migration in vitro and does not invade normal brain in vivo. When co-cultured, macrophages and granulocytes significantly increased U87 in vitro transwell migration compared to control. Finally, serum free media incubated for 48 hours with mouse macrophages or granulocytes was used as growth medium for U87 cells. Using a SRB cell proliferation assay, conditioned media from granulocytes but not macrophages increased glioma proliferation compared to controls. Conditioned media from both macrophages and neutrophils reduced the sensitivity of glioma cells to temozolomide in vitro. These results suggest that both macrophages and granulocytes may play an important role in promoting glioma angiogenesis, invasion and resistance to chemotherapy. However, granulocytes were more effective in promoting glioma proliferation. Targeting myeloid cells may be an effective approach to inhibiting the glioma malignant phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3291. doi:10.1158/1538-7445.AM2011-3291

Role of CD38 during ontogeny of murine B cells in bone marrow (153.23)

Abstract CD38 is a 45 KD transmembrane protein, expressed by immature and mature lymphocytes. Agonistic antibodies directed to this molecule induce several biological responses: cellular proliferation, protein phosphorylation, intracellular Ca2+ mobilization, rescue from and induction of apoptosis. CD38 ligation with a mAb anti-CD38 (NIM-R5) induces apoptosis of the murine pro-B cell line (Ba/F3); this phenomenon depends on tyrosine kinases activation and the MAP kinase ERK. The expression and function of CD38 during B-cell ontogeny has been poorly analyzed in mice. We analyzed the expression of CD38 during the ontogeny of B cells in murine bone marrow. CD38 is expressed from the preproB stage. ProB, preB and immature B cells from murine bone marrow were all positive for the expression of CD38, but immature B cells had the highest fluorescence intensity, and the proB stage shows a low and high expression of CD38 that suggest two probable populations. The results suggest that CD38 may play a role during the ontogeny of murine B cells. We are analysing whether CD38 ligation on these subpopulations, induces proliferation, maturation or apoptosis.