Enrichment of functionally distinct mouse hematopoietic progenitor cell populations using CD62L

The details of the bifurcation of the lymphoid and myeloid lineages following commitment by multipotent progenitor cells (MPP) remain a topic of controversy. We report that the surface glycoprotein CD62L can be characterized as a novel marker of this and other stages of early hematopoietic differentiation. Cell isolation and transplant studies demonstrated CD62L(neg/low) long-term hematopoietic stem cells and CD62L(high) MPP within the traditionally defined c-kit(pos)Lin(neg/low)Sca-1(pos) stem/progenitor cell population. Within the MPP population, previously defined as c-kit(pos)Lin(neg/low)Sca-1(pos)-Thy-1.1(neg)Flt3(pos), Sca-1 and CD62L resolved four populations and segregated Sca-1(high)CD62L(neg/low) MPP from Sca-1(high)CD62L(high) leukocyte-biased progenitors. Using a novel transplantation method that allows tracking of erythroid and platelet engraftment as an alternative to the classical method of in vitro colony formation, we characterized Sca-1(high)CD62L(neg/low) cells as MPP, based on transient engraftment of these lineages. These data establish CD62L as a useful tool in the study of early hematopoiesis and emphasize the power of trilineage-engraftment studies in establishing the lineage potential of MPP subsets.     

Healthy Neonates Possess a CD56-Negative NK Cell Population with Reduced Anti-Viral Activity

Background

Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.

Methods

NK cells were isolated from cord blood and from adult donors. NK subpopulations were identified as positive or negative for the expression of CD56 and characterized for expression of granzyme B and surface markers by multi-parameter flow cytometry. Cell function was assessed by viral suppression and cytokine production using autologous lymphocytes infected with HIV. Activating (NKp30, NKp46) and inhibitory (Siglec-7) markers in healthy infants and adults were compared with viremic HIV-infected adults.

Results

Cord blood contained increased frequencies of CD56 negative (CD56neg) NK cells with reduced expression of granzyme B and reduced production of IFNγ and the CC-class chemokines RANTES, MIP1α and MIP1β upon stimulation. Both CD56pos and CD56neg NK subpopulations showed impaired viral suppression in cord blood, with impairment most marked in the CD56neg subset. CD56neg NK cells from cord blood and HIV-infected adults shared decreased inhibitory and activating receptor expression when compared with CD56pos cells.

Conclusions

CD56neg NK cells are increased in number in normal infants and these effectors show reduced anti-viral activity. Like the expanded CD56neg population described in HIV-infected adults, these NK cells demonstrate functional impairments which may reflect inadequate development or activation.     

Characterization of lymphocyte subsets in patients with common variable immunodeficiency reveals subsets of naive human B cells marked by CD24 expression

Increased proportions of naive B cell subset and B cells defined as CD27(neg)CD21(neg)CD38(neg) are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27(neg)CD21(neg)CD38(neg) B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21(pos) naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27(neg)CD21(neg)CD38(neg) B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.     

CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Neonatal lung side population cells demonstrate endothelial potential and are altered in response to hyperoxia-induced lung simplification

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.     

Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.     

Plasmacytoid dendritic cells migrate in afferent skin lymph

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.     

Rapid, B lymphoid-restricted engraftment mediated by a primitive bone marrow subpopulation

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1. 1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1. 1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.     

CD45neg but not CD45pos human myeloma cells are sensitive to the inhibition of IGF-1 signaling by a murine anti-IGF-1R monoclonal antibody, mAVE1642

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.     

A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus

Human memory B cells comprise isotype-switched and nonswitched cells with both subsets displaying somatic hypermutation. In addition to somatic hypermutation, CD27 expression has also been considered a universal memory B cell marker. We describe a new population of memory B cells containing isotype-switched (IgG and IgA) and IgM-only cells and lacking expression of CD27 and IgD. These cells are present in peripheral blood and tonsils of healthy subjects and display a degree of hypermutation comparable to CD27+ nonswitched memory cells. As conventional memory cells, they proliferate in response to CpG DNA and fail to extrude rhodamine. In contrast to other recently described CD27-negative (CD27neg) memory B cells, they lack expression of FcRH4 and recirculate in the peripheral blood. Although CD27neg memory cells are relatively scarce in healthy subjects, they are substantially increased in systemic lupus erythematosus (SLE) patients in whom they frequently represent a large fraction of all memory B cells. Yet, their frequency is normal in patients with rheumatoid arthritis or chronic hepatitis C. In SLE, an increased frequency of CD27neg memory cells is significantly associated with higher disease activity index, a history of nephritis, and disease-specific autoantibodies (anti-dsDNA, anti-Smith (Sm), anti-ribonucleoprotein (RNP), and 9G4). These findings enhance our understanding of the B cell diversification pathways and provide mechanistic insight into the immunopathogenesis of SLE.     

A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34(+) cells isolated from umbilical cord blood

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ～15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.     

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Subpopulations of human dendritic cells display a distinct phenotype and bind differentially to proteins of the extracellular matrix

CD1a(pos) dendritic cells (DCs) and Langerhans cells (LCs) are highly specialized antigen-presenting cells mainly localized in the skin. Various cells have been identified as precursors of cutaneous DCs, but the definitive precursor subpopulations remain to be defined and characterized in detail. In this study, DCs were generated in vitro from monocytes (monocyte-derived DCs, MoDCs) and from CD34(pos) stem cells (CD34(pos) cell-derived DCs, CD34DCs). By virtue of their CD14 and CD1a expression, four CD34DC subpopulations were characterized while MoDCs contain three different subpopulations. Of these, CD14-expressing cells are considered to be precursors of fully differentiated DCs, which themselves are CD14(neg)CD1a(pos). Both, MoDCs and CD34DCs expressed the alpha integrins LFA-1, Mac-1, CR4, VLA-4, VLA-5 and the beta2 integrin CD18. CD34DCs and MoDCs were negative for VLA-3, whereas MoDCs, but not CD34DCs expressed VLA-6. Phenotypic and functional characterization of the cells generated herein at earlier time points revealed that DCs at day 3 of culture may reflect the in vivo situation more closely than at day 7. Adhesion of DC precursors to endothelial cells and to components of the extracellular matrix is a prerequisite for their migration towards the epidermis. To this end, we investigated adhesion of CD34DCs and MoDCs to components of the cutaneous extracellular matrix. Distinct DC subsets showed a differential binding pattern to proteins of the extracellular matrix. MoDCs and CD34DCs bound preferentially to laminin 332 via CD49f and to fibronectin via CD49e, but only weakly to laminin 111 or to collagens. While CD14(pos) cells preferentially bound to laminin 332, CD1a(pos) cells adhered to fibronectin. In summary, subpopulations of CD34DCs and MoDCs are phenotypically related to each other, but not identical and display differential binding to components of the extracellular matrix.     

Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is a valuable source for hematopoietic progenitor cell therapy. Moreover, it contains another subset of non-hematopoietic population referred to as mesenchymal progenitor cells (MPCs), which can be ex vivo expanded and differentiated into osteoblasts, chondrocytes and adipocytes. In this study, we successfully isolated the clonogenic MPCs from UCB by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblasts (majority) and spindle-shaped fibroblasts (minority). Both types of MPCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle-shaped clones possessed the positive CD90 expression and showed a greater tendency in adipogenesis, while the flattened clones were CD90 negative cells and showed a lower tendency in adipogenesis. The high number of flattened MPCs might be linked to the less sensitivity of UCB-derived MPCs in adipogenic differentiation.     

CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells

Experimental animals with myelin disorders can be treated by transplanting oligodendrocyte progenitor cells (OPCs) into the affected brain or spinal cord. OPCs have been isolated by their expression of gangliosides recognized by mAb A2B5, but this marker also identifies lineage-restricted astrocytes and immature neurons. To establish a more efficient means of isolating myelinogenic OPCs, we sorted fetal human forebrain cells for CD140a, an epitope of platelet derived growth factor receptor (PDGFR)α, which is differentially expressed by OPCs. CD140a(+) cells were isolated as mitotic bipotential progenitors that initially expressed neither mature neuronal nor astrocytic phenotypic markers, yet could be instructed to either oligodendrocyte or astrocyte fate in vitro. Transplanted CD140a(+) cells were highly migratory and robustly myelinated the hypomyelinated shiverer mouse brain more rapidly and efficiently than did A2B5(+)cells. Microarray analysis of CD140a(+) cells revealed overexpression of the oligodendroglial marker CD9, suggesting that CD9(+)/CD140a(+) cells may constitute an even more highly enriched population of myelinogenic progenitor cells.     

Characterization of dendritic cells subpopulations in skin and afferent lymph in the swine model

Transcutaneous delivery of vaccines to specific skin dendritic cells (DC) subsets is foreseen as a promising strategy to induce strong and specific types of immune responses such as tolerance, cytotoxicity or humoral immunity. Because of striking histological similarities between human and pig skin, pig is recognized as the most suitable model to study the cutaneous delivery of medicine. Therefore improving the knowledge on swine skin DC subsets would be highly valuable to the skin vaccine field. In this study, we showed that pig skin DC comprise the classical epidermal langerhans cells (LC) and dermal DC (DDC) that could be divided in 3 subsets according to their phenotypes: (1) the CD163(neg)/CD172a(neg), (2) the CD163(high)CD172a(pos) and (3) the CD163(low)CD172a(pos) DDC. These subtypes have the capacity to migrate from skin to lymph node since we detected them in pseudo-afferent lymph. Extensive phenotyping with a set of markers suggested that the CD163(high) DDC resemble the antibody response-inducing human skin DC/macrophages whereas the CD163(neg)CD172(low) DDC share properties with the CD8(+) T cell response-inducing murine skin CD103(pos) DC. This work, by showing similarities between human, mouse and swine skin DC, establishes pig as a model of choice for the development of transcutaneous immunisation strategies targeting DC.     

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120d a sustained production of cytokines (KC, IL-5, TNF-α, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A.