RhoH-Deficient Mice Show Altered B Cell Populations In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2307-2307
Author(s):  
Abel Sanchez-Aguilera ◽  
Jose Cancelas ◽  
David A. Williams
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Receptor ◽  
Wild Type ◽  
Marginal Zone B Cells

Abstract RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations. Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9) Bone marrow Spleen (*) indicates p<0.05; (**), p<0.01; (***), p<0.005 RhoH+/+ RhoH−/− RhoH+/+ RhoH−/− total B cells 7.8±1.8 11.0±2.4 (**) total B cells 31.7±10.1 25.4±8.8 pro-B 0.12±0.03 0.15±0.04 (*) transitional 8.7±1.2 8.6±2.8 pre-B 2.6±0.6 3.8±0.8 (***) B-2 11.6±4.1 7.6±2.5 (*) immature 1.5±0.4 2.1±0.5 (*) marginal 3.2±1.1 3.9±1.6 mature 1.4±0.7 1.7±0.9

scholarly journals Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help

2006 ◽  
Vol 203 (8) ◽  
pp. 1985-1998 ◽  
Author(s):  
Laura Mandik-Nayak ◽  
Jennifer Racz ◽  
Barry P. Sleckman ◽  
Paul M. Allen
Keyword(s):  
Lymph Node ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
B Cell Tolerance ◽  
Marginal Zone B Cells ◽  
T Cell Help

In K/BxN mice, arthritis is induced by autoantibodies against glucose-6-phosphate-isomerase (GPI). To investigate B cell tolerance to GPI in nonautoimmune mice, we increased the GPI-reactive B cell frequency using a low affinity anti-GPI H chain transgene. Surprisingly, anti-GPI B cells were not tolerant to this ubiquitously expressed and circulating autoantigen. Instead, they were found in two functionally distinct compartments: an activated population in the splenic marginal zone (MZ) and an antigenically ignorant one in the recirculating follicular/lymph node (LN) pool. This difference in activation was due to increased autoantigen availability in the MZ. Importantly, the LN anti-GPI B cells remained functionally competent and could be induced to secrete autoantibodies in response to cognate T cell help in vitro and in vivo. Therefore, our study of low affinity autoreactive B cells reveals two distinct but potentially concurrent mechanisms for their activation, of which one is T cell dependent and the other is T cell independent.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

2009 ◽  
Vol 417 (3) ◽  
pp. 673-683 ◽  
Author(s):  
Munetoyo Toda ◽  
Risa Hisano ◽  
Hajime Yurugi ◽  
Kaoru Akita ◽  
Kouji Maruyama ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Signalling ◽  
Negative Regulator ◽  
Mammary Adenocarcinoma ◽  
Marginal Zone B Cells ◽  
Tumour Bearing

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

Vav proteins regulate peripheral B-cell survival

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2391-2398 ◽  
Author(s):  
Elena Vigorito ◽  
Laure Gambardella ◽  
Francesco Colucci ◽  
Simon McAdam ◽  
Martin Turner
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Receptor ◽  
Cross Linking ◽  
Marginal Zone B Cells ◽  
Survival Signal ◽  
B Cell Survival ◽  
Follicular B Cells ◽  
Deficient Mice

AbstractMice lacking all 3 Vav proteins fail to produce significant numbers of recirculating follicular or marginal zone B cells. Those B cells that do mature have shortened lifespans. The constitutive nuclear factor-kappaB (NF-κB) activity of resting naive B cells required Vav function and expression of cellular reticuloendotheliosis (c-Rel). Rel-A was reduced in Vav-deficient B cells. Furthermore, expression of the NF-κB-regulated antiapoptotic genes A1 and Bcl-2 was reduced in mature Vav-deficient B cells. Overexpression of Bcl-2 restored the number of mature follicular B cells in the spleens of Vav-deficient mice. When activated by B-cell receptor (BCR) cross-linking, Vav-deficient B cells failed to activate NF-κB. Vav proteins thus regulate an NF-κB-dependent survival signal in naive B cells and are required for NF-κB function after BCR cross-linking.

Marginal Zone B Cells Mediate a CD4 T Cell Dependent Extrafollicular Antibody Response Following RBC Transfusion in Mice

Blood ◽  
2021 ◽  
Author(s):  
Patricia E Zerra ◽  
Seema R Patel ◽  
Ryan Philip Jajosky ◽  
Connie M Arthur ◽  
James W McCoy ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Antibody Response ◽  
B Cell ◽  
T Cell Activation ◽  
Antibody Formation ◽  
Marginal Zone ◽  
Cd4 T Cell ◽  
Marginal Zone B Cells ◽  
Rbc Transfusion

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme and ovalbumin fused to human Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they co-localize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited IgM and IgG anti-HOD antibody formation, while CD4 T cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild type or MZ B cell deficient recipients, suggesting that IgG formation is not dependent on MZ B cell-mediated CD4 T cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response and no increase in antigen specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Trisomy of the Down Syndrome Critical Region Suppresses Precursor B-Cell Differentiation and Promotes B-Cell Transformation Associated with Altered Expression of Polycomb Repressor Complex 2 Targets

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 115-115
Author(s):  
Andrew A. Lane ◽  
Diederik van Bodegom ◽  
Bjoern Chapuy ◽  
Gabriela Alexe ◽  
Timothy J Sullivan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Transformation ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Repressor Complex

Abstract Abstract 115 Extra copies of chromosome 21 (polysomy 21) is the most common somatic aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL), including >90% of cases with high hyperdiploidy. In addition, children with Down syndrome (DS) have a 20-fold increased risk of developing B-ALL, of which ∼60% harbor CRLF2 rearrangements. To examine these associations within genetically defined models, we investigated B-lineage phenotypes in Ts1Rhr mice, which harbor triplication of 31 genes syntenic with the DS critical region (DSCR) on human chr.21. Murine pro-B cell (B220+CD43+) development proceeds sequentially through “Hardy fractions” defined by cell surface phenotype: A (CD24−BP-1−), B (CD24+BP-1−) and then C (CD24+BP-1+). Compared with otherwise isogenic wild-type littermates, Ts1Rhr bone marrow harbored decreased percentages of Hardy fraction B and C cells, indicating that DSCR triplication is sufficient to disrupt the Hardy A-to-B transition. Of note, the same phenotype was reported in human DS fetal liver B-cells, which have a block between the pre-pro- and pro-B cell stages (analogous to Hardy A-to-B). To determine whether DSCR triplication affects B-cell proliferation in vitro, we analyzed colony formation and serial replating in methylcellulose cultures. Ts1Rhr bone marrow (B6/FVB background) formed 2–3-fold more B-cell colonies in early passages compared to bone marrow from wild-type littermates. While wild-type B-cells could not serially replate beyond 4 passages, Ts1Rhr B-cells displayed indefinite serial replating (>10 passages). Ts1Rhr mice do not spontaneously develop leukemia, so we utilized two mouse models to determine whether DSCR triplication cooperates with leukemogenic oncogenes in vivo. First, we generated Eμ-CRLF2 F232C mice, which express the constitutively active CRLF2 mutant solely within B-cells. Like Ts1Rhr B-cells, (but not CRLF2 F232C B-cells) Ts1Rhr/CRLF2 F232C cells had indefinite serial replating potential. In contrast with Ts1Rhr B-cells, Ts1Rhr/CRLF2 F232C B-cells also engrafted into NOD.Scid.IL2Rγ−/− mice and caused fatal and serially transplantable B-ALL. Second, we retrovirally transduced BCR-ABL1 into unselected bone marrow from wild-type and Ts1Rhr mice and transplanted into irradiated wild-type recipients. Transplantation of transduced Ts1Rhr cells (106, 105, or 104) caused fatal B-ALL in recipient mice with shorter latency and increased penetrance compared to recipients of the same number of transduced wild-type cells. By Poisson calculation, the number of B-ALL initiating cells in transduced Ts1Rhr bone marrow was ∼4-fold higher than in wild-type animals (1:60 vs 1:244, P=0.0107). Strikingly, transplantation of individual Hardy A, B, and C fractions after sorting and BCR-ABL1 transduction demonstrated that the increased leukemia-initiating capacity almost completely resides in the Ts1Rhr Hardy B fraction; i.e., the same subset suppressed during Ts1Rhr B-cell differentiation. To define transcriptional determinants of these phenotypes, we performed RNAseq of Ts1Rhr and wild-type B cells in methylcellulose culture (n=3 biologic replicates per genotype). As expected, Ts1Rhr colonies had ∼1.5-fold higher RNA abundance of expressed DSCR genes. We defined a Ts1Rhr signature of the top 200 genes (false discovery rate (FDR) <0.25) differentially expressed compared with wild-type cells. Importantly, this Ts1Rhr signature was significantly enriched (P=0.02) in a published gene expression dataset of DS-ALL compared with non-DS-ALL (Hertzberg et al., Blood 2009). Query of >2,300 signatures in the Molecular Signatures Database (MSigDB) C2 Chemical and Genetic Perturbations with the Ts1Rhr signature identified enrichment in multiple gene sets of polycomb repressor complex (PRC2) targets and H3K27 trimethylation. Most notably, SUZ12 targets within human embryonic stem cells were more highly expressed in Ts1Rhr cells (P=1.2×10−6, FDR=0.003) and the same SUZ12 signature was enriched in patients with DS-ALL compared to non-DS-ALL (P=0.007). In summary, DSCR triplication directly suppresses precursor B-cell differentiation and promotes B-cell transformation both in vitro and by cooperating with proliferative alterations such as CRLF2 activation and BCR-ABL1 in vivo. Pharmacologic modulation of H3K27me3 effectors may overcome the pro-leukemogenic effects of polysomy 21. Disclosures: No relevant conflicts of interest to declare.

Hepatic Stellate Cell Conditioned Myeloid Cells Provide a Novel Therapy for Prevention of Factor VIII Antibody Formation in the Hemophilia A Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4117-4117
Author(s):  
Sumantha Bhatt ◽  
Kathleen Brown ◽  
Feng Lin ◽  
Michael P Meyer ◽  
Margaret V. Ragni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Myeloid Cells ◽  
Gm Csf ◽  
Cox 2

Abstract Abstract 4117 Background: Hemophilia is an X-linked bleeding disorder resulting from a mutation in coagulation factor VIII (F.VIII). A major drawback of current plasma-derived or recombinant F.VIII therapy is the formation of F.VIII antibodies (inhibitors). Inhibitor formation is a T cell-dependent, B cell-mediated immune response to foreign infused F.VIII. Myeloid derived suppressor cells (MDSCs) are potent suppressors of T cell and B cell responses and are currently under study for therapeutic applications in transplantation and autoimmune diseases. However, the mechanisms of MDSC development and function remain unknown, and in vitro propagation of MDSCs has been a challenge. We hypothesized that MDSCs might be effective in inhibiting F.VIII inhibitor formation in the hemophilia A model. Methods: We developed a novel method for generating MDSCs in vitro by culturing bone marrow cells from hemophilia A mice with hepatic stellate cells (HSCs), hereafter referred to as HSC-conditioned myeloid cells (H-MCs). DCs were propagated from the bone marrow with GM-CSF and IL-4, whereas H-MCs were propagated from the bone marrow with GM-CSF and HSCs. Granulocyte contaminants were removed on day 2 and the remaining monocytic populations were harvested on day 5. Expression of cell surface antigens was analyzed by flow cytometry. Arginase1 and iNOS levels were compared by qPCR, with or without LPS stimulation. The in vitro suppressive capacity of the H-MCs was determined by a mixed leukocyte reaction culture. Splenic T cells from hemophilia A mice were stimulated by irradiated DCs (at a 1–20 ratio, APC to T cell) and recombinant F.VIII. Additional irradiated DCs or H-MCs were added in graded numbers as regulators. The proliferative response was determined by 3H-thymidine incorporation. The phenotype of cultured CD4+ T cells was characterized by intracellular staining for Foxp3 and IFN-gamma and analyzed by flow cytometry. Inhibition of B cells by H-MCs was determined by a CFSE dilution assay. Purified splenic B cells were labeled with CFSE and stimulated by Ig-M and IL-4. APCs (spleen cells) or H-MCs were added at a ratio of 1:10 (APC to B cell). The proportion of proliferating B cells was determined by CFSE dilution of B220 stained cells. In the COX-2 suppression assay, CFSE labeled B cells were treated with varying concentrations of the selective inhibitor of COX-2, NS398. The suppressive effect of H-MCs on B cells in vivo was determined by simultaneously administering H-MCs (I.V) and F.VIII (I.V.) to hemophila A mice on day 0 and rechallenging with recombinant F.VIII on days 2 and 4. WT B6 mice and hemophilia A mice without H-MC transfer served as controls. Plasma anti-F.VIII antibody titers were measured on day 12 by a modified ELISA assay. Results: H-MCs expressed low levels of costimulatory molecules but high levels of the inhibitory molecule B7-H1 and immunoregulatory enzyme arginase-1. In contrast, DCs expressed high levels of costimulatory molecules and MHC class II. In vitro studies demonstrated that the H-MCs markedly inhibited antigen specific T cell proliferation induced by dendritic cells in response to recombinant F.VIII (Fig. 1). H-MCs altered the T cell response in hemophilia A mice by promoting the expansion of regulatory T cells and inhibiting IFN-γ producing CD4+ T cells. When the H-MCs were cocultured with B cells isolated from hemophilia A mice, in the presence of Ig-M and IL-4, the H-MCs abrogated B cell activation and proliferation directly (Fig. 2). H-MCs may be modulating the B cell response through the Cox-2 pathway, as inhibition of Cox-2 through NS398 led to the restoration of B cell proliferation. More importantly, adoptive transfer of H-MCs into hemophilia Amice, at the time of F.VIII infusion, markedly suppressed anti-F.VIII antibody formation (Fig. 3). Conclusion: These results suggest that HSC conditioned myeloid cells may represent a potential therapeutic approach to induction of immune tolerance in patients with hemophilia A andother immune disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

2006 ◽  
Vol 26 (24) ◽  
pp. 9364-9376 ◽  
Author(s):  
Renren Wen ◽  
Yuhong Chen ◽  
Li Bai ◽  
Guoping Fu ◽  
James Schuman ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Wild Type ◽  
Interleukin 7

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2 −/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2 −/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

scholarly journals Outer Periarteriolar Lymphoid Sheath Arrest and Subsequent Differentiation of Both Naive and Tolerant Immunoglobulin Transgenic B Cells Is Determined by B Cell Receptor Occupancy

1997 ◽  
Vol 186 (5) ◽  
pp. 631-643 ◽  
Author(s):  
Matthew C. Cook ◽  
Antony Basten ◽  
Barbara Fazekas de St. Groth
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Receptor Occupancy ◽  
Cell Receptor ◽  
Antigen Concentration ◽  
Identical Manner ◽  
T Cell Help ◽  
Self Antigen

T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti–hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4+ TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

Role of FOXO1 in Germinal Center Development and Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3536-3536 ◽  
Author(s):  
David Dominguez-Sola ◽  
Jennifer Kung ◽  
Victoria A Wells ◽  
Antony B Holmes ◽  
Laura Pasqualucci ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nuclear Localization ◽  
Germinal Center ◽  
Target Genes ◽  
Cell Receptor ◽  
Missense Mutations ◽  
Wild Type ◽  
Foxo1 Gene

Abstract A significant fraction of B cell non-Hodgkin lymphomas (B-NHL) of germinal center origin carry heterozygous missense mutations in FOXO1, a member of the FOXO family of transcription factors. FOXO1 is a central component of the PI3K signaling cascade engaged by the B cell receptor and is essential for B cell homeostasis and survival (Dengler et al, Nat Immunol 2008; Srinivasan et al, Cell 2009; Lin et al, Nat Immunol 2010). In response to PI3K activation, AKT phosphorylates FOXO1 leading to its nuclear-cytoplasmic translocation and inactivation. Missense mutations of the FOXO1 gene are detectable in germinal center (GC)-derived B-NHL, including ~12% of Burkitt Lymphoma (BL) and ~9% of Diffuse Large B Cell Lymphoma (DLBCL) cases (Schmitz et al, Nature 2012; Trinh et al, Blood 2013; Pasqualucci et al, Cell Rep 2014). The role of FOXO1 in normal GC development as well as the contribution of its mutations to lymphomagenesis is unclear. We show that FOXO1 expression is restricted to the dark zone of GCs, where its nuclear localization is detectable in most B cells. Mice carrying the conditional inactivation of FOXO1 in GC B cells display normal GC in number and size. However, these GCs lack phenotypically defined (CXCR4hi/CD86lo) dark zones and are entirely composed by light zone B cells (CXCR4lo/CD86hi). FOXO1-/- GC B cells express AICDA and carry a normal number of mutations in their immunonoglobulin genes, but do not undergo affinity maturation, resulting in severely impaired antigen responses. In order to identify the biological program controlled by FOXO1 in GC B cells, we identified candidate transcriptional target genes by integrating ChIP-seq and gene expression data. These analyses showed that that the establishment of the dark zone fate relies on a FOXO1-dependent transcriptional network that is enriched for genes involved in immune signaling cascades triggered by the B cell receptor and by a variety of cytokines controlling GC polarity. Notably, a majority of these target genes are co-bound and co-regulated, in a FOXO1-dependent manner, by BCL6, a well characterized GC master regulator. To assess the role of BL- and DLBCL-associated mutations, we first investigated the subcellular localization of FOXO1 mutant proteins by transfecting wild type and mutant GFP-tagged FOXO1 alleles into HeLa cells. As previously shown (Trinh et al, Blood 2013), this analysis showed that mutant FOXO1 proteins, but not the wild-type one, readily localize in the nucleus. Analogously, immunofluorescence analysis of BL and DLBCL samples showed the presence of nuclear FOXO1 in all tumors carrying mutations in the FOXO1 gene. However, nuclear localization was also detectable in virtually all cases carrying normal FOXO1 genes. Accordingly, in vitro experiments testing the ability of normal and mutated FOXO1 proteins to respond to various signals activating the PI3K pathway in multiple BL and DLBCL cell lines, failed to display a correlation between the presence of mutations and responsiveness to these signals. Taken together, these results suggest that other mechanisms in addition to direct gene mutation are responsible for the constitutive nuclear localization of FOXO1 in tumors. We are now examining the consequences of FOXO1 missense mutations in vivo, by reconstituting FOXO1-/- GC B cells with FOXO1 mutants using bone marrow chimeras. Disclosures No relevant conflicts of interest to declare.

Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4507-4507 ◽  
Author(s):  
L. Laura Sun ◽  
Xiaocheng Chen ◽  
Yvonne Chen ◽  
Mark S. Dennis ◽  
Diego Ellerman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Antibody Fragments ◽  
Bispecific Antibodies ◽  
Cell Targeting ◽  
B Cell Malignancies

Abstract T-cell recruiting bispecific antibodies and antibody fragments have been used to harness the cytotoxic potential of T cells for cancer treatment. As an example, encouraging clinical responses have been reported with the B cell targeting Blinatumomab, a 55-kDa fusion protein composed of two single-chain antibody fragments (scFvs). However, the therapeutic promise of many reported bispecific antibodies and fragments is often limited by unfavorable pharmacokinetics and administration schedule, immunogenicity, and a propensity towards aggregation. We have adopted a knobs-into-holes (KIH) antibody format and produced T-cell dependent bispecific antibodies (TDB), which allow one arm to target various B cell antigens while the other arm recruits T cells by binding to the CD3e subunit of the T-cell receptor. These B cell targeting TDBs are full length, humanized IgG1 antibodies with natural antibody architecture. Single dose pharmacokinetic/pharmacodynamic studies in cynomolgus monkeys show the KIH format TDBs are well tolerated in life, result in potent B cell depletion in peripheral and lymphoid tissue, and demonstrate pharmacokinetic properties resembling conventional antibody therapy. One B cell antigen targeted is CD79b, a component of the B cell receptor complex. CD79b is restricted to B cells, is highly prevalent on B cell leukemia and lymphomas, and has been clinically validated by an anti-CD79b antibody-drug conjugate as a safe and effective therapeutic target for B cell malignancies (ASCO 2014 abstract#8519). In our present work, we show that anti-CD79b/CD3 TDB can be produced and purified from E.coli, free of homodimer and aggregates. Anti-CD79b/CD3 TDB is a conditional agonist, activating CD3+T cells only in the presence of CD79b expressing B cells. In vitro, it induces potent B cell killing in a T-cell dependent manner, and is broadly active against lymphoma cell lines with a wide range of CD79b antigen levels. Compared to bispecific antibodies targeting some other B cell antigens, anti-CD79b/CD3 TDB appears to be more potent in autologous B cell killing assays with human PBMCs isolated from healthy donors. Taking advantage of antibodies with a range of binding affinities, we show that the B cell cytotoxic potency of anti-CD79b/CD3 TDB can be enhanced with increased binding affinity of either the anti-CD79b arm or the anti-CD3 arm in vitro. To assess the therapeutic potential of anti-CD79b/CD3 TDB, we further demonstrate that it is active in killing B lymphoma cells isolated from leukemia and lymphoma patients. Collectively, these preclinical data suggest anti-CD79b/CD3 TDB may be a promising agent for clinical development in B cell malignancies. Disclosures Sun: Genentech: Employment. Chen:Genentech: Employment. Chen:Genentech: Employment. Dennis:Genentech: Employment. Ellerman:Genentech: Employment. Johnson:Genentech: Employment. Mathieu:Genentech: Employment. Oldendorp:Genentech: Employment. Polson:Genentech: Employment. Reyes:Genentech: Employment. Stefanich:Genentech: Employment. Wang:Genentech: Employment. Wang:Genentech: Employment. Zheng:Genentech: Employment. Ebens:Genentech: Employment.

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