Altered B-cell Research Articles

Endogenous Tim-1 promotes severe systemic autoimmunity and renal disease MRL-Faslprmice

The T-cell immunoglobulin mucin 1, also known as kidney injury molecule-1, modulates CD4+ T-cell responses and is also expressed by damaged proximal tubules within the kidney. Both Th subset imbalance (Th1/Th2/Th17) and regulatory T-cell and B-cell alterations contribute to the pathogenesis of autoimmune disease. This study investigated the effects of an inhibitory anti-T-cell immunoglobulin mucin 1 antibody (RMT1-10) in lupus-prone MRL-Fas(lpr) mice. MRL-Fas(lpr) mice were treated with RMT1-10 or a control antibody intraperitoneally twice weekly from 3 mo of age for 16 wk. RMT1-10 treatment significantly improved survival, limited the development of lymphadenopathy and skin lesions, preserved renal function and decreased proteinuria, reduced serum anti-DNA antibody levels, and attenuated renal leukocyte accumulation. Th1 and Th17 cellular responses systemically and intrarenally were reduced, but regulatory T and B cells were increased. RMT1-10 treatment also reduced glomerular immunoglobulin and C3 deposition and suppressed cellular proliferation and apoptosis. Urinary excretion and renal expression of kidney injury molecule-1 was reduced, reflecting diminished interstitial injury. As RMT1-10 attenuated established lupus nephritis, manipulating immune system T-cell immunoglobulin mucin 1 may represent a therapeutic strategy in autoimmune diseases affecting the kidney.

Targeted gene analysis: increased B-cell lymphoma 6 in preeclamptic placentas.

Preeclampsia is a leading cause for maternal and perinatal mortality and morbidity. Microarray-based transcriptional profiling has been widely used for identifying genes responsible for preeclampsia. These studies deliver multiple pictures of gene signatures, implying the complicated pathophysiology. In the present work, we designed our own gene array containing genes involved in various signaling transduction pathways and analyzed placental samples from patients with preeclampsia and controls. We verify that genes associated with angiogenesis and migration pathways are mostly altered in preeclamptic placentas. Interestingly, several genes including B-cell lymphoma 6 have been identified to be linked to preeclampsia. Increased expression of B-cell lymphoma 6 is correlated with enhanced FLT1 and LEPTIN, the hallmarks of preeclampsia. Moreover, the protein level of B-cell lymphoma 6 is elevated in preeclamptic placentas and is predominantly localized in the nucleus of villous cytotrophoblasts lying directly underneath the syncytial layer, suggestive of an involvement in the function of villous trophoblasts. Altered B-cell lymphoma 6, a key oncogene in B-cell lymphomagenesis, may be involved in the pathogenesis of preeclampsia, and further investigations are required to decipher the molecular mechanisms.

Bispecific Hexavalant Antibodies With Enhanced Trogocytosis For Treatment Of Lupus

BackgroundThe humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases (AIDs), treating currently over 1500 cases of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus (SLE). Because epratuzumab, which is currently in worldwide Phase III registration trials for SLE, reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity (ADCC) and negligible complement-dependent cytotoxicity (CDC) when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. Instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, activation, homing, and re-circulation, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and b7 integrin, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or SLE patients, and of NHL cells spiked into normal PBMCs (Rossi et al., Blood 2013 PMID: 23821660). Rituxmab has clinical efficacy in SLE, but failed to achieve primary endpoints in a Phase III trial. Here we show for the first time that a bispecific hexavalent antibody (bsHexAb), comprising epratuzumab and veltuzumab (humanized anti-CD20), exhibits enhanced trogocytosis compared to epratuzumab, with considerably less B-cell depletion than observed with anti CD20 mAbs. Methods and ResultsA pair of bsHexAbs were generated using DOCK-AND-LOCKTM (DNLTM) to comprise epratuzumab fused with four additional Fab fragments of either veltuzumab [designated 22*-(20)-(20)] or of a humanized anti-CD19 mAb [22*-(19)-(19)]. PBMCs were incubated with the bsHexAbs or the parental mAbs (10 µg/mL) overnight, and the relative surface levels of the key antigens were analyzed by flow cytometry. The 22*-(20)-(20) exhibited the broadest and most extensive trogocytosis, reducing each of CD22, CD20, CD19, CD21, CD79b, CD44, CD62L, and Beta-7 integrin more than epratuzumab, and to a similar extent as veltuzumab, except for CD22, which was much lower with the 22*-(20)-(20) (Table 1). In general, 22*-(19)-(19) showed intermediate trogocytosis, with less antigen reduction than 22*-(20)-(20), but more than epratuzumab. Veltuzumab and rituximab caused considerable (40-50%) B-cell depletion in the ex-vivo assay. Alternatively, epratuzumab, hA19, and both bsAbs did not significantly deplete B cells. ADCC, which is presumably, the primary mechanism of B-cell depletion in the ex-vivo assay, is less potent for 22*-(20)-(20), compared to veltuzumab. CDC, which along with ADCC is an important mechanism for B-cell depletion in vivo, is ∼25-fold less potent for 22*-(20)-(20) compared to veltuzumab. Epratuzumab has minimal CDC and ADCC. ConclusionThe bsHexAb 22*-(20)-(20) is an excellent candidate for treatment of SLE and other AIDs due to its ability to mediate potent trogocytosis without wholesale depletion of B cells, which leads to increased risk of serious infections associated with anti-CD20 therapy. Disclosures:Rossi:Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc: Employment, Stock option Other; IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties.

Reduced Hspa9 Expression Alters IL-7 Signaling In B-Cells

Myeloid and erythroid differentiation defects and cytopenias are most commonly described in myelodysplastic syndromes (MDS), however, a reduction in B-cell progenitors exists. The genetic events contributing to this reduction are poorly understood.Interstitial deletion or loss of one copy of the long arm of chromosome 5 (del5q) is the most common cytogenetic abnormality associated with MDS. Two commonly deleted regions on del(5q) have been described and no biallelic mutations have been identified implicating haploinsufficiency of genes on this interval as a driving mechanism. We, and others, have identified several del(5q) candidate genes, including RPS14, EGR1, CTNNA1, APC, NPM1, DIAPH1, miR145, miR146a, and HSPA9. Consistent with haploinsufficiency, HSPA9 mRNA levels are 50% reduced in del(5q) patients. We previously showed that knockdown of Hspa9by shRNA in a murine bone marrow transplant model resulted in a significant reduction in murine B-cells in the bone marrow, spleen and peripheral blood.To further characterize the role of Hspa9 in hematopoiesis, we created Hspa9 heterozygous mice (Hspa9+/-). Heterozygotes express 50% less Hspa9 protein and are born at normal Mendelian frequencies (N>100). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, Hspa9+/- mice show a significant reduction in CFU-PreB colonies compared to their wild-type littermates, indicating B-cell progenitor defects (14 vs. 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.001). Following long-term engraftment of transplanted bone marrow cells from Hspa9+/-or littermate controls into lethally irradiated recipients, we also observed a 5.8-fold reduction in bone marrow CFU-PreB colonies (N=7-9 mice/genotype, p=0.002), confirming the B-cell progenitor defect is hematopoietic cell-intrinsic.Despite the reduction in CFU-PreB colony numbers, frequencies of freshly isolated early B-cell progenitor and precursor populations in the bone marrow and spleen of Hspa9+/- mice are not different than wild-type littermate controls when assessed by flow cytometry (common lymphoid progenitor, Hardy fractions A-F). We hypothesized that these mice were able to compensate for B-cell alterations caused by loss of Hspa9 in vivo. Consistent with our hypothesis, the reduction in CFU-PreB colony numbers was partially rescued by increasing the concentration of IL-7 in the media. Hspa9+/- colony numbers increased 1.8 fold when the IL-7 concentration was increased from 10ng/mL to 50ng/mL compared to 0.80 fold for wild-type littermates (p=0.03, N=6 mice/genotype). This effect was unique to IL-7. Adding increasing concentrations of Flt-3 ligand, another cytokine that contributes to early B-cell development, did not alter CFU-PreB colony formation. We isolated B220+ cells from Day 7 CFU-PreB cultures for gene expression array analysis and observe reduced expression of genes promoting B-cell proliferation and activation in Hspa9+/- compared to Hspa9+/+ cells.Since IL-7 is the only supportive cytokine in the methylcellulose media, can partially rescue the reduced CFU-PreB phenotype, and is required for early B-cell development and survival, we hypothesized that Hspa9 haploinsufficiency inhibits transduction of IL-7 signaling. We tested this hypothesis using an IL-7 dependent mouse B-cell line (B7 cells; Ba/F3 cells that stably express the IL-7 receptor). Knockdown of Hspa9 by siRNAs resulted in a 8-fold reduction in cell number after 4 days in culture (p=0.004, confirmed with two independent siRNAs) and was associated with an increase in apoptosis and reduction in cells in S-phase of the cell cycle. Knockdown of Hspa9 in B7 cells resulted in reduced levels of phosphorylated Stat5, an immediate downstream target of IL-7 receptor stimulation, compared to cells treated with a non-targeting siRNA (measured at 5, 10, 15 and 30 minutes following 10ng/mL IL-7 stimulation, p≤0.03). Ongoing studies will further interrogate the effects of Hsap9 knockdown on Jak-Stat signaling.Collectively, these data implicate that loss of HSPA9 alters IL-7 signaling, potentially contributing to the reduction of B-cell progenitors observed in patients with del(5q)-associated MDS. Disclosures:No relevant conflicts of interest to declare.

The serine‐threonine kinase p90RSK is a new target of enzastaurin in follicular lymphoma cells

Follicular lymphoma is the second most common non-Hodgkin's lymphoma and, despite the introduction of rituximab for its treatment, this disease is still considered incurable. Besides genetic alterations involving Bcl-2, Bcl-6 or c-Myc, follicular lymphoma cells often display altered B-cell receptor signalling pathways including overactive PKC and PI3K/Akt systems. The effect of enzastaurin, an inhibitor of PKC, was evaluated both in vitro on follicular lymphoma cell lines and in vivo on a xenograft murine model. Using pharmacological inhibitors and siRNA transfection, we determined the different signalling pathways after enzastaurin treatment. Enzastaurin inhibited the serine-threonine kinase p90RSK which has downstream effects on GSK3β. Bad and p70S6K. These signalling proteins control follicular lymphoma cell survival and apoptosis; which accounted for the inhibition by enzastaurin of cell survival and its induction of apoptosis of follicular lymphoma cell lines in vitro. Importantly, these results were replicated in vivo where enzastaurin inhibited the growth of follicular lymphoma xenografts in mice. The targeting of p90RSK by enzastaurin represents a new therapeutic option for the treatment of follicular lymphoma.

THU0088 Abnormal memory B-cell reconstitution following anti-CD20 therapy for autoimmune diseases predicts clinical relapse

BackgroundThe advent of anti-CD20 therapy has significantly improved the management of various autoimmune conditions including rheumatoid arthritis (RA). The persistence of B-cells and/or plasmablasts in the immediate post-therapeutic phase (2...

B-cell subset alterations and correlated factors in HIV-1 infection

During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. B-cell phenotype and correlating factors were evaluated. Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. Significant differences were observed among patients' groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4 T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. Our results indicate that viremia and nadir CD4 T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

Abstract 4744: CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells.

Abstract Background. Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphoma and autoimmune diseases, demonstrating therapeutic activities in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy. Although epratuzumab is capable of depleting on average 35% of circulating B cells in patients, its in vivo mechanism of action (MOA) remains incompletely understood. We hypothesized that ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Methods. Peripheral blood mononuclear cells (PBMCs) from either healthy donors or lupus patients with flares, were incubated with epratuzumab, and the relative surface levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Results. Epratuzumab promptly induced a marked decrease of CD22 (&amp;gt;80%), CD19 (&amp;gt;50%), CD21 (&amp;gt;50%), and CD79b (&amp;gt;30%) on the surface of B cells in PBMCs obtained from normal donors or treatment-naïve lupus patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from its internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc-dependent and results from trogocytosis of epratuzumab-bound B cells to FcγR-expressing effector cells, including monocytes, NK cells, and granulocytes. Following incubation with epratuzumab, but not with an isotype control mAb, reduced staining of surface antigens on B cells coincided with positive CD19 and CD22 staining of the effector cells. In the absence of PBMCs, treatment of NHL cell lines (Daudi and Raji) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (&amp;lt;15%) reduction of CD19 and CD21. When these NHL cells (1 x 105 cells) were mixed with PBMCs (1 x 106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Under the conditions examined, rituximab at 10 μg/mL reduced the B-cell count by 50%, whereas epratuzumab did not cause significant B-cell depletion, either at 10 μg/mL or 1 mg/mL. Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. Whether the observed trogocytosis could be correlated with the depletion of malignant B cells in lymphoid tissues is currently under investigation. Citation Format: Edmund A. Rossi, David M. Goldenberg, Rosana Michel, Diane L. Rossi, Daniel J. Wallace, Chien-Hsing Chang. CD22-targeting epratuzumab mediates trogocytosis of multiple cell-surface markers on normal, malignant, and lupus B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2013-4744

Advances in basic and clinical immunology in 2012

Basic and clinical immunology articles published in the Journal in 2012 were mostly related to the expanding area of primary immunodeficiencies (PIDs). Novel forms of PID were identified by using whole-exome sequencing or after careful examination of flow cytometric data, as in the reports of lymphocyte-specific protein tyrosine kinase, CD27, and CD21 deficiencies. Absent IgG and IgA memory B cells were described in patients with hyper-IgE syndrome, which is consistent with defective antibody response and suggests a potential benefit of immunoglobulin replacement. Impaired production of antibodies to polysaccharide antigens by the human B-cell subset analog to murine B-1 cells was reported in a child with selective polysaccharide antibody deficiency. Increased production of inflammatory cytokines by monocyte-derived cells on Toll-like receptor activation was reported in patients with X-linked agammaglobulinemia, underscoring the important role of Bruton tyrosine kinase in modulation of inflammation. The mechanisms explaining susceptibility to yeast infections and development of chronic mucocutaneous candidiasis were extensively studied. Universal newborn screening for T-cell deficiencies is being implemented in several states, resulting in the diagnosis of a higher number of immunodeficient newborns than previously estimated. The use of laboratory testing to distinguish PIDs from HIV infection was clarified. In the management of PIDs, refinement of indication and strategies to hematopoietic stem cell transplantation resulted in improved outcomes. The use of anti-IL-6 mAbs showed promise as an alternative treatment in patients with Schnitzler syndrome.

The Characteristics and Significance of Locally Infiltrating B Cells in Lupus Nephritis and Their Association with Local BAFF Expression

Introduction. Dysfunction of the B lymphocyte is considered to be involved in the pathogenesis of lupus nephritis (LN). Intrarenal B cells have been found in several forms of inflammatory kidney disease. B-cell activating factor (BAFF) regulates B lymphocyte proliferation and survival, and contributes to human autoimmune disease. Their role in renal inflammation is not well defined. Methods. Clinical parameters and renal biopsies from 62 LN patients were prospectively analyzed. We performed standard immunohistochemistry on serial paraffin tissue sections using monoclonal antibodies to CD20 and BAFF to investigate the characteristics and significance of locally infiltrating B cells and local BAFF expression in patients with LN. Results. Intrarenal B cells and/or BAFF were mainly distributed in the renal interstitium. Compared to the LN-non-B-cell/BAFF expression group, proteinuria (g/24 hour), blood urea nitrogen, serum creatinine levels, LN renal activity, and chronicity indices, were all significantly greater in the LN-B-cell/BAFF expression groups. The expression of BAFF was strongly associated with the quantity of B-cell infiltrate in the interstitium. Conclusion. As BAFF expression was strongly associated with B-cell infiltration, we hypothesize that altered B-cell differentiation and tolerance induced by excess BAFF may be central to the pathogenesis of LN.

Exacerbation of lymphomatoid papulosis during rituximab therapy

We report two cases of lymphomatoid papulosis (LyP) occurring in conjunction with B-cell lymphoproliferative malignancies where the LyP was exacerbated during rituximab (anti-CD20 antibody)-based therapy. We postulate that the altered B-cell cytokine milieu acted to allow an exacerbation of the patient's concomitant T-cell disease and discuss the possible mechanisms by which this may have occurred.

CD22-Targeting Epratuzumab Down-Regulates Multiple BCR Regulators On the Surface of Normal, Lupus, and Malignant B Cells

AbstractAbstract 1357 Background.The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason its therapeutic activity may not result completely from B-cell depletion; instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results.PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab’)2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) suppression of CD19 and CD21. When these NHL cells (1×105 cells) were mixed with PBMCs (1×106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Importantly, in all these experiments, there was no evidence of depletion in B cells, whether malignant or not; nor had epratuzumab induced an increase in apoptotic (Annexin V+) B cells in PBMCs from any of 4 donors, compared to the untreated (22.9 ± 1.8% versus 22.1 ±1.2%; P=0.6864). Conclusions.This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures:Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Hspa9 Haploinsufficiency Induces a Cell-Intrinsic Defect in Mouse B-Cell Progenitors

AbstractAbstract 1714Patients with myelodysplastic syndromes (MDS) have a clonal hematopoietic stem cell disorder that results in dysplastic hematopoietic cells in their bone marrow as well as peripheral blood cytopenias. In addition to the commonly described erythroid and myeloid differentiation defects associated with MDS, a reduction in bone marrow B-cell progenitors exists in patients. The genetic events contributing to the reduction in B-cell progenitors remain poorly understood.The most common cytogentic abnormality identified in patients with MDS, occurring in approximately 35% of patients, is heterozygous interstitial deletion or loss of the long arm of chromosome 5 (5q). The interstitial deletions on chromosome 5 are single copy losses, and no biallelic disruptions of genes in deleted regions have been identified, implicating haploinsufficiency as the underlying genetic mechanism. We, and others, have shown that the levels of HSPA9 mRNA expression are reduced ∼50% in patients with del(5q) when compared to MDS patients without del(5q), consistent with a haploinsufficient phenotype. To model haploinsufficiency, we used shRNA to achieve ∼50% knockdown of Hspa9 in a murine bone marrow transplant model. This model showed a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, implicating HSPA9 haploinsufficiency may contribute to the B-cell alterations observed in MDS patients with del(5q).To further evaluate HSPA9 haploinsufficiency in vivo, we created a mouse model with a heterozygous deletion of Hspa9 (Hspa9+/−) and confirmed a 50% reduction in Hspa9 protein levels in bone marrow and spleen of these mice by Western blot. Hspa9+/− mice are born at normal Mendelian frequencies (N>100), however, breeding heterozygous mice suggests Hspa9−/− mice are embryonic lethal (24 Hspa9+/+:38 Hspa9+/−:0 Hspa9−/−). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, the numbers of bone marrow CFU-preB colonies as assessed by methylcellulose assay, are significantly reduced in Hspa9+/− mice compared to Hspa9+/+ littermates (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.0001). We performed noncompetitive bone marrow transplants of Hspa9+/− or Hspa9+/+ donor cells into Hspa9+/+ recipient mice and confirmed that the reduction of B-cell progenitors is a hematopoietic cell intrinsic phenotype (N=7–9 mice/genotype, p=0.002). We also confirmed that the Hspa9+/− bone marrow microenvironment did not contribute to the phenotype as transplantation of Hspa9+/+ donor bone marrow cells into Hspa9+/− recipients did not alter the number of CFU-preB colonies (N=5).Total frequencies of common lymphoid progenitors and B-cell precursors (Hardy fractions A, B/C, D, E and F) as assessed by flow cytometry are no different in Hspa9+/− and Hspa9+/+ mice. Therefore, we hypothesize that early Hspa9+/− B-cells may have an intrinsic signaling defect which can be compensated for in vivo. Early B-cell maturation is dependent on intracellular signaling mediated through cell surface receptors in response to environmental cytokines. Consistent with our hypothesis, we showed that Hspa9+/− CFU-preB in vitro colony formation is partially rescued by increasing concentrations of IL7 while Hspa9+/+ colony numbers remain unchanged (fold change in colony formation from 10ng/mL to 50ng/mL IL7 was 1.80 for Hspa9+/− vs. 0.80 for Hspa9+/+, p=0.03, N=6 mice/genotype). Supplementation of the media with another cytokine that contributes to early B-cell maturation, Flt3 ligand, does not alter Hspa9+/− or Hspa9+/+ CFU-preB colony formation, further implicating altered IL7 signaling. We are currently investigating the downstream responses to IL7 stimulation in B-cell progenitors from Hspa9+/− mice.Collectively, these data implicate loss of HSPA9 as a contributing factor in the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Disclosures:No relevant conflicts of interest to declare.

In Vivo Tungsten Exposure Alters B-Cell Development and Increases DNA Damage in Murine Bone Marrow

High environmental tungsten levels were identified near the site of a childhood pre-B acute lymphoblastic leukemia cluster; however, a causal link between tungsten and leukemogenesis has not been established. The major site of tungsten deposition is bone, the site of B-cell development. In addition, our in vitro data suggest that developing B lymphocytes are susceptible to tungsten-induced DNA damage and growth inhibition. To extend these results, we assessed whether tungsten exposure altered B-cell development and induced DNA damage in vivo. Wild-type mice were exposed to tungsten in their drinking water for up to 16 weeks. Tungsten concentration in bone was analyzed by inductively coupled plasma mass spectrometry and correlated with B-cell development and DNA damage within the bone marrow. Tungsten exposure resulted in a rapid deposition within the bone following 1 week, and tungsten continued to accumulate thereafter albeit at a decreased rate. Flow cytometric analyses revealed a transient increase in mature IgD(+) B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. DNA damage was increased in both whole marrow and isolated B cells, most notably at the lowest tungsten concentration tested. These findings confirm an immunological effect of tungsten exposure and suggest that tungsten could act as a tumor promoter, providing leukemic "hits" in multiple forms to developing B lymphocytes within the bone marrow.

PL07 The essential role of GH/IGF-I in the liver

Insulin-like growth factor I (IGF-I) signaling is mediated by insulin receptor substrate proteins (IRS). In turn, protein tyrosine phosphatase 1B (PTP1B) dephosphorylates and inactivates insulin and IGF-I receptors. IRS2-deficient mice present developmental defects in the nervous system, altered hepatic insulin signalling b-cell failure and develop type 2-like diabetes. IGF1 gene mutations cause syndromic sensorineural hearing loss in men and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. We have studied here, the hearing function and cochlear morphology of IRS2 null mice and the impact of PTP1B deficiency. Auditory brainstem responses and cochlear morphology of systemic Irs2−/−Ptpn1+/+, Irs2+/+Ptpn1-/and Irs2−/−Ptpn1−/− mice were studied at different postnatal ages. The results indicated that Irs2−/−Ptpn1+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology compared to wild type mice. Simultaneous PTP1B deficiency in Irs2−/−Ptpn1−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for the treatment of deafness associated to hyperglycemia and type 2-diabetes. We warmly thank the support of our colleagues Carlos Avendano, Deborah Burks and Lupe Camarero.

OR06-6 IRS2-deficient mice show sensorineural hearing loss that is delayed by concomitant PTP1B loss of function

Insulin-like growth factor I (IGF-I) signaling is mediated by insulin receptor substrate proteins (IRS). In turn, protein tyrosine phosphatase 1B (PTP1B) dephosphorylates and inactivates insulin and IGF-I receptors. IRS2-deficient mice present developmental defects in the nervous system, altered hepatic insulin signalling b-cell failure and develop type 2-like diabetes. IGF1 gene mutations cause syndromic sensorineural hearing loss in men and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. We have studied here, the hearing function and cochlear morphology of IRS2 null mice and the impact of PTP1B deficiency. Auditory brainstem responses and cochlear morphology of systemic Irs2−/−Ptpn1+/+, Irs2+/+Ptpn1-/and Irs2−/−Ptpn1−/− mice were studied at different postnatal ages. The results indicated that Irs2−/−Ptpn1+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology compared to wild type mice. Simultaneous PTP1B deficiency in Irs2−/−Ptpn1−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for the treatment of deafness associated to hyperglycemia and type 2-diabetes. We warmly thank the support of our colleagues Carlos Avendano, Deborah Burks and Lupe Camarero.

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)‐bisphosphate regulates B‐cell activation and autoantibody production

TAPP1 and TAPP2 (where TAPP is tandem PH domain containing protein) are dual PH domain adaptors that selectively bind PI(3,4)P2 (phosphatidylinositol (3,4)-bisphosphate). PI(3,4)P2 is a lipid messenger generated by phosphoinositide 3-kinase (PI3K) and SHIP, both of which are critical regulators of B-cell activation. To determine the functional role of TAPP-PI(3,4)P2 interactions, we utilized a double knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPP1 and TAPP2. TAPP KI mice show evidence of altered B-cell development, but generate phenotypically normal mature B-cell populations. Total serum immunoglobulin IgM and IgG levels were found to be markedly elevated in TAPP KI mice. B cells purified from TAPP KI mice were hyper-responsive to antigen receptor cross-linking, showing increased proliferation, CD86 expression, and Akt phosphorylation on Ser473 and Thr308. Female TAPP KI mice developed elevated levels of anti-DNA and antinuclear antibodies with age, associated with IgG deposition in kidneys and significant glomerulonephritis pathology. Together our results indicate that interaction of TAPPs with PI(3,4)P2 mediates feedback inhibition impacting on BCR signaling, with functional significance for control of autoreactive B cells.

Démarche diagnostique devant une lymphopénie : quand penser à la lymphopénie CD4+ idiopathique ?

Lymphocytopenia is defined by a lymphocyte count less than 1500/mm3 in adults and less than 4500/mm3 in children before the age of 8 months. Lymphocytopenia can be global or selectively affect a peculiar lymphocyte subpopulation. The patient's age, the context as well as the associated clinical manifestations and treatment prescribed must be taken into account in order to identify the etiology of lymphocytopenia. In adults, lymphocytopenia can be caused by: (1) insufficient thymic output (primary immune deficiencies, corticosteroid treatment, zinc deficiency, etc.), (2) increased lymphocyte catabolism (radiotherapy, chemotherapy, immunosuppressant, HIV infection, systemic lupus, etc.), (3) modified lymphocyte distribution (viral infections, septic shock, extensive burns, splenomegaly, granulomatosis, etc.), (4) multifactorial or unknown etiology (end-stage renal disease, lymphoid malignancies, solid tumor, ethnicity, etc.). In children, in addition to these etiologies, other immune deficiencies may be responsible for severe lymphocytopenia (thymocytes apoptosis, cytokine deficiencies, altered B-cell and T-cell receptor synthesis, signal transduction and cellular interactions deficiencies). Idiopathic CD4+ lymphocytopenia is a rare disorder. It is defined by a persisting lymphocyte CD4+ count less or equal to 300/mm3 or less or equal to 20% of total lymphocytes in the absence of alternative diagnosis. Clinical symptoms can be absent or include opportunistic infections, auto-immune manifestations, lymphoma or solid tumors. Treatment is similar to that of HIV-infected patients and sometimes relies on specific immunotherapy even though clinical benefit has not been evaluated.

Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome

AbstractThe immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)–deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.

Bioinformatics analysis reveals transcriptome and microRNA signatures and drug repositioning targets for IBD and other autoimmune diseases

Inflammatory bowel disease (IBD) is a complex disorder involving pathogen infection, host immune response, and altered enterocyte physiology. Incidences of IBD are increasing at an alarming rate in developed countries, warranting a detailed molecular portrait of IBD. We used large-scale data, bioinformatics tools, and high-throughput computations to obtain gene and microRNA signatures for Crohn's disease (CD) and ulcerative colitis (UC). These signatures were then integrated with systemic literature review to draw a comprehensive portrait of IBD in relation to autoimmune diseases. The top upregulated genes in IBD are associated with diabetogenesis (REG1A, REG1B), bacterial signals (TLRs, NLRs), innate immunity (DEFA6, IDO1, EXOSC1), inflammation (CXCLs), and matrix degradation (MMPs). The downregulated genes coded tight junction proteins (CLDN8), solute transporters (SLCs), and adhesion proteins. Genes highly expressed in UC compared to CD included antiinflammatory ANXA1, transporter ABCA12, T-cell activator HSH2D, and immunoglobulin IGHV4-34. Compromised metabolisms for processing of drugs, nitrogen, androgen and estrogen, and lipids in IBD correlated with an increase in specific microRNA. Highly expressed IBD genes constituted targets of drugs used in gastrointestinal cancers, viral infections, and autoimmunity disorders such as rheumatoid arthritis and asthma. This study presents a clinically relevant gene-level portrait of IBD subtypes and their connectivity to autoimmune diseases. The study identified candidates for repositioning of existing drugs to manage IBD. Integration of mice and human data point to an altered B-cell response as a cause for upregulation of genes in IBD involved in other aspects of immune defense such as interferon-inducible responses.