ALBERT

Description: Background Donor lymphocyte infusions (DLI) can produce lasting remissions in patients with relapsed chronic myeloid leukemia (CML), but are less effective in non-CML diseases. Chemotherapy-induced lymphodepletion prior to DLI, achieved with cyclophosphamide (Cy) and fludarabine (Flu), has been shown to enhance activation of donor lymphocytes and cause significantly more acute graft-versus-host disease (GVHD) than DLI alone. To safely balance the toxic versus beneficial effects of activated donor lymphocytes, we combined lymphodepleting chemotherapy with the infusion of donor T cells engineered to carry a suicide gene to treat patients with aggressive hematologic malignancies. Methods Donor T cells were transduced with a retroviral vector expressing the Herpes simplex thymidine kinase (TK) suicide gene and the human Thy-1 selection marker and expanded in culture for 15 to 19 days prior to their infusion. In this phase I/II trial, the safety and efficacy of Cy/Flu/DLI-TK was studied between February and December 2011 in 10 adults with relapsed multiple myeloma (n=5) or myelodysplasic syndrome/acute leukemia (n=5). One had previously failed to respond to at least one standard DLI while others had not received any DLI before inclusion. All were free of immunosuppressive therapy at inclusion. Patients were infused with a mean cell-dose of 5 (range: 1-10) x106 CD3+ cells/kg of which 98% were TK+ cells (range: 97%-99%). DLI products contained a mean ratio of 0.6% (range: 0.1-1.6) CD4+FoxP3+Helios+ regulatory T-cells. This trial was registered at www.clinicaltrials.gov as NCT 01086735. Results The Cy/Flu regimen was profoundly lymphodepleting and led to a mean 3-day period of neutropenia below 500 PMN/µL. Three patients developed acute GVHD following TK+ cell infusion. One patient had a grade I cutaneous GVHD resolving with local steroids. In another patient with grade III cutaneous and digestive involvement, intra-venous administration of ganciclovir (GCV) led to the complete and lasting resolution of symptoms correlated with clearance of TK+ cells in peripheral blood. A third patient had a grade III liver GVHD. Due to only partially controlled leukemia at the time of DLI, GCV treatment was postponed for one week after GVHD onset in order to support a putative GVL effect. Treatment with GCV led to the rapid disappearance of TK+ cells in peripheral blood and liver (see Figure) but without clinical improvement after 2 weeks, so that an additional immunosuppressive therapy should be instituted. Six patients, including one of the 2 treated with GCV, experienced relapse/progression of their malignancy and received additional anti-cancer therapy. With a median follow-up of 22 months after Cy/Flu/DLI-TK, 6 patients are alive, 5 of them being disease-free. In patients not treated with GCV, TK+ cells could still be detected in peripheral blood till at least 12 months after their infusion. Conclusion Lymphodepletion followed by suicide-gene-transduced T-cell infusion may help to safely enhance the immune activity of DLI, which could be further improved by regulatory T-cell depletion (Maury et al, Science Translat Medicine 2010). Disclosures: No relevant conflicts of interest to declare.

Description: Churg-Strauss syndrome (CSS) is characterized by systemic vasculitis and blood and tissue eosinophilia. Blood eosinophilia correlates with disease activity, and activated T cells from CSS patients are predominantly T helper 2 (Th2). Interleukin (IL)-25 has been shown to link innate and adaptive immunity by enhancing Th2 cytokine production. We sought to determine the involvement of IL-25 and its receptor IL-17RB in the pathogenesis of CSS. We found increased levels of IL-25 in the serum of active CSS patients (952 ± 697 vs 75 ± 49 pg/mL in inactive patients and 47 ± 6 pg/mL in healthy donors). IL-25 was correlated with disease activity and eosinophil level. Eosinophils were the main source of IL-25, whereas activated CD4+ memory T cells were the IL-17RB–expressing cells in CSS. IL-25 enhanced the production of IL-4, IL-5, and IL-13 by activated peripheral blood mononuclear cells. IL-25 and IL-17RB were observed within the vasculitic lesions of patients with CSS, and IL-17RB colocalized with T cells. Increased expression of IL-17RB, tumor necrosis factor receptor–associated factor 6, and JunB in vasculitic lesions of CSS underscored the IL-25–mediated activation, whereas up-regulation of GATA3 and IL-10 supported Th2 differentiation. Our findings suggest that eosinophils, through the production of IL-25, exert a critical role in promoting Th2 responses in target tissues of CSS.

Description: Key Points Tissue resident group 2 innate lymphoid cells are the main cells producing IL-5 and driving eosinophilia in response to low-dose IL-2 therapy. We described a novel cellular network activated during IL-2 treatment that may lead to a more efficient use of IL-2 in immunotherapy.

Description: Rituximab, an anti-CD20 monoclonal antibody, has been used to treat autoimmune disorders such as mixed cryoglobulinemia (MC). However, its mechanisms of action as well as the effects on cellular immunity remain poorly defined. We investigated the changes of peripheral blood B- and T-cell subsets, the clonal VH1–69 cells, as well as the cytokine profile following rituximab therapy. The study involved 21 patients with hepatitis C–related MC who received rituximab, of whom 14 achieved a complete response. Compared with healthy and hepatitis C virus (HCV) controls, pretreatment abnormalities in MC patients included a decreased percentage of naive B cells (P 〈 .05) and CD4+CD25+FoxP3+ regulatory T cells (P = .02) with an increase in memory B cells (P = .03) and plasmablasts (P 〈 .05). These abnormalities were reverted at 12 months after rituximab. Clonal VH1–69+ B cells dramatically decreased following treatment (32% ± 6% versus 8% ± 2%, P = .01). Complete responders of rituximab exhibited an expansion of regulatory T cells (P 〈 .01) accompanied with a decrease in CD8+ T-cell activation (P 〈 .01) and decreased production of interleukin 12 (IL-12; P = .02) and interferon-γ (IFN-γ; P = .01). Our findings indicate that in patients with MC, response to B-cell depletion induced by rituximab effectively normalizes many of the disturbances in peripheral B- and T-lymphocyte homeostasis.

Description: Expression of CD13/N-aminopeptidase may reflect cell activation and growth. We examined its role regarding cell growth in cultures of cord blood CD34+ cells with stem cell factor/Flt-3 ligand/granulocyte-macrophage colony-stimulating factor/tumor necrosis factor-. Indeed, 82% ± 6% of cells from culture day 5 were CD13hi, 25% ± 8% of which were still Lin−. About 50% of CD13hiLin− cells, which comprise progenitors of dendritic cells (DC), monocytes/macrophages and granulocytes, and 30% of CD13loLin− cells were CD34+. Sorted CD34+CD13hiLin− cells, cultured further for 7 days with the same cytokines, expanded 31-fold and CD34-CD13hiLin− cells 7-fold, but CD34+CD13loLin− and CD34−CD13loLin− cells did not grow. Thus, cell growth correlated with CD13 expression, all the more so that cells were CD34+. Actinonin, the most potent N-aminopeptidase inhibitor, was used to engage CD13 on sorted CD13hiLin− cells and on culture day-7 bulk cells. In both cases, this resulted in reversible cell growth arrest, with 30% to 60% fewer cells in the G2/S-M phase than in controls. Interestingly, similar effects were noted with CD13 monoclonal antibody TÜK1, which does not inhibit N-aminopeptidase activity, but not with N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells appeared susceptible to actinonin, which induced cell apoptosis at the same time as Bcl-2 was downregulated and caspase-3 activity increased, but finally percentages and yields of DC and macrophage precursors were affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an independent determinant, CD13 plays a role in the growth of DC/macrophage progenitors and precursors.

Description: Abstract 3416 The relationship between monocytes and mesenchymal stromal cells remains a controversial issue. During embryonic development, the two cell types emerge early and share a large pattern of tissue expression. Using human embryonic stem cells (hES), we recently reported that embryonic monocytes/macrophages were endowed mainly with anti-inflammatory and remodeling functions. Here, we show that a subset of embryonic monocytic cells can give rise to stromal cells. Mesoderm and hematopoietic specification of hES were achieved from embryoid bodies in Iscove's Modified Dulbecco's medium (IMDM) supplemented with 15% fetal bovine serum (FBS) in presence of Bone Morphogenetic Protein 4 (BMP-4, 10 ng/ml) and Vascular Endothelial Growth Factor (VEGF, 5 ng/ml), followed by fetal liver tyrosine kinase 3 ligand (FLT3-ligand, 10 ng/ml), stem cell factor (SCF, 50 ng/ml), interleukin-3 (IL-3, 100 U/ml) and thrombopoietin (TPO, 10 ng/ml). Between day 14–21 of culture, CD45+14+ cells were sorted, cultured for 4 days in presence of Monocyte-Colony Stimulating Factor (M-CSF, 50 ng/ml), Granulocyte-Macrophage CSF (GM-CSF, 20 ng/ml) and IL-3, and subsequently seeded on fibronectin. After culture in Endothelial Cell Growth Medium supplemented with Endothelial GF (EGF), VEGF (25 ng/ml) and bFibroblast GF (bFGF) (1 ng/ml) for 14 days, clones of adherent cells with typical fibroblast-like morphology emerged at a frequency of 2/104 plated embryonic monocytic cells. In order to eliminate contaminating stromal cells before seeding on fibronectin, CD34low43+ hematopoietic cells were sorted at day 10 and CD45+14+ cells were sorted 7 days later. These cells were cultured with the previously described growth factors, but in serum free medium that does not support stromal cell proliferation. In these conditions, we observed that stromal cell developed from CD45+CD14+ embryonic monocytes. These EM-SCs shared several phenotypic and functional characteristics with adult mesenchymal stem cells (MSCs). They could be expanded in vitro in complete alpha–modified Eagle's medium (MEMa) supplemented with 10% FBS, by successive cycles of dissociation. At a density of 500 and 1000 per cm2, EM-SCs formed small colonies of CFU-F. EM-SCs did not express the hematopoietic surface markers CD14 and CD45, nor the endothelial markers CD31 and KDR, and strongly expressed CD105, CD73, CD13 and CD90. In contrast with adult MSCs, they expressed CD133 and low levels of CD34. EM-SCs could not elicit a proliferative response in the presence of allogeneic lymphocytes, and exhibited a suppressive effect on T-cell proliferation in mixed lymphocyte reaction. Under appropriate conditions, EM-SCs displayed osteogenic, chondrogenic and adipogenic differentiation. They could also adopt a smooth muscle cell but not an endothelial or a cardiac phenotype. Compared to adult MSCs, EM-SCs did not expressed telomerase reverse transcriptase, but demonstrated longer telomeres and enhanced expression of genes encoding growth factors, adhesion proteins, tissue degrading enzymes, and anti-inflammatory chemokines. EM-SCs also secreted high amounts of proteins involved in tissue remodeling and angiogenesis. Thus, a rare subset of embryonic monocytic cells can give rise to a population of stromal cells with high immunosuppressive and remodeling functions. A large body of evidence shows that macrophages and stromal cells are involved in tumor development. It remains to be explored whether stromal cells with remodelling potential could derive from tumor-infiltrating macrophages as it can derive from embryonic macrophages. hES cells offer a valuable experimental model for in vitro studies of these differentiation pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Background- Mixed cryoglobulinemia (MC) vasculitis is an autoimmune disorder associated with chronic hepatitis C virus (HCV) infection. We previously reported that MC vasculitis is associated with a quantitative defect of peripheral blood regulatory T cells (Tregs). We aimed to prospectively evaluate the evolution of this defect during the course of anti-viral treatment. Methods- Treg frequencies and numbers were analyzed in 131 HCV chronically infected patients (including 66 with MC vasculitis) and 20 healthy volunteers. Measurements were taken before, during and after treatment with Pegylated Interferon and Ribavirin. Findings- At baseline, patients with MC vasculitis had significantly lower frequency and number of Tregs than patients without MC vasculitis. Complete remission of MC vasculitis following anti-viral treatment was associated with a significant increase in Treg levels compared to baseline levels. In contrast, Treg levels in non- or partial-responders, which did not differ from those of complete responders at baseline, remained unchanged over the course of the study, and where significantly lower that those of complete responders. Interpretation- The strong positive correlation between clinical responses and Treg levels provides additional support for the central role of Treg cells in the pathogenesis of HCV-induced MC-vasculitis. Furthermore, it emphasizes the dual role of Treg cells in chronic HCV infection: while they may hinder viral elimination, Treg cells also have a beneficial role in limiting autoimmune tissue injury. Correlations, in MC-vasculitis patients, between CD4+CD25 high levels and response to anti-viral treatment using clinical (A+B) or laboratory (C+D) measures. CR-complete response, NR/PR-no or partial response. Correlations, in MC-vasculitis patients, between CD4+CD25 high levels and response to anti-viral treatment using clinical (A+B) or laboratory (C+D) measures. CR-complete response, NR/PR-no or partial response.

Description: The study aim was to examine the B-Lymphocytes Stimulator (BLyS) receptor-ligand system in HCV induced B lymphocyte clonal disorders. 94 patients with chronic HCV [including 35 HCV+ mixed cryoglobulinemia (MC)-vasculitis and 9 HCV+ B-non Hodgkin’s lymphoma (B-NHL) patients] and 15 healthy volunteers were included. A 2-fold serum BLyS increase was associated with HCV-induced MC-vasculitis, and a three-fold increase with HCV-induced B-NHL, compared with HCV+ patients without vasculitis or healthy controls (p

Description: The aim of our study was to determine whether ex vivo expansion of umbilical cord blood (UCB) progenitor cells induces changes in their capacity to generate immune cells. CD34+ CB cells were cultured for 14 days with SCF, FLT3-l, TPO and G-CSF, inducing a total cells, CD34+ and LTC-ICs increase of 1500, 120 and 8 fold respectively. Non expanded (d0) and 14-day expanded (d14) CD34+ cells were compared for their capacity to produce T lymphocytes (TLs) using the fetal thymus organ culture system and DCs generated from d0 and d14 CD34+ cells were compared for their differentiation, phenotype and function. Total percentages of CD4+, CD4+CD8− and CD4+CD8+ TLs obtained from d0 and d14 CD34+ cells were comparable. In both fractions, most of the CD4+ T cells co-expressed iCD3 but a lower proportion of d14 derived TLs expressed sCD3. However, there was no significant difference between d0 and d14 derived TLs in term of Vb chain representation, all TCR-Vb chains examined being represented in each case. These data indicate that d0 and d14 CD34+ cells have a similar capacity to generate TLs and that expansion does not induced any skewing of the TCR-Vb repertoire. D0 and d14 CD34+ cells were next cultured with SCF, FL, GM-CSF and TNF-a to compare their capacity to differentiate into DCs. Similar percentages of CD1a+ DCs expressing the same levels of HLA-DR and co stimulatory molecules were obtained. DCs derived from d14 CD34+ cells were less potent to stimulate allogeneic TLs, but the pattern of cytokines produced by stimulated TLs was similar and no shift towards a predominant Th1 or Th2 response was observed. Moreover, in spite of a quantitative increase (15 fold) related to the CD34 pool amplification, we observed a decreased capacity (13-fold) of d14 cells to generate DCs compared to d0 CD34+ cells. Overall, these results indicate that ex vivo expansion of CD34+ cells doesn’t induce any major modification in T Lymphopoiesis capacity while alters somehow the capacity of the graft to generate DCs. We discuss in the context of UCB transplantation, the putative interest of co-infusion of expanded and non expanded fractions in view of improving myelopoiesis in the graft without subverting the immune reconstitution.