Elisabetta Bonifacio

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

Mesenchymal cells recruit and regulate T regulatory cells

OBJECTIVE Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. MATERIALS AND METHODS Human MSCs and different T cell populations (CD3(+), CD3(+)/CD45RA(+), CD3(+)/CD45RO(+), CD4(+)/CD25(+), CD4(+)/CD25(+)/CD45RO(+), CD4(+)/CD25(+)/CD45RA(+)) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. RESULTS We demonstrate MSC recruit Tregs from a fraction of CD3(+) and from immunoselected CD3(+)/CD45RA(+) and CD3(+)/CD45RO(+) fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4(+)/CD25(bright)/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25(+), CD4/CD25(+)/CD45RA(+), and CD4/CD25(+)/CD45RO(+)) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. CONCLUSIONS In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.

NOTCH1 PEST domain mutation is an adverse prognostic factor in B‐CLL

Prosper, J.Y., Campbell, K., Sutherland, D.R., Metcalfe, P., Horsfall, W. & Ouwehand, W.H. (2002) A tyrosine703serine polymorphism of CD109 defines the Gov platelet alloantigens. Blood, 99, 1692– 1698. Smith, J.W., Hayward, C.P., Horsewood, P., Warkentin, T.E., Denomme, G.A. & Kelton, J.G. (1995) Characterization and localization of the Gova/b alloantigens to the glycosylphosphatidylinositol-anchored protein CDw109 on human platelets. Blood, 86, 2807–2814.

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patients disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4+/CD25+ T regulatory cells (Tregs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated Tregs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4+/CD25+) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4+/CD25+bright cells were 28·4% ± 6·8. The CD4+/CD25+ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4+CD25+FoxP3+ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4+/CD25+ cells and CD4+/CD25‐ cells were not significantly different, indicating that Tregs had a broad T cell receptor repertoire. The inhibition assay showed that CD4+/CD25+ cells inhibited CD4+/CD25‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (Tresp) to Tregs, 1:2]; 60·8% ± 20·5 (ratio of Tresp to Tregs, 1:1); 25·6 ± 19·6 (ratio of Tresp to Tregs, 1:0·1)). Our study shows that negative/positive Treg selection, performed using the CliniMACS device and reagents, enriches significantly CD4+CD25+FoxP3+ cells endowed with immunosuppressive capacities. The CD4+CD25+FoxP3+ population is a source of natural Treg cells that are depleted of CD8+ and CD4+/CD25‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.

Are ongoing trials on hematologic malignancies still excluding older subjects

Hematologic malignancies are diseases that mainly affect older subjects. Multiple myeloma,[1][1] myelodysplastic syndromes[2][2] and chronic myeloid leukemia[3][3] are common in advanced age. Nevertheless, there is evidence that older patients with hematologic malignancies have often been excluded

Unique human CD133+ leukemia cell line and its modulation towards a mesenchymal phenotype by FGF2 and TGFβ1†

Immunological features of GM‐490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA‐DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM‐490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFβ1. When FGF2 and/or TGFβ1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription‐polymerase chain reaction (RT‐PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM‐490 cells differentiate towards the osteoblast pathway. GM‐490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM‐490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFβ1 on each others receptors make the GM‐490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases.

Chronic myeloproliferative disorders: the bone marrow stromal component is not involved in the malignant clone

Chronic myeloproliferative disorders: the bone marrow stromal component is not involved in the malignant clone