Gabriella Chirumbolo

Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.

Background. Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potential source of MSCs instead of bone marrow (BM). Methods. Flow cytometric analysis showed that DP-MSCs and BM-MSCs were equally SH2, SH3, SH4, CD29 and CD 166 positive. The in vitro proliferative kinetics of MSCs were measured by 3H-thymidine incorporation uptake. The immunosuppressive function of MSCs was then tested by coculturing PHA-stimulated allogeneic T cells with or without MSCs for 3 days. Results. BM-MSCs could be differentiated in vitro into osteogenic, chondrogenic and adipogenic lineages. DP-MSCs showed osteogenic and adipocytic differentiation, but did not differentiate into chondrocytes. Although DP-MSCs grow rapidly in vitro between day 3 and day 8 of culture and then decrease their proliferation by day 15, BM-MSCs have a stable and continuous proliferation over the same period of time. The addition of DP-MSCs or BM-MSCs resulted in 91 ± 4% and 75 ± 3% inhibition of T cell response, respectively, assessed by a 3H-thymidine assay. Conclusions. Dental pulp is an easily accessible and efficient source of MSCs, with different kinetics and differentiation potentialities from MSCs as isolated from the bone marrow. The rapid proliferative capacity together with the immunoregulatory characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation.

Role of plasmacytoid dendritic cells in immunity and tolerance after allogeneic hematopoietic stem cell transplantation

Dendritic cells (DC) may play an important role in the pathogenesis of alloimmune reactions, such as graft-vs.-host disease after allogeneic hematopoietic stem cell transplantation (HSCT). In humans, two types of DC-myeloid DC (mDC) and plasmacytoid DC (pDC) have been characterized and have distinct origins and functions. The data obtained from studies in vitro suggest that pDC are involved in the regulation of immunity, including the induction and maintenance of tolerance, as well as in the defence against viruses. The authors will review all the evidence currently available from reports exploring the role of pDC in clinical allogeneic HSCT.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage

OBJECTIVEnThe aim of this study was to determine whether expression of the CD86 costimulatory molecule in acute myeloid leukemia (AML) can identify blast cells committed to the monocytic/dendritic lineage.nnnMATERIAL AND METHODSnOne hundred ten consecutive AML patients observed at diagnosis were studied by flow cytometry. In selected experiments, in vitro cultures with CD34(+)CD86(+) or CD34(-)CD86(+) blasts were performed in the presence of granulocyte-macrophage colony-stimulating actor (GM-CSF) with or without tumor necrosis factor-alpha (TNF-alpha) or GM-CSF + interleukin-4 (IL-4), respectively, to induce a dendritic differentiation, documented by morphologic and immunophenotypic assays. T-cell alloreactivity to CD86(+) AML cells and leukemic dendritic cells (AML-DC) was tested in mixed leukocyte cultures and anti-leukemic cytotoxic assays.nnnRESULTSnCD86 was expressed in 54% AML, whereas CD80 and CD1a were only occasionally positive. CD86(+) AML samples included M5 and M4, but also 47% M0-M1 FAB types, and were more frequently CD14(+) (p < 0.00001) and CD34(-) (p = 0.00005) than CD86(-)AML. Six different patterns of CD86(+) AML were identified, according to CD34 or CD14 total or partial coexpression. Four samples enriched in CD34(+)CD86(+) AML cells differentiated into AML-DC CD86(+), CD80(+), CD40(+), CD11c(+), HLA-DR(++), CD14(+/-) that also were CD1a(+) or CD83(+), after 6 days of in vitro culture with GM-CSF +/- TNF-alpha. CD34(-)CD86(+) AML cells differentiated into AML-DC after 3 to 5 days (n = 5 experiments), and trisomy 8 was found in two AML and AML-DC samples by fluorescence in situ hybridization analysis. Finally, AML-DC induced more potent allo-T-cell proliferation, cytokine release, and anti-leukemic cytotoxicity than CD86(+) blasts.nnnCONCLUSIONSnIn AML, CD86 is a marker of monocytic/dendritic lineage. Because CD86(+) blasts may differentiate into DC rapidly, CD86(+)AML patients could be optimal candidates for immunotherapy studies, both in autologous and allogeneic settings.

Use of anti-BDCA-2 antibody for detection of dendritic cells type-2 (DC2) in allogeneic hematopoietic stem cell transplantation

TH2-inducing dendritic cells (DC2) are commonly identified as negative for lineage markers and positive for HLA-DR and CD123 expression. More recently, normal blood DC2 were shown also to be positive for BDCA-2 and BDCA-4 antigens. The aim of this study was to evaluate whether BDCA-2 expression on DC2 is impaired in patients undergoing an allogeneic hematopoietic stem cell transplantation (HSCT) and in healthy donors treated with G-CSF for HSC mobilization. Flow cytometry assays for DC2 detection using either a triple staining with anti-HLA-DR PerCP, anti-Lin+ anti-CD34 FITC and anti-CD123 PE monoclonal antibodies (mAbs), or a double staining with anti-HLA-DR PE and anti-BDCA-2 FITC mAbs were compared in blood samples from patients who underwent an allogeneic HSCT (n = 30) or from healthy donors before (n = 11) and after (n = 8) G-CSF mobilization, as well as in healthy donors’ leukapheresis products (n = 12) or bone marrow (n = 4). Staining of BDCA-2+ cells with other markers such as anti-CD38, anti-CD54 and anti-CD58 were also performed. Median values of CD123+ DC2 and BDCA-2+ DC2 were not statistically different in the blood of patients previously treated with chemotherapy, nor in the blood or bone marrow of heathy donors. Also, a 5 day G-CSF treatment did not affect BDCA-2 or adhesion molecule expression on healthy donors’ blood DC2 significantly. A correlation between all the results (n = 65) obtained with the two assays was demonstrated in a linear regression curve (r = 0.914) (P = 0.00001). BDCA-2 is a marker highly specific for DC2 that is not downregulated by chemotherapy or G-CSF treatment. Therefore, the anti-BDCA-2 mAb can be efficiently combined with other mAbs and used in studies addressing the role of DC2 in the allogeneic HSCT setting.

Selective apoptosis of monocytes and monocyte-derived DCs induced by bortezomib (Velcade)

Bortezomib, a proteasome inhibitor, has shown immunosuppressive activity in animal models of GVHD. In this study, we evaluated the effects of Bortezomib on the survival of monocytes, a major circulating source of DCs. PBMCs or purified CD14+ monocytes were cultured for 24u2009h with Bortezomib (0.1–100u2009ng/ml). Apoptosis was demonstrated on the basis of detection of phosphatydilserine. Bortezomib induced a significant dose-dependent depletion (P=0.008) of monocytes in PBMC preparations, with <1% CD14+ cells remaining at doses ⩾5u2009ng/ml. Moreover, Bortezomib decreased the survival of purified monocytes within 24u2009h (P=0.004) (n=6). Monocyte loss was due to apoptosis (effective dose 50%, ED50, 1–10u2009ng/ml). In addition, both immature and mature monocyte-derived DC underwent apoptosis following exposure to Bortezomib. Kinetic experiments showed that apoptosis increased at 16u2009h through 24u2009h of culture. However, short term (4u2009h) incubation with Bortezomib irreversibly committed monocytes to undergo apoptosis at 24, 72 and 144u2009h. Instead, Bortezomib induced no apoptosis of purified CD19+ B, CD3+ T lymphocytes and CD34+ progenitor cells (ED50 >50u2009ng/ml). The inhibitory effect of Bortezomib on professional APCs, such as monocytes and DCs, suggests its possible use in GVHD prophylaxis.

Increased donor CD86+CD14+ cells in the bone marrow and peripheral blood of patients with chronic graft-versus-host disease.

Background. Based on experimental models, chronic graft-versus-host disease (cGVHD) may depend on activated donor antigen-presenting cells presenting host antigens to donor T cells. Methods. Peripheral blood (PB) and marrow samples from 24 patients with cGVHD and 17 patients without GVHD after an allogeneic hematopoietic stem-cell transplant were analyzed by flow cytometry. The absolute number of myeloid dendritic cells (mDC) (BDCA1+CD19−), plasmacytoid DC (pDC) (BDCA2+CD123+) and monocytes (CD45+CD14+), and mean fluorescence intensity of costimulatory molecules and chemokine receptors were assessed in each antigen-presenting cell population. Results. Patients with cGVHD showed increased numbers of marrow monocytes when compared with patients without cGVHD (P=0.006). Moreover, monocytes of cGVHD patients had greater CD86 mean fluorescence intensity in marrow (P=0.02) and PB (P=0.04). Treatment with prednisone resulted in decreased CD86 expression in marrow (P=0.02) and PB (P=0.04) monocytes. The number and phenotype of mDC and pDC were similar in patients with or without cGVHD. Full-donor chimerism was detected in the PB of all patients, and in purified CD14+ monocytes from three cGVHD patients. Conclusion. Our results show an increased activation of donor-derived marrow and blood monocytes in patients with cGVHD, possibly suggesting the need to target monocytes in the treatment of cGVHD.

Allogeneic graft CD34+ cell dose correlates with dendritic cell dose and clinical outcome, but not with dendritic cell reconstitution after transplant

OBJECTIVEnThis study examined whether the CD34(+) cell dose in allografts correlates with the dose of myeloid dendritic cells (mDC) and plasmacytoid DC (pDC), and with DC reconstitution and clinical outcome after a myeloablative HLA-matched transplant.nnnPATIENTS AND METHODSnFifty-three patients were included in this study: 37 who had undergone a granulocyte colony-stimulating factor mobilized peripheral blood stem cells (PBSC) transplant from related donors and 16 who had undergone a marrow transplant from unrelated donors. The number of CD34(+) cells, lin(-)HLA-DR(+)CD11c(+) mDC, lin(-)HLA-DR(+)CD123(+) pDC, CD14(+) monocytes, and CD3(+)CD4(+), CD3(+)CD8(+), CD56(+), and CD19(+) lymphocytes was compared in the graft, as well as in the peripheral blood after transplant, in patients receiving more than versus less than or equal to the median number of CD34(+) cells in PBSC (5.78 x 10(6)/kg) or in marrow (2.8 x 10(6)/kg).nnnRESULTSnA higher CD34(+) cell dose was associated with larger numbers of mDC in PBSC (p=0.01) and pDC in marrow grafts (p=0.004). However, neither mDC nor pDC recovery after transplant correlated with the number of CD34(+) cells infused. Finally, higher doses of CD34(+) cells appeared to negatively affect (p=0.02) the overall survival in PBSC transplantation and were associated with a trend for higher acute graft-vs-host disease in PBSC and lower acute graft-vs-host disease in marrow transplant.nnnCONCLUSIONSnCD34(+) cell dose correlates with the dose of different DC subsets in PBSC and marrow grafts, but it does not affect DC reconstitution after transplant. Higher doses of CD34(+) cells in PBSC, but not in marrow, seem to adversely affect survival after transplant.

Human CD34+ blood cells induce T-cell unresponsiveness to specific alloantigens only under costimulatory blockade

OBJECTIVESnThe immunogenic role of human CD34(+) cells in allogeneic hematopoietic stem cell transplantation is controversial. In this study we tested the role of CD40 and CTLA4 ligands on CD34(+) cell costimulation of HLA-mismatched lymphocytes.nnnMATERIALS AND METHODSnAn anti-CD40L monoclonal antibody (hu5C8) and/or CTLA4-Ig molecule were used in primary mixed lymphocyte culture (MLC) with irradiated CD34(+) blood cells and allogeneic responders. Then, secondary MLC, cytotoxic activity, and effector cytokine expression and production were measured.nnnRESULTSnEach reagent was able to reduce anti-CD34(+) cell alloreactivity, but only the combination of the anti-CD40L monoclonal antibody and CTLA4-Ig induced greater than 90% inhibition of T-cell response in primary MLC and prevented generation of cytotoxic T cells when priming with purified CD34(+) cells. Importantly, responder cells activated by allogeneic CD34(+) cells in the presence of anti-CD40L monoclonal antibody and CTLA4-Ig entered a state of antigen-specific unresponsiveness while responding to third party antigen, tetanus toxoid, or phytohemagglutinin, and showed suppression of interferon-gamma and increase of interleukin-10 expression and release. Interestingly, addition of interleukin-2 in secondary MLC did not reverse T-cell anergy.nnnCONCLUSIONSnThe results demonstrate that human CD34(+) blood progenitors stimulate T-cell responses potently and can induce T-cell unresponsiveness only when both B7:CD28 and CD40:CD40L pathways are blocked, with an increase of interleukin-10-producing cells. Therefore, our data allow design of in vivo studies aimed at achieving T-cell tolerance across HLA barriers by using purified CD34(+) cells and costimulatory blockade.

Different immune reconstitution in multiple myeloma, chronic myeloid leukemia and acute myeloid leukemia patients after allogeneic transplantation of peripheral blood stem cells

In this study we compared the lymphocyte reconstitution in 13 multiple myeloma (MM), nine acute myeloid leukemia (AML) and 10 chronic myeloid leukemia (CML) patients after allogeneic G-CSF-mobilized PBSC transplantation from HLA-identical siblings. Conditioning regimens included standard total body irradiation + cyclophosphamide (CY), or busulphan + CY, whereas VP-16 was added in patients with advanced disease. Overall comparable numbers of mononuclear cells, CD34+ cells and CD3+ T cells were infused in each group. A significantly higher CD3+ T cell number was observed in MM and AML than in CML patients 1 month after transplant. However, MM patients showed a faster and better recovery of CD4+ T cells than both AML and CML patients at 3 months (Pu2009=u20090.01 and Pu2009=u20090.01, respectively) and 12 months (Pu2009=u20090.01 vs AML, while Pu2009=u2009NS vs cml) after transplant, and had a cd4:cd8 ratio >1 with a median CD4+ t cell value >400/μl 1 year after transplant. development of acute graft-versus-host disease (gvhd) did not affect cd4:cd8 ratios but patients who experienced acute gvhd >grade I had lower CD4+ and CD8+ t cell numbers at all time points. however, after excluding patients with gvhd >grade I, MM patients still showed a significantly higher CD4+ T cell value than patients with myeloproliferative diseases 1 year after transplant. These findings suggest that although allogeneic PBSC transplantation induces rapid immune reconstitution, different kinetics may occur among patients with hematological malignancies. In particular, the rapid reconstitution of CD4+ T cells in MM patients may contribute to the low transplant-related mortality achieved in this disease. Bone Marrow Transplantation (2000) 26, 1325–1331.

Enhancement of T cell activation by immobilized hu5C8 (anti‐CD40L) monoclonal antibody

Background:u2002 Soluble monoclonal antibodies (MoAb) targeting CD40L on T cells can partially block T cell alloreactivity by preventing the costimulatory signal of antigen presenting cells through CD40. However, it is not known if these MoAbs can also deliver inhibiting or stimulating signals through the CD40L receptor.