A Multiparameter Prognostic Risk Score of Chronic Graft-versus-Host Disease Based on CXCL10 and Plasmacytoid Dendritic Cell Levels in the Peripheral Blood at 3 Months after Allogeneic Hematopoietic Stem Cell Transplantation.

Chronic GVHD (cGVHD) is the major cause of long-term morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). There are no biomarkers that can consistently predict its occurrence. We aimed to evaluate whether numbers of antigen-presenting cell subsets in peripheral blood (PB) or serum chemokine concentrations are biomarkers of cGVHD occurrence. The study cohort comprised 101 consecutive patients undergoing allogeneic HSCT between January 2007 and 2011. cGVHD was diagnosed by both modified Seattle criteria and National Institutes of Health (NIH) criteria. Multicolor flow cytometry was used to determine the number of PB myeloid dendritic cells (DCs), plasmacytoid DCs, CD16+ DCs, and CD16+ and CD16- monocytes, as well as CD4+ and CD8+ T cells, CD56+ natural killer cells, and CD19+ B cells. Serum concentrations of CXCL8, CXCL10, CCL2, CCL3, CCL4, and CCL5 were measured by a cytometry bead array assay. At a median of 60 days after enrollment, 37 patients had developed cGVHD. Patients with cGVHD and those without cGVHD had comparable clinical characteristics. However, previous acute GVHD (aGVHD) was strongly correlated with later cGVHD (57% versus 24%, respectively; P = .0024). Each potential biomarker was screened for its association with cGVHD using the Mann-Whitney U test. Biomarkers that differed significantly (P < .05) between patients with cGVHD and those without cGVHD were analyzed by receiver operating characteristic (ROC) curve analysis to select the variables predicting cGVHD with an area under the ROC curve (AUC) >.5 and a P value <.05. A multivariate Fine-Gray model identified the following variables as independently associated with the risk of cGVHD: CXCL10 ≥592.650 pg/mL (hazard ratio [HR], 2.655; 95% confidence interval [CI], 1.298 to 5.433; P = .008), pDC ≥2.448/µL (HR, .286; 95% CI, .142 to .577; P < .001) and previous aGVHD (HR, 2.635; 95% CI, 1.298 to 5.347; P = .007). A risk score was derived based on the weighted coefficients of each variable (2 points each), resulting in the identification of 4 cohorts of patients (scores of 0, 2, 4, and 6). In a competing risk analysis to stratify patients at differing risk levels of cGVHD, the cumulative incidence of cGVHD was 9.7%, 34.3%, 57.7%, and 100% in patients with scores of 0, 2, 4, and 6, respectively (P < .0001). The score could nicely stratify the patients based on the risk of extensive cGVHD as well as NIH-based global and moderate to severe cGVHD. Based on ROC analysis, the score could predict the occurrence of cGVHD with an AUC of .791 (95% CI, .703 to .880; P < .001). Finally, a cutoff score ≥4 was identified as the optimal cutoff by Youden J index with a sensitivity of 57.1% and a specificity of 85.0%. A multiparameter score including a history of previous aGVHD, serum CXCL10 concentration, and number of pDCs in the PB at 3 months post-HSCT stratify patients at varying risk levels of cGVHD. However, the score needs to be validated in a much larger independent and possibly multicenter cohort of patients undergoing transplantation from different donor types and with distinct GVHD prophylaxis regimens.

Baseline cluster of differentiation 22 fluorescent intensity correlates with patient outcome after Inotuzumab Ozogamicin treatment.

Antigen-directed target therapy for B-cell acute lymphoblastic leukemia (B-ALL) is now the standard of care for relapsed/refractory (R/R) disease. A comprehensive determination of the target itself is mandatory to aid physician's choice. We determined baseline Cluster of differentiation 22 (CD22) expression percentage and fluorescent intensity on lymphoblasts of 30 patients with R/R B-ALL treated with anti-CD22 immunoconjugate drug Inotuzumab Ozogamicin (INO) and analyzed the impact of both parameters on patient outcome. Most patients (24/30, 80%) had a high leukemic blast CD22-positivity defined as ≥90%. We did not observe a benefit in terms of complete remission, overall survival (OS) and duration of response (DoR) for patients with CD22 ≥ 90% versus CD22 < 90%. Concerning CD22-FI quartile analysis we appreciated a trend for superior response rates in higher quartiles (Q2 -Q4 ) compared to Q1 and a significant benefit in terms of OS and DoR for patients with higher CD22-FI. INO demonstrates to be effective also in patients with lower CD22 expression, but therapeutical benefits are more evident in patients with higher CD22-FI. The evaluation of both CD22 percentage and CD22-FI of the leukemic blast may help physicians in therapeutic choices for R/R B-ALL patients when multiple treatment options are available, although no CD22 expression threshold can currently be identified below which INO should be considered not effective.

Long-Term Outcome After Adoptive Immunotherapy With Natural Killer Cells: Alloreactive NK Cell Dose Still Matters.

Recently, many reports were published supporting the clinical use of adoptively transferred natural killer (NK) cells as a therapeutic tool against cancer, including acute myeloid leukemia (AML). Our group demonstrated promising clinical response using adoptive immunotherapy with donor-derived alloreactive KIR-ligand-mismatched NK cells in AML patients. Moreover, the antileukemic effect was correlated with the dose of infused alloreactive NK cells ("functional NK cell dose"). Herein, we update the results of our previous study on a cohort of adult AML patients (median age at enrollment 64) in first morphological complete remission (CR), not eligible for allogeneic stem cell transplantation. After an extended median follow-up of 55.5 months, 8/16 evaluable patients (50%) are still off-therapy and alive disease-free. Overall survival (OS) and disease-free survival (DFS) are related with the dose of infused alloreactive NK cells (≥2 × 105/kg).

The timing of plerixafor addition to G-Csf and chemotherapy affects immunological recovery after autologous stem cell transplant in multiple myeloma.

Plerixafor inhibits CXCR4, thus inducing the mobilization of hematopoietic stem/progenitor cells in lymphoma and multiple myeloma (MM) patients eligible for autologous stem cell transplantation (ASCT). However, the kinetics of plerixafor-induced mobilization of lymphocyte subsets is poorly known. Here, we evaluated the graft content, the engraftment, and the immunological reconstitution of MM patients receiving plerixafor. Thirty-seven patients undergoing one or tandem ASCT were enrolled. After mobilization with cyclophosphamide plus G-CSF, plerixafor was added at hematological recovery regardless of CD34+ cell count. We evaluated the number of CD34+, CD34+/CD38-, CD3+, CD4+, CD8+, CD19+, CD56+/CD3-, CD4+/CD25+/FOXP3+, and CD138+/CD38+ cells on each apheresis. Hematological and immunological recovery were determined at 30 days, 3, 6, 9, and 12 months after ASCT. Overall, 34/37 patients mobilized a median of 10.1 × 106 CD34+ cells/Kg (IQ 7.7-13.4). Patients with <20/µL CD34+ cells at plerixafor administration (18/33) had a significantly higher CD34+ cell fold increase, but not a higher absolute number, than 16/33 patients with ≥20/µL CD34+ cells. A similar CD34+ and immune graft composition was reported. A higher number of CD3+ and CD8+ cells/µL was observed at 3 months after first ASCT (p < 0.05) in the group with ≥20 CD34+ cells/µL. Thus, in MM patients, the timing of plerixafor administration influences immunological recovery.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

INTRODUCTION: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. METHODS: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. RESULTS: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. CONCLUSION: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Human hematopoietic CD34+ progenitor cells induce natural killer cell alloresponses via NKG2D activation.

Human CD34+ cells cross-interact with allogeneic T lymphocytes. In this study we addressed the interaction between CD34+ cells and allogeneic natural killer (NK) cells. Purified NK cells were cultured with allogeneic KIR-permissive CD34+ or CD14+ blood cells, obtained from HLA group C homozygous donors, or with high-dose interleukin-2. A cytotoxicity assay was used to test the ability of NK cells to lyse NK-sensitive K562 or NK-resistant Daudi cells. Cytofluorometric assays were employed to assess cell phenotype and cytokine release. CD34+ cells induced greater lysis of K562 (p = 0.02) and Daudi cells (p = 0.01) than monocytes. CD34 cell stimulation resulted in upregulation of CD69 and CD25 on NK cells and in the production of interferon γ and tumor necrosis factor α. NK activation by CD34+ cells was inhibited by an anti-NKG2D antibody. However, NKG2D ligands such as MIC (MHC class I chain)-A/B and ULBP (UL16 binding protein)-1/3 were not detected on CD34+ cells. Cross-talk between NK and CD34+ cells also induced the upregulation of CD40 and CD86 co-stimulatory molecules on CD34+ cells. Our study indicates a direct NKG2D-dependent stimulatory effect of human CD34+ cells on allogeneic NK cells. These findings may be relevant to the NK-mediated rejection effect in HLA-mismatched hematopoietic stem cell transplantation.

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.

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

BACKGROUND: 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. MATERIALS AND METHODS: Blood mononuclear cells were stimulated by mitogens or allogeneic stimulator cells in the presence of hu5C8 MoAb, either in soluble form, or immobilized to the culture wells. T cell responses were evaluated by means of primary and secondary mixed lymphocyte culture (MLC), cytotoxic T lymphocyte (CTL) generation, immunophenotype, apoptosis assay and cytokine release. Also, the effect of hu5C8 on cells inhibited by CTLA4-Ig was tested. RESULTS: While the soluble hu5C8 inhibited T cell proliferation, the immobilized hu5C8 enhanced both mitogen and alloantigen-induced proliferative and cytotoxic T cell responses, without inducing further apoptotic T cell death. In the presence of CTLA4-Ig, immobilized hu5C8 increased the residual CD28-independent proliferation of alloantigen-specific T cells both in primary and secondary MLC, and prevented the inhibiting effect of CTLA4-Ig on the generation of CTL. Immobilized hu5C8 MoAb-stimulated T cells also showed a limited capacity of producing interleukin (IL)-10, even in the presence of CTLA4-Ig. CONCLUSIONS: We show that the hu5C8 MoAb has a strong mitogenic activity when immobilized, likely due to higher crosslinking capacity as compared to the soluble antibody. Strategies to induce ex-vivo T cell responses against tumor or viral antigens by means of hu5C8 MoAb antibody will be exploited based on these findings.

High dose rabbit antithymocyte globulin induction in living related liver transplantation.

BACKGROUND/AIMS: Induction with rabbit antithymocyte globulin (RATG) has been reported to be effective in cadaveric liver transplantation. The aim of this study was to compare two immunosuppressive protocols in adult living-related liver transplantation (LRLT). METHODOLOGY: From May 2001 through May 2003, 34 LRLT were performed. The first 17 patients (group 1) were treated with tacrolimus (TAC) and steroids. The next 17 patients (group 2) were treated with a steroid-sparing protocol using RATG. RESULTS: The one-year patient and graft survival was respectively 76.5% and 64.7% for group 1 and 88.2 and 76.5% for group 2 (p = 0.037 and p = NS, respectively). Incidence of acute cellular rejection was 41.2% in group 1 compared to 47% in group 2 (p = NS). Mean daily TAC dose at 6 months was 6.5 +/- 1.1 mg/day in group 1 and 3.2 +/- 0.9 mg/day in group 2 (p < 0.001). In group 1, 41.1% experienced CMV infection compared to 11.7% in group 2 (p = NS). CONCLUSIONS: These results suggest that this approach of RATG induction followed by postoperative, steroid-free, and low-dose TAC is safe and provides for adequate immunosuppression and similar outcome when compared to controls treated with standard TAC and steroid immunosuppression.

Higher numbers of blood CD14+ cells before starting conditioning regimen correlate with greater risk of acute graft-versus-host disease in allogeneic stem cell transplantation from related donors.

Host antigen-presenting cells (APCs) have been shown to induce acute graft-versus-host disease (aGVHD) in experimental models. In this study, we investigated whether pretransplantation blood levels of host APCs, such as plasmacytoid and myeloid dendritic cells and monocytes, correlate with the development of aGVHD. A total of 89 consecutive patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-matched related (n = 48) or unrelated (n = 41) donors were enrolled in the study. Blood samples were analyzed by flow cytometry before initiating the conditioning regimen. In related donor transplants, patient-donor sex mismatch and monocyte levels significantly correlated with aGVHD grade II-IV in both univariate and multivariate analyses. Similar results were not observed in recipients of matched unrelated transplants, possibly due to use of antithymocyte globulin (ATG) or differences in graft source in these patients. In conclusion, pretransplantation recipient monocyte levels are relevant to the development of GVHD in HSCT from related donors.

Dendritic cells of immune thrombocytopenic purpura (ITP) show increased capacity to present apoptotic platelets to T lymphocytes.

OBJECTIVE: Altered self-antigen processing/presentation of apoptotic cells by DCs and/or modifications of autoantigens may lead to the development of autoantibodies. Increasing evidence indicates that platelets may undergo apoptosis. Therefore, in the present study we investigated whether platelet apoptosis and/or dendritic cells (DCs) may play a role in the stimulation of the immuno-mediated anti-platelet response in chronic immune thrombocytopenic purpura (ITP). METHODS AND RESULTS: Twenty-nine patients with active ITP and 29 healthy adult volunteers were enrolled into the study. Freshly washed platelets and platelets aged in a plasma-free buffer for 72 hours at 37 degrees C were assessed by flow cytometry for phosphatidylserine exposure using annexin V-FITC, caspase activation, and platelet activation markers. CD14-derived DCs were characterized by immunophenotyping, cytokine production, and ability to present fresh and aged platelets to T lymphocytes. We demonstrated that platelets from ITP patients, either fresh or in vitro aged, show increased apoptosis (with low levels of activation) in comparison to their normal counterparts. We also found that immature DCs readily ingest apoptotic platelets. Furthermore, in ITP patients DCs, prepulsed with autologous/allogeneic fresh and aged platelets, are highly efficient in stimulating autologous T-cell proliferation as compared to DCs derived from healthy donors. This finding may be related to the upregulated expression of CD86 in DCs from ITP patients and not to higher phagocytic activity. CONCLUSION: These results suggest that DC dysfunction, together with increased propensity of platelets to undergo apoptosis, may play a role in the stimulation of the immune system in ITP.

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.

Acute graft-versus-host disease and steroid treatment impair CD11c+ and CD123+ dendritic cell reconstitution after allogeneic peripheral blood stem cell transplantation.

Human dendritic cells (DC) comprise 2 subsets-plasmacytoid CD123(+) and myeloid CD11c(+) DC-that may have distinct roles in the regulation of immunity after allogeneic hematopoietic stem cell transplantation. In this study, we analyzed the kinetics of CD123(+) DC and CD11c(+) DC reconstitution in 31 patients who underwent transplantation with allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood (PB) stem cells from HLA-identical sibling donors after myeloablative conditioning. Lineage marker-negative HLA-DR(+) CD11c(+) CD11c(+) DC and lineage marker-negative HLA-DR(+) CD123(+) CD123(+) DC, as well as monocytes and lymphoid subsets, were enumerated in donor grafts and in the PB of patients at various time points after transplantation. Reconstitution of both CD11c(+) DC and CD123(+) DC to normal levels occurred within 6 to 12 months and was not affected by the diagnosis, preparatory regimen, or graft composition. However, PB CD11c(+) DC and CD123(+) DC counts were significantly reduced in patients with acute GVHD grade II to IV (at 1 and 3 months) and grade I (at 1 month). Patients with chronic GVHD instead showed reduced CD123(+) DC counts only 6 months after transplantation. Moreover, treatment with steroids (>0.1 mg/kg) was significantly associated with reduced PB CD11c(+) DC and CD123(+) DC counts at all time points after transplantation. In multivariate analysis, only acute GVHD affected DC reconstitution early after transplantation. These results will prompt new studies addressing whether DC reconstitution correlates with immunity against infectious agents or with graft-versus-tumor reactions after PB stem cell allotransplantation.

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

OBJECTIVE: This 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. PATIENTS AND METHODS: Fifty-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). RESULTS: A 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. CONCLUSIONS: CD34(+) 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.

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.

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

OBJECTIVES: The 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. MATERIALS AND METHODS: An 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. RESULTS: Each 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. CONCLUSIONS: The 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.

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

OBJECTIVE: The 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. MATERIAL AND METHODS: One 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. RESULTS: CD86 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. CONCLUSIONS: In 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.