High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study

Background

Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34⁺CD38^low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34⁺CD38^low/−CD123⁺ phenotype.

Design and Methods

The percentage of CD34⁺CD38^low/−CD123⁺ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses.

Results

A proportion of CD34⁺CD38^low/−CD123⁺ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34⁺CD38^low/−CD123⁺ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells retained prognostic significance for both parameters after multivariate analysis.

Conclusions

The percentage of CD34⁺CD38^low/−CD123⁺ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.     

CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background

Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.

Design and Methods

The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.

Results

A cancer-testis antigen-specific CD8⁺ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8⁺ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.

Conclusions

Functional cancer-testis antigen-specific CD8⁺ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.     

Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

Background

The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis.

Design and Methods

An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis.

Results

Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34⁺ lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling.

Conclusions

Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34⁺ hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.     

Early measurement of CD34+ cells in peripheral blood after cyclophosphamide and granulocyte colony-stimulating factor treatment predicts later CD34+ mobilisation failure and is a possible criterion for guiding “on demand” use of plerixafor

Background

Early identification of predictive factors of failure to mobilise CD34+ cells could enable rational use of plerixafor during first mobilisation, avoiding the need for a second mobilisation course. However, “on demand” administration of plerixafor needs to be driven by established parameters to avoid inappropriate use.

Materials and methods

To address this issue, we studied the value of the peripheral blood CD34+ count, measured early (on days +10, +11, +12 and +13), in predicting the mobilisation outcome in the ensuing days. We retrospectively collected data from three Italian centres on 233 patients affected by multiple myeloma or lymphoma who underwent a first or second attempt at mobilisation with cyclophosphamide 4 g/m² and granulocyte colony-stimulating factor. To assess the diagnostic value of peripheral blood white blood cell and CD34+ cell counts with respect to “mobilisation failure”, we considered failed mobilisation as “disease” and the CD34+ cell count in peripheral blood, on a specific day, as a “diagnostic test”. For various thresholds, we measured sensitivity, false positive rate, specificity and positive predictive value (PPV) as well as the area under the receiver-operating characteristic curves (AUC).

Results

A CD34+ cell count <10×10⁶/L on day 13 had high sensitivity (1.00) and high specificity (1.00) for predicting subsequent mobilisation failure, with an AUC of 1.0. However, good prediction was also obtained using a lower threshold (CD34+ cell count: <6×10⁶/L) at an earlier time (day 12). The PPV of the day 13 threshold was 1.00 while that of the day 12 one was 0.87.

Discussion

We propose that patients with <6×10⁶/L CD34+ cells in peripheral blood on day 12 and <10×10⁶/L on day 13 following mobilisation with cyclophosphamide 4 g/m² and granulocyte colony-stimulating factor are candidates for “on demand” use of plerixafor, making the administration of this expensive agent more efficient and avoiding its inappropriate use.     

Monitoring of donor chimerism in sorted CD34+ peripheral blood cells allows the sensitive detection of imminent relapse after allogeneic stem cell transplantation

Analysis of donor chimerism is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. We prospectively investigated the predictive value of donor chimerism analyses in sorted CD34⁺ peripheral blood cells in 90 patients with acute leukemia and myelodysplastic syndrome. The cumulative incidence of relapse after four years was significantly increased in cases with decreasing or incomplete CD34⁺ donor chimerism (57% vs. 18%, p=0.0001). Multivariate analysis confirmed decreasing CD34⁺ donor chimerism as an independent predictor of relapse and inferior survival. The interval between a decrease of CD34⁺ chimerism of less than 80% and hematologic relapse was 61 days (range 0–567). Monitoring of CD34⁺ donor chimerism in the peripheral blood allows prediction of imminent relapse after allogeneic stem cell transplantation even when a disease-specific marker is lacking.     

Preferential expansion of human umbilical cord blood-derived CD34-positive cells on major histocompatibility complex-matched amnion-derived mesenchymal stem cells

Background

We previously found in a murine hematopoietic system that hematopoietic stem cells show high differentiation and proliferation capacity on bone marrow-derived mesenchymal stem cells/stromal cells (microenvironment) with “self” major histocompatibility complex (MHC).

Design and Methods

We examined whether amnion-derived adherent cells have the characteristics of mesenchymal stem cells, and whether these adherent cells can support the proliferation of umbilical cord blood-derived lineage-negative and CD34-positive cells (Lin^–CD34⁺ cells) obtained from the same fetus to a greater extent than those derived from other fetuses.

Results

Culture-expanded amnion-derived adherent cells expressed mesenchymal stem cell markers and HLA-ABC molecules and could differentiate into osteoblasts, adipocytes and chondrocyte-like cells, indicating that the cells have the characteristics of mesenchymal stem cells. The Lin^–CD34⁺ cells purified from the frozen umbilical cord blood were strongly positive for HLA-ABC, and contained a large number of hematopoietic stem cells. When the Lin^–CD34⁺ cells were cultured on the autologous (MHC-matched) or MHC-mismatched amnion-derived adherent cells in short-term assays (hematopoietic stem cell-proliferation) and long-term culture-initiating cell assays, greater expansion of the Lin^–CD34⁺ cells was observed in the MHC-matched combination than in MHC-mismatched combinations. The concentration of granulocyte-macrophage colony-stimulating factor in the culture supernatants of the long-term culture-initiating cell assays was significantly higher in the MHC-matched combination than in MHC-mismatched combinations.

Conclusions

It is likely that a MHC restriction exists between hematopoietic stem cells and mesenchymal stem cells/stromal cells in the human hematopoietic system and that granulocute-macropage colony-stimulating factor contributes to some extent to the preferential hematopoiesis-supporting ability of the MHC-matched amnion-derived adherent cells.     

Peripheral and bone marrow CD34+ cell levels on chronic myeloproliferative disease

Purpose: The aim of the present study was to examine the relationship between peripheral CD34⁺ and bone marrow CD34⁺ levels and the clinicopathologic characteristics and laboratory parameters of myeloproliferative disease (MPD) patients.

Patients and methods: A total of 103 MPD patients were enrolled in this study. We examined the relationship between bone marrow CD34⁺ and peripheral CD34⁺ levels and the patients’ clinicopathologic and laboratory parameters.

Results: There were no significant correlations between the peripheral CD34⁺ levels and the JAK-2 V617F mutation, thrombosis, white blood cells (WBC), lactate dehydrogenase (LDH), transferrin saturation (TS), ferritin, or bone marrow cellularity. In addition, there were no significant correlations between bone marrow CD34⁺ levels and the JAK-2 V617F mutation, thrombosis, WBC, LDH, TS, ferritin, or bone marrow cellularity (P?>?0.05). We did not identify any significant relationship between peripheral CD34⁺ and bone marrow CD34⁺ levels (P?>?0.05). However, there were significant correlations between peripheral CD34⁺ levels and bone marrow fibrosis (P?+ levels and constitutional symptoms (P?+ levels and bone marrow fibrosis (P?Conclusion: We did not find any significant relationship between the clinicopathologic and laboratory characteristics and peripheral and bone marrow CD34⁺ cells from bone marrow fibrosis patients. There was also no significant relationship between bone marrow CD34⁺ cells and peripheral CD34⁺ cells. Some peripheral CD34⁺ cells may originate from the spleen rather than the bone marrow, which may given us different result of some parameters.     

Thymic recovery after allogeneic hematopoietic cell transplantation with non-myeloablative conditioning is limited to patients younger than 60 years of age

Background

Long-term immune recovery in older patients given hematopoietic cell transplantation after non-myeloablative conditioning remains poorly understood. This prompted us to investigate long-term lymphocyte reconstitution and thymic function in 80 patients given allogeneic peripheral blood stem cells after non-myeloablative conditioning.

Design and Methods

Median age at transplant was 57 years (range 10–71). Conditioning regimen consisted of 2 Gy total body irradiation (TBI) with (n=46) or without (n=20) added fludarabine, 4 Gy TBI with fludarabine (n=6), or cyclophosphamide plus fludarabine (n=8). Stem cell sources were unmanipulated (n=56), CD8-depleted (n=19), or CD34-selected (n=5) peripheral blood stem cells. Immune recovery was assessed by signal-joint T-cell receptor excision circle quantification and flow cytometry.

Results

Signal-joint T-cell receptor excision circle levels increased from day 100 to one and two years after transplantation in patients under 50 years of age (n=23; P=0.02 and P=0.04, respectively), and in those aged 51–60 years (n=35; P=0.17 and P=0.06, respectively), but not in patients aged over 60 (n=22; P=0.3 and P=0.3, respectively). Similarly, CD4⁺CD45RA⁺ (naïve) T-cell counts increased from day 100 to one and two years after transplantation in patients aged 50 years and under 50 (P=0.002 and P=0.02, respectively), and in those aged 51–60 (P=0.4 and P=0.001, respectively), but less so in patients aged over 60 (P=0.3 and P=0.06, respectively). In multivariate analyses, older patient age (P<0.001), extensive chronic GVHD (P<0.001), and prior (resolved) extensive chronic graft-versus-host disease (P=0.008) were associated with low signal-joint T-cell receptor excision circle levels one year or more after HCT.

Conclusions

In summary, our data suggest that thymic neo-generation of T cells occurred from day 100 onwards in patients under 60 while signal-joint T-cell receptor excision circle levels remained low for patients aged over 60. Further, chronic graft-versus-host disease had a dramatic impact on thymic function, as observed previously in patients given grafts after myeloablative conditioning.     

Changes in peripheral CD19+Foxp3+ and CD19+TGFβ+ regulatory B cell populations in rheumatoid arthritis patients with interstitial lung disease

Objective

Although the role of regulatory B cells (Bregs) in rheumatic disease has gained increasing attention, two lesser-known Breg subsets that express either Foxp3 or transforming growth factor beta (TGFβ) are rarely examined in studies of rheumatic disease. This study investigates the association between the relative proportions of CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs, and clinical indicators of disease severity in rheumatoid arthritis (RA) patients with or without interstitial lung disease (ILD).

Methods

A total of 31 RA patients (14 with and 17 without ILD) and 26 healthy control subjects were included. All subjects did not have other autoimmune disease except RA, tumor, active infection, or a history of related drug administration. Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed by flow cytometry (FCM). The relationship between the relative proportions of CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs and their associations to RA and ILD incidence, as well as disease severity assessed by common clinical indicators, were then examined.

Results

Our analyses revealed RA patients had significantly lower proportions of peripheral CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs as compared to healthy controls. While no association was observed between CD19⁺Foxp3⁺ Bregs and ILD incidence, patients with ILD had a substantially lower percentage of CD19⁺TGFβ⁺ Bregs compared to RA patients without ILD. In addition, CD19⁺Foxp3⁺ Bregs were negatively correlated with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels in RA patients, whereas CD19⁺TGFβ⁺ Bregs were only correlated with CRP in RA patients with ILD. Furthermore, there was a negative association between CD19⁺Foxp3⁺ Bregs and disease severity scores, which was not found in analyses with CD19⁺TGFβ⁺ Bregs.

Conclusions

The proportions CD19⁺Foxp3⁺ and CD19⁺TGFβ⁺ Bregs were significantly decreased in RA patients, particularly in those with ILD complications, suggesting that Breg phenotypes may have different functions in the pathogenesis of RA and ILD.     

Identifying factors associated with changes in CD4+ count in HIV-infected adults in Saskatoon,Saskatchewan

OBJECTIVE:

To assess the impact of clinical and social factors unique to HIV-infected adults in Saskatoon, Saskatchewan, regarding the rate of CD4⁺ count change, and to identify factors associated with a risk of CD4⁺ count decline.

METHODS:

A retrospective longitudinal cohort study from medical chart reviews at two clinics was conducted in Saskatoon. Univariate and multivariate linear mixed effects models were used to assess the impact of selected factors on CD4⁺ count change.

RESULTS:

Four hundred eleven HIV-infected patients were identified from January 1, 2003 to November 30, 2011. Two hundred eighteen (53%) were male, mean (± SD) age was 35.6 ±10.1 years, 257 (70.8%) were First Nations or Métis, 312 (80.2%) were hepatitis C virus (HCV) coinfected and 300 (73.3%) had a history of injection drug use (IDU). In univariate models, age, ethnicity, HCV, IDU, antiretroviral therapy and social assistance were significant. Using ethnicity, HCV and IDU, three multivariate models (models 1, 2, 3) were built due to high correlation. First Nations or Métis ethnicity, HCV coinfection and a history of IDU were associated with significantly lower CD4⁺ counts in multivariate models. Older age and social assistance were associated with significantly lower CD4⁺ counts in models 1 and 3. Age was marginally significant in model 2 (P=0.055). Not prescribed antiretroviral therapy was associated with a significantly negative CD4⁺ count slope in all multivariate models.

CONCLUSION:

The unique epidemiology of this HIV-infected population may be contributing to CD4⁺ count change. Increased attention and resources focused on this high-risk population are needed to prevent disease progression and to improve overall health and quality of life.     

The role of matched sibling donor allogeneic stem cell transplantation in pediatric high-risk acute myeloid leukemia: results from the AML-BFM 98 study

Background

The role of allogeneic stem cell transplantation in post-remission management of children with high-risk acute myeloid leukemia remains controversial. In the multi-center AML-BFM 98 study we prospectively evaluated the impact of allogeneic stem cell transplantation in children with high-risk acute myeloid leukemia in first complete remission.

Design and Methods

HLA-typed patients with high-risk acute myeloid leukemia, who achieved first complete remission (n=247), were included in this analysis. All patients received double induction and consolidation. Based on the availability of a matched-sibling donor, patients were allocated by genetic chance to allogeneic stem cell transplantation (n=61) or chemotherapy-only (i.e. intensification and maintenance therapy; n=186). The main analysis was done on an intention-to-treat basis according to this allocation.

Results

Intention-to-treat analysis did not show a significantly different 5-year disease-free survival (49±6% versus 45±4%, P_{log rank}=0.44) or overall survival (68±6% versus 57±4%, P_{log rank}=0.17) between the matched-sibling donor and no-matched-sibling donor groups, whereas late adverse effects occurred more frequently after allogeneic stem cell transplantation (72.5% versus 31.8%, P_Fischer<0.01). These results were confirmed by as-treated analysis corrected for the time until transplantation (5-year overall survival: 72±8% versus 60±4%, P_Mantel-Byar 0.21). Subgroup analysis demonstrated improved survival rates for patients with 11q23 aberrations allocated to allogeneic stem cell transplantation (5-year overall survival: 94±6% versus 52±7%, P_log-rank=0.01; n=18 versus 49) in contrast to patients without 11q23 aberrations (5-year overall survival: 58±8% versus 55±5%, P_log-rank=0.66).

Conclusions

Our analyses defined a genetic subgroup of children with high-risk acute myeloid leukemia who benefited from allogeneic stem cell transplantation in the prospective multi-center AML-BFM 98 study. For the remainder of the pediatric high-risk acute myeloid leukemia patients the prognosis was not improved by allogeneic stem cell transplantation, which was, however, associated with a higher rate of late sequelae.     

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

Background

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34⁺ cells and mesenchymal stromal cells with regard to different spatial localizations.

Design and Methods

Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.

Results

In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.

Conclusions

We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.     

The role of 9-O-acetylated ganglioside D3 (CD60) and ��4��1 (CD49d) expression in predicting the survival of patients with S��zary syndrome

Background

Sézary syndrome is a rare and very aggressive leukemic variant of cutaneous T-cell lymphoma characterized by extensive skin involvement and a malignant circulating CD4⁺ T-cell clone which homes to the skin, over-expresses CD60, and lacks CD7, CD26 and CD49d. So far prognostic markers in this disease are limited to treatment with systemic steroids, age, serum lactate dehydrogenase, and a white blood cell count of 20×10⁹/L or higher: no other biological marker with prognostic value, especially related to malignant cells, has been described.

Design and Methods

We used flow activated cell sorting analysis to compare the distribution of the T-cell receptor-Vβ repertoire and several surface molecules (CD7, CD26, CD49d and CD60) within the circulating CD4⁺ T-cell population in 62 patients with Sézary syndrome, 180 with mycosis fungoides, 6 with B-cell lymphomas, and 19 with chronic eczema. We calculated the 5-year overall survival of patients with Sézary syndrome after first hospital admission using Kaplan–Meier product–limit estimates and hazard ratios from the Cox proportional hazards model.

Results

We found that both higher number of CD60⁺ and lower number of CD49d⁺ cells within circulating CD4⁺ T cells at disease presentation were significantly associated with a lower probability of survival. An exceedingly high risk of death was observed for patients with a combination of a high proportion of CD4⁺CD60⁺ cells (≥ 0.5×10⁹/L) and low proportion of CD4⁺CD49d⁺ cells (<0.5×10⁹/L) (hazard ratio = 12.303, 95% confidence interval 1.5–95.9; P<0.02). In addition, a skewed usage of T-cell receptor-Vβ subfamilies was observed in the circulating T-cell clone for 61.9% of all patients with Sézary syndrome, T-cell receptor-Vβ 2 and 5.1 subfamilies being the most frequently represented (42.8%), followed by T-cell receptor-Vβ 12 and 13.1.

Conclusions

In this study we showed that up-regulation of CD60 and down-regulation of CD49d on circulating CD4⁺ T cells are two useful markers for predicting a very poor outcome in patients with Sézary syndrome.     

Ex vivo expansion CD34+/AC133+ - selected autologous peripheral blood progenitor cells (PBPC) in high-risk breast cancer patients receiving intensive chemotherapy

AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for engraftment in transplant situations. We studied the effect of stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 11 (IL-11) on the ex vivo expansion of human CD34⁺/AC133⁺ progenitors isolated from leukapheresis products from chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) -mobilized adult donors. MiniMACS AC133⁺ isolated cells contained a mean of 85% (80-90) AC133⁺ cells. Enriched AC133⁺ cells co-expressed CD34⁺ 80%, CD71^low 36.6 % and CD33⁺ 6.6 %. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11, the number of cells increased 19 times. These cells expressed a mean 12% CD34⁺ and 74% CD71⁺ (23% CD 71^high) and 59% CD33⁺. This means that the absolute number of CD34⁺ cells increased slightly, the number of CD33⁺ increased 100 times and cells with high density CD71^high (23%) appeared. These cells represent the cells committed to erythroid differentiation. The increase in the number of CFU-GM with various combinations of cytokines SCF + Il-3 + IL-11 will be discussed. The number of CFU-GM in culture after a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11 increased 45 times.     

Imbalance of effector and regulatory CD4 T cells is associated with graft-versus-host disease after hematopoietic stem cell transplantation using a reduced intensity conditioning regimen and alemtuzumab

Background

A variety of immune pathways can lead to graft-versus-host disease. A better understanding of the type of immune response causing graft-versus-host disease in defined clinical hematopoietic stem cell transplant settings is required to inform development of methods for monitoring patients and providing them tailored care.

Design and Methods

Twenty-five patients were recruited presenting with myeloid malignancies and treated with a reduced intensity conditioning transplant regimen with graft-versus-host disease prophylaxis comprising in vivo lymphocyte depletion with alemtuzumab and cyclosporin. A prospective study was performed of lymphocyte subset reconstitution in peripheral blood in relation to the incidence of graft-versus-host disease.

Results

Acute graft-versus-host disease was associated with significantly higher numbers of natural killer cells and donor-derived effector CD4 T cells (CD45RO⁺ CD27⁻) early (day 30) after transplantation (p=0.04 and p=0.02, respectively). This association was evident before the emergence of clinical pathology in six out of seven patients. Although numbers of regulatory CD4 T cells (CD25^high Foxp3⁺) were similar at day 30 in all patients, a significant deficit in those who developed acute graft-versus-host disease was apparent relative to effector CD4 T cells (median of 41 effectors per regulatory cell compared to 12 to 1 for patients without graft-versus-host disease) (p=0.03). By day 180, a functional regulatory CD4 T-cell population had expanded significantly in patients who developed chronic graft-versus-host disease, reversing the imbalance (median of 3 effectors per regulatory cell compared to 9.6 to 1 for patients without graft-versus-host disease) (p=0.018) suggesting no overt absence of immune regulation in the late onset form of the disease.

Conclusions

Imbalance of effector and regulatory CD4 T cells is a signature of graft-versus-host disease in this transplantation protocol.     

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

Background

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy.

Design and Methods

We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d⁺ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice.

Results

The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d⁺ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d⁺ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules.

Conclusions

Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d⁺ hematologic malignancies.     

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34⁺ cells using a four-phase culture system. We first cultured CB CD34⁺ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34⁺ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34⁺ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 10¹² RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 10¹³ RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.     

CD69 is independently prognostic in chronic lymphocytic leukemia: a comprehensive clinical and biological profiling study

Background

CD69 is expressed in several hemopoietic cells and is an early activation marker in chronic lymphocytic leukemia. Chronic lymphocytic leukemia is a clinically heterogeneous disease which needs novel prognostic parameters which can be easily and efficiently managed.

Design and Methods

We investigated CD69 by flow cytometry in a series of 417 patients affected by chronic lymphocytic leukemia and compared this to other biological and clinical prognosticators.

Results

CD69 was associated with Rai stages (P=0.00002), β₂-microglobulin (P=0.0005) and soluble CD23 (P<0.0001). CD69 and ZAP-70 (P=0.018) or CD38 (P=0.00015) or immunoglobulin variable heavy chain gene mutations (P=0.0005) were also significantly correlated. Clinically, CD69 positive chronic lymphocytic leukemias received chemotherapy more frequently (74%; P<0.0001), and presented a shorter duration of response after fludarabine plus rituximab (P=0.010) as well as shorter progression free survival and overall survival (P<0.0001). CD69 demonstrated true additive prognostic properties, since the CD69⁺ plus ZAP-70⁺ or CD38⁺ or immunoglobulin variable heavy chain gene unmutated patients had the worst progression free survival and overall survival (P<0.0001). Interestingly, low CD69 expression was necessary to correctly prognosticate the longer progression free survival of patients with a low tumor burden of β₂-microglobulin (P=0.002), of soluble CD23 (P=0.020), or of Rai stages 0-I (P=0.005). CD69 was confirmed to be an independent prognostic factor in multivariate analysis of progression free survival (P=0.017) and overall survival (P=0.039).

Conclusions

Our data indicate that CD69 is significantly correlated with poor clinical and biological prognostic factors and is confirmed to be an independent disease prognosticator. This supports its introduction in a routine laboratory assessment and, possibly, in a prognostic scoring system for chronic lymphocytic leukemia, after an adequate standardization process.     

Rudimentary signs of immunosenescence in Cytomegalovirus-seropositive healthy young adults

Ageing is associated with a decline in immune competence termed immunosenescence. In the elderly, this process results in an accumulation of differentiated ‘effector’ phenotype memory T cells, predominantly driven by Cytomegalovirus (CMV) infection. Here, we asked whether CMV also drives immunity towards a senescent profile in healthy young adults. One hundred and fifty-eight individuals (mean ± SD; age 21 ± 3 years, body mass index 22.7 ± 2.7 kg m²) were assessed for CMV serostatus, the numbers/proportions of CD4⁺ and CD8⁺ late differentiated/effector memory cells (i.e. CD27⁻CD28⁻/CD45RA⁺), plasma interleukin-6 (IL-6) and antibody responses to an in vivo antigen challenge (half-dose influenza vaccine). Thirty percent (48/158) of participants were CMV⁺. A higher lymphocyte and CD8⁺ count (both p < 0.01) and a lower CD4/CD8 ratio (p < 0.03) were observed in CMV⁺ people. Eight percent (4/58) of CMV⁺ individuals exhibited a CD4/CD8 ratio <1.0, whereas no CMV⁻ donor showed an inverted ratio (p < 0.001). The numbers of CD4⁺ and CD8⁺CD27⁻CD28⁻/CD45RA⁺ cells were ~ fourfold higher in CMV⁺ people (p < 0.001). Plasma IL-6 was higher in CMV⁺ donors (p < 0.05) and showed a positive association with the numbers of CD8⁺CD28⁻ cells (p < 0.03). Finally, there was a significant negative correlation between vaccine-induced antibody responses to the A/Brisbane influenza strain and CMV-specific immunoglobulin G titres (p < 0.05). This reduced vaccination response was associated with greater numbers of total CD8⁺ and CD4⁺ and CD8⁺CD27⁻CD28⁻/CD45RA⁺ cells (p < 0.05). This study observed marked changes in the immune profile of young adults infected with CMV, suggesting that this virus may underlie rudimentary aspects of immunosenescence even in a chronologically young population.