Peripheral blood and BM CD34+ CD38- cells show better resistance to cryopreservation than CD34+ CD38+ cells in autologous stem cell transplantation

BACKGROUND: We and others have shown a critical role for CD34+ CD38- cells in hematopoietic recovery after autologous stem cell transplantation (ASCT), in particular for platelet reconstitution. Thus a routine assessment of CD34+ CD38- cells in freezing-thawing procedures for autografting could represent an important tool for predicting poor engraftment. METHODS: To compare the impact of cryopreservation on CD34+ CD38+ and CD34+ CD38- hematopoietic stem cell subsets, 193 autograft products collected in 84 patients with malignancies were assessed before controlled-rate cryopreservation in 10% DMSO and after thawing for autografting. RESULTS: Cell counts after thawing were significantly different from the pre-freezing counts for total CD34+ (P<0.0001) and CD34+ CD38+ (P<0.0001) cells, but not for CD34+ CD38- cells (P=0.252). Median losses for CD34+, CD34+ CD38+ and CD34+ CD38- cells were, respectively, 11.8%, 11.4% and 0.0%. The magnitude of fresh/post-thawing percentage cell variation was significantly different when comparing between the CD34+ CD38+ and CD34+ CD38- cell subsets (P<0.001). Moreover, CD34+ CD38- cells exhibited recovery values > or =100% in 85/160 graft products, compared with 51/193 in CD34+ CD38+ cells (P<0.0001). Also, recovery values > or =90% were significantly better in the CD34+ CD38- (98/160 grafts) than in the CD34+ CD38+ subsets (89/193 grafts) (P<0.01). DISCUSSION: In this work we have demonstrated that CD34+ cells that do not express the CD38 Ag show a significantly better resistance to cryopreservation. This could represent another example of the particular ability of less committed progenitor cells to overcome environmental injuries. Moreover, we consider routine assessment of CD34+ CD38- cells before freezing as clinically relevant, but post-thawing controls may be avoided because of their good resistance to freezing.     

CD34(+) CD38(-) and CD34(+) HLA-DR(-) cells in BM stem cell grafts correlate with short-term engraftment but have no influence on long-term hematopoietic reconstitution after autologous transplantation

BACKGROUND: Prior studies have demonstrated that relatively immature hematopoietic stem cells, including CD34(+) CD38(-) and CD34(+) HLA-DR(-) subsets, correlate with short-term hematopoietic reconstruction (SHR) after transplantation. The aim of this study was to investigate whether these immature CD34(+) subsets also correlate with long-term hematopoietic reconstitution (LHR) in recipients of ABMT. METHODS: We examined stem cell grafts from 58 patients with B-cell lymphoma or CLL who underwent ABMT after myeloablative conditioning. We determined whether total mononuclear cell dose (MNC), colony-forming unit-granulocyte-monocyte (CFU-GM), CD34(+) cell dose and CD34(+) cell subsets (CD34(+) CD38(-) and CD34(+) HLA-DR(-) were associated with SHR and/or LHR. Time to neutrophil engraftment (TNE) and time to platelet engraftment (TPE) were used to measure SHR, while platelet counts at day 100 and 1 year post-ABMT were used as indicators for LHR. RESULTS AND DISCUSSION: CD34(+) cell dose and CD34(+) cell subsets were significantly associated with SHR. However, at day 100 and 1 year post-transplant only total CD34(+) cell dose was associated with LHR. The association of total CD34(+) cell dose with LHR persisted after adjusting for age, sex and disease. None of the CD34(+) cell subsets analyzed showed evidence of significant association with LHR.     

A preliminary analysis of the balance between Th1 and Th2 cells after CD34+ cell-selected autologous PBSC transplantation

BACKGROUND: CD34+ cell-selected autologous PBSC transplantation (CD34+ APBSCT) is a procedure used for the treatment of patients with malignant disease that is intended to eliminate residual tumor cells from autologous grafts. However, frequent infectious complications after CD34+ APBSCT can occur. A delay of recovery of the absolute number of CD4+ T cells after transplantation was reported to be one disadvantageous factor. As data on T-cell function after CD34+ APBSCT are scanty, we analyzed changes in T-helper cell 1 (Th1) and T-helper cell 2 (Th2) after CD34+ APBSCT to evaluate immune reconstitution. METHODS: Twelve patients underwent APBSCT (CD34+APBSCT group, n=4, and unselected APBSCT, n=8). Peripheral blood (PB) samples were obtained at 2, 4, 8, 12 and 16 weeks after the transplantation. The dynamics of the Th1 and Th2 were analyzed at a single-cell level, using flow cytometry. RESULTS: In the CD34+ APBSCT group, not only the absolute count of CD4+ T cells but also the proportion of Th1 cells in CD4+ T cells and the ratio of Th1 to Th2 after transplantation were significantly decreased at 2 and 4 weeks after transplantation compared with findings in the unselected APBSCT group. DISCUSSION: We suggest that higher rates of infectious complications after CD34+ APBSCT may be due to the inability of residual T cells from the CD34+ cell selection to generate mature T cells that function adequately against infection. Although further study would be required, our preliminary data provide some information on the immune reconstitution after CD34+ APBSCT and differentiation of T lymphocytes into Th1 and Th2 in vivo.     

Aldehyde dehydrogenase as an alternative to enumeration of total and viable CD34(+) cells in autologous hematopoietic progenitor cell transplantation

We validated the correlation of aldehyde dehydrogenase ALDH(br) cells with total and viable CD34(+) cells in fresh and thawed hematopoietic progenitor cell (HPC) products, and looked for a correlation with time to white blood cell (WBC) and platelet engraftment after autologous transplantation, using simple linear regression analyzes. We found a significant correlation between pre-freeze ALDH(br) cell numbers and pre-freeze total CD34(+) (P < 0.001), viable CD34(+) (P < 0.001) and post-thaw viable CD34(+) (P < 0.001) cell numbers. We suggest that ALDH(br) may be substituted for CD34(+) cell numbers when evaluating HPC. As post-thaw viability testing apparently adds no significant information, we suggest that it may not be necessary. Finally, neither marker correlated with time to engraftment in our patients, supporting previous data suggesting the existence of a threshold dose for timely engraftment around 2.5 × 10(6) cells/kg.     

Wild-type measles virus interferes with short-term engraftment of human CD34+ hematopoietic progenitor cells

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.     

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.     

Mobilization of CD34~+CD38~- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.     

Autologous transplantation: the viable transplanted CD34+ cell dose measured post-thaw does not predict engraftment kinetics better than the total CD34+ cell dose measured pre-freeze in patients that receive more than 2x10(6) CD34+ cells/kg

BACKGROUND: The aim of the study was to investigate whether the number of viable CD34+ cells in cryopreserved PBPC autografts is a better predictor of engraftment than the total CD34+ cell number determined before freezing. METHODS: A total of 119 patients was treated with autotransplantation for various malignant disorders during the period 1996-2002. All patients were reinfused with at least 2x10(6)/kg total CD34 cells analyzed before programmed freezing in 10% DMSO. The total CD34 cell number determined before freezing was compared with the number of viable cells determined after cryopreservation for 51 of these patients. The number of viable cells was determined by a flow cytometric analysis including triple staining with anti-CD34, anti-CD45 and the viability marker 7-actinomycin D (7-AAD). RESULTS: Simple linear regression analyses showed that both the total transplanted CD34 cell dose measured before freezing and the viable CD34 cell dose determined after cryopreservation were significantly correlated with neutrophil and platelet engraftment. In a multiple regression model the prediction of engraftment was not improved when the transplanted viable CD34 cell dose was included as a variable in addition to the total CD34 cell dose measured immediately after collection. DISCUSSION: Routine estimation of viable CD34 cells after cryopreservation of PBPC autografts is not necessary as long as the total CD34 cell dose is determined before freezing and the patients are reinfused with at least 2x10(6) cells/kg body weight.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

Genetically modified CD34+ cells do not contribute to the mesenchymal compartment after autologous transplantation in the baboon

BACKGROUND: There is ongoing controversy about the transdifferentiation of hematopoietic stem cells (HSC) into different tissues such as mesenchymal cells. This transdifferentiation or 'plasticity' would be an appealing concept for many therapeutic strategies. While studies in the murine model show encouraging results, reports from clinical allogeneic stem cell transplantations do not support the concept of HSC plasticity. Our aim was to determine whether transplantation of transduced autologous marrow CD34+ cells leads to long-term engraftment of gene-marked cells with mesenchymal characteristics in the baboon. METHODS: We analyzed marrow of two baboons that had received green fluorescence protein (GFP)-marked CD34+ autologous marrow cells after myeloablative conditioning. Marrow was obtained 1 and 2.5 years after transplantation and adherent CD11a- (pan-leukocyte Ab) cells were cultured for 3 weeks. Cultures were then analyzed by flow cytometry and fluorescence microscopy for the presence of GFP+ cells. For further analysis fresh and cultured cells were also labeled with multiple Ab and functional analysis was performed. RESULTS: Both animals showed persistent and stable GFP marking by flow cytometry in peripheral blood leukocytes as well as in CD34+ marrow cells at 1 and 2.5 years after transplantation. There was no evidence of GFP+ mesenchymal cells by either flow cytometry or fluorescence microscopy, while functional and phenotypical analysis identified mesenchymal stem cells in these cultures. DISCUSSION: We conclude that genetically modified CD34+ cells do not contribute to the adherent marrow-derived mesenchymal cell population after autologous transplantation.     

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.     

Early CD4+ T cell reconstitution as predictor of outcomes after allogeneic hematopoietic cell transplantation

Background: An association between early CD4+ T cell immune reconstitution (CD4+ IR) and survival after T-replete allogeneic hematopoietic cell transplantation (HCT) has been previously reported. Here we report validation of this relationship in a separate cohort that included recipients of ex vivo T-cell-depleted (TCD) HCT. We studied the relationship between CD4+ IR and clinical outcomes.Methods: A retrospective analysis of children/young adults receiving their first allogeneic HCT for any indication between January 2008 and December 2017 was performed. We related early CD4+ IR (defined as achieving >50 CD4+ T cells/µL on two consecutive measures within 100 days of HCT) to overall survival (OS), relapse, non-relapse mortality (NRM), event-free survival (EFS) and acute graft-versus-host disease (aGVHD). Fine and Gray competing risk models and Cox proportional hazard models were used.Results: In this analysis, 315 patients with a median age of 10.4 years (interquartile range 5.0–16.5 years) were included. The cumulative incidence of CD4+ IR at 100 days was 66.7% in the entire cohort, 54.7% in TCD (N = 208, hazard ratio [HR] 0.47, P < 0.001), 90.0% in uCB (N = 40) and 89.6% in T-replete (N = 47) HCT recipients. In multi-variate analyses, not achieving early CD4+ IR was a predictor of inferior OS (HR 2.35, 95% confidence interval [CI] 1.46–3.79, P < 0.001) and EFS (HR 1.80, 95% CI 1.20–2.69, P = 0.004) and increased NRM (HR 6.58, 95% CI 2.82–15.38, P < 0.001). No impact of CD4+ IR on relapse or aGVHD was found. Within the TCD group, similar associations were observed.Conclusion: In this HCT cohort, including recipients of TCD HCT, we confirmed that early CD4+ IR was an excellent predictor of outcomes. Finding strategies to predict or improve CD4+ IR may influence outcomes.     

Providing a microenvironment for the development of human CD34+ hematopoietic cells in SCID mice

In order to develop a convenient small-animal model that can support the differentiation of human bone-marrow-derived CD34+ cells, we transplanted SCID mice with an immortalized human stromal cell line, Lof(11–10). The Lof(11–10) cell line has been characterized to produce human cytokines capable of supporting primitive human hematopoietic cell proliferation in vitro. Intraperitoneal injection of Lof(11–10) cells into irradiated SCID mice by itself resulted in a dose-dependent survival of the mice from lethal irradiation. The radioprotective survival was reflected by an increase in the growth and number of mouse bone-marrow-derived committed hematopoietic progenitors. The Lof(11–10) cells localized to the spleen, but not to the bone marrow of these animals and resulted in detectable levels of circulating human IL-6 in their plasma. Secondary intravenous injections of either human or simian CD34+ cells into the Lof(11–10)-transplanted SCID mice resulted in engraftment of injected cells within the bone marrow of these mice. The utility of this small-animal model that allows the growth and differentiation of human CD34+ cells and its potential use in clinical gene therapy protocols are discussed.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is overexpressed during G(2)/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34(+) hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34(+) cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34(+) cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G(0)/G(1). Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67(negative) and Cyclin D(negative) CD34(+) cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G(0) CD34(+) cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G(0) CD34(+) cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSE(bright) G(0) CD34(+) cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34(+) cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.