Ex vivo T-cell-depleted allogeneic stem cell transplantation for hematologic malignancies: The search for an optimum transplant T-cell dose and T-cell add-back strategy.

BACKGROUND: T-cell depletion (TCD) of allogeneic stem cell transplants (SCT) can reduce graft-versus-host disease but may negatively affect transplant outcome by delaying immune recovery. To optimize TCD in HLA-matched siblings with hematologic malignancies, we explored varying the transplant CD3+ T-cell dose between 2 and 50 × 104/kg (corresponding to 3-4 log depletion) and studied the impact of 0-6 × 107/kg CD3+ donor lymphocyte infusion (DLI) "add-back" on immune recovery post-SCT. METHODS: Two hundred seventeen consecutive patients (age range, 10-75 years) with hematologic malignancy (excluding chronic leukemias) underwent ex vivo TCD SCT from HLA-identical sibling donors from 1994-2015. Ninety-four patients had standard-risk disease (first remission acute leukemia [AL] and early stage myelodysplastic syndromes [MDS]) and 123 had high-risk disease (AL beyond first complete remission, advanced MDS or refractory B-cell malignancy). RESULTS: Median follow-up was 8.5 years. At 20 years post-SCT, overall survival (OS) was 40%, nonrelapse mortality (NRM) was 27% and relapse incidence was 39%. Factors affecting outcome in multivariate analysis were transplantation era, with OS increasing from 38% in the period 1994-2000 to 58% in 2011-2015, disease risk (hazard ratio [HR], 1.68 for high risk) and increasing age (HR, 1.19 per decade). Neither the T-cell dose or the add back of T cells in the first 100 days had any effect on OS, NRM and relapse. CONCLUSIONS: Outcomes for TCD SCT have greatly improved. However, our data do not support the need to precisely manipulate transplant CD3+ T-cell dose provided at least 3-log depletion is achieved or the use of T-cell add-back. Future improvements for TCD SCT await better strategies to prevent relapse, especially in high-risk recipients.

Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses.

Bone marrow mesenchymal stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 × 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system.

Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.

BACKGROUND AIMS: With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs). METHODS: Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression. RESULTS: The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression. CONCLUSIONS: This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay.

Ultra-low dose interleukin-2 promotes immune-modulating function of regulatory T cells and natural killer cells in healthy volunteers.

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m(2)/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56(bright) NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

Timing and intensity of exposure to interferon-γ critically determines the function of monocyte-derived dendritic cells.

A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects of interferon-γ (IFN-γ) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (moDC). Here, we report that following differentiation of monocytes into moDC with granulocyte-macrophage colony-stimulating factor and interleukin-4, IFN-γ induces moDC maturation and up-regulates the co-stimulatory markers CD80/CD86/CD95 and MHC Class I, enabling moDC to effectively generate antigen-specific CD4(+) and CD8(+) T-cell responses for multiple viral and tumour antigens. Early exposure of monocytes to high concentrations of IFN-γ during differentiation promotes the formation of macrophages. However, under low concentrations of IFN-γ, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN-γ or lipopolysaccharide and an inability to generate effective antigen-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that IFN-γ imparts differential programmes on moDC that shape the antigen-specific T-cell responses they induce. Timing and intensity of exposure to IFN-γ can therefore determine the functional capacity of moDC.

Genetically engineered fixed K562 cells: potent "off-the-shelf" antigen-presenting cells for generating virus-specific T cells.

BACKGROUND AIMS: The human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. METHODS: In order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. RESULTS: In preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8⁺ T cells in HLA-A∗0201⁺ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-ß blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. CONCLUSIONS: Fixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Evolution of the donor T-cell repertoire in recipients in the second decade after allogeneic stem cell transplantation.

After allogeneic stem cell transplantation (SCT), T lymphocyte function is reestablished from the donor's postthymic T cells and through thymic T-cell neogenesis. The immune repertoire and its relation to that of the donor have not been characterized in detail in long-term adult SCT survivors. We studied 21 healthy patients in their second decade after a myeloablative SCT for hematologic malignancy (median follow-up, 12 years). Immune profiles were compared with donor samples cryopreserved at transplant and beyond 10 years from SCT. Only one recipient was on continuing immunosuppression. Compared with the donor at transplant, there was no significant difference in CD4, CD8, natural killer, and B-cell blood counts. However, compared with donors, recipients had significantly fewer naive T cells, lower T-cell receptor excision circle levels, fewer CD4 central memory cells, more effector CD8(+) cells, and more regulatory T cells. TCR repertoire analysis showed no significant difference in complexity of TCRVß spectratype between recipients and donors, although spectratype profiles had diverged with both gain and loss of donor repertoire peaks in the recipient. In conclusion, long-term allogeneic SCT survivors have subtle defects in their immune profile consistent with defective thymic function but compatible with normal health. This study is registered at http://www.clinicaltrials.gov as NCT00106925.

Donor lymphocyte count and thymic activity predict lymphocyte recovery and outcomes after matched-sibling hematopoietic stem cell transplant.

Delayed immune recovery is a characteristic feature of allogeneic hematopoietic stem cell transplantation in adult recipients. Although recipient thymic T-cell neogenesis contributes to T-cell regeneration after transplantation, thymic recovery in the transplant recipient decreases with increasing age, and is diminished by intensive preconditioning regimens and graft-versus-host disease. In adult recipients, most events that determine transplant success or failure occur during the period when the majority of circulating T cells is derived from the donor's post thymic T-cell repertoire. As a result, the make-up of the donor lymphocyte compartment may strongly influence immune recovery and transplant outcomes. The aim of this study was to examine donor lymphocyte counts in a series of patients undergoing an allogeneic hematopoietic stem cell transplant to identify the potential contribution of donor regulatory and conventional T lymphocyte populations to immune recovery and transplant outcomes. We examined donor lymphocyte subset counts in relation to post-transplant lymphocyte recovery and transplant events in 220 consecutive myeloablative, T-cell-depleted, HLA-identical sibling hematopoietic stem cell transplant recipients with hematologic malignancies. In a multivariate analysis, absolute numbers of donor CD4(+) recent thymic emigrants were associated with overall survival (P=0.032). The donors' absolute lymphocyte count and thymic production of regulatory T cells were both associated with extensive chronic graft-versus-host disease (P=0.002 and P=0.022, respectively). In conclusion, these results identify donor immune characteristics that are associated with lymphocyte recovery, extensive chronic graft-versus-host disease, and survival in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using peripheral blood samples drawn from donors and patients enrolled in the ClinicalTrials.gov-registered trials NCT00001623, NCT00001873, NCT00353860, NCT00066300, NCT00079391, and NCT00398346.

Cytopenia and leukocyte recovery shape cytokine fluctuations after myeloablative allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation is associated with profound changes in levels of various cytokines. Emphasis has been placed on conditioning-associated mucosal damage and neutropenia and associated bacterial translocation as the initiating conditions predisposing to acute graft-versus-host disease. The post-transplant period is, however, also associated with increases in certain homeostatic cytokines. It is unclear how much the homeostatic drive to lymphocyte recovery and the production of cytokines from the engrafting donor immune system determine cytokine fluctuations in the peri- and immediate post-transplant period. The aim of this study was to examine the contributions of the conditioning regimen, donor engraftment, infections, and graft-versus-host disease to fluctuations in cytokines involved in homeostasis and inflammation. DESIGN AND METHODS: We examined the levels of 33 cytokines in relation to peri- and post-transplant events such as conditioning regimen, chimerism, and acute graft-versus-host disease in myeloablative, non-T cell-replete HLA-identical sibling donor stem cell transplantation for hematologic malignancies. RESULTS: We identified two cytokine storms. The first occurred following conditioning and reached peak levels when all the leukocytes were at their lowest concentrations. The second cytokine storm occurred concurrently with hematopoietic reconstitution and subsided with the achievement of full donor lymphocyte chimerism. CONCLUSIONS: Our results indicate that both recipient-related and donor-related factors contribute to the changes in cytokine levels in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using plasma samples drawn from patients enrolled in the ClinicalTrials.gov-registered trials NCT00467961 and NCT00378534.

Alloreactivity across HLA barriers is mediated by both naïve and antigen-experienced T cells.

T cell responses to allogeneic targets arise predominantly from the naïve pool. However, in humans, the risk of graft-versus-host disease is increased if the donor has circulating T cells recognizing multiple persistent DNA viruses, suggesting that memory T cells also contribute to the alloresponse. To examine HLA alloreactivity, we used flow cytometry-based proliferation and cytokine production assays. We identified the clonal identity of virus-specific T cells cross-reacting with HLA-disparate targets by sequencing the T cell receptor ß chains in virus-specific T cell lines restimulated with cognate and HLA-disparate targets and sorting these chains according to cytokine response. We confirmed that naïve T cells from cord blood and adult individuals responded to HLA-mismatched target cells. In addition, in adults, we identified memory T cells responding by cytokine release to HLA-mismatched targets both in direct assays and after 8 days of culture with allogeneic stimulator cells. Epstein-Barr virus-specific and cytomegalovirus-specific T cells, tested against a panel of 30 T cell antigen-presenting cells with a broad coverage of the most prominent HLA types, displayed specificity for certain mismatched HLA alleles. Sequencing of the T cell receptor ß chain demonstrated a clonotypic identity of cells that responded to both viral and allogeneic stimulation. These findings show conclusively that alloresponses in humans are not confined to the naïve T cell subset, and that memory viral antigen-specific T cells can cross-react with specific mismatched HLA-peptide complexes not presenting with cytomegalovirus or Epstein-Barr virus peptides.

The transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor.

The successful reconstitution of adaptive immunity to human cytomegalovirus (CMV) in hematopoietic stem cell transplantation (HSCT) recipients is central to the reduction of viral reactivation-related morbidity and mortality. Here, we characterized the magnitude, specificity, phenotype, function, and clonotypic composition of CMV-specific T-cell responses in 18 donor-recipient pairs both before and after HSCT. The principal findings were: (1) the specificity of CMV-specific T-cell responses in the recipient after HSCT mirrors that in the donor; (2) the maintenance of these targeting patterns reflects the transfer of epitope-specific T-cell clonotypes from donor to recipient; (3) less differentiated CD27(+)CD57(-) CMV-specific memory T cells are more likely to persist in the recipient after HSCT compared with more terminally differentiated CD27(-) CD57(+) CMV-specific memory T cells; (4) the presence of greater numbers of less differentiated CD8(+) CMV-specific T cells in the donor appears to confer protection against viral reactivation in the recipient after HSCT; and (5) CMV-specific T cells acquire a more differentiated phenotype and a restricted functional profile after HSCT. Overall, these findings define the immunologic factors that influence the successful adoptive transfer of antigen-specific T-cell immunity during HSCT, which enables the identification of recipients at particular risk of CMV reactivation after HSCT.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib.

Primitive quiescent CD34(+) chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34(+) cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34(+) populations. Quiescent CD34(+) cells from CML patients were less susceptible than their cycling CD34(+) and CD34(-) counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34(+) CML cells had higher surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34(+) CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell-mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

High avidity myeloid leukemia-associated antigen-specific CD8+ T cells preferentially reside in the bone marrow.

The activity of allogeneic CD8(+) T cells specific for leukemia-associated antigens (LAAs) is thought to mediate, at least in part, the curative effects of hematopoietic stem cell transplantation (HSCT) in myeloid malignancies. However, the identity and nature of clinically relevant LAA-specific CD8(+) T-cell populations have proven difficult to define. Here, we used a combination of coreceptor-mutated peptide-major histocompatibility complex class I (pMHCI) tetramers and polychromatic flow cytometry to examine the avidity profiles, phenotypic characteristics, and anatomical distribution of HLA A*0201-restricted CD8(+) T-cell populations specific for LAAs that are over-expressed in myeloid leukemias. Remarkably, LAA-specific CD8(+) T-cell populations, regardless of fine specificity, were confined almost exclusively to the bone marrow; in contrast, CD8(+) T-cell populations specific for the HLA A*0201-restricted cytomegalovirus (CMV) pp65(495-503) epitope were phenotypically distinct and evenly distributed between bone marrow and peripheral blood. Furthermore, bone marrow-resident LAA-specific CD8(+) T cells frequently engaged cognate antigen with high avidity; notably, this was the case in all tested bone marrow samples derived from patients who achieved clinical remission after HSCT. These data suggest that concomitant examination of bone marrow specimens in patients with myeloid leukemias might yield more definitive information in the search for immunologic prognosticators of clinical outcome.

Detection of low avidity CD8(+) T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8(+) T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8(+) T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) without impacting the TCR binding platform can detect cognate CD8(+) T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8(+) T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8(+) T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8(+) T cells.

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins.

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate post-allogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-gamma or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.

CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines.

In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

Autoreactive, cytotoxic T lymphocytes specific for peptides derived from normal B-cell differentiation antigens in healthy individuals and patients with B-cell malignancies.

PURPOSE: To investigate potential immunotherapeutic strategies in B lymphocytic malignancies we looked for CTLs recognizing CD19 and CD20 epitopes. EXPERIMENTAL DESIGN: Three CD19 and CD20 peptides binding to HLA-A*0201 were identified and used to detect peptide specific CTLs by a quantitative real-time PCR to measure IFN-gamma mRNA expression in 23 healthy individuals and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 follicular lymphoma, 2 acute lymphocytic leukemia, and 1 large cell lymphoma). Peptide-specific CTLs were expanded in culture with CD40-activated B cells to test lytic activity in three patients. RESULTS: In healthy individuals, CD8+ T-cell responses were detected in one to CD19(74-82), in three to CD20(127-135), and three to CD20(188-196). Seven of 27 patients (6 with CLL) had CD8+ T cells recognizing CD19(74-82). Seven patients responded to CD20(127-135) and three to CD20(188-196). All were CLL patients. CD19(74-82)-specific CTLs from three patients were expanded over 4 weeks. These cells were HLA-A*0201 specific and lytic for peptide-loaded antigen-presenting cells but not to malignant or unpulsed B cells. CONCLUSIONS: CTLs that recognize CD19 and CD20 epitopes exist in healthy individuals and may be increased in CLL patients. They are of low avidity and require high doses of peptide for activation. Strategies to increase T-cell avidity would be necessary for T-cell immunotherapeutic approaches using the peptides studied.

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte.

To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 +/- 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56(bright), mainly CD33- cells (7+/-10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5+/-2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-gamma. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.

Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6weeks. Four samples showed CD34(+)CD38(-) predominance, and four were predominantly CD34(+)CD38(+). CD34(+) CD38(-) predominant leukemia cells maintained the CD34(+) CD38(-) phenotype and were viable for 6weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34(+) CD38(+) predominant leukemic cells maintained the CD34(+)CD38(+) phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34(+) blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

Mitochondrial metabolism modulates differentiation and teratoma formation capacity in mouse embryonic stem cells.

Relatively little is known regarding the role of mitochondrial metabolism in stem cell biology. Here we demonstrate that mouse embryonic stem cells sorted for low and high resting mitochondrial membrane potential (DeltaPsi(m)L and DeltaPsi(m)H) are indistinguishable morphologically and by the expression of pluripotency markers, whereas markedly differing in metabolic rates. Interestingly, DeltaPsi(m)L cells are highly efficient at in vitro mesodermal differentiation yet fail to efficiently form teratomas in vivo, whereas DeltaPsi(m)H cells behave in the opposite fashion. We further demonstrate that DeltaPsi(m) reflects the degree of overall mammalian target of rapamycin (mTOR) activation and that the mTOR inhibitor rapamycin reduces metabolic rate, augments differentiation, and inhibits tumor formation of the mouse embryonic stem cells with a high metabolic rate. Taken together, our results suggest a coupling between intrinsic metabolic parameters and stem cell fate that might form a basis for novel enrichment strategies and therapeutic options.