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Delayed engraftment is a major limitation of cord blood transplantation (CBT), due in part to a defect in the cord blood (CB) cells' ability to home to the bone marrow. Because this defect appears related to low levels of fucosylation of cell surface molecules that are responsible for binding to P- and E-selectins constitutively expressed by the marrow microvasculature, and thus for marrow homing, we conducted a first-in-humans clinical trial to correct this deficiency. Patients with high-risk hematologic malignancies received myeloablative therapy followed by transplantation with 2 CB units, one of which was treated ex vivo for 30 minutes with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose to enhance the interaction of CD34(+) stem and early progenitor cells with microvessels. The results of enforced fucosylation for 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. The median time to neutrophil engraftment was 17 days (range, 12-34 days) compared with 26 days (range, 11-48 days) for controls (P = .0023). Platelet engraftment was also improved: median was 35 days (range, 18-100 days) compared with 45 days (range, 27-120 days) for controls (P = .0520). These findings support ex vivo fucosylation of multipotent CD34(+) CB cells as a clinically feasible means to improve engraftment efficiency in the double CBT setting. The trial is registered to www.clinicaltrials.gov as #NCT01471067., (© 2015 by The American Society of Hematology.)

In this issue of Blood, Zhou et al report long-term follow-up and detailed analysis of immune reconstitution associated with a different suicide gene strategy to abrogate graft-versus-host disease (GVHD).

The Wilms tumor protein, WT-1, is a widely recognized tumor antigen that is aberrantly expressed in myeloid and lymphoid leukemia and in this issue of Blood, Doubrovina et al report the most extensive catalog heretofore of HLA-restricted immunogenic peptides derived from WT-1, which are recognized by CD8 and CD4T cells.

Anti-HLA donor-specific Abs (DSAs) have been reported to be associated with graft failure in mismatched hematopoietic stem cell transplantation; however, their role in the development of graft failure in matched unrelated donor (MUD) transplantation remains unclear. We hypothesize that DSAs against a mismatched HLA-DPB1 locus is associated with graft failure in this setting. The presence of anti-HLA Abs before transplantation was determined prospectively in 592 MUD transplantation recipients using mixed-screen beads in a solid-phase fluorescent assay. DSA identification was performed using single-Ag beads containing the corresponding donor's HLA-mismatched Ags. Anti-HLA Abs were detected in 116 patients (19.6%), including 20 patients (3.4%) with anti-DPB1 Abs. Overall, graft failure occurred in 19 of 592 patients (3.2%), including 16 of 584 (2.7%) patients without anti-HLA Abs compared with 3 of 8 (37.5%) patients with DSA (P = .0014). In multivariate analysis, DSAs were the only factor highly associated with graft failure (P = .0001; odds ratio = 21.3). Anti-HLA allosensitization was higher overall in women than in men (30.8% vs 12.1%; P < .0001) and higher in women with 1 (P = .008) and 2 or more pregnancies (P = .0003) than in men. We conclude that the presence of anti-DPB1 DSAs is associated with graft failure in MUD hematopoietic stem cell transplantation.

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8(+) and CD4(+) T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a population of cells, in patients compared to controls. Patients with similar clinical leukemia types, particularly acute myeloid leukemia (AML), did not show a uniform pattern of sequence variation in single blasts. Some patients at relapse presented a complex shift of major haplotypes in single cells. Four patients showed high frequencies of cells containing mutations 189, 260, 16150, and 16488, respectively, as a result of clonal expansion and could be considered as potential markers for their respective disease progression. To our knowledge, this is the first large-scale study of mtDNA variation in single malignant cells. Our results suggest that the somatic mutation process in leukemia is complex, leading to diverse levels of genetic alterations due to either intrinsic aspects of leukemia pathophysiology or chemotherapy effects.

Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4(+) T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor alpha [TNF]alpha) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4(+) T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells.

Graft-versus-host disease (GVHD) occurs in an unpredictable fashion after 30% to 50% of matched-related transplantations. The presence of increased frequencies of CD4(+)CD25(+) regulatory T cells in donor grafts has been shown to ameliorate GVHD after allogeneic transplantation in murine models. To determine whether a similar relationship exists in humans, we quantitated the coexpression of CD25 on CD4(+) and CD8(+) T cells within 60 donor grafts infused into matched siblings and examined GVHD incidence in the respective recipients. Recipients in whom GVHD developed received donor grafts containing significantly higher frequencies of CD4(+) T cells coexpressing CD25 than those who did not (median, 9.26% vs 2.22%; P =.004). Frequencies of donor graft CD8(+) T cells coexpressing CD25 were also higher (0.65% vs 0.14%; P =.002). Furthermore, transplant recipients who received grafts containing fewer CD4(+)CD25(+) and CD8(+)CD25(+) T cells were less likely to acquire acute GVHD, even though these donor-recipient pairs were similar to others with respect to relevant clinical variables. These data suggest that the coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells in humans and that increased frequencies and numbers of CD25(+) T cells in donor grafts is associated with GVHD in transplant recipients.

Poor immune reconstitution after haploidentical stem cell transplantation results in a high mortality from viral infections and relapse. One approach to overcome this problem is to selectively deplete the graft of alloreactive cells using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient peripheral blood mononuclear cells (PBMCs), and this can result in graft versus host disease (GVHD). We have refined this approach using recipient Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as stimulators to activate donor alloreactive T cells. Our studies demonstrate that allodepletion with an anti-CD25 immunotoxin following stimulation with HLA-mismatched host LCLs more consistently depleted in vitro alloreactivity than stimulation with host PBMCs, as assessed in primary mixed lymphocyte reactions (MLRs). Allodepletion using this approach specifically abrogates cytotoxic T-cell responses against host LCLs. In interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assays, antiviral responses to adenovirus and cytomegalovirus (CMV) were preserved following allodepletion. Likewise, using HLA-A2-pp65 tetramers, we have shown that the frequency of CMV-specific T cells is unaffected by allodepletion. Moreover, the donor anti-EBV response is partially retained by recognition of EBV antigens through the nonshared haplotype. Finally, we studied whether allodepletion affects the response to candidate tumor antigens in myeloid malignancies. Using HLA-A2-PR1 tetramer analysis, we found that the frequency of T cells recognizing the PR1 epitope of proteinase 3 was not significantly different in allodepleted and unmanipulated PBMCs from patients with chronic myeloid leukemia (CML) undergoing transplantation. Based on these data, we have embarked on a phase 1 clinical trial of addback of allo-LCL-depleted donor T cells in the haplo-identical setting.

Cytomegalovirus (CMV) infection causes significant morbidity and mortality in the setting of immunodeficiency, including the immune reconstitution phase following allogeneic stem cell transplantation (SCT). We assessed CMV-specific CD4(+) and CD8(+) T-cell responses in 87 HLA-A*0201-positive (A2+) and/or B*0702-positive (B7+) allogeneic stem cell transplant recipients using HLA-peptide tetramer staining and cytokine flow cytometry (CFC) to examine the association of CMV-specific immune reconstitution and CMV antigenemia following SCT. Strong CMV-specific T-cell responses recovered in most subjects (77 of 87, 88%) after SCT. Frequencies of CMV-specific CD8(+) T cells were significantly higher in those subjects who experienced early antigenemia relative to those who did not (2.2% vs 0.33%, P =.0002), as were frequencies of CMV-specific CD4(+) T cells (1.71% vs 0.75%, P =.002). Frequencies of CMV-specific CD8(+) T cells were also higher in subjects experiencing late antigenemia (2.4% vs 0.57%). When we combined tetramer staining and an assessment of cytokine production in a single assay, we found that individuals who experienced CMV antigenemia had lower tumor necrosis factor-alpha (TNF-alpha)-producing fractions of tetramer-staining CMV-specific CD8(+) T cells than subjects who did not (25% vs 65%, P =.015). Furthermore, individuals at high risk for CMV reactivation, including patients with acute graft-versus-host disease and those receiving steroids, had low fractions of cytokine-producing CMV-specific CD8(+) T cells (25% and 27%, respectively). These data suggest that the inability to control CMV reactivation following allogeneic SCT is due to the impaired function of antigen-specific CD8(+) T cells rather than an inability to recover sufficient numbers of CMV-specific T cells.

Evidence suggests that T lymphocyte-mediated inhibition of hematopoiesis in myelodysplastic syndrome (MDS) contributes to cytopenia in some patients and can be reversed by treatment with immunosuppression. We examined the T-cell repertoires of 12 patients with MDS before and after antithymocyte globulin (ATG)-based treatment by T-cell receptor Vbeta (TCR-Vbeta) spectratype analysis. The average number of TCR-Vbeta families with skewed spectratypes, representative of clonal or oligoclonal T-cell populations, was 7.6 in MDS patients before treatment and 3.2 in healthy controls (P =.02). Four patients who recovered effective hematopoiesis after treatment lost prominent, skewed peaks on their spectratypes, suggesting loss or diminution of overrepresented clonal T-cell populations. In contrast, patients who did not recover effective hematopoiesis showed persistently skewed repertoires 3 to 6 months after treatment. In 3 patients with skewed repertoires, cDNA from the complementarity-determining region 3 (CDR3) of 4 TCR-Vbeta families was cloned and repetitively sequenced, confirming clonal T-cell dominance in each family. In one nonresponder, 16 of 19 CDR3 sequences were identical, demonstrating that 9.3% of the total T-cell population was made up of a single clone. By 6 months after treatment, this clone persisted on both spectratype and DNA sequence complementarity and when analyzed by flow cytometry was shown to be CD8(+)/CD45RA(+)/HLA-DR(-). T-cell clones were not anergic because they could be expanded 4-fold in vitro. Our results demonstrate that predominant clonal T cells that appear to be antigen-driven persist in patients with MDS unresponsive to immunosuppression, but predominant clones regress in responders to immunosuppression.

Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1-4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from chicken (CMIX) and mice (Mml/MIXL1), isolation of a human Mix-like gene has remained elusive. We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and Mml/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of Mml/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted to immature B- and T-lymphoid cells. Several acute leukemic cell lines of B, T, and myeloid lineage express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the alpha-globin gene, suggesting a functional conservation of the homeodomain. Isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation.

We previously showed that a peptide (PR1) derived from the primary granule enzyme proteinase 3 induced peptide specific cytotoxic T lymphocytes (CTL) in a normal HLA-A2.1+ individual. These CTL showed HLA-restricted cytotoxicity to myeloid leukemias (which overexpress proteinase 3). To further investigate their antileukemic potential, we studied the ability of PR1-specific CTL, derived from two HLA-A2.1+ normal individuals, to inhibit colony-forming unit granulocyte-macrophage (CFU-GM) from normal and leukemic individuals. CTL from 20 day PR1 peptide-pulsed lymphocyte cultures showed 89% to 98% HLA-A2.1-restricted colony inhibition of chronic myeloid leukemia targets. Colony formation in normal HLA-A2.1+ bone marrow or HLA-A2.1- CML cells was not inhibited. Sequencing of the exon encoding PR1 showed that colony inhibition was not caused by polymorphic differences in proteinase 3 between effectors and targets. Analysis by flow cytometry showed that proteinase 3 was overexpressed in the leukemia targets compared with normal marrow targets (median channel fluorescence 1,399 v 298, P = .009). These results show that PR1-specific allogeneic T cells preferentially inhibit leukemic CFU-GM based on overexpression of proteinase 3, and that proteinase 3-specific CTL could be used for leukemia-specific adoptive immunotherapy.