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. IntroductionUmbilical cord blood (CB), first demonstrated to have clinical utility by Gluckman et al as a source of hematopoietic stem cells in the setting of Fanconi anemia, 1 was later demonstrated to have utility as a source of unrelated donor stem cells for patients lacking matched-sibling donors. [2][3][4][5] Over the past decade, a large number of studies have demonstrated the clinical utility of CB transplantation (CBT) as a treatment for both malignant and nonmalignant diseases of children and adults. 4,6 The establishment of international cord blood banks, advances in supportive care and donor graft selection, and novel clinical approaches aimed at improving engraftment (eg, ex vivo expansion of CB-derived progenitors 7,8 and the infusion of pooled unrelated units 9 ) have improved outcomes and led to a dramatic increase in the number of CBTs performed worldwide.CB grafts obtained from matched unrelated donors offer advantages over bone marrow or peripheral blood stem cells (PBSC) such as noninvasive procurement, more rapid availability without the need for the more prolonged process of screening and obtaining stem cells from a matched unrelated donor (MUD), and the apparently greater tolerance for incompletely human leukocyte antigen (HLA)-matched products. 10 These advantages are paramount for recipients in historically underrepresented minority groups, for whom the prospect of locating a MUD registry donor remains relatively diminished. At our institution, more than twice the proportion of CB transplant recipients are minorities relati...

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-␣ (TNF-␣)-producing fractions of tetramer-staining CMVspecific 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 antigenspecific CD8 ؉ T cells rather than an inability to recover sufficient numbers of CMV-specific T cells. (Blood. 2002;100: 3690-3697)

Aspergillus fumigatus (AF) is a ubiquitous mold and is the most common cause of invasive aspergillosis, an important source of morbidity and mortality in immunocompromised hosts. Using cytokine flow cytometry, we assessed the magnitude of functional CD4 ؉ and CD8 ؉ T-cell responses following stimulation with Aspergillus antigens. Relative to those seen with cytomegalovirus (CMV) or superantigen stimulation, responses to Aspergillus antigens were near background levels. Subsequently, we confirmed that gliotoxin, the most abundant mycotoxin produced by AF, was able to suppress functional T-cell responses following CMV or staphylococcal enterotoxin B (SEB) stimulation. Additional studies demonstrated that crude AF filtrates and purified gliotoxin inhibited antigen-presenting cell function and induced the preferential death of monocytes, leading to a marked decrease in the monocyte-lymphocyte ratio. Analysis of caspase-3 activation confirmed that gliotoxin preferentially induced apoptosis of monocytes; similar effects were observed in CD83 ؉ monocyte-derived dendritic cells. Importantly, the physiologic effects of gliotoxin in vitro were observed below concentrations recently observed in the serum of patients with invasive aspergillosis. These studies suggest that the production of gliotoxin by AF may constitute an important immunoevasive mechanism that is mediated by direct effects on antigenpresenting cells and both direct and indirect effects on T cells. IntroductionAspergillus fumigatus (AF) is the most common cause of invasive aspergillosis (IA) and a major source of infection-related mortality in immunocompromised patients, such as allogeneic stem cell transplant (SCT) recipients. 1,2 In these patients prophylactic antifungal therapy has been found to have little effect on disease incidence. 1,[3][4][5] Despite advances in early diagnosis and new antifungal agents, 3,6,7 IA remains a leading cause of death in this patient population, with an attributable mortality rate ranging from 30% to 80%. 8 AF is among the most ubiquitous of those fungi with airborne conidia (spores) and is commonly found in human domiciles. Pulmonary infection by AF, the predominant type of IA, is acquired through the inhalation of Aspergillus conidia, while the invasion stage of the disease is characterized by hyphal destruction of pulmonary tissue. 1 The mediocre efficacy of antifungals in the setting of profound immunosuppression contributes to the poor prognosis of this opportunistic infection. The development of effective strategies to improve AF-specific immune reconstitution should greatly influence the natural history of IA.Historically, there was a biphasic distribution of IA following bone marrow transplantation (BMT); IA was most common in the pre-engraftment period associated with neutropenia, with a second peak in incidence associated with acute and/or chronic graft-versushost disease (GVHD). 5 However, recent reports have indicated late-onset IA predominates after allogeneic SCT, often in concomitance with the occurrence o...

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemiaassociated 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)- IntroductionCD8 T cells specific for the human leukocyte antigen-A2 (HLA-A2)-restricted peptides WT1 and PR1, which are derived from the endogenous leukemia-associated antigens Wilms' tumor antigen [1][2][3] and proteinase 3 (P3), respectively, mediate cytotoxicity against acute myeloid leukemia (AML). PR1-specific T cells also contribute to cytogenetic remission of chronic myeloid leukemia (CML) in patients treated with interferon, 4,5 and vaccination with WT1 and PR1 6,7 can induce specific CD8 immunity in patients with myeloid malignancies. These results validate endogenous self-peptides as targets for immunotherapy, including vaccination, adoptive cell therapy, or antibodies that bind peptide/MHC. Such T-cell receptor (TCR)-like monoclonal antibodies (mAbs) may have selective activity against leukemia if target peptide/MHC complexes are aberrantly expressed on leukemia. Furthermore, mAbs are easy to administer and can be dosed frequently, which may increase their effectiveness against high leukemia burdens.Eliciting TCR-like mAbs has been technically challenging, 8 primarily because of the high immunogenicity of HLA molecules in mice. Phage-display libraries, 9 peptide/MHC immunization, 10,11 and the combination of both strategies 8,12 have been used to produce TCR-like mAbs targeting peptides derived from solid-tumor antigens (eg, MAGE, ␤-HCG, TARP, and NY-ESO-1) in the context of HLA-A1 or HLA-A2. [9][10][11]13,14 Although antibody activity against primary tumors has not been well studied, complement-dependent cytotoxicity (CDC) against tumor cell lines has been reported. 11 Some toxin-conjugated antibodies also show activity against tumor cells. 14-16 However, to eradicate cancer, these antibodies must be active against cancerinitiating cells, and TCR-like mAb-induced cytolysis of cancer stem cells has not been reported. Nevertheless, because PR1-specific CTLs suppress leukemia progenitor cells in vitro 17 and because Lin Ϫ CD34 ϩ CD38 Ϫ cells are enriched for leukemia stem cells (LSCs) 18 and can be easily studied, we hypothesized that if an anti-PR1/HLA-A2 antibody could be produced, it may be active against blasts and LSCs from HLA-A2 ϩ AML patients.We report the discovery of 8F4, a novel mAb that binds with high affinity to a conformational epitope of PR1/HLA-A2 and induces dose-dependent cytolysis of myeloid leukemia cells but not normal hematopoietic cells. 8F4 mediates CDC against Lin Ϫ CD34 ϩ CD38 Ϫ LSCs and preferentially inhibits the growth of leukemia progenitor cells. These results justify further study of TCR-like antibodies to verify the differential effects against normal stem cells and LSCs. Biologically significant differences may justify further study of a humanized form o...