Impact of post-transplant minimal residual disease on the clinical outcome of pediatric acute leukemia.

This retrospective study examined the clinical significance of FCM-MRD in 36 patients with ALL and 29 patients with AML after their first allogeneic HSCT. Hematological (FCM-MRD ≥5.0%) and molecular relapse (FCM-MRD <5.0%) were first detected in 10 and two patients with ALL and in seven and eight patients with AML, respectively. Eight of 10 patients with molecular relapse eventually progressed to hematological relapse, although most were treated with immunological intervention by aggressive discontinuation of immunosuppressive therapy or donor lymphocyte infusion. Among these 12 patients, four of seven patients that obtained MRDneg CR following post-transplant chemotherapy remain alive and disease-free after their second HSCT; however, all five patients who underwent a second HSCT in non-CR died of disease or treatment-related complications. As the FCM-MRD monitoring system used in the current study was probably not sensitive enough to detect MRD, which could be elucidated by immunological intervention, more sensitive diagnostic tools are mandatory for post-transplant MRD monitoring. Additional studies are required to address the impact of presecond transplant MRD on the clinical outcome of second HSCT.

Roles of transferrin receptors in erythropoiesis.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

Reduced Numbers and Proapoptotic Features of Mucosal-associated Invariant T Cells as a Characteristic Finding in Patients with Inflammatory Bowel Disease.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. METHODS: Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohn's disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. RESULTS: Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. CONCLUSIONS: The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

Effect of intensive granulocyte and monocyte adsorptive apheresis in patients with ulcerative colitis positive for cytomegalovirus.

BACKGROUND AND AIM: Cytomegalovirus (CMV) exacerbates ulcerative colitis (UC) refractory to immunosuppressive therapies. The conditions under which CMV reactivation occurs in patients with UC, however, is unclear. In addition, the diagnostic and treatment strategies for UC positive for CMV have not been established. Granulocyte and monocyte adsorptive apheresis (GMAA) is natural biological therapy for UC in which the granulocytes/macrophages producing inflammatory cytokines are removed. We investigated the rate of colonic CMV reactivation and the efficacy of GMAA in active UC patients positive for CMV without concomitant corticosteroid (CS) therapy. METHODS: Fifty-one active UC patients without concomitant CS therapy were enrolled. Colonic CMV reactivation was examined by real-time polymerase chain reaction (PCR) using biopsy specimen and/or histological examination. All patients were treated with intensive GMAA (twice per week). Rates of clinical remission and mucosal healing were compared between UC patients positive and negative for CMV. RESULTS: Of 51 patients, 15 (29.4%) were diagnosed as CMV positive. The clinical remission rates following intensive GMAA did not differ between UC patients positive and negative for CMV (73.3% vs 69.4%, p=0.781). Proportion of patients achieving mucosal healing was also similar between these two groups. CMV-DNA became negative in all UC patients positive for CMV who achieved clinical remission 1 week after completion of intensive GMAA. CONCLUSIONS: Intestinal inflammation might trigger CMV reactivation in a subpopulation of active UC patients without CS treatment. GMAA could be a promising option for active UC positive for CMV.

Sialic acid cyclization of human Th homing receptor glycan associated with recurrent exacerbations of atopic dermatitis.

BACKGROUND: The molecular pathogenesis underlying recurrent exacerbations of atopic dermatitis (AD) is unclear. Some peripheral CCR4(+) and CCR7(+) helper memory T cells express the specific homing receptor, sialyl 6-sulfo Lewis X (G152 glycan). This glycan loses receptor activity via cyclization of its sialic acid moiety, thus becoming cyclic sialyl 6-sulfo Lewis X (G159 glycan). These findings suggest that the disordered expression of G152 and G159 glycans may be associated with recurrent exacerbations of AD. OBJECTIVE: To assess the possible association of G152 and G159 glycans, which are expressed on peripheral helper T (Th) cells, with frequency of exacerbations. METHODS: The percentage of glycan-expressing cells among peripheral blood CD4(+)CD45RO(+) lymphocytes was determined by flow cytometry. The association of glycans with the frequency of exacerbations determined by recurrence scores as well as with current disease activity was statistically tested. RESULTS: Current disease activity was significantly associated with CCR4(+)CCR7(-) memory Th cells expressing CSLEX-1 glycan, the conventional skin-trafficking receptor without sialic-acid-cyclization activity. In contrast, the frequency of exacerbations was positively and negatively associated with CCR4(+)CCR7(+) memory Th cells expressing G152 and G159 glycans, respectively. Receiver operating characteristics analyses indicated that the ratio of the G152(+)/G159(+) cell percentages discriminated patients with highly recurrent AD with the best accuracy. CONCLUSION: Flow cytometric determination of G159 and G152 glycans on peripheral helper memory T cells may be clinically useful for identifying patients with highly recurrent AD. Disordered sialic acid cyclization of G152 glycan may underlie highly recurrent AD, which may provide a novel therapeutic approach.

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9.

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.

Multiple reversions of an IL2RG mutation restore T cell function in an X-linked severe combined immunodeficiency patient.

Reversion mosaicism is increasingly being reported in primary immunodeficiency diseases, but there have been few cases with clinically improved immune function. Here, a case is reported of X-linked severe combined immunodeficiency (SCID-X1) with multiple somatic reversions in T cells, which restored sufficient cell-mediated immunity to overcome viral infection. Lineage-specific analysis revealed multiple reversions in T cell receptor (TCR) αß+ and TCRγδ+ T cells. Diversity of the TCRVß repertoire was comparable to normal and, furthermore, mitogen-induced proliferation of the patient's T cells was minimally impaired compared to healthy controls. In vivo and in vitro varicella antigen-specific T cell responses were comparable to those of healthy controls, although a reduced level of T cell receptor excision circles suggested that recent thymic output was low. During long-term evaluation of the patient's immunologic status, both the number of CD4+ and CD8+ T cells and T cell proliferation responses were stable and the patient remained healthy. This case demonstrates that multiple but restricted somatic reversions in T cell progenitors can improve the clinical phenotype of SCID-X1.

Increase in circulating endothelial progenitor cells predicts response in patients with advanced non-small-cell lung cancer.

Previous reports have shown that circulating endothelial progenitor cells (CEPs) are released in response to cytotoxic chemotherapy. We investigate the relationship between the kinetics of CEPs during one cycle of chemotherapy and the response to cytotoxic chemotherapy and prognostic impacts. Previously untreated patients (n = 38) receiving cytotoxic chemotherapy for non-small-cell lung cancer were included. Blood sampling was carried out on day 1, day 8, and just before the second cycle of chemotherapy. The mononuclear cell fraction was analyzed for CEPs by FACS analysis. We evaluated the relationship between the kinetics of CEPs, each independent clinicopathological variable, the response to chemotherapy, and the risk factors associated with prognosis. On the eighth day after chemotherapy, a significant decrease in CEPs was observed. In contrast, CEP counts before the second cycle of chemotherapy were significantly increased. The high percentage change in CEPs between day 1 and before the second cycle of chemotherapy is an independent predictive factor for response to chemotherapy. However, the change in CEP levels did not predict progression-free survival. These findings indicate that the late release of CEPs is a common phenomenon after chemotherapeutic treatment. The correlation with clinical response to chemotherapy provides further support for the biologic relevance of these cells in patients' prognosis and highlights the potential use of CEPs as therapeutic targets.

Cross-priming of CD8(+) T cells in vivo by dendritic cells pulsed with autologous apoptotic leukemic cells in immunotherapy for elderly patients with acute myeloid leukemia.

OBJECTIVE: The prognosis for elderly patients with acute myeloid leukemia (AML) remains dismal. To explore the potential of immunotherapy for improving clinical outcomes for these patients, we performed a phase I clinical trial of dendritic cell (DC)-based immunotherapy for elderly patients with AML. MATERIALS AND METHODS: Autologus monocytes were obtained after reducing tumor burden by chemotherapy. Immature DCs induced with granulocyte-macrophage colony-stimulating factor and interleukin-4 were pulsed with autologous apoptotic leukemic cells as antigens. DCs were administered intradermally to four patients five times at 2-week intervals. To facilitate DC migration to lymph nodes, injection sites were pretreated with killed Streptococcus pyogenes OK-432 one day before. DCs were coinjected with OK-432 to induce maturation and interleukin-12 production in vivo. RESULTS: Antileukemic responses were observed by an interferon-γ enzyme-linked immunospot assay or a tetramer assay in two of four patients. In a human leukocyte antigen-A∗2402-positive patient, induction of CD8(+) T-cell responses to WT1- and human telomerase reverse transcriptase - derived peptides were observed, indicating cross-priming in vivo. The two patients with antileukemic immunity showed longer periods of disease stabilization than the other two patients. CONCLUSIONS: This study demonstrates the immunogenicity of autologous DCs that cross-present leukemia-associated antigens from autologous apoptotic leukemic cells in vivo in elderly patients with AML.

B7-H1 expression is regulated by MEK/ERK signaling pathway in anaplastic large cell lymphoma and Hodgkin lymphoma.

B7-H1 is a member of the B7 family that inhibits the function of T-cells through its receptor programmed death-1 (PD-1). We examined B7-H1 expression in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) and found that it was constitutively expressed in both clinical samples and cell lines. In anaplastic lymphoma kinase-positive (ALK(+)) ALCL cells, B7-H1 expression was suppressed by the blocking of extracellular signal-regulated kinase (ERK) signaling and upregulated by the augmentation of ERK activity by phorbol 13-myristate 12-acetate stimulation, suggesting that B7-H1 expression is regulated by ERK signaling pathway in ALCL. ERK is one of the downstream mediators of nucleophosmin (NPM)/ALK signaling in ALK(+)ALCL, and pharmacological inhibition of ALK was shown to dephosphorylate ERK and down-regulate B7-H1. The involvement of NPM/ALK in B7-H1 expression was also demonstrated by introducing the construct into human non-ALCL lymphoid cell lines, which resulted in B7-H1 expression. In the case of HL, B7-H1 expression was shown to be dependent on the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results suggest that B7-H1 expression is controlled by common ERK signaling pathways in ALCL and HL cells. Our findings provide a potentially effective immunotherapeutic strategy for these B7-H1-expressing tumors.

Anti-oligosaccharide antibodies as tools for studying sulfated sialoglycoconjugate ligands for siglecs and selectins.

Several sulfated sialoglycoconjugates have recently been shown to serve as ligands for selectins and siglecs. For instance, alpha2-->3 sialylated 6-sulfo-Lewis x was found to serve as a ligand for selectins on skin-homing helper memory T cells, and alpha2-->6 sialylated 6-sulfo-LacNAc to be a preferred ligand for CD22/siglec-2 on human naïve B cells. Monoclonal antibodies specific to sulfated sialoglycoconjugates are effective tools to dissect these ligands on minor subsets of human leukocytes.

Large cell transformation mimicking regional lymphomatoid papulosis in a patient with mycosis fungoides.

A 57-year-old Japanese man with tumor-stage mycosis fungoides suddenly presented multiple small papules on the right chest. Histopathology of a biopsy specimen from the papules revealed medium-to-large pleomorphic lymphoid cells throughout the entire dermis but not in the epidermis, and the large cells expressed CD30 antigen. These newly-developed papules underwent spontaneous remission in the following 3 months. We reviewed the reported cases of mycosis fungoides, which showed CD30-positive large cell transformation and those of CD30-positive lymphoproliferative disorders associated with mycosis fungoides.