克罗恩病相关的NOD2及β-防御素-2基因多态性对β-防御素-2表达的影响

目的探讨克罗恩病（Crohndisease,CD）相关的NOD2及人β-防御素-2（humanbeta—defensin2,hBD-2）基因多态性对hBD-2转录活性的影响及主要机理。方法将hBD-2报告基因质粒和NOD2真核表达载体共转染至HEK293T细胞,用脂多糖（LPS）和TNF—α分别孵育刺激后测定hBD-2转录活性变化。结果LPS对hBD-2转录活性有抑制作用（P=0．020）,TNF—α可相对升高hBD-2转录活性,呈剂量依赖性（P=0．004）;LPS作用时,NOD2（P268S）改变前后hBD-2的转录活性差异有统计学意义（P=0．008）;hBD-2（-233）改变前后hBD-2的转录活性差异无统计学意义（P=0．053）。在TNF-α（5ng／m1）刺激时,NOD2（P268S）改变前后hBD-2的转录活性差异无统计学意义（P=0．064）;hBD-2（-233）改变前后hBD-2的转录活性差异有统计学意义（P=0．006）;NF—κB抑制剂可显著下调hBD-2转录的激活（P〈0．001）。结论NOD2（P268S）改变能够降低hBD-2的内源性表达;hBD-2（-233）改变导致hBD-2诱生性表达的减少;NF—κB通路可能是hBD-2诱导表达的主要路径。     

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.     

The androgen-regulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin

Spermatozoa bind a variety of proteins as they pass through the proximal regions of the epididymis, where they acquire forward motility and fertilizing ability. Recent evidence indicates that certain epididymis-specific secretory proteins that bind sperm have antibacterial activity and may function as part of the innate immune system. We reported earlier that ESC42, now designated human beta-defensin 118 (DEFB118), is a sperm-binding protein. In this study, we demonstrate that DEFB118 has potent antibacterial activity that is dose, time, and structure dependent. Incubation of Escherichia coli for 60 min with 10 microg/ml DEFB118 reduced bacterial survival to 20% of the control, and 25 microg/ml reduced survival to 5% of the control. DEFB118 concentrations of 50 and 100 microg/ml further reduced survival to less than 2 and 1%, respectively. A biphasic effect of salt concentration on the antibacterial activity of DEFB118 was observed. Reduction of disulfide bonds and alkylation of cysteines resulted in the complete loss of antibacterial activity. DEFB118 caused rapid permeabilization of both outer and inner membranes of E. coli and striking morphological alterations in the bacterial surfaces visible by scanning electron microscopy consistent with a membrane-disruptive mechanism of bacterial killing. In contrast, eukaryotic cell membranes were not permeabilized by DEFB118, as indicated by the rat erythrocyte hemolytic assay. Studies on DEFB118 inhibition of macromolecular synthesis and membrane permeability in E. coli were consistent with a primary effect at the cell membrane level. DEFB118 may contribute to epididymal innate immunity and protect the sperm against attack by microorganisms in the male and female reproductive tracts.     

Phage display generation of a novel human anti-CD1A monoclonal antibody with potent cytolytic activity

CD1A is a cell surface protein expressed on Langerhans cells and cortical thymocytes that could potentially be used as an immunotherapeutic target in Langerhans Cell Histiocytosis (LCH), the cortical subtype of T-cell acute lymphocytic leukaemia (T-ALL) and other CD1A-positive tumours. The monoclonal antibody (mAb) CR2113 was selected from a panel of six fully human mAbs isolated from a semi-synthetic phage display library, based on specificity and avidity against cells expressing CD1 antigen variants. CR2113 recognized CD1A in T-ALL cell lines and patient samples. Confocal microscopy revealed that the CR2113-CD1A complex was internalized at 37°C. Furthermore, while CR2113 induced moderate complement-dependent cytotoxicity (CDC), potent antibody-dependent cell cytotoxicity (ADCC) activity was observed against CD1A expressing cell lines as well as T-ALL cell lines and T-ALL patient samples. In vivo experiments showed that CR2113 as a naked antibody has modest but specific anti-tumour activity against CD1A-expressing tumours. CR2113 is a high-affinity human anti-CD1A mAb with significant ADCC activity. These properties make CR2113 a candidate for clinical diagnostic imaging and therapeutic targeting of LCH as well as potential use in other clinical applications.     

Hexachlorocyclohexanes, potent stimuli of O2- production and calcium release in human polymorphonuclear leukocytes

alpha-, gamma-, and delta-Hexachlorocyclohexane (HCCH), but not the beta-isomer, are shown to be potent stimuli for the production of superoxide anion (O2-) and the release of calcium in human polymorphonuclear leukocytes (PMNs). Although O2- production occurs in the absence of exogenous divalent cations, calcium (0.5 mmol/L) enhances O2- production by 50%. In addition, gamma-HCCH-induced O2- production is sensitive (IC50-30-40 mumol/L) to inhibition by the putative intracellular antagonist, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8). Furthermore, it is shown that gamma-HCCH induces a marked loss of membrane-associated calcium as monitored by chlortetracycline fluorescence. This pool of calcium appears to encompass the same pool released by the formylated tripeptide, formylmethionylleucylphenylalanine (FMLP). We suggest that the HCCHs represent a new class of stimuli of O2- production and calcium mobilization in PMNs.     

Altered serum levels of human neutrophil peptides (HNP) and human beta-defensin 2 (hBD2) in Wegener's granulomatosis

Defensins are highly conserved peptides with antimicrobial and immunomodulatory functions. Due to their chemotactic properties on mononuclear cells, including dendritic cells and macrophages, defensins may contribute to granuloma formation in Wegener’s granulomatosis (WG). In order to explore whether these peptides might be involved in WG pathogenesis, sera of patients were screened to detect altered defensin levels. For this purpose, serum and EDTA-blood of patients with WG (n = 17; aged 54.8 ± 15.5 years) and age- and sex-matched healthy controls (n = 24; aged 55.5 ± 16.8 years) were collected. Levels of neutrophil α-defensins (human neutrophil peptides, HNP) and β-defensin (hBD) 2 and 3 in serum were measured by ELISA. By this means, WG patients displayed higher serum levels of hBD2 and HNP when compared to controls. Furthermore, serum hBD2 was raised in patients with meningeal granulomas (n = 4) or in those undergoing treatment with cyclophosphamide (n = 4). In order to detect whether increased gene expression in polymorphonuclear cells is responsible for the elevated defensin levels, real-time polymerase chain reaction with gene-specific primers was performed. Expression of specific mRNA in polymorphonuclear cells was observed for HNP only, but did not parallel HNP serum levels, suggesting that degranulation rather than increased gene expression may be responsible for increased HNP serum levels in WG. In conclusion, elevated serum levels of HNP and hBD2 in WG patients suggest a role for both defensins in WG pathogenesis.     

High concentrations of human β-defensin 2 in gastric juice of patients with Helicobacter pyloriinfection

AIM: Human beta-defensin (HBD)-1 and HBD-2 are endogenous antimicrobial peptides. Unlike HBD-1, the HBD-2 expression is augmented by Helicobacter pylori (H pylori). We sought to determine HBD-1 and HBD-2 concentrations in gastric juice during H pylori infection. METHODS: HBD-1 and HBD-2 concentrations were measured by radioimmunoassay in plasma and gastric juice of 49 H pylori-infected and 33 uninfected subjects and before and after anti-H pylori treatment in 13 patients with H pylori-associated gastritis. Interleukin (IL)-1beta and IL-8 concentrations in gastric juice were measured by enzyme-linked immunosorbent assay (ELISA). Histological grades of gastritis were determined using two biopsy specimens taken from the antrum and corpus. Reverse phase high performance liquid chromatography (RP-HPLC) was used to identify HBD-2. RESULTS: HBD-2 concentrations in gastric juice, but not in plasma, were significantly higher in H pylori-positive than -negative subjects, albeit the post-treatment levels were unchanged. Immunoreactivity for HBD-2 was exclusively identified in H pylori-infected mucosa by RP-HPLC. HBD-2 concentrations in gastric juice correlated with histological degree of neutrophil and mononuclear cell infiltration in the corpus. IL-1beta levels correlated with those of IL-8, but not HBD-2. Plasma and gastric juice HBD-1 concentrations were similar in H pylori-infected and uninfected subjects. CONCLUSION: Our results place the beta-defensins, especially HBD-2, in the front line of innate immune defence. Moreover, HBD-2 may be involved in the pathogenesis of H pylori-associated gastritis, possibly through its function as immune and inflammatory mediator.     

Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of angiogenesis

Platelet factor-4 (PF-4)/CXCL4 was the first chemokine described to inhibit neovascularization. Here, the product of the nonallelic variant gene of CXCL4, PF-4var1/PF-4alt, designated CXCL4L1, was isolated for the first time from thrombin-stimulated human platelets and purified to homogeneity. Although secreted CXCL4 and CXCL4L1 differ in only three amino acids, CXCL4L1 was more potent in inhibiting chemotaxis of human microvascular endothelial cells toward interleukin-8 (IL-8)/CXCL8 or basic fibroblast growth factor (bFGF). In vivo, CXCL4L1 was also more effective than CXCL4 in inhibiting bFGF-induced angiogenesis in rat corneas. Thus, activated platelets release CXCL4L1, a potent regulator of endothelial cell biology, which affects angiogenesis and vascular diseases.     

Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5′ flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme''s capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions.The Dna2 enzyme functions at the crossroads of key DNA metabolic processes. It was initially identified in screens for Saccharomyces cerevisiae mutants deficient in DNA replication (1, 2), and its importance was underscored by the finding that dna2Δ mutants are not viable (3). When the DNA2 gene was cloned, it was shown to be conserved in evolution from yeast to humans and found to contain conserved nuclease and helicase motifs (4). Further work identified a number of genetic and physical interactions of Dna2 with factors required for the synthesis and maturation of the lagging strand during DNA replication, including Rad27 [homolog of human Flap endonuclease 1, FEN1 (5)]. Overexpression of Rad27 rescued growth defects of some dna2 point mutants, and, conversely, overexpression of Dna2 suppressed rad27Δ defects (5). Subsequent work established that Dna2 functions together with Rad27 in the removal of single-stranded (ss) flaps generated at the 5′ termini of Okazaki fragments by polymerase δ-catalyzed strand displacement. Although Rad27 seems to have a more general role in flap processing, Dna2 is required to cleave only a subset of longer flaps bound by Replication Protein A (RPA), which stimulates its nuclease activity while inhibiting cleavage by Rad27. In this process, Dna2 and Rad27 were proposed to function in a sequential manner, with Dna2 loading first onto the flap termini and shortening them with its 5′–3′ nuclease. Rad27 was then proposed to further cleave the shortened flaps at the ss/dsDNA junctions, creating thus ligatable substrates for Cdc9 (DNA ligase 1), which completes Okazaki fragment maturation (6–8). The role of Dna2 in Okazaki fragment processing is now generally accepted although it still remains somewhat puzzling why dna2Δ cells are inviable whereas rad27Δ mutants are not.More recently, it has been shown that Dna2 has an independent and conserved function in dsDNA break repair (9). Specifically, Dna2 belongs to one of the pathways that resect 5′ ends of dsDNA breaks to initiate homologous recombination. This process leads to the formation of long 3′ overhangs, which become coated by the strand exchange protein Rad51, and which also prime DNA synthesis during the downstream steps in homologous recombination. Genetic and later biochemical work established that Dna2 functions together with the vigorous Sgs1 helicase (Bloom in human cells) downstream of the Mre11-Rad50-Xrs2 (MRX) complex (9–14). MRX first recognizes dsDNA breaks and is likely involved in their initial processing, and subsequently helps recruit Sgs1 and Dna2. Sgs1 helicase then unwinds the DNA from the break and the ssDNA is coated by RPA, which stimulates the 5′–3′ nuclease activity of Dna2. The specific degradation of the 5′ end of the unwound DNA ensures the correct polarity of resection, which is required for homologous recombination (12, 13). However, the roles of Dna2 are not limited to Okazaki fragment and dsDNA break processing. Dna2 was also shown to function in telomere maintenance (15), aging (16), long-patch base excision repair (17), and prevention of reversal of stalled replication forks (18). The expression of human DNA2 was increased in human cancers and negatively correlated with disease outcome, indicating that DNA2 function is relevant for human health (19). However, the role of Dna2/DNA2 in these latter processes remains poorly defined.The potent nuclease activity of Dna2 seems to be critical for all of its functions, including replication and recombination. Point mutants lacking the nuclease activity are inviable as a dna2Δ strain (20). The nuclease activity of Dna2 is ssDNA-specific, and shows both 5′–3′ and 3′–5′ polarities. Because RPA stimulates the 5′–3′ nuclease and inhibits the 3′–5′ activity, it is likely that only the 5′–3′ directionality is important in vivo (12, 13). It has been shown that the Dna2 nuclease can load only on a free ssDNA tail, and that it subsequently cleaves DNA endonucleolytically into short oligonucleotides (21, 22).Much less is known about the function of the helicase activity of Dna2. To date, very weak 5′–3′ unwinding capacity has been demonstrated for the yeast enzyme (4, 7, 23). Interestingly, similarly to the Dna2 nuclease, also the helicase domain requires a free DNA end (22). In contrast, no unwinding activity could be detected in the Xenopus laevis Dna2. Whether human DNA2 possesses helicase activity remains controversial (24–27). Yeast point mutants lacking helicase activity show impaired growth but are viable under most conditions (23). Overexpression of Rad27 partially rescues dna2 helicase-deficient mutants, suggesting that the helicase activity might play a supportive but nonessential role in DNA replication (28). The helicase-deficient mutants show a dramatic sensitivity to the DNA alkylating drug methylmethanesulfonate (MMS), pointing to an as-yet uncharacterized role of Dna2 helicase in the repair of DNA damage (29). In contrast, Dna2 helicase activity had no detectable effect on DNA end resection (9). Due to the limited unwinding capacity of Dna2 observed in vitro, the motor was proposed to function as an ssDNA translocase to aid positioning of the nuclease domain on ssDNA and to remove secondary structures from ssDNA, rather than as a DNA helicase to unwind dsDNA (23).In this work, we expressed S. cerevisiae Dna2 and its variants and optimized the purification of these polypeptides. We now show that Dna2 is a potent but cryptic DNA helicase. It functionally interacts with RPA, which enables it to unwind tens of kilobases of dsDNA. Surprisingly, the nuclease of wild-type Dna2 interferes with this remarkable helicase capacity by cleaving ssDNA tails that the helicase requires for loading onto DNA to initiate unwinding. The interplay between the two main biochemical activities of Dna2 might fine-tune its behavior to suit its distinct cellular roles.     

Adenosine A2A receptor agonists with potent antiplatelet activity

Selected adenosine A_2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC₅₀ 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC₅₀ 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC₅₀ 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC₅₀ 0.97±0.07 µmol/L) and equipotent to NECA (EC₅₀ 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K_i values determined in radioligand-binding studies were not predictive of the A_2A agonists’ antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A_2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A_2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A_2A receptor agonists.     

Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity

BACKGROUND AND AIMS: The gastrointestinal microflora exerts a barrier effect against enteropathogens. The aim of this study was to examine if bifidobacteria, a major species of the human colonic microflora, participates in the barrier effect by developing antimicrobial activity against enterovirulent bacteria. METHODS: Antibacterial activity was examined in vitro against a wide range of Gram negative and Gram positive pathogens. Inhibition of Salmonella typhimurium SL1334 cell association and cell invasion was investigated in vitro using Caco-2 cells. Colonisation of the gastrointestinal tract in vivo by bifidobacteria was examined in axenic C3/He/Oujco mice. Antimicrobial activity was examined in vivo in axenic C3/He/Oujco mice infected by the lethal S typhimurium C5 strain. RESULTS: Fourteen human bifidobacterium strains isolated from infant stools were examined for antimicrobial activity. Two strains (CA1 and F9) expressed antagonistic activity against pathogens in vitro, inhibited cell entry, and killed intracellular S typhimurium SL1344 in Caco-2 cells. An antibacterial component(s) produced by CA1 and F9 was found to be a lipophilic molecule(s) with a molecular weight of less than 3500. In the axenic C3/He/Oujco mice, CA1 and F9 strains colonised the intestinal tract and protected mice against S typhimurium C5 lethal infection. CONCLUSION: Several bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity, suggesting that they could participate in the "barrier effect" produced by the indigenous microflora.     

Prostacyclin: a potent activator of human colonic adenylate cyclase activity.

Prostacyclin and its stable catabolite 6-keto-prostaglandin F1 alpha were tested on the human colonic mucosal adenylate cyclase which represents the key enzyme in the secretory process in this organ. Prostacyclin was able to activate dose-dependently the human cyclase system. Saturating concentrations of prostacyclin and 6-keto-prostaglandin F1 alpha (each 0.28 mM) increased the enzyme activity 2.5-fold and only 1.4-fold, respectively. The presence of prostacyclin receptor sites in human colonic mucosa suggests a physiological role of this novel prostaglandin in human colonic function.     

Interleukin 2-dependent release of interleukin 3 activity by T4+ human T-cell clones.

We have isolated and studied two alloreactive, T4+, human lymphocyte clones that release interleukin 2 (IL-2) and interleukin 3 (IL-3) bioactivities upon stimulation with IL-2, alloantigen, or Sepharose-conjugated antibodies directed against the T3 protein. Anti-IL-2 receptor monoclonal antibodies block IL-2-, alloantigen-, or anti-T3-stimulated IL-3 release. Hence, release of IL-3 activity in each circumstance is rigorously dependent upon activation of the IL-2 receptor. Even low, nonmitogenic concentrations of recombinant IL-2 stimulated IL-3 release.     

Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release

Apelin, a recently isolated neuropeptide that is expressed in the supraoptic and the paraventricular nuclei, acts on specific receptors located on vasopressinergic neurons. The increased phasic pattern of these neurons facilitates sustained antidiuresis during dehydration or lactation. Here, we investigated whether apelin interacts with arginine vasopressin (AVP) to maintain body fluid homeostasis. We first characterized the predominant molecular forms of endogenous hypothalamic and plasma apelin as corresponding to apelin 13 and, to a lesser extent, to apelin 17. We then demonstrated that, in lactating rats, apelin was colocalized with AVP in supraoptic nucleus magnocellular neurons and given intracerebroventricularly inhibited the phasic electrical activity of AVP neurons. In lactating mice, intracerebroventricular administration of apelin 17 reduced plasma AVP levels and increased diuresis. Moreover, water deprivation, which increases systemic AVP release and causes depletion of hypothalamic AVP stores, decreased plasma apelin concentrations and induced hypothalamic accumulation of the peptide, indicating that AVP and apelin are conversely regulated to facilitate systemic AVP release and suppress diuresis. Opposite effects of AVP and apelin are likely to occur at the hypothalamic level through autocrine modulation of the phasic electrical activity of AVP neurons. Altogether, these data demonstrate that apelin acts as a potent diuretic neuropeptide counteracting AVP actions through inhibition of AVP neuron activity and AVP release. The coexistence of apelin and AVP in magnocellular neurons, their opposite biological effects, and regulation are likely to play a key role for maintaining body fluid homeostasis.     

Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity

Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2-mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2(+) progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.