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Originally published online as doi:10.1189/jlb.0105026 on April 7, 2005

Published online before print April 7, 2005
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(Journal of Leukocyte Biology. 2005;78:27-36.)
© 2005 by Society for Leukocyte Biology

T cells from paroxysmal nocturnal haemoglobinuria (PNH) patients show an altered CD40-dependent pathway

Giuseppe Terrazzano*, Michela Sica*, Cristina Becchimanzi{dagger}, Silvia Costantini{dagger}, Bruno Rotoli{dagger}, Serafino Zappacosta*, Fiorella Alfinito{dagger} and Giuseppina Ruggiero*,1

* Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, and
{dagger} Cattedra di Ematologia, Dipartimento di Medicina Clinica e Sperimentale, Università di Napoli "Federico II," Naples, Italy

1 Correspondence: Cattedra di Immunologia-Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II", Via Pansini 5, 80131 Naples, Italy. E-mail: giruggie{at}unina.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoiesis disorder characterized by the expansion of a stem cell bearing a somatic mutation in the phosphatidylinositol glycan-A (PIG-A) gene, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. A number of data suggest the inability of the PIG-A mutation to account alone for the clonal dominance of the GPI-defective clone and for the development of PNH. In this context, additional immune-mediated mechanisms have been hypothesized. We focused on the analysis of T lymphocytes in three PNH patients bearing a mixed GPI+ and GPI– T cell population and showing a marked cytopenia. To analyze the biological mechanisms underlying the control of T cell homeostasis in PNH, we addressed the study of CD40-dependent pathways, suggested to be of crucial relevance for the control of autoreactive T cell clones. Our data revealed significant, functional alterations in GPI+ and GPI– T cell compartments. In the GPI– T cells, severe defects in T cell receptor-dependent proliferation, interferon-{gamma} production, CD25, CD54, and human leukocyte antigen-DR surface expression were observed. By contrast, GPI+ T lymphocytes showed a significant increase of all these parameters, and the analysis of CD40-dependent pathways revealed a functional persistence of CD154 expression on the CD48+CD4+ lymphocytes. The alterations of the GPI+ T cell subset could be involved in the biological mechanisms underlying PNH pathogenesis.

Key Words: GPI-defective clones • cytopenia • immune response


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paroxysmal nocturnal haemoglobinuria (PNH) is a hematological syndrome characterized by the emergence of a haematopoiesis progenitor bearing somatic mutations in the phosphatidylinositol glycan-A (PIG-A) gene, which is involved in the synthesis of the glycosylphosphatidylinositol (GPI) anchor [1 , 2 ]. A clonal population not expressing molecules needing a GPI anchor to be mounted on the cell membrane rises from the PIG-A-defective progenitor in PNH patients. A defective clonal haematopoiesis, together with a residual polyclonal haematopoiesis, develops through several lineages and accounts for the mixed (GPI+ and GPI–) phenotype, commonly present in peripheral blood of PNH patients [3 , 4 ].

Hemolytic anemia, thrombophylia, and cytopenia are clinical hallmarks of PNH [1 2 3 4 5 ]. The lack of the GPI-linked molecules CD55 (decay-accelerating factor) and CD59 (membrane inhibitor of reactive lysis), involved in the protection of red cells from the lytic attack of activated complement fractions [6 ], causes a chronic hemolysis with capricious exacerbations. Platelet surface activation by small amounts of complement and urokinase plasminogen activator receptor deficiency [7 ] might explain thrombophylia; however, the origin of the underlying bone marrow failure is still unknown [8 9 10 ].

GPI-defective clones have been identified in healthy individuals [11 ] and pig-a knockout (KO) chimeric mice are unable to mimic a PNH syndrome [12 , 13 ]. These observations strongly suggest that an isolated PIG-A mutation is insufficient to account for the clonal dominance of the GPI-defective clone and for the development of PNH. The possibility that some extrinsic, selective pressure could favor the preferential expansion of the GPI-defective clone has been hypothesized [8 9 10 11 12 13 14 ]. In this context, the absence of GPI-linked proteins might provide the GPI-defective clone with a survival advantage in a background that impairs normal haematopoiesis.

A relationship between PNH and aplastic anemia (AA), whose autoimmune origin is generally accepted, has been reported [15 ]. Immune-suppressive treatments have been observed to be effective in PNH as well as in AA [16 , 17 ]. The sequential occurrence of a clinical onset of PNH in AA patients, the evidence of GPI-defective clones in AA [18 ], and recent evidences of an oligoclonal T cell repertoire in PNH patients [19 , 20 ] strongly support the involvement of T cell-mediated mechanisms in the pathogenesis of PNH. In this regard, a general perturbation of the biological mechanisms underlying the control of T cell homeostasis and the function of autoreactive T cell clones can be hypothesized.

T cells require additional signals, besides that generated by the T cell receptor (TCR) complex, to decide whether and how to respond to antigens. Moreover, the interactions between costimulatory ligands and their receptors are critical for activation of T lymphocytes, prevention of tolerance and development of T cell-dependent immunity [21 22 23 ].

CD40-dependent costimulatory pathways were observed to orchestrate the response of T cells during their development as well as during their encounter with the antigen [24 25 26 27 ]. Murine and human forms of a ligand for CD40, namely CD154, were cloned and found to be a membrane glycoprotein with a molecular weight of ~39 kDa induced on T cells following activation [28 ]. In normal T cells, the expression of CD154 is tightly regulated [29 ]. Indeed, a transient surface up-regulation of the CD154 molecule after 6–8 h from TCR triggering is accompanied by a rapid down-modulation of the structure.

The analysis of GPI-defective lymphocyte functions in PNH patients is still controversial. pig-a gene KO chimeric mice revealed normal TCR-dependent immune responses by GPI-defective T lymphocytes [30 ]. In contrast, alterations in T cell activation have been described by using enzymatic cleavage [31 , 32 ] or antisense inhibition of GPI-linked molecules in murine models [33 ]. Moreover, defects in T cell memory phenotype [34 ], lectin-dependent T cell proliferation [35 ] and TCR-dependent signaling [36 ] have been described in GPI-defective lymphocytes in PNH. Notably, a functional analysis of GPI+ T cell compartment in PNH patients is still lacking.

To address the biological mechanisms underlying PNH pathogenesis, our study analyzed the functional effectiveness of the GPI– and GPI+ T cell compartments in three PNH patients showing a marked cytopenia. This clinical feature, suggesting an active bone marrow failure condition, is expected to provide a useful model to investigate the potential involvement of T cell-mediated processes in PNH pathogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and controls
Three PNH patients, two males and one female, aged 30–40 years, were enrolled in the study (Table 1 ). PNH diagnosis was documented in all cases by flow cytometry analysis of peripheral blood, using labeled monoclonal antibodies (mAb) against GPI-linked molecules, as described [37 , 38 ]. All patients had a percentage of GPI-defective T lymphocytes >15%, as detected by double-labeling with anti-CD3 and anti-CD48 mAb and showed a marked cytopenia. None of the enrolled patients was receiving any medical treatment along the study. Patients 1 and 3 received transfusional therapy at least 3 weeks before the blood sample collection used for the experiments. Informed consent was obtained from individual patients before each blood sample collection. Ten sex- and age-matched, healthy donors were used as controls.


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  		Table 1. Patient Characteristics

 
mAb, immunofluorescence, and flow cytometry
Fluorescein isothiocyanate (FITC), phycoerythrin (PE), cychrome, and allophycocyanin-labeled mAb against CD3, CD4, CD25, CD45, CD48, CD54, human leukocyte antigen (HLA)-DR, CD154, interferon-{gamma} (IFN-{gamma}), interleukin (IL)-4, and isotype-matched controls were purchased from BD PharMingen (San Jose, CA). The PE-labeled CD48 mAb was purchased from Serotec Ltd. (London, UK).

To analyze the production of IFN-{gamma} and IL-4, intracellular staining with the specific mAb was performed by using the fixing/permeabilization kit purchased from Caltag (Burlingame, CA) following the manufacturer’s instructions. To avoid extracellular cytokine export, the cultures were performed in the presence of 5 µg/ml brefeldin-A (Sigma-Aldrich, St. Louis, MO), as described [39 ].

All phenotypes referred to flow cytometry analysis of the lymphocyte population gated by using forward-scatter and side-scatter parameters, as well as CD45 labeling. Flow cytometry and data analysis were performed by using a two-laser-equipped FACSCalibur apparatus and the CellQuest analysis software (Becton Dickinson, San Jose, CA).

Analysis of lymphocyte proliferation
Peripheral blood mononuclear cells (PBMC) were isolated from patients and controls by centrifugation of peripheral blood on Lymphoprep cushion (Nycomed, Oslo, Norway) gradient.

PBMC (1x106/ml) were cultured in 24-well, flat-bottomed plates (Falcon) with anti-CD3 mAb CLB-CD3/E, a gift of Dr. René van Lier (Central Laboratory of the Blood Transfusion Service, Amsterdam), phorbol 12-myristate 13-acetate (PMA), and ionomycin, all purchased from Sigma-Aldrich Italia (Milan).

To analyze the proliferation of GPI+ and GPI– T lymphocytes, PBMC were labeled with 5, 6-carboxyfluorescein-diacetate-succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) before the culture with the anti-CD3 CLB-CD3/E mAb. This technique has been described already as a reliable replacement of 3H thymidine incorporation for the evaluation of lymphocyte proliferation [40 ]. Indeed, CFSE fluorochrome spontaneously and irreversibly couples to intracellular proteins and is equally distributed to the daughter cells after mitosis. Proliferating cells can be tracked by flow cytometry, based on the sequential loss of fluorescence intensity [41 ]. Furthermore, multiparametric flow cytometry analysis allows the simultaneous assessment of normal and GPI-defective T cell subsets within the dividing cell population, also monitoring their phenotype changes associated with activation and cell division. All tests were performed in the presence of RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (Gibco, Grand Island, NY).

Statistical analysis
The statistical analysis for P calculation was performed by using Student’s t-test. Results were considered significant when a Pvalue <0.05 was obtained.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TCR-dependent T cell activation shows alterations in the GPI– and GPI+ compartments in PNH patients
The analysis of 3H thymidine incorporation after 72 h culture in the presence of anti-CD3, phytohemagglutinin, and PMA + ionomycin did not reveal significant differences in the proliferation levels of the T lymphocytes obtained from PNH patients, as compared with the age–sex-matched, healthy control group (data not shown).

To focus on a comparative analysis between GPI+ and GPI– T cells, we used a CFSE-labeling technique combined with multiparametric flow cytometry analysis. The deficiency of the GPI-linked CD48 molecule on defective T lymphocytes allowed a precise identification of the GPI– and GPI+ T cell subsets in the PNH-derived blood samples, maintaining the biological complexity of PNH and avoiding any functional interference as a result of the separation procedures. Figure 1 shows the results obtained by CD3 triggering of the two cell compartments in one representative PNH patient after 72 h of incubation with the anti-CD3 CLB-CD3/E mAb. This antibody overcomes the need for cross-linking by Fc receptors [42 ] as the GPI-linked CD16 molecule known to be defective in PNH. As shown, a significant impairment of the proliferative response of the GPI-defective (CD48–) T cell compartment was revealed in the representative PNH patient. By contrast, the percentage of proliferating CD3+CD48+ T lymphocytes was increased significantly in comparison with the CD48– counterpart as well as with the healthy control T lymphocytes (Fig. 1A) . Notably, 35% of CD3+CD48+ healthy control-derived cells (from 35% to 54% in the control group) underwent no more than one cell division as compared with 24% (from 23% to 38%) of the CD3+CD48+ and 56% (from 56% to 72%) of the CD3+CD48– from the PNH patient. In addition, 61% of the healthy control cells (from 39% to 61% in the control group), 70% (from 54% to 70%) of the CD3+CD48+ and 41% (from 28% to 41%) of the CD3+CD48– from the PNH patient were shown as having undergone at least two cell divisions. This difference is statistically significant when comparing the results obtained in all the recruited PNH patients with the group of 10 age–sex-matched, healthy controls for the more proliferating (P<0.05) and the less proliferating fractions (P<0.05). Similar data were obtained by analyzing the ability of the GPI-defective T lymphocytes to up-regulate CD25, HLA-DR, as well as CD54 (intercellular adhesion molecule-1) molecules in comparison with the GPI+ counterpart and the healthy control-derived T cell population (Fig. 1B) . Table 2 summarizes the results obtained in all the tested PNH patients for proliferation levels and CD25 surface expression after 72 h of incubation in the presence of anti-CD3 mAb. The results are referred to as the mean value obtained in the three concordant experiments performed for each single individual. The reference values obtained in the healthy control group, divided in male and female subgroups, also reported the statistical analysis. As shown, in the three patients, a mean of 35 ± 6, 31 ± 5, and 29 ± 9 CD3+CD48– cells underwent at least two cell divisions after CD3 triggering, as compared with the 70 ± 11, 68 ± 9, and 62 ± 6 of the CD3+CD48+ counterpart. These differences have been found significant (P<0.05) also in comparison with the reference values obtained in the control group, considered as a whole or with the relative homologous male or female subgroups. Similar data were referred to for CD25 expression. The presence of normal, accessory cells, provided by using PBMC from healthy donors, was unable to modify the impaired, TCR-dependent activation of PNH GPI-defective T cells in CD3 triggering experiments (data not shown).



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  		Figure 1. Analysis of TCR-dependent activation of the GPI+ and GPI– T cell compartments in PNH patients. PBMC from one representative healthy control and one PNH patient were collected and analyzed after 72 h of incubation with anti-CD3 mAb. (A) The CFSE-staining profiles as a measure of lymphocyte proliferative effectiveness (see Materials and Methods). GPI+ and GPI– T cells were gated on FITC anti-CD48 and cychrome anti-CD3 mAb fluorescences (R1 and R2, respectively). A defective proliferation of the CD3+CD48– subset can be observed. Indeed, 56% of cells undergo none or only one replication cycle in comparison with 24% of the CD3+CD48+ population and 35% of the CD3+CD48+ healthy control-derived counterpart. Notably, a significant increased proliferation of the CD3+CD48+ lymphocytes from PNH patients in comparison with the CD3+CD48+ healthy control-derived subset can be observed. Indeed, 70% of CD3+CD48+ cells from PNH patients undergo two or more replication cycles in comparison with 61% of the CD3+CD48+ healthy control-derived cells (P<0.05). (B) The analysis of CD25, HLA-DR, and CD54 surface levels, evaluated as mean fluorescence intensity (MFI) of the staining profiles obtained from the above populations. Bold lines indicate co-staining in the presence of the PE-labeled anti-CD25, HLA-DR, and CD54 mAb; dotted lines refer to the staining in the presence of the isotype control mAb. A significant decrease of all parameters was revealed in the CD3+CD48– population. Notably, increased expression of all these molecules was observed in the GPI+ T cell compartment from the PNH patient. Data are representative of one of three independent experiments and were confirmed in all the PNH patients recruited in the study.

 

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  		Table 2. Comparative Analysis of Proliferation Levels and CD25 Expression in PNH Patients and Healthy Controls

 
The comparison between PNH GPI+ T cells and healthy donor lymphocytes revealed a significant alteration in all the parameters considered. Indeed, proliferation, CD25, CD54, and HLA-DR up-regulation were increased significantly.

To address the cytokine production in PNH patients, we analyzed IFN-{gamma} and IL-4 production after PMA + ionomycin activation. This approach has been described as useful in the study of an established cytokine profile in human models [43 ]. Figure 2 shows that a severe impairment of IFN-{gamma} production can be revealed in the GPI-defective T cells after a 6 h culture in the presence of PMA + ionomycin. A slight but consistent decrease of IL-4 production was also observed in the GPI-defective T lymphocytes, confirming their impaired activation ability (not shown). By contrast, the comparison of the GPI+ T cells from PNH patients with the healthy control population revealed a significant increase (P<0.05) in their IFN-{gamma} production.



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  		Figure 2. Flow cytometry analysis of IFN-{gamma} production by GPI+ and GPI– T lymphocytes from a PNH patient. Plots show the percentage of anti-IFN-{gamma} mAb-stained cells incubated with medium alone or with PMA + ionomycin. The GPI+ and GPI– CD3 lymphocyte compartment was identified by gating on PE anti-CD48 and cychrome anti-CD3 mAb fluorescences (R1 and R2, respectively). As shown, a significant decrease in the intracellular cytokine level was observed in the GPI-defective population. Data are representative of one of three independent experiments and were confirmed in all the PNH patients recruited in the study.

 
TCR-triggered CD4+CD48+ lymphocytes from PNH patients show functional persistence of surface CD154 expression
To investigate more deeply the alterations observed in the T cell functions of PNH patients, we addressed the study of their CD40-dependent pathways. With this aim, we analyzed the kinetics of CD154 induction occurring in GPI– and GPI+ T lymphocytes from PNH patients. As shown in Figure 3 , after 8 h of incubation with anti-CD3 CLB-CD3/E mAb, a significant up-regulation of the CD154 molecule was observed in healthy control CD4+ lymphocytes, as well as in the GPI+ and GPI-defective compartments of PNH patients (Fig. 3) . CD48+CD4+ cells, which represent the GPI+ population in PNH, showed a significant increase in their surface CD154 expression when compared with the GPI-defective CD4+ counterpart and the CD4+ cells from the healthy controls (Fig. 3) .



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  		Figure 3. Analysis of TCR-dependent CD154 expression on the GPI+ and GPI– CD4+ populations from two PNH patients compared with healthy donors. The plots refer to the staining of CD48+CD4+ and CD48–CD4+ (R1 and R2, respectively) with anti-CD154 mAb after 8 h of incubation in the presence of anti-CD3 mAb. A significant impairment in CD154 induction was observed in the GPI-defective population, as compared with the GPI+ counterpart. Notably, an increased surface level of this molecule was observed in the GPI+ CD4 population from the PNH patients in comparison with the healthy controls. Data are representative of one of four concordant experiments.

 
To assess the functional relevance of the increased CD154 up-regulation on the surface of anti-CD3-treated T cells from PNH patients, we evaluated the ability of these populations to up-regulate the CD23 molecule on the surface of B lymphocytes from healthy controls. Indeed, the up-regulation of this molecule has been described as a relevant target for CD40 triggering [44 , 45 ]. Therefore, we cocultured normal PBMC in the presence of irradiated healthy donor or PNH-derived PBMC, previously incubated with anti-CD3 mAb for 8 or 48 h. After overnight culture, the level of CD23 was measured on B lymphocytes, as identified by CD19 co-staining. As shown (Fig. 4A ), a significantly increased up-regulation of this molecule was observed in the presence of PNH as compared with control PBMC collected after 8 h from CD3 triggering (P<0.05). Notably, also, the medium-cultured PNH cells, but not the healthy control, showed significant ability to up-regulate the CD23 molecule on B lymphocytes. Moreover, lymphocytes from PNH patients, collected after 48 h of anti-CD3 incubation (Fig. 4B) , revealed a persistent ability to up-regulate CD23. The healthy control cells, collected after the same culture period, were substantially unable to modify CD23 expression on the surface of the target population (P<0.05). The results were confirmed by using the CD48+CD4+ enriched lymphocyte fraction (not shown). The observed effects were partially (from 30% to 50%) inhibited by anti-CD40 mAb treatment of the target population, demonstrating the involvement of CD40-dependent pathways (data not shown).



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  		Figure 4. CD3-triggered PBMC from PNH patients are able to up-regulate CD23 surface levels on control B cells. The graph refers to the level of CD23 antigen expression on control B lymphocytes, identified by costaining with anti-CD19 mAb, after overnight incubation in the presence of 30 Gy-irradiated PNH (shaded columns) or healthy control (open columns) PBMC pretreated with anti-CD3 mAb or medium, as indicated. PNH cells significantly up-regulated the CD23 molecule on control B cells after 8 (A) and 48 h (B) of CD3 triggering. Notably, healthy control-derived PBMC showed a significant ability to up-regulate B lymphocyte CD23 only after 8 h of anti-CD3 incubation. Data refer to MFI levels obtained in three independent experiments. Standard deviations are depicted on the top of each bar. *, Statistical significant differences (P<0.05) in comparison with the normal control group.

 
To assess whether the increased CD23 up-regulation on B cells could correlate with the expression of CD154 on the lymphocytes of PNH patients, we analyzed CD154 kinetic expression on the CD4+ population after incubation with the anti-CD3 mAb for 8, 24, and 48 h. As shown in Figure 5 , a significant persistence of the CD154 molecule on the CD48+CD4+ cells from PNH patients was observed after 24 and 48 h from anti-CD3 treatment. In addition, a slight but consistent expression of CD154 was observed after 48 h treatment with medium in the CD48+CD4+ cells from the PNH patients (not shown).



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  		Figure 5. Kinetics of TCR-dependent CD154 induction on CD4+CD48+ lymphocytes of one representative healthy control and two PNH patients. Staining profile of anti-CD154 mAb on CD4+CD48+ lymphocytes. Plain and dotted lines indicate anti-CD3 and medium incubation, respectively. The CD154 up-regulation after 8 h is accompanied by a consistent persistence of CD154 surface expression after 24 and 48 h of anti-CD3 incubation in the PNH patients but not in the representative healthy control. Data refer to one of three concordant experiments and were confirmed in all the PNH patients recruited for the study.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study reveals the occurrence of significant functional alterations in the GPI+ as well as the GPI-defective T cell compartments in three cytopenic PNH patients. A significant increase and the functional persistence of CD154 on TCR-triggered CD4+CD48+ lymphocytes from PNH patients were observed. Severe defects in TCR-dependent proliferation, CD25, CD54, and HLA-DR surface expression in the GPI-defective T cell population were also demonstrated. By contrast, a significant increase in the same parameters was revealed in the GPI+ T cells of PNH patients, as compared with the healthy donors.

TCR-dependent proliferation, expression of activation molecules and cytokine production were all significantly decreased in GPI-defective T cells. In this regard, the inability of healthy donor monocytes to overcome such impairment strongly supports the occurrence of some defects in the TCR-dependent activation machinery of the GPI-defective T lymphocytes. These data confirm and extend previous reports showing severe dysregulation of the immune system in a pig-a–/Rag–/– chimeric murine model [46 ], impaired lectin-triggered proliferation [31 32 33 , 35 ], TCR-dependent signaling in GPI– T cells [36 ] and defects of the monocyte/granulocyte GPI-defective subsets [47 , 48 ] in PNH patients. The divergence with the competence of the GPI-defective T lymphocyte population referred to by Takahama et al. [30 ] could be a result of the inability of the Cre P-lox approach to mimic the PNH pathophysiology [9 , 13 , 14 ].

Several evidences suggest that lipid rafts regulate dynamic interactions between T cell signaling structures [49 ]. These membrane microdomains harbor hydrophobic lipid-membrane proteins that provide saturated acyl/alkyl chains, as represented by proteins with a GPI anchor, in the outer leaflet of the plasma membrane [50 ]. In addition, most inner leaflet-anchored raft proteins, as represented by the Src-related tyrosine kinases, carry a C16-saturated palmitoyl fatty acid, which inserts into the lipid bilayer. Thus, raft microdomains could represent key platforms that couple events outside the cell with signaling pathways inside the cell, also through GPI-linked, protein-dependent interactions [50 51 52 53 ]. Therefore, the involvement of the lack of GPI-linked proteins, observed in PNH, in the pathogenesis of the impaired TCR-triggered activation could be hypothesized.

Our observations, as a whole, strongly support the possibility that GPI-defective clones have to evolve under permissive conditions in a background that impairs normal haematopoiesis. Moreover, a number of data indicate that nonmutated PIG-A+ stem cells are markedly impaired in their growth in PNH patients [54 ].

The possibility that immune-mediated mechanisms might account for the clonal dominance of the PIG-A mutation and for the development of PNH has been already suggested [8 , 9 , 13 , 14 , 19 , 20 ]. In this regard, PNH patients showing a marked cytopenia, likely suggesting the occurrence of an active bone marrow failure process, are expected to represent optimal models to address such hypothesis. Indeed, in these patients, a possible defective regulation of T cell homeostasis, also affecting the functional asset of T lymphocytes, could be consequent to a perturbation of immune tolerance. Such condition could promote the generation of hyperactivated T cell effectors accounting for the increased response to TCR-triggering by us observed in vitro. In our experimental model, the use of a multiparametric immune fluorescence technique allows the analysis of the GPI+ compartment, maintaining the biological complexity of PNH and avoiding any functional interference as a result of the separation procedures. Therefore, the increased activation level reached ex vivo by the GPI+ T cell compartment of PNH patients might suggest the presence of a T cell population, which is altered even in vivo. In this context, the presence of a chronic stimulation, likely consequent to a defective control of activated complement components, as demonstrated for PNH platelets [7 ], cannot be ruled out.

Interactions between costimulatory ligands and their receptors are critical for activation of T lymphocytes, prevention of tolerance and development of T cell-dependent immunity. Moreover, only a few costimulatory molecules are expressed on T cells in a constitutive manner, whereas a majority is induced following cell activation subsequent to antigen recognition by TCR [21 , 23 , 29 ]. The CD40-dependent pathways have been described as relevant for the control of innate and adaptive immune responses and for the achievement and maintenance of immune tolerance [26 , 27 ]. Given the relevance of these pathways, a tightly regulated expression of CD154 could be necessary to maintain the antigen specificity of T cell activation.

CD23 up-regulation has been demonstrated already as a relevant target for CD154-dependent triggering on B cells [44 , 45 ]. Our data indicate that 8 h anti-CD3-treated lymphocytes from PNH patients showed increased ability to up-regulate surface CD23 levels on B cells, in comparison with the healthy control-derived cells. Moreover, we observed that 48 h anti-CD3-cultured lymphocytes from PNH patients but not from healthy controls are able to up-regulate such a molecule. These effects were significantly but not completely inhibited by anti-CD40 mAb target treatment. The observed CD154 persistence on 48 h anti-CD3-treated CD4+CD48+T cells from PNH patients is expected to likely account for such effect. The observation that 8 h medium-treated CD48+CD4+ lymphocytes from PNH patients show a significant ability to up-regulate CD23 surface levels in the absence of detectable CD154 expression points to the involvement of CD40-dependent and -independent pathways in mediating such effects.

Altered persistent expression of CD154, the CD40 ligand, has been described in pathological conditions characterized by a defective control of autoreactive clones [55 , 56 ]. In this context, the presence of a skewed T cell repertoire in PNH [19 , 20 ] and aplastic anemia patients [20 , 57 ] has been already associated with a possible dysregulation of the biological mechanisms underlying tolerance maintenance. Therefore, the persistence of a functional CD154 molecule on the TCR-triggered CD48+CD4+ lymphocytes could be suggested as relevant for the possible immune-mediated pathogenesis of the disease in PNH.


   ACKNOWLEDGEMENTS
 
This study was supported by the Italian Ministero dell’Università e Ricerca Scientifica, PRIN Grant 2002. F. A. and G. R. contributed equally to this study. We are in debt to our patients for their generous collaboration to the study. We also thank Dr. G. Matarese for helpful discussion and critical reading of the manuscript.

Received January 17, 2005; revised February 25, 2005; accepted March 9, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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