Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0904500 on February 22, 2005

Published online before print February 22, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904500v1
77/5/661    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martín-Fernández, J. M.
Right arrow Articles by Regueiro, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martín-Fernández, J. M.
Right arrow Articles by Regueiro, J. R.
(Journal of Leukocyte Biology. 2005;77:661-668.)
© 2005 by Society for Leukocyte Biology

Herpesvirus saimiri-transformed CD8+ T cells as a tool to study Chediak-Higashi syndrome cytolytic lymphocytes

José M. Martín-Fernández*, Juan A. Cabanillas*, Miguel Rivero-Carmena*, Esther Lacasa{dagger}, Julián Pardo{dagger}, Alberto Anel{dagger}, Pedro R. Ramírez-Duque{ddagger}, Fernando Merino§, Carlos Rodríguez-Gallego and José R. Regueiro*,1

* Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain;
{dagger} Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain;
{ddagger} Hematología, Hospital Central, Tachira, Venezuela;
§ Fisiología, Facultad de Medicina y Odontología, Universidad del País Vasco, Bilbao, Spain; and
Inmunología, Hospital Universitario de Gran Canaria Doctor Negrín, Las Palmas, Spain

1 Correspondence: Inmunología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. E-mail: regueiro{at}med.ucm.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytolytic CD8+ T lymphocytes are the main cell type involved in the fatal lymphoproliferative-accelerated phase of the Chediak-Higashi syndrome (CHS). To generate a cellular tool to study the defects of this T cell subset in vitro, we have used Herpesvirus saimiri, a lymphotropic virus that transforms human T lymphocytes into extended growth and in addition, endows them with natural killer (NK) features. Transformed CHS CD8+ T cells were generated and characterized in comparison with healthy controls. The results showed that transformed CHS T cells maintained the defects described in primary CHS lymphocytes, such as giant secretory lysosomes and impaired NK and T cell receptor/CD3-induced, perforin-mediated cytolytic activity [which, however, could be restored after extended culture in the presence of interleukin-2 (IL-2)]. Upon activation with phorbol ester plus calcium ionophore or upon extended culture with IL-2, transformed CHS T cells showed normal, perforin-independent plasma membrane CD178/CD95L/FasL-mediated cytolytic activity but negligible secretion of microvesicle-bound CD95L. Transformed (and primary) CHS T cells were otherwise normal for cytolysis-independent activation functions, such as proliferation, surface expression of several activation markers including major histocompatibility complex class II, and cytokine or surface activation-marker induction. Therefore, the CHS protein [CHS1/LYST (for lysosomal traffic regulator)] can be dispensable for certain NK and T cell cytolytic activities of activated CHS CD8+ T lymphocytes, but it seems to be required for microvesicle secretion of CD95L. We conclude that transformed CHS T cells may be useful as a tool to study in vitro the relative role of CHS1/LYST in NK and T lymphocyte cytolysis and antigen presentation.

Key Words: T lymphocyte • immunodeficiency diseases • cytolytic cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chediak-Higashi syndrome (CHS) is a lethal, autosomal, recessive human disorder characterized by impaired immunity and pigmentation [1 ]. The disease is caused by mutations in CHS1, which encodes a large, ubiquitous phosphoprotein termed CHS1 or LYST (for lysosomal traffic regulator) [2 , 3 ]. The biological role of CHS1 is unknown [4 ], but it is required for the normal membrane targeting of secretory lysosomes, specialized organelles present in all cells but critical for the normal function of leukocytes and melanocytes. These cell types share part of the molecular machinery involved in the regulation of secretory lysosome metabolism, causing the coexistence or independence of immunodeficiency (ID) and albinism in certain genetic disorders [1 ]. The lack of CHS1 impairs immune cytolysis by T and natural killer (NK) lymphocytes, as their secretory lysosomes (termed lytic granules) fuse and enlarge, and their contents cannot be released properly on their targets. As a consequence, it is believed that cytolytic responses against viral infections [notably Epstein-Barr virus (EBV)] are not effective or cannot be extinguished by fratricide and cause uncontrolled T cell activation, lymphoproliferation, and tissue infiltration by activated CD8+ T cells [1 , 5 ].

Current in vitro models of CHS are restricted to interleukin-2 (IL-2)-dependent T cells lines, which may or may not display NK activity [6 7 8 ]. Herpesvirus saimiri (HVS) strain C488 is a common lymphotropic virus of squirrel monkeys, which has been used to transform into extended growth human mature T lymphocytes. Transformed cells remain IL-2-dependent and acquire a constitutively activated [major histocompatibility (MHC) class II+, CD25+, CD69+, CD45R0+] T lymphocyte phenotype (CD3+, CD2+, CD7+, and CD4+ or CD8+) [9 ]. In addition, the transformed T cells display NK-like features, such as CD56 expression and lysis of the MHC class I-negative cell lines K562 [10 ], L721.221, and C1R, which can be inhibited by CD1d [11 ]. In contrast to untransformed T cells, they are antigen- and mitogen-independent, they can express CD178/CD95L/FasL and CD30 [12 ], and they acquire a functional T helper cell type 1 profile [9 ].

It has been shown [13 14 15 16 17 18 19 20 21 22 ] that transformed T cells from congenital ID preserve the original defects. We therefore reasoned that transformed T cells from CHS patients might be useful as a tool to study the role of CHS1/LYST in cytolytic lymphocytes. To this end, we transformed T cells from CHS patients that lack a functional CHS1 protein and compared them to transformed T cells from healthy individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and controls
Four patients, from Venezuela (CH1, -6, and -8) or Spain (CHC), with well-documented CHS, according to the diagnostic criteria of the International Union of Immunological Societies Scientific Group for Primary Immunodeficiency Diseases [23 ], two healthy female obligate carriers (CH3 and -9), four healthy family members (CH2, -4, -5, -7), and six unrelated, healthy controls (CTC5, CTLT, CTDRD, CC1–3) were included in the study. Endemic CHS has been reported previously in the Tachira State of Venezuela [24 ]. Informed consent was obtained from all individuals. In addition, five X-linked agammaglobulinemia patients (XLA1–5) were included as an irrelevant ID control. Mutation analysis by polymerase chain reaction and simultaneous, single-stranded sequence polymorphism heteroduplex assays in all patients and several healthy family members failed to detect pathological mutations in CHS1, as reported in almost half of the patients in the larger series, although a K590N heterozygous polymorphism was detected in CH2 [25 ].

Cell lines
HVS-transformed T cell lines were derived from peripheral blood lymphocytes (PBL) as described [26 ]. PBL (2x106) in 1 ml were exposed (or not) to 1 ml infective HVS supernatant (final concentration, 1x106 cells/ml) in 24-well plates (Costar, Cambridge, MA) in the presence or absence of 1 µg/ml phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI). Thereafter, cells were grown in Panserin/RPMI (RPMI-1640 medium from Biochrom, Berlin, Germany, and Panserin medium from Pan Biotech, Aidenbach, Germany) supplemented medium with 10% fetal calf serum (FCS; Flow Laboratories, Rockville, MD), 1% L-glutamine (Gibco-BRL Paisley, UK), and 1% antibiotic and 50 IU/ml human recombinant (hr)IL-2 [kindly provided by Frederick Cancer Research and Development Center, National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD]. A transformed phenotype was indicated by the death of unexposed HVS cultures as compared with the sustained growth and T lymphoblast cell morphology of HVS-exposed cultures as described [27 ]. HVS-exposed T cells had been maintained in long-term culture for more than 8 months when the experiments reported here were performed.

Phenotypical analyses
The following conjugated monoclonal antibodies (mAb) were used for cytofluorometric analyses: Leu4 (anti-CD3), Leu-3a (anti-CD4), Leu 2a (anti-CD8{alpha}), Leu23 (anti-CD69), FastImmune anti-hIL-2, FastImmune anti-human tumor necrosis factor {alpha} (TNF-{alpha}), and FastImmune anti-human interferon-{gamma} (IFN-{gamma}) from Becton Dickinson (Mountain View, CA); mouse immunoglobulin G (used as negative control) from Caltag (Burlingame, CA); CD154 from PharMingen (San Diego, CA); human leukocyte antigen-DR, CD2, CD25, and CD71 from Caltag Laboratories (Burlingame, CA). For single- and two-color immunofluorescence, 1 x 106 cells were incubated for 30 min at 4°C with appropriate fluorescein isothiocyanate- or phycoerythrin-conjugated mAb in phosphate-buffered saline (PBS)/EDTA buffer containing 1% FCS. After two washes with PBS buffer, the cells were analyzed in an Epics Elite Analyzer cytofluorometer (Coulter, Hialeah, FL). Isotype-matched, irrelevant antibodies were used to define background fluorescence [15 ]. Data were collected on 104 viable cells as determined by electronic gating from a tight region around the lymphocyte population on the forward-scatter/side-scatter parameters dot-plot. For histochemical analysis, cytospin slides (400 rpm, 4 min, room temperature) were stained with haemotoxylin/eosin.

Cytolysis assays
NK lytic activity was measured using a standard 51Cr release assay with K562 as target cells as described previously [26 ]. Briefly, transformed effector cells were IL-2-starved (or not, 40 IU/ml) for 1 week before incubation in triplicate at different effector:target (E:T) cell ratios (from 1:1 to 50:1) with 51Cr-labeled K562 targets (5x103 cells/well) in U-bottom, 96-well plates. For (perforin-mediated) CD3-redirected cytolysis, effector cells were cultured for 4 h with Fas/CD95–, FcR+ 3H-thymidine-labeled mouse leukemia L1210 cells in the presence or absence of 5 µg/ml mouse anti-human CD3 mAb UCHT-1, essentially as described [28 ]. For Fas/CD95-mediated cytolysis, effector cells were stimulated (or not) for 3 h with 10 ng/ml phorbol myristate acetate (PMA; Sigma Chemical Co., St. Louis, MO) plus 600 nM ionomycin (Sigma Chemical Co.) in the presence or absence of 1 µg/ml cycloheximide (CHX). Cells were then cultured for 16 h with 3H-thymidine-labeled, Fas-transfected murine L1210 cells (termed L1210Fas) or as a negative control, untransfected L1210 cells. Experiments were done in the presence of 1 mM EGTA plus 1.5 mM MgCl2 to block perforin-mediated cytolysis. For cell-free cytolysis, supernatants of effector cells stimulated as above were collected and tested for growth inhibition of L1210 and L1210Fas by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction method [29 ]. Lysis was measured as median cpm in a {gamma}- or ß-counter (Packard, Meridien, CT). For spontaneous and maximum lysis controls, target cells were incubated alone or in 2% sodium dodecyl sulfate, respectively. The percentage of specific lysis was calculated as follows: experimental lysis – spontaneous lysis x 100/maximum lysis – spontaneous lysis. Spontaneous lysis was typically below 10%.

Proliferation assays
Proliferation was measured by standard 3H-thymidine uptake assays [30 ]. Briefly, transformed cells were IL-2-starved for 7 days before stimulation with immobilized anti-CD3 mAb (IOT3b, Immunotech, Marseille, France) at a concentration of 1 µg/ml, PMA (10 ng/ml) plus ionomycin (750 ng/ml), or hrIL-2 (50 IU/ml, kindly provided by Frederick Cancer Research and Development Center, NCI, NIH). PBL (80,000/well) were resuspended in RPMI 1640 (Cambrex Bio Science, Walkersville, MD), supplemented with 10% FCS (ICN Biomedicals Inc., Irvine, CA). Cells were stimulated with 12.5 ng/ml soluble anti-CD3 (HIT3a, Becton Dickinson), 50 IU/ml hrIL-2 (R&D Systems, Minneapolis, MN), or 10 ng/ml PMA (Sigma Chemical Co.) plus 2 µg/ml ionomycin (Sigma Chemical Co.). Cells were cultured in U-bottom, 96-well plates (0.2 ml/well) for 72 h (PBL) or 48 h (HVS-transformed cells) before adding 10 µl/well 3H-thymidine (1 µCi/well, Amersham, Buckinghamshire, UK) overnight, harvested onto glass fiber filters, and analyzed in a ß-counter (Wallac, Turku, Finland, or Packard, respectively).

Induction of cytokines
To analyze intracellular cytokine induction, transformed cells were collected, washed twice in PBS, and starved overnight in Panserin/RPMI medium supplemented with 2% FCS. Cells were then resuspended at 2.5 x 106 cells/ml in 96-well plates and stimulated for 9 h (peak production time-point) with or without 1 µg/ml immobilized anti-CD3 mAb (IOT3b) or in the presence of 10 ng/ml PMA plus 750 ng/ml ionomycin. For the last 2 h, 10 µg/ml Brefeldin A (Sigma Chemical Co.) was added to the cultures to block secretion. Cells were harvested, washed twice in PBS buffer, and fixed with 500 µl 1% formaldehyde in PBS for 10 min at room temperature. Then, the cells were stained intracellularly for cytokine content as described [15 ]. In some experiments, supernatants from cells cultured and stimulated as above (Brefeldin treatment excluded) were used to analyze cytokine secretion as indicated below for PBL.

To analyze cytokine secretion by PBL, whole blood was diluted 1:1 in RPMI 1640 (Cambrex Bio Science) and activated with PHA (5 µg/ml, Sigma Chemical Co.), soluble anti-CD3 (25 ng/ml HIT3a, Becton Dickinson), or PMA (10 ng/ml, Sigma Chemical Co.) plus ionomycin (1 µg/ml, Sigma Chemical Co.). Serial dilutions of supernatants collected after 48 h of culture were used to determine cytokine levels (IL-2, IFN-{gamma}, TNF-{alpha}) by flow cytometry (FACSCalibur, Becton Dickinson) using the cytometric bead array system (Becton Dickinson) following the manufacturer’s protocols.

Induction of surface-activation molecules
To measure CD25, CD71, and CD154 (CD40L) induction after stimulation, cells were IL-2-starved overnight in Panserin/RPMI medium supplemented with 2% FCS. Cells were then resuspended at 2 x 105 cells/ml in 96-well plates in the absence of stimuli, in the presence of 1 µg/ml plastic-bound anti-CD3 mAb (IOT3b), or in the presence of 10 ng/ml PMA (Sigma Chemical Co.) plus 750 ng/ml ionomycin (Sigma Chemical Co.) and incubated for 9 h at 37°C. The 9-h time-point was chosen in kinetic experiments, as at that time, the difference between stimulated and unstimulated cells is largest [16 ]. Then, cells were washed twice in PBS, stained with specific mAb for 30 min at 4°C, washed twice in PBS, and analyzed by flow cytometry as described above.

Statistical analysis
Student’s t-tests were performed using SPSS (Chicago, IL) 11.5.1 statistical program software. Only P values below 0.05 were considered significant. Data are presented as mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypical characterization of transformed CHS T cells
PBL were incubated with infective HVS supernatant to transform them into extended growth. CHS and control, transformed T cells were then characterized comparatively. After exposure to HVS, 11 different transformed T cell lines were derived, three of them from patients and eight from healthy individuals (six family members, including obligate carriers, and two unrelated controls). All of them showed sustained growth by day 30 (doubling times ranged 2–3 days) and lymphoblastoid cell morphology, whereas the unexposed cultures were lost by day 40 (Fig. 1A ). Once established, the T cell lines displayed the typical activated (CD69+, CD25+, MHC class II+, CD71+) T/NK lymphocyte surface phenotype, and they were all CD2+ CD8+ T cell receptor (TCR){alpha}ß+ (Fig. 1B) . The histochemical analysis of the patients’ T cell lines revealed the structural CHS hallmark described in primary cells, namely, the presence of giant, secretory lysosomes, which are not observed in carriers or controls (Fig. 2 ).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 1. Transformed CHS T cell lines show normal growth and a CD8+-activated T cell surface phenotype. (A) PBL exposed (solid lines) or unexposed (dashed lines) to infective HVS supernatant were grown as indicated in Materials and Methods. Representative growth kinetics of a line from a CHS patient (CH1), an obligate carrier (CH3), and a healthy control (CTC5) are shown. Similar results were obtained with all other samples. (B) Representative surface phenotype of an established T cell line (more than 90 days in culture) from a CHS patient (CH8), an obligate carrier (CH9), and a healthy control (CTLT). Similar results were obtained with all other samples. For CD71 expression, see Figure 5 .

 


View larger version (132K):
[in this window]
[in a new window]
  		Figure 2. Transformed CHS T cell lines show giant secretory lysosomes. Cytospin slides were stained as indicated in Materials and Methods. Cells (35–65%) from CHS samples (CH1, -6, -8) but <10% from healthy individuals (CTC5 and CH9) contained giant lysosomes (arrows). Original magnification, 100x.

 
Cytolytic activity of transformed CHS CD8+ T cells
Primary T and NK lymphocytes from CHS patients show impaired cytolytic activity (reviewed in ref. [31 ]). It was important to show if transformed T cells preserved these functional features. Thus, we first tested the transformed T cell lines for spontaneous, NK-like cytotoxicity by a standard K562 51Cr release assay and for CD3-dependent (perforin-mediated) cytolysis of Fas-negative mouse L1210 cells. The results showed that despite transformation, patients’ T cell lines behaved as their primary counterparts, in that they were unable to exert normal cytolysis on the susceptible targets as compared with T cell lines from healthy controls or carriers (Fig. 3 , upper). However, activation by extended culture in the presence of exogenous IL-2 normalized the cytolytic activity of the CHS T cell lines (Fig. 3 , lower), as also described in primary cells [32 ].



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Transformed CHS T cell lines show impaired NK and CD3-dependent cytolysis, which can be restored by IL-2. Cell lines cultured in the absence (upper, CH1–3, 6–8, CTC5, CTDRD, CTLT) or in the presence of IL-2 for 1 week (lower, CH1–3, CTC5) were tested for NK cytolytic activity using K562 as targets (left) or for perforin-mediated, redirected cytolysis of L1210 cells using the mouse anti-CD3 mAb UCHT-1 (right; see Materials and Methods). Results are depicted as mean ± SD for different cell lines (left) or mean ± SD of at least three different experiments with two cell lines (CH1 and -2, right). In the absence of anti-CD3, lysis was always below 10% (not shown). P values were below 0.05 for patients versus controls or carriers only in the upper panels. {alpha}CD3, Anti-CD3.

 
In addition to perforin-dependent cytolysis, T and NK cells can exert, upon proper activation, CD95/Fas-mediated cytolysis through the expression of CD95L/FasL, which has been shown to be normal in IL-2-activated, primary CHS T cells [32 ]. This type of cytolytic activity may be selectively analyzed on CD95/Fas-transfected L1210 murine cells in the presence of EGTA (which blocks perforin-mediated cytolysis). We therefore compared CHS and normal, transformed T cells for CD95L/FasL-dependent cytolytic activity. The results showed that unstimulated, transformed CHS T cell lines (as well as controls) exerted little spontaneous cytolysis on the transfectants but were induced to do so by a 3-h PMA plus ionomycin stimulation or by extended culture with exogenous IL-2, reaching levels that were comparable with those of healthy controls (Fig. 4 , top and middle). The cytolytic activity induced by PMA + ionomycin stimulation was independent of de novo protein synthesis, as it was resistant to CHX treatment. Thus, activation induced the functional expression of preformed CD95L/FasL by transformed CHS and control T cells.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Transformed CHS T cell lines show normal, cell-mediated but not cell-free CD95L/FasL-dependent cytolysis. T cells from a patient (CH1) or a healthy relative (CH2) cultured in the absence (top) or in the presence (middle) of IL-2 for 1 week were stimulated (P+I) or not (basal) for 3 h and tested for cell-mediated, CD95/Fas-induced cytolytic activity in the presence or absence of CHX using CD95/Fas-transfected mouse L1210 cells as targets at a fixed 5:1 E:T ratio. P + I, PMA plus ionomycin. Lysis of untransfected L1210 cells was always below 15% (not shown). Supernatants from P + I-stimulated, transformed T cell lines were collected and tested for cell-free, CD95/Fas-induced growth inhibition of L1210 and L1210 Fas cells, as indicated in Materials and Methods (bottom). All results are the mean ± SD of at least three different experiments. P values were below 0.05 for patient versus control lines only in the bottom panel.

 
It has been reported that activated human T cell blasts can secrete preformed CD95L/FasL-loaded microvesicles with potent cytolytic activity [33 ]. The microvesicles originate from multivesicular bodies [34 ], although their relationship with secretory lysosomes has not been established. To analyze the relative contribution of microvesicles to the observed CD95/Fas-mediated cytolysis by transformed CHS T cells after activation, effector cell-free cytolysis was measured in the supernatants of the stimulated cultures by the MTT reduction test, using untransfected and CD95/Fas-transfected L1210 cells as targets. The results showed that transformed CHS T cells cannot release CD95L/FasL-loaded microvesicles (Fig. 4 , bottom), despite their normal, CD95/Fas-mediated cytolysis of susceptible targets (Fig. 4 , top and middle).

Activation features of transformed CHS CD8+ T cells
Deregulated T cell activation is present in CHS patients [5 ]. We therefore measured several T cell activation features in transformed (or fresh) CHS T cells in response to TCR/CD3-dependent and -independent stimuli.

First, their proliferative response to several mitogens was assayed and found to be similar to healthy controls or carriers (Fig. 5A , top left). Similar results were obtained in fresh CHS PBL (Fig. 5A , top right). Second, we evaluated intracellular cytokine induction (IFN-{gamma} and TNF-{alpha}) in response to membrane (anti-CD3) and transmembrane (PMA+ionomycin) stimuli. No differences were observed among normal, X-linked agammaglobulinemia (used as a further control), carrier, and CHS T cells for any of the stimuli (Fig. 5A , middle and bottom left). Again, similar results were obtained in fresh CHS PBL with these and other stimuli (Fig. 5A , middle and bottom right), confirming that transformation does not lead to functional changes in T cells for these assays. As the CHS mutation impairs certain secretory functions in affected cells, cytokine secretion was measured in transformed CHS T cells. The results showed that cytokine secretion was, if anything, higher than controls by this assay (Fig. 5B) .



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Transformed and fresh CHS T cells show normal proliferation and cytokine induction and secretion. (A) T cell lines (HVS, left) or fresh PBL (right) from CHS patients (CH1, CH8, CHC), XLA patients (XLA1–5), obligate carriers (CH3, -9), or healthy individuals (CTC5, CTDRD, CTLT, CH2, CC1–3) were stimulated as indicated, and 3H-thymidine incorporation (top) or cytokine content or secretion (middle and bottom) were measured as described in Materials and Methods. Results are depicted as mean ± SD for different control or XLA donors or as mean ± SD of the indicated patient or carrier cell lines when several experiments were performed (left panels). The actual value of each control cell line is included for further reference. {alpha}CD3 or CD3, Anti-CD3; P + I, PMA plus ionomycin; CPM, counts per minute. (B) Cytokine secretion was measured in transformed T cells from a patient (CH1) and a healthy relative (CH2) after stimulation, as indicated.

 
Activated CHS T cells show impaired secretion of their giant granules’ contents [8 ]. Proteins that traffic through secretory lysosomes may be affected and contribute to the lymphoproliferation observed in vivo, as described for cytotoxic T-lymphocyte antigen (CTLA)-4/CD152 [5 , 35 ]. Thus, we third screened for the induction of several surface proteins that might be relevant to lymphoproliferation or T cell activation (CD25, CD154/CD40L, and CD71). The results showed that they were normal, indicating that these proteins are not dependent on the secretory lysosome pathway for their surface induction (Fig. 6 ).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 6. Transformed CHS T cell lines show normal induction of surface-activation molecules. (A) T cell lines from CHS patients (CH1, -8), obligate carriers (CH3, -9), and healthy individuals (CTC5, CTDRD, CTLT, CH2, -4, -5) were stimulated as indicated, and the induction of several activation molecules was measured as mean fluorescence intensity (MFI). Results are depicted as mean ± SD for different control cell lines or mean ± SD of the indicated patient or carrier cell lines when several experiments were performed. The actual value of each control cell line is included for further reference. (B) Representative staining results for CD71.

 
Together, these results fit well with the accepted, selective role of CHS1/LYST in membrane targeting of specialized secretory lysosomes but not of other secretory vesicles carrying surface or soluble proteins through the direct exocytosis pathway, such as MHC class I or cytokines [36 ].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present report, we have shown that transformed CD8+ T lymphocytes from CHS patients are a useful tool to study in vitro the defects that may result in their observed, massive lymphoproliferation in vivo.

The biological role of CHS1/LYST remains to be determined. Despite the fact that it is ubiquitous, only leukocytes (particularly cytolytic lymphocytes) and melanocytes appear to be affected. Studies in T and NK cells are hampered by insufficient primary cells from patients and would benefit from a suitable in vitro model. Untransformed, CHS IL-2-dependent CD8+ T cell lines are scarce, short-lived, and antigen-dependent and in contrast to primary cells, show normal NK and lectin-dependent cytolysis [6 ]. The HVS-transformed CHS CD8+ T cell lines described here show a homogeneous phenotype and extended growth (Fig. 1) yet display the defects described in primary CHS PBL, such as giant lysosomes (Fig. 2) and impaired (perforin-dependent) NK and CD3-induced cytolytic activity, which are nevertheless restored by extended culture with exogenous IL-2 (Fig. 3 and refs. [37 , 38 ]). The mechanism whereby IL-2-activated, fresh CHS PBL secrete perforin is unclear, although they can also exert CD95L/FasL-dependent cytolysis [32 ]. We have shown that transformed CHS T cells show impaired, perforin-dependent cytolysis (Fig. 3) yet can be induced to exert normal, perforin-independent cytolysis (Fig. 4) . Thus, they may help to unravel this paradox.

The fatal lymphoproliferative syndrome in CHS is characterized by the peripheral expansion and tissue infiltration of activated (MHC class II+) CD8+ T cells. Two hypotheses have been proposed to explain this feature [1 ]: Impaired killing of infected cells would result in antigen persistence and sustained stimulation, and expansion of lymphocytes, or impaired fratricide would block self-down-regulation of lymphocyte clone size. However, human CD8+ T cells showed normal cytolytic activity against EBV-transformed, autologous targets in vitro [39 ], and our results indicate that there are no intrinsic differences between CHS and normal CD8+ T cells in growth rate (Fig. 1) , activation-induced cytolysis (Fig. 3) , functional CD95L/FasL expression (Fig. 4) , cell proliferation (Fig. 5) , or cytokine and activation marker expression (Figs. 5 and 6) . Recent results in mice demonstrate that termination of immunity occurs via the interaction of Fas/CD95+ lymphoid cells with FasL/CD95L+ nonlymphoid cells at the site of antigen challenge, and FasL/CD95L is absent in quiescent tissue but is rapidly up-regulated during the local immune reaction [40 ]. Although we have not addressed this issue directly, as CHS1/LYST is ubiquitous, it could be speculated that lymphoproliferation and tissue infiltration in CHS might be a result of defects for the induction of indirect killing by FasL/CD95L vesicles in nonlymphoid, rather than lymphoid, cells. This hypothesis requires further experimental characterization.

CD95L/FasL follows the constitutive secretory pathway and is expressed at the plasma membrane in several human cell types (HeLa, Sertoli) [41 ] and in murine cytolytic T lymphocyte clones [28 ]. In normal human T cell blasts, in contrast, CD95L is mainly sorted to and stored in secretory lysosomes until an appropriate stimulus occurs. Once activated, preformed CD95L is expressed at the plasma membrane within the secretory domain of the immunological synapse [41 ], or it is secreted on the surface of microvesicles [34 ]. Our results suggest that microvesicle secretion, but not surface expression, of preformed CD95L is strongly dependent on CHS1/LYST (Fig. 4) . Impaired intracellular trafficking has been reported in transformed CHS B lymphocytes, which show poor peptide presentation despite normal surface MHC class II expression [35 , 42 ]. The biological role of MHC class II expression by activated human T cells is still unknown, but it may provide down-regulatory signals to other T cells and/or anergy or apoptosis signals to the bearer [43 ]. Also, defective signaling via TCR/CD3 has been reported in MHC class II-deficient, transformed CD8+ T cells [14 ]. Therefore, poor peptide presentation by MHC class II in infiltrating activated CD8+ T cells in CHS could be relevant to their expansion. The role of CHS1/LYST in peptide presentation by T lymphocytes can now be addressed using transformed T cells, which express MHC class II strongly.

Human CHS CD8+ (but not CD4+) T cells store CTLA-4/CD152 exclusively in giant lytic granules, causing poor trafficking to the cell surface after activation [5 ]. This differential behavior may be more general, as normal CD8+ (but not CD4+) T cells build a short-lived, immunological synapse with a specialized secretory domain in the outer adhesion ring close to TCR/CD3 complex clusters [44 ]. Also, expansion of CD8+ (but not CD4+) T cells in MHC class II-deficient mice was CTLA-4-independent, pointing to a critical, down-regulatory role for CD152 in MHC class II-independent CD8+ cell activation in vivo [45 ]. CD8+ HVS-transformed CHS T cells may help to explore in vitro these and further differential features in a clinically important T cell subset.

Transformed CD8+ T cells display NK [11 ] as well as T cell activities. Therefore, they may be useful to compare both types of cytolysis in vitro within the same cell background in the absence of CHS1/LYST. This was not possible to date [44 ] but may be relevant in view of recent results in mice, where mutations affecting NK and NKT perforin function cause tissue infiltration by polyclonal lymphocytes [46 ], as reported in other human syndromes affecting lytic granule traffic (Griscelli syndrome) or content (perforin deficiency) [1 ].

Last, lentiviral transduction of HVS-transformed CHS T cells may now be used to map CHS1/LYST functional domains in vitro, following published procedures [47 , 48 ].


   ACKNOWLEDGEMENTS
 
This work was supported in part by the Programa de Cooperación Internacional con Iberoamérica from the Ministerio de Educación y Cultura Grant PR264/97-7415, Fondo de Investigaciones Sanitarias Grant 00/936 and RedRespira-ISCiii-RTIC-03/11, Comunidad Autónoma de Madrid Grants 8.3/13/97 and 8.3/21/01, and Ministerio de Ciencia y Tecnología Grants SAF2001-1774 and BMC2002-3247. J. M. M-F. and M. R-C. were supported by Comunidad Autónoma de Madrid. We thank Fernando Setién, Yolanda Campos-Martín, and Ramón M. Rodriguez-López from Inmunología (Universidad Complutense de Madrid), Drs. C. Alarcón Zurita and A. González from Hematología (Hospital Clínico, Madrid), and Dr. Richard A. Spritz (Human Medical Genetics Program, University of Colorado Health Sciences, Denver) for technical support. Dr. Craig W. Reynolds (Frederick Cancer Research and Development Center, NCI, Frederick, MD) is gratefully acknowledged for the continuous supply of recombinant human IL-2. Drs. José Manuel Calvo Villas and Elena Carreter de Granda (Hematología, Hospital General de Lanzarote, Canarias, Spain) were instrumental in obtaining blood samples from patient CHC and healthy controls.

Received September 8, 2004; revised December 30, 2004; accepted January 24, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Trambas, C. M., Griffiths, G. M. (2003) Delivering the kiss of death Nat. Immunol. 4,399-403[CrossRef][Medline]
  2. Barbosa, M. D., Nguyen, Q. A., Tchernev, V. T., Ashley, J. A., Detter, J. C., Blaydes, S. M., Brandt, S. J., Chotai, D., Hodgman, C., Solari, R. C., Lovett, M., Kingsmore, S. F. (1996) Identification of the homologous beige and Chediak-Higashi syndrome genes Nature 382,262-265[CrossRef][Medline]
  3. Nagle, D. L., Karim, M. A., Woolf, E. A., Holmgren, L., Bork, P., Misumi, D. J., McGrail, S. H., Dussault, B. J., Jr, Perou, C. M., Boissy, R. E., Duyk, G. M., Spritz, R. A., Moore, K. J. (1996) Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome Nat. Genet. 14,307-311[CrossRef][Medline]
  4. Tchernev, V. T., Mansfield, T. A., Giot, L., Kumar, A. M., Nandabalan, K., Li, Y., Mishra, V. S., Detter, J. C., Rothberg, J. M., Wallace, M. R., Southwick, F. S., Kingsmore, S. F. (2002) The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins Mol. Med. 8,56-64[Medline]
  5. Barrat, F. J., Le Deist, F., Benkerrou, M., Bousso, P., Feldmann, J., Fischer, A., de Saint, B. G. (1999) Defective CTLA-4 cycling pathway in Chediak-Higashi syndrome: a possible mechanism for deregulation of T lymphocyte activation Proc. Natl. Acad. Sci. USA 96,8645-8650[Abstract/Free Full Text]
  6. Merino, F., Collins, M., Purtilo, D. T. (1987) Chediak-Higashi syndrome-derived T cell lines manifest giant lysosomal granules, normal natural killer cell, and lectin-mediated cytotoxicity J. Clin. Lab. Anal. ,72-76
  7. Stinchcombe, J. C., Page, L. J., Griffiths, G. M. (2000) Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients Traffic 1,435-444[CrossRef][Medline]
  8. Baetz, K., Isaaz, S., Griffiths, G. M. (1995) Loss of cytotoxic T lymphocyte function in Chediak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis J. Immunol. 154,6122-6131[Abstract]
  9. Meinl, E., Hohlfeld, R. (2000) T cell transformation with Herpesvirus saimiri: a tool for neuroimmunological research J. Neuroimmunol. 103,1-7[CrossRef][Medline]
  10. Biesinger, B., Muller-Fleckenstein, I., Simmer, B., Lang, G., Wittmann, S., Platzer, E., Desrosiers, R. C., Fleckenstein, B. (1992) Stable growth transformation of human T lymphocytes by Herpesvirus saimiri Proc. Natl. Acad. Sci. USA 89,3116-3119[Abstract/Free Full Text]
  11. Campos-Martin, Y., Gomez del Moral, M., Gozalbo-Lopez, B., Suela, J., Martinez-Naves, E. (2004) Expression of human CD1d molecules protects target cells from NK cell-mediated cytolysis J. Immunol. 172,7297-7305[Abstract/Free Full Text]
  12. Kraft, M. S., Henning, G., Fickenscher, H., Lengenfelder, D., Tschopp, J., Fleckenstein, B., Meinl, E. (1998) Herpesvirus saimiri transforms human T-cell clones to stable growth without inducing resistance to apoptosis J. Virol. 72,3138-3145[Abstract/Free Full Text]
  13. Rodriguez-Gallego, C., Corell, A., Pacheco, A., Timon, M., Regueiro, J. R., Allende, L. M., Madrono, A., Arnaiz-Villena, A. (1996) Herpes virus saimiri transformation of T cells in CD3 {gamma} immunodeficiency: phenotypic and functional characterization J. Immunol. Methods 198,177-186[CrossRef][Medline]
  14. Alvarez-Zapata, D., de Miguel Olalla, S., Fontan, G., Ferreira, A., Garcia-Rodriguez, M. C., Madero, L., van den Elsen, P., Regueiro, J. R. (1998) Phenotypical and functional characterization of Herpesvirus saimiri-immortalized human major histocompatibility complex class II-deficient T lymphocytes Tissue Antigens 51,250-257[Medline]
  15. Rivero-Carmena, M., Porras, O., Pelaez, B., Pacheco-Castro, A., Gatti, R. A., Regueiro, J. R. (2000) Membrane and transmembrane signaling in Herpesvirus saimiri-transformed human CD4(+) and CD8(+) T lymphocytes is ATM-independent Int. Immunol. 12,927-935[Abstract/Free Full Text]
  16. Cabanillas, J. A., Cambronero, R., Pacheco-Castro, A., Garcia-Rodirguez, M. C., Martin-Fernandez, J. M., Fontan, G., Regueiro, J. R. (2002) Characterization of Herpesvirus saimiri-transformed T lymphocytes from common variable immunodeficiency patients Clin. Exp. Immunol. 127,366-373[CrossRef][Medline]
  17. Gallego, M. D., Santamaria, M., Pena, J., Molina, I. J. (1997) Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation Blood 90,3089-3097[Abstract/Free Full Text]
  18. Broker, B. M., Kraft, M. S., Klauenberg, U., Le Deist, F., de Villartay, J. P., Fleckenstein, B., Fleischer, B., Meinl, E. (1997) Activation induces apoptosis in Herpesvirus saimiri-transformed T cells independent of CD95 (Fas, APO-1) Eur. J. Immunol. 27,2774-2780[Medline]
  19. Altare, F., Durandy, A., Lammas, D., Emile, J. F., Lamhamedi, S., Le Deist, F., Drysdale, P., Jouanguy, E., Doffinger, R., Bernaudin, F., Jeppsson, O., Gollob, J. A., Meinl, E., Segal, A. W., Fischer, A., Kumararatne, D., Casanova, J. L. (1998) Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency Science 280,1432-1435[Abstract/Free Full Text]
  20. Allende, L. M., Hernandez, M., Corell, A., Garcia-Perez, M. A., Varela, P., Moreno, A., Caragol, I., Garcia-Martin, F., Guillen-Perales, J., Olive, T., Espanol, T., Arnaiz-Villena, A. (2000) A novel CD18 genomic deletion in a patient with severe leucocyte adhesion deficiency: a possible CD2/lymphocyte function-associated antigen-1 functional association in humans Immunology 99,440-450[CrossRef][Medline]
  21. Nakamura, H., Zarycki, J., Sullivan, J. L., Jung, J. U. (2001) Abnormal T cell receptor signal transduction of CD4 Th cells in X-linked lymphoproliferative syndrome J. Immunol. 167,2657-2665[Abstract/Free Full Text]
  22. Garcia-Perez, M. A., Paz-Artal, E., Corell, A., Moreno, A., Lopez-Goyanes, A., Garcia-Martin, F., Vazquez, R., Pacho, A., Romo, E., Allende, L. M. (2003) Mutations of CD40 ligand in two patients with hyper-IgM syndrome Immunobiology 207,285-294[CrossRef][Medline]
  23. . No authors listed (1999) Primary immunodeficiency diseases. Report of an IUIS Scientific Committee. International Union of Immunological Societies Clin. Exp. Immunol. 118(Suppl. 1),1-28
  24. Ramirez-Duque, P., Arends, T., Merino, F. (1982) Chediak-Higashi syndrome: description of a cluster in a Venezuelan-Andean isolated region J. Med. 13,431-451[Medline]
  25. Karim, M. A., Suzuki, K., Fukai, K., Oh, J., Nagle, D. L., Moore, K. J., Barbosa, E., Falik-Borenstein, T., Filipovich, A., Ishida, Y., Kivrikko, S., Klein, C., Kreuz, F., Levin, A., Miyajima, H., Regueiro, J., Russo, C., Uyama, E., Vierimaa, O., Spritz, R. A. (2002) Apparent genotype-phenotype correlation in childhood, adolescent, and adult Chediak-Higashi syndrome Am. J. Med. Genet. 108,16-22[CrossRef][Medline]
  26. Zapata, D. A., Pacheco-Castro, A., Torres, P. S., Ramiro, A. R., Jose, E. S., Alarcon, B., Alibaud, L., Rubin, B., Toribio, M. L., Regueiro, J. R. (1999) Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3{gamma} J. Biol. Chem. 274,35119-35128[Abstract/Free Full Text]
  27. Mittrucker, H. W., Muller-Fleckenstein, I., Fleckenstein, B., Fleischer, B. (1992) CD2-mediated autocrine growth of Herpes virus saimiri-transformed human T lymphocytes J. Exp. Med. 176,909-913[Abstract/Free Full Text]
  28. Anel, A., Buferne, M., Boyer, C., Schmitt-Verhulst, A. M., Golstein, P. (1994) T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A Eur. J. Immunol. 24,2469-2476[Medline]
  29. Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czerwinski, M. J., Fine, D. L., Abbott, B. J., Mayo, J. G., Shoemaker, R. H., Boyd, M. R. (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48,589-601[Abstract/Free Full Text]
  30. Perez-Aciego, P., Alarcon, B., Arnaiz-Villena, A., Terhorst, C., Timon, M., Segurado, O. G., Regueiro, J. R. (1991) Expression and function of a variant T cell receptor complex lacking CD3-{gamma} J. Exp. Med. 174,319-326[Abstract/Free Full Text]
  31. Ward, D. M., Griffiths, G. M., Stinchcombe, J. C., Kaplan, J. (2000) Analysis of the lysosomal storage disease Chediak-Higashi syndrome Traffic 1,816-822[CrossRef][Medline]
  32. Nakazawa, T., Agematsu, K., Yasui, K., Onodera, T., Inoue, R., Kaneko, H., Kondo, N., Yamamoto, M., Kayagaki, N., Yagita, H., Okumura, K., Komiyama, A. (1999) Cytolytic mechanisms involved in non-MHC-restricted cytotoxicity in Chediak-Higashi syndrome Clin. Exp. Immunol. 118,108-114[CrossRef][Medline]
  33. Martinez-Lorenzo, M. J., Anel, A., Gamen, S., Monle n, I., Lasierra, P., Larrad, L., Pineiro, A., Alava, M. A., Naval, J. (1999) Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles J. Immunol. 163,1274-1281[Abstract/Free Full Text]
  34. Monleon, I., Martinez-Lorenzo, M. J., Monteagudo, L., Lasierra, P., Taules, M., Iturralde, M., Pineiro, A., Larrad, L., Alava, M. A., Naval, J., Anel, A. (2001) Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells J. Immunol. 167,6736-6744[Abstract/Free Full Text]
  35. Lem, L., Riethof, D. A., Scidmore-Carlson, M., Griffiths, G. M., Hackstadt, T., Brodsky, F. M. (1999) Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases J. Immunol. 162,523-532[Abstract/Free Full Text]
  36. Young, S. L., Murphy, M., Zhu, X. W., Harnden, P., O’Donnell, M. A., James, K., Patel, P. M., Selby, P. J., Jackson, A. M. (2004) Cytokine-modified Mycobacterium smegmatis as a novel anticancer immunotherapy Int. J. Cancer 112,653-660[CrossRef][Medline]
  37. Klein, M., Roder, J., Haliotis, T., Korec, S., Jett, J. R., Herberman, R. B., Katz, P., Fauci, A. S. (1980) Chediak-Higashi gene in humans. II. The selectivity of the defect in natural-killer and antibody-dependent cell-mediated cytotoxicity function J. Exp. Med. 151,1049-1058[Abstract/Free Full Text]
  38. Merino, F., Rodríguez, F., Biondo-Bracho, M., Amesty, C., Ramirez-Duque, P. (1984) Altered lectin-mediated cytotoxicity in Chediak-Higashi syndrome patients Clin. Immunol. Immunopathol. 33,139-143[CrossRef][Medline]
  39. Merino, F., Pauza, M., Purtilo, D. T. (1986) Anti-Epstein-Barr virus memory T-cell response in Chediak-Higashi syndrome patients Immunol. Lett. 12,51-54[CrossRef][Medline]
  40. Barreiro, R., Luker, G., Herndon, J., Ferguson, T. A. (2004) Termination of antigen-specific immunity by CD95 ligand (Fas ligand) and IL-10 J. Immunol. 173,1519-1525[Abstract/Free Full Text]
  41. Bossi, G., Griffiths, G. M. (1999) Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells Nat. Med. 5,90-96[CrossRef][Medline]
  42. Faigle, W., Raposo, G., Tenza, D., Pinet, V., Vogt, A. B., Kropshofer, H., Fischer, A., Saint-Basile, G., Amigorena, S. (1998) Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome J. Cell Biol. 141,1121-1134[Abstract/Free Full Text]
  43. Holling, T. M., Schooten, E., van Den Elsen, P. J. (2004) Function and regulation of MHC class II molecules in T-lymphocytes: of mice and men Hum. Immunol. 65,282-290[CrossRef][Medline]
  44. Stinchcombe, J. C., Griffiths, G. M. (2003) The role of the secretory immunological synapse in killing by CD8+ CTL Semin. Immunol. 15,301-305[CrossRef][Medline]
  45. Stohl, W., Xu, D., Kim, K. S., David, C. S., Allison, J. P. (2004) MHC class II-independent and -dependent T cell expansion and B cell hyperactivity in vivo in mice deficient in CD152 (CTLA-4) Int. Immunol. 16,895-904[Abstract/Free Full Text]
  46. Lacorazza, H. D., Miyazaki, Y., Di Cristofano, A., Deblasio, A., Hedvat, C., Zhang, J., Cordon-Cardo, C., Mao, S., Pandolfi, P. P., Nimer, S. D. (2002) The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells Immunity 17,437-449[CrossRef][Medline]
  47. Matheux, F., Ikinciogullari, A., Zapata, D. A., Barras, E., Zufferey, M., Dogu, F., Regueiro, J. R., Reith, W., Villard, J. (2002) Direct genetic correction as a new method for diagnosis and molecular characterization of MHC class II deficiency Mol. Ther. 6,824-829[CrossRef][Medline]
  48. Toscano, M. G., Frecha, C., Ortega, C., Santamaria, M., Martin, F., Molina, I. J. (2004) Efficient lentiviral transduction of Herpesvirus saimiri immortalized T cells as a model for gene therapy in primary immunodeficiencies Gene Ther 11,956-961[CrossRef][Medline]



This article has been cited by other articles:


Home page
 BloodHome page
M. Del-Rey, J. Ruiz-Contreras, A. Bosque, S. Calleja, J. Gomez-Rial, E. Roldan, P. Morales, A. Serrano, A. Anel, E. Paz-Artal, et al.
A homozygous Fas ligand gene mutation in a patient causes a new type of autoimmune lymphoproliferative syndrome
Blood, August 15, 2006; 108(4): 1306 - 1312.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904500v1
77/5/661    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martín-Fernández, J. M.
Right arrow Articles by Regueiro, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martín-Fernández, J. M.
Right arrow Articles by Regueiro, J. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS