Published online before print February 22, 2005
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* Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain;
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain;
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
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Key Words: T lymphocyte • immunodeficiency diseases • cytolytic cells
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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.
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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
), Leu23 (anti-CD69), FastImmune anti-hIL-2, FastImmune anti-human tumor necrosis factor (TNF-), and FastImmune anti-human interferon-
(IFN-) 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 - 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-, TNF-) 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.
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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
).
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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
.
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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.
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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. CD3, Anti-CD3.
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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.
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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- and TNF-) 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)
.
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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. 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.
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 ].
Received September 8, 2004; revised December 30, 2004; accepted January 24, 2005.
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immunodeficiency: phenotypic and functional characterization J. Immunol. Methods 198,177-186[CrossRef][Medline] J. Biol. Chem. 274,35119-35128 J. Exp. Med. 174,319-326This article has been cited by other articles:
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