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*
First Department of Internal Medicine and
Department of Pediatrics, Ehime University School of Medicine, Shigenobu, Ehime, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies using various mutant and knockout mice have clearly demonstrated the detailed mechanisms of CTL-mediated cytotoxicity. The availability of lpr and gld mutant mice, which have a defective Fas gene and a point mutation in the Fas ligand gene, respectively, provides an excellent means of assessing the importance of the Fas/Fas ligand pathway. The largely normal primary in vitro response of CD8+ CTLs from gld mice to Fas+ target cells (8) suggests that the Fas/Fas ligand pathway plays a minor role in CD8+ CTL-mediated cytotoxicity, whereas the data obtained from experiments using perforin-deficient mice suggest that the granule exocytosis pathway is dominant in CD8+ CTL-mediated cytotoxicity (9). By contrast, it has been reported that the cytotoxicity of CD4+ CTLs from gld mice is markedly defective, suggesting that the Fas/Fas ligand system is the major pathway of CD4+ CTL-mediated cytotoxicity (10). The importance of Fas to the cytotoxicity of murine CD4+ CTLs has also been reported by other investigators (11, 12).
In contrast to these recent advances in knowledge of the mechanisms of CTL-mediated cytotoxicity in the murine system, the mechanism of human CTL-mediated cytotoxicity is still obscure due to the lack of suitable experimental systems. Recently, an inherited autoimmune lymphoproliferative disorder caused by Fas gene mutations has been described (13, 14, 15). Because this human disease seems to reflect the situation in lpr mice, this disorder is expected to provide a useful experimental system for studying the role of the Fas/Fas ligand system in human CTL-mediated cytotoxicity.
Recent studies have shown that CD4+ CTLs as well as CD8+ CTLs play an important role in protection against and recovery from viral infections. For example, cytotoxicity mediated by CD4+ CTLs is important in HSV infection (16, 17), because HSV infection results in the down-regulation of surface MHC class I expression (18, 19). In the present study, we attempted to clarify the mechanisms of cytotoxicity mediated by HSV-specific human CD4+ CTLs using lymphocytes collected from family members with a Fas gene mutation. This novel experimental system for the study of human CTLs gave results that differ from those reported previously for murine systems; the Ag-specific and HLA class II-restricted cytotoxicity exerted by HSV-specific human CD4+ CTLs was completely independent of the Fas/Fas ligand system and was mediated mainly via granule exocytosis, which was also the main pathway of CD8+ CTL-mediated cytotoxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was performed after obtaining the informed consent of the patient’s parents. The details of the patient, who has a homozygous Fas gene mutation, have been reported previously (20). The patient is the daughter of parents who are first cousins. The patient showed marked lymphadenopathy, hepatosplenomegaly, pancytopenia, and hypergammaglobulinemia, but has not been suffering from severe viral infections. A homozygous point mutation was present in the splice acceptor site of intron 3 of the Fas gene of this patient. This mutation results in the skipping of exon 4 and the complete loss of Fas expression. The patient’s parents were both heterozygotes with the same Fas gene mutation. B lymphoblastoid cell lines (B-LCLs)3 were established by in vitro EBV transformation using peripheral blood B lymphocytes collected from the patient and her parents. The expression of Fas was determined by direct immunofluorescence using the FITC-conjugated anti-Fas mAb (MBL, Nagoya, Japan). Stained cells were analyzed with a flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA).
Generation of HSV-specific CD4+ T cell lines and clones
Human CD4+ T cell bulk lines and clones directed against HSV-infected cells were generated as reported previously (21) with a slight modification. Briefly, PBMCs collected from the patient and her mother were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated human AB serum (this medium will be referred to as "culture medium"). Thereafter, UV light-inactivated HSV type 1 (HSV-1) was added to the cells, which were then seeded in round-bottomed microtiter wells at 1 x 105 cells/well and cultured at 37°C in a 5% CO2 incubator for 6 days. Then CD4+ cells were isolated using magnetizable polystyrene beads coated with an anti-CD4 mAb (Dynal, Oslo, Norway) and were cultured in medium containing IL-2. The CD4+ T lymphocytes were stimulated with mitomycin C (MMC)-treated PBMCs, collected from the patient’s mother, and HSV Ag every 10–14 days and were used for the experiments as HSV-specific CD4+ T cell bulk lines after at least three cycles of HSV Ag stimulation. Uncloned bulk cell lines were selected from the mixture of CD4+ T lymphocytes collected from eight microtiter wells, and each line was cultured separately. HSV-specific CD4+ T cell clones were established by cloning HSV Ag-stimulated CD4+ T lymphocytes using the limiting dilution method as reported previously (21). HSV-specific CD4+ T cell bulk lines and clones were used for each experiment after 3–5 days of HSV Ag stimulation when they were activated.
Alloantigen-specific CD8+ CTLs were also generated for use as controls that cause granule exocytosis-mediated and DNA fragmentation-inducing cytotoxicity. PBMCs from an individual whose HLA class I and class II were nonidentical to those of the patient and her mother were cocultured with an MMC-treated B-LCL established from the patient’s PBMCs at a responder-to-stimulator ratio of 5:1. After 5 days, CD8+ T cells were isolated using anti-CD8 mAb-coated magnetic beads (Dynal). CD8+ T cells were then cultured in IL-2-containing culture medium and restimulated with MMC-treated B-LCL cells. By these procedures, CD8+ CTL lines directed against allogeneic B-LCLs were established.
Cytotoxicity assays
Cytotoxicity was determined by 51Cr release assays, which were performed as described previously (22). Briefly, to prepare HSV-infected target cells, B-LCL cells were inoculated with HSV-1 at a multiplicity of infection of 10 and were cultured for 16 h. Various numbers of effector cells and 1 x 10451Cr (Na251CrO4; New England Nuclear, Boston, MA)-labeled target cells were incubated together in 0.2 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FCS in round-bottomed microtiter wells. Target cells were also added to wells containing medium alone and to wells containing 1% Triton X-100 to determine the spontaneous and maximal levels of 51Cr release, respectively. After 4 h, 0.1 ml of supernatant was collected from each well. The percentage of specific 51Cr release was calculated as (cpm experimental release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release) x 100. To examine the Ca2+ dependency of the cytotoxicity, cytotoxicity assays were performed in the presence of EGTA (Sigma, St. Louis, MO) at various concentrations. To evaluate the role of perforin in CD4+ CTL-mediated cytotoxicity, effector CTLs were pretreated with an inhibitor of the perforin-based cytotoxic pathway, concanamycin A (CMA; Wako Pure Chemical Industries, Osaka, Japan) at various concentrations for 2 h before being incubated with the target cells in the presence of CMA. Treatment with CMA at the concentrations used in the present study showed no toxic effect against T lymphocytes and B-LCL cells as determined from cell growth curves and 51Cr release assays (data not shown). Effector CTLs were used for experiments after 3–5 days of Ag stimulation when they were activated. Each cytotoxicity assay was performed at least twice, and similar data were obtained.
DNA fragmentation assays
DNA fragmentation in the target cells was determined by [125I]UdR release assays as described previously (23). Briefly, target cells were incubated with [125I]UdR (Amersham, Arlington Heights, IL) for 2 h at 37°C and washed twice. Various numbers of effector cells and 1 x 104 [125I]UdR-labeled target cells were incubated together in 0.2 ml of assay medium in round-bottomed microtiter wells. After 4 h, 0.1 ml of supernatant was removed from each well and 0.1 ml of 0.2% Triton X-100 was added to each well and mixed by pipetting. The solubilized samples were centrifuged at 250 x g for 10 min, 0.1 ml of supernatant was collected from each well, and the radioactivity was counted. The total radioactivity of the target cells was estimated by directly counting the radioactivity of the cell suspension without solubilization. The specific [125I]UdR release was estimated as described for 51Cr release.
Detection of cytolytic mediator mRNA expression
Expression of the mRNAs for various cytolytic mediators in HSV-specific CD4+ CTLs was investigated using RT-PCR. Total RNA was extracted from CTLs that had been stimulated with HSV Ag 5 days earlier, and cDNA was synthesized by RT with Moloney murine leukemia virus reverse transcriptase as described previously (24). Amplification of the cDNAs by PCR was performed using the following primers: perforin, 5'-ACCAGCAATGTGCATGTGTCTGTG-3' and 5'-GAAGGAGGCCGTCATCTTGTGCTT-3'; granzyme B, 5'-TGCAGGAAGATCGAAAGTGCG-3' and 5'-GAGGCATGCCATTGTTTCGTC-3'; Fas ligand, 5'-ATAGGATCCATGTTTCTGCTCTTCCACCTACAGAAGGA-3' and 5'-ATAGAATTCTGACCAAGAGAGAGCTCAGATACGTTGAC-3'. The expected lengths of the amplified cDNAs for the cytolytic mediators were 459 bp, 180 bp, and 506 bp for perforin, granzyme B, and Fas ligand, respectively.
Flow cytometric analysis
Expression of surface and intracytoplasmic molecules was examined by immunofluorescence using a flow cytometer. To detect surface Fas ligand expression, cells were incubated with biotin-conjugated hamster anti-human Fas ligand mAb (MBL). After washing twice, the cells were incubated with FITC-conjugated streptavidin (Immunotech, Marseille, France). To detect intracytoplasmic perforin expression, cells were fixed with 0.6% paraformaldehyde for 1 h at 4°C, washed, then incubated in PBS with 0.2% Tween 20 for 15 min at 37°C. After a further wash, the cells were stained with FITC-conjugated mouse anti-human perforin mAb (Pharmacell, Paris, France) or FITC-conjugated control mouse IgG.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fas expression on B-LCLs established from peripheral blood B
lymphocytes collected from the patient (Fas-/-), the
patient’s parents (Fas+/-), and an unrelated individual
without a Fas gene mutation (Fas+/+) is shown in
Fig. 1
. B-LCL from the patient
completely lacked Fas expression and the levels of Fas expression
on the B-LCLs from the patient’s parents were slightly reduced
compared with that of the control B-LCL without Fas gene
mutation.
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. The patient’s father and mother both
shared HLA-A24, B48, DRB1*0401, DQA1*0301, DQB1*0301, and DPB1*0201
with the patient. The HLA alleles of the unrelated donor, MY, differed
from those of the patient except for HLA-DQB1*0301.
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Four and five CD4+ CTL clones directed against
HSV-infected cells were generated from PBMCs collected from the patient
and her mother, respectively. Four CD4+ uncloned bulk T
cell lines were also generated from both the patient and her mother.
Flow cytometric analysis of their surface phenotypes revealed that
>98% of all bulk line and clone cells were CD3+,
CD4+, and CD8-. Similar data were obtained
from all bulk lines and clones, so the data for one representative bulk
line and one clone established from the patient’s PBMCs, designated
PT-bulk-1 and PT-clone-1, and for one bulk line and one clone
established from the mother’s PBMCs, designated MO-bulk-1 and
MO-clone-1, are presented. The cytotoxicities of these bulk
CD4+ T lymphocyte lines and clones against HSV-1-infected
and mock-infected target cells from the patient, the patient’s
parents, and an unrelated HLA-DR-mismatched donor are shown in Table II. Cytotoxicity against HSV-infected
target cells, but not against the HSV-uninfected cells, was detected,
suggesting that the CD4+ T lymphocytes possessed
HSV-specific cytotoxic activity. The cytotoxicity of the
CD4+ T lymphocyte clones against HSV-infected cells seemed
to be restricted by HLA class II as reported previously (22), because
the HSV-infected target cells of the HLA-DR-mismatched donor were not
lysed and the cytotoxicity was inhibited by an anti-HLA-DR mAb
(data not shown). The low level of HLA-DR-unrestricted cytotoxicity of
the uncloned CD4+ T lymphocyte lines against HSV-infected
target cells might have been due to HLA-DQ-restricted cytotoxicity (16)
and to the sensitivity of HSV-infected B-LCL to NK cell-mediated
cytotoxicity (25), and also by some of the HSV-specific
CD4+ T lymphocytes, which may also possess NK cell-like
HLA-unrestricted cytotoxic activity (21), as reported previously. It
appeared that the degrees of cytotoxicity of the bulk cell lines and
clones against HSV-infected Fas-/- cells and HSV-infected
Fas+/- cells were almost the same, suggesting that the
Fas/Fas ligand pathway is not essential for cytotoxicity mediated by
HSV-specific CD4+ CTLs. The levels of cytotoxicity against
HSV-infected target cells mediated by alloantigen-specific
CD8+ CTLs were slightly lower than those against
mock-infected target cells. These results may reflect the
down-regulation of HLA class I expression that is induced by HSV-1
infection (18, 19).
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Expression of cytolytic mediators
Since cytotoxicity mediated by HSV-specific human CD4+
CTLs seemed to be Fas independent, we examined the expression of Fas
ligand in these CD4+ CTLs. Expression of perforin and
granzyme B, which are important cytolytic mediators in CD8+
CTL- and NK cell-mediated cytotoxicity, was also examined by RT-PCR. As
shown in Fig. 2A, it appeared
that mRNAs for Fas ligand, perforin, and granzyme B were all expressed
in HSV-specific CD4+ bulk lines and clones. Expression of
surface Fas ligand and intracytoplasmic perforin was confirmed by flow
cytometry (Fig. 2B).
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As the perforin/granzyme pathway is known to be
Ca2+-dependent, we examined the cytotoxic activity of
HSV-specific CD4+ CTLs in the absence of extracellular
Ca2+. No cytotoxicity against either HSV-infected
Fas-/- or Fas+/- target cells was observed
in the presence of the Ca2+-chelating agent EGTA at
concentrations of more than 1 mM (Table III), indicating that HSV-specific
CD4+ CTL-mediated cytotoxicity is completely
Ca2+-dependent.
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It has been reported that treatment of CTLs with CMA results in
the complete inhibition of perforin-based cytotoxic activity (27). In
the light of this previous finding, we examined the significance of
granule exocytosis in HSV-specific CD4+ CTL-mediated
cytotoxicity using CMA. As shown in Table IV, the cytotoxicities of
CD4+ bulk cell lines and clones generated from both the
patient and her mother against HSV-infected target cells were almost
completely inhibited by pretreatment of the effector cells with CMA at
concentrations of more than 10 nM, as has been reported for
perforin-dependent CD8+ CTL-mediated cytotoxicity. Taken
together with the results of the RT-PCR, which showed the
expression of perforin and granzyme B mRNAs in the effector cells,
these data strongly suggest that granule exocytosis is the main pathway
of cytotoxicity mediated by HSV-specific human CD4+ CTLs.
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Because it is well known that DNA fragmentation in target cells is
induced by CTLs via either granule exocytosis or the Fas/Fas ligand
system, we expected that the DNA in HSV-1-infected target cells
incubated with HSV-specific CD4+ CTLs would be fragmented.
The results of the [125I]UdR release assay, which
reflects DNA fragmentation in target cells, are shown in Table V. Unexpectedly, the level of DNA
fragmentation in the HSV-1-infected target cells that had been
incubated with HSV-specific CD4+ CTLs was low compared with
the level of 51Cr release. Although alloantigen-specific
CD8+ CTLs induced marked DNA fragmentation in
mock-infected LCL cells, the level of DNA fragmentation in
HSV-1-infected LCL cells incubated with alloantigen-specific
CD8+ CTLs was low, as was the case for HSV-specific
CD4+ CTL-mediated DNA fragmentation in HSV-1-infected
target cells. These data strongly suggest that HSV-1 renders infected
cells resistant to CTL-induced DNA fragmentation, as has been reported
recently (28).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytotoxicity mediated by HSV-specific human CD4+ CTLs seems to be Ca2+-dependent, because addition of the Ca2+-chelating agent EGTA to the assay medium resulted in complete abrogation of cytotoxicity. Fas-dependent cytotoxicity has been reported to be Ca2+-independent (29), and EGTA has been used to discriminate between the granule exocytosis and Fas/Fas ligand pathways. However, recent studies of perforin-deficient T lymphocyte clones have provided controversial results. Subsequent studies have revealed that even though the interaction between Fas ligand and Fas is Ca2+-independent, TCR-dependent up-regulation of Fas ligand on the cell surface requires extracellular Ca2+ (30). Therefore, we could not conclude that cytotoxicity mediated by human CD4+ CTLs is Fas independent on the basis of the results obtained from the experiments using EGTA.
The involvement of the Fas/Fas ligand system in cytotoxicity mediated by murine CTLs has been studied in detail using the cells of the Fas mutant lpr and Fas ligand mutant gld mice. In contrast to murine experimental systems, previous studies on the cytotoxic mechanisms of human CTLs have been conducted indirectly by studying the correlation between cytotoxicity and the expression levels of Fas on the target cells, and by using antagonistic anti-Fas mAbs (31, 32). However, these experimental systems do not seem to be suitable, because treatment with an anti-Fas mAb may not completely inhibit the binding of Fas ligand to Fas and may affect the effector cell in various ways as well as the target cell. To clarify the role of the Fas/Fas ligand system in human CD4+ CTL-mediated cytotoxicity, we performed experiments for the first time using the cells of a patient with Fas deficiency, which can be regarded as the human counterpart of lpr mice (15), and demonstrated clearly that cytotoxicity mediated by HSV-specific human CD4+ CTLs is completely independent of the Fas/Fas ligand system. This result seems to differ from previous evidence suggesting that in the murine system the major mechanism of cytotoxicity mediated by CD8+ CTLs is granule exocytosis, whereas that mediated by CD4+ CTLs is based almost solely on the Fas/Fas ligand system (10, 11, 12).
The cause of the difference between the main pathways in murine and human CD4+ CTLs is obscure. The possibility that Fas-independent cytotoxicity reflects only a small subpopulation of human CD4+ CTL is unlikely, because all of the HSV-specific CD4+ CTL bulk lines as well as the clones established from lymphocytes collected from the Fas-/- patient and her Fas+/- mother lysed HSV-infected Fas-/- target cells. This discrepancy may be explained by the activation of different pathways by murine and human CD4+ CTLs, because the signal transduction events that trigger perforin-mediated cytotoxicity and the signaling requirements for induction of Fas ligand expression are distinct (33).
In the present study, we examined the role of the granule exocytosis pathway in human CD4+ CTL-mediated cytotoxicity using CMA, which is a specific inhibitor of vacuolar type H+-ATPase. CMA inhibits the activity of perforin in dense granules, due mostly to accelerated degradation of perforin caused by an increase in the pH within the lytic granules, and therefore it is used to discriminate between the granule exocytosis and Fas/Fas ligand systems in CTL-mediated cytotoxicity (27, 34). It has also been reported that expression of perforin mRNA is not induced in CD4+ T lymphocytes even after stimulation with IL-2 (35). However, recent studies have shown that perforin is expressed in CD4+ T lymphocytes that have been stimulated with various viral Ags (36, 37, 38). Taken together with evidence from in vivo studies in perforin-deficient mice suggesting that perforin-mediated cytotoxicity plays a major protective role against viral infections (9), the present study strongly suggests that granule exocytosis is a major pathway for cytotoxicity mediated by CD4+ CTLs as well as CD8+ CTLs in viral infections.
Although various types of CD4+ CTLs have been identified, the role of cytotoxicity mediated by CD4+ CTLs in vivo is still obscure. It has been proposed that CD4+ CTLs have an immunomodulatory role, that is, they may preferentially eliminate MHC class II-positive APCs to terminate the immune response, and thus prevent an overreaction of the ongoing immune response (39). Because the Fas/Fas ligand system is important for eliminating autoreactive lymphocytes, this hypothesis seems to explain the role of Fas-dependent CD4+ CTLs. On the other hand, it seems more likely that perforin-dependent CD4+ CTLs have a direct role in protection against viral infections. The immediate early protein ICP47 of HSV inhibits the transporter for Ag processing-mediated transport of Ag-derived peptide across the endoplasmic reticulum membrane. This interference prevents the assembly of peptides with MHC class I molecules in the endoplasmic reticulum, and ultimately prevents recognition of HSV-infected cells by MHC class I-restricted CD8+ CTLs (18, 19, 40). Therefore, MHC class II-restricted CD4+ CTLs are thought to play an important role in protection against and recovery from HSV infection. Indeed, HLA class II-restricted HSV-specific CD4+ CTL clones have been isolated from the lesions of HSV infection (41, 42). In addition, it has been reported that HSV infection induces Fas-independent apoptosis (43). Other investigators have reported that HSV-1 renders infected cells resistant to CTL-mediated DNA fragmentation, which is the essential mechanism of Fas-mediated cell death (28). This finding was confirmed in our present study. When considering these findings, perforin-dependent, rather than Fas-dependent, cytotoxicity mediated by CD4+ CTLs seems likely to be more effective for lysis of HSV-infected cells. The observation that our patient with Fas deficiency has not been suffering from severe viral infections seems to support this hypothesis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masaki Yasukawa, First Department of Internal Medicine, Ehime University School of Medicine, Shigenobu, Ehime 791-0295, Japan. E-mail address:
3 Abbreviations used in this paper: LCL, lymphoblastoid cell line; CMA, concanamycin A; MMC, mitomycin C.
Received for publication September 8, 1998. Accepted for publication February 18, 1999.
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References |
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pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL. J. Immunol. 158:5283.[Abstract]
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C. M. Dobbs and K. Haskins Comparison of a T Cell Clone and of T Cells from a TCR Transgenic Mouse: TCR Transgenic T Cells Specific for Self-Antigen Are Atypical J. Immunol., February 15, 2001; 166(4): 2495 - 2504. [Abstract] [Full Text] [PDF]
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E. Chang, L. Galle, D. Maggs, D. M. Estes, and W. J. Mitchell Pathogenesis of Herpes Simplex Virus Type 1-Induced Corneal Inflammation in Perforin-Deficient Mice J. Virol., December 15, 2000; 74(24): 11832 - 11840. [Abstract] [Full Text]
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D. M. Koelle, S. N. Reymond, H. Chen, W. W. Kwok, C. McClurkan, T. Gyaltsong, E. W. Petersdorf, W. Rotkis, A. R. Talley, and D. A. Harrison Tegument-Specific, Virus-Reactive CD4 T Cells Localize to the Cornea in Herpes Simplex Virus Interstitial Keratitis in Humans J. Virol., December 1, 2000; 74(23): 10930 - 10938. [Abstract] [Full Text]
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C. Traidl, S. Sebastiani, C. Albanesi, H. F. Merk, P. Puddu, G. Girolomoni, and A. Cavani Disparate Cytotoxic Activity of Nickel-Specific CD8+ and CD4+ T Cell Subsets Against Keratinocytes J. Immunol., September 15, 2000; 165(6): 3058 - 3064. [Abstract] [Full Text] [PDF]
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M. Yasukawa, H. Ohminami, J. Arai, Y. Kasahara, Y. Ishida, and S. Fujita Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans Blood, April 1, 2000; 95(7): 2352 - 2355. [Abstract] [Full Text] [PDF]
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