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Essential Roles of Perforin in Antigen-Specific Cytotoxicity Mediated by Human CD4⁺ T Lymphocytes: Analysis Using the Combination of Hereditary Perforin-Deficient Effector Cells and Fas-Deficient Target Cells ¹

Fumio Yanai^*, Eiichi Ishii, Kensuke Kojima, Atsuhiko Hasegawa, Taichi Azuma, Shinichi Hirose^*, Naohiro Suga^||, Akihisa Mitsudome^*, Masafumi Zaitsu, Yasushi Ishida, Yuji Shirakata^¶, Koji Sayama^¶, Koji Hashimoto^¶ and Masaki Yasukawa^2,

^* Department of Pediatrics, Fukuoka University School of Medicine, Fukuoka, Japan; Department of Pediatrics, Saga Medical School, Saga, Japan; First Department of Internal Medicine and Departments of Pediatrics and ^¶ Dermatology, Ehime University School of Medicine, Ehime, Japan; and ^|| Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the cytotoxic mechanisms of murine CTLs have been investigated extensively using various mutant and knockout mice, those of human CTLs, especially CD4⁺ CTLs, are still obscure. To clarify the roles of perforin in Ag-specific cytotoxicity mediated by human CD4⁺ CTLs, alloantigen-specific and HSV-specific human CD4⁺ T lymphocyte bulk lines and clones were established from a patient with hereditary perforin deficiency and her healthy father, and their cytotoxic activities were investigated. Alloantigen-specific CD4⁺ T lymphocytes expressing perforin exerted cytotoxicity against Fas-negative as well as Fas-positive allogeneic B lymphoblastoid cell lines established from members of a family with hereditary Fas deficiency. Perforin-deficient, but not perforin-expressing, CD4⁺ T lymphocytes failed to show strong cytotoxicity against HSV-infected autologous B lymphoblastoid cells. Perforin-deficient CD4⁺ T lymphocytes could exert relatively low level cytotoxicity against allogeneic IFN--treated keratinocytes. Although cytotoxicity mediated by perforin-expressing CD4⁺ CTLs was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, cytotoxicity against IFN--treated keratinocytes mediated by perforin-deficient CD4⁺ T lymphocytes was inhibited only partially by concanamycin A, but was inhibited significantly by antagonistic anti-Fas Ab and anti-Fas ligand Ab. The combination of perforin-deficient effector T lymphocytes and Fas-negative target cells used in the present study provides a novel experimental system for studying the detailed mechanisms of human CTL-mediated cytotoxicity. The present data demonstrate that perforin-negative CD4⁺ CTLs can exert cytotoxicity against Fas-sensitive target cells; however, perforin plays essential roles in Ag-specific cytotoxicity mediated by human CD4⁺ as well as CD8⁺ CTLs.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Because CTLs undoubtedly play an important role in resistance against virus infections and cancers, understanding the mechanisms of CTL-mediated cytotoxicity is extremely important. Although conventional CTLs are CD8⁺ and MHC class I-restricted, CD4⁺ and MHC class II-restricted CTLs specific for alloantigens, viruses, and tumor-associated Ags have also been identified in vitro and in vivo (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). The mechanisms of cytotoxicity mediated by CTLs have been examined extensively, and various pathways, including perforin/granzymes, Fas/Fas ligand, membrane-bound TNF-, membrane-bound lymphotoxin, and TNF-related apoptosis-inducing ligand (TRAIL),³ have been identified (11). Among these, granule exocytosis mediated by perforin/granzymes and Fas/Fas ligand pathways are thought to be the main mechanisms of CTL-mediated Ag-specific cytotoxicity (12, 13, 14, 15, 16, 17). The former pathway involves pore formation in the target cell membrane through extracellular Ca²⁺-dependent polymerization of perforin, accompanied by release of serine proteases, granzymes, responsible for downstream activation of caspase and subsequent DNA fragmentation, resulting in target cell apoptosis. The latter pathway is mediated by the interaction of Fas ligand expressed on the surface of the CTLs with the apoptosis-inducing receptor Fas expressed on the target cells. The cytoplasmic tail of Fas contains a motif called the death domain. In addition to these main cytotoxic pathways, it has been reported recently that TRAIL is important for CTL-mediated cytotoxicity (18); however, the precise roles of TRAIL-mediated cytotoxicity in resistance against viral infections and malignancies are still obscure.

The cytotoxic mechanisms of murine CTLs have been examined extensively using various mutant and knockout mice, including Fas-mutant lpr, Fas ligand-mutant gld, perforin-deficient, and granzyme-deficient mice. The data obtained from experiments using perforin-deficient mice suggest that the granule exocytosis pathway is dominant in murine CD8⁺ CTL-mediated cytotoxicity (19). 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 murine CD4⁺ CTL-mediated cytotoxicity (20, 21, 22). In contrast to our detailed knowledge of the mechanisms of cytotoxicity mediated by murine CTLs, the mechanism of human CTL-mediated cytotoxicity is still obscure because of the lack of suitable experimental systems.

The recent finding that autoimmune lymphoproliferative syndrome (ALPS) is a Fas deficiency disorder (23, 24) led us to use Fas-deficient cells from a patient with ALPS to examine the mechanisms of cytotoxicity mediated by human CTLs. We recently reported that granule exocytosis may be the main pathway of Ag-specific cytotoxicity mediated by human CD4⁺ as well as CD8⁺ CTLs on the basis of the finding that human CTLs exert similar cytotoxicity against Fas-expressing and Fas-deficient target cells (25, 26). This experimental system is the human counterpart of the murine system using lpr and gld mutant mice, which have a defective Fas gene and a point mutation in the Fas ligand gene, respectively (27, 28). Recently, it has been reported that some cases of familial hemophagocytic lymphohistiocytosis (FHL) are caused by perforin gene defects (29). T lymphocytes of patients with this disease are expected to provide a useful tool for studying perforin-based effector mechanisms.

On the basis of this concept, in the present study we examined the mechanisms of Ag-specific cytotoxicity mediated by human CD4⁺ CTLs using a combination of T lymphocytes from a patient with perforin deficiency and Fas-deficient target cells from a patient with ALPS. The data obtained from this novel experimental system revealed that perforin plays an essential role in Ag-specific cytotoxicity mediated by human CD4⁺ as well as CD8⁺ CTLs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Perforin-deficient and Fas-deficient families

The present study was performed after obtaining the informed consent of the patients’ parents. The donor of perforin-deficient T lymphocytes was a 6-mo-old female who showed typical clinical manifestations of hemophagocytic lymphohistiocytosis, such as fever, pancytopenia, and hepatosplenomegaly, at 2 mo of age. Her parents were both healthy. FHL was suspected, and analysis of the perforin gene was performed as described previously (30).

The details of the patient with a homozygous Fas gene mutation have been reported previously (31). The patient is the daughter of parents who are first cousins. 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 healthy and appeared to be heterozygotes with the same Fas gene mutation. B lymphoblastoid cell lines (B-LCLs) were established by in vitro EBV transformation of peripheral blood B lymphocytes of the patient and her parents.

Flow cytometric analysis

Expression of cell surface CD3, CD4, and CD8 was examined by flow cytometric analysis with FITC-conjugated anti-CD3 mAb (BD PharMingen, San Diego, CA), FITC-conjugated anti-CD4 mAb (BD PharMingen), and FITC-conjugated anti-CD8 mAb (BD PharMingen), respectively. Detection of intracellular perforin expression was performed as follows. 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 anti-perforin mAb (BD PharMingen) or FITC-conjugated mouse IgG as the control. After being washed, the cells were analyzed with a FACSCalibur (BD Biosciences, San Jose, CA) equipped with CellQuest software (BD Biosciences). Two-color flow cytometry with PE-conjugated anti-CD8 mAb and FITC-conjugated anti-perforin mAb was performed as described previously (30).

Culture and IFN- treatment of human keratinocytes

Normal human keratinocytes were cultured as previously described (32). Briefly, normal human skin was cut into 3- to 5-mm pieces and incubated with 250 U/ml dispase (Godoshusei, Tokyo, Japan) in DMEM overnight at 4°C. After separation of the epidermis from the dermis by forceps, the epidermal sheets were rinsed with PBS, incubated in 0.25% trypsin solution for 10 min at 37°C, and teased with forceps. Epidermal cells were rinsed and collected by centrifugation, then resuspended in MCDB153 medium supplemented with insulin (5 µg/ml), hydrocortisone (0.5 µM), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), and bovine hypothalamic extract (100 µg/ml). When cells reached semiconfluence, culture medium was replaced by fresh medium containing recombinant human IFN- (Genzyme, Boston, MA) at a concentration of 1000 U/ml. The cells were cultured for 3 days in the presence of IFN- and were then collected to use for experiments. As described previously (33), the expression of surface HLA-DR on keratinocytes was induced strongly by treatment with IFN- (data not shown).

Generation of alloantigen-specific T lymphocyte bulk lines and clones

Alloantigen-specific CD4⁺ and CD8⁺ T lymphocyte bulk lines and clones were generated as described previously (26). Briefly, PBMCs from the patient with perforin deficiency (perforin^-/-) and her father (perforin^+/-) were cocultured with a mitomycin C (MMC)-treated B-LCL established from the patient with ALPS (Fas^-/-) or from the patient’s father (Fas^+/-) in RPMI 1640 medium supplemented with 10% heat-inactivated human AB-type serum (this medium will be referred to as culture medium). Magnetizable polystyrene beads coated with anti-CD4 and anti-CD8 mAb (Dynal, Oslo, Norway) were used to isolate CD4⁺ and CD8⁺ T lymphocytes, respectively, from PBMCs that had been stimulated with an allogeneic B-LCL for 6 days. Uncloned CD4⁺ and CD8⁺ T lymphocytes were further cultured in culture medium supplemented with 10 U/ml recombinant human IL-2 (Genzyme), stimulated with the allogeneic B-LCL three times at 1-wk intervals, and then used as T lymphocyte bulk lines. Alloantigen-specific CD4⁺ T lymphocyte clones were established by limiting dilution from PBMCs that had been stimulated with an allogeneic B-LCL and were cultured continuously in culture medium supplemented with IL-2 with repeated stimulation by the allogeneic B-LCL as described previously (26).

Allogeneic keratinocyte-specific CD4⁺ and CD8⁺ T lymphocytes were generated by stimulating PBMCs from the patient with perforin deficiency and her father with MMC-treated allogeneic keratinocytes that had been cultured in the presence of IFN- for 3 days as described above. Allogeneic keratinocyte-specific CD4⁺ and CD8⁺ T lymphocyte bulk lines and clones were established as described above for B-LCL-specific T lymphocytes.

Generation of HSV-specific CD4⁺ T lymphocyte bulk lines and clones

HSV-specific CD4⁺ T lymphocyte bulk lines and clones were generated as described previously (34). Briefly, PBMCs from the patient with perforin deficiency and her father were suspended in culture medium. Then, UV light-inactivated HSV-1 was added to the cells, which were seeded in round-bottom microtiter wells at 1 x 10⁵ cells/well and cultured at 37°C in a 5% CO₂ incubator for 6 days. CD4⁺ cells were isolated by the use of magnetizable polystyrene beads coated with anti-CD4 mAb (Dynal) and were cultured in culture medium containing IL-2. The CD4⁺ T lymphocytes were stimulated with MMC-treated PBMCs from the patient’s father and HSV Ag every 10–14 days and were used for experiments as HSV-specific CD4⁺ T lymphocyte bulk lines after at least three cycles of HSV Ag stimulation. HSV-specific CD4⁺ T lymphocyte clones were established by cloning HSV Ag-stimulated CD4⁺ T lymphocytes by the limiting dilution method as reported previously (35).

Cytotoxicity assays

Cytotoxicity was determined by ⁵¹Cr release assays, performed as described previously (36). 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 10^{4 51}Cr (Na₂⁵¹CrO₄; NEN, Boston, MA)-labeled target cells were incubated together in 0.2 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FCS in round-bottom 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 evaluate the role of perforin in CD4⁺ CTL-mediated cytotoxicity, effector cells were pretreated with an inhibitor of the perforin-based cytotoxic pathway, concanamycin A (CMA) (Wako Pure Chemical, Osaka, Japan), at a concentration of 10 nM for 2 h before being incubated with the target cells in the presence of CMA. Treatment with CMA at the concentration 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). In some experiments effector T lymphocytes and target cells were preincubated with anti-Fas ligand mAb (NOK1; BD PharMingen) and anti-Fas mAb (ZB4; MBL, Nagoya, Japan), respectively, for 30 min before performing cytotoxicity assays. To determine HLA-DR restriction, target cells were preincubated with anti-HLA-DR mAb (L243; American Type Culture Collection, Manassas, VA) for 30 min and then cocultured with effector T lymphocytes. Effector T lymphocytes 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.

Detection of IFN- production

T lymphocytes were washed twice with RPMI 1640 medium to remove cytokines present in culture medium. For alloantigen-specific T lymphocyte bulk lines and clones, 1 x 10⁵ T lymphocytes were cocultured with or without 5 x 10⁴ MMC-treated B-LCL cells or MMC-treated keratinocytes in 0.2 ml of RPMI 1640 medium supplemented with 10% FCS in a flat-bottom microtiter well. For HSV-specific CD4⁺ T lymphocyte bulk lines and clones, 1 x 10⁵ T lymphocytes and 2 x 10⁵ MMC-treated autologous PBMCs were suspended in 0.2 ml of RPMI 1640 medium supplemented with 10% FCS and cultured in a flat-bottom microtiter well in the presence or the absence of HSV Ag. In some experiments anti-HLA-DR mAb (L243) was added to wells at an optimal concentration as described previously. After 72 h the supernatant was collected from each well and assayed for the production of IFN- by ELISA (Endogen, Woburn, MA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mutation of perforin genes and defect of perforin protein in lymphocytes of the patient

Analysis of perforin genes revealed that the patient with FHL is a compound heterozygote for mutations of exon 2 (del 207C) and exon 3 (del 1090–91CT). Her father appeared to be a heterozygote for mutation of exon 3 (del 1090–91CT). Expression of perforin protein in peripheral blood lymphocytes of the patient and a healthy individual was examined by two-color flow cytometry. As shown in Fig. 1, perforin was detectable in peripheral blood lymphocytes of the patient’s father, but was completely absent from lymphocytes of the patient.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis of perforin expression in PBL. Perforin expression was detected in PBL from the patient’s father, but not in those from a patient with perforin gene mutations.

The HLA types of each individual are shown in Table I. The patient with perforin deficiency and her father shared HLA-A33, B58, C3, and DRB1*0301. On the other hand, the patient with Fas deficiency and her father shared HLA-A24, B48, Cw8, and DRB1*0401. The HLA-DRB1 alleles of the patient with perforin deficiency and those of her father were different from those of the unrelated donor of keratinocytes.

IFN- production of alloantigen-specific T-lymphocytes in response to stimulation with allogeneic B-LCLs

Four CD4⁺ T lymphocyte uncloned bulk lines, seven CD4⁺ T lymphocyte clones, three CD8⁺ T lymphocyte bulk lines, four CD4⁺ T-lymphocyte bulk lines, four CD4⁺ T lymphocyte clones, and three CD8⁺ T-lymphocyte bulk lines were established from perforin-positive T lymphocytes (perforin-deficient patient’s father) by stimulation with Fas-positive and Fas-negative allogeneic B-LCL cells, respectively. Similarly, four CD4⁺ T lymphocyte uncloned bulk lines, five CD4⁺ T lymphocyte clones, three CD8⁺ T lymphocyte bulk lines, four CD4⁺ T lymphocyte bulk lines, six CD4⁺ T lymphocyte clones, and three CD8⁺ T lymphocyte bulk lines were established from perforin-deficient T lymphocytes (perforin-deficient patient) by stimulation with Fas-positive and Fas-negative allogeneic B-LCL cells, respectively. The Ag specificities of T lymphocyte bulk lines and clones generated from the perforin-deficient patient and her healthy father by stimulation with Fas-deficient or Fas-positive allogeneic B-LCL were examined by measuring IFN- production. As the same data were obtained from all bulk lines and clones examined, representative data for one CD4⁺ T lymphocyte bulk line, two CD4⁺ T lymphocyte clones, and one CD8⁺ T lymphocyte bulk line are shown in Fig. 2. All CD4⁺ T lymphocyte bulk lines and clones generated from perforin-deficient and perforin-positive donors that had been stimulated by Fas-positive or Fas-negative allogeneic B-LCL produced a large amount of IFN- in response to stimulation with allogeneic cells. The production of IFN- by these T lymphocyte bulk lines and clones was significantly inhibited by anti-HLA-DR mAb, indicating that their Ag specificities were alloantigen-specific and HLA-DR-restricted. CD8⁺ T lymphocyte lines also produced IFN- in response to stimulation with allogeneic B-LCL; however, their IFN- production was not inhibited by anti-HLA-DR mAb, since their Ag specificity was not restricted by HLA class II, but was restricted by HLA class I.

View larger version (42K): [in this window] [in a new window]   FIGURE 2. IFN- production by alloantigen-specific CD4+ and CD8+ T lymphocytes in response to stimulation with autologous or allogeneic B-LCL. Responder cells were cocultured with or without autologous or allogeneic B-LCL in the presence or the absence of anti-HLA-DR mAb for 3 days. IFN- production was determined by ELISA as described in the text. A, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father by stimulation with allogeneic Fas+/- B-LCL. B, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father by stimulation with allogeneic Fas-/- B-LCL. C, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin-/- patient by stimulation with allogeneic Fas+/- B-LCL. D, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin-/- patient by stimulation with allogeneic Fas-/- B-LCL.

Representative data for the cytotoxic activities mediated by perforin-positive and perforin-negative CD4⁺ and CD8⁺ T lymphocytes specific for alloantigen are shown in Fig. 3. As we reported previously (26), perforin-positive CD4⁺ CTLs exerted similar cytotoxicity against Fas-positive and Fas-negative target cells. The cytotoxicity mediated by CD4⁺, but not CD8⁺, CTLs was inhibited by anti-HLA-DR mAb. In contrast, the cytotoxicity of perforin-negative CD4⁺ and CD8⁺ T lymphocytes against Fas-positive target cells was much less than that of perforin-positive T lymphocytes. The cytotoxicity of perforin-negative CD4⁺ and CD8⁺ T lymphocytes against Fas-negative B-LCL was almost undetectable. These data suggest that the main pathway of cytotoxicity mediated by alloantigen-specific CD4⁺ as well as CD8⁺ CTLs is Fas-independent and perforin-dependent.

View larger version (47K): [in this window] [in a new window]   FIGURE 3. Cytotoxicity of alloantigen-specific CD4+ and CD8+ T lymphocytes against autologous and allogeneic B-LCL. A, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father by stimulation with allogeneic Fas+/- B-LCL. Their cytotoxicity against allogeneic Fas+/- B-LCL in the absence () or the presence () of anti-HLA-DR mAb and against autologous B-LCL (•) was determined. B, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father by stimulation with allogeneic Fas-/- B-LCL. Their cytotoxicity against allogeneic Fas-/- B-LCL in the absence () or the presence () of anti-HLA-DR mAb and against autologous B-LCL (•) was determined. C, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin-/- patient by stimulation with allogeneic Fas+/- B-LCL. Their cytotoxicity against allogeneic Fas+/- B-LCL in the absence () or the presence () of anti-HLA-DR mAb and against autologous B-LCL (•) was determined. D, CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin-/- patient by stimulation with allogeneic Fas-/- B-LCL. Their cytotoxicity against allogeneic Fas-/- B-LCL in the absence () or the presence () of anti-HLA-DR mAb and against autologous B-LCL (•) was determined.

IFN- production by HSV-specific CD4⁺ T lymphocyte bulk lines and clones in response to stimulation with HSV Ag

Four CD4⁺ T lymphocyte bulk lines and seven CD4⁺ T lymphocyte clones directed against HSV were generated from the perforin-positive father, and four CD4⁺ T lymphocyte bulk lines and five CD4⁺ T lymphocyte clones directed against HSV were generated from the perforin-deficient patient. Representative data for IFN- production by these CD4⁺ T lymphocyte lines and clones are shown in Fig. 4. All CD4⁺ T lymphocyte bulk lines and clones examined appeared to secrete a large amount of IFN- in response to stimulation with HSV Ag in the presence of autologous APCs. Their IFN- production was inhibited significantly by anti-HLA-DR mAb, suggesting that the CD4⁺ T lymphocyte bulk lines and clones shown here were HSV-specific and HLA-DR-restricted.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. IFN- production by HSV-specific CD4+ T lymphocytes in response to stimulation with HSV Ag. HSV-specific CD4+ bulk lines and clones were generated from PBMCs of the perforin+/- father (A) and the perforin-/- patient (B). HSV-specific CD4+ T lymphocytes and autologous APCs were cocultured with or without HSV Ag in the presence or the absence of anti-HLA-DR mAb for 3 days. IFN- production was determined by ELISA as described in the text.

We reported previously that HSV-specific human CD4⁺ T lymphocytes can exert cytotoxicity against HSV-infected cells in an HLA class II-restricted manner (2). As in our previous reports, perforin-positive CD4⁺ T lymphocytes specific for HSV lysed HSV-infected, but not mock-infected, autologous B-LCL (Fig. 5). Cytotoxicity against HSV-infected autologous B-LCL mediated by HSV-specific CD4⁺ CTLs was inhibited by anti-HLA-DR mAb. In contrast, the cytotoxicity against HSV-infected autologous B-LCL of HSV-specific CD4⁺ T lymphocytes generated from the perforin-deficient patient was significantly lower than that of perforin-positive CD4⁺ CTLs. These data strongly support our previous report that the main pathway of cytotoxicity against HSV-infected cells mediated by HSV-specific CD4⁺ CTLs is granule exocytosis.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Cytotoxicity of HSV-specific CD4+ T lymphocytes. HSV-specific CD4+ bulk lines and clones were generated from PBMCs of the perforin+/- father (A) and the perforin-/- patient (B). Their cytotoxicity against HSV-infected autologous B-LCL in the absence () or the presence () of anti-HLA-DR mAb and mock-infected autologous B-LCL (•) was determined.

IFN- production by alloantigen-specific T lymphocytes in response to stimulation with allogeneic keratinocytes

We next attempted to examine the cytotoxic mechanisms of CD4⁺ CTLs against IFN--treated keratinocytes, because IFN--treated keratinocytes are known to be sensitive to Fas-mediated cytotoxicity (37). Four CD4⁺ T lymphocyte bulk lines, four CD4⁺ T lymphocyte clones, and three CD8⁺ T lymphocyte bulk lines, four CD4⁺ T lymphocyte bulk lines, three CD4⁺ T lymphocyte clones, and three CD8⁺ T lymphocyte bulk lines were established from T lymphocytes of the perforin-positive father and the perforin-deficient patient, respectively. Representative data are shown in Fig. 6. Similar to allogeneic B-LCL-specific T lymphocytes, established CD4⁺ and CD8⁺ T lymphocyte lines and clones produced IFN- in response to stimulation with allogeneic IFN--treated keratinocytes. The addition of anti-HLA-DR mAb resulted in the reduction of IFN- production by CD4⁺, but not CD8⁺, T lymphocytes.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. IFN- production by allogeneic keratinocyte-specific CD4+ and CD8+ T lymphocytes in response to stimulation with allogeneic keratinocytes. Allogeneic keratinocyte-specific CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father (A) and the perforin-/- patient (B). Allogeneic keratinocyte-specific CD4+ and CD8+ T lymphocytes were cocultured with or without allogeneic keratinocytes in the presence or the absence of anti-HLA-DR mAb for 3 days. IFN- production was determined by ELISA as described in the text.

The cytotoxic activities of allogeneic keratinocyte-specific CD4⁺ and CD8⁺ CTLs are shown in Fig. 7. CD4⁺ as well as CD8⁺ CTLs exerted strong cytotoxicity against IFN--treated allogeneic keratinocytes. The cytotoxicity of CD4⁺, but not CD8⁺, CTLs against keratinocytes was inhibited by anti-HLA-DR mAb, as it was for allogeneic B-LCL-specific CTLs. It was noteworthy that perforin-negative CD4⁺ T lymphocytes exerted cytotoxicity against IFN--treated keratinocytes, although at a level significantly lower than that of perforin-positive T lymphocytes. In contrast, the cytotoxicity of perforin-negative CD8⁺ T lymphocytes against allogeneic keratinocytes was extremely low.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Cytotoxicity of allogeneic keratinocyte-specific CD4+ and CD8+ T lymphocytes. Allogeneic keratinocyte-specific CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father (A) and the perforin-/- patient (B). Their cytotoxicity against allogeneic keratinocytes in the absence () or the presence () of anti-HLA-DR mAb was determined.

As shown in Fig. 7, allogeneic keratinocyte-specific CD4⁺ T lymphocytes generated from the patient with perforin deficiency showed a low level of cytotoxicity against IFN--treated keratinocytes. It has been reported that IFN- treatment makes keratinocytes more sensitive to Fas-mediated apoptosis (37). On the basis of this previous report we examined whether cytotoxicity of CD4⁺ T lymphocytes against IFN--treated keratinocytes is mediated by the Fas-dependent pathway. As shown in Fig. 8, the cytotoxicity of perforin-positive CD4⁺ CTLs against allogeneic keratinocytes was almost completely inhibited by CMA, which is a potent inhibitor of the perforin-mediated cytotoxic pathway (38). Interestingly, however, cytotoxicity mediated by perforin-deficient CD4⁺ T lymphocytes against keratinocytes appeared to be only minimally inhibited by CMA, whereas addition of antagonistic anti-Fas Ab and anti-Fas ligand Ab to the assay medium resulted in significant inhibition of cytotoxicity. These data suggest that the perforin-dependent cytotoxic pathway is superior to the Fas-dependent pathway in perforin-expressing CD4⁺ T lymphocytes, and that Fas-dependent cytotoxicity can be generated by perforin-deficient CD4⁺ T lymphocytes, albeit to a relatively low extent.

View larger version (28K): [in this window] [in a new window]   FIGURE 8. Inhibitory effect of anti-Fas and Fas ligand mAbs and CMA on cytotoxicity mediated by allogeneic keratinocyte-specific CD4+ and CD8+ T lymphocytes. Allogeneic keratinocyte-specific CD4+ bulk lines, CD4+ clones, and CD8+ bulk lines were generated from PBMCs of the perforin+/- father (A) and perforin-/- patient (B). Theircytotoxicity against allogeneic keratinocytes in the absence () or the presence of anti-Fas and Fas ligand mAbs () or CMA () was determined.

Finally, we confirmed the absence and the presence of perforin expression in CD4⁺ T lymphocyte clones established from the perforin-deficient patient and her healthy father, respectively. Representative data from the flow cytometric analysis are shown in Fig. 9. As expected, perforin was completely absent from CD4⁺ T lymphocyte clones established from the patient with perforin gene mutations. In contrast, perforin was apparently expressed in all CD4⁺ CTL clones examined that were established from the patient’s father.

View larger version (40K): [in this window] [in a new window]   FIGURE 9. Flow cytometric analysis of perforin expression in CD4+ T lymphocyte clones. Intracellular perforin in CD4+ T lymphocyte clones specific for allogeneic B-LCL (A), CD4+ T lymphocyte clones specific for HSV (B), and CD4+ T lymphocyte clones specific for allogeneic keratinocytes (C) was stained with FITC-conjugated anti-perforin mAb () or FITC-conjugated control Ab () and was analyzed by flow cytometry as described in the text.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanisms of cytotoxicity mediated by CD4⁺ and CD8⁺ CTLs have been examined extensively in murine systems using perforin-knockout mice and Fas-deficient and Fas ligand-deficient mutant mice. Previous studies of the cytotoxic mechanisms of murine CTLs have revealed that the granule exocytosis pathway is dominant in CD8⁺ CTL-mediated cytotoxicity, whereas 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 (19, 20, 21, 22). We examined the cytotoxic mechanisms of human CTLs for the first time using a novel experimental system involving a combination of hereditary perforin-deficient and Fas-deficient cells; this seems to be the counterpart of the murine system using perforin-knockout and Fas gene mutant mice. Interestingly, the results obtained in the present study differed from those reported previously for the murine system. Specifically, the Fas/Fas ligand system plays a minor role, and perforin plays an essential role in Ag-specific cytotoxicity mediated by human CD4⁺ and CD8⁺ CTLs.

The reason for the discrepancy between the mechanism of murine CD4⁺ CTL-mediated cytotoxicity and that of human CD4⁺ CTL-mediated cytotoxicity is unknown. It was reported previously that murine CD4⁺ CTLs possessing perforin-dependent cytotoxic activity can be generated in short term in vitro mixed lymphocyte cultures only in the absence of activated CD8⁺ T lymphocytes (39). This finding suggests that perforin-dependent cytotoxicity mediated by CD4⁺ CTLs is merely an in vitro phenomenon observed only in unusual situations, such as the absence of CD8⁺ T lymphocytes. However, Yoshimi et al. (40) reported that >20% of CD4⁺ T lymphocytes in the peripheral blood of patients with lymphoproliferative disorders developed after allogeneic bone marrow transplantation expressed perforin in their cytoplasm and exerted cytotoxicity through the perforin-dependent pathway. In addition, Appay et al. (41) reported recently that a population of perforin-expressing CD4⁺ T lymphocytes is markedly expanded in patients with chronic viral infections, in particular, HIV infection, strongly suggesting that perforin-dependent cytotoxicity mediated by CD4⁺ CTLs is not merely an artifact of in vitro culture, but is important for the immunoregulatory system in vivo.

It is noteworthy that perforin-deficient CD4⁺ T lymphocytes exerted cytotoxicity against IFN--treated keratinocytes, although to a relatively low extent. Understanding the mechanisms of cytotoxicity against keratinocytes mediated by alloantigen-specific CTLs is important, since epidermal cells are the major targets for graft-vs-host disease (GVHD). Indeed, it has been reported that the reactivity of donor T lymphocytes against recipient keratinocytes, rather than against recipient hemopoietic cells, is associated with the severity of acute GVHD in HLA-identical bone marrow transplantation (42). Cytotoxicity against IFN--treated keratinocytes mediated by perforin-deficient CD4⁺ CTLs was significantly inhibited by anti-Fas and anti-Fas ligand mAbs. Taken together with the evidence that treatment with IFN- causes keratinocytes to be more sensitive to Fas-mediated apoptosis, this finding indicates that perforin-deficient CD4⁺ T lymphocytes are capable of exerting cytotoxicity against Fas-sensitive target cells via the Fas/Fas ligand pathway. It is also interesting that treatment of perforin-deficient CD4⁺ T lymphocytes with an inhibitor of vacuolar type H⁺-ATPase, CMA, which inhibits perforin-based cytotoxicity mostly due to accelerated degradation of perforin by an increase in the pH of lytic granules (38), appeared to partially inhibit their cytotoxicity against keratinocytes. It has been reported recently that some EBV-specific CD4⁺ CTLs express granulysin, a recently identified cytolytic mediator associated with exocytotic cytolytic granules (43). Although we did not examine the expression of granulysin in perforin-deficient CD4⁺ T lymphocytes, it seems likely that the granule exocytosis pathway mediated by granulysin is involved in perforin-deficient CD4⁺ CTL-mediated cytotoxicity. However, granulysin-mediated exocytosis seems to be a minor cytolytic pathway, since CMA only slightly inhibited cytotoxicity mediated by perforin-deficient CD4⁺ T lymphocytes.

In addition to the perforin/granzyme and Fas/Fas ligand pathways, TRAIL has been reported to be involved in cytotoxic mechanisms mediated by CD4⁺ CTLs (18). Our present data, however, indicate that the TRAIL-mediated cytotoxic mechanism is not an important pathway for Ag-specific cytotoxicity against B-LCLs and keratinocytes mediated by human CD4⁺ CTLs. Khanolkar et al. (44) reported recently that EBV-specific CD4⁺ CTLs can mediate bystander lysis of susceptible target cells through both the Fas/Fas ligand and the TRAIL pathways, but that the dominant mechanism of cytotoxicity following cognate HLA class II-restricted recognition of EBV-transformed cells is the perforin/granzyme pathway. These findings support our present data and suggest that the Fas/Fas ligand and TRAIL pathways are involved mainly in bystander cytotoxicity against susceptible targets mediated by CD4⁺ T lymphocytes following Ag-specific recognition.

Comparison of the clinical manifestations of ALPS, a hereditary disorder involving Fas gene mutations, and FHL, involving perforin gene mutations, may provide important information for understanding the roles of Fas and perforin in resistance against viral infections in vivo. Patients with ALPS usually present with enlargement of lymphoid organs resulting from accumulation of CD3⁺TCR⁺CD4^-CD8^- T lymphocytes and with autoimmune disorders; however, severe viral infections are rare in these patients (45, 46). In contrast, FHL is a fatal disorder characterized by fever, skin rashes, hepatosplenomegaly, pancytopenia, hypertriglyceridemia, coagulopathy, and neurological involvement. Severe viral infections, especially herpesvirus infections, occur frequently and may cause serious complications in patients with FHL (47, 48). Indeed, our patient died from disseminated human CMV and human herpesvirus 6 infections. Taken together with these clinical observations, the present data strongly suggest that the perforin/granzyme-dependent cytotoxic pathway, which is used by CD4⁺ as well as CD8⁺ CTLs, plays an important role in resistance against viral infections and in rejection and GVHD in recipients of allogeneic organ transplantation.

	Footnotes

 
¹ This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Welfide Medicinal Research Foundation; the Sagawa Cancer Research Foundation; the Cancer Research Foundation; the Yamanouchi Foundation for Research on Metabolic Disorders; the Uehara Memorial Foundation; the Takeda Science Foundation; and the Japan Medical Association.

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: yasukawa{at}m.ehime-u.ac.jp

³ Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; ALPS, autoimmune lymphoproliferative syndrome; CMA, concanamycin A; FHL, familial hemophagocytic lymphohistiocytosis; GVHD, graft-vs-host disease; LCL, lymphoblastoid cell line; MMC, mitomycin C.

Received for publication October 15, 2002. Accepted for publication December 6, 2002.

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