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International Immunology, Vol. 12, No. 7, 1095-1103, July 2000
© 2000 Japanese Society for Immunology

Peripheral blood extrathymic CD4+CD8+ T cells with high cytotoxic activity are from the same lineage as CD4+CD8– T cells in cynomolgus monkeys

Ki-Hoan Nam1,3, Hirofumi Akari2, Keiji Terao1, Hiroaki Shibata1, Seiji Kawamura3 and Yasuhiro Yoshikawa3

1 Tsukuba Primate Center, National Institute of Infectious Diseases, 1 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan
2 Department of Virology, School of Medicine, University of Tokushima, 3 Kuramoto, Tokushima 770-8503, Japan
3 Department of Biomedical Science, Faculty of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Correspondence to: K. Terao


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Reference
 
We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin–granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-{gamma} and produced a high level of IFN-{gamma} when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR Vß with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.

Keywords: function, non-human primate, origin


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Reference
 
There exist two major subsets of human T lymphocytes identified by the expression of the differentiation antigens, CD4 and CD8, which direct TCR to MHC class II and I respectively. In human peripheral blood lymphocytes (PBL), most of the T lymphocytes express either CD4 or CD8 and a low percentage (<3%) of double-positive (DP) T cells with blastoid morphology is usually found (1–3). However, some individuals with neoplastic infectious diseases or without obvious diseases were shown to possess a much higher percentage of DP T lymphocytes in blood, although such cases were very rare (4–7).

In contrast, higher percentages of peripheral blood DP T lymphocytes were found in rhesus monkeys (Macaca mulatta) (8,9), cynomolgus monkeys (Macaca fascicularis) (10–12) and African green monkeys (Cercopithecus aethiops) (13). Our previous works have revealed marked differences of DP T cells in their phenotype, in vivo distribution and age-dependent increase of DP T cells compared with CD4 single-positive (SP) or CD8 SP T cells in cynomolgus monkeys (11,12,14). The DP T cells were of the resting memory phenotype expressing CD2hi, CD3+CD28–, CD29hi, CD49dhi, CD69– and CD80lo. In addition, DP T cells were CD8{alpha}+ß– and CD1b–, while thymic DP T cells were CD8{alpha}+ß+ and CD1b+. These results strongly suggested that the DP T cells are a novel extrathymic T lymphocyte lineage (11). However, the precise characteristics of the DP T cells in rhesus monkeys and African green monkeys are still unknown.

Here we show, for the first time, the functional characteristics of DP T cells in cynomolgus monkeys. DP T cells showed a similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells, and showed dual functions of helper and strong cytotoxic activities mainly using the perforin–granzyme pathway. In addition, some expanded in vivo DP T cell clones shared TCR Vß with corresponding expanded CD4 SP T cell clones, strongly suggesting that these two corresponding clones in DP and CD4 SP T cells are derived from the same cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Reference
 
Preparation of cells
Fresh blood was obtained from healthy cynomolgus monkeys aged 10–20 years who were bred and reared at Tsukuba Primate Center, National Institute of Infectious Diseases. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque (Pharmacia, Milton Keynes, UK) and washed with RPMI 1640, then suspended in RPMI 1640 supplemented with 50 IU penicillin, 50 µg streptomycin, 2 mM L-glutamine, 50 µM 2-mercaptoethanol and 10% heat- inactivated FCS (we will refer to this medium as medium).

Antibodies and reagents
Antibodies used were FITC-labeled or purified anti-CD3 mAb (clone FN18; Biosorce, Camarillo, CA), FITC- or phycoerythrin (PE)-labeled anti-CD4 mAb (clone NU-TH/1; Nichirei, Tokyo, Japan), PE- or PE–Cy5 (Cy5)-labeled anti-CD8 (clone Leu-2a; Becton Dickinson, San Jose, CA), purified or FITC-labeled anti-CD28 (clone L293 Becton Dickinson), PE-labeled anti-human CD95 mAb (clone DX2; PharMingen, San Diego, CA), FITC- or PE-labeled anti-CD20 (clone Leu-16; Becton Dickinson), and isotype-matched control mAb (Becton Dickinson) and purified or horseradish peroxidase-labeled goat anti-monkey IgG mAb (Nordic Immunological, Tiburg, Netherlands). Purified anti-CD20 mAb (clone 2H7) was kindly provided from Dr Edward Clark (University of Washington, Seattle, WA). The standard monkey IgG was purified and recombinant monkey Fas ligand (rmFasL) (Kirii et al., manuscript in preparation) was synthesized in our laboratory. Pokeweed mitogen (PWM), concanavalin A (Con A) and phytohemagglutinin (PHA) were purchased from Sigma (St Louis, MO).

Cell sorting
The PBMC were reacted with mAb (FITC-labeled anti-CD4, PE-labeled anti-CD16 and Cy5-labeled anti-CD8 mAb) at 4°C for 30 min for isolation of CD4 SP, DP and CD16/CD8 SP T cells. Stained PBMC were then washed with medium and the desired cells were sorted out by an Epics Elite (Coulter, Hialeah, FL). The purity of sorted cells was always >98 %.

B cells were isolated using MiniMACS (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. In brief, PBMC were stained with anti-CD20 mAb (2H7) and then reacted with microbead-labeled anti-mouse IgG antibody (Miltenyi Biotec). The cells were passed through a MiniMACS magnetic column. The trapped cells were recovered; >96% of the recovered cells were CD20+ B cells, while CD3+ T cells were <1%.

Cell stimulation with mitogens or anti-CD3 antibody
Ninety-six-well flat-bottom plates were coated with 100 µl of anti-CD3 mAb at the indicated concentrations by overnight incubation at 4°C and then washed with PBS 3 times. The sorted DP, CD4 SP and CD8 SP T cells (5x104 cells/well) were cultured in the plates in total volumes of 100 µl. After indicated culture periods which included pulsing with [3H] thymidine (1 µCi/well) during the last 12 h, these cells were harvested on a Filter Mat (Skatron Instrument, Sterling, VA). The [3H]thymidine incorporation was measured with a liquid scintillation counter (Aloka, Tokyo, Japan). All samples were performed in triplicate.

Fas ligation and MTT assay
To examine whether DP T cells are apoptotic, sorted T cells (1x105 cells/200 µl medium/well) were treated with various concentrations of rmFasL in a 96-well flat-bottom plates for 20 h, then further incubated for 4 h with 10 µl (5 mg/ml in PBS) MTT (Sigma) per well. Optical density at 450 nm was measured after adding 100 µl HCl–isopropanol solution per well. Percentage of live cells was calculated by a standard curve which was obtained at the same time with graded numbers of sorted cells.

Helper activity for B cell Ig production
Various numbers of sorted T cells were added to 3x104 autologous B cells on 96-well flat-bottom plates. The mixtures were cultured in the medium containing PWM (10 µg/ml) for 1 week. The amount of IgG released into the culture supernatant was measured by ELISA using goat anti-monkey IgG antibody and peroxidase-labeled goat anti-monkey IgG antibody. After colorizing with o-phenylenediamine (Sigma), optical density at 450 nm was measured by a Microplate Reader (Molecular Devices, Sunnyvale, CA).

Anti-CD3 antibody-redirected cytotoxicity activity
The mouse mastocytoma cell line, P-815, cells were labeled with 51Cr for 3 h and then washed 3 times with medium. The labeled cells were reacted with anti-CD3 mAb at a concentration of 20 µg/ml at 4°C for 2 h. After washing with medium, 100 µl of the P-815 cells (5x103 cells) was added to each well of 96-well round-bottom plates. One hundred microliters of sorted T cells was added to target cells at various E:T ratios. After incubation for 4 h at 37°C, released 51Cr was measured with a {gamma}-counter (Aloka, Tokyo, Japan). Specific cytotoxic activity was calculated by the formula: (measured c.p.m. – spontaneous c.p.m.)/(total c.p.m. – spontaneous c.p.m.). All samples were performed in triplicate.

Semiquantitative RT-PCR
mRNA was isolated from fresh PBMC or sorted T cells (1x106 cells) using a QuickPrep micro mRNA Purification kit (Pharmacia Biotech, Uppsala, Sweden). The cDNA was synthesized from mRNA using a First-strand cDNA synthesis kit (Pharmacia Biotech). Published PCR primer pairs (15) for IFN-{gamma} (rhesus monkey), IL-2 (cynomolgus monkey), IL-4 (human), perforin (human), granzyme B (human) and GAPDH (human) were used with some modifications (see Table 1Go). PCR was performed in 50 µl reaction containing 5 µl of 10xEx Taq Buffer (Takara, Shiga, Japan), 4 µl of dNTPs (Takara), 0.5 µl (2.5 U) of Ex Taq DNA polymerase (Takara), 3 µl of 10 µM of cytokine primers and 3 µl of 10 µM of GAPDH primers (30 s at 94°C, 30 s at 60°C and 1 min at 72°C, 28 cycles). The predicted lengths of amplified DNA products were confirmed by staining the gel with ethidium bromide after 5% PAGE and the signal density was analyzed by Quantity One software (pdi, Huntiton Station, NY).


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  		Table 1. Oligoncleotide primers used
 
ELISA for IFN-{gamma} detection
To detect IFN-{gamma} produced by DP T cells, sorted T cell subsets were stimulated with immobilized anti-CD3 antibody (5 µg/ml) on 24-well flat-bottom plates with a total volume of 800 µl (5x105 cells/well). After incubation in a humidified incubator at 37°C for 2 days, the supernatant was subjected to detection of IFN-{gamma} using an ELISA kit specific for the macaque monkey (Biosource, Camarillo, CA) according to the manufacturer's instruction.

Single-strand conformation polymorphism (SSCP) for TCR analysis
cDNA from each sorted T cell population (1x106 cells) was synthesized as described above. Primers for each TCR family in cynomolgus monkeys were described in our previous study (16). PCR were performed in 50 µl reactions (30 s at 94°C, 30 s at 60°C and 1 min at 72°C, 35 cycles). SSCP clonotype analysis was performed under essentially the same conditions as described by Illés et al. (17). In brief, the amplified products for each Vß (3 µl) were diluted (1:1) with denaturing solution comprising 95% formamide, 10 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol, and heat-denatured at 94°C for 3 min. The product was then loaded (4 µl) onto a non-denaturing 4% polyacrylamide gel containing 10% glycerol. After electrophoresis, the DNA was transferred onto nylon membrane (Biodyne A; PAL, Washington, NY) and hybridized with a biotinylated Cß-specific internal probe after fixation with UV light (50 mJ/cm2). The DNA was visualized by a chemiluminescent substrate system (Phototope Detection kit; New England Biolabs, Beverly, MA).

Direct TCR DNA sequencing
A small area of the SSCP gel corresponding to the band was cut out and then DNA was extracted by incubating with water at 95°C for 20 min. The extract was submitted to a second amplification by PCR (30 s at 94°C, 30 s at 60°C and 1 min at 72°C, 32 cycles) with a corresponding Vß and a Cß primer and then, the PCR product was purified using a Concert Rapid PCR Purification System (Gibco/BRL, Egglestein, Germany). The purified PCR product was subjected to sequencing PCR (30 s at 95°C, 30 s at 60°C and 1 min at 72°C, 30 cycles) using a Thermo Sequenase cycle sequencing kit (Amersham Life Science, Cleveland, OH) with a IRD41-labeled Cß primer (Aloca, Tokyo, Japan). The product was loaded onto sequencing gel containing 50% urea. Electrophoresis and detection was performed with LI-COR sequencing apparatus (Aloca).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Reference
 
Stimulation of DP T cells with mitogens and anti-CD3 mAb
We previously showed that most DP T cells do not express CD28 (11). To investigate whether DP T cells are anergic, we stimulated DP T cells with mitogens, PHA or Con A. As shown in Fig. 1Go(A), DP T cells were activated by mitogenic stimulations, indicating they have the ability to proliferate. Next, to test whether DP T cells can proliferate in response to CD3–TCR signaling, DP T cells were stimulated with immobilized anti-CD3 mAb. DP T cells could proliferate by ligation of CD3 with anti-CD3 mAb at the same level as CD4 SP or CD8 SP T cells (Fig. 1BGo). The proliferation was dose dependent (Fig. 1CGo). The kinetics of proliferation of DP T cells was similar to those of CD4 SP or CD8 SP T cells (data not shown). In addition, when T cell subsets were stimulated with either mitogen [Con A (5 µg/ml) or PHA (10 µg/ml)] or plate bound anti-CD3 mAb (5 µg/ml), the expressions of CD4 and CD8 on three different T cell subsets was stable and most DP T cells did not express CD28 after stimulation (data not shown).



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  		Fig. 1. Responsiveness of DP T cells. DP, CD4 SP, CD8 SP and PBMC (5x104 cells/well) were cultured with mitogen [Con A (5 µg/ml) or PHA (10 µg/ml)] (A) or on anti-CD3 antibody-coated 96-well flat-bottom plates (B and C) for 4 and 5 days respectively with a final 12 h [3H] thymidine pulse. Aliquots of 1 µg/ml anti-CD3 antibody (B) or indicated concentrations of CD3 antibody (C) were used to coat the plate. The c.p.m. of [3H]thymidine incorporated was measured with a liquid scintillation counter. All experiments were performed in triplicate and data shown are the means. Representatives are shown from three independent experiments.

 
Sensitivity of DP T cells to Fas ligation with FasL
Since DP T cells show a memory phenotype (11) and express Fas on their surface (Fig. 2AGo), we compared the effects of Fas ligation on T cell subsets. DP, CD4 SP and CD8 SP T cell subsets were incubated with medium-supplemented graded concentration of rmFasL for 20 h. After an additional culture for 4 h with MTT, surviving cell numbers were measured by colorimetry. As shown in Fig. 2Go(B), there was no difference in induction of apoptosis by ligation of Fas with rmFasL among DP, CD4 SP and CD8 SP T cells.



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  		Fig. 2. Fas expression levels in T cell subsets did not affect the sensitivity of apoptosis induced by rmFasL ligation. (A) DP, CD4 SP and CD8 SP T cells in PBMC (left) expressed different levels of Fas antigen on their surface (right). (B) Apoptosis was induced by incubating sorted T cells (1x105 cells/well) with various concentrations of rmFasL in 96-well flat-bottom plates for 20 h, then further incubated for 4 h after adding 10 µl (5 mg/ml in PBS) MTT to each well. Live cells were calculated by standard curves which were obtained with graded numbers of sorted cells. All experiments were performed in triplicate and data shown are the means. Representatives are shown from three independent experiments.

 
Helper activity of DP T cells
It has been reported that CD4 cells co-expressing CD8 showed helper function in swine (18) or African green monkeys (13). We, therefore, tested whether cynomolgus monkey DP T cells also show the helper activity. DP T cells were co-cultured with autologous B cells with PWM stimulation for 1 week. The cultured supernatant was collected and IgG content in supernatant was measured. As shown in Table 2Go, B cells produced a significant level of IgG in the case of co-culture with DP T cells (100%), although only CD4 SP T cells showed helper activity in wells co-cultured with smaller number of T cells (25%). The highest IgG level was observed when B cells were co-cultured with CD4 SP T cells. On the other hand, B cells co-cultured with CD8 SP T cells did not produce IgG.


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  		Table 2. Helper activity associated with the IgG production of peripheral DP, CD4 SP and CD8 SP T cells
 
Anti-CD3 antibody-redirected cytotoxic activity of DP T cells
DP T cells in cynomolgus monkeys express CD8 which were composed of only the {alpha} chain (11). However, expression of the {alpha} chain alone has been found to be sufficient for the function of the CD8 molecules—enhancing specific antigen recognition of TCR restricted to MHC class I (19,20). Therefore, we tested whether DP T cells have cytotoxic activity. Anti-CD3 mAb-redirected cytotoxic activity was measured, because that specific antigens for DP T cells are unknown at present. A representative result is shown in Fig. 3Go. DP T cells showed comparable cytotoxic activity with that of CD8 SP T cells. On the other hand, CD4 SP T cells did not show significant cytotoxic activity. This cytotoxic activity was specific for anti-CD3 mAb signal, because labeling of P-815 cells with control antibody, anti-CD20 mAb (2H7), did not induce any cytotoxic activity (Fig. 3BGo). The cytotoxic activity obtained was unrelated to NK activity, because DP T cells and CD8 SP T cells did not kill P-815 target cells without antibody (Fig. 3BGo).



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  		Fig. 3. DP T cells showed comparable anti-CD3 antibody-redirected cytotoxic activity with CD8 SP T cells. P-815 cells were labeled with 51Cr and then reacted with either anti-CD3 (A and B), anti-CD20 (B) or no antibodies (B). The P-815 cells (5x103 cells/well) were added to each well of 96-well round bottom plates. DP, CD4 SP, CD8 SP and PBMC were added to target cells at the indicated E:T ratios (A) and at 25:1 (B). Then, a standard 51Cr-release assay was performed. Specific cytotoxic activity was calculated by a formula: (measured c.p.m. – spontaneous c.p.m.)/(total c.p.m. – spontaneous c.p.m.). All experiments were performed in triplicate and data shown are the means. Representatives are shown from four independent experiments.

 
Mechanism of cytotoxic activity used by DP T cells
To determine whether DP T cells use the perforin–granzyme pathway, highly purified (>99.5%) DP, CD4 SP and CD8 SP T cells were subjected to semiquantitative RT-PCR to detect mRNA signals for perforin and granzyme B. As shown in Fig. 4Go, the mRNA signal for perforin was detected in all T cell subsets at a similar level. However, a stronger signal for granzyme B was detected in DP T cells than that in CD4 SP T cells. Although there was an individual difference, the signal density for granzyme B in DP T cells was comparable with that in CD8 SP T cells (Fig. 4Go). These results suggest that DP T cells might kill target cells through the perforin–granzyme pathway.



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  		Fig. 4. mRNA levels for effector molecules of DP T cells. mRNAs from highly purified (>99%) fresh DP, CD4 SP and CD8 SP T cells were subjected to RT-PCR (28 cycles) for IFN-{gamma}, perforin and granzyme B (A). The relative density of each signal, which was normalized to GAPDH signals in (A), was compared with CD8 SP T cells (B). A representative is shown from four independent experiments. M, size marker (bp); 4, CD4 SP T cells; DP, DP T cells; 8, CD8 SP T cells.

 
Cytokine production by DP T cells
To investigate the profile of cytokines produced by DP T cells, mRNA for cytokines from fresh DP T cells was measured. Fresh DP, CD4 SP and CD8 SP T cell subsets expressed comparable levels of mRNA for IFN-{gamma} (Fig. 4Go), However, we could not detect mRNA for IL-2 or IL-4 in fresh T cell subsets (data not shown).

To detect IFN-{gamma} produced from the T cell subsets, DP, CD4 SP and CD8 SP T cells were stimulated for 48 h with immobilized anti-CD3 mAb on 24-well plates. The supernatant was used for measuring of IFN-{gamma} by ELISA. In contrast to the mRNA expression in fresh T cell subsets, activated DP T and CD8 SP T cells produced much higher levels of IFN-{gamma} than CD4 SP T cells (Table 3Go). However, when T cells were cultured in medium only, IFN-{gamma} was hardly been detected in all T cell subsets (Table 3Go).


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  		Table 3. IFN-{gamma} (pg/ml) production by DP T cells stimulated with immobilized anti-CD3 antibody
 
Comparison of TCR usage between DP, CD4 SP and CD8 SP T cell subsets
To investigate the relationship of the ancestors among DP, CD4 SP and CD8 SP T cells, TCR Vß usage was compared by SSCP clonotype analysis (17) using macaque monkey Vß family-specific primers (16). The mRNAs from T cell subsets including PBMC were isolated, and then subjected to RT-PCR and SSCP analysis for each Vß family. The SSCP band patterns from T cell subsets were compared each other and representative Vßs are shown in Fig. 5Go(A). There were several bands in each T cell subset, indicating the presence of expanded T cell clones. Between CD4 SP and CD8 SP T cells, and between DP and CD8 SP T cells, there were no clones that migrated to identical position. In contrast, several bands in DP T cells that migrated to identical positions to the bands in CD4 SP T cells (Fig. 5AGo, in Vß2, Vß4 and Vß16), suggesting that some clones in DP T cells use the same Vß with the corresponding clones in CD4 SP T cells, and thus the two corresponding clones in DP and CD4 SP T cells may be derived from the same T cell. However, there were also unique bands appearing only in DP T cells (Fig. 5AGo, in Vß2, Vß4 and Vß12/13), indicating that the persistence of clones in DP T cells is independent of CD4 SP or CD8 SP T cells.



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  		Fig. 5. Use of identical TCR chains in DP T cells to CD4 SP T cells. mRNAs from highly purified (>99%) fresh DP, CD4 SP and CD8 SP T cells were subjected to SSCP analysis after RT-PCR. Representative Vßs from a monkey are shown (A). Similar results were obtained from four monkeys. (B) TCR sequence from a DP T cell clone was compared with that from a corresponding CD4 SP T cell clone (upper part, Vß2, anti-sense) and the sense sequences are shown (lower part). A representative pair is shown from three pairs. PB, PBMC; 4, CD4 SP T cells; DP, DP T cells; 8, CD8 SP T cells. Asterisks indicate the bands that migrated to the identical position in CD4 and DP T cells. Dashes indicate the bands present in DP T cells but not in CD4 SP and CD8 SP T cells.

 
To confirm the TCR sequence of DP and CD4 SP T cell clones, both of which migrated to the same position in SSCP analysis, we cut out the gel corresponding to the bands from the same position from the DP or CD4 SP clone and sequenced. The TCR sequence of all the three analyzed clones from DP T cells (Vß1, Vß2 and Vß5) was consistent with that in the corresponding CD4 SP T cell clone including the CDR3 region (Fig. 5BGo and data not shown).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Reference
 
The concept of extrathymic T cell differentiation is widely accepted for the origin of T cells in elder individuals (21,22). Clear evidence, however, has not yet been reported for the origin and function of the extrathymic T cell lineage, because no suitable marker is available to detect them (22). Our previous studies have demonstrated that (i) the levels of DP T cells rapidly increase after 10 years of age when thymic involution is completed, (ii) the phenotype of DP T cells differs from those of thymic DP cells in terms of expression of CD8ß and CD1b, and (iii) DP cells show memory phenotypes and distribute mainly in peripheral blood and spleen but rarely in lymph nodes in cynomolgus monkeys. These findings strongly suggest that DP T cells are from an extrathymic T cell lineage (11,12).

Swine is another animal which possesses peripheral DP T cells that have been extensively studied (18,23–25). Swine DP T cells are thought to be derived from CD4 SP T cells by activation (25) and they showed class II MHC-restricted memory Th cell function (18) but no evidence of cytotoxic activity (26–29). The present results of SSCP analysis of TCR clearly showed that the cynomolgus monkey DP T cells shared the same TCR ß chain with CD4 SP T cells. This result strongly suggests that DP T cells are derived from the same ancestors as CD4 SP T cells, although further studies on antigens and MHC restriction for DP T cells are needed. However, monkey DP T cells showed dual functions: low helper and high cytotoxic activities. The lower helper activity in B cell IgG production indicates that DP T cells do not have a professional Th2 function. Further study is needed on the cytokine profile of DP T cells in the activated state.

The most striking result is that DP T cells showed strong cytotoxic activity which was comparable to CD8 SP T cells. This result is completely different from swine DP T cells which show no evidence of cytotoxic activity (26–28). In addition, DP T cells in cynomolgus monkeys decreased in percentages after infection with pathogenic simian immunodeficient virus (30). On the other hand, DP T cells were not changed in absolute number when experimental autoimmune encephalitis was induced by immunization with monkey brain white matter in complete Freund's adjuvant (our unpublished data). These data are consistent to the result that DP T cells have cytotoxic activity.

There are three well-known cytotoxic mechanisms: Fas-mediated, degranulation-mediated, and surface tumor necrosis factor (TNF)-{alpha} mediated pathways (25). Among these, the TNF-{alpha} pathway cannot be detected by standard 4 h 51Cr-release assay (25). DP T cells expressed strong perforin and granzyme B mRNAs. The perforin mRNA level in DP T cells was comparable with those from CD4 SP or CD8 SP T cells. As to the role of perforin in cytotoxic activity, it has been shown that the levels of perforin do not correlate well with overall cytotoxic activities (31) and that the constitutional presence of CD4 SP cells expressing perforin was also demonstrated in healthy humans (32). Meanwhile, only DP and CD8 SP T cells expressed strong message for granzyme B which plays a critical role in cytotoxic T lymphocyte (CTL)-induced apoptosis (33). These results indicate that DP T cells preferentially kill their targets via the degranulation pathway. If the cytotoxic activity we obtained is related with the Fas–FasL pathway, the P-815 target cells should express Fas. However, only <15% of P-815 cells expressed Fas on their surface (data not shown). Furthermore, when 51Cr labeled P-815 cells were cultured for 4 h with 200 µg rmFasL/ml, a dose which can induce maximum apoptosis in PBMC and T cell subsets (data not shown and Fig. 2BGo), the released 51Cr level was not higher than spontaneous release. In addition, CD4 SP T cells, which are considered to use the Fas–FasL pathway to induce target cell death (34), did not show any significant cytotoxic activity (Fig. 3Go). Considering these results, the Fas–FasL pathway is likely minor in the cytotoxicity of DP T cells in cynomolgus monkeys.

Fresh DP T cells produced a high level of mRNA for IFN-{gamma} but not IL-2 or IL-4. This result is consistent with the fact that CD4+ CTL are associated with the Th1, but not with the Th2 phenotype (32). IFN-{gamma} is responsible for directing the cell-mediated immune response leading to the eradication of intracellular pathogens (35,36). IFN-{gamma} as well as IL-12 are important to Th1 development from naive precursor T cells. In particular, if DP T cells are class II MHC restricted, DP T cells could eliminate activated APC or T cells which express class II MHC in order to prevent an over-reaction of the ongoing immune response or to remove potentially hazardous cells. Thus, DP T cells may have regulatory roles in host immunity. In mouse and human, the MHC class II-restricted CD4+ CTL play an important role in viral infections (37) or tumor killing where CD4+ CTL becomes powerful mediators of autologous tumor recognition through significant lysis and cytokine release, including IFN-{gamma} (38).

In addition to difference in functions, there are other differences between DP T cells of macaque and swine. DP T cells in cynomolgus monkeys did not show any increase at least the first 5 years of life and increased abruptly at ~10 years of age when thymic involution had been completed (12), while DP T cells in swine increase in percentage soon after birth (25). We could not find as in vivo or in vitro condition which induced DP T cells from CD4 SP T cells or CD4 SP T cells from DP T cells in cynomolgus monkeys. These differences may be a reflection of the difference in their origin. The role of the CD8 molecule on DP T cells from swine and macaque is still open to question.

The intestinal intraepithelial lymphocyte (IEL) population is interesting. IEL are suggested to be extrathymic originated T cells (39–41). Some CD4+ IEL express CD8 chain (39–40) and expression of CD8 is found to mediate anti-CD3-redirected cytolytic activity (42). Therefore, it is also possible that CD4+CD8+ IEL with CTL activity migrate into blood, then compose the peripheral DP T cell population, showing the unique role(s) of CD8 molecules on DP T cell functions in cynomolgus monkeys. Furthermore, recently, Helgeland et al. described that CD4 SP IEL and CD4/CD8 DP IEL which increase with age, expressed overlapping Vß chain repertoires in rats (43). This result supports another possibility that peripheral blood CD4 SP or DP T cells expressing overlapping Vß chains are released from the IEL pool. Comparison of TCR Vß usage between peripheral DP T cells and DP IEL might give some clues to clarify the origin of peripheral DP T cells.

Finally, some characteristics of DP T cells in cynomolgus monkeys are shared with NKT cells (44) in other species, including phenotype (45,46) and functions (47), although there are differences, including the age of appearance of the cells. We are investigating the relation of DP T cells with NKT cells.

Cynomolgus monkey peripheral DP T cells are a unique T cell population with dual functions which overlap with CD4 SP and CD8 SP T cells. They might have regulatory roles in secondary immune responses in cynomolgus monkeys.


   Acknowledgments
 
We thank Mr T. Ono and Mr H. Narita for animal care and blood collection. This study was supported by funds for Comprehensive Research on Aging and Health from the Japan Foundation for Aging and Health.


   Abbreviations
 
Con A concanavalin A
CTL cytotoxic T lymphocyte
Cy5 phycoerythrin–Cy5
DP double-positive
FasL Fas ligand
IEL intestinal intraepithelial lymphocyte
PBL peripheral blood lymphocyte
PBMC peripheral blood mononuclear cell
PE phycoerythrin
PHA phytohemagglutinin
PWM pokeweed mitogen
SSCP single-strand conformation polymorphism
SP single positive
TNF tumor necrosis factor

   Notes
 
Transmitting editor: K. Okumura

Received 21 January 2000, accepted 31 March 2000.


   Reference
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Reference
 

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