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Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China; and Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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9-fold that of
Th1-prone C57BL/10 mice. More than 90% of activated
NK1-CD4+8- thymocytes did not use
the invariant V
14-J281 chain characteristic of typical
CD1-restricted NK1+CD4+ T cells.
Stat6-null
NK1-CD4+8- thymocytes produced
bioactive IL-4, with induction of IL-4 mRNA expression within 1 h
of stimulation. Our results support the possibility that TCR
repertoire-diverse conventional NK1-CD4+ T
cells are a potential IL-4 source for directing naive T cells toward
Th2/type 2 CD8+ T cell (Tc2) effector
development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Two likely characteristics can be expected of a cell capable of providing IL-4 used for priming naive T cells into IL-4-secreting effectors: 1) IL-4-independent IL-4 gene inducibility and 2) rapid induction kinetics. Cells that fulfill both these criteria include NK1 T cells, mast cells, basophils, and eosinophils (5, 6, 7). However, it is difficult to envisage how allergens of such widely diverse structures can all activate IL-4 production by either NK1 T cells that express a highly limited TCR repertoire (reviewed in Ref. 8) or by mast cells, basophils, and eosinophils that express no Ag-specific receptor at all. Considering the large structural repertoire of allergens and the highly Ag-specific nature of the response they induce, it would seem most straightforward to propose conventional T cells, cells known to express a diverse TCR repertoire, as the source of IL-4 that is used to initiate the cascade of events that lead ultimately to IgE production. The major stumbling block to this idea is that little, if any, IL-4 can be detected for naive T cells upon TCR stimulation (1, 2, 3). We re-examined relevant literature on this subject because 1) the origin of IL-4 that is used to direct Th2/Tc2 development potentially lies at the most upstream regulatory point of IgE response and 2) Th2 responses independent of NK1 CD4+ T cells have been reported (9, 10, 11, 12, 13, 14). In the absence of intentional IL-4 priming, CD4+ cells that have been reported to produce IL-4 include neonatal spleen and thymus CD4+ T cells (15, 16), adult thymus heat-stable Ag (HSA)lowCD4+8- thymocytes (15, 17, 18), and NK1+CD4+8- thymocytes (19, 20). Two key reports are noteworthy. First, IL-4 production by adult thymus CD4+8- T cells was confined to the NK1+ subset with nearly no demonstrable IL-4 activity within the NK1- subset (20). The second is the readily demonstrable IL-4-producing cells for the CD44- subset of thymus HSAlowCD4+8- thymocytes, although its frequency was 3-fold lower than the CD44+ subset (17). If indeed all IL-4 production is attributed to NK1+ T cells (20), one would have expected a much lower frequency of IL-4-producing cells among HSAlowCD44-4+8- thymocytes than that reported (17), due to the uniformly CD44high nature of NK1+CD4+ T cells (21). Although it is not immediately clear as to the reason for the inconsistent results concerning IL-4 production by NK1-CD4+8- thymocytes, a major difference was the age of the mice used. While Bendelac et al. (17) used 6-wk-old mice to demonstrate IL-4 production by HSAlowCD44-4+8- thymocytes, Arase et al. (20) found no IL-4 production by NK1-CD4+8- thymocytes from mice 10 to 13 wk of age. This age difference, together with the observation that neonatal T cells are able to produce IL-4 (15, 16) and mount Th2 responses (22, 23), raised the possibility of an age-dependent decline of IL-4 inducibility in NK1-CD4+8- thymus T cells. In this paper we present supporting data for the age-dependent nature of IL-4 inducibility in NK1-CD4+8- thymus T cells. In addition, other characteristics consistent with NK1-CD4+8- thymus T cells as a potential source of IL-4 used for Th2/Tc2 priming are also presented.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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C57BL/10ScN breeders originally obtained from the Division of Research Service, National Cancer Institute, National Institutes of Health (Frederick, MD) were bred and housed under specific pathogen-free conditions at the Institute of Molecular Biology Animal Facility, Academia Sinica (Taipei, Taiwan). BALB/cByJ mice were obtained from the National Laboratory of Animal Breeding and Research Center (Taipei, Taiwan). Stat6-null mice on a mixed B6/129 background were kindly provided by Cynthia Watson and Dr. William E. Paul (Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).
T cell isolation
Single-cell thymocyte suspensions were twice depleted of
CD8+ cells by adherence to culture plates coated
with anti-CD8 mAb by a modified procedure used to deplete spleen
CD8+ T cells (24). For the
first-cycle CD8+ cell depletion, 5 x
107 to 7.5 x 107
total thymocytes in 5 ml of HBSS + 5% FCS were added to each 100
x 20 mm Nunclone culture plate (Nunc, Roskilde, Denmark) that was
previously coated with 10 µg of anti-CD8 (clone 3.155, Ref.
25), at room temperature for 50 min. Nonadherent cells
from two to three first-cycle anti-CD8-coated plates were pooled
and added to another culture plate that had been coated with 20 µg of
anti-CD8, again at room temperature for 50 min. Two-cycle
CD8-depleted C57BL/10 thymocytes were stained with a combination of
FITC-anti-NK1 (clone PK136, Ref. 26), PE-anti-CD4
(clone GK1.5, Ref. 27), Cy5-anti-CD44 (clone IM7, Ref.
28), and Texas Red (TR)-anti-CD8 (clone 2.43, Ref.
25). The stained cells were subjected to electronic cell
sorting using a FACStarPlus
fluorescence-activated cell sorter equipped with dual laser excitation
(Becton Dickinson, San Jose, CA).
NK1-CD4+8- phenotype was used as
the sorting criteria for conventional C57BL/10
CD4+ T cells used in Figs. 1
and 2.
NK1-CD44low4+8-
was used as the sorting criteria for C57BL/10 and Stat6-null
CD4+ T cells used in
Figs. 3–6. For BALB/c mice,
CD44lowCD4+8-
phenotype was used as the sorting criteria for cells throughout. Sort
purity was monitored by reanalysis of sorted cells and was always
>98%.
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T cells were stimulated by plate-bound anti-CD3 + anti-CD28. Individual wells of 96-well culture plates (Nunc) were coated overnight with 50 µl of anti-CD3 (clone 500A.A2, Ref. 29) + anti-CD28 (clone 37.51, Ref. 30), each at 10 µg/ml in PBS. The indicated number of T cells was added per well in 0.1 ml of Mishell-Dutton medium (31) containing 5% FCS (HyClone, Logan, UT), 50 mM HEPES, and 5 x 10-5 M 2-ME. Where indicated, rIL-2 (Biogen, Cambridge, MA; expressed in weight amounts equivalent to a reference standard provided by Dr. Craig Renolds, Biological Response Modifiers Program, National Cancer Institute, National Institutes of Health, Frederick, MD) was added at 0.5 ng/ml.
Purification and fluorochrome conjugation of mAbs
Purified mAbs were obtained from hybridoma culture supernatant
by affinity chromatography. Columns containing Sepharose 4B beads
conjugated with mouse anti-rat 
mAb (clone MAR18.5, Ref.
32) were used to purify 3.155, 2.43, and IM7 as previously
described (24). Protein A-Sepharose columns were used to
purify PK136 and 500A.A2 as previously described (24).
Anti-CD28 mAb was similarly purified except that a polyvalent goat
anti-hamster Ig-conjugated Sepharose 4B column was used. FITC
conjugation was performed as previously described (33).
Cy5 (Amersham, Buckinghamshire, U.K.) and TR (Molecular Probes, Eugene,
OR) conjugations were performed according to manufacturer’s
suggestions. Staining by all fluorochrome-conjugated Abs was specific
because >98% of the observed fluorescence was inhibited by relevant
unlabeled Ab but unaffected by irrelevant control Ab.
Cytokine bioassay
Culture supernatant from activated T cells was 2-fold serially
titrated and assayed for IL-4 using the CT.4S indicator cells
(34). One unit of IL-4 was defined as the amount that
induced half maximal proliferation as assessed by
[3H]thymidine incorporation of CT.4S indicator
cells. Because all IL-4 bioassys were performed in microwells
containing 0.1 ml of culture medium, 1 U of IL-4 under our assay
condition is equivalent to 10 U/ml. The lower limit of detection was
arbitrarily defined by 3 SD above the mean of
[3H]thymidine incorporated by CT.4S cells
cultured in medium alone. IL-4 detected by the CT.4S bioassay was
verified by addition of 11B11 anti-IL-4 mAb (35) which
blocked the activity to background levels. Using recombinant IL-4
(provided by Dr. William E. Paul) as a reference standard, 1 ng/ml of
IL-4 was found to be equivalent to 2000 U/ml derived from the CT.4S
assay as performed in our laboratory.
Cytokine quantitation by competitive RT-PCR
Expression of IL-2 and IL-4 mRNA was analyzed by competitive RT-PCR essentially as described (36), except that ß-actin was used as a control instead of hypoxanthine phosphoribosyltransferase. The ß-actin competitive DNA was constructed as follows. A 285-bp PFU (Stratagene, La Jolla, CA)-amplified PCR product (forward, 5'-AAGGTGTGATGGTGGGAATG-3' and reverse, 5'-ATGGCTACGTACATGGCTGG-3') was blunt-end cloned into HincII-digested pBlueScript II KS(+/-) (Stratagene). A 100-bp spacer DNA derived from an intron of mouse IL-12 p35 gene was cloned into the BglII site internal of the 285-bp actin fragment. Total RNA from indicated cells was extracted (Ultraspec RNA Isolation System; Biotecx, Houston, TX). Escherichia coli tRNA (Sigma, St. Louis, MO) was added at 3 µg/sample. RNA samples in 7 µl of diethyl pyrocarbonate water were denatured for 5 min at 65°C, quickly chilled on ice, and reverse transcribed into cDNA in a total volume of 21.5 µl reverse transcription (RT) buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2) containing 100 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD), 20 U RNasin (Promega, Madison, WI), 50 µg/ml random hexamer primers (Amersham Pharmacia, Uppsala, Sweden), 0.3 mM dNTP (Amersham Pharmacia), and 10 mM DTT. The reaction was stopped by incubation at 95°C for 5 min. Quantitation of mRNA expression was performed in a two-stage procedure. In the first stage, rough estimates of gene expression were determined using 10-fold serial titrating amounts of competitive DNA. Based on stage 1 results, competitive DNA was added in small increments to cDNA from a fixed number of activated T cells to determine equivalence points (36). The PCR was run for 35 cycles (94°C for 30 s, 56°C for 45 s, and 72°C for 40 s) and the products analyzed by 2% agarose gel electrophoresis. Primers for IL-4 were as previously described (36). Actin primers were as described above. The amount of competitive DNA at the equivalence point was arbitrarily normalized to the amount of wild-type mRNA of 500 activated T cells. Relative cytokine mRNA expression was then determined by taking the ratio of cell number-normalized cytokine equivalence to control ß-actin equivalence.
Single-cell RT-PCR
Using the automated cell deposition unit attachment of
FACStar+ cell sorter (Becton Dickinson),
activated T cells were deposited one cell into each Terasaki well
(Robbins Scientific, Sunnyvale, CA) containing 4 µl of RT buffer (50
mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 (pH 8.3)
containing 2% Triton X-100, 0.1 mg/ml BSA, 0.5 mM spermidine, 10
ng/µl oligo(dT12–18) (Amersham Pharmacia), 0.5
mM dNTP, 0.8 U/µl RNasin, and 5 U/µl murine leukemia reverse
transcriptase). Identical sources of dNTP, RNasin, and murine leukemia
reverse transcriptase as described above were used. The RT reaction
mixture was incubated for 90 min at 37°C and stored frozen at
-70°C until PCR was performed. Nested IL-4 PCR was first started
using the external primer set to amplify one-third cell equivalent of
the RT reaction product. One-tenth of the first PCR product was
subjected to a second PCR using internal primers. PCR products were
analyzed by 2% agarose gel electrophoresis. Single cells that were
identified to be IL-4 mRNA+ were subjected to
analysis of V14-J281 and TCR -chain constant region (C)
expression, also by nested PCR. Identical conditions (40 cycles at
94°C for 30 s, at 55°C for 60 s, and at 72°C for
90 s) were used for all nested PCR amplification. The
nested PCR product sizes for IL-4, V14-J281, and C were 159,
173, and 289 bp, respectively, and the primer sequences were IL-4
external forward, 5'-CATCGGCATTTTGAACGAGGTCA-3' and IL-4 external
reverse, 5'-CTTATCGATGAATCCAGGCATCG-3'; IL-4 internal forward,
5'-CCTCACAGCAACGAAGAAC-3' and IL-4 internal reverse,
5'-AAGCCCGAAAGAGTCTCTG-3'; V14-J281 external forward,
5'-GGTGGTTCAAACAGGACAC-3' and V14-J281 external reverse,
5'-GTTTTGTCAGTGATGAACGT-3'; V14-J281 internal forward,
5'-GTCAAATGGGAGATACTCAGC-3' and V14-J281 internal reverse,
5'-CAGGTATGACAATCAGCTGAGTCC-3'; C external forward,
5'-CAGAACCTGCTGTGTACCAG-3' and C external reverse,
5'-GATTCGGAGTCCCATAACTG-3'; C internal forward,
5'-CCCTCTGCCTGTTCACCGACTT-3' and C internal reverse,
5'-CGGAGTCCCATAACTGACAG-3'.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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C57BL/10
NK1-CD4+8-
thymus T cells were stimulated by plate-bound anti-CD3 +
anti-CD28 mAb. Significantly more IL-4 was produced by
NK1-CD4+8-
thymocytes from young adolescent than older mice, displaying an inverse
correlation for mice within the 25- to 50-day age window (Fig. 1
A). For mice >50 days of
age, IL-4 production was uniformly low. Consistent with already
published results (21, 37, 38), an age-dependent increase
in
NK1+CD4+8-
T cells, expressed either as percentage of total
CD4+8- T cells (Fig. 1B) or as total cell number per thymus (Fig. 1C),
was observed. Therefore, the age-dependent nature of IL-4 inducibility
in NK1-CD4+ T cells
follows a pattern distinct and opposite that of
NK1+CD4+ T cell
development.
The nonobese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus spontaneously. Although periinsulitis characterized by mononuclear cell infiltration is detectable as early as 3 wk of age, clinical diabetes marked by islet destruction does not develop until quite some time later and is most evident in 3- to 4-mo-old NOD mice (39). In view of our observed age-dependent decline of IL-4 production by thymus NK1-CD4+ T cells in mice <50 days (or 7 wk) of age, and the protection of autoimmune diabetes by systemic IL-4 administration (40) or by pancreatic ß cell-specific IL-4 transgene expression (41), it is tempting to speculate that the failure of young adolescent NOD mice to develop clinical symptoms, despite periinsulitis, is due to the IL-4 production by recent thymic emigrants that have infiltrated the pancreas.
Heightened IL-4 production by CD4+8- thymus T cells from Th2-prone BALB/c mice
For
NK1-CD4+8-
T cells to be considered as the source of IL-4 that directs Th2/Tc2
development, their IL-4-producing capacity might be expected to be
significantly higher in the prototypic Th2-prone BALB/c mice than the
Th1-prone C57BL/10 mice (reviewed in Ref. 42). Because
PK136 anti-NK1.1 mAb does not detect NK1+ T
cells in BALB/c mice and because virtually all
NK1+CD4+ T cells are
CD44high (21), we used
CD44low4+8-
phenotype as the criteria for isolation of
NK1-CD4+-equivalent T
cells. For mice under 2 mo of age, the average IL-4 production by
BALB/c thymus CD44lowCD4+ T
cells was 1842 U/ml (n = 5; Fig. 2B), a value 9-fold higher
than the 197 U/ml observed for their counterparts in C57BL/10 thymuses
(n = 18; Fig. 2A). For mice >2 mo of age,
IL-4 production by C57BL/10 and BALB/c thymus
CD44lowCD4+ T cells was 43
U/ml (n = 14) and 776 U/ml (n = 5),
respectively (Fig. 2, A and B). Age-associated
change indexed by (<2 mo) ÷ (> 2 mo) average IL-4 production
values revealed a more significant decline factor of 4.6 for C57BL/10
than the 2.4 for BALB/c. Although the BALB/c genetic background may be
intrinsically more resistant to an age-associated decline, the
absorption/utilization of IL-4 by activated T cells can be expected
to bias ratio determination of bioactivity in culture supernatant
(production - consumption), especially for low IL-4-producing
cells.
Our observation of heightened IL-4 production by BALB/c thymus CD44low4+8- T cells correlates well with elevated IL-4 production during the early phase (3–4 days) of Leishmania infection of nonhealing BALB/c but not healing C3H or B6 strains (43, 44), supporting a role for thymus NK1-CD4+ T cells in the initiation of Th2 immune response.
TCR stimulation of <2 mo BALB/c spleen
CD44low4+8-
T cells resulted in IL-4 production that ranged from 62 to 524 U/ml,
with an average value of 173 U/ml (Fig. 2D). IL-4 production
by similarly stimulated C57BL/10
NK1-CD44low4+8-
T cells ranged from 10 to 20 U/ml with an average of 15 U/ml (Fig. 2C). Despite the generalized low IL-4 production levels by
spleen CD4+ T cells, Th2-prone BALB/c spleen
CD4+ T cells nevertheless produced on average
>10-fold more IL-4 than their Th1-prone C57BL/10 counterparts. This
relationship was not seen in mice >2 mo of age as uniformly low 24
U/ml of IL-4 production was observed for both BALB/c and C57BL/10
spleen CD4+ T cells (Fig. 2, C and
D).
Because immune responses take place in specialized peripheral lymphoid
compartments such as the spleen and not the thymus, it is difficult to
explain our finding of much lower IL-4 production by spleen than thymus
NK1-CD4+8-
T cells if peripheral
NK1-CD4+ T cells indeed
are the IL-4 source for Th2/Tc2 priming. On the other hand, although
the amount of IL-4 produced by spleen
NK1-CD44lowCD4+
T cells is rather low, it is nevertheless detectable, particularly in
spleens of young BALB/c mice (Fig. 2D). The critical
question is whether the relatively low level IL-4 production by spleen
NK1-CD44lowCD4+
T cells we observed is sufficient to direct Th2/Tc2 development.
Although our results do not address this question directly,
understanding the nature of the depressed IL-4 inducibility by spleen
NK1-CD44lowCD4+
T cells in comparison with their immediate thymic precursors may shed
some light on this seeming paradox. First, it is possible that T cells
possessing prompt IL-4 gene inducibility do not exit the thymus,
implicating an intrathymic IL-4 role. If this were the case, one would
expect to find IL-4 expression in freshly isolated thymus
NK1-CD4+ T cells. Because
no IL-4 was detected in freshly isolated
NK1.1-CD44lowCD4+
T cells (see Figs. 4 and 6), it appears unlikely that IL-4 plays an
intrathymic role. This conclusion is also consistent with the reported
normal intrathymic T cell development in IL-4-null mice
(45). Alternatively, prompt IL-4 gene inducibility in
thymus
NK1-CD44lowCD4+
T cells is temporally expressed, due either to its developmental
stage-specific nature or to the rapid death rate of cells that express
this phenotype. Elegant work by Bendalac et al. (17)
showed that IL-4-producing
CD4+8- thymocytes can and
do leave the thymus, a process associated with a rapid loss of IL-4
inducibility. Although it is likely that a substantial fraction of
recent thymic emigrants capable of IL-4 production are
NK1+CD4+ T cells, this was
not formally established because markers specific for NK1
CD4+ T cells were not used in the
identification of recent thymic emigrants (17). Related to
this was the finding of IL-4-producing cells among
CD44lowHSAlowCD4+8-
thymocytes, although its frequency was 3-fold less than that of
CD44highHSAlowCD4+8-
thymocytes (17). Because NK1 CD4 T cells are uniformly
CD44high (21) and Arase et al.
(20) reported that all the IL-4-producing activity was
found within the minor subset of
NK1+CD4+8-
thymocytes, it is not clear as to why IL-4-producing cells were so
readily identified for
CD44lowHSAlowCD4+8-
thymocytes in the Bendelac report. One major difference between these
reports was the age of the mice used. While Bendelac et al. used
5-wk-old mice, 10- to 13-wk-old mice were used for the Arase study. It
is therefore possible that the IL-4-producing
CD44lowHSAlowCD4+8-
thymocytes from 5-wk-old mice reported by Bendelac et al. contained a
significant number of
NK1-CD4+ T cells, in
addition to NK1 (CD44high)
CD4+ T cells. Indeed, this possibility is
directly supported by our finding of age-associated decline in IL-4
inducibility among
NK1-CD4+8-
thymus T cells (Fig. 1A). Whether the previously
demonstrated postthymic down-regulation of IL-4 inducibility
(17) can also be applied to
NK1-CD4+ T cells in
addition to the nonconventional CD1-restricted NK1
CD4+ T cells is now an open question.
Minimal V14-J281 TCR usage by IL-4-producing
NK1-CD4+ T cells
Because the pattern of age-dependent IL-4 inducibility in
NK1-CD4+ T cells is
opposite that of NK1+CD4+ T
cell development (Fig. 1), it is possible that
NK1-CD4+ T cells with the
potential to make IL-4 are the direct precursors of
NK1+CD4+ T cells and the
age-dependent nature is simply a reflection of
NK1- 
NK1+ conversion.
If this were the case, one would expect a strong bias toward invariant
TCR V14-J281 chain usage, a known characteristic of
NK1+CD4+ T cells (reviewed
in Ref. 8). To address this possibility,
CD44low4+8-
T cells were stimulated by plate-bound anti-CD3/CD28. Day 2
activated single cells were subjected to RT-PCR analysis using IL-4,
V14-J281, and C-specific primer sets. Of the 600 activated
C57BL/10
NK1-CD44low4+8-
T cells analyzed, 11 (1.8%) expressed IL-4 mRNA. Of these 11 IL-4
producers, only one (9%) was V14-J281+,
whereas all were positive for C mRNA (Fig. 3B). For activated BALB/c
conventional
CD44low4+8-
thymocytes, a higher frequency of IL-4-producing cells (15 IL-4
producers or 8.3% of 180 analyzed single cells) was observed (Fig. 3A), consistent with the higher IL-4 production levels in
bulk cultures reported in Fig. 2. Of the 15 BALB/c IL-4 producers, one
(6.7%) was V14-J281+, whereas 15 of 15
(100%) were positive for C mRNA (Fig. 3B). Consistent
with previously published results (8), 90% (9 of 10)
IL-4-producing NK1+CD4+ T
cells expressed V14-J281 mRNA, whereas all expressed C.
Therefore, the vast majority of thymus
NK1-CD4+8-
T cells capable of producing IL-4 do not use V14-J281 and are
therefore unrelated to the
NK1+CD4+ T cell
lineage.
Stat6-independent and prompt IL-4 production by thymus NK1-CD4+8- T cells
Two criteria expected of the IL-4 source for Th2/Tc2 priming
response are 1) rapid production kinetics and 2) Stat6 independence
(46, 47). The kinetics of IL-4 gene activation by
NK1-CD44low4+CD8-
thymocytes was analyzed for Stat6-null and wild-type
C57BL/10 control mice by competitive RT-PCR (Fig. 4A) and by CT.4S bioassay
(Fig. 4B). Extremely rapid and similar induction patterns
were observed for both C57BL/10 (wild-type) and Stat6-null
NK1-CD44lowCD4+8-
thymocytes such that within 1 h of TCR stimulation, IL-4 mRNA
induction reached 31-fold (0.373 ÷ 0.0121) for C57BL/10 and
33-fold (0.299 ÷ 0.0091) for Stat6-null cells. This
rapid IL-4 gene induction kinetics for in vitro-activated
NK1-CD4+8-
T cells was similar to that of in vivo activated NK1 T cells
(19) and qualifies thymus
NK1-CD4+8-
T cells as a potential source of IL-4 for Th2/Tc2 priming. IL-4 mRNA
expression continued to increase after 1 h and peak induction
levels of 82-fold (0.995 ÷ 0.0121) for C57BL/10 and 91-fold
(0.829 ÷ 0.0091) for Stat6-null cells were similarly
observed at 16 h poststimulation. IL-4 mRNA expression
was clearly detectable for the entire 40-h observation period, although
slightly lower induction levels (62-fold for C57BL10 and 68-fold for
Stat6-null) were observed by the 40-h time point. Similar
levels of IL-4 bioactivity were detected by 16 h after
anti-TCR stimulation for both C57BL/10 and Stat6-null
NK1-CD44low4+8-
thymocytes. Time-dependent and steadily increasing amounts
of IL-4 bioactivity in culture supernatant is consistent with the
finding of significant IL-4 mRNA induction levels at all time points
studied during the entire 40-h observation period (Fig. 4A).
Our finding of much more rapid IL-4 production kinetics by in
vitro-stimulated
NK1-CD44low4+8-
thymus T cells than NK1 T cells (19) is most consistent
with a lack of relatedness between these two types of IL-4-producing
cells, a conclusion also supported by the infrequent TCR V14-J281
chain usage by IL-4-producing
NK1-CD4+ T cells
(Fig. 3).
Minimal V14-J281 TCR usage by IL-4-producing
Stat6-null thymus NK1-CD4+ T
cells
The nearly identical magnitude and kinetics of IL-4 gene
activation for C57BL/10 and Stat6-null
NK1-CD44low4+8-
T cells (Fig. 4) is suggestive of similar (non-V14-J281) TCR
usage. However, it is nevertheless important to formally demonstrate
the lack of V14-J281 usage by Stat6-null
NK1-CD44low4+8-
T cells. To test this, Stat6-null
NK1-CD44lowCD4+8-
thymocytes were compared against wild-type C57BL/10 thymocytes and
V14-J281-biased
NK1+CD4+ thymocytes (Fig. 5). Day 2 anti-CD3/CD28-activated
single cells were subjected to RT-PCR analysis using IL-4,
V14-J281, and C-specific primer sets. Of the 600
activated Stat6-null
NK1-CD44lowCD4+8-
T cells analyzed, 11 (1.8%) expressed IL-4 mRNA. All of the 11 IL-4
producers expressed C mRNA, but only one (9%) was
V14-J281+. Similar to results shown in Fig. 3, control C57BL/10
NK1-CD44lowCD4+8-
T cells contained 2.0% (12 positives of a total of 600 single cells
analyzed) IL-4 mRNA+ cells. Although all 12
IL-4+ cells expressed C mRNA, only
one (8.3%) used V14-J281. Consistent with previously published
results (8) and results shown in Fig. 3, 83% (10 of 12)
IL-4-producing NK1+CD4+ T
cells expressed V14-J281 mRNA and were all
C+. Because the vast majority (>90%) of
IL-4-producing Stat6-null thymus
NK1-CD44lowCD4+8-
T cells did not use V14-J281, they are unrelated to the typical
V14-J281-biased lineage of
NK1+CD4+ T cells.
IL-4 production by mature HSAlow subset of thymus NK1-CD44low4+8- T cells
Because
NK1-CD44lowCD4+8-
thymocytes contain a continuum of immature to mature T cells,
IL-4-producing potential may be maturation stage specific. To address
this possibility,
NK1-CD44lowCD4+8-
T cells were separated into mature (HSAlow) and
immature (HSAhigh) subsets (48, 49)
and examined for their abilities to produce IL-4 (Fig. 6). Because immature
(HSAhigh) thymus CD4+ T
cells are known to be poor IL-2 producers (50), exogenous
IL-2 was supplied to ensure T cell activation induced by
anti-CD3/CD28 (51). Competitive RT-PCR (Fig. 6A) and bioassay (Fig. 6B) both showed IL-4 gene
activation mainly within the mature (HSAlow)
subset of thymus
NK1-CD44lowCD4+8-
T cells, with minimal IL-4 induction in the
HSAhigh subset. The slight gain in IL-4
expression by HSAlow over
HSAall cells is consistent with a relatively low
percentage (3.2%) of immature HSAhigh cells
among thymus
NK1-CD44lowCD4+8-
T cells. Restriction of IL-4 inducibility to within
HSAlow mature thymus CD4+ T
cells, along with a relatively low frequency of IL-4-producing cells,
raises an interesting possibility of a distinct
NK1-CD4+ T cell
subset/lineage as the IL-4 source for Th2/Tc2 priming. It should be
noted that the relatively low percentage of immature
HSAhigh cells in our preparation of thymus
NK1-CD44lowCD4+8-
T cells appears to differ from the previously reported higher frequency
of HSAhigh cells among unfractionated thymus
CD4+8- T cells (49, 50). The most likely explanation for this apparent discrepancy
is that our method of isolating thymus CD4+ T
cells involved depletion of CD8+ cells on plates
coated with 3.155 anti-CD8 mAb, a procedure that has been shown to
deplete the immature (HSAhigh) subset of thymus
CD4+8- T cells
(51).
Taken together, the results presented here demonstrate the existence of NK1-CD4+ T cells within the thymus capable of rapid and Stat6-independent IL-4 production. The more prominent IL-4 inducibility in young adolescent mice is consistent with the relative ease and frequent development of allergic symptoms in young children (52, 53). The heightened IL-4 inducibility in NK1--equivalent BALB/c thymus CD4+ T cells correlates well with its Th2-prone nature and raises the possibility that genetically linked allergies may stem from prompt IL-4 release by recent thymic emigrant NK1-CD4+ T cells. Furthermore, the likely potential for the IL-4-producing NK1-CD4+ T cells to express a diverse TCR repertoire is also consistent with the widely varying structures of allergic substances.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John T. Kung, Institute of Molecular Biology, Academia Sinica, Nankang 11529, Taiwan, Republic of China. E-mail address:
3 Abbreviations used in this paper: HSA, heat-stable Ag; TR, Texas Red; C, TCR -chain constant region; RT, reverse transcription.
Received for publication April 9, 1999. Accepted for publication August 16, 1999.
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References |
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