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Introduction |
The T cell antigen receptor (TCR) is a multimeric
complex consisting of subunits that function primarily
either in antigen recognition (
/
or 
/
) or signal transduction (CD3-, -, and -
, and a 
family dimer) (1). The
family members constitute a group of structually and
functionally related proteins that include , 
(an alternatively spliced form of ), and FcRI (Fc; references 1, 2).
Thymocytes from mice lacking expression of both and
chain (/
/) are reduced in number and express extremely low levels of surface TCR relative to /+/+ mice
(3). Nevertheless, T cell development is not completely arrested in // mice as they contain both CD4+CD8+,
or double positive (DP),1 and CD4+ CD8 and CD4
CD8+, or single positive (SP) thymocytes and peripheral
SP T cells (3). In contrast, expression of the CD3 (//)
complex is absolutely required for thymocyte development, as mice lacking expression of CD3- subunits fail to
develop beyond the the most immature CD4 CD8, or
double negative (DN), stage (6). The transition of DN thymocytes into DP thymocytes is regulated by the pre-TCR,
a signaling complex composed of chain, pre-T, and
CD3 subunits, and which is also thought to include a family dimer (7). The fact that low numbers of DP thymocytes (10-30% of normal) are generated in // mice
indicates that, though important, and are not essential for pre-TCR function. Likewise, the presence of low
numbers of SP thymocytes and peripheral T cells in //
mice (3) demonstrates that / chains are not absolutely
required for /-TCR expression or for promoting T cell
development. Because of the extremely low levels of surface expression in // mice, the subunit composition of
surface pre-TCR and TCR complexes has not been accurately determined. One possibility is that the pre-TCR and
TCR can be expressed in the absence of a family dimer,
and function, albeit inefficiently, to promote thymocyte development. Another possibility is that in // mice
the pre-TCR and/or /-TCR complexes associate with
Fc chain homodimers, since Fc is reported to be expressed during early thymocyte development (8). In
mice lacking Fc, thymocyte development is unaffected,
and therefore Fc normally does not play a significant role
in the development of thymus-dependent T cells (11).
Nevertheless, Fc, together with chain, functions as a
component of the TCR complex expressed on restricted
populations of T cells ("thymus-independent" T cells), and
in both Fc/ and // mice these T cells express relatively high levels of surface TCR (4, 5, 10). In addition,
overexpression of Fc chain (or chain) in thymocytes
restores TCR surface expression and /-T cell development in // mice (12). Therefore, all of the family
proteins are capable of independently supporting /-TCR
surface expression and promoting the development of /
-TCR+ thymocytes.
In this study, we have generated mice lacking all three family proteins (//-Fc/ mice) and compared the T
cell populations present in these animals to those found in
mice lacking either / or Fc alone. The results provide
direct evidence that pre-TCR and /-TCR complexes
lacking a family dimer are capable of supporting T cell
development, positive selection, and T cell activation.
Moreover, they reveal that, in the absence of specific stimuli, Fc is not normally expressed in thymus-dependent T
cell populations, whereas both / and Fc are expressed
in thymus-independent T cells. A possible function for the
restricted expression of different family proteins may be
to modify the TCR signaling response in distinct populations of T cells.
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Materials and Methods |
Mice.
The generation of // mice and Fc/ mice has
been previously described (3, 11). // (Fc+/+) mice were
mated to (/+/+) Fc/ mice and F1 progeny from these matings (i.e., /+/-Fc+/) were then mated. Since the / and
Fc loci map to the same region of mouse chromosome 1 (2), F2
progeny were screened for crossover events that resulted in either
a //-Fc+/ or /+/-Fc/ genotype. One //+-
Fc/ mouse, identified among the first 100 F2 progeny analyzed,
served as a founder line for the generation of //-Fc/
mice. Genotypes were identified initially by Southern blotting as
previously described (3, 11) and were subsequently screened by
PCR. Screening of / was performed with oligonucleotides Z1: 5'-GAAGAGAGGAATATGACGTCTTGGAGAAGA-3'; Z2:
5'-AAGGACGATCTGAGTACTGAG-3'; and ZNEO: 5'-TTCTGGATTCATCGACTGTGG-3'. PCR parameters were: 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s × 35 cycles. Screening of
Fc was performed with oligonucleotides 4081: 5'-CTCACGGCTGGCTATAGCTGCCTT-3'; 4087: 5'-ACCCTACTCTACTGTCGACTCAAG-3'; and 2969: 5'-CTCGTGCTTTACGGTATCGCC-3'. PCR parameters were: 94°C for 20 s, 55°C
for 20 s, and 72°C for 30 s × 35 cycles. PCR products were resolved on a 2% agarose gel and visualized by staining with ethidium bromide.
Antibodies and Multicolor Flow Cytometry.
mAbs used for flow
cytometric analysis were purchased from PharMingen (San Diego,
CA) and included fluorochrome (FITC or PE) or biotin-conjugated anti-CD4 (RM4.5); anti-TCR- (H57-597); anti-CD8- (53-6.7); anti-CD8- (53-5.8); anti-CD3-, (145-2C11); anti-CD25 (7D4); anti-CD44 (IM7); anti-CD69; anti-B220 (RA3-6B2); anti-CD24 (M1/69); anti-NK1.1 (PK136); anti-IL-2R
(TM-1); and anti-/-TCR (GL3). Ascites from hybridoma
2.4G2 was a kind gift of J. Titus (National Institutes of Health)
and was purified in our laboratories. Streptavidin red 670 (GIBCO BRL, Gaithersburg, MD) was used in conjunction with
biotinylated antibodies. Cells were stained with specific antibodies and analyzed on a Becton Dickenson Immunocytometry Systems FACScan® using standard Cell Quest software as previously
described (13).
Cell Preparations and Stimulation.
Thymus, spleen, and lymph
nodes were excised from mice and single cell suspensions were
prepared. Intestinal intraepithelial lymphocytes (i-IELs) were prepared from the small intestine as previously described (13). Dendritic epithelial T cells (DETCs) were obtained from trunk skin
and were prepared as previously described (15). For thymocyte
activation, DP thymocytes were isolated as previously described
(16) and incubated at 37°C on plates that had been previously
coated with anti-TCR- (H57-597). After 12-16 h, the stimulated cells were assessed for CD69 or CD5 surface expression by
flow cytometric analysis (FCM) as previously described (14).
Lymph node T cells were purified from total lymph node cell
suspensions by magnetic bead separation (Miltenyi Biotec, Auburn, CA) using biotinylated anti-CD4 and anti-CD8 and streptavidin microbeads. Purity of magnetically separated T cell populations assessed by FCM was 
90% for all samples.
Cytokine Assay
For cytokine assays, purified T cells (106)
were either cultured with media alone, media containing PMA
(10 ng/ml; Sigma Chemical Co., St. Louis, MO) and ionomycin
(1 µM; Sigma Chemical Co.), or plate-bound anti-CD3 (145-2C11) for 18 h. Supernatants were collected and elaboration of
IFN-, IL-4, and IL-2 were measured by ELISA. Quantitation of
IFN- and IL-4 were determined according to manufacturer's
instructions (Endogen, Cambridge, MA). IL-2 ELISAs were performed using the reagents and protocol obtained from PharMingen.
For semiquantitative reverse transcription (RT) PCR, cells
were pelleted and RNA was extracted with STAT-60 reagent
(Tel-Test Inc., Friendswood, TX) and treated with DNase to remove contaminating genomic DNA. The quality of RNA preparations was assessed by gel electrophoresis, and RNA was reverse
transcribed using the Superscript Preamplification system
(GIBCO BRL). Serial dilutions (1:2 to 1:128) of the RT reaction
were made and then amplified by PCR using oligonucleotides
corresponding to cyclophilin A: 5'-GGGTGGTGACTTTACACGCCATAATG-3' and 5'-TCAAAAGAAATTAGAGCTGTCCACAGTCGG-3'; and CD3: 5'-TACAAAGTCTCCATCTCAGG-3' and 5'-TGGCCGCTCCTTGTTTTG-3'. PCR
reactions were stopped after 25 cycles and again after 35 cycles
then run on a 2% agarose gel. Bands were visualized by staining
with ethidium bromide and quantitated by densitometry. Dilutions of the RT reaction that yielded equivalent control bands
were then amplified for 35 cycles using primers specific to IL-2,
IL-4, and IFN- (Clontech, Palo Alto, CA). For all PCR reactions, parameters were: 94°C for 3 min, then 35 cycles of 94°C
for 45 s, 60°C for 45 s, 72°C for 2 min, then 72°C for 5 min.
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Results |
Thymocyte Development in Mice Lacking Expression of All Family Proteins (,, and Fc).
Mice lacking expression
of all family proteins (//-Fc/) were generated as
described in Materials and Methods. Examination of thymocytes from //-Fc/ mice by FCM revealed a
phenotype essentially identical to that of // (Fc+/+)
mice (Fig. 1 A). In addition, // and //-Fc/
mice contained similar total numbers of thymocytes (10-
30% of normal, data not shown), which consisted almost
entirely of DN and DP cells. DP thymocytes from //
mice express extremely low but detectable levels of TCR
as assessed by staining with anti-CD3- (3-5, 14, and Fig.
1 A) and anti-TCR- mAbs (3, 14). A similarly low but
discernable level of TCR surface expression was observed
on DP thymocytes from //-Fc/ mice by FCM
(Fig 1 A). Significantly, although CD4+CD8 and
CD4CD8+ SP cells were not readily detectable in the
thymus, SP T cells were present in the lymph nodes (data
not shown) and spleen (Fig. 1 B) of //-Fc/ mice
in numbers similar to those observed in // mice (data
not shown). Together, these findings demonstrate that the
low but detectable level of TCR expression on DP thymocytes from // mice, and the ability to generate
"mature" SP T cells are not dependent upon the expression
of endogenous Fc chain.
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Fig. 1.
Phenotypic analysis of thymocytes and T cells from /+/+-Fc+/+, //-Fc/ , //-Fc+/+, and /+/+-Fc/ mice. (A) CD4,
CD8, and CD3 expression on total thymocytes. (B) CD4 versus CD8 expression on ungated splenocytes. CD3 expression is shown on CD4+ and CD8+
gated splenocytes (CD4+/CD8+). (C) CD25 versus CD44 expression on gated (CD4CD8CD3B220) thymocytes. Immunofluorescence and flow
cytometric analysis was performed with thymocytes and splenocytes from young adult (4-8 wk-old) mice. Shaded areas represent staining with experimental antibodies and solid lines represent staining with negative control antibody. Numbers in quadrants of dual parameter histograms represent the percentage of cells contained within that quadrant. (D) Induction of CD4+CD8+ thymocytes in RAG1/, // and in RAG1/, //-Fc/ mice
by anti-CD3- antibodies. 6-12-wk-old mice were injected intraperitoneally with 200 µl PBS containing 75 µg of affinity purified CD3- specific antibody 145-2C11. Control animals were injected with 200 µl PBS. 6 d after injection, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8. Results shown are representative of three separate experiments for each genotype.
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Thymocyte development has been shown to be severely
compromised at the DN stage in // mice (17). Whereas
the most mature subset of DN thymocytes (CD44CD25)
constitutes 10-20% of the DN population in normal adults
and in mice lacking only Fc (Fig. 1 C), these cells are
nearly absent in // mice (reference 17 and Fig. 1 C).
Examination of DN thymocyte subsets from both //
and //-Fc/ mice revealed a block at the identical
(CD44/loCD25+ 
CD44CD25) stage of maturation
(Fig. 1 C), indicating that neither Fc nor / chain is required
for thymocyte development before the CD44/loCD25+
stage. Since the generation and subsequent expansion of
CD44CD25 DN thymocytes is thought to be controlled
by signaling through the pre-TCR complex (7, 18), the
paucity of CD44CD25 DN thymocytes in // mice
implies a function for (and/or ) as components of the pre-TCR. However, the fact that some DP thymocytes are
generated in both // and //-Fc/ mice indicates that the pre-TCR is capable, albeit inefficiently, of
transducing signals that promote the development of DN
thymocytes to the DP stage in the absence of all family
proteins. To further evaluate the ability of surface complexes expressed on DN thymocytes in //-Fc/ mice
to transduce signals that promote the formation of DP thymocytes, we generated //-Fc/ RAG-1/ mice.
Thymocytes from mice deficient in RAG-1 or RAG-2 are
blocked in their development at the DN stage but can be
induced to differentiate to the DP stage upon stimulation
with anti-CD3 mAb (19). Injection of anti-CD3- mAb
into both //-Fc+/+ RAG-1/ (Fig. 1 D and reference 20) and //-Fc/ RAG-1/ mice (Fig. 1 D)
resulted in increased thymic cellularity (5-20× control)
and the generation of large numbers of DP thymocytes. Together these data demonstrate that both early and late
stages of thymocyte development are not absolutely dependent on the expression of family proteins.
TCR Complexes Expressed on DP Thymocytes and T Cells
from //-Fc/ Mice Transduce Activating Signals.
Maturation of DP thymocytes into SP T cells is controlled by
TCR-mediated signals that are received during positive selection in the thymus (21). Although DP thymocytes from
// mice express barely detectable levels of surface TCR,
these TCR complexes are nevertheless capable of transducing signals that result in the positive selection of low numbers of thymocytes (3). Cross-linking of surface TCR
complexes on DP thymocytes from // mice results in
the upregulation of CD69 and CD5 (reference 14 and Fig.
2), events which are associated with positive selection in
vivo (22). To determine if a similar TCR-mediated signaling response could be elicited in thymocytes from //-
Fc/ mice, DP thymocytes were stimulated in vitro with
antibodies directed against either CD3- or TCR-. Significantly, in vitro cross-linking of the TCR complexes on DP
thymocytes from //-Fc/ mice with anti-TCR-
(Fig. 3) or anti-CD3- (data not shown) induced upregulation of both CD69 and CD5. Moreover, the extent of CD5
and CD69 upregulation was similar in thymocytes from / /-Fc/ and //mice after stimulation (Fig. 2).
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Fig. 2.
CD69 and CD5 upregulation on DP thymocytes in response
to TCR engagement. DP thymocytes were purified and stimulated for
12-16 h on plates coated with either PBS or anti-TCR-. Cells were
then stained with anti-CD69 or anti-CD5 and analyzed by FCM. Shaded
areas depict cells stained with anti-CD69 or anti-CD5 after anti-TCR-
stimulation. Solid lines depict cells stained with anti-CD69 or anti-CD5
after incubation in media without antibody stimulation.
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Fig. 3.
Surface phenotype of
peripheral T cells from //-
Fc/ mice. Activation-memory
phenotype of splenic T cells. Cells
were stained with anti-CD4-PE versus anti-CD44-FITC, anti-CD62L-FITC, anti-CD2-FITC, or anti-CD5-FITC. Single color histograms
show staining on CD4+ T cells.
Data were collected on 5 × 103
gated cells. Shaded areas represent
staining with experimental antibodies and solid lines depict staining
with negative control antibodies.
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To determine if the TCR complexes expressed on SP T
cells from //-Fc/ mice were also capable of transducing activating signals, we examined lymph node T cells for
expression of cell surface molecules associated with activation and memory. Surprisingly, although T cells from //-
Fc/ mice express extremely low levels of surface TCR,
a high percentage of these cells appeared to have an activated or memory phenotype (i.e., CD44hi, CD62Llo; Fig.
3). A high percentage of SP T cells from // were also
CD44hi, CD62Llo, whereas the majority of T cells from
both control (/+/+-Fc+/+) and Fc/ mice displayed
a naive phenotype (CD44lo, CD62Lhi; Fig. 3). Nevertheless,
T cells from both //-Fc/ and // mice were
largely refractory to direct TCR stimulation in vitro as they
did not appreciably increase levels of CD69 or IL-2R and
proliferated poorly in response to treatment with cross-linking anti-TCR antibodies or anti-TCR plus anti-CD28
(data not shown).
We next examined the ability of T cells from //
and //-Fc/ mice to produce cytokines after stimulation for 18 h with either PMA and ionomycin or anti-
CD3- mAb. Stimulation of purified T cells from //
and //-Fc/ mice (as well as from Fc/ and /
+/+-Fc+/+ mice) resulted in production of IL-2 (Table
1), but in all samples IL-4 and IL-10 remained undetectable
(data not shown). However, although T cells from /+/+-
Fc+/+ and Fc/ mice produced only low levels of IFN-
after stimulation, T cells from both // and //-
Fc/ mice produced large quantities of IFN- after stimulation (Table 1). A similar cytokine profile was observed
when cytokine production was assessed by RT-PCR (Fig.
4). IFN- mRNA was also detectable in freshly isolated ex
vivo (unstimulated) T cells from //-Fc/ mice and
// mice by RT-PCR (Fig. 4). Since purified populations of T cells were used for these experiments, it is unlikely that IFN- was derived from contaminating cell
populations, such as NK cells. Together, these findings indicate that despite their low levels of surface TCR, a high
percentage of T cells from both // and //-Fc/
mice appear to be endogenously activated and exhibit a
Th1 memory cell phenotype.
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Fig. 4.
Semiquantitative RT-PCR for detection of IFN-, IL-2, or
IL-4 mRNA. RNA obtained from unstimulated purified splenic CD4+
and CD8+ T cells (ex vivo) or after 18 h of stimulation with PMA + ionomycin was reverse transcribed and amplified with oligonucleotide primers specific for IL-2, IL-4, and IFN-. Reactions were standarized by performing PCR with oligonucleotides corresponding to cyclophilin and
CD3-, whose mRNAs should be equivalent in purified T cell populations. +, indicates homozygosity for the wild-type / or Fc alleles;  indicates homozygosity for the mutant / or Fc alleles, as indicated.
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i-IEL Populations in //-Fc+/+, /+/+-Fc/,
and //-Fc/ Mice.
Distinct populations of lymphocytes have been defined within the intestinal epithelium (i-IELs, references 23, 24). Athough some of these
cells (CD4+ and/or CD8-/+) appear to be dependent
on the thymus for their generation, those expressing a homodimer of CD8- (CD8-/) are thought to arise through a thymus-independent developmental pathway
(24). Similar to peripheral (lymph node and spleen) CD4+
and CD8-/+ /-T cells, thymus-dependent i-IEL
populations are unaffected in Fc/ mice (11) but are reduced in number and are TCRlo/ in both // mice (4,
5, 25) and //-Fc/ mice (data not shown). These
results are consistent with the idea that thymus-dependent
i-IELs express / but not Fc during their development.
On the other hand, thymus-independent i-IELs have been
shown to express both and Fc chains (8), and mice lacking either / or Fc contain /-TCR+ CD8-/+ and
/-TCR+ i-IELs that express only moderately reduced
levels of surface TCR when compared with similar populations of i-IELs from /+/+-Fc+/+ mice (references 4, 5,
and Fig. 5). Significantly, in the absence of , , and Fc
chains, all population of i-IELs (thymus-dependent and -independent) are TCRlo/ (Fig. 5).
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Fig. 5.
i-IEL development in //-Fc/ mice i-IELs were prepared from mice as described (13) and three-color FCM was performed.
For internal staining, cells were first stained with anti-CD4 and anti-
CD8- externally, then treated with intracellular staining buffer followed
by staining with anti-CD3, anti-TCR-, or anti-TCR- mAbs. Data
depict two-color analysis of CD3 versus TCR- or CD3 versus TCR-
on software-gated CD4 CD8- cells. Numbers reflect the percentage
of gated CD4CD8- cells in that quadrant.
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To examine the lineage of TCR i-IELs in //-
Fc/ mice, cells were analyzed for the presence of intracellular TCR- and TCR- chains. Interestingly, intracellular staining for TCR- chains revealed that TCR-/
lineage T cells are virtually absent in //-Fc/ mice
whereas TCR-/ lineage T cells are readily detected in the same animals (Fig. 5, bottom). Notably, TCR-/+ cells
are also markedly reduced in number in // mice, but
not Fc/ mice, despite the fact that i-iELs from these animals express comparable levels of surface TCR (references
4, 5 and Fig. 5, 4th and 5th columns). Together, these findings indicate that either (a) the generation and/or survival
of TCR-/+ T cells is particularly dependent on expression of / chain, or (b) that Fc is poorly expressed in developing / lineage T cells. Finally, these results demonstrate that expression of a family dimer is required for
efficient TCR surface expression on all T cell populations
including both thymus-dependent and thymus-independent TCR-/+ and TCR-/+ cells.
/+-TCR DETCs and NK1.1+ T Cells Use Chain for
TCR Surface Expression.
We next examined mice lacking
expression of /, Fc, or all family proteins for the presence of DETCs that express /-TCR and are thymically-derived (26). We observed that though present, DETCs
from both // mice and //-Fc/ mice express
extremely low or undetectable levels of surface TCR, whereas
DETCs from Fc/ mice express high levels of /-TCR
(Fig. 6 A). These results were unexpected as it had been previously reported that / mice contain DETCs that express relatively high levels of /-TCR (27).
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Fig. 6.
Phenotype of DETC and DN NK1.1+ thymocytes from /+/+-Fc+/+, //-Fc+/+, /+/+-Fc/, and //-Fc/ mice. (A)
DETCs were purified as described in Materials and Methods and stained with antibodies directed against TCR- and CD3- or Thy 1 and CD45. (B)
Expression of surface TCR on NK1.1+ thymocytes from /+/+-Fc+/+, //-Fc+/+ and //-Fc/ mice. Shown are two-color plots of
NK1.1 versus. /-TCR or /-TCR on gated (CD24) thymocytes. Numbers in quadrants refer to percent of NK1.1+ thymocytes that express TCR
(/-TCR or /-TCR). Single-color plots show expression of /-TCR on gated NK1.1+ thymocytes. Shaded areas in single color histograms represent staining with negative control antibodies.
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We also examined thymocytes from // and //-
Fc/ for the presence of NK1.1+ T cells that are also
thymically derived but not necessarily thymus-dependent
(30). Although both // and //-Fc/ mice
contained thymocytes of the expected "activation-NK"
phenotype (i.e., NK1.1+, IL-2R+, CD44+, MEL-14)
TCR+ cells were detectable only in // mice and these
cells were exclusively /-TCRlo (Fig. 6 B). Significantly,
although an earlier study had reported the presence of large
numbers of NK1.1+ /-TCR+ thymocytes in / mice
(31), we found that NK1.1+ /-TCR+ T cells were virtually undetectable in both // and //-Fc/
mice (Fig. 6 B) . The most likely explanation for the striking variance between our results and those of previous
studies is that the latter analyzed / mice in which chain is expressed (28); thus chain most likely contributed to the TCR surface expression observed on DETCs and NK1.1+ thymocytes from these mice.
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Discussion |
The results of this study demonstrate that "partial" TCR
complexes that lack family proteins (, , and Fc) can
promote the maturation of at least some thymocytes. Indeed, thymocytes from //-Fc/ mice appear to undergo a relatively normal developmental program; originating from precursor DN thymocytes they develop to the DP stage, undergo positive selection, and emerge as SP T cells.
T cells generated in //-Fc/ mice express a functionally active TCR such that stimulation of these complexes by direct engagement results in the production of
specific cytokines. Collectively, these observations indicate that pre-TCR and TCR complexes that contain CD3 subunits but not a family dimer can transduce signals normally associated with fully assembled TCR complexes.
Although some thymocytes are capable of developing in
// and
Family Members (
, and Fc
RI
)
Top
Abstract
Introduction
