Differential effects of manipulating signaling in early T cell development in intestinal intraepithelial lymphocytes and thymocytes
Stephanie T. Page1,2,
Lisa Y. Bogatzki1,2,
Jessica A. Hamerman1,2,
Marie Malissen4,
Roger M. Perlmutter1,2,3,5 and
Ann M. Pullen1,2,6
1 Howard Hughes Medical Institute, and
2 Departments of Immunology, and
3 Biochemistry and Medicine (Medical Genetics), University of Washington, Seattle, WA 98195, USA
4 Centre d'Immunolgie, INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseilles Cedex 9, France
Correspondence to:
A. M. Pullen, Department of Pathology and Microbiology, School of Medical Sciences, Bristol University, University Walk, Bristol BS8 1TD, UK
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Abstract
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A pre-TCRCD3 signal is required for the efficient maturation of CD4CD8 thymocytes to the CD4+CD8+ stage. This study addressed whether a similar signal is required for maturation of intestinal intraepithelial lymphocytes (IEL) that may develop extrathymically. We have shown previously that IEL from mice deficient for CD3-associated
chains include an immature population of CD3CD8
+ cells expressing cytoplasmic TCR ß chains but lacking detectable surface TCR
ß, CD16 and B220. Here we stimulated the appearance of such IEL in
+/
/ mice by expression of an activated Lck transgene or in vivo treatment with anti-CD3
. Anti-CD3
treatment of RAG-deficient animals also yielded CD16B220 IEL. In contrast, expression of a TCRß transgene in rag-1/ mice did not stimulate the appearance of CD3CD8
+CD16B220 cells. Taken together these data indicate that although anti-CD3
treatment and LckF505 assist in catalyzing a CD16+B220+
CD16B220 transition, these manipulations are not equivalent to a pre-TCR signal in IEL lymphocytes.
Keywords: CD3
, development, Fyn, intraepithelial lymphocyte, Lck, thymocyte
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Introduction
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Intraepithelial lymphocytes (IEL) of the small intestine include both TCR
ß- and TCR
-bearing T cells. A portion of these cells expresses an unusual form of the CD8 co-receptor, a CD8
homodimer (1,2), which distinguishes these IEL from conventional CD8
ß-expressing T cells that mature within the thymus. TCR
ß+ and TCR
+ IEL expressing CD8
exhibit several additional characteristics which have led to the view that these cells differ from thymically derived T cells and may mature within the intestine (3,4). Firstly, they are able to utilize the Fc receptor
chain in place of the CD3-associated
chain for TCR signaling. Thus, both TCR
ß+ and TCR
+ CD8
+ IEL develop in mice deficient for
, while no mature thymically derived CD4+ or CD8
ß+ T cells expressing high TCR
ß levels are present in these animals (58). Secondly, these IEL are dependent on the IL-2 receptor ß chain for their development (9). Moreover, conventional thymic positive and negative selection processes apparently do not affect the repertoire of TCR
ß+CD8
+ IEL (1,1016).
A signal from the pre-TCRCD3 complex is proposed to result in development of CD4CD8 thymocytes to the CD4+CD8+ stage and the subsequent proliferation of these cells (1719). Mutant mouse strains deficient for components of the pre-TCR, pre-TCR
(pT
), TCRß or CD3
generate only a few CD4+CD8+ thymocytes and exhibit a 10- to 100-fold reduction in thymocyte number (1820). The src-family non-receptor protein tyrosine kinases (PTK) p56lck (Lck) or p59fyn (Fyn) are thought to mediate the pre-TCR signal during thymopoiesis. In mice lacking both these PTK, TCR
ß+ thymocyte development is blocked at the CD4CD8CD44CD25+ stage (21,22). Subsequent positive selection of CD4+CD8+ thymocytes into mature CD4+ or CD8
ß+ T cells requires a TCR signal resulting from recognition of a self peptideMHC ligand within the thymic cortex (23).
In contrast to the signals associated with thymocyte development, little is known regarding the lymphopoiesis which may commence in the recently described cryptopatches within the lamina propria and give rise to TCR
ß+ intestinal IEL (24,25). Kinetic studies (26), as well as analyses of IEL from RAG-deficient and SCID mice (2,3), animals lacking both Lck and Fyn (27), and young wild-type mice (26,28), suggest that CD3CD8
CD44+ IEL and CD3CD8
+ IEL may include precursors of TCR
ß+ and TCR
+ IEL. RAG and pre-T
transcripts are detected in IEL from SCID and nude mice respectively (2931), and this also supports the scenario that immature IEL rearrange their TCR genes and express pre-TCR in the intestine. Our recent data indicating that cells among the CD3CD8
+ IEL express pre-T
transcripts and exhibit VDJß gene rearrangement further support the view that some of these cells are committed to the T cell lineage (31).
To pursue the nature of the CD8
+ IEL maturation process, we utilized mice expressing a constitutively active Lck transgene (32), as well as mice deficient for components of the TCRCD3 signaling pathway (5,19,33), to characterize possible precursors of TCR
ß+CD8
+ IEL and to determine whether mimicking a pre-TCR signal would drive IEL development. We have reported recently the appearance of a unique maturational intermediate for TCR
ß+CD8
+ IEL in
-deficient mice, that expresses CD8
homodimers and cytoplasmic TCR ß chains, but lacks detectable surface TCR
ß expression (31). Here we show that expression of an active Lck transgene or in vivo anti-CD3
treatment of
+/
/ mice triggers the appearance of such CD3CD8
+ cytoplasmic TCRß+ IEL. After these manipulations, as well as upon anti-CD3
treatment of RAG-deficient animals, the intermediate population that appears lacks CD16 and B220 expression. In contrast, expression of a TCRß transgene in rag-1/ mice does not result in the appearance of CD3CD8
+CD16B220 IEL. Together these data indicate that in contrast to their effects on thymocyte development, expression of active Lck and anti-CD3
treatment do not mimic a pre-TCR signal in intestinal IEL.
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Methods
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Mice
Mice deficient for CD3
and
have been reported previously (5,19).
/ animals were crossed with mice bearing a constitutively active mutant of Lck, LckF505 (32), and their progeny were backcrossed with
/ animals to generate
/LckF505 offspring.
/LckF505 mice were then crossed to
/ animals and the F1 progeny were intercrossed to generate
+/
/LckF505 mice. Progeny were screened for CD3
and
disruption and for the LckF505 transgene by PCR. lck/fyn/ mice (21) were used in collaboration with Drs Nicolai van Oers and Arthur Weiss (Howard Hughes Medical Institute, UCSF Medical Center, San Francisco, CA). Vß8.2 (34) and 2B4ßEH [Vß3 (35)] TCRß chain transgenic mice were kindly provided by Harold von Boehmer (Institute Necker, INSERM 373, Paris) and Mark Davis (Howard Hughes Medical Institute, Stanford University, CA) respectively, and were crossed with rag-1/ mice (33) purchased from the Jackson Laboratory (Bar Harbor, ME). F1 progeny were backcrossed to rag-1/ animals, and F2 progeny were screened by PCR for the TCRß transgene and the wild-type rag-1 allele. C57BL/6J mice were purchased from the Jackson Laboratory. All mice were maintained under specific pathogen-free conditions.
mAb
The following mAb were used for flow cytometry: anti-CD4FITC [GK1.5 (36)] and anti-CD8
FITC [53-6.72 (37)]. Anti-Fc
RII/III supernatant [2.4G2 (38)] was used as a primary blocking reagent where detection for CD16/32 expression was not performed. Anti-CD8
TriChrome (TC), anti-TCR
TC [GL3 (39)], streptavidinTC used to detect biotinylated reagents and anti-B220phycoerythrin (PE) were purchased from Caltag Labs (San Francisco, CA). Anti-TCRßFITC and PE [H57-597 (40)], anti-CD8
PE, anti-CD44FITC, anti-CD16/32FITC and PE, biotinylated anti-CD16/32, anti-CD3
FITC, and anti-CD8ßFITC were obtained from PharMingen (San Diego, CA).
Surface and cytoplasmic staining for flow cytometric analyses
IEL were isolated and purified as described (27,41). IEL and thymocytes were surface stained as reported (27). For cytoplasmic staining, surface stained cells were washed and fixed by incubation in 100 µl of 4% paraformaldehyde in PBS for 20 min in the dark. Following three washes, the cells were permeabilized in FACS buffer (balanced salt solution + 2% FCS + 0.08% sodium azide) + 0.1% saponin and blocked for 5 min on ice with anti-Fc
RII/III in saponin. Intracellular staining for TCRß was performed by incubation on ice with anti-TCRßFITC, followed by three washes in 0.1% saponin in FACS buffer.
Flow cytometric analysis and sorting
Multi-parameter cytometric analysis was performed on a FACScan flow cytometer using Lysys II software (Becton Dickinson, San Jose, CA) and flow cytometric analyses were plotted using ReproMan software (Truefacts Software, Seattle WA). All histograms are plotted on a logarithmic scale.
In vivo anti-CD3
treatment
Mice were injected i.p. with 200 or 300 µg of anti-CD3
antibody [145-2C11 (42)] purchased from Pharmingen, or hamster Ig, and were sacrificed 6 days later.
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Results
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CD3CD8
+ IEL expressing cytoplasmic TCR ß chains are detected in
+/
/ mice expressing a constitutively active form of Lck
We have previously shown that TCR
ß+CD8
+ IEL development is ablated in mice deficient for both Lck and Fyn, and that IEL in these mice are enriched for CD3CD8
CD44+CD16+ and CD3CD8
+CD16+B220+ putative IEL precursors (27). Our data suggested that these src-family PTK control development of the TCR
ß+ IEL population in situ and, therefore, we posited that a constitutively active mutant of Lck might facilitate development of more mature cells in this IEL lineage. To test this hypothesis, we analyzed IEL from
+/
/ animals in the presence or absence of the LckF505 transgene. Due to the lack of CD3-associated
chains,
+/
/ mice have no mature TCR
ß+ IEL expressing CD4 or CD8
ß heterodimers (57,31). However, the immature T lymphocytes in these mice do express low levels of the CD3 component
and therefore can transmit CD3-mediated signals. Thus, these animals provide an ideal source of IEL in which to study the development of TCR
ß+CD8
+ cells that putatively mature within the small intestine.
The LckF505 transgene, expressed under control of the lck proximal promoter, encodes a constitutively active mutant of Lck that is altered at the negative regulatory residue, Y505 (32). In the thymus of RAG-deficient mice, overexpression of this LckF505 transgene facilitates maturation of CD4CD8 thymocytes to the CD4+CD8+ stage and expansion of the pool of CD4+CD8+ thymocytes to wild-type levels (43). As shown in Fig. 1
,
+/
/ mice have only a small proportion of CD4+CD8+ thymocytes and expression of the LckF505 transgene on this background promotes the CD4CD8 to CD4+CD8+ transition.

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Fig. 1. CD3CD8 + IEL express cytoplasmic TCR ß chains in +/ /LckF505 mice. Thymocytes and IEL were isolated from +/ /, +/ /LckF505, C57BL/6 (B6) and rag-1/ mice, and were stained for three-color flow cytometry. For the thymus, surface staining was performed using anti-CD4PE, anti-CD8 TC and anti-TCRßFITC. To stain cytoplasmic TCRß, thymocytes were stained on the surface with anti-CD8 TC and anti-TCRßPE, permeabilized in saponin, and stained intracellularly with anti-TCRßFITC. Purified IEL were surface stained with anti-CD8 TC, anti-B220PE or anti-TCRßPE, and anti-CD16/32FITC. Intracellular IEL staining was performed using anti-TCRßFITC in saponin. IEL staining is shown after gating on lymphocytes by forward and side scatter, and for CD8 expression. Data are presented on a logarithmic scale. cTCRß and sTCRß represent cytoplasmic and surface TCRß expression respectively. The percentage of CD8 + cells falling within each rectilinear gate is shown. Results are representative of analyses performed in at least three separate experiments using 4- to 8-week-old mice. The data from six similar experiments are shown in Table 1 .
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Mature TCR
ß+ and TCR
+ IEL were absent in
+/
/ mice whether or not they expressed the LckF505 transgene (Fig. 1
and data not shown). However, cytoplasmic staining revealed an increase in the representation of IEL expressing TCR ß chains in the
+/
/ LckF505 mice compared to their
+/
/ littermates (Fig. 1
and Table 1
), whereas the majority of CD8
+ IEL in
+/
/ and rag-1/ mice express CD16 and B220, a significant fraction of the CD8
+ cells in the
+/
/ LckF505 mice lack expression of these markers (Fig. 1
and Table 1
). The CD8
+ IEL pool in the C57BL/6 mice includes both mature TCR
ß and TCR
cells, some of which express these two markers, and this results in the continuum for both CD16 and B220 expression (Fig. 1
and data not shown). In these wild-type mice the IEL precursors represent an insignificant proportion of the total CD8
+ IEL.
Since CD3CD8
+cytoTCRß+CD16B220 IEL are present in
+/
/ LckF505 mice but not in their
+/
/ littermates, expression of a constitutively active mutant of Lck appears to drive the development of cells in the TCR
ß+CD8
+ IEL lineage.
CD3 signaling drives maturation of IEL in rag-1/ and
+/
/ mice but not in lck/fyn/ animals
It has been reported previously that thymocyte maturation in RAG- or TCRß-deficient mice can be driven from the CD4CD8 to the CD4+CD8+ stage by in vivo treatment with anti-CD3
(17,4446). This antibody treatment has been suggested to mimic the pre-TCR signal in CD4CD8 thymocytes. We postulated that if a signal from the pre-TCR, mediated by CD3 components and the src-family PTK Lck or Fyn, is required for the appearance of CD8
+ IEL expressing detectable levels of cytoplasmic TCR ß chains, it might be possible to mimic such a signal using anti-CD3
treatment in vivo.
As expected, anti-CD3
treatment of rag-1/ and
+/
/ mice resulted in the appearance of significant numbers of CD4+CD8+ thymocytes (Fig. 2
). The thymocytes of RAG-deficient mice could not rearrange their TCR genes and therefore their CD4+CD8+ cells lacked expression of TCRß protein (Fig. 2
). In contrast, in anti-CD3
-treated
+/
/ mice, many of the CD4+CD8+ thymocytes expressed cytoplasmic TCRß although they lacked surface TCR
ß (Fig. 2
and data not shown).
In the control rag-1/ mice treated with hamster Ig the CD8
+ IEL expressed CD16 and B220 (Fig. 2
and Table 2
). However, 6 days after anti-CD3
treatment, CD8
+ IEL lacking CD16 and B220 expression were detected in the rag-1/ mice (Fig. 2
and Table 2
). Moreover, the CD8
+ IEL in
+/
/ mice expressed CD16 and B220, but lacked surface TCR, and anti-CD3
treatment of these mice also resulted in the appearance of CD3CD8
+CD16B220 IEL, a fraction of which expressed cytoplasmic TCR ß chains (Fig. 2
, Table 2
and data not shown).
It is noteworthy that these CD3CD8
+cytoTCRß+CD16B220 IEL and CD3CD8
+CD16B220 found in anti-CD3
treated
+/
/ and rag-1/ animals respectively were indistinguishable in size from those present in
+/
/ LckF505 mice, and were intermediate between the larger CD3CD8
+CD16+B220+ IEL and the smaller, mature CD3+ cells (data not shown). All told, anti-CD3
treatment of
+/
/ and rag-1/ mice appears to drive the development of cells in the TCR
ß+CD8
+ IEL lineage.
In marked contrast, CD3CD8
+cytoTCRß+ CD16B220 IEL did not appear in anti-CD3
treated lck/fyn/ mice (Fig. 3
), although the disappearance of the TCR
+ cells in these animals clearly indicated that the anti-CD3
reagent reached the IEL compartment (Fig. 3
), either causing down-regulation of surface TCR
or elimination of TCR
+ IEL. This result is compatible with a role for Lck and Fyn acting downstream of CD3, and mediating a signal necessary for the appearance of the CD3CD8
+ cytoplasmic TCRß+ IEL population.
Rearranged TCRß transgenes are expressed in CD3CD8
+CD16+B220+ IEL in rag-1/ mice
Previous analyses of thymic development in RAG-deficient mice expressing a rearranged TCRß chain transgene demonstrated that thymocytes in these animals advance to the CD4+CD8+ stage (20,45,47) and that Lck is involved in mediating a signal required for this transition (43,46,48,49). These results support the hypothesis that a signal generated via the pre-TCR is required for the CD4CD8 to CD4+CD8+ transition during thymopoiesis. One possible interpretation of our results outlined above is that a similar pre-TCR-generated signal is required for the development of CD3CD8
+cytoTCRß+ CD16 B220 IEL in situ. We therefore analyzed IEL from rag-1/ mice which bore either a Vß8 (34) or Vß3 (35) rearranged TCRß transgene.
As previously reported, thymocyte development proceeds to the CD4+CD8+ stage in rag-1/+TCRß animals (45,47) (Fig. 4
). Surprisingly, while intracellular staining of IEL from rag-1/+TCRß mice clearly shows expression of the transgene in CD3CD8
+ IEL, these cells retain expression of CD16 and B220 (Fig. 4
). This was the case whether the Vß3 or Vß8 transgenes, which include different promoter elements, were introduced (Fig. 4
and data not shown). These results suggest that the expression of a rearranged TCRß transgene in CD3CD8
+CD16+B220+ IEL in rag-1/ animals is insufficient to drive the generation of CD3CD8
+CD16 B220 IEL from their putative IEL precursors.
 |
Discussion
|
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The maturation of thymocytes proceeds via a well-characterized pathway. In mice deficient for RAG, TCRß, pre-T
, CD3
or both Lck and Fyn, thymocyte development is blocked at the CD4CD8CD44CD25+ stage (1921,33,50). During thymopoiesis, the pre-TCR signal, necessary for the CD4CD8 to CD4+CD8+ transition and expansion of the CD4+CD8+ thymocyte pool, can be mimicked by anti-CD3
treatment (17,4446) or by overexpression of a constitutively active mutant of Lck (43). To determine whether a parallel developmental scheme operates for the extrathymic maturation of IEL residing in the small intestine, these cells have been examined from mice deficient in various TCR signaling components.
A compilation of published data and our results lead us to propose one possible scheme for the maturational pathway underlying the emergence of TCR
ß+CD8
+ IEL, which is shown in Fig. 5
. Briefly, analyses of IEL from RAG-deficient and SCID mice indicate a block in TCR
ß+CD8
+ IEL development (2,3). Moreover, our recent studies of mice deficient for both Lck and Fyn (27), or lacking CD3
(31), imply a similar perturbation in maturation of IEL of the TCR
ß+ lineage. IEL from all these mice include CD3CD8
CD44+ and CD3CD8
+CD16+B220+ IEL, which may include precursors of both TCR
ß+ and TCR
+ CD8
+ IEL (Figs 1 and 3
) (27,31,51). Evidence suggests that
-deficient IEL substitute the FcR
chain for TCR signaling (57), and perhaps because
includes only one immunoreceptor tyrosine-based activation motif (ITAM) while
has three, these
/ IEL mature only inefficiently. We have reported that
/ animals include CD3CD8
+cytoTCRß+ IEL lacking CD16 and B220 expression (31), which could be intermediates in IEL development as suggested (52). IEL from wild-type mice include negligible numbers of these cells, which might rapidly rearrange their TCR
genes and mature to express surface TCR.
Here we demonstrate that partial maturation of RAG-deficient IEL, as judged by the loss of CD16 and B220 expression, can be stimulated in vivo by anti-CD3
antibodies (Fig. 2
and Table 2
). A similar finding has been reported recently by Terhorst et al. (52). Moreover, if CD3
is made limiting by gene dosage in mice also lacking
, the appearance of CD3CD8
+cytoTCRß+CD16B220 IEL can be stimulated by expression of active Lck (Fig. 1
and Table 1
) or by anti-CD3
treatment (Fig. 2
and Table 2
). However, complete elimination of CD3
results in the absence of this putative immature IEL population, and it cannot be rescued by the LckF505 transgene (Page and Pullen, unpublished data).
Our results using the active LckF505 transgene and anti-CD3
antibody treatment suggest that CD4+CD8+ thymocytes and CD3CD8
+cytoTCRß+CD16B220 IEL require similar signals for their generation; however, the data from analyses of RAG-deficient mice expressing two different TCRß transgenes (Fig. 4
) indicate that the developmental requirements for these two immature T cells can be distinguished. These studies demonstrate that the condition of TCR components and their associated signaling structures can influence the cell surface phenotype of CD3CD8
+ IEL, in a manner that suggests that CD16+B220+ IEL down-regulate these markers and subsequently up-regulate levels of cytoplasmic TCRß protein (Fig. 2
). Although anti-CD3
and LckF505 may assist in catalyzing a transition from CD16+B220+ to CD16B220, this does not reflect a signaling process wholly analogous to that entrained by the pre-TCR since expression of TCRß cannot, by itself, reconstitute the defect observed in RAG-deficient mice (Fig. 4
). It is unlikely that pre-TCR cannot be expressed in these IEL, since pre-T
transcripts are detectable in these rag-1/ mice (31), and TCRß is expressed in the cytoplasm of the 2B4ß transgenic IEL and also on the surface, perhaps as ßß dimers, of the HY transgenic IEL.
In addition, it should be noted that LckF505 promotes the appearance of cytoplasmic TCRß+ cells in the IEL compartment, but antagonizes ß chain gene rearrangement and hence expression in thymocytes (Fig. 1
) (53,54).
Taken together, these data suggest that the CD3CD8
+CD16B220 IEL expressing cytoplasmic TCR ß chains may represent developmental intermediates of the TCR
ß lineage maturing within the intestinal epithelium. Cells of this phenotype are not found in the thymus or lymph nodes (data not shown). Our analyses of IEL from rag-1/ + TCRß mice (Fig. 4
) and ZAP70/ mice (Page et al., unpublished observations) indicate that these animals lack CD3CD8
+cytoTCRß+CD16B220 IEL despite abundant populations of CD4+CD8+ thymocytes. Hence these data suggest that CD3CD8
+cytoTCRß+CD16B220 IEL are not derived from CD4+CD8+ thymocytes which have turned off CD4 and CD8ß expression. Moreover, our earlier study of the methylation state of the CD8ß promoter among CD8
+ IEL also suggests these cells do not arise from CD8ß-expressing precursors (55).
Overall, our data are consistent with the model presented in Fig. 5
for the extrathymic maturation sequence that underlies the emergence of TCR
ß+CD8
+ IEL in the small intestine. They indicate that the signals derived from TCR components, including the Lck PTK, participate in regulating the relative representation of the maturation intermediates. Furthermore, our results provide additional evidence that signaling during IEL maturation differs from that regulating thymocyte development. Although expression of a constitutively active Lck transgene, anti-CD3
treatment and expression of a TCRß transgene in RAG-deficient mice all apparently mimic a pre-TCR signal during thymopoiesis, these manipulations are not equivalent in driving TCR
ß+CD8
+ IEL development.
 |
Acknowledgments
|
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We thank Deborah Wilson, Katherine Forbush, Ethan Ojala and Xiao Cun Pan for maintaining our animal colony at the University of Washington, and Edward Chung for technical assistance. We are grateful to Drs Arthur Weiss and Nicolai van Oers (HHMI, UCSF) for their helpful discussions and for providing access to lck/fyn/ mice. S. T. P. has been supported by National Institutes of Health training grants GM-07226 and CA-09537, and by a Poncin Scholarship. J. A. H. is a predoctoral fellow of the Howard Hughes Medical Institute. M. M. is supported by grants from CNRS, INSERM, and ARC. R. M. P. and A. M. P. were Investigators of the Howard Hughes Medical Institute, and A. M. P. was a Pew Scholar in the Biomedical Sciences.
 |
Abbreviations
|
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Fyn | p59fyn |
IEL | intraepithelial lymphocytes |
Lck | p56lck |
PE | phycoerythrin |
pT | pre-TCR |
PTK | protein tyrosine kinase |
rag | recombination activating gene |
TC | TriChrome |
 |
Notes
|
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5 Present address: Merck Research Laboratories, PO Box 2000, RY80-A1, 126 East Lincoln Avenue, Rahway, NJ 07065, USA 
6 Present address: Department of Pathology and Microbiology, School of Medical Sciences, Bristol University, University Walk, Bristol BS8 1TD, UK 
Transmitting editor: E. Simpson
Received 20 July 1998,
accepted 19 October 1998.
 |
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