By
From the * Department of Microbiology and Immunology, and Department of Medicine, Howard
Hughes Medical Institute, University of California, San Francisco, California 94143; and The
Skirball Institute of Biomolecular Medicine, Howard Hughes Medical Institute, New York University
Medical Center, New York, New York 10016
Itk is a member of the Btk/Tec/Itk family of nonreceptor protein tyrosine kinases (PTKs), and has been implicated in T cell antigen receptor (TCR) signal transduction. Lck and Fyn are the Src-family nonreceptor PTKs that are involved in TCR signaling. To address the question of how these members of different families of PTKs functionally contribute to T cell development and to T cell activation, mice deficient for both Itk and either Lck or Fyn were generated. The Itk/Lck doubly deficient mice exhibited a phenotype similar to that of Lck-deficient mice. The phenotype of the Itk/Fyn doubly deficient mice was similar to that of Itk deficient mice. However the Itk/Fyn doubly deficient mice exhibited a more severe defect in TCR-induced proliferation of thymocytes and peripheral T cells than did mice deficient in either kinase alone. These data support the notion that Itk and Fyn both make independent contributions to TCR-induced T cell activation.
Multiple nonreceptor protein tyrosine kinases (PTKs)
are activated after TCR stimulation. These PTKs are
responsible for the tyrosine phosphorylation of the TCR- Recently, a new family of nonreceptor PTKs, expressed
specifically in the hematopoietic system (4), has been implicated in antigen receptor-mediated responses. Members of
this family include Btk, Itk, and Tec. Itk is highly expressed
in T cells and mast cells. Mice deficient in Itk expression
have a reduced number of mature T cells, which also are
compromised in proliferation after TCR stimulation (5).
Thus, this third class of nonreceptor PTKs, in addition to
those of the Src and Syk/ZAP-70 families, is involved in T
cell development and activation.
The mechanism by which Itk is regulated in T cells is
not known. Activation of Btk by Src family PTKs has been
observed (6, 7). This observation raises the possibility that
an Src family kinase might interact functionally with Itk in
T cells. Thymic development and T cell functional responses in mice deficient in Lck or Fyn do not mimic the
defects observed in Itk-deficient mice. Lck-deficient mice
exhibit a prominent but incomplete developmental arrest at
the transition from the CD4 Generation of Itk/Fyn and Itk/Lck Doubly Deficient Mice.
Itk-deficient mice have been previously reported (5). Fyn-deficient
mice were obtained from Paul L. Stein and Philippe Soriano (10)
at Fred Hutchinson Cancer Research Center (Seattle, WA). Lck-deficient mice were obtained from Tak W. Mak (Amgen Institute, Toronto, Canada; reference 8). Itk/Fyn and Itk/Lck doubly
deficient mice were obtained by breeding Itk-deficient mice with
Fyn-deficient mice and Lck-deficient mice, respectively. Genotyping of these mice was carried out using PCR. The oligonucleotides for genotyping the itk locus are as follows: Itk knockout
(KO): ASneo+, 5
and CD3 chains, the kinases themselves, and a number of
cellular substrates (1, 2). Among these cytoplasmic PTKs
are the Src-family members such as Fyn and Lck, and the
ZAP-70 and Syk kinases. Functionally important interactions as well as physical interactions between these two families of PTKs have been well studied (for review see reference 3).
CD8
(double negative, DN)
to the CD4+CD8+ (double positive, DP) stage of development. The few peripheral T cells that do develop have
markedly impaired TCR functional responses (8). In mice
lacking Fyn, thymic development occurs normally, but
markedly reduced thymocyte proliferation and some diminution in peripheral T cell proliferation in response to TCR stimulation were observed (9, 10). A complete arrest in the transition of the DN cells to DP cells in mice deficient in
both Lck and Fyn has been observed (11, 12). These studies
suggest that Lck and Fyn have some redundant roles in
TCR signal transduction. To address whether Itk and the
Src family members expressed in T cells have overlapping
or nonoverlapping functions in T cell development and activation, we generated mice deficient in both Itk and Fyn,
as well as mice deficient in both Itk and Lck. This study
provides evidence that Fyn and Itk both make important
independent contributions to the functional responses of thymocytes and T cells.
ATTGAACAAGATGGATTGCAC 3
; ASneo
,
5
CGTCCAGATCATCCTGATC 3
; Itk WT: itk420, 5
TGGGTGCTGACCCTTAAAGAAG 3
; itkintron3a, 5
GCCGTAAATGAACAGGTGGTGA 3
. The expected size of Itk KO is
475 bp, and that of Itk WT is 218 bp. The ASneo+ and ASneo
primers are located in the neo gene, and thus will also amplify from the neo insertions of the Fyn-deficient mice and Lck-deficient mice. Additional PCR reactions using the itk KO-specific
oligoes were performed to distinguish itk+/+ and itk+/
in certain
genotype combinations (5).
, 5
GCATCGCCTTCTATCGCCTTCTTGACG 3
; fynR, 5
GCAAAACAACCCACACAGAG 3
; Fyn WT:
fynF, 5
TTACCCTCTGAGCATCTGAC 3
; fynR, see above.
The expected size of Fyn KO is ~400 bp, and that of Fyn WT is
~550 bp. The oligonucleotides for genotyping the lck locus are as
follows: Lck KO: neo.lck, 5
TATCAGGACATAGCGTTGGCTACCC 3
; lckR, 5
CTTAGACTCACGTGCTCCACAGGTA
3
; Lck WT: lckF, 5
TGGTGAGACCTGACAACTGTCCGGA 3
; lckR, see above. The expected size of Lck KO is 560 bp, and that
of Lck WT is 375 bp. Each PCR reaction was carried out individually under similar conditions. The typical conditions are 93°C
for 30 min for denaturation, 55°C for 45 min for annealing, and
74°C for 45 min for amplification, with a total of 35 cycles of the above three steps, followed by a 74°C for 3-h step to finish up the
reaction.
Flow Cytometric Analysis.
Flow cytometry was performed by
staining 106 freshly isolated thymocytes and lymph node cells in
100 µl of PBS supplemented with 0.3% BSA and 0.01% sodium
azide on ice for 30 min. For three-color analysis, cells were first
stained with various biotin-conjugated antibodies against different
lymphocyte surface proteins, followed by staining with a cocktail
of streptavidin-TRICOLOR (Caltag Labs, San Francisco, CA),
CD4-PE (YTS191.1; Caltag), and CD8-FITC (CT-CD8, Caltag). Cells were analyzed on a FACScan® or FACSCalibur using
the Lysis II or the CellQuest softwares. Viable cells were gated on
the basis of forward- and side-light scattering. Additional antibodies used included anti-CD69 (H1.2F3), anti-heat stable antigen (M1/69), anti-TCR
(H57-597), and anti-IgM (R6-60.2),
all from PharMingen (San Diego, CA).
Cell Proliferation Assays.
Complete medium (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 50 µM -mercaptoethanol, 100 µM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin) was used for cell culture and
preparation. Responder cells were isolated from thymi or spleens of
age-matched wild-type, itk+/
fyn+/+, itk+/
fyn
/
,itk
/
fyn+/+,
and itk
/
fyn
/
mice (8-10-wk old, H-2b/b). Splenocytes from
BALB/c (H-2d/d) and C57BL/6 (H-2b/b) were inactivated by irradiation (~2,000 rad). Proliferation assays were set up in a volume of 200 µl in flat-bottomed wells of microtiter plates. For the
MLR, serial dilutions of 4 × 105, 2 × 105, 1 × 105, and 0.5 × 105 splenocytes were mixed with 5 × 105 stimulator cells of various types in each well, and were incubated for 96 h before pulsing
for 16 h with 1 µCi of [3H]thymidine per well. For other cell
proliferation assays, 5 × 104 cells were mixed with 5 × 105 irradiated C57BL/6 splenocytes plus anti-CD3 (purified 145-2C11 at
1.5, 0.5, 0.167, and 0.056 µg/ml final concentration) in the presence of PMA (1 ng/ml). Cells were incubated for 48 h before pulsing for 16 h with 1 µCi of [3H]thymidine per well. Cellular
radioactivity was harvested using a microtiter plate cell harvester
(Tomtec, Orange, CT). All assays were done in triplicates, and
the mean and standard deviation are presented.
Maturation of thymocytes takes place in an ordered sequence of developmental events (13). Early immature thymocytes do not express either the CD4 or the CD8 coreceptors (DN). As a result of pre-TCR signaling events, the DN cells acquire expression of both CD4 and CD8 to become DP cells. Subsequently, expression of one of the coreceptors is extinguished, and the thymocytes become mature CD4+ and CD8+ single positive T cells. Mice lacking Fyn, an Src family nonreceptor PTK have normal numbers of thymocyte and T cell subpopulations as well as normal levels of surface expression of the TCR chains (Table 1 and Fig. 1), consistent with previous reports (9, 10). On the other hand, mice deficient in Itk have a partial block in T cell development (Table 1 and Fig. 1) in agreement with our previous studies (5). Itk-deficient mice have reduced numbers of mature lymph node T cells as well as a reduced ratio of CD4 to CD8 single positive cells in the thymus and the periphery.
|
To address whether Itk and Fyn might functionally interact, we examined the developmental consequences of a defect in both of these kinases. Mice deficient for both Fyn and Itk expression were derived by breeding. Analysis of the Itk/Fyn doubly deficient mice demonstrated similar thymic and peripheral T cell phenotypes as the Itk-deficient mice (Table 1 and Fig. 1). Hence, the absence of Fyn did not further enhance the developmental consequences of Itk-deficiency.
A few mice deficient in both Lck and Itk were also obtained. The developmental consequences of this breeding revealed that mice deficient in both Itk and Lck had thymic phenotypes indistinguishable from Lck-deficient mice (data not shown). A partial developmental arrest at the DN to DP transition and a 10-fold reduction in thymocyte number were observed, consistent with studies of the Lck-deficient mice previously reported (8). As observed in the Lck-deficient mice, only a few peripheral T cells were detectable in the spleens and lymph nodes of the Itk/Lck doubly deficient animals. Thus, the absence of Itk does not further augment the already severe phenotype observed in the Lck deficient mice.
Thymocyte Proliferation in the Absence of Both Itk and Fyn.To address whether Itk and Fyn make independent
contributions to the processes that lead to T cell activation,
thymocytes and splenocytes were prepared from Itk/Fyn
doubly deficient mice and compared with those from mice
lacking either kinase alone in TCR-mediated proliferative
responses. Thymocytes lacking either Fyn or Itk consistently showed a reduction in proliferation to anti-CD3 mAb
over a broad dose range when compared to control Itk+
Fyn+ thymocytes (Fig. 2 A), consistent with previous studies (9, 10). Interestingly, thymocytes lacking both Itk and
Fyn exhibited a much more severe proliferative defect, detectable over all doses of anti-CD3 mAb used. These results
suggest that both Fyn and Itk contribute to the proliferative
response induced by TCR stimulation in thymocytes.
Proliferation of Mature T cells in Response to anti-TCR and Allogeneic MHC Stimulations.
To examine the response of
mature peripheral T cells, splenocytes from Itk/Fyn doubly
deficient mice as well as from mice deficient in either kinase
alone or control mice were stimulated with anti-CD3 mAb
in the presence or absence of PMA. As has been previously
reported (9, 10), the proliferation of Fyn splenocytes was
similar to that of wild-type (Itk+Fyn+) splenocytes. On the
other hand, splenocytes lacking Itk showed a proliferative
response of 15-30% of that of the Itk+Fyn+ cells (Fig. 2 B
and data not shown). Consistent with the observation using
thymocytes, Itk
Fyn
splenocytes had a much more profound reduction in their proliferative response. Taken together, these results suggest that Itk and Fyn both make at
least some independent contributions to TCR-regulated pathways that lead to T cell proliferative responses.
To examine the impact of these kinases on antigen-dependent responses, mature T cells from mutant mice
were also examined in MLR. Consistent with the results
with anti-CD3 induced proliferation, ItkFyn
splenocytes
responded very poorly to allogeneic MHC stimulation, whereas Itk-deficient splenocytes had an intermediate level
of proliferation (Fig. 3). On the other hand, splenocytes
lacking Fyn exhibited near normal alloantigen-induced
proliferation (data not shown), consistent with previous
studies (9, 10). These results support the notion that both
Itk and Fyn functionally contribute to T cell responses to
alloantigens.
We describe here the characterization of Itk/Fyn doubly deficient mice in T cell development and activation. Our data failed to reveal an additive effect upon thymocyte and T cell development in mice that lack both Fyn and Itk. On the other hand, we observed a synergistic loss of proliferative responses of these cell populations in the absence of both Itk and Fyn. The severe defect in proliferative responses induced with TCR stimulation suggests that these kinases provide at least some independent functions in the events leading to T cell and thymocyte proliferation.
Itk-deficient mice and Fyn-deficient mice have distinctive phenotypes. Mice lacking Itk have a reduced ratio of CD4 to CD8 single positive thymocytes (5). The absolute numbers of mature CD4+ T cells are also reduced by about twofold compared to wild-type mice expressing Itk (Table 1). Fyn-deficient mice, on the other hand, do not exhibit an obvious developmental phenotype (9, 10). The main immune system defect of Fyn-deficient mice is in mature thymocytes that have diminished proliferative responses as well as reduced tyrosine phosphorylation of cellular proteins upon TCR stimulation (9, 10). Overall, the developmental consequences resulting from a lack of either Itk or Fyn is quite modest.
Itk/Fyn doubly deficient mice resembled Itk-deficient mice in their T cell developmental phenotypes, suggesting that even in the absence of Itk, Fyn is not critically required for T cell development, nor does it play a function that is redundant with Itk. In contrast, thymocytes and mature T cells in Itk/Fyn doubly deficient mice exhibited a severe defect in proliferative responses upon TCR stimulation. This defect was much more pronounced than would be predicted by an additive effect of lacking either kinase alone. These data suggest that both Itk and Fyn are important in T cells activation and are likely to play partially distinct functions.
To address whether Fyn is the only specific and functional partner of Itk in T cell activation, we also generated mice deficient for both Itk and Lck, another Src family member expressed in T cells. The developmental phenotypes of Itk/Lck double-deficient mice resembled those of Lck-deficient mice (data not shown; reference 8). There was an early block of thymocyte maturation resulting in reduced numbers of CD4+CD8+ double-positive thymocytes, and very few mature T cells. Because of this early block in development, it is difficult to assess whether Lck deficiency exacerbates the later defect due to the absence of Itk. The phenotype of Itk/Lck-deficient mice is also distinct from Fyn/Lck doubly deficient mice (11, 12), which have a complete lack of double-positive thymocytes. The results of Fyn/ Lck doubly deficient mice indicate that there is a certain degree of functional redundancy between Fyn and Lck in promoting transition of thymocytes from double-negative to double-positive stages. The results also suggest that Lck-deficiency provides a good platform to reveal Fyn's contribution to thymocyte development. The lack of similarity between Itk/Lck double-deficient mice and Fyn/Lck doubly deficient mice, however, suggests that Itk is not the sole molecule mediating signals from Fyn at these specific stages of thymocyte maturation. This interpretation is consistent with our studies of Itk/Fyn doubly deficient mice demonstrating that both Itk and Fyn contribute to T cell activation; lack of either kinase alone is not sufficient to abolish T cell activation.
In B lymphocytes, Btk is thought to have functions similar to those of Itk in T cells. Mice deficient in Btk exhibit defects in B cell development and in BCR-mediated proliferative responses. The Src-family PTK Lyn has been shown to regulate the activity of Btk, and mice lacking Lyn have decreased proliferative responses. In T cells, Lck and Fyn may have functions similar to Lyn in B cells. Thus, these PTKs may be involved in activating Itk. The finding that the absence of Fyn exacerbates the proliferative defect of Itk-null T cells suggests that Fyn is additionally involved in pathways distinct from those involving Itk. At least one of these Fyn-specific pathways may involve yet another cytoplasmic PTK. Recent studies have shown that in T cells, Fyn specifically regulates TCR-mediated activation of Pyk2, a PTK homologous to the focal adhesion kinase (14). Thus, Fyn and Itk are likely to regulate at least some distinct events involved in the proliferative response resulting from TCR stimulation. Further work will be required to define these distinct events.
Address correspondence to Dr. Arthur Weiss, UCSF, Box 0724, 3rd and Parnassus, San Francisco, CA 94143. Phone: 415-476-1291; FAX: 415-502-5081; email: aweiss{at}itsa.ucsf.edu
Received for publication 3 October 1997 and in revised form 21 October 1997.
X.C. Liao is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (1992-1995) and by a Special Fellowship from the Leukemia Society of America, Inc. (1995-1996). D.R. Littman and A. Weiss are investigators of the Howard Hughes Medical Institute.We thank Paul Stein (Wistar Institute, Philadelphia, PA) and Philippe Soriano (Fred Hutchinson Cancer Research Institute, Seattle, WA) for providing the Fyn-deficient mice and Clifford Lowell (University of California San Francisco) for advice on genotyping them using the PCR reactions; Tak Mak (Amgen Institute, Toronto) for providing the Lck-deficient mice; N. Killeen, D. Qian, and N. van Oers for valuable discussions.
1. | Wange, R.L., and L.E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity. 5: 197-205 [Medline]. |
2. | Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell. 76: 263-274 [Medline]. |
3. | Qian, D., and A. Weiss. 1997. T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 9: 205-212 [Medline]. |
4. | Desiderio, S., and J.D. Silicano. 1994. The Itk/Btk/Tec family of protein-tyrosine kinases. Chem. Immunol. 59: 191-210 [Medline]. |
5. | Liao, X.C., and D.R. Littman. 1995. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity. 3: 757-769 [Medline]. |
6. | Rawlings, D.J., A.M. Scharenberg, H. Park, M.I. Wahl, S. Lin, R.M. Kato, A.-C. Fluckiger, O.N. Witte, and J.-P. Kinet. 1996. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science. 271: 822-825 [Abstract]. |
7. | Alexandropoulos, K., G. Cheng, and D. Baltimore. 1995. Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc. Natl. Acad. Sci. USA. 92: 3110-3114 [Abstract]. |
8. | Molina, T.J., K. Kishihara, D.P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C.J. Paige, K.-U. Hartmann, A. Veillette, et al . 1992. Profound block in thymocyte development in mice lacking p56lck. Nature. 357: 161-164 [Medline]. |
9. | Appleby, M.W., J.A. Gross, M.P. Cooke, S.D. Levin, X. Qian, and R.M. Perlmutter. 1992. Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell. 70:751-763. |
10. | Stein, P.L., H.-M. Lee, S. Rich, and P. Soriano. 1992. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell. 70: 741-750 [Medline]. |
11. |
van Oers, N.S.C.,
B. Lowin-Kropf,
D. Finlay,
K. Connolly, and
A. Weiss.
1996.
![]() ![]() |
12. | Groves, T., P. Smiley, M.P. Cooke, K. Forbush, R.M. Perlmutter, and C.J. Guidos. 1996. Fyn can partially substitute for Lck in T lymphocyte development. Immunity. 5: 417-428 [Medline]. |
13. | Tanaka, Y., L. Arbouin, A. Gillet, S.-Y. Lin, A. Magnan, B. Malissen, and M. Malissen. 1995. Early T-cell development in CD3-deficient mice. Immunol. Rev. 148: 172-199 . |
14. |
Qian, D.,
S. Lev,
N.S.C. van Oers,
I. Dikic,
J. Schlessinger, and
A. Weiss.
1997.
Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling.
J. Exp.
Med.
185:
1253-1259
|