Address correspondence to Harald von Boehmer, Dept. of Pathology, Harvard Medical School, Dana-Farber Cancer Institute, Smith Building Room 736, 1 Jimmy Fund Way, Boston, MA 02115. Phone: (617) 632-6880; Fax: (617) 632-6881; email: harald_von_Boehmer{at}dfci.harvard.edu
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Abstract |
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Key Words: T lymphocyte subsets cell lineage receptor-mediated signal transduction receptor antigen T-cell
The present address of I. Aifantis is The University of Chicago, Dept. of Medicine, Section of Rheumatology, Chicago, IL 60637.
The present address of F. Gounari is Tufts University, New England Medical Center, Molecular Oncology Research Institute, Boston, MA 02115.
Abbreviations used in this paper: DN, CD4-8- double negative; EGFP, enhanced green fluorescent protein; FTOC, fetal thymic organ culture; HSA, heat-stable antigen; pT
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Introduction |
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In sharp contrast, TCRß complexes localize to membrane rafts, from where they trigger signal transduction cascades only in response to stimulation with costimulatory molecules and agonist peptides presented by MHC molecules (8, 9). Like the pre-TCR, TCR
ß complexes are constitutively internalized. However, in contrast to the pre-TCR, unstimulated TCR
ß complexes are recycled back to the cell surface (10, 11). Only in response to stimulation are internalized TCR
ß complexes degraded (12).
The lack of pre-TCR-induced survival, proliferation, and differentiation is evident in pT-/- mice, even though some cells still pass the pre-TCR-controlled checkpoint by virtue of their ability to form TCR
and TCR
ß complexes (13, 14). Whether or not a TCR
molecule can functionally replace pT
in the pre-TCR complex is a matter of considerable controversy.
One view suggests that the cell-autonomous nature of pre-TCR signaling is dependent on the identity of the TCRß partner component. Several papers document that, in contrast with pre-TCR expression, premature expression of a TCRß complex promotes a "
-like" T cell lineage fate, and results in impaired proliferation and differentiation to the CD4+8+ stage of thymocyte development (1518). More specifically, some evidence has implicated the cytoplasmic domain unique to pT
as essential for pre-TCR function. Provision of a wild-type pT
transgene to pT
-/- mice restored pre-TCR-induced proliferation, survival, and differentiation better than a transgene lacking the pT
cytoplasmic domain or the proline-rich regions thereof (19). Pulse chase experiments documented cell-autonomous internalization and degradation of TCR and CD3
surface receptor components in cell lines expressing pre-TCR components, but not in those expressing TCR
or TCR
ß components (7). Mutagenesis studies identified the cytoplasmic domain of human pT
as essential for cell-autonomous receptor internalization and degradation (6). These latter studies further implied that cell-autonomous constitutive internalization might represent one mechanism by which pre-TCR surface expression and signaling is self-regulated. An apparent requirement for strict regulation of surface expression level may also exist in the analogous cell-autonomous preB cell receptor signaling cascade (20).
The alternative view implies that the cell-autonomous nature of pre-TCR signaling depends on the developmental stage at which the receptor is expressed rather than on qualities inherent to the TCRß partner component itself. This view suggests that pT represents merely a "surrogate TCR
molecule," whose function is restricted to stabilizing surface expression of a productively rearranged TCRß molecule. Indeed, a recent paper implicated elevated raft content, stronger capacitative Ca2+ entry, and increased extracellular signal-related kinase activation as factors generating a unique developmental environment in CD4-8- double negative (DN) 3 thymocytes (21).
Thus far, observations concerning the potential interchangeability of pT and TCR
have been inconclusive. Utilization of TCR
and TCRß transgenes (14, 1618), or a TCR
transgene expressed at developmental stages different from those of endogenous pT
(15, 22), has precluded a direct assessment of the performance of a TCR
molecule expressed at precisely the same developmental stage as that of endogenous pT
. Although controlled by the p56Lck proximal promoter, the wide variation among founders in the expression of transgenes encoding either pT
or pT
substituted with the connecting peptide, transmembrane, and cytoplasmic domains of TCR
made difficult a comparison between these two transgenes (23). The proliferative potential of pT
-/- fetal thymocytes retrovirally transduced with either pT
or TCR
may have been obscured by analysis of their proliferation in fetal thymic lobes (21), which is much reduced when compared with that of thymocytes developing in situ. Finally, analyses of the reconstitution of empty adult or fetal thymi in which there is no competition for resources or niche space may grossly overestimate the ability of TCR
molecules to mimic the functions of pT
.
Here, we analyze the interchangeability of WTpT (WTpT
) with TCR
, and with a TCR
/pT
hybrid molecule consisting of the extracellular domain of TCR
joined to the transmembrane and cytoplasmic domains of pT
. By placing each transgene under the control of the p56Lck proximal promoter, we ensured that each potential TCRß partner component is expressed at an equal and relevant stage of thymocyte development. By crossing each transgene onto the pT
-/- genetic background, we allowed each receptor to perform in the absence of endogenous pT
. Furthermore, by introducing equal numbers of precursors expressing different receptors into a single thymus, we forced them to compete for available space and resources, revealing differences in receptor function that may be obscured by analyses of the developmental potential of cells that express only a single type of receptor in a noncompetitive environment.
We found that the TCR molecule was unable to fully restore the proliferation, survival, differentiation and
ß T cell lineage commitment induced by the WTpT
molecule. When placed in direct competition, the superiority of WTpT
became more dramatically apparent with regard to proliferation and progression to the CD4+8+ stage of thymocyte development. Substitution of the transmembrane and cytoplasmic domains of TCR
with those of pT
generated a TCR
/pT
hybrid molecule exhibiting enhanced performance in competition with WTpT
when compared with TCR
, supporting the view that some of the cell-autonomous nature of pre-TCR signaling is bestowed on the complex by properties unique and intrinsic to the pT
molecule.
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Materials and Methods |
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Generation of Transgenic Mice.
WTpT and pT
-PRO
transgenic mice were generated as described previously (19, 24). The TCR
/pT
hybrid construct was generated by PCR of an N15 TCR
cDNA template with the primers TCRaBamup, 5'-ACGGATCCTTTCCACCATGAACATGCGTCC-3', and TCRalo, 5'-AGGAATTCTGAAAGTTTAGGTTCATATCTGT-3'; and a pT
cDNA template with the primers pTaEcoup, 5'-CAGAATTCCTGGCTGAGCCTACTGCGCCTGCT-3', and pTaBamlo, 5'-TGGGATCCAGGGGTGGGTAAGATCTAA-3'. The two amplification products were cut with EcoRI, and the cohesive EcoRI sites were fused. cDNA encoding the TCR
chain of the N15 TCR (provided by L. Clayton, Dana-Farber Cancer Institute, Boston, MA) was inserted pUC1017 vector, 3' to the p56Lck proximal promoter. p56Lck proximal promoter TCR
or TCR
/pT
hybrid-hGH minigene fragments released by NotI digestion were microinjected into fertilized eggs. Founders were screened for transgene insertion by amplification of tail DNA with the primers lckp, 5'-AACCCAGTCAGGAGCTTGAA-3'; mus3, 5'-CATCGAGCAGAAGCAGTTTGA-3'; and TCRalo. pT
deficiency was assessed by amplification of tail DNA with the primers pTaF, 5'-TCACAGTGCTGGTAGATGGAAGG-3'; pTaKOR, 5'-GTTTGCTCGACATTGGGTGG-3'; and pTaWTR, 5'-GGCTCAAGAGATAACCTGAACCATG-3'.
Antibodies and Reagents.
Anti-CD8, anti-CD4, anti-TCR, anti-TCRß, anti-CD25, anti-CD44, anti-CD24 (heat-stable antigen [HSA]), antiGr-1, antiTer-119, anti-Dx5, anti-CD19, anti-CD3
, anti-CD45.1, anti-CD45.2, and Annexin V were purchased from BD Biosciences. Each mAb was either biotinylated or directly conjugated to FITC, PE, cychrome, or allophycocyanin fluorophores. Cychrome- or allophycocyanin-conjugated streptavidin (BD Biosciences) was used to reveal staining with biotinylated mAbs. Surface staining of thymocytes and BM was performed as described previously (14, 19). Cells were analyzed using a FACSCaliburTM flow cytometer (Becton Dickinson) and sorted using a MoFlo cell sorter (DakoCytomation). Propidium iodide was obtained from Sigma-Aldrich.
RT-PCR.
High pure RNA isolation kit (Roche) was used to isolate thymocyte RNA. SuperScript First-strand Synthesis System for RT-PCR (Invitrogen) was used to generate cDNA. Fivefold serial dilutions of cDNA were used for semi-quantitative PCR analysis of transgene and actin levels with the primers lckp; mus3; A2, 5'GGCACCCCCTTTCCGTCTCT-3'; TCRalo; actinup, 5'-TGGAATCCTGTGGCATCCATGAAAC-3'; and actinlo, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'.
Competition Assay.
BM cells lacking the lineage markers Gr-1, Ter-119, Dx5, CD19, TCRß, and CD3 were sorted from WTpT
, TCR
, TCR
/pT
hybrid, pT
-PRO
, and pT
-/- mice. 12 x 105 cells of each population were mixed and injected intravenously into the tail vein of each irradiated (500 rad) Rag-/-
c-/- mouse. Thymic reconstitution was analyzed 5 wk after injection.
Retroviral Infection.
WTpT, TCR
, or TCR
/pT
hybrid constructs, upstream of an internal ribosomal entry site and enhanced green fluorescent protein (EGFP; CLONTECH Laboratories, Inc.), were cloned into a modified Moloney murine leukemia virus-based retroviral vector (provided by R. Mulligan, Children's Hospital, Boston, MA). Retroviral supernatants were generated as described previously (19). 105 58
-ß+ T cell hybridoma cells were infected on ice for 3 h in the presence of 8 µg/ml polybrene.
Online Supplemental Material.
Surface expression of TCRß protein on CD4-CD8-CD25+CD44- (DN3) thymocytes from WTpT and transgenic mice was analyzed using anti-TCRß from BD Biosciences using a FACSCaliburTM flow cytometer (Becton Dickinson). Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20031973/DC1.
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Results |
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Neither TCR nor TCR
/pT
Hybrid Molecules Functionally Replace pT
in the pre-TCR Complex.
When compared in noncompetitive conditions with WTpT, neither TCR
nor the TCR
/pT
hybrid molecule successfully recapitulated pre-TCR function. In contrast with that of the endogenous pT
promoter, the activity of the p56Lck proximal promoter persists in the CD4+8+ stage of thymocyte development. Expression of a WTpT
transgene controlled by the p56Lck proximal promoter resulted in a copy numberdependent increase in apoptosis of CD4+8+ thymocytes, and a concomitant decrease in thymic cellularity (24). Although this effect was severe in transgenic founders containing >20 copies of the WTpT
transgene, little or no reduction in thymic cellularity was apparent in low copy number transgenic founders. Accordingly, the WTpT
transgenic founder used in the present work contained two to three transgene copies (24). Cellularity of thymi in TCR
and TCR
/pT
hybrid transgenic mice was only one third of that in WTpT
mice (Fig. 1 D). The fraction of thymocytes remaining at the CD4-8- stage of development was increased three- to fourfold in TCR
and TCR
/pT
hybrid thymi, whereas the proportion of thymocytes in the CD4+8+ compartment was decreased (Fig. 2 A).
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Pre-TCR signaling in DN3 thymocytes induces down-regulation of CD25 surface expression, survival, entry into the cell cycle, and progression through the DN4 stage, as well as acquisition of CD4 and CD8 surface expression (5). Propidium iodide staining of DN4 thymocytes sorted from TCR and TCR
/pT
hybrid mice revealed a threefold reduction in the proportion of this population in the S/G2/M phases of the cell cycle (Fig. 2 B), whereas Annexin V staining identified a three- to fourfold increase in the proportion of cells expressing the apoptotic surface phenotype characterized by externalized phosphatidyl serine (Fig. 2 C). Collectively, these data implicate deficits in cell cycle entry and survival initiated by TCR complexes containing TCR
or TCR
/pT
hybrid molecules as two reasons for the observed thymic hypocellularity.
Defective ß T Cell Lineage Commitment Induced by TCR
and TCR
/pT
Hybrid Molecules.
In accordance with the behavior of endogenous pT (13, 25), provision of the WTpT
transgene to pT
-/- mice reduced the proportion and absolute number of thymocytes expressing surface TCR
complexes (19). When compared with the CD4-8- compartment in WTpT
thymi, both the proportion (Fig. 3 A) and absolute number (Fig. 3 B) of TCR
+ thymocytes were elevated in mice expressing TCR
or TCR
/pT
hybrid transgenes. In contrast to the pre-TCR, receptors substituted with TCR
and TCR
/pT
hybrid molecules lack the ability to rescue CD4-8- thymocytes from commitment to the
TCR lineage.
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In addition to the abnormally increased population of CD4-8-TCRß+TCR+ thymocytes, an unusually large population of CD4-8-TCRß+TCR
- thymocytes was observed in mice expressing TCR
and TCR
/pT
hybrid transgenes (Fig. 3 D). In contrast to the HSAloCD4-8-TCRß+TCR
- population observed in C57Bl/6 (26) and WTpT
mice, the CD4-8-TCRß+ TCR
- thymocytes in TCR
and TCR
/pT
hybrid mice bore an immature HSAhi surface phenotype (Fig. 3 E). In fact, their HSAhi surface phenotype most closely resembled that of thymic HSAhi TCR
+ thymocytes (Fig. 3 F). Collectively, these observations raise the possibility that TCR
ß complexes may substitute more effectively for the TCR
complex than for the pre-TCR.
Differences in Pre-TCR and TCRß Functionality Become Much More Apparent under Competitive Conditions.
Analysis of the ability of precursors expressing each transgene merely to fill empty thymi, in an environment of unlimited resources and niche space, might obscure functional differences between each TCR complex. To directly assess the ability of TCR and TCR
/pT
hybrid molecules to function as the partner of TCRß in the pre-TCR complex, we forced precursors expressing each of these molecules to compete with those expressing WTpT
. To this end, 12 x 105 lineage-negative BM cells from mice expressing TCR
TCR
/pT
hybrid transgenes or pT
-/- mice were sorted and mixed with equal numbers of lineage-negative BM cells sorted from WTpT
mice. Crossing TCR
, TCR
/pT
hybrid, and WTpT
transgenes onto the pT
-/- Ly5.1+Ly5.2- or Ly5.1+Ly5.2+ background allowed us to identify thymocytes derived from each donor. Each BM cell mixture was injected into the tail vein of irradiated Rag-/-
c-/- recipients. Reconstituted thymi were analyzed 5 wk after injection.
The differences visible between thymi of mice expressing WTpT and TCR
transgenes became much more apparent in the competition assay. Although in noncompetitive assays, WTpT
transgenic thymi contained on average only threefold more thymocytes than did TCR
transgenic thymi, WTpT
-derived thymocytes dominated TCR
-derived thymocytes in the competitive reconstitution assay by an average of >60-fold (Fig. 4, A and C). The proportion of WTpT
-derived donor thymocytes that progressed to the CD4+8+ developmental stage was also far greater than that of the competing TCR
-derived population (Fig. 4 B). pT
-/--derived thymocytes were dominated by WTpT
-derived thymocytes by an average factor of thirty (Fig. 4, A and C). This slight (twofold) difference in performance between thymocytes derived from TCR
and pT
-/- BM could be the consequence of transgenesis and was not statistically significant (P > 0.09). Therefore, provision of a TCR
transgene failed to give any competitive advantage to a pT
-/- population.
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Specifically, the more efficient pairing of TCRß with TCR than with pT
(27) is attributed to the presence of a second extracellular V
domain in TCR
, which is absent in pT
. In addition, pairing with TCRß is influenced by differences in the position of the interchain cystine residue within the connecting peptide regions of TCR
and pT
. The addition of a V
domain to the extracellular portion of pT
generated a molecule capable of outcompeting wild-type pT
for pairing with TCRß molecules in the ER (27). The distance between the transmembrane domain and the cystine residue involved in covalent linkage with TCRß is longer in TCR
than in pT
. Relocation of this TCR
cystine to the position of the pT
cystine was accompanied by loss of efficiency in pairing with TCRß, to the degree that this mutant TCR
molecule, when expressed in the same cell as a wild-type TCR
molecule, could not compete for pairing with limited TCRß molecules (27). By virtue of its TCR
extracellular domain and connecting peptide sequence, the hybrid may outcompete pT
for pairing to TCRß in the ER. This higher efficiency of pairing may better protect TCRß monomers from degradation in the ER, resulting in higher levels of hybrid TCR complexes at the thymocyte surface. The apparent strict regulation of pre-TCR surface expression, mediated by its constitutive internalization and ubiquitin-mediated proteasome-dependent degradation (6, 7), highlights the potential link between receptor surface level and function. Thus, deviation from the exquisitely low level of surface receptor expression characteristic of the pre-TCR checkpoint could alter the intensity of signals received by DN3 thymocytes.
Indeed, symmetrical TCR complexes containing TCR and TCR
/pT
hybrid molecules, both of which contain a second extracellular V
domain as well as TCR
connecting peptide sequences, were expressed at levels higher than those of normal pre-TCR complexes on the surface of the 58
-ß+ T cell hybridoma (28), which is a variant of a DO-11.10.7 mouse T cell hybridoma that does not express functional TCR
or TCRß chains (Fig. 6). Thus, the possibility exists that higher surface levels might impair the pT
-like function of TCR
/pT
hybrid TCR complexes.
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Discussion |
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Although the TCR/pT
hybrid performance was superior to that of the TCR
and pT
-PRO
molecules, it remained inferior to that of WTpT
. One possible explanation for this discrepancy is that the higher surface expression levels of TCR
/pT
hybrid TCR complexes may impair their pT
-like function (Fig. 6). The strict regulation of pre-TCR surface expression, mediated by its constitutive internalization and ubiquitin-mediated proteasome-dependent degradation (6, 7), highlights the significance of the potential link between receptor surface level and function. Alternatively, the pT
-like function of TCR
/pT
hybrid TCR complexes may be affected by the strength of association between TCR
/pT
hybrid molecules and TCR
chains. Sequences unique to the connecting peptide of TCR
, which are present in the TCR
/pT
hybrid but absent in WTpT
, are required for strong association with TCR
chains (29). Their potentially stronger association with TCR
chains might allow TCR
/pT
hybrid TCR complexes to activate or augment signaling pathways that are silent or attenuated during normal pre-TCR signaling. A final possibility is that the second extracellular V
domain in the TCR
/pT
hybrid molecule may allow, in addition to higher levels of surface expression, recognition of MHC molecules in the thymic microenvironment. In contrast, the asymmetrical nature of the pre-TCR ectodomain precludes recognition of MHC molecules. The observed spontaneous segregation of pre-TCR complexes to glycolipid-enriched microdomains, which contain a high concentration of signaling molecules (2), may be involved in the cell-autonomous nature of pre-TCR signal initiation. Recognition of thymic MHC molecules might interfere with spontaneous membrane segregation processes, resulting in altered receptor signaling capacity.
Whether or not TCR can substitute for pT
in the pre-TCR complex has been a matter of some controversy and discussion. Conflicting views placing significance either on the receptor components themselves or on the developmental stage at which they are expressed have been the subject of numerous investigations. However, three factors have precluded a complete assessment of the interchangeability of pT
and TCR
at this point of development. These factors are as follows: (a) expression of TCRß and/or TCR
transgenes at developmental stages different than that of endogenous pT
, either in the presence (1518, 22) or absence (14) of endogenous pT
; (b) analyses of the ability of precursors to fill empty thymi with no competition for niche space or resources; and (c) use of fetal thymic organ culture (FTOC) systems that may not reveal the full proliferative potential of precursors (21). Here, we observe the performance of a TCR
molecule expressed in the absence of pT
, at the same relevant developmental stage and RNA levels as endogenous pT
, in adult thymi, both by itself and in direct competition for thymic space and resources.
Utilization of p56Lck proximal promoter elements ensured a uniform temporal regulation of WTpT, TCR
, TCR
/pT
hybrid, and pT
-PRO
transgene expression. Thus, our results fail to support the notion that the developmental stage at which the pre-TCR is expressed, rather than the identity of the receptor components themselves, is the factor on which pre-TCR function is most dependent. A recent work (21), reaching the opposite conclusion, compared the development of pT
-/- fetal thymocytes retrovirally transduced with either WTpT
or TCR
in FTOC. The short time of incubation in FTOC (2 d), which may have obscured differences in the full proliferative potential of precursors retrovirally transduced with each receptor component, raises a question regarding the validity of the conclusion that these two molecules are functionally equivalent at the pre-TCR checkpoint.
Its failure to rescue CD4-8- thymocytes from commitment to the T cell lineage may be perhaps the most significant deficiency in the repertoire of "pre-TCR-like" skills possessed by a TCR
ß complex. Specifically, this defect highlights a potential reason for the precise temporal segregation of pT
and TCR
expression during thymocyte development. By virtue of its failure to direct immature CD4-8- thymocytes through the normal ß selection processes vital to the development of mature CD4+ and CD8+ T lymphocytes, a TCR
molecule expressed at the pre-TCR checkpoint could instead direct young CD4-8- thymocytes into a pathway leading to an immature
-like fate.
Our data in TCR transgenic mice and previous data in pT
-/-TCR
-/- mice (14) indicate that a prematurely expressed TCR
chain can generate some limited numbers of TCRß-selected CD4+8+ thymocytes and argues that the
ß TCR, rather than a putative TCR
4/TCR
heterodimer (22), is involved in the selection of CD4+8+ thymocytes.
The requirement for the cytoplasmic domain of pT at the pre-TCR checkpoint might reside in its ability to recruit the pre-TCR complex to glycolipid-enriched membrane microdomains rich in signaling molecules. An interaction of the proline-rich region of the pT
cytoplasmic domain with Src homology 3 domains of raft-localized Src kinases could result in both raft localization and, via disruption of the inhibitory Src kinase intramolecular loop, in cell-autonomous activation of Src kinase activity. Alternatively, the pT
cytoplasmic domain might be required for constitutive internalization and degradation of pre-TCR complexes (6, 7), thereby regulating and maintaining the exquisitely low levels of pre-TCR complexes normally observed on the thymocyte surface.
Future studies analyzing differences in genetic and biochemical profiles induced by expression of WTpT and TCR
at the pre-TCR checkpoint may provide insight as to why TCR
is unable to substitute for pT
at this crucial stage of thymocyte development.
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Acknowledgments |
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This work was supported by National Institutes of Health grants AI47281 and AI45846 (to H. von Boehmer), and a National Science Foundation Fellowship (to C. Borowski). I. Aifantis is supported by the Cancer Research Institute and the Howard Hughes Medical Institute Research Resource Program at The University of Chicago. The authors declare that they have no competing financial interests.
Submitted: 14 November 2003
Accepted: 31 December 2003
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References |
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