The Biological Activity of Natural and Mutant pT{alpha} Alleles

Deena Gibbonsa, Nataki C. Douglasd, Domingo F. Barberd, Qiang Liua, Renee Sullod, Liping Gengd, Hans-Joerg Fehlingc, Harald von Boehmerb, and Adrian C. Haydaya,d
a Guy's King's St. Thomas' Medical School, Guy's Hospital, London Bridge, London SE1 9RT, United Kingdom
b Dana Farber Cancer Institute, Boston, MA 02115
c Department of Immunology, Medical Faculty/University Clinics Ulm, D-89070 Ulm, Germany
d Yale University, Department of Molecular, Cell and Developmental Biology, New Haven, CT 06520

Correspondence to: Adrian C. Hayday, Peter Gorer Department of Immunobiology, GKT School of Medicine, King's College, London, 3rd Floor New Guy's House, Guy's Hospital, London SE1 9RT, United Kingdom. Tel:44-(0)20-7955-8768 Fax:44-(0)20-7955-4961 E-mail:adrian.hayday{at}kcl.ac.uk.


  Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

ß selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRß chain, CD3 proteins, and a surrogate TCR{alpha} chain, pT{alpha}. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pT{alpha} gene encodes two RNAs, pT{alpha}a, and a substantially truncated form, pT{alpha}b. This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pT{alpha}-/- mice. Further active alleles of pT{alpha} include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR C{alpha}. Thus, very little of the pT{alpha} chain is required for function. These data support a hypothesis that the primary role of pT{alpha} is to stabilize the pre-TCR, and that much of the conserved structure of pT{alpha} probably plays a critical regulatory role.

Key Words: pre-TCR, thymocyte development, {alpha}/ß T cells, allelic exlusion, transgenic


  Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Most {alpha}/ß thymocytes develop and mature within the thymus. Progression through this intrathymic differentiation can be defined by the sequential expression of particular cell surface markers. Thus, whereas most mature {alpha}/ß T cells express either CD4 or CD8 coreceptors, their earliest immature thymic progenitors are CD4-CD8- double negative (DN). The earliest such DN progenitors express high levels of heat stable antigen (HSA) and CD44 (DN subset I), whereafter the cells acquire CD25 (DN II), then lose CD44 (DN III) and subsequently CD25 (DN IV) (1).

Although these subset classifications are purely operational and mask additional heterogeneity within each subset, the onset of TCR ß gene rearrangement can be largely attributed to DN II and DN III. Only those thymocytes that succeed in generating a functional TCRß chain selectively survive through the transition from DN III to DN IV (2) (3). As a result of such "ß selection," cells survive, become activated, expand in size, and proliferate rapidly before acquiring CD4 and CD8 (4). Such CD4+CD8+ double-positive (DP) cells account for the majority (~80%) of thymocytes (5).

ß selection is mediated by the pre-TCR which consists of—at minimum—the ß chain, CD3 components, and a surrogate TCR{alpha} chain termed pre-T{alpha} (pT{alpha}) (6) (7) (8). Thymocyte differentiation in mice deficient in components of the pre-TCR, or in signaling molecules downstream of it, are inhibited in their transition across "ß selection" (9) (10). Therefore, in pT{alpha}-/- mice there are very few DP cells, and the total number of thymocytes is usually only 1–10% of normal. Those DP cells that develop are largely driven by the contribution of TCR{gamma}/{delta} or the precocious expression of TCR{alpha}/ß (11) (12). Furthermore, it has been reported that thymocytes in pT{alpha}-/- mice more commonly express two productively rearranged TCRß chain genes, suggesting a defect in allelic exclusion (13).

By contrast, {gamma}/{delta} cell differentiation does not depend on pT{alpha}, and in pT{alpha}-/- mice, {gamma}/{delta} cell numbers are conspicuously increased. In particular, there occurs a subset of {gamma}/{delta} cells coexpressing CD4 that is not readily detected in normal mice (14). Such observations are consistent with the proposal that {alpha}ß/{gamma}{delta} lineage determination in thymocytes is somewhat flexible, strongly affected by the expression of TCR chains and of the pre-TCR (15) (16). In sum, the pre-TCR promotes a maturational program that includes cell survival; cell activation and growth; proliferation; differentiation to DP cells; the arrest of further ß chain gene rearrangement; and possibly fate determination. Hence, the mechanism of action of the pre-TCR is of considerable interest.

The pT{alpha} genes in mice and humans each give rise to two transcripts (17) (18). One, pT{alpha}a, encodes a transmembrane protein comprising a single Ig-like extracellular domain of ~110 amino acids, a transmembrane domain containing two charged amino acids, and a COOH terminal cytoplasmic region of ~30 amino acids (7). This COOH tail distinguishes pT{alpha} from other TCR or Ig chains that lack cytoplasmic regions of anything greater than a few amino acids. The second transcript, pT{alpha}b, splices out the second exon encoding the Ig-like ectodomain, and could therefore produce a significantly smaller isoform with very limited capacity to interact with molecules extracellularly (17). One important question is whether both naturally occurring alleles of pT{alpha} can promote thymocyte development, or whether the smaller form might be an inactive isoform that might, for example, perform a regulatory role. In this paper, a genetic complementation approach has been used to answer this question. Additionally, this paper examines which regions of the pT{alpha} protein are required for biological activity.

Because the pre-TCR is expressed in vivo at very low levels, and because there are few cell lines representative of immature thymocytes, there has been little biochemical characterization of the pre-TCR. Likewise, there has been no identification of a pre-TCR ligand. Instead, information on the active form of the pre-TCR has been gained primarily from genetic or cell biological experiments. Such experiments have provided some seemingly contradictory observations. First, a truncated pT{alpha} chain which lacks the highly conserved extracellular Ig-like loop can, together with a truncated TCRß chain, restore the development of DP cells in mice that are deficient in recombinase activating genes (RAG) 1 or 2, and that as a result cannot rearrange their endogenous TCRß chain genes (19). This result seems consistent with studies indicating that the pre-TCR can aggregate in the plasma membrane with lck in detergent insoluble glycolipids (DiGs), commonly termed rafts, even in the absence of any overt ligand (20). By this view, the more important components of pT{alpha} would appear to be the charged transmembrane domain that facilitates pairing with CD3; a juxtamembrane cysteine in the intracellular tail that may contribute to raft association; and any other components of the cytoplasmic tail that regulate signaling or pre-TCR stability. In particular, there is a proline-rich motif, occurring once in human pT{alpha} and repeated in murine pT{alpha} that has similarities to protein kinase C substrate sites (7), and to regions in CD2 that mediate signal transduction by binding to CD2BP2 (21). Indeed, in somatic cell transfection studies, mutants lacking the proline motifs show differences in properties compared with full length versions (unpublished data). However, other experiments indicate that thymocyte development in pT{alpha}-/- mice can be rescued by a pT{alpha} allele lacking the bulk of the cytoplasmic tail (14). Combining these data, one might hypothesize that only a very small portion of pT{alpha} (~80 amino acids), lacking much of the ectodomain and its intracellular region, is required for biological activity. This hypothesis is confirmed in this report. The implications of these findings and of the transgenic methodologies commonly used in such studies are discussed.


  Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Generation of Transgenic Mice.
cDNAs of the different pT{alpha} forms were generated by PCR. The full length pT{alpha}a, "p600," and the second isoform, pT{alpha}b, "p300," have been made previously (17). Truncated forms of pT{alpha}a and pT{alpha}b, named p600{Delta}P and p300{Delta}P, respectively, in which the last cytoplasmic 16 amino acids (containing the two proline rich regions and the two potential PKC phosphorylation sites) were deleted, were generated by PCR using the following primers: for p600{Delta}P, 5'-AATAGATCTCTACCATCAGGCATCGCT-3' and 5'-AATCCGCGGCTACTGGAGGTGCTGGCCCGC-3'. For p300{Delta}P, 5'-AATAGATCTCTACCATCAGGGGAATCT-3' and 5'-AATCCGCGGCTACTGGAGGTGCTGGCCCGC-3'. The pT{alpha}C{alpha} construct, in which the connecting peptide, transmembrane region, and cytoplasmic tail of p600 were substituted with those of TCR-C{alpha}, was generated in two fragments using two sets of primers. For the 5' part of pT{alpha}C{alpha}, 5'-AATAGATCTCTACCATCAGGCATCGCT-3' and 5'-AGCACACACCCCCTCCAGCTGTCAGACGTTCCCTGTGATGCCACGTTGACCGAG-3'. For the 3' part of pT{alpha}C{alpha}, 5'-CTCGGTCAACGTGGCATCACAGGGAACGTCTGACAGCTGGAGGGGGTGTGTGCT-3' and 5'-AATCCGCGGTCAACTGGACCACAGCCTCAGCGT-3'. Both PCR products were annealed for 30 min at 45°C, and subsequently amplified using the primers 5'-AATAGATCTCTACCATCAGGCATCGCT-3' and 5'-AATCCGCGGTCAACTGGACCACAGCCTCAGCGT-3'.

In each case, products were cloned in-frame (via BglII/SacII) into the expression vector pDisplay (Invitrogen). The Ig{kappa} leader sequence, HA tag, and cDNA were then subcloned from pDisplay into the BamH1 site of the T cell lineage–specific expression vector, p1017 (22) (23). A Not1-Not1 fragment from p1017 was then microinjected into C57.BL6 x SJL zygotes, that were implanted into pseudopregnant females. Progeny were screened by PCR and Southern, and transgenic lines crossed to pT{alpha}2/-.

Isolation of Lymphocytes.
Intraepithelial lymphocytes were harvested as described (24). Single thymocyte suspensions were obtained by crushing whole thymi between the edges of frosted glass slides into FACS® buffer (1x PBS/2% FCS/0.1% azide). Live cells were determined by trypan blue exclusion.

Flow Cytometry.
Thymocytes or intestinal intraepithelial lymphocytes at <=2 x 107/ml were stained with the antibodies listed below and analyzed, on either a FACStarPlusTM (Becton Dickinson) or a FACS VantageTM (Becton Dickinson) flow cytometer. Data were analyzed with CELLQuestTM software. Monoclonal antibody reagents obtained from BD PharMingen were: {alpha}CD4-FITC (GK1.5), {alpha}CD8{alpha}-PE (53–6.7), {alpha}CD25-FITC (7D4), {alpha}CD44-APC (IM7), {alpha}CD44-cy-Chrome (IM7), {alpha}HSA-PE (M1/69), {alpha}TCR {alpha}ß-PE (H57–597), {alpha}TCR {gamma}{delta}-PE (GL3), {alpha}CD3{epsilon}-biotin (145–2C11). Other antibodies and reagents for flow cytometry included avidin Red670 (GIBCO BRL) and rat normal serum (GIBCO BRL).

RNA Isolation, cDNA Synthesis, and Semiquantitative PCR.
RNA isolated using Trizol reagent (GIBCO BRL) was DNase treated (GIBCO BRL) and quantitated by spectrophotometry. AMV reverse transcriptase (Roche) reactions were primed with Pd(N) (Amersham Pharmacia Biotech). Standard reverse transcription (RT)-PCRs using 200 ng thymocyte RNA from pT{alpha}-/- transgenic, and nontransgenic littermates used primers as follows: HA-For, 5'-CCA TAT GAT GTT CCA GAT TAT GCT-3'; {Delta}P1{Delta}P2-Rev, 5'-CTG GAG GTG CTG GCC CGC-3'; PTA REV, 5'-CTA TGT CCA AAT TCT GTG GGT-3'; HGH REV, 5'-GGA TAT AGG CTT CTT CAA AC-3'.

Immunoprecipitation and Immunoblotting.
For immunoprecipitations, 2 x 107 freshly isolated thymocytes or pT{alpha} transfectants (17) were washed in cold PBS buffer, lysed in 300 µl ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCI, 1% NP-40, 0.25% Na-deoxycholate, 10 mM NaF, 10 mM Na4P2O7, 1 mM EGTA, 1 mM MgCl2, 1 mM Na3VO4, 1 mM PMSF, 20 µg/ml aprotinin, 20 µg/ml leupeptins, 10 µg/ml Pepstatin A, 20 µg/ml antipain). Lysates were incubated for 20 min on ice before centrifugation at 13,000 rpm for 15 min at 4°C. Postnuclear lysates were agitated for 1 h at 4°C with 2 µl anti-HA monoclonal antibody HA.11 (BabCO). 25 µl protein A-Sepharose beads (Amersham Pharmacia Biotech), swollen and washed in lysis buffer, were added and incubated overnight at 4°C. The beads were washed 3x in cold lysis buffer, and proteins eluted by boiling for 5 min in SDS sample buffer, separated by 15% SDS-PAGE gel, and transferred to nitrocellulose for immunoblotting. The membranes were blocked with 4% nonfat milk in TBS (10 mM Tris-HCl, pH 7.6, 150 mM NaCl) and incubated with HA.11 (1:3,000). Bound Ab was revealed with 1:8,000 diluted horseradish peroxidase (HRP)-conjugated donkey anti–mouse IgG (Jackson ImmunoResearch Laboratories) using Western blot chemiluminescence reagent (NEN Life Science Products).

Allelic Exclusion.
Single CD4+CD8+ double-positive thymocytes were sorted using a MoFlo high speed cell sorter (Cytomation) into a 96-well plate, containing 10 µl of PCR buffer containing proteinase K (250 µg/ml). Plates were incubated at 55°C for 1 h and then the proteinase K was inactivated by heating at 95°C for 15 min. Plates were then stored at -20°C. TCRß rearrangements were amplified by a seminested two-step PCR using primers as described by Aifantis and colleagues (13) (25). Briefly, 40 µl of a mixture containing dNTPs, buffer, 3 pmol of each Vß, Dß, and Jß primer, and 0.1 U of Taq polymerase (QIAGEN) was added to each well. Amplification comprised five cycles in which the denaturing step was 96°C and the annealing temperature decreased from 68–60°C, followed by a further 25 cycles (30 s at 94°C, 1 min at 58°C, 1 min at 72°C) and finally 7 min at 72°C. For the second round of amplification, 1 µl of the first round product was transferred into a fresh tube containing a single 5' primer in conjunction with the nested Jß1 or Jß2 primer (10 pmol of each), dNTPs, buffer, and Taq polymerase (0.1 U) in final volume of 25 µl. Amplification was for 35 cycles following the same procedure as the first round PCR (except denaturing was always at 94°C). Rearrangements were detected by migration of the PCR product on a 1.5% ethidium bromide stained agarose gel and positives purified using QIAGEN purification kits. Direct sequencing of the PCR products was performed using the BigDye Ready Reaction sequencing mix (ABI) and automated sequencing performed on a 96 lane ABI 377 sequencer.


  Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Generation of Transgenic pT{alpha}2/- Mice Expressing Different Forms of pT{alpha}b.
To determine whether the naturally occurring pT{alpha}b gene and derivatives thereof could functionally promote thymocyte development, numerous lines of transgenic mice were generated expressing either pT{alpha}b (p300) or a truncated form lacking the proline-rich regions and much of the remainder of the cytoplasmic tail (p300{Delta}P). As a positive control, mice were also generated that expressed pT{alpha}a (p600) or a corresponding cytoplasmic tail deletion mutant (p600{Delta}P). Transgenic mice of the latter type have previously been reported (14). Additionally, transgenic mice were generated that expressed a chimeric cDNA encoding the extracellular region of pT{alpha} joined to the connecting peptide, transmembrane, and intracellular regions of the mature C{alpha} gene (pT{alpha}C{alpha}, Fig 1 A). In each case, relevant cDNAs were cloned into the p1017 vector, containing the lck proximal promoter to ensure early T lineage expression ((22) (23); Fig 1 B). Transgenic lines were established (genotyping not shown) and backcrossed onto the pT{alpha}-/- background, generating F2 animals that expressed the different transgenes in the absence of endogenous pT{alpha}.



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Figure 1. Schematic representation of different pT{alpha} transgenes and their expression. (A) Schematic representation of the five different pT{alpha} cDNAs constructed as described in Materials and Methods. (B) Each of the pT{alpha} cDNAs was cloned (together with a HA tag and Ig{kappa} leader sequence) into the BamH1 site of the p1017 vector. The purified Not1 fragment of p1017 recombinants was microinjected into the pronuclei of fertilized eggs. (C) Reverse transcription (RT)-PCR detection of pT{alpha} mRNA expression from thymocytes of the different pT{alpha} transgenics. Transgenics positive by genotyping (+) gave a positive mRNA expression when compared with those that were negative by genotyping (-). GAPDH was positive in all samples. (D) The expression of p300 and p300{Delta}P protein in thymocytes from transgenic mice compared with protein expression in a p300 transfected cell line. 7.1 kD denotes the migration of a protein size marker on the same gel.

In every case, pT{alpha}-/- mice that genotyped positively for a transgene showed transgene RNA expression in the thymus (Fig 1 C). As the emphasis of this manuscript is on the biological activity of pT{alpha}b, the expression of wild-type and mutant forms of pT{alpha}b protein was additionally examined (Fig 1 D). Levels detected by Western analysis of HA-tagged protein in thymocyte lysates were comparable to those in a pT{alpha}b-transfected cell line (17) (26). Not surprisingly, both RNA and protein expression varied markedly among different founder transgenics, limiting the degree to which one should cross-compare phenotypes of transgenic mice generated with different pT{alpha} alleles.

pT{alpha}b Transgenes Induce DP Cell Representation.
The surface phenotypes of thymocytes in the pT{alpha} transgenic mice were analyzed by flow cytometry. The majority of pT{alpha}-/- thymocytes are blocked as DN cells (Fig 2 A). Conversely, pT{alpha}b, as well as the smallest transgene, p300{Delta}P, could completely restore DP thymocyte representation (DP cells are 84% of the total thymocyte number in 300{Delta}P mice, compared with 7% in pT{alpha}2/- mice; Fig 2 A). The lack of any essential functional requirement for the pT{alpha} cytoplasmic tail was confirmed by the restored phenotypes of pT{alpha}-C{alpha}, and p600{Delta}P transgenic mice (Fig 2 B). In all, substantive restoration of DP differentiation was shown by at least one line of transgenic mice generated with each of the alleles (p300, p300{Delta}P, p600, p600{Delta}P, and pT{alpha}-C{alpha}; Fig 2 B).



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Figure 2. Restoration of phenotype by expression of pT{alpha} transgenes. (A) CD4 versus CD8 expression on thymocytes from pT{alpha}-/- (left) and p300{Delta}P.pT{alpha}-/- (right). The percentage of thymocytes in each quadrant are shown. (B) The percentage of DP (i.e., CD4+CD8+) cells in the thymi of mice expressing different pT{alpha} transgenes. Individual vertical lines represent independent lines of mice; individual data points on those vertical lines represent the percentage of DP cells in the thymi of individual mice. (C and D) The percentage of DN {gamma}/{delta} cells (C) or CD4+ {gamma}/{delta} cells (D) in the thymi of mice expressing different pT{alpha} transgenes. Individual vertical lines represent independent lines of mice; individual data points on those vertical lines represent the percentage of {gamma}/{delta} cells in the thymi of individual mice. (E) Total cellularity of the thymus (x106 cells) was established by trypan blue exclusion. Individual vertical lines represent independent lines of mice; individual data points on those vertical lines represent the cell numbers in individual mice.

pT{alpha}b Transgenes Reduce {gamma}/{delta} Cell Representation in the Thymus and Gut.
Normally, thymic {gamma}/{delta} cells compose ~1% of thymocytes. These levels are increased to 5–20% of thymocytes in pT{alpha}-/- mice, and comprise both DN cells and CD4+ {gamma}/{delta} cells, a subset rarely detected in wild-type thymi (Fig 2C and Fig D). Conversely, the conventional, low level of {gamma}/{delta} cell representation (both DN and CD4+) was restored in transgenic pT{alpha}2/- mice expressing the full length or the truncated forms of pT{alpha}b (Fig 2C and Fig D). Indeed, the normal phenotype was restored in at least one line of transgenic mice generated with each of the five pT{alpha} alleles under study (Fig 2C and Fig D). In all cases the influence of pre-TCR expression over the percentages of {gamma}/{delta} cells correlated reasonably well with the absolute numbers of {gamma}/{delta} cells (Table 1), indicating that changes in the percentages of {gamma}/{delta} cells are real changes and not simply an indirect effect of changes in other cell subsets, e.g., DP thymocytes.


 
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Table 1. Percentage of {gamma}/{delta} Cells Correlates Well with Absolute Cell Numbers

Consistent with the block in {alpha}/ß T cell development in pT{alpha}-/- mice, the intestinal intraepithelial lymphocytes (IELs) are depleted of {alpha}/ß T cells, and instead comprise ~90% {gamma}/{delta} cells (Table 2). Because the representation of {gamma}/{delta} cells in the pT{alpha}-/- thymus was significantly suppressed by the expression of the small p300{Delta}P construct, the question arose as to whether {gamma}/{delta} cells would be similarly reduced to normal levels in the gut. This was indeed the case: IELs from pT{alpha}-/- mice expressing the p300{Delta}P transgene included fewer {gamma}/{delta} cells than the pT{alpha}-/- gut, with a representation (~50% of CD3+ IELs) that was comparable to normal (Table 2).


 
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Table 2. p300{Delta}P Transgene Expression Recovers {alpha}/ß IELs

Hypocellularity of the pT{alpha}2/- Thymus Is Rescued.
The thymi of 6–8-wk-old wild-type mice contain an average of 1.2 x 108 thymocytes, with a range of ~0.9–1.9 x 108 cells. pT{alpha}-/- mice contain 10–50-fold fewer thymocytes. Two transgenic lines, one expressing p300 (pT{alpha}b) and one expressing p300{Delta}P showed rescue of an approximately normal range (Fig 2 E). In other lines, thymic cellularity was often not well rescued even where there was substantial and parallel restoration of normal DP and TCR{gamma}/{delta}1 thymocyte phenotypes (Fig 3). However, this does not reflect a failure of pT{alpha} transgenes to regulate cellularity because there is an additional effect whereby transgenes expressing either TCR chains or pre-TCR chains commonly reduce thymus cellularity, even on a wild-type background (unpublished data). As an example, two p600{Delta}P mice on a pT{alpha}+/+ background contain 4.8 x 107 and 6.2 x 107 thymocytes, respectively, while one p300{Delta}P pT{alpha}+/- strain contained 6.6 x 107 cells. The fact that these cell numbers, albeit lower than normal, are comparable in transgenic mice on a pT{alpha}-/- or a pT{alpha}+ background indicates that each of the trangenes has overcome the negative influence of pT{alpha} deficiency on thymocyte numbers (see Discussion).



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Figure 3. Schematic comparison of different transgenic lines. Two parameters of thymocyte phenotype (DP or {gamma}/{delta} cell numbers) segregate together in different transgenic lines, but segregate variably with thymus size. Gradation of shading represents movement away from wild-type phenotype (see key).

Allelic Exclusion Occurs in p300{Delta}P Mice.
One component of normal thymocyte development is allelic exclusion. To test whether this occurs in the DP thymocyte compartment that is restored in mice expressing the smallest pT{alpha} transgene, p300{Delta}P, TCRß rearrangements were examined by single cell PCR. Multiple primers were used that amplify many (but not all) Vß segments, and that distinguish rearrangements of V or D to Jß1 or Jß2. Products were obtained from all cells analyzed. Conspicuously, the biased Vß usage seen in immature thymocytes of normal mice (e.g., preferential usage of Vß8) was also seen in the p300{Delta}P pT{alpha}-/- mice (27).

Many cells gave more than two PCR products but when these rearrangements were analyzed they corresponded to DNA excision loops formed alongside correct chromosomal rearrangements. 25% of cells exhibited a single in-frame VDJ rearrangement together with a DJ rearrangement. A further 1/3 of cells showed only one rearrangement which was an in-frame VDJ junction. The remaining cells showed one in-frame VDJ rearrangement together with a nonproductive VDJ rearrangement (Table 3). No cell tested produced more than one in frame VDJ rearrangement, indicating that allelic exclusion operates in the p300{Delta}P transgenic mouse.


 
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Table 3. Allelic Exclusion Occurs in the p300{Delta}P Transgenic Line


  Discussion
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Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

This study has investigated whether the second naturally occurring pT{alpha} transcript, pT{alpha}b encodes a biologically active pT{alpha} isoform. It has also asked whether thymocyte development can be sustained by an even smaller pT{alpha}b allele that lacks both the major Ig-like ectodomain and the bulk of the cytoplasmic tail. Finally, it has asked whether thymocyte development can be functionally sustained by a pT{alpha} allele in which the cytoplasmic tail has been exchanged for the short tail of C{alpha}. The measurement of biological activity was the functional complementation of the pT{alpha}-/- mouse. The four primary defects reported in pT{alpha}-/- mice are: the paucity of DP thymocyte differentiation (correlated with a relative increase in the percentage of DN thymocytes); increases in TCR{gamma}/{delta}1 cells of both DN and CD4+ phenotypes; the failure of allelic exclusion at the TCRß locus; and decreases in thymocyte numbers. Both the full length and truncated alleles of pT{alpha}b as well as the pT{alpha}-C{alpha} allele showed the capacity to largely or fully restore these phenotypes, although allelic exclusion was assessed in only one of the strains (p300{Delta}P). Thus, highly truncated forms of pT{alpha} are biologically active, including the naturally occurring form, pT{alpha}b that is conserved in humans and mice (17) (18). Interestingly, a targeted mutation of the pT{alpha} locus that would be predicted to leave intact the coding potential of pT{alpha}b showed a very mild phenotype in terms of altered thymocyte differentiation, consistent with the idea that pT{alpha}b is biologically active in vivo (10).

There is some contention over which of the four primary thymocyte defects reported in pT{alpha}-/- mice reflect direct targets of pT{alpha}. More than one of these events may be directly downstream of pT{alpha}, but some may have greater dependence on pT{alpha} function than do others. Data presented here show that all four parameters are rescued in at least one line expressing either the full length or the heavily truncated pT{alpha} alleles. Nonetheless, in several mice in which thymocytes could be classified into largely normal subset distributions, cellularity was not always normal (Fig 3). Similar variability in thymus cellularity is evident when data from other mice transgenic for pre-TCR components are considered (14) (19). At minimum, these data demonstrate that appropriate thymocyte differentiation can occur independent of extensive proliferation. A similar situation appears to characterize the IL-7-/- mouse (28).

The lack of normal cellularity seems in part due to an inhibitory effect of the expression of pT{alpha} transgenes, as a similar reduction, relative to normal, was seen in the transgenic mice on a pT{alpha}+ background. Similar observations have been made in mice expressing TCR transgenes. This may reflect the inappropriate prolonged expression of the pre-TCR that is a variable characteristic of transgenic mice. It is known that constitutively activated lck provokes loss of DP cells (29) possibly because the cells interpret continued signaling as a negative selection stimulus. Sustained expression of the pre-TCR may do likewise. Ordinarily, the pre-TCR is expressed at very low levels, and may be easily displaced by TCR{alpha}/ß after TCR{alpha} gene rearrangement. In transgenic mice, the physiologic expression of pT{alpha} will not be precisely mimicked because of the heterologous promoter elements, and because of integration sites that will vary from founder to founder. Additionally, the displacement of pT{alpha} from TCRß will likely depend on active signaling mechanisms that regulate the stability and intracellular localization of the pT{alpha} protein (30). These mechanisms may not function properly in the context of mutant pT{alpha} transgenes. For these various reasons, the transgenic mice may harbor a situation similar to that reported in lck transgenic mice. These are significant qualifications that must be applied to the interpretation of such transgenic studies.

The simplest explanation for the biological activity of very small forms of pT{alpha} is that pT{alpha} functions merely to stabilize the ß chain (aiding interaction with CD3 and other downstream signaling molecules), and that a minimal peptide of pT{alpha} is sufficient to accomplish this. This is consistent with other instances of expression of truncated pT{alpha} or TCRß alleles. For example, mice transgenic for a TCRß chain lacking the Vß region showed appropriate DN to DP transition (31). The biological activity of truncated versions of pT{alpha} is consistent with evidence that, independent of any ligand, the pre-TCR spontaneously clusters and associates with signaling molecules such as p56lck, CD3 molecules, and Zap-70 via sequestration in lipid rafts (20).

Presumably all active forms of pT{alpha} will elicit signaling to nuclear factor (NF)-{kappa}B implicated in promoting cell survival (26), and Vav-1 and Rac-1 (32) (33) implicated in modulating actin dynamics that are important in spatially orienting signaling molecules to coordinate and sustain signal transduction (for a review, see reference (34)). Indeed, electrophoretic mobility shift assays indicate high levels of NF-{kappa}B activity in DN thymocytes from pT{alpha} transgenic mice (unpublished data). Nonetheless, it may be that signaling from the physiologic pre-TCR; signaling from a pre-TCR containing the pT{alpha}-C{alpha} chimeric molecule, and signaling in the absence of the pre-TCR but via cross-linking CD3, each activate the same signaling molecules, but by different means. For example, the palmitoylation of the juxtamembranous Cys residue present in pT{alpha} that is implicated in raft association, cannot obviously occur in the pT{alpha}-C{alpha} chimeric protein that lacks the juxtamembrane cysteine. Yet, both of the pT{alpha}-C{alpha} transgenic lines showed comparable restoration of thymocyte phenotypes, and were not obviously less effective than the other transgenes. Possibly, the pT{alpha}-C{alpha} protein facilitates signal transduction largely independent of raft association, because the connecting peptide of C{alpha} (as opposed to the equivalent domain in pT{alpha}) has a much stronger association with CD3{zeta} (35). This would be consistent with the hypothesis that regulated activation of common downstream signaling pathways is the rate determining step to ß-selection, and can be achieved by distinct, albeit related mechanisms.

If only a very small part of pT{alpha} is required for its function, the question arises as to why pT{alpha} shows conservation of its structure. One explanation is that the biological activity of the pre-TCR is so potent that many of its structural features are essential for downregulation, ensuring that the pre-TCR is expressed only at appropriate levels only during the appropriate time window. In addition to the low level and restricted time frame of pre-TCR expression, several other experiments are suggestive of this. For example, sustained expression of a transgenic TCRß allele that lacks the V region led to thymic lymphomas (36). Although such lymphomas did not characterize any of the several pT{alpha} transgenic mice reported here, this may be because ectopically expressed pT{alpha} can be displaced, albeit inefficiently, by TCR{alpha}, whereas a mutant TCRß chain would not be, thus sustaining ligand-independent signaling. Likewise, severe lymphomas with some characteristics of pre-T cells (including sustained pT{alpha} expression) developed with >80% penetrance in several lines of mice transgenic for an activated form of Notch 3, the regulated expression of which normally characterizes the ß-selection stage (for a review by Rothenberg, see reference (37)). In striking illustration of the potency of sustained pre-TCR signaling, the development of Notch 3–induced tumors is dependent on pT{alpha} expression (unpublished data).

According to this view, the pre-TCR is a potent agent of cell growth and survival that must be downregulated after ß-selection. Therefore, there are likely to be active signaling components of the pre-TCR pathway, and possibly an as yet unidentified ligand that may target regions of pT{alpha} in order to negatively regulate its expression and activity. This would lead to conservation of those regions of pT{alpha}. This remains to be tested biochemically, although the recent report that the pT{alpha} tail can serve as an endoplasmic reticulum retention signal is consistent with this outlook (38). Studies of pre-B cell receptor signaling have indicated that the expression level of pre antigen receptors is a product both of the structure of pre-antigen receptor chains and the cell biological characteristics unique to the immature cells in which the pre-antigen receptors are expressed (39).


  Footnotes

D. Gibbons, N.C. Douglas, and D.F. Barber contributed equally to this work.
D.F. Barber's present address is Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852.
L. Geng's present address is Dana Farber Cancer Institute, Boston, MA 02115.
Q. Liu's present address is Brigham and Women's Hospital, Boston, MA 02115.


  Acknowledgements
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
References

Grants from the Wellcome Trust, the Charitable Foundation of Guy's and St. Thomas' Hospitals, and the National Institutes of Health (GM37759) (A.C. Hayday), and the MSTP program [N.C. Douglas]. Support provided (D.F. Barber) by Spanish government fellowship (Ministerio de Educacion y Ciencia).


  References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgements
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