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The Journal of Immunology, 2002, 169: 4913-4919.
Copyright © 2002 by The American Association of Immunologists

Positive Selection by the Pre-TCR Yields Mature CD8+ T Cells1

Yuriko Ito2,*, Satoko Arai2,*,{dagger}, Nicolai S. C. van Oers*, Iannis Aifantis{ddagger}, Harald von Boehmer{ddagger} and Toru Miyazaki3,*

* Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390; {dagger} Department of Applied Biological Chemistry, University of Tokyo, Tokyo, Japan; and {ddagger} Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been of much interest whether there is functional redundancy between the constitutively signaling pre-T{alpha}/TCR{beta} (pre-TCR) and ligated TCR{alpha}{beta} complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in {beta}-selection at the CD4-CD8- double-negative stage and the TCR{alpha}{beta} being crucial for positive/negative selection at the CD4+CD8+ double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCR{alpha}-deficient (TCR{alpha}-/-) mice produced a small number of mature CD8+ T cells. Surprisingly, when pre-T{alpha} was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8+ T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8+ T cells was also observed in TCR{alpha}-/- mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8+ T cells drastically decreased in the absence of pre-T{alpha} on a TCR{alpha}-/- background. Thus, the pre-TCR appears to functionally promote positive selection of CD8+ T cells. The biased production of CD8+ T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of T cells in the thymus is controlled at two consecutive checkpoints (1, 2). At the first checkpoint, termed {beta}-selection, CD4-CD8- double-negative thymocytes with a productive TCR{beta} rearrangement are rescued from programmed cell death by the pre-TCR consisting of the varied TCR{beta} and the invariant pre-TCR{alpha} (pT{alpha})4 chain. As a result, the cells proliferate, and begin to coexpress CD4 and CD8 and to rearrange the TCR{alpha} locus. Some evidence suggests that the pre-TCR is also involved in channeling cells into the {alpha}{beta}-lineage because it reduces the number of {gamma}{delta} T cells with productive TCR{beta} rearrangements (3, 4). At the second checkpoint, the engagement of the TCR{alpha}{beta} by appropriate MHC ligands rescues CD4+CD8+ double-positive (DP) thymocytes from apoptotic death, resulting in the generation of mature CD4+CD8- or CD4-CD8+ single-positive (SP) thymocytes. Very interestingly, there exists an obvious similarity of proximal signaling events between the constitutively signaling pre-TCR and the ligated TCR{alpha}{beta}, resulting in tyrosine phosphorylation of CD3{zeta} and CD3{epsilon} subunits, recruitment and phosphorylation of ZAP-70 and syk protein kinases, activation of the ras/mitogen-activated protein kinase pathway, and rapid increase in intracellular calcium levels (5, 6, 7, 8, 9). Because of these similarities, we asked the question whether in certain situations the pre-TCR can partially or even entirely substitute for the ligated TCR{alpha}{beta}, resulting in the generation of "{alpha}{beta}-less" mature T cells. In this study, we approached this question by qualifying different strains of mice expressing the pre-TCR on the DP precursor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic mice

A 1.3-kb fragment of pT{alpha} cDNA (kindly provided by H. J. Fehling, Ulm, Germany) was subcloned into the blunt-ended BamHI site of the expression vector controlled by the lck-proximal promoter (10). The complete plasmid (plck-pT{alpha}) was digested with NotI, and the transgene fragment no longer containing vector sequence was purified by Geneclean II kit (Bio 101, Vista, CA). DNA was microinjected into fertilized eggs of C57BL/6 mice. Resulting founders were screened for transgene by PCR and Southern blotting. Five independent founders carried the transgene. Two of the five mice that expressed transgene at relatively higher levels, as assessed by Northern blotting using thymus RNA, were backcrossed within TCR{alpha}-/- background. All animals used in the experiments were cared in accordance with institutional guidelines.

RNA analysis

Total RNA was isolated from thymus and lymph nodes (LNs) by using RNAzolB (Tel-Test, Friendswood, TX) from various mice. A quantity amounting to 15 µg total RNA was denatured, electrophoresed in a 1% agarose gel, blotted on a nylon membrane, and then hybridized with 32P-labeled either pT{alpha} or {beta}-actin cDNA fragment.

Immunoprecipitation and Western blotting

Thymocytes from various mice (6.0 x 107) were lysed at a concentration of 1 x 108 cells/ml in a Triton X-100-containing lysis buffer (20 mM Tris-Cl, pH 7.6, 150 mM NaCl, 2.0 mM EDTA, and protease and phosphatase inhibitors). The TCR{zeta} subunit was specifically immunoprecipitated with an anti-{zeta} mAb (6B10.2). The immunoprecipitates were separated on SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were immunoblotted with anti-phosphotyrosine or anti-{zeta} mAb, as previously described (7).

Antibodies

Abs used in this study: anti-mouse CD4 (GK1.5), anti-mouse CD8{alpha} (53-6.7), anti-mouse TCR{beta} chain (H57-597), anti-mouse TCR{gamma}{delta} (GL-3), anti-mouse CD8{beta} (H35-17.2), anti-pan NK cells (DX-5), anti-Thy-1.2 (30-H12), anti-mouse CD25 (3C7), anti-mouse CD69 (H1.2F3), anti-mouse heat-stable Ag (HSA) (30-F1), anti-phosphotyrosine (4G10), and anti-CD3{zeta} (6B10.2) were purchased from BD PharMingen (San Diego, CA). The anti-CD3{epsilon} mAb (145-2C11) was purchased from eBioscience (San Diego, CA). Intracellular staining was performed by using the Cytofix/Cytoperm kits (BD PharMingen).

T cell activation

For IL-2 production, LN cells (1.0 x 106 cells/well) either from wild-type (WT) or pT{alpha}-transgenic (TG)/TCR{alpha}-/- mice were cultured in the presence or absence of 10 ng/ml of PMA + 5 x 10-7 M of ionomycin (Sigma-Aldrich, St. Louis, MO) in a 96-well plate for 16 h. The cells were then stained for intracellular IL-2 and cell surface CD8. CD8+ cells were analyzed for IL-2 levels by FACSCalibur cytometer (BD Biosciences, San Jose, CA). For expression of activation markers, LN cells (1.0 x 106 cells/well) either from WT or pT{alpha}-TG/TCR{alpha}-/- mice were cultured in the presence or absence of 1 µg/well of immobilized anti-CD3{epsilon} mAb or 1 µg/ml Con A in a 96-well plate for 16 h. The anti-CD3{epsilon} mAb was immobilized by preincubation of wells at 4°C overnight. The cells were then stained for CD8, {gamma}{delta}, intracellular TCR{beta} (int{beta}), and either for CD25 or CD69, and then analyzed by cytometer. CD8+{gamma}{delta}-int{beta}+ cells were analyzed for expression of either CD25 or CD69.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Positive selection of mature CD8+ T cells by the pre-TCR

First, we analyzed a set of mice expressing either low or high levels of the pre-TCR in the absence of TCR{alpha} chain, i.e., TCR{alpha}-deficient (TCR{alpha}-/-) mice (11) or TCR{alpha}-/- mice expressing a transgenic pT{alpha} chain under the regulation of lck-proximal promoter. In thymocytes of TCR{alpha}-/- mice, the pre-TCR should be expressed at some level in DP thymocytes because most of the cells express the TCR{beta} chain due to {beta}-selection, and still express the pT{alpha} chain that in this situation cannot be outcompeted by the TCR{alpha} chain (12). In the newly developed pT{alpha}-transgenic mice on a TCR{alpha}-/- background (pT{alpha}-TG/TCR{alpha}-/- mice), the pT{alpha} expression was much higher than in TCR{alpha}-/- mice in the thymus, although it was at undetectable level in the peripheral lymphoid organs by Northern blotting, which is compatible with the characteristic of the transgene prompter (10) (Fig. 1GoA). To assess whether the amount of signals mediated via the pre-TCR in each cell at the DP stage was actually strengthened by pT{alpha} overexpression in transgenic mice, we analyzed the tyrosine phosphorylation of CD3{zeta} chains in thymocytes, which are known to be functionally coupled to both the pre-TCR as well as the TCR{alpha}{beta} (7, 13, 14, 15). To this end, CD3{zeta} chains were immunoprecipitated from lysates of thymocytes from either pT{alpha}-TG/TCR{alpha}-/-, TCR{alpha}-/-, or WT mice, and their phosphorylation status was examined. In pT{alpha}-TG/TCR{alpha}-/- and TCR{alpha}-/- mice, CD3{zeta} chain should associate only with the pre-TCR, because of the lack of TCR{alpha}{beta} in the absence of {alpha}-chain. As illustrated in Fig. 1GoB, there was much more CD3{zeta} chain phosphorylation in thymocytes from pT{alpha}-TG/TCR{alpha}-/- as compared with those in TCR{alpha}-/- (highlighted as {zeta}-PO4), whereas total amounts of precipitable {zeta}-chains were almost equal in both strains (bottom panel, CD3-{zeta}). Thus, pT{alpha} overexpression appeared to achieve efficiently increased signals through the pre-TCR in transgenic thymi.



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  		FIGURE 1. Positive selection of CD8+ T cells by the pre-TCR. A, The pT{alpha} expression was markedly enhanced in the thymus (Thy) of pT{alpha}-TG/TCR{alpha}-/- mice, but not in the LN, as assessed by Northern blotting. B, Increased CD3{zeta} chain phosphorylation under the overexpression of pT{alpha} in TCR{alpha}-/- mouse thymocytes. Lysates of thymocytes from WT, TCR{alpha}-/-, and pT{alpha}-TG/TCR{alpha}-/- mouse thymi were immunoprecipitated with an anti-CD3{zeta} mAb and probed sequentially on Western blots with anti-phosphotyrosine mAb 4G10 (top panel) and anti-CD3{zeta} mAb 6B10.2 (bottom panel). C, LN (mesenteric and inguinal) cells from either TCR{alpha}-/- or pT{alpha}-TG/TCR{alpha}-/- mice were stained with anti-CD4, anti-CD8{alpha}, and anti-TCR{beta} chain (for intracellular staining) and analyzed by FACSCalibur cytometer (BD Biosciences). CD4/CD8{alpha} profiles of the cells pregated on int{beta}+ population are displayed. The int{beta}+CD8+ cells were remarkably increased in pT{alpha}-TG/TCR{alpha}-/- mice (right panel) as compared with those in TCR{alpha}-/- mice (left panel). These int{beta}+CD8+ cells were analyzed for TCR{gamma}{delta} (D), and for CD8{beta}, Thy-1.2, and DX-5 (E) by using specific mAbs. For expression of CD8{beta}, Thy-1.2, or DX-5 presented in E, the int{beta}+CD8+ cells gated on {gamma}{delta}-negative population were analyzed. Dashed lines present positive or negative controls: {gamma}{delta}+ populations within CD4-CD8- cell population (for D), B220 (B cell marker)-positive cells within LN cells stained for CD8{beta} or Thy-1.2, and DX-5-positive NK cell population within CD4-CD8- cell population (for E).

 
Surprisingly, peripheral LNs of pT{alpha}-TG/TCR{alpha}-/- mice contained a large number (2.7 x 105 cells in all of collected mesenteric and inguinal LNs: 4.5% of total LN cells) of CD8+ cells that harbored int{beta} (Fig. 1GoC, right). A small, but clearly detectable number of the int{beta}+CD8+ cells also did exist in LNs from TCR{alpha}-/- mice (1.2 x 104 cells in LNs, 0.2% of total LN cells; Fig. 1GoC, left). These int{beta}+CD8+ cells were TCR{gamma}{delta}- (Fig. 1GoD), Thy-1.2+, and CD8{alpha}+{beta}+, and lacked expression of DX-5, a pan-NK cell marker (Fig. 1GoE), thus satisfying the criteria for {alpha}{beta}-lineage T lymphocytes despite the absence of TCR{alpha}{beta} complexes. These findings strongly implicate that the pre-TCR harbors some potential of mediating signals required for positive selection of CD8+ SP cells. The presence of a small number of int{beta}+CD8+ cells even in LNs from nontransgenic TCR{alpha}-/- mice is consistent with the very low, but significant amount of CD3{zeta} chain phosphorylation detectable in TCR{alpha}-/- thymocytes (Fig. 1GoB). Consistently, a far increased number of mature CD8+ SP thymocytes, displaying a low level of the HSA (CD24) expression, were detected in the thymus of pT{alpha}-TG/TCR{alpha}-/- mice than in the thymus of TCR{alpha}-/- mice (Table IGo). In LNs from both TCR{alpha}-/- and pT{alpha}-TG/TCR{alpha}-/- mice, a small number of int{beta}+CD4+ cells was also detected (Fig. 1GoC). Such int{beta}+CD4+ cells had been previously reported as CD4+TCR{beta}low+ cells, and it was not clear whether they were derived from the thymus (16, 17, 18). In contrast to the int{beta}+CD8+ cells, the number of these CD4+ cells was comparable in both types of mice (1.8 x 104 in LNs, 1.3 x 105 in the spleen in TCR{alpha}-/-, vs 1.6 x 104 in LNs, 1.1 x 105 in the spleen in pT{alpha}-TG/TCR{alpha}-/-), suggesting that the level of the pre-TCR complexes expressed in the thymus does not influence the development of the cells. Both strains also had int{beta}+CD4-CD8- cells, which consisted of {gamma}{delta} cells (3) and a proportion of DX-5+-NK T-like cells (data not shown).


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  		Table I. Absolute numbers of mature CD8+ thymocytes in TCR{alpha}-/- and pT{alpha}-TG/TCR{alpha}-/- micea

 
Egr-1 enhances the positive selection of CD8+ T cells by the pre-TCR

We further analyzed whether lowering the threshold for signaling required for positive selection by overexpressing of the Egr-1, a zinc-finger transcription factor, would increase the generation of int{beta}+CD8+ T cells in TCR{alpha}-/- mice (19, 20). We previously reported that overexpression of Egr-1 in thymocytes markedly lowered the threshold of signal strength through the TCR{alpha}{beta} (or the avidity of TCR/MHC interaction) required for positive selection of mature SP thymocytes (19), and thus one might expect that Egr-1 could also lower the signal strength of the pre-TCR required for positive selection of int{beta}+CD8+ T cells. The result illustrated in Fig. 2GoA shows that this is in fact true: the Egr-1 transgene expression on the TCR{alpha}-/- background (Egr-TG/TCR{alpha}-/-) resulted in a phenotype very similar to that of pT{alpha}-TG/TCR{alpha}-/- mice, in which a large (even larger than in pT{alpha}-TG/TCR{alpha}-/-) proportion (5.5 x 105 in LNs, 7.5% of total LN cells) of int{beta}+CD8+ cells with a {gamma}{delta}-, Thy-1.2+, CD8{alpha}+{beta}+, DX-5- phenotype was present in peripheral LNs. As shown in Fig. 2GoB, their thymi also harbored a proportion of the HSAlow CD8+ mature SP thymocytes. Likewise, the absolute number of the HSAlow CD8+ mature SP cells in Egr-TG/TCR{alpha}-/- thymus was larger (~2.5 times) than that in pT{alpha}-TG/TCR{alpha}-/- thymus (see Table IGo and Fig. 2GoB).



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  		FIGURE 2. Peripheral CD8+ T cells were also increased under the Egr-1 overexpression in TCR{alpha}-/- mice. A, LN (mesenteric and inguinal) cells from Egr-1 transgenic mice on a TCR{alpha}-/- background (Egr-TG/TCR{alpha}-/- mice) were analyzed for the presence of int{beta}+CD8+ T cells by staining, as described in the legend for Fig. 1Go, C–E. B, Thymocytes from WT or Egr-TG/TCR{alpha}-/- mice were stained for CD4, CD8, and HSA, then analyzed by cytometer. Demonstrated are the CD4/CD8 profiles of mature thymocytes, which were pregated on HSAlow populations.

 
Direct evidence for the involvement of the pT{alpha} chain

Further evidence for the involvement of the pre-TCR in positive selection of int{beta}+CD8+ T cells was observed in TCR{alpha} enhancer-deficient (E{alpha}-/-) mice (21): They likewise possessed a comparable (or even a slightly larger) proportion of int{beta}+CD8+ cells in the LNs as TCR{alpha}-/- mice (1.9 x 104 in LNs, 0.4% of total LN cells; Fig. 3Go, left panel). Moreover, in mice doubly deficient for E{alpha} and pT{alpha} (22) (pT{alpha}-/-/E{alpha}-/- mice), the number of the CD8+ cells drastically decreased (<5 x 103 in LNs; Fig. 3Go, right panel), confirming the indispensable requirement of the pT{alpha} chain for the selection event. Again, the presence of a similar proportion of the int{beta}+CD4+ cells in E{alpha}-/- and pT{alpha}-/-/E{alpha}-/- mice (3.6 x 104 in LNs of E{alpha}-/-, vs 3.4 x 104 in LNs of pT{alpha}-/-/E{alpha}-/-) supports the notion that the development of the int{beta}+CD4+ cells in the absence of TCR{alpha} is pre-TCR independent. Interestingly, the number of the int{beta}+CD4+ cells was larger in E{alpha}-/- mice than in TCR{alpha}-/- mice (see Fig. 1GoC). In addition, the proportion of the int{beta}+CD4-CD8- cells in E{alpha}-/- mice was much smaller than in TCR{alpha}-/- mice (compare Figs. 1GoC and 3). The precise reason for this difference is unclear. E{alpha} might also influence the development of the int{beta}+CD4-CD8- cells, although further study will be required. All facts considered, it is very likely that signals mediated by the pre-TCR and the ligated TCR{alpha}{beta} are functionally equivalent with regard to positive selection of CD8+ cells from DP precursors.



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  		FIGURE 3. Peripheral CD8+ T cells were remarkably decreased in the absence of pT{alpha}. The presence of the CD8+ T cells was comparably analyzed in the LNs from TCR{alpha} enhancer-deficient (E{alpha}-/-) mice and E{alpha}, pT{alpha} doubly deficient (pT{alpha}-/-/E{alpha}-/-) mice by staining, as in the previous experiments (see legends for Figs. 1Go and 2Go). The int{beta}+CD8+ cells were drastically decreased in pT{alpha}-/-/E{alpha}-/- mice.

 
CD8+ T cells selected by the pre-TCR are functional

Having int{beta}+CD8+ cells undergo positive selection by the pre-TCR, we then addressed whether these cells are functionally equivalent to the T cells from WT mice. First, we addressed the IL-2 production by the CD8+ cells in response to ionomycin + PMA. LN cells from either pT{alpha}-TG/TCR{alpha}-/- or WT mice were stimulated in the presence or absence of ionomycin + PMA for 16 h, then IL-2 production in the CD8+ cells were determined by intracellular staining analysis for IL-2. As shown in Fig. 4GoA, CD8+ cells from both pT{alpha}-TG/TCR{alpha}-/- and WT mice efficiently produced IL-2 upon stimulation. Second, the activation status of the CD8+ cells in response to either CD3{epsilon} cross-linking or Con A stimulation was also studied. LN cells were stimulated by immobilized anti-CD3{epsilon} Ab or Con A, then the expression levels of T cell activation markers, CD25 and CD69, on the CD8+ cells were analyzed. As illustrated in Fig. 4GoB, CD8+ cells from pT{alpha}-TG/TCR{alpha}-/- mice functionally responded to either stimulation, demonstrating up-regulation of these markers. Probably due to the less amount of CD3{epsilon} on the cell surface, as demonstrated in Fig. 4GoC, CD69 and CD25 expression levels after stimulation were lower in pT{alpha}-TG/TCR{alpha}-/- than in WT. Together, the CD8+ cells selected by the pre-TCR were functionally competent.



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  		FIGURE 4. CD8+ T cells selected by the pre-TCR are functionally competent. A, LN cells either from WT or pT{alpha}-TG/TCR{alpha}-/- mice were cultured in the presence or absence of 10 ng/ml of PMA + 5 x 10-7 M of ionomycin. The cells were then stained for intracellular IL-2 and CD8. Intracellular IL-2 levels in CD8+ cells are present. The solid lines represent the level of intracellular IL-2 when incubated with stimulation, whereas the dashed lines represent those on cells incubated without stimulation. B, LN cells either from WT or pT{alpha}-TG/TCR{alpha}-/- mice were cultured in the presence or absence of 1 µg/well of immobilized anti-CD3{epsilon} mAb or 1 µg/ml of Con A. The cells were then stained for CD8, {gamma}{delta}, int{beta}, and either for CD25 or CD69, and then analyzed by cytometer. CD25 and CD69 expression on CD8+{gamma}{delta}-int{beta}+ cells are presented. The solid lines demonstrate the expression of CD25 or CD69 when incubated with stimulation, whereas the dashed lines represent those on cells incubated without stimulation. C, Lower expression of CD3{epsilon} on CD8+ cells from pT{alpha}-TG/TCR{alpha}-/- mice. LN cells either from WT or pT{alpha}-TG/TCR{alpha}-/- mice were stained for CD3{epsilon}, CD8, {gamma}{delta}, and int{beta}+, and analyzed by cytometer. CD3{epsilon} expression on CD8+{gamma}{delta}-int{beta}+ cells is presented. Bold line, WT; solid line, pT{alpha}-TG/TCR{alpha}-/-; dashed line, negative control (CD3{epsilon} expression on B220+ LN B cells).

 
MHC molecules are not required for the positive selection events by the pre-TCR

Positive selection mediated by TCR{alpha}{beta} strictly requires interaction between the TCR and appropriate class I or class II MHC/Ag-peptide complex. We wondered whether positive selection of CD8+ cells operated by the pre-TCR is also dependent on MHC molecules. We investigated this question by analyzing the proportion of the int{beta}+CD8+ T cells in mice doubly deficient for class I MHC and TCR{alpha}, or for class II MHC and TCR{alpha}, which were generated by cross-breeding of TCR{alpha}-/- mice with either class I MHC-/- (K-/-D-/-) (23, 24) or class II MHC-/- (A{beta}-/-) (25) mice. No obvious difference in the size of the int{beta}+CD8+ T cell population was detected between the MHC/TCR{alpha} doubly deficient vs TCR{alpha}-/- mice (Table IIGo), suggesting that the recognition of MHC Ags by the pre-TCR is not required for the selection event. We also studied {beta}2-microgloblin ({beta}2m)-/-TCR{alpha}-/- and TAP-/-TCR{alpha}-/- mice (26) to test possible involvement of nonclassical MHC molecules (most of which are dependent on {beta}2m and/or TAP for their cell surface expression). Again, a comparable fraction of the int{beta}+CD8+ T cells was observed in {beta}2m-/-TCR{alpha}-/- and TAP-/-TCR{alpha}-/- when compared with TCR{alpha}-/- mice (Table IIGo). In addition, we also analyzed {beta}2m-/-A{beta}-/- TCR{alpha}-/- mice, which lack both class I and class II MHC as well as most of nonclassical MHC molecules. Likewise, the number of int{beta}+CD8+ T cells in these mice was equivalent to that in TCR{alpha}-/- mice (Table IIGo). Thus, recognition of MHC molecules (either classical or nonclassical) is not necessary for the pre-TCR to mediate positive selection. At the transition stage from the double-negative to the DP ({beta}-selection), it has been shown that pre-TCR localizes cell autonomously to membrane rafts, where it appears to signal in a constitutive and ligand-independent manner (4, 27, 28). Likewise, the recognition of an extracellular ligand might not be required for positive selection by the pre-TCR, while we cannot mutually exclude an involvement of non-MHC cell surface ligand(s).


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  		Table II. Absolute numbers of int{beta}+ CD8+ cells in the LNs of various micea

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that the pre-TCR can promote positive selection of CD8+ T cells in the thymus, by comparatively analyzing various strains of mice. Our observation strongly implies that the pre-TCR signals bear functional resemblance to those transduced by the TCR{alpha}{beta} following ligand engagement.

One may wonder why the pre-TCR appeared to select predominantly CD8+ cells. One possible explanation might be insufficient signal strength to induce CD4+ cells in TCR{alpha}-/- mice, and even in pT{alpha}-TG/TCR{alpha}-/- or Egr-TG/TCR{alpha}-/- mice. Accumulating evidence suggests that the strength and/or duration of TCR signaling appear to influence CD4/CD8 lineage commitment (29, 30, 31, 32, 33). Indeed, positive selection of CD8+ cells by weak TCR signals has been demonstrated in various experiments (34, 35). The lower level of CD3{zeta} chain phosphorylation in thymocytes from pT{alpha}-TG/TCR{alpha}-/- (selecting predominantly CD8+ cells) as compared with WT animals (selecting both CD4+ and CD8+ cells) as shown in Fig. 1GoB might be sufficient for positive selection of CD8+ cells, but not for CD4+ cells. Recently, a new perspective, referred to as kinetic signaling model, was proposed based on a precise in vitro analysis of DP cells undergoing TCR signals (36, 37, 38). This model postulates that: DP cells terminate CD8 transcription and convert into CD4+CD8low+ intermediate cells in response to TCR (+coreceptor) signals; these CD4+CD8low+ intermediate cells are not yet lineage committed and retain the potential to differentiate into either CD4 or CD8 mature SP cells; sustained signaling in these cells results in their differentiation into CD4 SP cells, whereas cessation of signaling results in coreceptor reversal and differentiation into CD8 SP cells. In TCR{alpha}-/- as well as Egr-TG/TCR{alpha}-/- mice, the biased CD8+ cell production might fit with the model, because endogenous pT{alpha} expression is rapidly down-regulated during the transition from the DP to the SP stage (lower in late DP cells than in early DP cells, then almost undetectable in mature SP thymocytes) (39), perhaps resulting in a decreased expression level of the pre-TCR in CD4+CD8low+ intermediate as compared in DP cells. However, the potential difference of the pT{alpha} transgene expression in DP and CD4+CD8low+ intermediate cells will need to be determined to extend this explanation to the priority of CD8+ cell production in pT{alpha}-TG/TCR{alpha}-/- mice as well. The alternative view of course is that pre-TCR signals never have the strength required for CD4+ T cell production.

In summary, our observations provide new insights on thymocyte development, two of which may be worth re-emphasizing. First, the fact that the pre-TCR can promote positive selection of CD8+ T cells further suggests the functional resemblance of the signaling pre-TCR and the ligated TCR{alpha}{beta}. Second, the biased production of CD8+ T cells by the pre-TCR signals may support the propriety of hypothesis for the involvement of signal strength in CD4/CD8 lineage commitment.


   Acknowledgments
 
We thank Drs. H. J. Fehling (Ulm, Germany) for providing pT{alpha} cDNA; F. Lemonnier (Paris, France) for K-/-D-/- mice; F. W. Alt (Boston, MA) for E{alpha}-/- mice; C. Minjares and S. Hall for creating transgenic mice and help with care of the mice; and Drs. S. Ward and J. D. Farrar (Dallas, TX) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by research grant of Juvenile Diabetes Foundation (1-2001-581), American Liver Foundation Herman Lopata Memorial Liver Scholar Award, and Howard Hughes Medical Institute research support (to T.M.); National Institutes of Health Grant RO1AI42953 (to N.S.C.v.O.); National Institutes of Health Grants RO1A14728 and RO1A145846 (to H.v.B.); and the "Eugenia Spanopolou" (to I.A.). Back

2 Y.I. and S.A. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Toru Miyazaki, Center for Immunology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard NA 7200, Dallas, TX 75390-9093. E-mail address: Toru.Miyazaki{at}UTSouthwestern.edu Back

4 Abbreviations used in this paper: pT{alpha}, pre-TCR{alpha}; DP, double positive; {beta}2m, {beta}2-microglobulin; HSA, heat-stable Ag; int{beta}, intracellular TCR{beta}; LN, lymph node; SP, single positive; TG, transgenic; WT, wild type. Back

Received for publication June 11, 2002. Accepted for publication August 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rodewald, H. R., H. J. Fehling. 1998. Molecular and cellular events in early thymocyte development. Adv. Immunol. 69:1.[Medline]
  2. Malissen, B., M. Malissen. 1996. Functions of TCR and pre-TCR subunits: lessons from gene ablation. Curr. Opin. Immunol. 8:383.[Medline]
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