A critical lineage-nonspecific role for pTα in mediating allelic and isotypic exclusion in TCRβ-transgenic mice

TCRβ transgenes mediate pTα-dependent allelic exclusion in immature DN3 thymocytes

Although it is well established that early expression of TCRβ transgenes in the thymus leads to efficient inhibition of endogenous TCRβ rearrangement (otherwise known as allelic exclusion), the role of pTα in this process remains controversial 7, 8. One technical problem with previous studies has been that allelic exclusion in the thymus has been assessed either at the population level (via endogenous TCRβ rearrangements) or at the single-cell level in mature T cells (via surface staining for endogenous TCRVβ chains). Since allelic exclusion is first detectable at the immature DN3 stage of thymus development (where endogenous TCRβ rearrangement occurs), we have adapted a previously described double ic staining protocol for TCRβ and Vβ 9, in order to optimize analysis of allelic exclusion in individual DN3 thymocytes. Moreover, since there is some indication that expression levels or developmental timing of TCRβ transgenes may influence the efficiency of allelic exclusion 10, we have utilized three independent strains of TCRβ Tg mice that express icTCRβ very early in development but differ widely in icTCRβ expression. Thus, TCRVβ8.2 and TCRVβ3 Tg mice express approximately six- and twofold more icTCRβ than WT mice, whereas TCRVβ8.1 Tg mice express about tenfold less (according to mean fluorescence intensity of icTCRβ expression; Fig. 1A).

As expected from previous studies 11, 12, all three TCRβ Tg mice exhibited virtually complete inhibition of endogenous TCRβ rearrangements, as evidenced by the absence of icTCRVβ5- (Fig. 1B) or icTCRVβ6-expressing cells (data not shown) in DN3 thymocytes. In order to address the role of pre-TCR signaling in allelic exclusion, the three TCRβ Tg mice were backcrossed onto a pTα^–/– 13 or CD3ϵ^–/– 14 genetic background. Consistent with an earlier report using CD3ϵ^–/– TCRVβ8.1 Tg mice 15, endogenous TCRVβ5 expression was restored to control levels in DN3 thymocytes from all three TCRβ Tg strains on a CD3ϵ^–/– background (data not shown), confirming the critical role of the CD3 complex in signaling allelic exclusion. Interestingly, a similar result was obtained when the three TCRβ Tg strains were crossed to pTα^–/– mice (Fig. 1C), although restoration of endogenous TCRVβ5 expression was incomplete in pTα^–/– TCRVβ8.2 Tg mice expressing the highest levels of transgenic icTCRβ. These data clearly demonstrate that pTα is essential to mediate allelic exclusion by TCRβ transgenes in DN3 thymocytes over a wide range of icTCRβ expression.

TCRβ transgenes mediate pTα-dependent “isotypic” exclusion at the TCRγ locus

In addition to their ability to mediate allelic exclusion, TCRβ transgenes have been shown to efficiently block endogenous TCRγ rearrangements and reduce γδ cell numbers in the thymus 10, 16, 17, a process referred to as “isotypic” exclusion 16. The role of pTα in isotypic exclusion is not clear, since a single study using pTα^–/– TCRVβ8.2 Tg mice demonstrated only a modest restoration of endogenous Vγ rearrangements and γδ T cell numbers 8. As shown in Fig. 2A, all three TCRβ transgenes strongly reduced thymic γδ T cell numbers, although the greatest reduction was observed with the highly expressed TCRVβ8.2. As noted previously 13, absolute γδ T cell number increased significantly (threefold) in pTα^–/– mice (Fig. 2A, B). Interestingly, γδ cell numbers were increased to an even greater extent (20–60-fold) in the three TCRβ Tg mice in the absence of pTα (Fig. 2A), reaching levels close to those in nontransgenic pTα^–/– controls (Fig. 2B). It should be noted that icTCRβ expression was significantly reduced in DN3 thymocytes on the pTα^–/– background in both WT and TCRβ Tg mice (see Fig. 1B, C). This effect has been noted previously 18 and may reflect a reduced stability of the TCRβ protein in the absence of pTα. In any event, these data demonstrate that pTα also plays a critical role in mediating isotypic exclusion by TCRβ transgenes.

TCRβ transgenes mediate pTα-dependent allelic exclusion in γδ cell precursors

The ability of pTα to simultaneously mediate allelic and isotypic exclusion by TCRβ transgenes in immature thymocytes suggests that pTα may be active in precursors of both αβ and γδ lineages. Previous studies using pTα ic staining 6, single-cell PCR 19 and reporter mice 20 are consistent with the possibility that most DN3 thymocytes, which have recently been shown to contain a very small subset of γδ precursors 21, express pTα. However, no functional evidence for the presence of pTα in γδ precursors has been reported. To address this issue directly, we took advantage of the fact that a small but distinct subset (10–15%) of thymic γδ cells undergo functional TCRβ rearrangement, resulting in the expression of icTCRβ 6, 22. As shown in Fig. 3A, the TCRβ locus is in fact allelically excluded in γδ cells from all three TCRβ Tg mouse strains, since endogenous TCRVβ5-expressing cells are completely absent. Importantly, allelic exclusion is lost in TCRβ Tg γδ cells in the absence of pTα (Fig. 3B) although restoration of endogenous TCRVβ5 cells is incomplete in TCRVβ8.2 Tg mice, as observed previously for DN3 thymocytes (Fig. 1C). These data clearly demonstrate that pTα is expressed and potentially functional in the γδ cell lineage.

Similar levels of pTα mediate allelic and isotypic exclusion in αβ and γδ lineages

The preceding data indicate that pTα can potentially mediate allelic exclusion in both αβ and γδ cell lineages, as well as isotypic exclusion. However, it is not clear whether the efficiency of pre-TCR signaling differs in each of these situations. To address this important issue, we took advantage of the fact that the TCRVβ8.1 transgene, although expressed at very low levels (Fig. 1A), was nevertheless capable of mediating efficient pTα-dependent allelic and isotypic exclusion in both αβ and γδ lineages (Fig. 1–3). In this situation, the ability of the weakly expressed TCRVβ8.1 transgene to function in pre-TCR signaling might be expected to depend upon pTα gene dosage. Indeed, as shown in Fig. 4A, haploinsufficient pTα^+/– TCRVβ8.1 Tg mice showed an intermediate phenotype with respect to their ability to mediate allelic exclusion in both αβ and γδ lineages as well as in their capacity to suppress γδ cell numbers via isotypic exclusion (Fig. 4B). Thus, it appears that inhibition of TCRβ and TCRγ rearrangements in TCRβ Tg mice can occur as a result of similar levels of pre-TCR signaling, independently of lineage.

pTα-dependent allelic and isotypic exclusion occur despite normal levels of RAG expression

The ability of the pre-TCR to mediate allelic and isotypic exclusion at similar pTα doses in TCRβ Tg mice raises the possibility that a similar mechanism may be involved in shutting off TCRβ and TCRγ rearrangement. One obvious candidate for such a mechanism would be down-regulation of expression of the RAG genes, as has been shown for DP thymocytes undergoing positive selection 23–25. However, as shown in Fig. 5A, expression of RAG1 mRNA in DN3 thymocytes (where the vast majority of TCRβ and TCRγ rearrangements occur) is not affected by the presence of a TCRVβ8.1 transgene that efficiently inhibits endogenous TCRβ and TCRγ rearrangement (10 and Fig. 5B). Furthermore, RAG2 protein, which can be regulated either at the mRNA level 25 or via phosphorylation and subsequent degradation in cycling cells 26, 27, was not down-regulated in DN (Fig. 5C) and DN3 (data not shown) thymocytes from all three strains of TCRβ Tg mice. Control experiments using DP thymocytes from TCRαβ Tg P14 mice confirmed that both RAG1 mRNA (Fig. 5A) and RAG2 protein (Fig. 5C) were dramatically down-regulated upon TCR engagement, as expected from previous reports 23–25. Collectively, these data indicate that allelic and isotypic exclusion in DN3 thymocytes can occur independently of regulation of RAG expression.

Mice

C57.BL/6 WT mice were purchased from Harlan Netherlands (Horst, The Netherlands). C57.BL/6 pTα^–/– mice 13 were originally provided by Dr. H.-J. Fehling (University Clinics, Ulm, Germany). C57.BL/6 Tg Vβ8.1 11 and C57.BL/6 Tg P14 40 mice were originally obtained from Dr. H.-P. Pircher (University of Freiburg, Germany). C57.BL/6 Tg Vβ3 41 and C57.BL/6 Tg Vβ8.2 12 mice were originally obtained from Dr. M. Dohlsten (Pharmacia, Sweden) and Dr. H. Bluethman (Hoffmann-La Roche AG, Switzerland), respectively. C57.BL/6 Tg Vβ8.1, C57.BL/6 Tg Vβ3 and and C57.BL/6 Tg Vβ8.2 mice were backcrossed to pTα^–/– mice. C57.BL/6 RAG2^–/– mice were purchased from Taconic. All mice were used at 4–8 wk of age. This study has been reviewed and approved by the Service Vétérinaire Cantonal of Etat de Vaud.

Cell preparation, flow cytometry and sorting

For the analysis of icTCRβ expression on CD25⁺ CD44^– DN (DN3) thymocytes, or DN3 sorting, WT DN-enriched 22 or total pTα^–/– thymocyte cell suspensions were incubated with a cocktail of FITC-conjugated Ab to CD4, CD8, CD3ϵ, TCRβ, TCRδ, B220, CD11c, Ter119, CD161, Gr-1 and F4/80, together with anti-CD44-Alexa647, anti-CD25-APC-Cy7 and anti-CD117-PE-Cy7 Ab. DN3 thymocytes were defined as cocktail^– CD117^– CD25⁺ CD44^– cells. Staining for γδ T cells was performed by incubating WT DN-enriched or total pTα^–/– thymocyte cell suspensions with a cocktail of PE-Cy7-conjugated Ab to CD4, CD8, B220, Ter119, TCRβ, GR-1, NK1.1 and CD11b, together with anti-CD3ϵ-Alexa647 and anti-TCRδ-FITC Ab. γδ T cells were defined as cocktail^– CD3ϵ⁺ TCRδ⁺ cells. icTCRβ staining was performed as described 9, 22. For the isolation of DP thymocytes, WT or P14 Tg thymocyte cell suspensions were incubated with a cocktail of FITC-conjugated Ab to B220, CD11c, Ter119, CD161, Gr-1 and F4/80, together with anti-CD4-Alexa647 and anti-CD8-PE-Cy5 Ab. DP cells were defined as cocktail^– CD4⁺ CD8⁺. For the isolation of B cells, lymph node cell suspensions were incubated with anti-F4/80-FITC, anti-CD19-PE and anti-CD3-PE-Cy5 Ab. The B cell population was sorted as F4/80^– CD3^– CD19⁺ cells.

Cells were analyzed on a FACSCanto flow cytometer using FACSDiva software (Becton Dickinson). Dead cells were gated out by their forward and side scatter profile. Sortings were performed on a FACSAria flow cytometer (Becton Dickinson).

Real-time PCR

Real-time PCR using SYBR Green was performed on a LightCycler (Roche, Rotkreuz, Switzerland) according to the manufacturer's instructions. Total RNA from sorted DN3 cells was purified using a High Pure RNA tissue kit (Roche) that includes DNase treatment. Total RNA was reverse-transcribed using random nonamers and AMV reverse transcriptase (Roche). Genomic DNA was extracted from sorted DN3 cells and quantification of Vγ2-Jγ1 rearrangements was performed as described 4. For the PCR reaction, the LightCycler-FastStart DNA Master SYBR Green I kit (Roche) was used following the instruction manual. RAG1 transcripts were normalized to TATA-binding protein (TBP) and Vγ2-Jγ1 rearrangement was normalized to Thy-1. All primer sequences are available upon request. Amplification plots were analyzed using the second-derivative method with LC data analysis 3.5 software (Roche), and the relative quantification was determined using the LightCycler relative quantification software 1.0 (Roche). Sextuplet analysis showed that measurement errors were always <9%.

Immunoblot analysis

Total thymocyte and isolated DP or DN populations were lysed in lysis buffer (Sigma) and the protein concentration was determined by BCA assay (Pierce). Protein extracts (14 µg for total thymus and DN cells, 10 µg for DP cells) were applied to SDS-PAGE and analyzed by Western blot for RAG2 expression. To ensure that equal amounts of protein were loaded, the membrane was reprobed with anti-β-actin Ab. The anti-RAG2 (C-19) Ab was from Santa Cruz Biotechnology and the anti-β-actin Ab from Sigma.