Eur. J. Immunol. 1992. 22: 635-639
Fredrick Ivars. The Unit for Applied Cell and Molecular Biology, Umeh University, u m e z
T cell subset-specific expression of antigen receptor p chains in a chain-transgenic mice* A large number of Vp8 gene-encoded cDNA were analyzed from peripheral CD4+ and CD8+ T cell subsets of T cell receptor (TcR) a chain-transgenic mice. This analysis demonstrates that a limited repertoire of TcR fi chains are co-expressed with the transgenic u chain. Most importantly, certain v p 8 - J ~ combinatons were found exclusively in one of the subsets and, in some cases, subset-specific differences were localized to the VDJ junctional region of the chain genes. In contrast, CD4-CD8- transgenicT cells, as well as CD4+ and CD8+ T cells from normal littermate controls, were found to express diverse p chain repertoires, The present study suggests that p chains with distinct structural characteristics are expressed in the CD4+ and CD8+ subsets, respectively. Moreover, the data suggest that the same structural constraints do not apply to the population of CD4-CD8- transgenic T cells.
These data are consistent with the view that CD4+ and CD8+ express distinct sets of TcR.
Most T cells in peripheral lymphoid organs carry antigenspecific receptors (TcR) composed of a and p chains. A large number of different a and fi chain variable regions can potentially be generated from germ-line genes and genetic mechanisms that modify the junctions between these genes during the rearrangement process (reviewed in [l]). The repertoire of T cell specificities that is generated in this way is both positively and negatively selected, before the exit of the mature CD4+ or CD8+ T cells from the thymus [2-91. This results in a peripheral Tcell repertoire that is self MHC restricted and tolerant to self-components encountered in the thymus. Furthermore, theT cell repertoire may be further positively [lo] and negatively selected [11-131 in peripheral lymphoid tissues. The molecular mechanisms of intrathymic selection are still poorly understood. Recent studies using TcR-transgenic mouse models have, however, indicated that the MHC restriction specificity of the transgenic TcR, together with the CD4 or CD8 molecules, directs positive selection of the CD4+CD8+ precursor cells into either the CD4+ or CD8+ mature Tcell subsets [8, 14-17]. Thus, as a result of intrathymic positive selection mature CD4+ and CD8+ T cells are class I1 and class I MHC restricted, respectively  and would be expected to express largely different TcR repertoires. In the present report this issue was studied using a novel approach. The TcR fi chain repertoire expressed in CD4+ and CD8+ cells of TcR ci chain-transgenic mice were analyzed and found to be different. Moreover, a remarkable conservation of amino acid motifs was observed in the junctional regions of the chains isolated from CD8+ cells.
Fellow of the Knut and Alice Wallenberg Foundation. This work was supported by a grant from the Swedish Natural Science Research Council.
Correspondence: Fredrik Ivars, The Unit for Applied Cell and Molecular Biology, Ume5 University, S-901 85 Umei, Sweden 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
2 Materials and methods 2.1 Mice Transgenic mice expressing the rearranged CI chain gene of theT cell hybridoma 2B4  have been described in detail elsewhere [20, 211.The mice used in here were offsprings of the fifth or sixth generation of backcross to C57BL/6 mice and were H-2b homozygotes.
2.2 Flow cytometry and cell sorting Single-cell suspensions were prepared from spleen and lymph nodes and washed in PBS.The cells were stained with antibodies and analyzed (lo4 gated events per sample) on a FACScan flowcytometer (Becton Dickonson, Mountain View, CA), as suggested by the manufacturer. Stained cells were sorted into CD4+ and CD8+ fractions using a FACS Star Plus cell sorter (Becton Dickinson). The following reagents were used for stainings: FITC-conjugated 2B4 u chain-specific antibody A2B4-2 , FITC-conjugated anti-CD3 antibody 145.2C.11 , biotin-or FITC-conjugated anti-CD8 antibody 53.6.72 , PE-conjugated anti-CD4 antibody GK 1.5 , FITC-conjugated goat anti-mouse Ig and streptavidin-PE. All these reagents except FITC-A2B4-2, biotin-A2B4-2 and FITC-145.2C. 11 were bought from Becton Dickinson. 2.3 Cell cultures
Pooled lymph node and spleen cells were prepared and suspended in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 5 % FCS, glutamine, 2-mercaptoethanol and antibiotics. The cells (1 x lo6 - 2 x 106/ml) were cultured for 4 days in tissue culture bottles that had been precoated with A2B4-2 (15 ml of a 2.5 pg/ml solution for 2 h at room temperature) or 145.2C.11 antibodies (10 pg/ml). To enrich for CD4-CD8- cells, the activated cells were treated with anti-CD4 (RL 172.4; ) and 0014-2980/92/0303-0635$3.50+ .2510
Eur. J. Immunol. 1992. 22: 635-639
anti-CD8 antibodies (3.155; ) and rabbit complement (Cedarlane, Hornby, Ontario). Aliquots of the activated cells were cultured for about 24 h in medium containing 5 YO supernatant of Con A-activated rat spleen cells (2.5 pg/ml Con A for 24 h in vitro), to promote reexpression of TcR molecules (see legend to Fig. 2).
oligonucleotide primers used were: Cpl 5'-CACGAGGGTAGCCTTTTGTT-3' ; (2132 5'-CTAAGCTTTTGATGGCTCAAA-3'; Cp3 5 '-GGAGACCTTGGGTGGAGTCAC-3'; Vp8 S'-ATGGATCCAAGGCCTCCAGA-3'.
2.5 Statistical calculations 2.4 cDNA synthesis and polymerase chain reaction (PCR) Approximately lo5sorted CD4+ and CD8+ cells were lysed in isotonic buffer containing 200 pg/ml yeast tRNA and fresh diethylpyrocarbonate 1/1000. The nuclei were removed, the supernatant was boiled for 5 min, phenol extracted and the RNA precipitated with ethanol. cDNA was synthesized using oligo-dT priming [2Y] and thereafter precipitated with spermine . The PCR was performed essentially according to Saiki et al. , on a thermal cycler (Hybaid Ltd,Teddington, GB) using0.4 pM each of theVg8 and Cpl primers and Taq polymerase (Perkin-Elmer Cetus, Emeryville, CA). The amplification protocol consisted of five cycles of 0.5 min at 93 "C, 0.5 min at 45 "C and 1 min at 72 "C, followed by 20 cycles of 0.5 rnin at 93 "C, 0.5 rnin at 55 "C and 0.5 min at 72°C. The reaction product was gel-purified and reamplified as above, using the Vp8- and Cg2-specific primers. This reaction product was digested with Bam HI and Hind 111 restriction endonucleases and cloned into the Bluescript SK-vector (Stratagene, La Jolla, CA). Each library was screened by colony hybridization  using radioactively labeled Cp3 oligonucleotide. Both strands of plasmid DNA, were sequenced by the dideoxymethod using Sequenase (U.S Biochemicals, Cleveland, OH) or T7 polymerase (Pharmacia, Uppsala, Sweden).The
Figure 1. Selection for and isolation of 2B4 a chain-expressing cells. A2B4-2 antibody-activated cells were analyzed for expression of CD4 and CD8 markers A) before and after sorting into B) CD4+ and C) CD8+ populations, respectively. In a separate experiment, cells activated in the same manner, were analyzed for expression of 2B4 a chain and CD3 markers D) before and E) after reculture in vitro for 26 h. Before activation 6.2 % CD4+ and 5.8 YO CD8+ cells were detected (data not shown).
Maximum likelihood estimates, and 95 YO confidence bounds, of the size of the CD4+ and CD8+ populations (see Fig. 3) were calculated as described by Litwin and Schlomchik .The probability of observing