Subset-specific TcR
Eur. J. Immunol. 1992. 22: 635-639
Fredrick Ivars. The Unit for Applied Cell and Molecular Biology, Umeh University, u m e z
expression
635
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.
1 Introduction
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 [18] 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.
[I 957.51
*
p chain
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 [19] 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 [22], FITC-conjugated anti-CD3 antibody 145.2C.11 [23], biotin-or FITC-conjugated anti-CD8 antibody 53.6.72 [24], PE-conjugated anti-CD4 antibody GK 1.5 [25], 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; [26]) and 0014-2980/92/0303-0635$3.50+ .2510
636
Eur. J. Immunol. 1992. 22: 635-639
F. Ivars
anti-CD8 antibodies (3.155; [27]) 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 [28]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 [30]. The PCR was performed essentially according to Saiki et al. [31], 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 [30] 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
CD8
Oh
E
26h
CD3
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 [32].The probability of observing
Eur. J. Immunol. 1992. 22: 635-639
Fredrick Ivars. The Unit for Applied Cell and Molecular Biology, Umeh University, u m e z
expression
635
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.
1 Introduction
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 [18] 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.
[I 957.51
*
p chain
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 [19] 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 [22], FITC-conjugated anti-CD3 antibody 145.2C.11 [23], biotin-or FITC-conjugated anti-CD8 antibody 53.6.72 [24], PE-conjugated anti-CD4 antibody GK 1.5 [25], 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; [26]) and 0014-2980/92/0303-0635$3.50+ .2510
636
Eur. J. Immunol. 1992. 22: 635-639
F. Ivars
anti-CD8 antibodies (3.155; [27]) 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 [28]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 [30]. The PCR was performed essentially according to Saiki et al. [31], 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 [30] 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
CD8
Oh
E
26h
CD3
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 [32].The probability of observing
