Eur. J. Immunol. 1990. 20: 417-424
Hanspeter Pircheroa, Pamela OhashiO, Guido Miescher+, Rosemarie LangO, Athanasios ZikopoulosO, Kurt Burki., Tak W. MakA, H. Robson MacDonald+ and Hans HengartnerO Institute of Pathology, Department of Experimental Pathology, University Hospitalo, Zurich, Ludwig Institute for Cancer Research+, Lausanne Branch, Epalinges, Department of Preclmical Research, Pharmaceutical Division, Sandoz Ltd.., Basel and The Ontario Cancer Institute, Department of Biophysics and Immunology, University of TorontoA, Toronto
Allelic exclusion in TcR
p chain
transgenic mice
417
T cell receptor (TcR) fJ chain transgenic mice: studies on allelic exclusion and on the TcR+ y/6 population To study allelic exclusion of TcR genes we analyzed two types (I and 11) of TcR p transgenic mice. Tcells derived from both types of mice contained similar amounts of transgenic RNA transcripts; however, surface expression of the transgenic p chain was drastically reduced in type I1 compared to type I. In type I transgenic mice, productive rearrangements and expression of endogenous TcR p genes were suppressed whereas on T cells of type I1 mice, both transgenic and endogenous TcR p chains were expressed on the surface of the same cell. These findings suggest that allelic exclusion of TcR genes in p transgenic mice depends on amount and/or onset of transgene expression during thymic development. Furthermore, TcR y rearrangements and the population of TcR yla-bearing double-negative CD4-CD8- thymocytes were reduced fivefold in type I transgenic animals. However, the V, usage and the y/6+ dendritic epidermal cell populations appeared normal. RNase protection analysis further revealed low levels of transgenic TcR p chain transcripts in TcR+ y/6 CD4-CD8- thymocytes. These results suggest that the p transgene only quantitatively influences the y/6 T cell compartment, and supports the independence of the y/6 population.
1 Introduction In an immune response the specific recognition of foreign antigens is mediated via distinct receptors on the surface of B and T lymphocytes. Both the Ig H and L chain heterodimer, and theTcR a@ heterodimer are composed of VDJ gene segments that are assembled during differentiation by somatic DNA rearrangements [ l , 21. Although individual B and Tcells have the potential to generate one functional receptor chain from each chromosome, it is generally believed that a mechanism known as allelic exclusion occurs in each cell, which allows the expression of a single unique heterodimer on the cell surface [3,4]. Two mechanisms for allelic exclusion have been proposed. A feedback mechanism may exist where productive rearrangement regulates subsequent recombination events on the other homologous allele [5].The second mechanism predicts that the probability of generating two functional rearrangements is very low and therefore only one heterodimer is produced [6].
transgenes have shown that rearrangements of endogenous genes are inhibited in the transgenic mouse, thus supporting the model of feedback inhibition [7-111. However, a complication arises in T cells since four rearranging gene families have been identified (a,p, y and 6) (reviewed in [2]).Thus far, it has been demonstrated that either a a@ or a y/6 heterodimer is expressed on the the cell surface and it appears that these heterodimers are present on functionally distinct populations of T cells (reviewed in [2, 121). Therefore,T cells must exhibit exclusion at the level of the individual a,p, y or 6 chain alleles, and also at the level of the type of receptor (a@ or y/6) that is generated. In our work we used a hybrid transgene construct (MHC class I promoter/TcR p cDNA/Ig enhancer) and describe here two types of TcR p transgenic mouse lines which differently regulate endogenous TcR p genes. We also examined the effect of the p transgene on y rearrangements and the effect on y/S cell populations.
2 Materials and methods Over the past years, many groups have used transgenic mice carrying a variety of functional rearranged immune receptor genes to study effects on allelic exclusion. Studies using functional Ig H and L chain genes or the TcR p chain as
[I 78141
*
This work was supported by SNF grants 3.295-0.85 and 3.2130.85 and the Radiumstiftung Zurich.
Correspondence:Hans Hengartner, Institut fur Pathologie, Sternwartstr. 2, CH-8091Zurich, Switzerland Abbreviations: DEC: Dendritic epidermal cells DN: Double negative 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
2.1 Transgenic mice The construction of the transgene (Fig. 1) and the generation of transgenic mice has been described recently [13]. Transgenic mice were identified by Southern blot analysis of tail DNA. The animals used in this study were second or third generation offspring from transgenic founder animals bred with C57BL/6 mice. The transgenic TcR p chain was composed of Vg8.1, Dg, Jg2.4 and Cg gene segments [14], and originated from an LCMV-specific, H-2Db-restricted cytotoxic T cell clone [15]. According to Southern blot analysis, the copy number of the transgene was 10-20 in lines 128 and 132 (type I) and 5 in line 126 (type 11). The transgenes were stably inherited and transmitted in a Mendelian fashion, indicating integration in a single chromosomal site.
-
0014-2980/90/0202-0417$02.50/0
418
Eur. J. Immunol. 1990.20: 417-424
H. Pircher, €! Ohashi, G. Miescher et al.
K z K b promotor
TCR 0 Emu
Wobln exon 2 + 3
POly A signal
lmmunoglobulln heavy chain enhancer
Figure 1. Scheme of the transgene construct. The detailed construction of the plasmid has been described recently [13].
2.2 DNA and RNA isolation Tail DNAwas isolated from approximately 1cm of tail.The bone was removed and the skin was minced into small pieces with a razor blade. The skin was then incubated overnight at 55 "C in 0.7 ml of proteinase K buffer (100 mM EDTA, 100 mM NaCl, 50 mM Tris pH 7.5, 1% SDS and 2 mg/ml proteinase K; Merck, Darmstadt, FRG). Cells (lo7) were lysed in 5 ml buffer. DNA was isolated by phenol-chloroform extraction and ethanol precipitation. Total RNA was prepared by the guanidinium isothiocyanate extraction procedure and purified on CsCl gradients [16, 171. 2.3 DNA and RNA analysis For Southern Blot analysis, 20 pg of genomic DNA was digested, separated on a 0.8% agarose gel and transferred to a Biodyne membrane (Pall, Glen Cove, NY). Twenty micrograms of total RNA was separated on 1% agarose glyoxal gels [18] and transferred to a Biodyne membrane. Filters were prehybridized at 42 "C for 3 h with 50% formamide, 5 x SSC, 50mM KH2P04, pH 6.5, 5 x Denhardt's, 1% SDS and SO0 pg/ml salmon sperm DNA. Hybridization was performed overnight at 42 "C with 32P-oligolabeledprobes in 50% formamide, 5 X SSC, 20 mM KH2P04,5 x Denhardt's, 1% SDS and 100 pg/ml salmon sperm DNA.The following probes were used: PL5 (cDNA, mouse Cg, a gift of D. Loh, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis); pP14P (cDNA,Vg8Cg) [14]; pP14, (cDNA,Va2Ca) [14]; p8/lOy (cDNA Vy1.2q2) [19]; pUC-C1A (a 6.4-kb genomic Bam HI-Hind III fragment of mouse Cpl loci) [20]; Dgl (a 1.4-kb Pst I fragment upstream of Dpl gene segment) [20]; pUC-Jb2 (a 2.2-kb Eco RI fragment of the Jg2-gene segment) [20]. The SP6 probes containing Vy1.2, Vy2,Vy3,and Vy4inserts of, respectively, 310,158,213 and 182 bp were provided by Dr. D. Raulet (MIT, Cambridge, MA).They are described elsewhere [21], together with the RNase protection procedure. A 173-bp Bgl I1 Pst I p-actin cDNA restriction fragment was cloned into pSP64 and used as an internal control. 2.4 Inmunofluorescence
Single-cell suspensions were prepared from mesenteric LN and thymus. Spleen cells (2 x 106 cells/ml) were cultured for 3 days in the presence of 3 pg/ml Con A (Pharmacia, Uppsala, Sweden) and purified using a Ficoll-Hypaque gradient. Double-negative thymocytes were obtained by
treatment with anti-CD4 and anti-CD8 antibodies plus C, as described in detail elsewhere [22]. TcR y/Bexpressing cells were obtained by culturing CD4-CD8- thymocytes for 7 days with Con A (2 pg/ml) and IL 2 (200 U/ml) as described [22]. The following mAb were used: KJ16, rat IgG, specific for Vp8.1-t 8.2 [23]; 44.22.1 rat IgG, specific for Vp6 [24]; KT11, rat IgG specificforVpll[25]; B2A2, rat IgM; F23.1, mouse IgGza specific for Vg8 [26]; F23.2, mouse IgGl specific for Vp8.2 [27]; 145.2Cl1, hamster IgG specific for murine CD3 [28]; anti-CD4-PE and antiCDS-FITC (Becton Dickinson, Mountain View, CA); The following second-stage reagents were used: FITC-coupled goat anti-rat (Tago, Burlingame, CA), FITC-coupled goat anti-hamster IgG (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD), FITC-coupled anti-mouse IgGza and PE-coupled goat anti-rat IgG (Southern Biotechnology, Birmingham, AL) Ig antibodies. FCM was performed on an Epics Profile Analyzer (Coulter Electronics, Hialeah, FL) .
2.5 T cell hybridomas Spleen cells were stimulated at 2 x 106cells/ml with 3 pg/ml Con A for 3 days. Cells (2 x lo7)were fused with 1 x lo7of the thioguanine-resistant,TcRa-P- variant of the BW5147 lymphoma a (a kind gift of Dr. W. Born, National Jewish Hospital, Denver, Co) according to established procedures [29], and selected in HAT medium for 7 days. T cell hybridomas used for TcR rearrangement studies were distributed in 96-well plates under LD conditions to ensure clonality. T cell hybridomas used for immunofluorescence were pooled and analyzed 7 to 10 days after fusion. 2.6 Isolation of dendritic epidermal cells (DEC)
DEC were isolated from the trunk epidermis using a modified procedure reported by [30]. Epidermal sheets were obtained by incubating the skin with 1% porcine trypsin (Gibco Laboratories, Paisley, Scotland) for 1h at 37 "C, and single-cell suspensions were made by forcing the sheets through a wire mesh. The cells were cultured overnight in Iscove's modified Dulbecco's medium, 10% FCS, 5 x lop5 M 2-ME, penicillin, streptomycin, and 3 pg/ml Con A , purified over Lympholyte M gradient and analyzed by FCM.
3 Results 3.1 Allelic exclusion of TcR fi genes in type I, but not in type I1 TcR fi transgenic mice
To investigate the molecular mechanism that regulates TcR gene rearrangements, we studied TcR transgenic mice. Cell surface expression of the transgenicTcR P chain (Vp8.1) was analyzed by immunofluorescence with TcR Vp8-specific antibodies. Virtually all splenic T cells from the transgenic mouse lines 128,132 and 126wereVg8+ and CD3+ (Fig. 2).This indicated that the majorityof Tcells expressed the transgene on the cell surface. However, the Vg8 staining profile showed that the transgene product was expressed on the cell surface at a lower density on Tcells from line 126 (which will be designated as type 11) when
Eur. J. Immunol. 1990. 20: 417-424
Allelic exclusion in TcR
p chain transgenic mice
419
Table 1. TcR Vg analysis of peripheral Tcells from transgenic mice with mAb
Mice
% of Con A blast cellsa)stained with mAb KJ16 44.22.1 F23.2 KT11 Vg8.1 + 2 VP6 Vp8.2 Voll
Control Type I Type I1
14.1 f 0.8 96.1 4.7 70h)
6.1 f 0.2 0.6 f 0.3 7.3 k 1.1
8.8 f 1.4 0.3 f 0.2 4.8 f 1.0
compared t o lines 128 and 132 (type I). Additionally, amongT cells derived from type I1 mice, a small percentage of cells expressing TcR Vg8 at normal density was observed. The CD3 staining was similar in both transgenic lines and comparable to normal mice, indicating that the overall TcR densities of the examined T cells were comparable. This finding suggested that T cells from type I1 mice also
TCR Vf38
CD3
log fluorescence Figure 2. Surface expression of TcR Vp8 and CD3 on peripheral T cells of transgenic mice. Splenic Con A blast cells from transgenic mice and a negative littermate controls were stained by indirect immunofluorescence with mAb KJ16 (Vg8.1+ 2) and 145.2.Cll (CD3), followed by FITC-labeled goat anti-rat or goat anti-hamster Ig and analyzed by FCM. Negative controls with the fluorescent conjugate alone are shown as dotted lines.
$4
6.0 k 1.0 0.2 f 0.2 3.1 f 1.3
a) T cells from 3-day Con A-activated spleen cells were stained with the indicated mAb. Data are presented as average percentages (after subtraction of background staining with the fluorescent conjugate alone) of at least three separate mice per group. b) Due to the low staining intensity, only approximate percentages are given (see Fig. 2).
expressed endogenous $ chains in addition to the transgenic $ chain.To test this,T cells from type I and type I1 lines were examined for endogenous Vg gene expression. Antibodies specific for other TcR Vg chains (Vg6; 8.2, 11) stained T cells from transgene-negative littermates whereas T cells expressing these chains from type I mice were drastically reduced (Table 1). In contrast,Vg6+,Vg8.2+ and Vpll+ T cells were found among transgenicTcells from type I1 mice. The frequency of Vg6+ Tcells in these mice was similar to normal mice (6.7%) and the percentage of Vg8.2+ (4.2%) and Vgll+ (3.9%) cells was slightly reduced. This data demonstrates that the expression of endogenous TcR $ genes was strongly suppressed in type I but not in type I1 transgenic mice. These results were confirmed by Northern blot analysis of Tcell RNA from type I and I1 transgenic mice using a Cg probe. Because the transgene construct contains the 3' portion of the P-globin gene (Fig. l ) , the transgenic $ transcripts were slightly longer than endogenous $ chain transcripts. As shown in Fig. 3, both types of transgenic T cells expressed similar amounts of transgenic transcripts; however, RNA molecules from completely rearranged endogenous $ genes were detected only inTcells of type I1 mice. Short transcripts from incomplete $ gene rearrangements were found in transgenic and control T cells. TcR p gene rearrangements of 8 transgenic Tcell hybridomas from type I mice were studied by Southern blot analysis using Cg-, Jg2- and Dgl-specific probes. Of the 16TcR $ loci analyzed, we found 7 Dgl-Jgl, 2 Dgl-Jg2 and 3 Dp2-Jp2 rearrangements (not shown). This result indicates that completeVDJ recombination events rarely occurred in type I TcR $ transgenic mice in contrast to the high number of Dg-Jg rearrangements and confirms previous studies of transgenic mice carrying rearranged genomic TcR $ genes [ l l , 311. 3.2 'Qpe I1 mice express two different TcR on the cell surface
Control type I typell
Figure3. Transcription of TcR p genes in transgenic mice. Northem blot analysis of total RNA isolated from splenic Con A blast cells from normal and transgenic mice. The blot was hybridized with a Cp cDNA probe. Closed head arrow indicates transgenic transcripts and open head arrow indicates transcripts from completely rearranged endogenous p genes.
Since endogenous $ chains were detected in type I1 mice, T cells expressing both transgenic (Vg8.1) and endogenous chains may be present. This was examined by performing two-color immunofluoresence with Vg6 (PE labeled; red) and V@(fluorescein labeled; green) specific antibodies. T cells of normal control mice expressed either Vg6 or Vg8 (Fig. 4, left). I n contrast, Vg6+ T cells of type I1 animals were also stained with Vg8-specific antibodies as indicated by the shift of the Vg6+ population in the two parameter histogram towards the green fluorescence (Fig. 4, right). This indicated that T cells coexpressed Vg6 and transgenic Vg8 in type I1 mice.
420
Eur. J. Immunol. 1990. 20: 417-424
H. Pircher, I? Ohashi, G. Miescher et al.
type II
control 21.9 I
0.4 I
30.8 I
2.8 I
V06 Figure 4. Coexpression of TcR Vp6 and Vp8. LN cells from normal and type I1 transgenic mice were double stained with mAb against Vp8 (F23.1) and Vp6 (44.22.1). The cells were first stained with mAb F23.1 and goat anti-mouse FITC-IgG2,. The staining was followed by mAb 44.22.1 and finally by goat anti-rat PE-IgG. Cytograms represent 5 x 104 viable cells accumulated on a logarithmic scale of fluoresence intensity.
3.3 Expression of the transgene is turned off after cell fusion
To examine allelic exclusion, we generated T cell hybridomas by fusing Con A-activated T cells from type I transgenic mice with the a-P- variant of the BW5147 lymphoma. We made the surprising observation that T cell hybridomas derived from transgenic Vg8+ T cells were all Vg8and CD3- (Fig. 5). In contrast,Tcell hybridomas derived Con A blast T cells
T cell hybridomas
control
from normal Tcells were CD3+ and the frequency of Vp8+ hybridomas was similar to that of normal activated Tcells (Fig. 5). This finding suggested that transcription of the transgene was turned off after fusion with BW5147. To further examine this phenomenon, DNA and RNA from cloned T cell hybridomas were analyzed. Although the transgene was detected in the DNA of all hybridomas, only short endogenous p chain RNA transcripts from incomplete P gene rearrangements were found (not shown). These results explained the absence of the transgene product on the cell surface of most Tcell hybridomas and further confirmed our data that functional endogenousTcR p chains were not produced in T cells derived from type I mice. 3.4 The TcR f3 transgene also affects TcR y genes It was of interest to examine the effect of the TcR p transgene on endogenous TcR y genes. DNA from peripheral Tcells of type I transgenic and control mice was digested with Eco RI and the blots were hybridized to a VY1.2C,,2probe. In both transgenic and control Tcells a 15-kb fragment (closed head arrow) was observed representing y rearrangements (Fig. 6). In transgene-negative T cells the signal intensity of rearranged vs. the 10.5-kb germ-line band was approx. 5 : 1. In transgene-positive T cells, the ratio was about 1: 1indicating that the frequency of y rearrangements was decreased in TcR P transgenic animals. Similar results were obtained when thymus DNA was used (Fig. 6). Interestingly, inhibition of y rearrangements was also observed with type 11 transgenic mice. Northern blot analysis of thymus W A further showed a slightly reduced level of y transcripts in the type I and I1 transgenic mice (not shown). Analysis of y rearrangements
thymus n-
oocv
cvm transgenic
z
P
E a
-E
3
control
kb -13.4 -10.8
transgenic
-7.5
log fluorescence Figure 5. Surface expression of CD3 and TcR Vp8 on Con A blast T cells and Tcell hybridomas derived from transgenic mice. Splenic Con A blast T cells and Tcell hybridomas from control and from type I transgenic mice were stained by indirect immunofluorescence with anti-CD3 and with anti-Vp8-specific mAb.The samples were analyzed by FCM.
Figure 6. TcR y rearrangements in p transgenic mice. DNA from splenic T cells and thymus of the indicated transgenic lines were digested with Eco RI and hybridized with aVy1.2q2 cDNA insert as described in Sect. 2.9.This probe hybridized to Cyl,q 2 , q 3 and V,l genes. Rearranged bands are indicated by the closed head arrow, germ-line bands by open head arrows.
Eur. J. Immunol. 1990.20: 417-424
Allelic exclusion in TcR
p chain transgenic mice
421
in T cell hybridomas revealed that 4 out of 8 derived from type I transgenic and 7 out of 8 from control mice had undergone y rearrangements (data not shown).
3.5 The population of TcR y/S expressing thymocytes is reduced in p transgenic mice We have recently demonstrated that TcR a$ and y/6 are expressed on phenotypically distinct populations of doublenegative (DN) CD4-CD8- thymocytes [22]. By two-color flow microfluorometry two distinct CD3+ subsets may be resolved in control mice: brightly staining CD3+B2A2+ cells representing y/6 cells and a a@-bearing B2A2- subset expressing CD3 at a lower level (Fig. 7). In type I TcR p transgenic mice, the yl6 subset was reduced 4-5-fold when compared to control littermates, (2.2% vs. 10.1%), suggesting that the p transgene affected the population of
-
0,
Control littermate
v 0
VP8.,Transgenic
(A1
w l
(81
(Cl
Figure 9. RNase protection analysis of TcR Vp8 (A), C, (B) and C6 (C) transcripts in Con A-stimulated TcR+ y/6 CD4-CD8- thymocytes from type I transgenic mice (TG+) and control littermates (TG-) compared to cultured spleen and L N cells. The expected protected fragments are indicated. C, indicates the homologues protected fragment whereas the fragment indicated by Ci is due to strain polymorphism in the TcR C, locus. -
1
I
s m
( u '
CD3 FLUORESCENCE (log)
Figure 7. The TcR y/S expressing subset of DN thymocytes is reduced in transgenic animals. Freshly isolated adult DN CD4-CD8- thymocytes from transgene-negative siblings and type I transgenic mice were double stained with mAb B2A2 and 145-2C11 (anti-CD3). Cytograms represent 105 viable cells accumulated on a logarithmic scale of fluoresence intensity.
ylb-bearing cells in adult thymus. Interestingly, the TcR a@ bearing subset of B2A2- CD4-CD8- thymocytes was also significantly reduced in the transgenic animals (1.4% vs. 4.0% in littermates; Fig. 7). The DN, y/Benriched population of Tcells were selectively expanded as described previously [22] and theV, usage was e ~ a m in e d . V~ l. 22-(nomenclature by Garman et al. [21].) have been shown to be preferentially expressed over V,3 4 in adult thymocyte populations [32,33]. By RNase protection experiments, V 4 . 2 + 2 were present in the transgenic and nontransgenic DN thymocytes at comparable levels, whereasVy3 4 were absent (Fig. 8).These data indicate that although the p transgene affects the number of DN y/6 thymocytes, the V, gene usage remains the same in type I transgenic mice. By RNase protection experiments, low levels of TcR Vg8.1 transcription were also detected in these y/6 expressing DN thymocytes derived from transgenic mice (Fig. 9A, lane 2). It is unlikely that this signal was due to contaminating a@ expressing DN thymocytes since TcR C, message was undetectable in these expressing cells (Fig. 9B). Furthermore, cell surface expression of the transgenic p chain was not detected on these cells (not shown).This suggested that the transgenic p chain could not pair with y or 6 chains and further indicated that the transgenic transcripts were not derived from contaminating a / p DN thymocytes.
+
+
Protected fragments
c v y 1.2 cvy3 c vy4
p actin +
c v y 2
~
L
probes
A
Vy1.2
h
A
Vy2
Vy3
J
Vy4
+ t3 actin
Figure 8. RNase protection analysis of V, transcripts in Con Astimulated TcR+ y/6 CD4-CD8- thymocytes derived from type I transgenic (TG+) and control littermates (TG-) compared to cultured C57BLI6 LN cells. The expected protected fragments are indicated by arrows, the single-stranded RNA hybridization probes used are indicated below the autoradiogram. In the first series of lanes, a p-actin probe has been included as an internal standard.
+
3.6 ylS populations in the skin of transgenic mice
Several studies have shown that Thy-l+ DEC express TcR ylS [34-361. In order to determine if the p transgene had an effect on the DEC cell population and to determine if any transgenic TcR Vb8+ T cells could be identified in this
422
H. Pircher, F! Ohashi, G. Miescher et al. control
transgenic type I
41.7 %
36.5 %
Thy 1 DEC
40.3%
h
29.3 %
CD3
DEC
TCR VD8 DEC
16.0%
I’i
90.6 %
T;c;
compartment, trunk epidermal cells were isolated. Lympholyte M enriched cells from transgenic and nontransgenic littermates were stained with CD3 Thy-1 and TcR Vp8specific mAb. FCM analysis indicated that approximately 30%-40% of the lymphocyte-enriched population was Thy-l+ and approximately 30%40% also expressed CD3 in both transgenic and nontransgenic mice. In addition, none of the skin cells expressed TcR Vg8 (Fig. 10). Mice ranging from 12 to 70 days of age were examined several times, but consistent variations in the numbersof Thy-l+ or CD3+ DEC were not seen. Therefore, although the thymocyte population of y/6 cells was reduced in type I mice, the “mature” peripheral y/6 DEC exist in comparable numbers. The lack of Vg8+ cells supports previous work that suggests that the epidermal compartment is comprised exclusively of y/6-bearing cells [36].
4 Discussion In the present report we describe two types of TcR p transgenic mice which expressed normal (type I; two transgenic lines) and low (type 11; one line) amounts of transgenic p chains on the surface of T cells. Allelic exclusion was demonstrated on T cells from type I transgenic mice in the following ways. FCM analysis using mAb specific for various Vg segments indicated that other Vg chains could not be detected on the surface of peripheral T cells. Northern analysis of thymocytes as well as Con Astimulated peripheral Tcells detected the larger transcript from the transgenic p chain and not the endogenous 1.3-kb p chain. In addition, hybridomas made from peripheral T cells showed incomplete VDJ p chain rearrangements.
Eur. J. Immunol. 1990.20: 417-424
Therefore, in type 1 transgenic mice functional rearrangements and expression of endogenous p chain were virtually inhibited. However, unusual results were seen in similar studies with type I1 transgenic mice. Using Vg-specific mAb, FCM analysis showed a low level of Vp8 on the majority of T cells, while the overall population expressed essentially normal levels of the other Vg families. The expression of endogenous genes was confirmed by Northern blot analysis. Further examination of peripheral T cells from the type I1 transgenic mice surprisingly showed T cells that simultaneously expressed two different types of p chains on the cell surface. Although both type I and I1 mice carry the identical transgene, type I mice exhibit allelic exclusion whereas type I1 mice not only rearranged and expressed endogenousTcR p genes, but also permitted the expression of two different TcR p chains on the cell surface. The reasons for the completely different effects on the endogenous p genes in type I and I1 transgenic mice are unclear. Northern blot analysis of both thymocytes and peripheral Tcells demonstrated that similar RNA levels of the transgene are transcribed in both type I and I1 animals. Both transgenic lines have been made by injecting the identical construct, and therefore, the immediate transcriptional and translational regulation and processing should be identical. Because of this, a similar amount of protein should be made in both types of transgenic mice.Therefore, varying amounts of transgenic transcripts or protein is an unlikely explanation for the differences found at the endogenous fi chain locus. Only two notable differences are apparent in these transgenic lines. Type I1 mice contain fewer copies of the integrated transgene, and the integration site of the transgene would also vary between the three examined lines. It is possible that these two factors in combination or alone could alter the stage at which the transgene is activated during Tcell ontogeny. For example, it is possible that transcription of the transgene in type I1 mice (compared to type I) is expressed later in thymocyte differentiation. This could allow endogenous p chain rearrangement. Therefore, competition between the transgenic and endogenous p chains for pairing with the a chain may occur, leading to the observed reduced cell surface expression of the transgenic chain. In the course of generating Tcell hybridomas, we made the observation that the expression of the transgene was turned off after cell fusion with the BW5147 lymphoma.This result was observed with three independent transgenic mouse lines (not shown). In these mice transcription of the transgene was regulated by a class I promoter (H-2Kb) in the presence of the Ig enhancer [13]. Since the transgenic BW5147 hybridomas expressed class I molecules on the cell surface, transcription driven by the class I promoter was not generally affected in these cells (not shown). Furthermore, we were also able to establish transgenicTcell lines in vitro which continuously expressed the transgenic p chain. It has been recently shown that genes activated by the Ig H chain enhancer are extinguished upon fusion with the T cell lymphoma BW5147 [37]. Therefore, it is possible that a similar mechanism led to the extinction of transgenic transcripts in BW5147 hybridomas. During T cell ontogeny, it appears that two distinct populations of Tcells are generated that express either the TcR
Eur. J. Immunol. 1990. 20: 417-424
a l p or y / 6 (reviewed in [2, 121). The mechanism or differentiation events that determine the type of receptor that is expressed on the Tcell are not understood. Thus, in order to determine if the transgenic p chain altered theTcell lineages, the y16 populations were studied in the following ways. Southern blot analysis of total thymocytes DNA, as well as peripheral T cell hybridomas demonstrated a decrease in y chain rearrangements. In addition, the level of y chain message detected by Northern blot analysis was also decreased. However, since a / p Tcells have been shown to rearrange and transcribe y genes [32,38,39],it is not clear if the decrease in y rearrangement and RNA expression that is detected in these analyses originates from the a / p or y/6 T cell populations. Therefore, the y/S DN thymocyte population was examined and the relative number of y/6 double-negative cells was found to be decreased approximately fivefold. However, the relative use of V,1.2,2, 3 and 4) genes (nomenclature by Garman et al. [21]) remained comparable between transgenic and nontransgenic lines. This latter result is in contrast to a recent study with TcR p transgenic mice which showed selective suppression of the V,2 gene segment [40].
In order to determine if other populations of y/6 cells in these p chain transgenic mice were likewise decreased, the skin dendritic epidermal cell population was examined.The trunk epidermis from mice ranging from 12 days old to 2 months old was analyzed. A difference in Thy-l+ CD3+ cells was not detected in transgenic mice compared to their control littermates. It is noteworthy that none of the dendritic epidermal cells expressed the transgene on the cell surface, demonstrating the presence of only y/6 cells in the epidermal compartment .This also suggests that y/6 cells have a unique function in the skin which cannot be substituted by a@ cells. A somewhat unexpected finding in the present study was the reduction in a l p TcR+ CD4-CD8- thymocytes in type I transgenic mice. This subset (whose developmental status remains enigmatic) is characterized by over-expression of TcR utilizing the Vg8 gene family, particularly Vg8.2 [41, 421. It will be interesting to examine this subset in detail in Vg8.2 transgenic mice to determine whether the relative lack of such cells in Vg8.1 transgenic animals reflects a general developmental block or, alternatively, is due to the absence (via allelic exclusion) of aTcRVp chain (Vg8.2) that is appropriate to undergo some obligatory selective event. The observation that the p transgene could inhibit both endogenous p and y TcR rearrangements suggests a similar molecular mechanism which controls TcR rearrangements. It also agrees with two recent studies [31, 401 which demonstrated suppression of y rearrangements in transgenic mice bearing genomicTcR p genes. In one study [40], the transgenic p chain was prematurely expressed with an uncharacterized polypeptide on the cell surface of all thymocytes including the CD4-CD8- subset. In our own studies, surface expression of the transgenic chain was not detected on the CD4-CD8- thymocyte subset in the absence of CD3 (not shown). This demonstrates that premature chain expression on CD4-CD8- thymocytes is not a prerequisite for suppression of y rearrangements or reduction in the number of y/6+ CD4-CD8- thymocytes.
Allelic exclusion in TcR
p chain transgenic mice
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Our data concerning V, usage and y/S dendritic epidermal cells also suggest that the yl6 populations in these mice are relatively normal. Therefore, we believe that the observed decrease in y rearrangement and expression in thymus and peripheral Tcells of transgenic mice may be due (at least in part) to a decreased number of y rearrangements in the a / p T cell population. This may be a consequence of accelerated T cell ontogeny, resulting in a shorter time span wh’ich y can undergo rearrangement. The fact that the transgenic TcR p chain was expressed at the RNA level in y/6 DN thymocytes demonstrates that precursor cells were able to differentiate into the y/6 lineage despite the presence of a functional TcR p chain. This supports a recent study by Winoto et al. [43] which suggested that a/p and y/6 Tcells are separate lineages.The reduced.number of DN, y / 6 cells in the thymus probably reflects the accelerated development of alp Tcells because of the presence of a functional p chain, thus resulting in an relative decrease of y/6 population. We thank M. Condraufor technical advice at the FACS and Beatrice Borter for secreterial help. Received July 24, 1989; in revised form November 10, 1989.
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