hamster IgG specific for murine CD3 (Leo et al.,1987); 15.1.5 and 15.5.5 .... Leo,O., Foo,M., Sachs,D.H., Samelson,L.E. and Bluestone,J.A. (1987). Proc. Natl.
The EMBO Journal vol.8 no.3 pp.71 9 - 727, 1 989
T cell tolerance to MIsa encoded antigens in T cell receptor V38.1 chain transgenic mice
Hanspeter Pircher1'2, Tak W.Mak2, Rosmarie Lang1, Wolfgang Ballhausen2, Edith Ruedi', Hans Hengartner', Rolf M.Zinkernagel1 and
K.Burki3 Institute of Pathology, Department of Experimental Pathology, University Hospital, 8091 Zurich, Switzerland, -The Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Canada and 3Department of Preclinical Research, Pharmaceutical Division, Sandoz Ltd, 4001 Basel,
Switzerland Communicated by R.M.Zinkernagel
To study T cell tolerance, transgenic mice were generated that expressed the Mls5-reactive T cell receptor (TCR) 3 chain V(8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on 90% of peripheral T cells. In transgenic mice bearing Mls', thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V,B-specific antibodies revealed that expression of endogenous TCR (3 genes was suppressed. Both Mls' and N4lsb TCR ( chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR ( chain transgenic Mlsa, but not in transgenic Mlsb mice. Key words: MlSa/T cell receptor/tolerance/transgenic mice -
Introduction T lymphocytes recognize antigens in association with major histocompatibility complex (MHC) encoded molecules by a clonally expressed receptor (for review see Zinkernagel and Doherty, 1979; Schwartz, 1985). This heterodimeric T cell receptor (TCR) is composed of a and ( polypeptides which determine both antigen and MHC specificity (for review see Marrack and Kappler, 1987; Toyonaga and Mak, 1987; Davis and Bjorkman, 1988). The challenge remains, however, to understand the mechanism of how bonemarrow-derived precursor cells mature in the thymus into functional T lymphocytes which show MHC-restricted recognition and tolerance to self. Recently, the production of TCR VA-specific monoclonal antibody (mAb) and the discovery of simple correlations between TCR V( use and antigen reactivity has allowed the course of single maturing T cells with known antigen reactivity to be followed in vivo. Kappler et al. (1987) provided the first direct evidence that tolerance to self MHC products was due to clonal deletion of self-reactive T cells. These authors were able to show that T cells expressing the ©IRL Press
TCR V, 17 segment recognize the MHC class II molecule I-E, and that in mice expressing I-E, no or few mature V,(17 T cells were found. Further studies (Kappler et al., 1988; MacDonald et al., 1988a) extended these observations to non-MHC antigens. Both groups demonstrated that T cells expressing the TCR variable gene segment V(6 or V38. 1 reacted predominantly with Mlsa-encoded antigens and that these T cells were selectively deleted in Mlsa-bearing mice. In this report we describe the production of TCR ( chain transgenic mice which express the variable gene segment V(8. 1 on a large fraction of their T cells. We examined the influence of reduced TCR variability due to the transgenic TCR (3 chain on T-cell-dependent immune responses and its effect on endogenous TCR , expression. These mice also permitted further analyses of the mechanisms leading to self-tolerance, because almost all T cells maturing in these mice were predisposed to react with Mlsa-encoded antigens. Very recently, Kisielow et al. (1988) have used a similar approach with o( TCR transgenic mice expressing a receptor for the male (HY) antigen on their T cells. Our results with V(8. I-Mlsa transgenic mice will be discussed in the context of their findings.
Results Transgenic mice The transgenic TCR ( gene was derived from a lymphocytic choriomeningitis virus (LCMV)-specific, H-2Db-restricted cytotoxic T cell clone, called P14 (Pircher et al., 1987b). DNA sequence determination revealed that the TCR ( chain of P14 was composed of V(8. 1, Do(, J(2.4 and C(2 gene segments (Pircher et al., 1987a). P14 TCR ( chain cDNA was set under the control of the H-2Kb promoter and a genomic fragment of the human ( globin gene was joined 3' to introduce RNA splice sites and a poly(A) signal (construct A, Figure 1). Additionally, a second construct was generated by inserting the immunoglobulin heavy chain (IgH) enhancer element into construct A (construct B, Figure -
x
0.5kb I
a
H-2Kb promotor
C
o
C
C0
IjIW
WM TCR B cDNA
83-globin exon 2 + 3
poly A signal
x
o
iC
Immunoglobulln heavy chain enhancer
CONSTRUCT A
CONSTRUCT B
Fig. 1. The H-2Kb promoter/T cell receptor 3 chain cDNA construct. The detailed construction of the hybrid genes is described in Materials and methods. The restriction sites marked with an asterisk (*) were destroyed during plasmid construction.
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H.Pircher et al.
1). The chimeric genes were excised from the vector sequences and 500 copies were microinjected into the male pronucleus of (C57BL/6 x DBA/2)F2 fertilized eggs. A total of 14 DNA-positive founder mice were obtained, ten with construct A and four with construct B. -
The IgH enhancer element is active in T cells of transgenic mice Transgenic founder mice were crossed with C57L or C57BL/6 mice and lymph node cells of transgenic offspring were analyzed for cell surface expression of the transgenic TCR V38. 1 chain with the V38. 1 + 8.2-specific mAb KJ 16 (Haskins et al., 1984; Behlke et al., 1987) (Table I). The transgenic 3 chain should be expressed only on the surface of T cells, because functional TCRat and CD3 polypeptides present only in T cells are required for cell surface expression (Ohashi et al., 1985). In nine out of ten independent transgenic lines carrying construct A (without IgH enhancer), 10-20% of the T cells were KJ16+. Since in these mice 10-20% KJ16+ T cells are expected due to endogenous Vf8+ expression, nine out of ten transgenic mice with construct A probably did not express the transgene. In mouse 140, 57% of the T cells were V,B8+, indicating expression of the introduced gene. In marked contrast, offspring from four out of four independent transgenic founder mice carrying 5-20 copies of construct B (with IgH enhancer) expressed the introduced TCR 3 chain since 70-90% of the T cells were stained with KJ16. The offspring of both founder mice 126 and 128 Table I. The immunoglobulin heavy chain enhancer enables expression of the transgenic TCR3 cDNA in T cells Mouse number
Construct Gene copya
% of peripheral T cells expressing
Vf8 137 182.5 138.3 140 143.2 148.5 170.3 180.7 153.2 153.6 162.2 125.4 126.6 126.5 128.10 128.4 132.7 C57BL/6 (C57BL/6 x C57L)F 1
A A A A A A A A A A A B B B B B B
+ + ++ ++ ++ ++ ++ ++ ++ +++ +++ ++ + + ++ +
++
14 10 11 56 10 12 10 9 11 9 10 71 75 dull 12 93 10 87 18 10
Transgenic founder mice were mated with C57BL/6 or C57L mice. Lymph node cells from transgenic Fl mice were stained by indirect immunofluorescence with mAb KJ16 (anti-TCR V,B8.1+8.2) and mAb 30-H12 (anti-Thy 1.2) and analyzed on a FACS. These results were then used to calculate the percent TCR V,B8 + cells of Thy 1.2 + T cells. aThe copy number of the transgene was roughly estimated by blot hybridization with a TCR ,B chain probe. +, 1-5 copies; ++, 5-20 copies; +++, >20 copies.
720
demonstrated a segregation of the transgene into 1 - 3 copies (mice 126.5 and 128.4) and 5-20 copies (mice 126.6 and 128.10). The percentage of KJ16+ T cells in mice which carried only 1 -3 copies of construct B was not increased. The KJ16 staining intensity of T cells from mouse 126.6 ( - 5 copies) was lower when compared to T cells from mice 125.4, 128.10 and 132.7 (-.10-20 copies). Therefore,
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EXPRESSION Fig. 2. Southern blot analysis of EcoRI-digested tail DNA from transgenic offspring. This blot shows the analysis of offspring from founder mice 125, 126, 128 and 132. The transgene is indicated by the 2 kb band, and the 2.3 kb band represents the endogenous CO locus. Additionally, the expression of the transgene based on immunofluorescence data is shown: -, 10-20% endogenous Vf8' T cells; +, 50-70% dull V,B8+ T cells; 4+, 70-90% bright V38+ T cells. Southern blot analysis was carried out as described in Materials and methods with a cDNA probe containing the Vf8. 1 and Cf2 segment.
CD8
TCR
CD4
V,
CD3
A
a 0
B
0
E z C
Fluorescence Intensity
(log scale)
Fig. 3. Surface expression of CD8, CD4, TCR V,B8 and CD3 on thymocytes from transgenic and normal mice. Untreated thymocytes were stained by indirect immunofluorescence with mAb 53.6.72 (anti-CD8), H 129.19 (anti-CD4), KJ16 (anti-TCR V38. 1+8.2) and mAb 145-2C I (anti-CD3) and analyzed by flow microfluorometry (solid line). Negative controls with the fluorescent conjugate alone are shown as dotted lines. (A) C57BL/6. (B) Transgenic (128.6 x C57BL/6)FI; H-2hb; Mlsb. (C) Transgenic (128.6 x DBA/2)FI; H-2bd; Mlsba.
T cell tolerance in T cell receptor f transgenic mice
these results suggest a correlation between copy number and transcriptional activity among transgenic mice carrying construct B (Figure 2). We report here the analysis of second-generation offspring from the male founder 128. Analysis of these mice revealed that the introduced DNA was transmitted in a simple Mendelian manner. Thymocytes expressing the TCR at a high density are deleted in V,38. 1-transgenic Ml5a mice Recent studies (Kappler et al., 1988) showed that T cells with the Vf38. 1 TCR reacted with an extraordinarily high frequency with Mlsa-encoded antigens and that mice expressing MlSa have few or no Vf38. 1 + T cells. Since our transgene contained the Vf38. 1 TCR ,B variable gene segment, it was of interest to examine the fate of T cells
TCR V
CD4
CD8
fP8
-1
II
t
1) (I)
en
in V,B8. 1 transgenic mice expressing Mlsa. The male mouse 128.6 was an offspring of founder 128 x C57L mouse. This mouse was crossed with female C57BL/6 (H-2b, Mlsb) and with female DBA/2 (H-2d, Mlsa) mice. Mls typing of the C57BL/6 offspring was performed with the Mlsa_specific T cell line CgCBJ100.16 (a kind gift of Dr H.Festenstein, London Hospital Medical College, UK; data not shown). Thymocytes of second-generation offspring were analyzed for cell surface expression of CD3, CD4, CD8 and TCR V38 with mAbs (Figure 3). The thymus size and the CD4 and CD8 staining profiles of thymocytes from transgenic mice and negative littermate controls were very similar, independent of the Mls haplotype. In both Mlsb and Mlsa transgenic mice, we observed a high percentage of V,38+ thymocytes; however, the staining intensity profile revealed a striking difference between MlSa and Mlsb mice. In transgenic offspring of C57BL/6 (H-2bb, Mlsbb) mice, a subset with high KJ 16 receptor density (10-15 %) could be distinguished from a population of dully staining thymocytes (Figure 3B). The number of thymocytes with a high receptor density was clearly reduced in transgenic offspring of DBA/2 (H-2bd, Mlsab) mice (Figure 3C). Analogous results were obtained with anti-CD3 mAb. As shown by Roehm et al. (1984) the density of the ao3 receptor on mature thymocytes is significantly higher than on immature cells. Therefore, our data indicated that either the generation of mature thymocytes was severely suppressed or the receptor density was reduced on mature thymocytes in transgenic MlSa offspring.
'-
1-
--A
1/1\,
,
I
E z
Fluorescence intensity (log scale) Fig. 4. Surface expression of CD8, CD4 and TCR V38 on lymph node cells from transgenic mice and negative littermates. Lymph node cells were incubated with the mAb 53.6.73 (anti-CD8), H129.19 (anti-CD4) and KJ16 (anti-TCR V38. 1+8.2), followed by FITC-labeled goat anti-rat immunoglobulin and analyzed on a FACS. Controls with the fluorescent conjugate alone are shown as dotted lines. The FACS profile of one representative example from each group is shown. (A) Negative littermate (128.6 x C57BL/6)F1; H-2bb; MIsh; (B) Transgenic (128.6 x C57BL/6)FI; H-2bb Mlsb. (C) Negative littermate (128.6 x DBA/2)F1; H-2bd: Mlsab. (D) Transgenic (128.6 x DBA/2)FI; H-2Kd; Mlsah.
The proportion of Thy 1.2+ lymph node cells is decreased in V,B8. 1-transgenic Misa mice To examine peripheral T cells from transgenic mice, lymph node cells were stained with Thy 1.2, CD4, CD8, TCR V36, Vfl8 or V(31 1 specific mAb. Sample fluorescence histograms for the CD8, CD4 and TCR Vf8 staining are shown in Figure 4 and the complete data are summarized in Table II. About 50-70% of total lymph node cells isolated from transgenic C57BL/6 (Mlsb) offspring and from control negative littermates were stained with the Thy 1.2 specific mAb. In marked contrast, only 20-25% of total lymph node cells derived from transgenic DBA/2 (Mlsa) offspring were Thy 1.2+. This result correlated well with our finding that the generation of mature T cells was inhibited in the thymus of these mice. In transgenic C57BL/6 offspring, a large population (-60 %) of total lymph node cells reacted with the mnAb KJ16. The average KJ16 staining intensity of transgenic T cells in these mice was slightly reduced when compared to normal T cells.
Table II. FACS analysis of lymph node cells from transgenic mice and negative litterrnates H-2
Mice
(128.6 (128.6 (128.6 (128.6
x x
x x
C57BL/6)FI C57BL/6)FI DBA/2)FI DBA/2)FI
b/b b/b b/d b/d
Mls
b/b b/b b/a b/a
Transgene
+ +
% of lymph node cell stained with mAba H-129.19 53.6.72 30-H12 anti-IgM CD8 CD4 B cells Thy 1.2
60 66 51 21
10 14 3 3
34 12 31 ± 12 31 ±2 52 2
33 ±8 23 5 39 3 9 2
28 42 12 13
5 10 1 5
KJ16 Vf38.1+8.2 7 60 6 17
3 6 2 6
44.22.1 V,B6
F23.2 V38.2
KTIl
2 6 1 1 1 ± 1 1 1
4 1 4 1
1 1 1 1
1.7 0.4 n.d. n.d.
VOIll
aTotal lymph node cells were incubated with the indicated mAb, followed by FITC-labeled goat anti-rat immunoglobulin or FITC-labeled avidin as described in Materials and methods and analyzed by flow microfluorometry. The average percentages + SEM (after subtraction of staining with the fluorescent conjugate alone) are shown based on the analysis of at least three separate mice per group.
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To distinguish between V38. 1 and V/8.2+ T cells, we used mAb F23.2 (V/8.2) together with mAb KJ 16 (V38.1 + 8.2) (Kappler et al., 1988). As shown in Table II, mAb F23.2 did not stain lymph node cells from transgenic offspring. This indicated that all KJ16+ T cells were expressing V,B8. 1. By two-colored fluorescence, we observed a slightly more frequent Vf38.1 usage on CD8+ (93%) compared to CD4+ (84%) peripheral T cells in transgenic C57BL/6 offspring. Most of the CD8+ T cells (87%) and -40% of the few remaining CD4+ T cells were V,B8. 1+ in DBA/2 offspring. These results indicated that some V38. 1 T cells must have survived tolerance induction in Mlsab, H-2bd mice. This may have resulted from pairing with a TCR a chain which did not allow Mlsa recognition. The transgenic TCR,/ chain influences the CD4/CD8 ratio of T cells T cells have been divided into two categories based on their
expression of CD4 and CD8 molecules. Generally, cytotoxic T cells are MHC class I restricted and CD8+, whereas helper T cells recognize antigen in association with MHC class II molecules and are CD4+. Since the transgenic ,B chain was derived from a H-2Db restricted, CD8+ cytotoxic T cell, it was of interest to determine the ratio of CD4/CD8 T cells in these transgenic mice. To analyze the effect of the transgene, the CD4/CD8 values of the transgenic mice were always compared with those of transgene-negative littermates. In transgenic C57BL/6 offspring, we found a Table III. Expression of TCR V,B8. 1 by subsets of T cells of transgenic mice Mice
% of total % of population population lymph node cells stained with KJ16
T cell
(128.6 x C57BL/6)Fl CD8 CD4 MlSb/b (128.6 x DBA/2)F1 CD8
Mlsb/a
CD4
93 ± 2 84 i 3 87 I1 38 15
45 i 11 22 ± 7 18 ± 6 8i 1
Lymph node cells were stained with mAb KJ16 (anti-V38.1+8.2) and goat anti-rat IgG-FITC. The staining was followed by biotinylated mAb YTS 169.4 (anti-CD8) and mAb 191.1 (anti-CD4), and finally phycoerythrin-conjugated avidin. The percentage of F23.2 + (TCR V,B8.2) lymph node cells was 1 i 1 %. The average percentages 4 SEM established by FACS analysis (after subtraction of staining with the fluorescence conjugate alone) of two separate mice per group are shown.
decrease of the CD4/CD8 ratio of peripheral T cells from 1.2 to 0.6 (Table II). A similar analysis of transgenic DBA/2 offspring revealed a marked reduction of the percentage of CD4+ lymph node cells from -40% to 10% (Table II); the percentage of CD8 ± T cells was not changed in transgenic Mlsa mice. This resulted in a considerable decrease of the CD4/CD8 ratio from 3.2 to 0.7 in transgenic Mlsa mice. The CD4 and CD8 staining intensity of transgenic versus normal T cells was the same, independent of the Mls type of the mouse (Figure 4). To examine the influence of MHC and Mls on the skewing of the CD4/CD8 ratio, transgenic H-2k,Mlsb and H-2, Mlsa mice were included in the analysis. They were obtained by crossing the transgenic line 128.6 (H-2b,Mlsb) with B10.BR (H-2k,Mlsb) and DBA/2 (H-2d.Mlsa) mice and selecting transgenic offsprings that were homozygous at the MHC. For this analysis cortisone-resistant thymocytes (CRT) were used as a source of mature T cells to avoid any postthymic effects such as preferential expansion of T cell subsets in the periphery. Surprisingly, we observed in all transgenic mice a strong predominance of CD8+ CRT independent of MHC and Mls type (Table IV). Since the sizes of the thymi and the overall numbers of CRT were comparable, these results suggest that the transgenic TCR / chain selectively favored the maturation of CD8+ T cells in the thymus apparently in a non-MHC-restricted fashion. Influence of the transgene on TCR , chain expression Our observation that V/6 +, V'/1 + and V/8.2 + (Table II) T cells were not found amongst transgenic T cells suggested that the expression of endogenous / chains was suppressed. This result was confirmed by Northern blot analysis: we found that the transgenic / chain transcript was slightly longer than endogenous / chain transcripts due to the truncated / globin gene, which was incorporated in the transgene. Therefore, we were able to distinguish endogenous and transgenic transcripts on a gel using a C/ probe. As shown in Figure 5, long transgenic transcripts were detected exclusively in the spleens of transgenic mice 128.6 and 132.2 mice.
The functional activity of T cells in transgenic mice As shown above, one TCR /3 chain formed the antigen receptor virtually exclusively together with endogenous a chains in most T cells of the transgenic line 128. The antigen repertoire of these mice was examined as follows.
Table IV. CD4/CD8 ratio of cortisone-resistant thymocytes in transgenic mice Mice
(128.6 x C57BL/6)Fl x C57BL/6
(128.6 x BlO.BR)Fl x BlO.BR (128.6 x DBA/2)Fl x DBA/2
H-2
b/b b/b k/k k/k d/d d/d
MIs
b/b b/b b/b b/b
Transgenic
+ +
a/a/-
+
% of CRT stained with mAba H-129.19 53.6.72 CD4 CD8 61 28 71 22 66 40
± 3
±3 i 4 6 i 7 ± 8
21 i 3 54 3 19 ± 3 65 8 ± 19 3 37 8
KJ16
CD4/CD8 ratio
Vf8 10 i 2 92 2 21 ± 2 94 1 ± 16 3 74 10
2.9 0.5 3.7 0.3 3.4 1.0
aCortisone-resistant thymocytes (CRT) were obtained 48 h after a single injection of 4 mg hydrocortisone acetate. The cells were stained by indirect immunofluorescence with the indicated mAb and analyzed by flow microfluorometry. H-2 typing was done by immunofluorescence. The average percentages ± SEM (after subtraction of staining with the fluorescent conjugate alone) are shown based on the analysis of three separate mice per group.
722
T cell tolerance in T cell receptor 3 transgenic mice
First, we assayed the cytotoxic T cell response against allogeneic target cells in vitro. Spleen cells from transgenic and negative littermates were sensitized in vitro with allogeneic BlO.BR (H-2k) spleen cells and assayed after 5-6 days on BW 5147 (H-2k) target cells. The cytotoxic responses of transgenic mice derived from C57BL/6 matings were normal and comparable to those of non-transgenic controls (Figure 6A). The lysis of syngeneic EL-4 target cells was always < 5 %. The cytolytic activity of transgenic T cells could be completely blocked with the anti-V38-specific mAb F23. 1 (Figure 6B). The same antibodies did not affect the activity of effector T cells derived from non-transgenic mixed lymphocyte cultures. This result demonstrated that the transgenic f chain was part of the functional TCR recognizing H-2k target cells and also confirmed our observation that endogenous f chain did not significantly contribute to the effector T cell pool. In contrast to negative of DBA/2 mice did not littermates, transgenic generate an alloreactive H-2 -specific response (Figure 6A, lower panel). However, the response was partially restored by increasing the initial number of responder cells in the mixed lymphocyte reaction or by adding Con A conditioned T cell supernatant to the culture media (not shown). Second, we examined the ability of these transgenic mice to mount a MHC-restricted cytotoxic T cell response against
viral antigens in vivo. As shown in Figure 7A, transgenic mice from C57BL/6 matings and all negative control littermates were able to generate vaccinia-virus-specific cytotoxic T cells. In marked contrast, spleen cells from vaccinia-primed transgenic DBA/2 offspring did not show significant virus-specific cytolytic activity. Third, we tested the strictly T-cell-dependent neutralizing IgG response against vesicular stomatitis virus (VSV). Transgenic mice from both C57BL/6 and DBA/2 matings mounted a normal T-cell-independent IgM response to VSV (not shown). Surprisingly, transgenic MlSa mice generated a neutralizing anti-VSV IgG response within normal ranges
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Fig. 5. Transcription of TCR 3 chain genes in transgenic mice. Total RNA was prepared from spleen cells of transgenic mice 128.10, 132.7 and of normal C57BL/6 mice, separated on a 1% agarose gel using glyoxal and transferred to nylon filters. The blot was hybridized with a cDNA probe containing Vf8 and C32 segments as described in Materials and methods.
Fig. 6. Induction of alloreactive cytotoxic T cells in transgenic mice in vitro. (A). The indicated number of responder spleen cells from transgenic (128.6 x C57BL/6)F1. -A-; (128.6 x DBA/2)FI, V -), were - -; and from negative control littermates (- -A stimulated with 1 irradiated (2000 rad) BlO.BR (H-2k) spleen cells in vitro and assayed after 5 days for cytolytic activity against BW 5147 (H-2k) target cells. Lysis of EL-4 (H-2b) target cells was c5%; spontaneous release was 10- 15%. (B) Inhibition of alloreactive cytolytic T cell activity by TCR V38-specific mAbs. Cultures from transgenic mice (128.6 x C57BL/6(F1 (-A-) and negative littermates (- A -) were assayed for cytolytic activity against BW 5147 (H-2k) target cells in the presence of the indicated concentration of F23.1 mAb.
723
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20'48 10'24 5'12
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TIME AFTER VSV INFECTION (DAYS)
Fig. 7. Induction of vaccinia-virus-specific cytotoxic T cells in in vivo (A) and of a vesicular stomatitis virus (VSV)-specific humoral response (B) in transgenic mice. (A) Transgenic offspring from C57BL/6 (-A-) and DBA/2 (-7-) matings, and negative littermate control mice (- A -, - V -) were immunized with vaccinia virus i.v. (2 x 106 p.f.u./mouse). After 6 days the cytolytic activity of spleen cells was determined on vaccinia-virus-infected MC57G (H-2b) fibroblast cells. Spontaneous release was 10-15%. (B) Transgenic offspring of C57BL/6 (-A-) and DBA/2 (-V-) matings, and negative littermate controls (-A -, -V -) were injected on day 0 with 106 p.f.u. of VSV and bled on days 8 and 12. Anti-VSV neutralizing IgG titers were determined with a standard neutralization assay as described previously (Charan and Zinkernagel, 1986). Results were derived from two or three individual mice per group; maximum ranges were a 1 2-fold dilution step.
(Figure 7B). These results also demonstrated that the transgene did not affect B cell function.
Discussion We have injected a chimeric gene consisting of a class I (V,B8. 1) cDNA and IgH promoter, T cell receptor enhancer into embryos of mice. The cloned DNA was incorporated into the germline and inherited in a normal Mendelian fashion. Expression of the transgenic TCR chain and its influence on the T cell subset was analyzed in the context of the recently established correlation between MHC-Mlsa and TCR V(8.1 expression (Kappler et al., 1988). The IgH enhancer functions in T cells of transgenic mice
We observed that offspring from four out of four founder mice carrying 5-20 copies of construct B (with IgH enhancer, Figure 1) expressed the transgenic (3 chain on most T cells. Surprisingly, offspring from nine out of ten founder mice harboring the identical construct without the IgH enhancer did not reveal detectable expression. Therefore, we conclude 724
that the DNA fragment containing the IgH enhancer core and 5' and 3' flanking sequences strongly increased the probability of transgenic TCR ( chain expression in T cells. It has been shown for the immunoglobulin genes that the IgH enhancer element is only one of the elements conferring tissue specificity (Grosschedl and Baltimore, 1985). Other experiments have demonstrated that in immunoglobulin It transgenic mice, mature T and B cells transcribed the It-gene almost equally (Grosschedl et al., 1984). Therefore, it is very likely that as well as B cells, T cells also contain specific trans-acting factors required for it-gene activation. Thus, it is possible that in our transgenic mice the IgH enhancer element associated with the transgene was capable of counteracting negative chromosomal position effects. Thymocytes expressing the a/5 TCR at a high density are deleted in transgenic Mlsa mice Our experimental approach is based on the development of transgenic mice expressing the TCR variable region Vf8. 1 on a large fraction of their T cells and on the discovery that T-cell-bearing receptors with the variable region V,B8. 1 are reactive with Mlsa-encoded antigens (Kappler et al., 1988). FACS analysis with V38- and CD3-specific mAb revealed that mature thymocytes expressing the afi TCR at a high density were almost completely eliminated in transgenic Mls5nmice, whereas the population of immature thymocytes with lower TCR density was not affected. This suggested that tolerance to Mlsa in these transgenic mice occurred at a point of transition between immature and mature thymocytes. These data are compatible with the findings in unmanipulated animals (Kappler et al., 1987, 1988), with the extension that the whole T cell population and not only a subset of cells could be examined in transgenic mice. They also agree with studies using V,B6-specific mAb in Mlsa mice, which showed that tolerance to Mlsa was signaled by absence of V,B6+ T cells on CD4 and CD8 single positive cells (MacDonald et al., 1988a). They differ, however, from the very recently reported experiments with transgenic mice, which expressed a distinct ao3 TCR specific for the male (HY) antigen (Kisielow et al., 1988). These authors observed that the male transgenic thymus was severely depleted of double positive CD4+ CD8+ cells. In our case, the size of the thymus, the number of thymocytes and their CD4 and CD8 expression was within normal ranges in both transgenic
and normal Mlsa mice. The expression pattern of the ca3 TCR on thymocytes in V,(8.1 transgenic Mlsb mice assayed with an anti-CD3 mAb was comparable to unmanipulated mice with a bright (10-15%) and a dull CD3+ subset (-50%) (Bluestone et al., 1987; Richie et al., 1988). In contrast, female thymocytes of the reported aoj-HY transgenic mice exhibited an unusual distribution of TCR since >95% of the cells were stained with CD3- and Vf38-specific mAb. One may therefore argue that the deletion of the CD4+ CD8+ thymocyte subset in male aof-HY transgenics, but not in V(8. 1-Mlsa transgenics was due to the differing pattern of receptor expression. The varying receptor distribution could be explained by the nature of the transgene: in TCRao4-HY transgenic mice already functional a and a TCR genes were introduced, whereas in V(8. 1 transgenic mice, the expression of the TCR was dependent upon the functional rearrangement of endogenous a chain genes. In addition, transgenic receptor expression was set under control of different promoters (a MHC class I promoter in the case
T cell tolerance in T cell receptor f transgenic mice
of TCR V,38.1 and its own a: TCR promoter in the case of HY transgenic mice). T cells in MIsb mice
Several groups have recently introduced a functionally rearranged genomic TCR(3 gene (V(8.2) into the germline of mice (Fenton et al., 1988; Sha et al., 1988; Uematsu et al., 1988). The transgene was expressed on most peripheral T lymphocytes and in two reports (Sha et al., 1988; Uematsu et al., 1988) did not influence the ratio of CD4/CD8 T cells, whereas in one study (Fenton et al., 1988) the CD4/ CD8 ratio was reversed. Our own results showed that the transgenic TCR ( chain (V38. 1) clearly skewed the CD4/CD8 ratio of mature T cells towards CD8+ to a similar extent to that observed in ao3 TCR transgenic mice (Sha et al., 1988; Teh et al., 1988) (Table IV). However, the predominance of the CD8+ T cell subset in transgenic mice was independent of the MHC or Mls type of the mice. Since the transgenic (3 chain was derived from a class I (H-2Db) restricted CD8 + T cell clone, these findings suggest that the TCR ( chain studied bears an inherent affinity for class I molecules which then leads to a preferential selection of CD8+ cells in the thymus. In this context, it is noteworthy that in normal mice the CD8+ subset also exhibits a slightly more frequent V,B8 use when compared to the CD4+ subset (Roehm et al., 1984). T cells in MIsa mice The population of mature, high receptor density expressing thymocytes was significantly reduced in transgenic DBA/2 offspring (H-2bd,Mlsba). Therefore, it was of interest to examine peripheral T cells of these mice. We found a decrease of the Thy 1.2+ T cell subset. This result signaled a mechanism of T cell tolerance against MlSa in these mice: most T cells were apparently not allowed to leave the thymus because they bore the transgenic V(38.1 receptor. This resulted in the observed reduction of T cells in the periphery of transgenic Mlsa mice. The finding that the number of Thy 1+ CD8+ lymph node cells increased in these mice with age (> 3 months; not shown) supports the interpretation that permissible T cells may slowly accumulate with time in the periphery. The presence of V,B8.1+ T cells with normal CD4 and CD8 expression in transgenic DBA/2 offspring suggested that pairing of certain endogenous a chains with the transgenic ( chain may circumvent MlSa reactivity. Analyses of the Va usage in these T cells will provide more information about the influence of the a chain in this system. What is the explanation for the apparently selective deletion of the CD4 subset in these V,B8.1 transgenic Mlsa mice? It has been shown that Mlsa is mainly recognized by CD4+ T cells (Janeway et al., 1980). Therefore, one may argue that it is principally the CD4 subset which has to be eliminated in these mice to maintain tolerance. This interpretation would imply that negative selection does not occur at the double positive CD4+ CD8+ state of thymocyte differentiation in the transgenic mice examined (Fowlkes et al., 1988; MacDonald et al., 1988b). This would be in contrast to the fact that both CD4+ and CD8 + T cells expressing V,B8.1 were deleted in normal Mlsa mice (Kappler et al., 1988). Alternatively, one may argue that both CD4+ and CD8+ subsets were equally affected by negative selection due to Mlsa; however, the CD8+ subset
was restored by the observed skewing of the CD4/CD8 ratio towards CD8 in mice expressing the transgenic TCR 3 chain. Our results discussed in this section differ in at least two aspects from the recently reported a(3-HY transgenic males (Kisielow et al., 1988). First, we found in V,B8.1 transgenic Mlsa mice a reduction of the Thy 1.2+ T cell subset in lymph node cells, whereas the numbers of Thy 1 + lymph node cells reported from ox,-HY transgenic males were within normal ranges. Second, T cells from V,B8.1 transgenics expressed normal amounts of CD8 or CD4 per cell in contrast to the high number of double negative CD4CD8- T cells and to the low level of CD8 detected in the male af3-HY transgenics. Two main differences in the experimental system may explain these findings. First, different antigenic specificities were examined which are recognized in the context of either class I (HY) or class II (Mls). Second, the HY transgenic system is more rigid since both a and (3 chains are encoded by the transgene compared to the V,B8.1 transgenic mice used here where other variable components of the receptor (endogenous Va, Ja) could influence specificity. Endogenous A chain expression The present report does not focus on the effect of the transgene on endogenous (3 chain genes. However, three findings suggested that endogenous (3 chain genes were not expressed in the transgenic line 128. (i) V(6+, V38.2+ and V(31 1 + T cells were not found in these mice. (ii) Northern blot analysis of spleen RNA revealed no detectable endogenous (3 chain transcripts. (iii) The cytolytic activity of transgenic, alloreactive T cells could be completely blocked with V(38-specific mAb. These data agree with the reported findings that introduction of functional genomic TCR ( chain genes into the germline of mice prevented endogenous ( chain expression (Uematsu et al., 1988). However, preliminary analysis of another transgenic line, i.e. 126, revealed expression of endogenous ( chain genes. In these mice, the introduced TCR ( chain was expressed at a lower level on most T cells (not shown). The transgenic,6 chain forms a functional TCR The examined H-2b, Mlsb transgenic mice showed a normal allogeneic and vaccinia virus CTL response and a good IgG anti-VSV antibody response. This demonstrated that the same transgenic ( chain was able to form a functional TCR recognizing various MHC and viral antigens. Further experiments with a variety of antigens will show whether holes in the T cell repertoire can be found in those mice with a variability only in the a chains. Surprisingly, the CTL response, but not the T-cell-dependent IgG anti-VSV antibody response, was affected in transgenic H-2bd, Mlsab mice. The inability to generate cytotoxic T lymphocytes may be explained by a reduced T cell precursor frequency in these mice. Our observation that the alloreactive CTL response in vitro could be partially restored by increasing either the number of responder cells in the MLR or by adding Con A supernatant would be compatible with such a conclusion. Alternatively, these data may indicate that the few CD4+ T cells provided sufficient T help for the VSV response, but that the available T help may have been limiting for induction of a T-cell-mediated cytolytic response in vivo. This report and similar experiments by others (Fenton et al., 1988; Kisielow et al., 1988; Sha et al., 1988; Teh et
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al., 1988; Uematsu et al., 1988) reflect the enormous analytical possibilities of TCR transgenic mice in investigating basic immunological questions. During the past few years, TCR cDNA genes from several characterized T cells have been cloned. Therefore, the described expression vector (construct B), which allows expression of cDNA in lymphoid cells at a high frequency, will facilitate further studies with TCR transgenic mice. Additionally, expression of other cloned genes (cDNA) in lymphocytes of transgenic mnice can be envisaged.
Materials and methods Plasmid construction The isolation and characterization of the TCR chain cDNA clone of the T cell line P14 has been described previously (Pircher et al., 1987a). To eliminate the AATAAA polyadenylation signal of the cDNA insert, - 150 bp of the 3' untranslated region were removed at the HindII site. XhoI and sites were introduced at the 5' and 3' ends, respectively, of the TCR,3 cDNA. The single HindlIl site in the polylinker of the pUC H2 plasmid (a kind gift of Dr E.Wagner, EMBL, Heidelberg, FRG), which contained the HindIII-Nrul 2 kb fragment of the H-2Kb promoter (Kimura et al., 1986), was converted into a XAoI site. The 1.7 kb BamHI-BglII fragment of the 3' half (BamHI site to 3' end) from the human adult 3-globin gene (Lawn et al., 1980) was subcloned into the polylinker BamHI site of the plasmid that contained the /3-globin gene was pUC H2 plasmid. The a gift of Dr S.Hedrick (University of California, San Diego, CA). Finally, the second XhoI site was introduced in the plasmid at the 3' end of the fglobin fragment (pUC H2XXS). The modified P14 $ cDNA was then cloned into the Sal - BamHI sites of pUC H2XXS and vector sequences were completely removed by XhoI digestion (construct A). Construct B was generated by subcloning the XhoI fragment of construct A into the polylinker Sall site of the plasmid pME-13 (H.Pircher, unpublished), which contained the 1.5 kb EcoRI-HindIII fragment of the murine IgH enhancer (Banerji et al., 1983; Gillies et al., 1983). The construct was then excised from this plasmid by cleavage at the XhoI and SnaI sites in the polylinkers.
BamHI
pBe-
Transgenic mice The inserts were separated from the vector by preparative agarose gel electrophoresis and elution from DE81 ion-exchange paper (Whatman Ltd, Maidstone, UK). The DNA was extracted twice with phenol -chloroform and chloroform and precipitated with ethanol. The DNA was diluted in 10 mM Tris-HCI (pH 7.5), 0.1 mM EDTA at a final concentration of 100 ng/pl. Approximately 500 molecules were injected into the male pronucleus of fertilized eggs derived from (C57BL/6 x DBA/2)F2 hybrids following the procedure described by Brinster et al. (1981) and Hogan et al. (1986). Surviving microinjected eggs were transferred into the oviducts of pseudopregnant outbred CDl foster females. Positive mice were identified by tail blot hybridization using the P14,B cDNA insert as a probe.
Immunofluorescence The following mAbs were used in these experiments: KJ 16, rat IgG, specific for V38. 1 and V38.2 (Haskins et al., 1984); 30-H 12, biotinylated mouse IgG, specific for Thy 1.2 (Becton Dickinson, Mountain View, CA); 53.6.72 and YTS 169.4, rat IgG specific for murine CD8 (Ledbetter and Herzenberg, 1979; Cobbold et al., 1984); H129.19 and YTS 191.1, rat IgG specific for murine CD4 (Cobbold et al., 1984; Pierres et al., 1984); 44.22.1 rat IgG specific fpr V36 (Acha-Orbea et al., 1983); KTI 1, rat IgG specific for VI3 1I (a kind gift of Dr K.Tomonari, Transplantation Biology Section, MRC, Middlesex UK); F23. 1, mouse IgG specific for Vf8 (Staerz et al., 1985); F23.2, mouse IgG specific for V,B8.2 (Staerz et al., 1985); 145.2Cl 1, hamster IgG specific for murine CD3 (Leo et al., 1987); 15.1.5 and 15.5.5 mouse IgG specific for H-2kd (Ozato et al., 1980); B8.24.3, mouse IgG specific for H-2Kb (Kohler et al., 1981). Single cell suspensions were prepared from lymph nodes and thymus, resuspended in PBS containing 1% bovine serum albumin and 0.2 % NaN3, and then were incubated with hybridoma supernatant (1: 1) of the various mAbs at room temperature for 30 min (mAb KJ 16 was incubated at 37°C). Cells were washed twice and incubated again with fluorescein isothiocyanate (FITC)-coupled goat anti-rat immunoglobulin antibodies (TAGO, Burlingame, CA) or phycoerythrin (PE) labeled goat anti-mouse IgG antibodies (Southern Biotechnology Birmingham AL). Fluorescence-activated cell sorter (FACS) analysis was performed on an Epics Profile Analyzer.
Generation of cytotoxic T cells in vitro and in vivo and induction of VSV-specific antibodies Spleen cells (5 x 106, 1.7 x 106, 6 x 105) were cultured with 107 X-irradiated (2000 rad) allogeneic stimulator spleen cells in 1.5 ml culture medium. After 5 days, viable cells were recovered, counted and tested on 5tCr-labeled target cells in a 3-h assay as described elsewhere (Acha-Orbea et al., 1983). The results are presented as specific lysis = [(exp. release spontaneous release)/(total release-spontaneous release)] x 100. Vaccinia-virus-specific T cells were generated by i.v. injection of 2 x 106 p.f.u./mouse vaccinia virus (Lancy isolate, Schweiz, Serum & Impfinstitut, Bern). After 6 days, spleen cells were assayed against vacciniavirus-infected (5 p.f.u./cell) fibroblast MC57G target cells in a 5-h assay. VSV-specific antibodies were generated by injection of 106 p.f.u. VSV-IND (Mudd-Summers isolate) i.v. into mice. Neutralizing IgM and IgG titers (resistant to ,B-mercaptoethanol) were determined in a plaque-inhibition assay as described (Charan and Zinkernagel, 1986).
Acknowledgements We thank M.Condrau and R.Schneider for technical advice on the FACS and Beatrice Borter and Susi Grossman for secretarial help, and acknowledge the technical assistance of U.Steinmann, S.Cooper and M.Vollenweider. We thank A.Althage and Dr M.Schulz for reviewing the manuscript. H.P.P. obtained his postdoctoral fellowship at the initial phase of the project from the Swiss National Foundation. This work was supported by SNF grants 3.295-0.85 and 3.213-0.85, The Radiumstiftung Zurich, the National Cancer Institute of Canada, The Medical Research Council of Canada, Natural Science and Engineering Research Council of Canada and the Kanton of
Zurich. DNA and RNA isolation For the isolation of tail DNA, -1 cm of the tail was cut off, the bone was removed and the skin was chopped into small pieces with a razor blade. ,ul of 0.1 M EDTA, The skin was then incubated overnight at 55°C in 700 0.05 M Tris-HCI (pH 8), 1% SDS and 500 Ag/ml proteinase K. The resulting homogenate was centrifuged and the DNA was isolated by phenol -chloroform extraction and ethanol precipitation. Total RNA was prepared by the guanidium isothiocyanate extraction procedure (Chirgwin et al., 1979) and purified on CsCl gradients (Glisin et al., 1974). DNA and RNA analysis For Southern blot analysis, 20ug of total genomic tail DNA was digested with EcoRI, separated on agarose gel and transferred to Biodyne membranes (PALL, Glen Cove, NY). Total RNA (20yg) isolated from spleen cells was separated on 1 % agarose glyoxal gels (Maniatis et al., 1982) and transferred to Biodyne membranes. Filters were prehybridized with 50% formamide,S x SSC, 0.05 M KH2PO4 (pH 6.5), 5 x Denhardt's, 1% SDS and 500 itg/ml salmon sperm DNA and hybridized overnight to the 32P-oligolabeled P14 fi cDNA insert probe in 50% formamide, 5 x SSC, 0.02 M KH2PO4, 5 x Denhardt's,1 % SDS and 100 ILg/ml salmon sperm DNA. The filters were washed twice in 2 x SSC at room temperature and for30 min at S0C in0.3 x SSC.
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Received on November 14, 1988
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