Hiroaki Takimoto*A, Takao NakamuraA, Makoto Takeuchi*, Yukiko Sumi+, Toshiyuki Tanaka., Kikuo Nomoto. and Yasunobu Yoshikai+
Age-associated increase in number of CD4+CDS+ intestinal intraepithelial lymphocytes in rats*
Laboratory of Germfree Life*, Research Institute for Disease Mechanisms and Control, Nagoya University School of Medicine, Nagoya, Department of ImmunologyA, Medical Institute of Bioregulation, Kyushu University, Fukuoka and Department of Immunology., The Tokyo Metropolitan Institute of Medical Science, Tokyo
A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344.The site of the CD4+CD8+population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+IEL and some of CD8+ IEL in aged rats were of CD5+CD45RW phenotype. In germ-free Wistar rats, age-associated increase in the number of CD4+CD8+CDSf IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CDSfCD45RB- IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD4+CD8+thymocytes bearing a low intensity of CD3/T cell receptor (TcR) alp, the CD4+CD8+T cells in IEL expressed a high intensity of CD3/TcRu//3 on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the E L in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8' IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in thk epithelium.
1 Introduction Intestinal intraepithelial lymphocytes (IEL) represent a unique population expressing CD8 and able to exhibit non-MHC restricted cytolytic activity [1-7].The CD8+ IEL in mice consist of heterogeneous populations bearing or not Thy-1 [l],TcRy/6 or a/p [ 3 , 4, 6, 71, and the CD8u homodimer or the CD8 alp heterodimer [2]. Athymic nude mice contain a significant number of IEL expressing TcR y/6, suggesting that murine IEL expressing TcR y/6 may develop along an extrathymic pathway [ 8 , 91. Furthermore, there has been convincing evidence that a significant fraction of IEL bearing TcRulp also develop outside thymus. TcR u/@+IEL bearing the CD8 u homodimer have been reported to be detected in nude mice, scid mice and irradiated and thymectomized mice repopulated by T celldepleted BM cells [lo]. Rocha et al. have found that the forbidden T cell clones bearing TcR a/@capable of recognizing the self-superantigens, which are normally deleted in the thymus, are detected in the IEL bearing the CD8u
[I 96801
*
This work was supported in part by grants to Y.Y. from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, Sapporo Bioscience Foundation and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
Correspondence: Yasunobu Yoshikai, Laboratory of Germ-free Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya 466, Japan Abbreviations: IEL: Mesenteric LN
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Eur. J. Immunol. 1992. 22: 159-164
Intraepithelial
lymphocytes
MLN:
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
homodimer [11]. Thus, TcRy/6 IEL and TcRu/p IEL bearing the CD8u homodimer can be considered as thymus-independent populations. On the other hand, TcR a/p IEL bearing the CD8 a/@heterodimer and a small minority of CD4+ IEL are thought to be thymus-dependent populations derived from dividing precursors in Peyer's patches under antigenic stimulation [ 121. Recently, a unique population of CD4+CD8+T cells bearing TcR u/p has been detected in both murine IEL and rat IEL [13, 141. Although the CD4+CD8+IEL are thought to be involved in the epithelial immunity, the characteristics of the unique population in IEL, including their origin and functions remain obscure. Unlike murine IEL, most rat IEL expressTcR u/p [15].The fact that athymic nude rats contain noTcR u/@IEL suggests that rat IEL may be exclusively thymus dependent [15, 161. In the present study, we have characterized the IEL in rats with different genetic backgrounds and different ages and found that there was an age-related increase in the number of CD4"CD8+ IEL, irrespective of their genetic backgrounds. The CD4+CD8+ IEL, which were of the CD3/TcR u/phlgh,CD5+ and CD45RB- phenotype, appear to proliferate spontaneously. The unique population was not detected either in the IEL of aged nude rats or in those of aged germ-free rats. The implication of these findings for the roles of CD4+CD8+ Tcells in IEL in epithelial immunity are discussed.
2 Materials and methods 2.1 Animals WKA/Sea, BN/Sea and LEW/Sea rats were obtained from Seiwa Experimental Animal Institute (Fukuoka, Japan). ACI/N Jcl rats were obtained from Clea Japan, Inc. (Tokyo,
+
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Japan). F344/Bu Crj rats were obtained from Charles River Japan, Inc. (Atsugi, Japan) and athymic nude rats (F344/N nu/nu) were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine. Germfree Wistar rats and conventional Wistar rats were maintained in our laboratory.
2.2 Preparation of lymphocytes IEL were prepared according to a modification of a method of Cerf-Bensussan et al. [17]. Briefly, small intestine was flushed with SO ml of PBS, fat and Peyer's patches were removed.The intestine was opened longitudinally, it was cut into 2-cm pieces, and then washed again in PBS.The pieces were stirred in 199 medium (Gibco, Grand Island, NY) supplemented with 20% Nu serum (Collaborative Research Inc., Bedford, MA) and 1mM dithioerythritol (Sigma Chemical Co., St. Louis, MO) at 37°C for 20 min and then spun down. The pieces were resuspended in RPMI 1640 (Gibco) supplemented with 20% Nu serum and agitated in a 37°C for 20 min, and then debris were removed. This cell suspension was centrifuged, resuspended in 8 ml of 44% Percoll (Sigma), and layered on 5 ml of a 67.5% Percoll solution.The gradient was centrifuged at 600 x g at 20°C for 20 min. Lymphocytes at the interface were harvested, and washed twice with HBSS.
2.3 Antibodies The following mAb were used in this work: PE-conjugated W3/25 (anti-rat CD4 mAb), FITC-conjugated OX8 (antirat CD8 mAb), PE-conjugated OX19 (anti-rat CD5 mAb), FITC-conjugated OX22 (anti-rat CD45RE3 mAb), FITCconjugated OX12 (anti-rat 3t chain mAb; Serotec, Oxford, GB), FITC-conjugated or biotinylated R73 (anti-rat TcRa/P mAb; kindly provided by Dr. T. Hiinig [HI, biotin-conjugated 1F4 (anti-rat CD3 mAb, [19]).They were conjugated with FITC or biotin according to standard procedures. Secondary antibodies were PE-conjugated anti-mouse IgM, biotinylated anti-mouse IgM, biotinylated anti-mouse IgG (Caltag Laboratories, San Francisco, CA). streptavidin-PE and streptavidin-Duochrom was obtained from Becton Dickinson (San Jose, CA).
2.4 FCM analysis For two-color FCM analysis were stained with FITCconjugated mAb and PE-conjugated mAb or biotinylated mAb followed by streptavidin-PE. For three-color FCM analysis were stained with FITC-conjugated mAb, PEconjugated mAb, and biotinylated mAb followed by streptavidin-DuoChrom. The lymphocytes were analyzed on a FACScan flow cytometer (Becton Dickinson).
2.5 MLR IEL (1 x lo5) from 4-week-old or 30-week-old WKA rats were cultured with irradiated (33 Gy) syngeneic or allogeneic (ACI rats) spleen cells (1 x lo5) in flat-bottom microtiter plates. After 3 , 5 or 7 days of culture, 37 kBq of tritiated thymidine was added for the final 6 h and the cultures were harvested onto filters and counted.
Eur. J. Immunol. 1992. 22: 159-164
3 Results and discussion 3.1 Cell surface phenotype of IEL in young and aged rats of various strains IEL were characterized phenotypically by FCM with anti-CD4 mAb and anti-CD8 mAb. As shown in Fig. l a , in young rats (4-5 weeks of age), IEL were composed of a large number of CD8+ cells, CD4-CD8- cells and only a few CD4+ cells in Wistar, F344, WKA, BN and LEW rats. CD4+CD8+cells were hardly detected in IEL at this stage. An appreciable number of CD4+ IEL and CD4+CD8+IEL could be detected 10 weeks after birth in most strains. In aged rats (6 months old), the number of CD4+CD8+ IEL increased to a considerable level in all strains examined. Conversely, the proportions of CD8+ cells and CD4-CD8cells decreased in the IEL of the aged rats.The CD4+CD8+ cells were hardly detected in the peripheral lymphoid tissues such as spleen and mesenteric LN, Peyer's patch or liver (data not shown). Next we examined age-related changes in the expression of theTcR/CD3 complex on rat IEL. IEL were double-stained with anti-CD3 mAb and anti-TcR ci/P mAb. Consistent with a previous report [ 141, a large number of rat IEL expressed TcR alp in all strains we examined. However, a significant number of CD3+TcRalp- IEL, presumably TcRy/6+ IEL, were also detected in the IEL of young strains, ranging from 20% to 45% of CD3+ IEL (Fig. lb). The number of CD3+TcRa/p- IEL decreased with aging. This subset was hardly detected at any stage in the peripheral lymphoid tissues (data not shown). The intensity of the CD3EcR complex was compared between IEL and thymocytes. Thymocytes are divided into three groups as accessed by the intensity of the TcR/CD3 complex; CD3/TcR-, CD3/TcR1OW and CD3/TcRhigh[20, 211. Approximately half of CD4+CD8+ thymocytes are known to express low amounts of the TcR/CD3 complex but an appreciable number of T c R / C D ~ thymocytes ~'~~ is also present in the CD4+CD8+population in mice [22]. Consistent with these reports, CD4+CD8+ thymocytes of young rats contained not only a high proportion of TcR/CD3loWcells but also an appreciable level of T c R / C D ~ ~ ' cells. ~~ The CD4+CD8+CD3highthymocytes expressed a lower intensity of CD8 than CD8+ thymocytes. In terms of relative amounts of TcR surface expression, the CD4+CD8+IEL of aged rats expressed a high intensity of TcR/CD3 complex, similar to some fractions of CD4+CD8+ thymocytes and the single-positive T cells in thymus and the peripheral lymphoid tissues. CD5 is known to be a panT cell marker [23].To examine the expression of CD5 on IEL, we stained IEL with anti-CD5 mAb and anti-CD3 mAb. As shown in Fig. 2a, most of CD3 cells in MLN expressed CD5, while CD5+ cells were hardly detected in the IEL of young rats of all strains. The CD5+ IEL increased gradually to a substantial level by 6 months after birth. To determine whether the CD4+CD8+ IEL in aged rats expressed CD5, we examined the IEL in aged rats by three-color staining with antLCD5 mAb, anti-CD4 mAb and anti-CD8 mAb. As shown in Fig. 2b, all CD4+CD8+ cells expressed the CDS molecule, whereas most CD4-CD8+ cells did not express CD5 in aged rats. In terms of relative expression of CD5 molecule, CD4+CD8+ IEL expressed lower amounts of CD5 than CD4+ IEL, all of
CD4+CD8+ IEL in rat
Eur. J. Immunol. 1992. 22: 159-164
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Figure I . Expression of CD4 and CD8 of theTcR/CD3 complex on IEL of various rat strains. (a) IEL were stained with FITC-conjugated anti-CD4 mAb (W3/25) and PE-conjugated anti-CD8 mAb (0x8).(b) IEL were stained with anti-rat CD3 mAb (1F4) followed by PE-conjugated anti-mouse IgM, and stained with FITC-conjugated anti-TcR a/fi mAb (R73). Analysis was performed on a FACScan. Results are from one of three similar experiments.
which expressed high amounts of CD5. CD45RE3 expression determined by OX22 mAb, a marker of virgin Tcells [24], was also examined in the IEL. In young rats, CD45RB+ cells were to be found CD8+ or CD4-CD8IEL. In aged rats, CD4+ IEL did not express CD45Rl3, suggesting that CD4+CD8+ IEL expressed CD45RJV (data not shown).
3.2 Absence of CD4+CD8+ IEL in aged nude rats and in aged germ-free rats Our results showed an age-related increase in the number of CD4+CD8+cells and CD5+ cells in rat IEL, irrespective of their genetic backgrounds. To investigate the origin of the CD4+CD8+IEL in aged rats, we analyzed the cell surface phenotype of the IEL in aged athymic nude F344 rats. Young nude rats contained IEL with the CD8+CD3TcR ulp- phenotype and which had large granules in their cytoplasm (data not shown). As shown in Fig. 3a, aged nude rats (12 month old), appreciable levels of CD8+ IEL and CD4+ IEL bearing CD3/TcRu/P were detected, suggesting that some IEL may be able to differentiate outside the thymus in rats. However, CD4+CDS+CDS+IEL were hardly detected in the IEL of aged nude rats. It would thus appear that CD4+CD8+CDSf IEL may be derived from Tcell precursors of thymic origin.
To determine whether the intestinal microflora plays an important role in development of the CD4+CD8+CDSf IEL in aged rats, we examined the IEL of Wistar rats bred under germ-free condition. As shown in Fig. 3b , aged germ-free rats contained a significant number of CD8+CD3+TcRu//B+but not CDSfCD4+CD8+1EL.These results suggested that stimulation with the intestinal microflora may be important for the expansion of the CD5+CD4+CD8+IEL in aged rats. Our data presented here revealed that CD4+ IEL appear before CD4+CD8+ IEL, raising the possibility that CD4+CD8+ IEL may originate from CD4+ IEL that acquire CD8. In mice, thymic y/6 cells negative for CD4 or CD8 are reported to express the CD8 cx homodimer following in vitro activation by Con A [25]. Paliard et al. reported that activation and culturing of human CD4+ T cell clones in IL 4 resulted in the acquisition of CD8 due to its de ylovo synthesis [26]. The CD4+CD8+ cells are also detected in LN and spleen after antigen or mitogen activation [27] or in the lamina propria of the intestine in rats [28]. Taken together, expression of CD8 might be induced on CD4+ IEL by external stimuli. We cannot at present define what cytokines mediate CD8 induction on the IEL. Guy-Grand et al. has recently proposed that the gut epithelium is a site that plays an attractive and a differen-
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Eur. J. Immunol. 1992. 22: 159-164
(a)
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CD5 Figure 2. Expression of CD5 on IEL of various rat strains. (a) IEL were stained with anti-CD3 mAb followed by FITC-conjugated anti-mouse IgM, and PE-conjugated anti-CD5 mAb (0x19). (b) IEL were also stained with anti-CD4 mAb followed by biotinylated anti-mouse IgM and streptavidin-DuoChrom,FITC-conjugated anti-CD8 mAb, and PE-conjugated antLCD5 mAb, and expression of CD5 was displayed after setting the gates on CD4+CD8+IEL. Results are from one of three similar experiments.
tiation-inducing role on the appearance of a significant fraction of CD8+ IEL [lo]. Therefore, alternatively precursors of IEL might differentiate into CD4+CD8+ IEL via a CD8+ stage at a local site, outside of the thymus, under the influence of the intestinal epithelial environment. Immature CD8+ Tcell precursors in the tyhmus are known t o acquire CD4 after overnight culture in medium only [29, 301. Our results, however, revealed that following overnight incubation of IEL of young rats in vitro, no CD4+CD8+ IEL were generated (data not shown). Furthermore, the CD4+CD8+ IEL were hardly detected in aged nude mice, which contained an appreciable level of CD4+ IEL and CD8+ IEL bearingTcR a/P.Taken together, it is unlikely that the CD4+CD8+IEL are generated locally in the intestine without thymic influence. Although our results with aged nude rats revealed that a significant fraction of CD4+ IEL and CD8+ IEL can develop extrathymically in rats (Fig. 3a), CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD3+CD4+CDSf thymocytes, the CD4+CD8+ IEL express a high intensity of CD3/TcRdP, similar to single-positive T cells in the peripheral lymphoid tissues. Recently, a significant fraction of CD4+CD8+ thymocytes have been reported to express a high intensity of CD3/TcR on their surface [22]. We can also assume that the CD4+CD8+ IEL may be derived from this population of the CD4+CD8+ thymocytes, which are intermediates between immature CD4+CD8+ thymocytes and mature,
single-positiveT cells. It remains, however, to be elucidated whether the CD3highCD4+CD8+thymocytes can migrate directly to the periphery including the intestine.
3.3 Spontaneous proliferation of the IEL in aged rats To elucidate the specificity and function of the CD4+CD8+ IEL, whole IEL of young or aged rats were incubated at 37 "C in RPMI 1640 medium supplemented with 10% FCS in the absence of added mitogens or growth factors, or in the presence of syngeneic APC or allogeneic APC. Surprisingly, the IEL in aged rats proliferated spontaneously on the third day of the in vitro culture (Fig. 4a, p < 0.001), while whole IEL in young rats showed little, if any, DNA synthesis at any stage in the in vitro culture. To determine the phenotype of the spontaneously proliferating cells in the IEL of aged rats, FCM analysis was carried out on the blastoid cells in the 3-day in vitro culture of the IEL. As shown in Fig. 4b, the size of the CD4+CD8+ subset significantly increased, whereas CD4+ IEL and CD4-CD8- IEL decreased conversely after 3 days of in vitro culture. O n the other hand, a shift in subpopulations of the IEL was not observed in the in vitro culture of the IEL of young rats (data not shown).
So far, the relevance of CD4 and CD8 expression of IEL to the function and specificity of the IEL remains unclear.
Eur. J. Immunol. 1992. 22: 159-164
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Figure 3. Surface phenotype of IEL from aged nude rats and aged germ-free rats. IEL were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated anti-CD4 mAb, FITC-conjugated antiTcR a / p mAb and biotinylated anti-CD3 mAb and streptavidinPE, or FITC-conjugated anti-TcR alp mAb and PE-conjugated anti-CD5 mAb. Results are from one of three similar experiments.
CD1, a family of MHC class I-like molecules, is reported to be expressed on intestinal epithelium in mice [31]. In rats IEL are also reported to start expressing MHC class XI with age [17]. CD4+CD8+ IEL may be involved in recognizing antigens in the context of MHC class I or class I1 expressed on the intestinal epithelium. The cytolytic activity of IEL is known to be regulated by externally derived stimuli via a specific functional interaction between IEL and gutassociated antigens [32].Expression of CD8 is found to mediate anti-CD3-directed cytolytic activity by the CD4+CD8+ Tcell clones. Our results showing that the CD4+CD8+IEL expressing CD5 but no CD45RB prolife-
CD8 1 1 1 1 1 1 ) Figure 4. Proliferative response of IEL from aged WKA rats upon syngeneic stimulation. (a) IEL (1 :< los) from 4-week-old (00 A) or 30-week-old (W 0 A) WKA rats were cultured with irradiated (33 Gy) syngeneic (0W) or allogeneic (ACI rats) (00 ) spleen cells (1 X lo5), or medium only (AA)in flat-bottom microtiter plates. After 3 , 5 or 7 days of culture, 37 kBq of tritiated thymidine was added for the final 6 h. (b) Surface phenotype of IEL after syngeneic stimulation. After 3 days of culture, IEL were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated antiCD4 mAb.
rate spontaneously, suggest that the CD4+CD8+ IEL may be in an activated state. The CD4+CD8+ IEL may play important roles in the first line of defense against various pathogens in the epithelium by recognizing these pathogens once presented by MHC class I and I1 and by becoming cytolytic. Further functional analysis is required to elucidate the roles of the CD4+CD8+ IEL in the epithelial immunity.
4 Concluding remarks Age-related increase in C D 4 - T D 8 + IEL was evident in rats. A unique population of CD4+CD8+ IEL expressed CD5, a high intensity of CDYTcRdP but not CD45RB.
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CD4+CD8+IEL originate from a thymus-dependent population and expand by external stimuli such as the intestinal microflora. CD4+CD8+ IEL may participate in immunosurveillance at the first line of defense in aged animals. We thank Dr. 7: Hiinig for providing the anti-rat E R d p (R73) mAb.
Received June 13, 1991; in final revised form September 23, 1991.
5 References 1 Klein, J. R., J. Exp. Med. 1986. 164: 309. 2 Parrot, D. M. V , Tait, C., Muckenzie, S., Momat, A. MCI., Davies, M. D. J. and Micklem, H. S., Ann. N X Acad. Sci. 1983. 409: 307. 3 Goodman, T. and Lefranqois, L., Nature 1988. 333: 855. 4 Bonneville, M., Janeway, C. A., Ito, K., Haser,W., Ishida, I., Nakanishi, N. and Tonegawa, S., Nature 1988. 336: 479. 5 Viney, J. L., Kilsham, I? J. and MacDonald, T. T., Eur. J. Immunol. 1990. 20: 1632. 6 Takagaki,Y, DeCloux, A., Bonneville, M. and Tonegawa, S., Nature 1989. 339: 712. 7 Goodman, T. and Lefranqois, L., J. Exp. Med. 1989. 1870: 1569. 8 Bandeira, A . , Mota-Santos, T., Itohara, S.,Degerman, S., Hawser, C. ,Tonegawa, S. and Coutinho, A . ,J. Exp. Med. 1990. 172: 239. 9 Bandeira, A , , Itohara, S., Bonneville, M., Burlen-Defranoux, O., Mota-Santos, T., Coutinho, A. and Tonegawa, S., Proc. Natl. Acad. Sci. U S A 1991. 88: 43. 10 Guy-Grand, D., Cerf-Bensussan, N., Malissen, B., MalassisSeris, M . , Briottet, C. and Vassalli, F!, J. Exp. Med. 1991. 173: 471. 11 Rocha, B.,Vassalli, P. and Guy-Grand, D., J. Exp. Med. 1991. 173: 483. 12 Guy-Grand, D., Griscelli, C. and Vassalli, I?, J. Exp. Med. 1978. 148: 1661
Eur. J. Immunol. 1992. 22: 159-164 13 Mosley, R. L., Styre, D. and Kleim, J. R., Int. Imrnunol. 1990. 2: 361. 14 Fangmann, J., Schwinzer, R. and Wonigeit, K., Eur. J. Immunol. 1991. 21: 753. 15 Vaage, J. T.,Tissen, E., Ager, A., Roberts, I., Fossum, S. and Rolstad, B., Eur. J. Irnrnunol. 1990. 20: 1193. 16 Viney, J. L., MacDonald, T. T. and Kilsham, F! J., Irnrnunol. Lett. 1990. 22: 49. 17 Cerf-Bensussan, N., Quaroni, A., Kurnik, J. T. and Bhan, A. K., J. Immunol. 1984. 132: 2244. 18 Hiinig, T., Wallng, H.-J., Hartley, J. K., Lawetzkg, A. and Tiefenthaler, G., J. Exp. Med. 1989. 169: 73. 19 Tanaka,T., Masuko,T.,Yagita, H., Tamura,T. and Hashimoto, Y , J. Immunol. 1989. 142: 2791. 20 Havran,W. L., Poenie, M., Kimura, J.,Tsien, R.,Weiss, A. and Allisan, J. €?,Nature 1987. 330: 170. 21 Shortman, K., Egerton, M., Spangrude, G. J. and Scollay, R., in Sprent, J. (Ed.), Semin. Immunol., vol. 2, W. B. Saunders, London 1990, p. 3. 22 Shortman, K.,Vremec, D. and Egerton, M., J. Exp. Med. 1991. 173: 323. 23 Ledbetter, J. A , , Evans, R. L., Lipinski, M., CunninghamRundles, C., Good, R. A. and Herzenberg, L. A.,J. Exp. Med. 1981. 153: 310. 24 Barclay, A. N., Jackson, D. I. ,Williams, A. C. and Williams, A. E , EMBO J. 1987. 6: 1259. 25 MacDonald, H. R., Schreger, M., Home, R. C. and Bron, C., Eur. J. Irnrnunol. 1990. 20: 927. 26 Paliard, X., Malefijt, R.W., DeVries, J. E. and Spits, H., Nature 1988. 335: 642. 27 Bevan, D. J. and Chisholm, I? M., Immunology 1986. 59:
64. 28 Van der Heijden, F. L., Immunology 1986. 59: 397. 29 Paterson, D. J. and Williams, A. F., J. Exp. Med. 1987. 166: 1603. 30 MacDonald, H. R., Budd, R. C. and Home, R. C., Eur. J. Irnrnunol. 1988. 18: 519. 31 Bleicher, F! A., Balk, P. S., Hagen, S. J., Blumberg, R. S., Flotte, T. J. and Terhorst, C., Science 1990. 250: 679. 32 LefranGois, L. and Goodman, T., Science 1989. 243: 1716.
Age-associated increase in number of CD4+CDS+ intestinal intraepithelial lymphocytes in rats*
Laboratory of Germfree Life*, Research Institute for Disease Mechanisms and Control, Nagoya University School of Medicine, Nagoya, Department of ImmunologyA, Medical Institute of Bioregulation, Kyushu University, Fukuoka and Department of Immunology., The Tokyo Metropolitan Institute of Medical Science, Tokyo
A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344.The site of the CD4+CD8+population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+IEL and some of CD8+ IEL in aged rats were of CD5+CD45RW phenotype. In germ-free Wistar rats, age-associated increase in the number of CD4+CD8+CDSf IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CDSfCD45RB- IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD4+CD8+thymocytes bearing a low intensity of CD3/T cell receptor (TcR) alp, the CD4+CD8+T cells in IEL expressed a high intensity of CD3/TcRu//3 on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the E L in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8' IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in thk epithelium.
1 Introduction Intestinal intraepithelial lymphocytes (IEL) represent a unique population expressing CD8 and able to exhibit non-MHC restricted cytolytic activity [1-7].The CD8+ IEL in mice consist of heterogeneous populations bearing or not Thy-1 [l],TcRy/6 or a/p [ 3 , 4, 6, 71, and the CD8u homodimer or the CD8 alp heterodimer [2]. Athymic nude mice contain a significant number of IEL expressing TcR y/6, suggesting that murine IEL expressing TcR y/6 may develop along an extrathymic pathway [ 8 , 91. Furthermore, there has been convincing evidence that a significant fraction of IEL bearing TcRulp also develop outside thymus. TcR u/@+IEL bearing the CD8 u homodimer have been reported to be detected in nude mice, scid mice and irradiated and thymectomized mice repopulated by T celldepleted BM cells [lo]. Rocha et al. have found that the forbidden T cell clones bearing TcR a/@capable of recognizing the self-superantigens, which are normally deleted in the thymus, are detected in the IEL bearing the CD8u
[I 96801
*
This work was supported in part by grants to Y.Y. from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, Sapporo Bioscience Foundation and Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
Correspondence: Yasunobu Yoshikai, Laboratory of Germ-free Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya 466, Japan Abbreviations: IEL: Mesenteric LN
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Intraepithelial
lymphocytes
MLN:
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992
homodimer [11]. Thus, TcRy/6 IEL and TcRu/p IEL bearing the CD8u homodimer can be considered as thymus-independent populations. On the other hand, TcR a/p IEL bearing the CD8 a/@heterodimer and a small minority of CD4+ IEL are thought to be thymus-dependent populations derived from dividing precursors in Peyer's patches under antigenic stimulation [ 121. Recently, a unique population of CD4+CD8+T cells bearing TcR u/p has been detected in both murine IEL and rat IEL [13, 141. Although the CD4+CD8+IEL are thought to be involved in the epithelial immunity, the characteristics of the unique population in IEL, including their origin and functions remain obscure. Unlike murine IEL, most rat IEL expressTcR u/p [15].The fact that athymic nude rats contain noTcR u/@IEL suggests that rat IEL may be exclusively thymus dependent [15, 161. In the present study, we have characterized the IEL in rats with different genetic backgrounds and different ages and found that there was an age-related increase in the number of CD4"CD8+ IEL, irrespective of their genetic backgrounds. The CD4+CD8+ IEL, which were of the CD3/TcR u/phlgh,CD5+ and CD45RB- phenotype, appear to proliferate spontaneously. The unique population was not detected either in the IEL of aged nude rats or in those of aged germ-free rats. The implication of these findings for the roles of CD4+CD8+ Tcells in IEL in epithelial immunity are discussed.
2 Materials and methods 2.1 Animals WKA/Sea, BN/Sea and LEW/Sea rats were obtained from Seiwa Experimental Animal Institute (Fukuoka, Japan). ACI/N Jcl rats were obtained from Clea Japan, Inc. (Tokyo,
+
OO14-298O/92/0101-0159$~.50 .25/0
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Japan). F344/Bu Crj rats were obtained from Charles River Japan, Inc. (Atsugi, Japan) and athymic nude rats (F344/N nu/nu) were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine. Germfree Wistar rats and conventional Wistar rats were maintained in our laboratory.
2.2 Preparation of lymphocytes IEL were prepared according to a modification of a method of Cerf-Bensussan et al. [17]. Briefly, small intestine was flushed with SO ml of PBS, fat and Peyer's patches were removed.The intestine was opened longitudinally, it was cut into 2-cm pieces, and then washed again in PBS.The pieces were stirred in 199 medium (Gibco, Grand Island, NY) supplemented with 20% Nu serum (Collaborative Research Inc., Bedford, MA) and 1mM dithioerythritol (Sigma Chemical Co., St. Louis, MO) at 37°C for 20 min and then spun down. The pieces were resuspended in RPMI 1640 (Gibco) supplemented with 20% Nu serum and agitated in a 37°C for 20 min, and then debris were removed. This cell suspension was centrifuged, resuspended in 8 ml of 44% Percoll (Sigma), and layered on 5 ml of a 67.5% Percoll solution.The gradient was centrifuged at 600 x g at 20°C for 20 min. Lymphocytes at the interface were harvested, and washed twice with HBSS.
2.3 Antibodies The following mAb were used in this work: PE-conjugated W3/25 (anti-rat CD4 mAb), FITC-conjugated OX8 (antirat CD8 mAb), PE-conjugated OX19 (anti-rat CD5 mAb), FITC-conjugated OX22 (anti-rat CD45RE3 mAb), FITCconjugated OX12 (anti-rat 3t chain mAb; Serotec, Oxford, GB), FITC-conjugated or biotinylated R73 (anti-rat TcRa/P mAb; kindly provided by Dr. T. Hiinig [HI, biotin-conjugated 1F4 (anti-rat CD3 mAb, [19]).They were conjugated with FITC or biotin according to standard procedures. Secondary antibodies were PE-conjugated anti-mouse IgM, biotinylated anti-mouse IgM, biotinylated anti-mouse IgG (Caltag Laboratories, San Francisco, CA). streptavidin-PE and streptavidin-Duochrom was obtained from Becton Dickinson (San Jose, CA).
2.4 FCM analysis For two-color FCM analysis were stained with FITCconjugated mAb and PE-conjugated mAb or biotinylated mAb followed by streptavidin-PE. For three-color FCM analysis were stained with FITC-conjugated mAb, PEconjugated mAb, and biotinylated mAb followed by streptavidin-DuoChrom. The lymphocytes were analyzed on a FACScan flow cytometer (Becton Dickinson).
2.5 MLR IEL (1 x lo5) from 4-week-old or 30-week-old WKA rats were cultured with irradiated (33 Gy) syngeneic or allogeneic (ACI rats) spleen cells (1 x lo5) in flat-bottom microtiter plates. After 3 , 5 or 7 days of culture, 37 kBq of tritiated thymidine was added for the final 6 h and the cultures were harvested onto filters and counted.
Eur. J. Immunol. 1992. 22: 159-164
3 Results and discussion 3.1 Cell surface phenotype of IEL in young and aged rats of various strains IEL were characterized phenotypically by FCM with anti-CD4 mAb and anti-CD8 mAb. As shown in Fig. l a , in young rats (4-5 weeks of age), IEL were composed of a large number of CD8+ cells, CD4-CD8- cells and only a few CD4+ cells in Wistar, F344, WKA, BN and LEW rats. CD4+CD8+cells were hardly detected in IEL at this stage. An appreciable number of CD4+ IEL and CD4+CD8+IEL could be detected 10 weeks after birth in most strains. In aged rats (6 months old), the number of CD4+CD8+ IEL increased to a considerable level in all strains examined. Conversely, the proportions of CD8+ cells and CD4-CD8cells decreased in the IEL of the aged rats.The CD4+CD8+ cells were hardly detected in the peripheral lymphoid tissues such as spleen and mesenteric LN, Peyer's patch or liver (data not shown). Next we examined age-related changes in the expression of theTcR/CD3 complex on rat IEL. IEL were double-stained with anti-CD3 mAb and anti-TcR ci/P mAb. Consistent with a previous report [ 141, a large number of rat IEL expressed TcR alp in all strains we examined. However, a significant number of CD3+TcRalp- IEL, presumably TcRy/6+ IEL, were also detected in the IEL of young strains, ranging from 20% to 45% of CD3+ IEL (Fig. lb). The number of CD3+TcRa/p- IEL decreased with aging. This subset was hardly detected at any stage in the peripheral lymphoid tissues (data not shown). The intensity of the CD3EcR complex was compared between IEL and thymocytes. Thymocytes are divided into three groups as accessed by the intensity of the TcR/CD3 complex; CD3/TcR-, CD3/TcR1OW and CD3/TcRhigh[20, 211. Approximately half of CD4+CD8+ thymocytes are known to express low amounts of the TcR/CD3 complex but an appreciable number of T c R / C D ~ thymocytes ~'~~ is also present in the CD4+CD8+population in mice [22]. Consistent with these reports, CD4+CD8+ thymocytes of young rats contained not only a high proportion of TcR/CD3loWcells but also an appreciable level of T c R / C D ~ ~ ' cells. ~~ The CD4+CD8+CD3highthymocytes expressed a lower intensity of CD8 than CD8+ thymocytes. In terms of relative amounts of TcR surface expression, the CD4+CD8+IEL of aged rats expressed a high intensity of TcR/CD3 complex, similar to some fractions of CD4+CD8+ thymocytes and the single-positive T cells in thymus and the peripheral lymphoid tissues. CD5 is known to be a panT cell marker [23].To examine the expression of CD5 on IEL, we stained IEL with anti-CD5 mAb and anti-CD3 mAb. As shown in Fig. 2a, most of CD3 cells in MLN expressed CD5, while CD5+ cells were hardly detected in the IEL of young rats of all strains. The CD5+ IEL increased gradually to a substantial level by 6 months after birth. To determine whether the CD4+CD8+ IEL in aged rats expressed CD5, we examined the IEL in aged rats by three-color staining with antLCD5 mAb, anti-CD4 mAb and anti-CD8 mAb. As shown in Fig. 2b, all CD4+CD8+ cells expressed the CDS molecule, whereas most CD4-CD8+ cells did not express CD5 in aged rats. In terms of relative expression of CD5 molecule, CD4+CD8+ IEL expressed lower amounts of CD5 than CD4+ IEL, all of
CD4+CD8+ IEL in rat
Eur. J. Immunol. 1992. 22: 159-164
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Figure I . Expression of CD4 and CD8 of theTcR/CD3 complex on IEL of various rat strains. (a) IEL were stained with FITC-conjugated anti-CD4 mAb (W3/25) and PE-conjugated anti-CD8 mAb (0x8).(b) IEL were stained with anti-rat CD3 mAb (1F4) followed by PE-conjugated anti-mouse IgM, and stained with FITC-conjugated anti-TcR a/fi mAb (R73). Analysis was performed on a FACScan. Results are from one of three similar experiments.
which expressed high amounts of CD5. CD45RE3 expression determined by OX22 mAb, a marker of virgin Tcells [24], was also examined in the IEL. In young rats, CD45RB+ cells were to be found CD8+ or CD4-CD8IEL. In aged rats, CD4+ IEL did not express CD45Rl3, suggesting that CD4+CD8+ IEL expressed CD45RJV (data not shown).
3.2 Absence of CD4+CD8+ IEL in aged nude rats and in aged germ-free rats Our results showed an age-related increase in the number of CD4+CD8+cells and CD5+ cells in rat IEL, irrespective of their genetic backgrounds. To investigate the origin of the CD4+CD8+IEL in aged rats, we analyzed the cell surface phenotype of the IEL in aged athymic nude F344 rats. Young nude rats contained IEL with the CD8+CD3TcR ulp- phenotype and which had large granules in their cytoplasm (data not shown). As shown in Fig. 3a, aged nude rats (12 month old), appreciable levels of CD8+ IEL and CD4+ IEL bearing CD3/TcRu/P were detected, suggesting that some IEL may be able to differentiate outside the thymus in rats. However, CD4+CDS+CDS+IEL were hardly detected in the IEL of aged nude rats. It would thus appear that CD4+CD8+CDSf IEL may be derived from Tcell precursors of thymic origin.
To determine whether the intestinal microflora plays an important role in development of the CD4+CD8+CDSf IEL in aged rats, we examined the IEL of Wistar rats bred under germ-free condition. As shown in Fig. 3b , aged germ-free rats contained a significant number of CD8+CD3+TcRu//B+but not CDSfCD4+CD8+1EL.These results suggested that stimulation with the intestinal microflora may be important for the expansion of the CD5+CD4+CD8+IEL in aged rats. Our data presented here revealed that CD4+ IEL appear before CD4+CD8+ IEL, raising the possibility that CD4+CD8+ IEL may originate from CD4+ IEL that acquire CD8. In mice, thymic y/6 cells negative for CD4 or CD8 are reported to express the CD8 cx homodimer following in vitro activation by Con A [25]. Paliard et al. reported that activation and culturing of human CD4+ T cell clones in IL 4 resulted in the acquisition of CD8 due to its de ylovo synthesis [26]. The CD4+CD8+ cells are also detected in LN and spleen after antigen or mitogen activation [27] or in the lamina propria of the intestine in rats [28]. Taken together, expression of CD8 might be induced on CD4+ IEL by external stimuli. We cannot at present define what cytokines mediate CD8 induction on the IEL. Guy-Grand et al. has recently proposed that the gut epithelium is a site that plays an attractive and a differen-
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(a)
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CD5 Figure 2. Expression of CD5 on IEL of various rat strains. (a) IEL were stained with anti-CD3 mAb followed by FITC-conjugated anti-mouse IgM, and PE-conjugated anti-CD5 mAb (0x19). (b) IEL were also stained with anti-CD4 mAb followed by biotinylated anti-mouse IgM and streptavidin-DuoChrom,FITC-conjugated anti-CD8 mAb, and PE-conjugated antLCD5 mAb, and expression of CD5 was displayed after setting the gates on CD4+CD8+IEL. Results are from one of three similar experiments.
tiation-inducing role on the appearance of a significant fraction of CD8+ IEL [lo]. Therefore, alternatively precursors of IEL might differentiate into CD4+CD8+ IEL via a CD8+ stage at a local site, outside of the thymus, under the influence of the intestinal epithelial environment. Immature CD8+ Tcell precursors in the tyhmus are known t o acquire CD4 after overnight culture in medium only [29, 301. Our results, however, revealed that following overnight incubation of IEL of young rats in vitro, no CD4+CD8+ IEL were generated (data not shown). Furthermore, the CD4+CD8+ IEL were hardly detected in aged nude mice, which contained an appreciable level of CD4+ IEL and CD8+ IEL bearingTcR a/P.Taken together, it is unlikely that the CD4+CD8+IEL are generated locally in the intestine without thymic influence. Although our results with aged nude rats revealed that a significant fraction of CD4+ IEL and CD8+ IEL can develop extrathymically in rats (Fig. 3a), CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD3+CD4+CDSf thymocytes, the CD4+CD8+ IEL express a high intensity of CD3/TcRdP, similar to single-positive T cells in the peripheral lymphoid tissues. Recently, a significant fraction of CD4+CD8+ thymocytes have been reported to express a high intensity of CD3/TcR on their surface [22]. We can also assume that the CD4+CD8+ IEL may be derived from this population of the CD4+CD8+ thymocytes, which are intermediates between immature CD4+CD8+ thymocytes and mature,
single-positiveT cells. It remains, however, to be elucidated whether the CD3highCD4+CD8+thymocytes can migrate directly to the periphery including the intestine.
3.3 Spontaneous proliferation of the IEL in aged rats To elucidate the specificity and function of the CD4+CD8+ IEL, whole IEL of young or aged rats were incubated at 37 "C in RPMI 1640 medium supplemented with 10% FCS in the absence of added mitogens or growth factors, or in the presence of syngeneic APC or allogeneic APC. Surprisingly, the IEL in aged rats proliferated spontaneously on the third day of the in vitro culture (Fig. 4a, p < 0.001), while whole IEL in young rats showed little, if any, DNA synthesis at any stage in the in vitro culture. To determine the phenotype of the spontaneously proliferating cells in the IEL of aged rats, FCM analysis was carried out on the blastoid cells in the 3-day in vitro culture of the IEL. As shown in Fig. 4b, the size of the CD4+CD8+ subset significantly increased, whereas CD4+ IEL and CD4-CD8- IEL decreased conversely after 3 days of in vitro culture. O n the other hand, a shift in subpopulations of the IEL was not observed in the in vitro culture of the IEL of young rats (data not shown).
So far, the relevance of CD4 and CD8 expression of IEL to the function and specificity of the IEL remains unclear.
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CD4+CD8+ IEL in rat
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Figure 3. Surface phenotype of IEL from aged nude rats and aged germ-free rats. IEL were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated anti-CD4 mAb, FITC-conjugated antiTcR a / p mAb and biotinylated anti-CD3 mAb and streptavidinPE, or FITC-conjugated anti-TcR alp mAb and PE-conjugated anti-CD5 mAb. Results are from one of three similar experiments.
CD1, a family of MHC class I-like molecules, is reported to be expressed on intestinal epithelium in mice [31]. In rats IEL are also reported to start expressing MHC class XI with age [17]. CD4+CD8+ IEL may be involved in recognizing antigens in the context of MHC class I or class I1 expressed on the intestinal epithelium. The cytolytic activity of IEL is known to be regulated by externally derived stimuli via a specific functional interaction between IEL and gutassociated antigens [32].Expression of CD8 is found to mediate anti-CD3-directed cytolytic activity by the CD4+CD8+ Tcell clones. Our results showing that the CD4+CD8+IEL expressing CD5 but no CD45RB prolife-
CD8 1 1 1 1 1 1 ) Figure 4. Proliferative response of IEL from aged WKA rats upon syngeneic stimulation. (a) IEL (1 :< los) from 4-week-old (00 A) or 30-week-old (W 0 A) WKA rats were cultured with irradiated (33 Gy) syngeneic (0W) or allogeneic (ACI rats) (00 ) spleen cells (1 X lo5), or medium only (AA)in flat-bottom microtiter plates. After 3 , 5 or 7 days of culture, 37 kBq of tritiated thymidine was added for the final 6 h. (b) Surface phenotype of IEL after syngeneic stimulation. After 3 days of culture, IEL were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated antiCD4 mAb.
rate spontaneously, suggest that the CD4+CD8+ IEL may be in an activated state. The CD4+CD8+ IEL may play important roles in the first line of defense against various pathogens in the epithelium by recognizing these pathogens once presented by MHC class I and I1 and by becoming cytolytic. Further functional analysis is required to elucidate the roles of the CD4+CD8+ IEL in the epithelial immunity.
4 Concluding remarks Age-related increase in C D 4 - T D 8 + IEL was evident in rats. A unique population of CD4+CD8+ IEL expressed CD5, a high intensity of CDYTcRdP but not CD45RB.
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CD4+CD8+IEL originate from a thymus-dependent population and expand by external stimuli such as the intestinal microflora. CD4+CD8+ IEL may participate in immunosurveillance at the first line of defense in aged animals. We thank Dr. 7: Hiinig for providing the anti-rat E R d p (R73) mAb.
Received June 13, 1991; in final revised form September 23, 1991.
5 References 1 Klein, J. R., J. Exp. Med. 1986. 164: 309. 2 Parrot, D. M. V , Tait, C., Muckenzie, S., Momat, A. MCI., Davies, M. D. J. and Micklem, H. S., Ann. N X Acad. Sci. 1983. 409: 307. 3 Goodman, T. and Lefranqois, L., Nature 1988. 333: 855. 4 Bonneville, M., Janeway, C. A., Ito, K., Haser,W., Ishida, I., Nakanishi, N. and Tonegawa, S., Nature 1988. 336: 479. 5 Viney, J. L., Kilsham, I? J. and MacDonald, T. T., Eur. J. Immunol. 1990. 20: 1632. 6 Takagaki,Y, DeCloux, A., Bonneville, M. and Tonegawa, S., Nature 1989. 339: 712. 7 Goodman, T. and Lefranqois, L., J. Exp. Med. 1989. 1870: 1569. 8 Bandeira, A . , Mota-Santos, T., Itohara, S.,Degerman, S., Hawser, C. ,Tonegawa, S. and Coutinho, A . ,J. Exp. Med. 1990. 172: 239. 9 Bandeira, A , , Itohara, S., Bonneville, M., Burlen-Defranoux, O., Mota-Santos, T., Coutinho, A. and Tonegawa, S., Proc. Natl. Acad. Sci. U S A 1991. 88: 43. 10 Guy-Grand, D., Cerf-Bensussan, N., Malissen, B., MalassisSeris, M . , Briottet, C. and Vassalli, F!, J. Exp. Med. 1991. 173: 471. 11 Rocha, B.,Vassalli, P. and Guy-Grand, D., J. Exp. Med. 1991. 173: 483. 12 Guy-Grand, D., Griscelli, C. and Vassalli, I?, J. Exp. Med. 1978. 148: 1661
Eur. J. Immunol. 1992. 22: 159-164 13 Mosley, R. L., Styre, D. and Kleim, J. R., Int. Imrnunol. 1990. 2: 361. 14 Fangmann, J., Schwinzer, R. and Wonigeit, K., Eur. J. Immunol. 1991. 21: 753. 15 Vaage, J. T.,Tissen, E., Ager, A., Roberts, I., Fossum, S. and Rolstad, B., Eur. J. Irnrnunol. 1990. 20: 1193. 16 Viney, J. L., MacDonald, T. T. and Kilsham, F! J., Irnrnunol. Lett. 1990. 22: 49. 17 Cerf-Bensussan, N., Quaroni, A., Kurnik, J. T. and Bhan, A. K., J. Immunol. 1984. 132: 2244. 18 Hiinig, T., Wallng, H.-J., Hartley, J. K., Lawetzkg, A. and Tiefenthaler, G., J. Exp. Med. 1989. 169: 73. 19 Tanaka,T., Masuko,T.,Yagita, H., Tamura,T. and Hashimoto, Y , J. Immunol. 1989. 142: 2791. 20 Havran,W. L., Poenie, M., Kimura, J.,Tsien, R.,Weiss, A. and Allisan, J. €?,Nature 1987. 330: 170. 21 Shortman, K., Egerton, M., Spangrude, G. J. and Scollay, R., in Sprent, J. (Ed.), Semin. Immunol., vol. 2, W. B. Saunders, London 1990, p. 3. 22 Shortman, K.,Vremec, D. and Egerton, M., J. Exp. Med. 1991. 173: 323. 23 Ledbetter, J. A , , Evans, R. L., Lipinski, M., CunninghamRundles, C., Good, R. A. and Herzenberg, L. A.,J. Exp. Med. 1981. 153: 310. 24 Barclay, A. N., Jackson, D. I. ,Williams, A. C. and Williams, A. E , EMBO J. 1987. 6: 1259. 25 MacDonald, H. R., Schreger, M., Home, R. C. and Bron, C., Eur. J. Irnrnunol. 1990. 20: 927. 26 Paliard, X., Malefijt, R.W., DeVries, J. E. and Spits, H., Nature 1988. 335: 642. 27 Bevan, D. J. and Chisholm, I? M., Immunology 1986. 59:
64. 28 Van der Heijden, F. L., Immunology 1986. 59: 397. 29 Paterson, D. J. and Williams, A. F., J. Exp. Med. 1987. 166: 1603. 30 MacDonald, H. R., Budd, R. C. and Home, R. C., Eur. J. Irnrnunol. 1988. 18: 519. 31 Bleicher, F! A., Balk, P. S., Hagen, S. J., Blumberg, R. S., Flotte, T. J. and Terhorst, C., Science 1990. 250: 679. 32 LefranGois, L. and Goodman, T., Science 1989. 243: 1716.
