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Development of intraepithelial T lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice
Journal of Hepatology 1999; 30: 68 l-688 Printed in Denmark . All rights reserved Munksgaard Copenhagen
Copyright 8 European Association for the Study of the Liver I999
Journalof Hepatology ISSN 0168-8278
Development of intraepithelialT lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice Satoshi Yamagiwa l, Shuhji Seki*, Katsuaki Department
of Immunology
Shirai, Yuhei Yoshida, Chikako Toru Abo
Miyaji, Hisami Watanabe
and
and ‘Third Department of Internal Medicine, Niigata University School of Medicine, Niigata and =Division of Basic Traumatology. National Defense Medical College Research Institute, Tokorozawa, Japan
Background/Aims: We recently reported the adult mouse liver to contain c-kit+ stem cells that can give rise to multilineage leukocytes. This study was designed to determine whether or not adult mouse liver stem cells can generate intraepithelial T cells in the intestine as well as to examine the possibility that adult liver c-kit+ stem cells originate from the fetal liver. Met/&s: Adult liver mononuclear cells, hone marrow (BM) cells, liver c-kit+ cells or bone BM c-kit+ cells of BALB/c mice were i.v. transferred into 4 Gy irradiated CB17/-SCID mice. In other experiments, fetal liver cells from Ly5.1 C57BL/6 mice and T cell depleted adult BM cells from Ly5.2 C57BL/6 mice were simultaneously transferred into irradiated C57BW6 SCID mice (Ly5.2). At 1 to 8 weeks after cell transfer, the SCID mice were examined.
Results: Not only BM cells and BM c-kit+ cells hut also liver mononuclear cells and liver c-kit+ cells reconstituted y8T cells, CD4+CD8+ double-positive T cells and CDSa+~- T cells of intestinal intraepithelial lymphocytes of SCID mice. Injection of a mixture of fetal liver cells from Ly5.1 C57BL/6 mice and adult BM cells from Ly5.2 C57BL/6 mice into Ly5.2 C57BL/6 SCID mice induced both Ly5.1 and Ly5.2 T cells, while also generating c-kit+ cells of both Ly5.1 and Ly5.2 origins in the liver. Conchsions: Adult mouse liver stem cells were able to generate intestinal intraepithelial T cells of the SCID mice, and it is thus suggested that some adult liver stem cells may indeed be derived from the fetal liver.
I
the liver and periphery could develop (4). On the other hand, certain T cells among the intraepithelial lymphocytes (IEL) of the intestine have also been reported to differentiate extrathymically because these T cells exist in the IEL of athymic nude mice (although their number is reduced compared to normal mice) and in thymectomized radiation chimera mice (7-12). IEL T cells contain T cells with unique phenotypes which are rare in the periphery and are different from conventional T cells. These IEL-specific T cells include CD4+ CD8+ (double positive, DP) T cells, CD8ola (CD8afb-) @I cells and yST cells (7-12). IEL T cells also contain selfminor lymphocyte stimulatory Ag specific (potentially autoreactive) V/3T cells which are normally deleted during thymic T cell development (10). The present study was carried out to determine whether adult liver hematopoietic stem cells have the potential to produce these IEL T cells. Our study demonstrates that liver mononuclear cells (MNC) or liver
been believed that the liver ceases to be a hematopoietic organ after birth. However, we previously reported that intermediate T cell receptor (TCR) cells differentiate extrathymically in the liver of mice (l-3), and that adult mouse livers still contain ckit+ hematopoietic stem cells (4). c-kit is an essential molecule for constitutive hematopoiesis and pluripotent stem cells are surface c-kit+ (5,6). When c-kit+ stem cells of the adult liver as well as bone marrow (BM) c-kit+ cells were transferred into irradiated SCID (severe combined immunodeficient) mice that genetically lack T and B cells, it was shown that the SCID mice could be rescued and virtually all lineage cells in T HAS LONG
Received 17 August: revised 5 October: accepted 17 October 1998
Correspondence: Shuhji Seki, Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa 359-85 13, Japan. Fax: 8142 991 1613. E-mail: [email protected]
Key words: Adult liver stem cells; Fetal liver; Intraepithelial T cells of the intestine.
681
S. Yumugiwn et al 33% Percoll solution containing 100 U/ml hepdrin and were centrifuged at 2000 rpm for I5 min at room temperature. The pellet was resuspended in an RBC lysis solution, then was washed twice with medium. The BM cells were obtained by flushing the femurs with medium. The cell suspensions were filtered through a 200-gauge nylon mesh to remove any debris. Th~mocytes were obtained by forcing the thymus through a 200-gauge steel mesh. The small intestine was removed and flushed with PBS to eliminate any luminai contents. The mesentery, Peyer’s patches or lymph follicles were then resected. The intestine was opened longitudinally and cut into I-2-cm long fragments, which were incubated for 15 min in 20 ml Ca?‘- and Mg”+free HBSS containing 5 mM EDTA while shaking in a 37°C water bath. The supernatant was then collected and washed. This procedure for removing the epithelium from gut fragments was performed twice. Pellets containing IEL were resuspended. passed through a glass wool column to remove any aggregates and dead cells, and then were washed twice before use. After removing the epithelium. gut fragments were incubated in 25 ml of PBS containing 2% FCS and 0.15 mgiml collagenase for 75 min while shaking in a 37°C water bath. The supernatant and debris were collected and filtered through a steel
c-kit+ hematopoietic stem cells can generate IEL T cells that are similar to those induced by BM cells. Furthermore, we also discuss the origin of adult liver ckit+ cells and host tolerance after liver transplantation.
Materials and Methods Mice Male CB17/-SCID mice (H-2d) (13), BALBic mice (H-2d) and Ly5.2 C57BLi6 (B6) (H-2’) mice were purchased from CLEA Japan Inc. (Tokyo, Japan). Ly5.1 B6 mice (H-zb) (14) were kindly provided by Dr. K. Kishihara (Medical Institute of Bioregulation, Kyushu University, Fukuoka. Japan). Ly5.2 B6 SCID (H-2b) mice were purchased from the Central Institute for Experimental Animals (Kanagawd, Japan). Cell prepurations All mice were euthanized by exsanguination from the subclavian artery and vein, and then the liver and spleen were removed. The spleen was pressed on a 200-gauge sterile mesh and washed. The pellet was treated with a red blood cell (RBC) lysis solution (I 55 mM NH,CI, 10 mM KHC03, 1 mM EDTA, 170 mM Tris, pH 7.3). Hepatic MNC were prepared as previously described (3). Briefly, the liver was pressed through a stainless steel mesh and suspended in Eagle’s MEM medium supplemented with 5 mM HEPES and 2% FCS. After one washing, the cells were resuspended in an osmolarity-and pH-adjusted
Control
Monocionul Abs (mAbs,i, jlowq~tometric anal~~,sis and cell ,sorting Anti-CD4 (RM4-5). anti-CD8 (53-6.7), anti-CD3 (145-2Cl I), antiaIEL (M290, an IEL specific marker) (15). anti-c-kit (3Cl. a stem cells marker), B220 (RA3%6B2, a B cell marker). anti-Mac-l (Ml/70. a myeloid cell marker), anti-Cr.1 (RA3%8CS. a granulocyte marker).
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Fig. 1. Induction of DP T ~11s in the tl~ynzus und IEL as 12~11as CD4+ or CDS-+ T cells in every organ of‘ the irradiated CBI 7/-SCID mice by the trmsfer qf rrdult liver MNC or BM cells. Control duta WC’ ,from normnl BA LB/‘c mice.
Liver stem cells and T cells of the intestine
Fig. 2. Induction of yST cells, CD8aa (CDSa’~-) T cells and a-IEL+ T cells in IEL of the irradiated SCID mice by the transfer of adult liver MNC or BM cells.
Control I7- l2.6 .I
Liver transfer
BM transfer
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Small intestine + IEL F I
Small intestine g IEL 8 I CD8a
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TERl19 (an erythroid lineage marker) and anti-CD45 (30Fll .l, common leukocyte Ag), Ly5.1 (CD45.1) and Ly5.2 (CD45.2) Abs were all purchased from PharMingen (San Diego, CA, USA). All mAbs were used in an FITC, PE or biotin conjugated form. The biotinylated reagents were developed with FITC or PE-conjugated streptavidin (Be&on-Dickinson Co., Mountain View, CA, USA) or TRI-COLORconjugated streptavidin (CALTAG Lab., San Francisco, CA, USA). To prevent nonspecific staining of mAbs, antiCD32/16 Ab (2.4G2, PharMingen) was added before staining with labeled mAbs. The cell suspensions were stained with mAbs and then were analyzed by FACScan (Becton-Dickinson). Any dead cells were excluded by a forward scatter, side scatter and PI gating. The c-kit+ lineage negative (CD3B220- Mac-lGr-ll TER119-) cells of hepatic MNC and BM cells were sorted by FACS Vantage (Becton-Dickinson).
Cell transfer After 4 Gy-irradiation (4), either 1 x 10’ of BM cells or hepatic MNC from BALBlc mice were injected intravenously into CB17/-SCID mice. Fivex lo4 sorted c-kit+ lineage negative cells of hepatic MNC or BM c-kit+ lineage negative cells were also injected into 4 Gy-irradiated CB17/-SCID mice. In some experiments, 5X lo6 fetal liver cells (day 20) from B6 Ly5.1 mice and 5~ lo6 T-cell depleted adult BM cells from B6 Ly5.2 mice were simultaneously injected into 4 Gyirradiated B6 Ly5.2 SCID mice. The T cell depletion of BM cells was done by staining the BM cells with anti-CD3 Ab and then sorting CD3 negative cells.
Results Liver MNC as well as BM cells reconstituted T cells in IEL of irradiated SCID mice
Four weeks after transferring liver MNC or BM cells from BALB/c mice into 4 Gy irradiated CB17/-SCID mice, the SCID mice were examined. As a result, CD4+ or CD8+ single positive T cells were found to appear in all organs tested, while CD4+8+ DPT cells emerged in the IEL and thymocytes (Fig. 1). In addition, liver MNC were able to develop $T cells, CD8a+P(CD8aa) T cells (conventional CD8+ T cells express CD8atj3+) and a-IEL (an IEL specific marker) positive T cells (Fig. 2). Reconstitution of T cells in IEL of irradiated SCID cells from BALB/c mice FiveX lo4 liver c-kit+ cells or BM c-kit+ cells of BALB/ c mice developed DP T cells (Fig. 3) $T cells (Fig. 4) CD8afpT cells and a-IELf T cells in IEL of CB17/-SCID mice (Fig. 5). Furthermore, liver and BM c-
mice by adult liver c-kit?
683
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et al.
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Fig. 3. Induction of DP T cells in IEL by the transfer of 5 X IO4 adult liver ckit+ cells as well as BM c-kit+ cells of irradiated SCID mice. SCID mice were examined 4 weeks ufter cell transfer. The control duta are ,from normal BALB/c mice.
Liver c-kit*cell transfer
BM c-kit’cell transfer
-CD8a------
day 7 r
Control
Liver c-kit+cell transfer
BM c-kit*cell
transfer
684
day 14
day 21
Fig. 4. Induction of uPT cells as well as $T cells in the IEL qf irrudiated SCID mice by the transfer of adult liver c-kit+ cells.
Liver stem cells and T cells of the intestine
day 14 7”i/
day 21
day 14
day 21
-1o”Tq-3q
Control
Liver c-kit*cell transfer
BM c=kit*cell transfer CD3 Fig. 5. Induction of CD8cza (CDSa’~-) liver c-kit+ cells.
T cells and u-IEL+ T cells in IEL of irradiated SCID mice by the transfer of adult
kit+ cells were able to induce IEL-specific T cells within 14 days after cell transfer when thymus could not yet provide T cells into the periphery (4) (Fig. 35), thus indicating that IEL can directly differentiate from stem cells in a thymus independent manner. The transfer of fetal liver cells from B6 Ly.5.1 mice together with BM cells from B6 Ly5.2 mice induced both Ly5.1 and Ly.5.2 c-kit+ cells in the liver of B6 SCID mice When a mixture of 5X IO6fetal liver cells from B6 Ly5.1
mice and the same number of T cell-depleted adult BM cells from B6 mice were transferred into 4 Gy-irradiated B6 SCID (Ly5.2) mice, both Ly5.1 cells and Ly5.2 cells (including T and B cells, not shown) appeared in the liver, thymus and small intestine at 8 weeks after the transfer (Fig. 6). In addition, both Ly5.1 and Ly5.2 ckit+ cells were also recognized in the liver (Fig. 7). Thus, liver c-kit+ cells of irradiated B6 SCID mice were derived from both adult BM and the fetal liver.
Discussion Among mouse T cells, liver IL-2Rj?+ T cells with intermediate TCR (l-4) and certain T cells in IEL (7-12)
can differentiate extrathymically. Extrathymic T cells in IEL reportedly include CD8a+P$T cells, CD8a+j3- @3T cells and DPT cells. Although we recently reported that adult liver hematopoietic stem cells can reconstitute both extrathymic T cells and conventional thymus-derived T cells (4), it is still not known whether or not adult liver stem cells can reconstitute IEL T cells. In addition, if they can reconstitute IEL T cells, it is important to determine whether IEL T cells induced by the liver stem cells’are same as those induced by BM stem cells. The present study has demonstrated that the adult liver contains pluripotent stem cells that can develop not only thymic T cells or peripheral T cells, but also IEL T cells. The IEL T cells induced by liver c-kit+ stem cells were essentially the same as those induced by BM c-kit+ cells. The transfer of both fetal liver hematopoietic cells from Ly5.1 mice and BM cells from adult Ly5.2 mice into irradiated B6 SCID mice induced IEL T cells, thymic T cells, peripheral T cells and B cells in B6 SCID mice. Furthermore, c-kit+ cells in the liver of these SCID mice contained both Ly5.1 c-kit+ cells and Ly5.2 c-kit+ cells, thus suggesting that at least some c-kit+ cells in the livers of adult mice originated from the fetal liver.
68.5
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Fig. 6. Induction of LyS.l+ cells and Ly5.2+ cells in irradiated B6 SCID mice by the transfer of a mixture of Ly5.1i fetal liver hematopoietic cells and L~5.2~ BM cells. 5X IO6fetal liver cells of Ly5.1 86 mice and same number of T cell depleted BM cells ,from adult Ly5.2 86 mice were simultaneously transferred into irradiated B6 SCID (Ly5.2) mice. Eight weeks after the cell transfer, the mice were examined.
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IEL T cells are believed to differentiate extrathymically (7-12). The main reasons for this interpretation are as follows: 1) athymic nude mice have CD8a+p- $T cells, CDXa+p- @I’ cells and DPT cells; 2) the IEL of thymectomized and irradiated mice which received BM cells from normal mice contain T cells; 3) CD801tP- c&I cells in IEL contain potentially autoreactive self-Mls Ag-specific VP cells which are generally deleted during normal thymic T cell development (so-called negative selection) (7-12). Similarly, liver intermediate TCR cells develop in a thymus-independent manner because athymic nude mice have intermediate TCR cells (both $ and q!JTCR cells) in the liver, and liver intermediate @TCR cells also include 686
Fig. 7. Appearance of both Ly5.1 and Ly5.2 c-kit+ cells in irradiated B6 SCID mice by the transfer of the mixture of LyS. I + .fetal liver hematopoietic cells and Ly,5.2+ BM cells. Eight weeks after the cell transfer, the mice were examined. Ly5.1’ cells or L,y5.2’ cells were gated and their CD45 (common leukocyte Ag) and c-kit expressions were anulyzed by a 3-color flowcytometric analvsis.
self-minor lymphocyte stimulatory Ag-specific T cells (2,3). Moreover, thymectomized and irradiated mice which received T cell depleted BM cells can also develop intermediate TCR cells (1). Another notable finding is that adult liver c-kit+ stem cells likely include descendants of fetal liver stem cells. Although we did not rule out the possibility that stem cells of the adult mouse liver might come from BM, our findings suggest that adult liver stem cells are also derived from the fetal liver. In line with these lindings, it was also reported that a number of c-kit+ cells or hematopoietic cells still remain after birth in the liver, while their number gradually decreases with age (l&17). In fact, liver MNC of both neonatal mice and
Liver stem cells and T cells of the intestine
young mice successfully reconstitute lymphoid cells in lethally irradiated hosts (our unpublished observation). Thus, certain stem cells of fetal liver remain in the liver after birth. Therefore, although the degree of hematopoiesis in the liver decreases after birth, it is reasonable to think that liver hematopoiesis continues after birth into adulthood. Since hepatocytes (but not Kupffer cells) are essential for the development of liver intermediate TCR cells (18) and liver perfusion from the portal vein does not affect the number of c-kit’ cells (4), stem cells in the adult liver may indeed reside and differentiate in Disse’s space. We earlier reported that liver MNC gradually increase with age because of the increase in the number of extrathymic intermediate TCR cells (2,3,19). In addition, these intermediate TCR cells gradually increase in the periphery with age, whereas the thymus rapidly involutes with age and therefore the number of the thymus-derived high TCR cells decreases in the periphery (2,3,19). This phenomenon (the increase and activation of liver MNC and the involution of the thymus) is also almost always observed in bacterial infections, malignancies, autoimmune diseases and pregnancy (see review 2). These phenomena can also be regarded as liver hematopoiesis. Furthermore, this study has also shown an additional capacity of the adult liver, namely that it can provide IEL T cell precursors to the intestine. It has also been recognized that a successful liver transplantation facilitates other organ engraftments (20,21), including the kidney, pancreas and the intestine. This phenomenon can be explained by the fact that either donor liver MNC or precursors migrate to other organs and the periphery of the host, and results in a donor-host chimera (the presence of both donor and host leukocytes in the host) (20) inducing resultant host tolerance, and these phenomena are quite consistent with the present results. Liver parenchymal cells also seem to be important because the soluble class I Ags produced by donor liver (but not by other organs) may play a role in inducing host tolerance (21). Bone marrow cells from athymic nude mice generate IEL T cells in thymectomized and irradiated mice, although the number of IEL T cells is considerably smaller than in radiation chimera mice without a thymectomy (12). It has also been suggested that thymic hormones (or cytokines including IL-7) or thymic epithelial cell factors are needed for the effective expansion of extrathymic T cells in the intestine (12,22) and the liver (23,24) of nude mice. Therefore, although liver stem cells from normal mice quickly migrate to the intestine and the liver of SCID mice and generate IEL T cells (as demonstrated in this study) as well as liver intermediate TCR cells (4) within 2 weeks after trans-
fer (before thymic reconstruction), the effective expansion of extrathymic T cells may thereafter be affected by the either thymus or its factors (22-34). Cryptopaches of the intestine were recently reported to have a large number of lymphocytes from 1417 days after birth and thereafter (25). These lymphocytes contain a large fraction of c-kit+ TCR/CD3- IL-7R+ stem cells. Moreover, these intestinal c-kit+ cells from nude mice (which are apparently thymus independent) could also generate IEL T cells without thymic reconstitution when injected into irradiated SCID mice (26). However, it should be noted that liver c-kit+ cells (up to 30% are IL-7R+, our unpublished data), but not intestinal c-kit+ cells, are pluripotent because only the former can generate thymocytes (regular T cells), B cells, erythroid and myeloid lineage cells (4,26,27). Although c-kit+ cells in the intestine are probably provided from the fetal liver and develop in situ into T cells after birth, the possibility cannot be ruled out that intestinal c-kit+ cells originate in the liver and/or the bone marrow after birth. The present results may partly explain why liver transplantation leads to the successful engraftment of the small intestine, since the engrafted liver provides donor leukocytes to the recipients and also creates a donor-host chimera while inducing immune tolerance. Finally, it is interesting to note that bacterial Ags including LPS (endotoxin) are continuously brought from the intestine to the liver via the portal vein (28) and activate liver MNC (29). Therefore, the present results suggest the possibility that lymphoid systems of the liver and the intestine may affect each other.
References 1. Sato K, Ohtsuka K, Hasegawa K, Yamagiwa S, Watanabe H, Asakura H, et al. Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation. J Exp Med 1995; 182: 759-67. 2. Abo T, Watanabe H, Iiai T, Kimura M, Ohtsuka K, Sato K, et al. Extrathymic pathways of T-cell differentiation in the liver and other organs. Intern Rev Immunol 1994; 11: 61-102. 3. Tsukahara, A, Seki S, Iiai T, Moroda T, Watanabe H, Suzuki S, et al. Mouse liver T cells: their change with aging and in comparison with peripheral T cells. Hepatology 1997; 26: 301-9. 4. Watanabe H, Miyaji C, Seki S, Abo T. c-kit+ stem cells and thymocyte precursors in the livers of adult mice. J Exp Med 1996; 184: 687-93. 5. Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi S, Kunisada T, Sudo T, et al. Expression and function of c-kit in hematopoietic progenitor cells. J Exp Med 1991; 174: 63-71. 6. Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Nishikawa SI, Miura Y, et al. Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule. Blood 1991; 78: 170612. 7. Mosley RL, Styre D, Klein JR. Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras. J Immunol 1990; 145: 1369-75.
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S. Yamagiwa et al. 8. Bandeira A, Itohara S, Bonneville M, Burlen-Defranoux 0, Mota-Santos T, Coutinho A, et al. Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor gamma delta. Proc Nat1 Acad Sci USA 1991; 88: 43-7. 9. Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991; 173: 471-81. 10. Rocha B. Vassalli P Guy-Grand D. The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta’ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J Exp Med 1991; 173: 483-86. 11. Poussier P, Edouard P, Lee C, Binnie M, Julius M. Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J Exp Med 1992; 176: 187-99. 12. Rocha B. Vassalli P, Guy-Grand D. Thymic and extrathymic origins of gut intraepithelial lymphocyte populations in mice. J Exp Med 1994; 180: 68 1-6. 13. Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature 1983: 301: 527-30. 14. Morse HC, Shen FW, Hammerling U. Genetic nomenclature for loci controlling mouse lymphocyte antigens. Immunogenetics 1987; 25: 71-8. 15. Kilshaw PJ, Murant SJ. A new surface antigen on intraepithelial lymphocytes in the intestine. Eur J Immunol 1990; 20: 2201-7. 16. Taniguchi H, Toyoshima T. Fukao K, Nakauchi H. Presence of hematopoietic stem cells in the adult liver. Nat Med 1996; 2: 1988 203. 17. Kawamura T, Toyabe S, Moroda T, Iiai T, Takahashi-Iwanaga H, Fukuda M, et al. Neonatal granulocytosis is a postpartum event which is seen in the liver as well as in the blood. Hepatology 1997; 26: 157672. 18. Kawachi Y, Arai K, Moroda T, Kawamura T, Umezu H, Naito M. et al. Supportive cellular elements for hepatic T cell differentiation: T cell expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma, and are lost after hepatocyte damage. Eur J Immunol 1995; 25: 345229. 19. Ohteki T, Okuyama R, Seki S, Abo T, Sugiura K. Kusumi A, et
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Copyright 8 European Association for the Study of the Liver I999
Journalof Hepatology ISSN 0168-8278
Development of intraepithelialT lymphocytes in the intestine of irradiated SCID mice by adult liver hematopoietic stem cells from normal mice Satoshi Yamagiwa l, Shuhji Seki*, Katsuaki Department
of Immunology
Shirai, Yuhei Yoshida, Chikako Toru Abo
Miyaji, Hisami Watanabe
and
and ‘Third Department of Internal Medicine, Niigata University School of Medicine, Niigata and =Division of Basic Traumatology. National Defense Medical College Research Institute, Tokorozawa, Japan
Background/Aims: We recently reported the adult mouse liver to contain c-kit+ stem cells that can give rise to multilineage leukocytes. This study was designed to determine whether or not adult mouse liver stem cells can generate intraepithelial T cells in the intestine as well as to examine the possibility that adult liver c-kit+ stem cells originate from the fetal liver. Met/&s: Adult liver mononuclear cells, hone marrow (BM) cells, liver c-kit+ cells or bone BM c-kit+ cells of BALB/c mice were i.v. transferred into 4 Gy irradiated CB17/-SCID mice. In other experiments, fetal liver cells from Ly5.1 C57BL/6 mice and T cell depleted adult BM cells from Ly5.2 C57BL/6 mice were simultaneously transferred into irradiated C57BW6 SCID mice (Ly5.2). At 1 to 8 weeks after cell transfer, the SCID mice were examined.
Results: Not only BM cells and BM c-kit+ cells hut also liver mononuclear cells and liver c-kit+ cells reconstituted y8T cells, CD4+CD8+ double-positive T cells and CDSa+~- T cells of intestinal intraepithelial lymphocytes of SCID mice. Injection of a mixture of fetal liver cells from Ly5.1 C57BL/6 mice and adult BM cells from Ly5.2 C57BL/6 mice into Ly5.2 C57BL/6 SCID mice induced both Ly5.1 and Ly5.2 T cells, while also generating c-kit+ cells of both Ly5.1 and Ly5.2 origins in the liver. Conchsions: Adult mouse liver stem cells were able to generate intestinal intraepithelial T cells of the SCID mice, and it is thus suggested that some adult liver stem cells may indeed be derived from the fetal liver.
I
the liver and periphery could develop (4). On the other hand, certain T cells among the intraepithelial lymphocytes (IEL) of the intestine have also been reported to differentiate extrathymically because these T cells exist in the IEL of athymic nude mice (although their number is reduced compared to normal mice) and in thymectomized radiation chimera mice (7-12). IEL T cells contain T cells with unique phenotypes which are rare in the periphery and are different from conventional T cells. These IEL-specific T cells include CD4+ CD8+ (double positive, DP) T cells, CD8ola (CD8afb-) @I cells and yST cells (7-12). IEL T cells also contain selfminor lymphocyte stimulatory Ag specific (potentially autoreactive) V/3T cells which are normally deleted during thymic T cell development (10). The present study was carried out to determine whether adult liver hematopoietic stem cells have the potential to produce these IEL T cells. Our study demonstrates that liver mononuclear cells (MNC) or liver
been believed that the liver ceases to be a hematopoietic organ after birth. However, we previously reported that intermediate T cell receptor (TCR) cells differentiate extrathymically in the liver of mice (l-3), and that adult mouse livers still contain ckit+ hematopoietic stem cells (4). c-kit is an essential molecule for constitutive hematopoiesis and pluripotent stem cells are surface c-kit+ (5,6). When c-kit+ stem cells of the adult liver as well as bone marrow (BM) c-kit+ cells were transferred into irradiated SCID (severe combined immunodeficient) mice that genetically lack T and B cells, it was shown that the SCID mice could be rescued and virtually all lineage cells in T HAS LONG
Received 17 August: revised 5 October: accepted 17 October 1998
Correspondence: Shuhji Seki, Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa 359-85 13, Japan. Fax: 8142 991 1613. E-mail: [email protected]
Key words: Adult liver stem cells; Fetal liver; Intraepithelial T cells of the intestine.
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S. Yumugiwn et al 33% Percoll solution containing 100 U/ml hepdrin and were centrifuged at 2000 rpm for I5 min at room temperature. The pellet was resuspended in an RBC lysis solution, then was washed twice with medium. The BM cells were obtained by flushing the femurs with medium. The cell suspensions were filtered through a 200-gauge nylon mesh to remove any debris. Th~mocytes were obtained by forcing the thymus through a 200-gauge steel mesh. The small intestine was removed and flushed with PBS to eliminate any luminai contents. The mesentery, Peyer’s patches or lymph follicles were then resected. The intestine was opened longitudinally and cut into I-2-cm long fragments, which were incubated for 15 min in 20 ml Ca?‘- and Mg”+free HBSS containing 5 mM EDTA while shaking in a 37°C water bath. The supernatant was then collected and washed. This procedure for removing the epithelium from gut fragments was performed twice. Pellets containing IEL were resuspended. passed through a glass wool column to remove any aggregates and dead cells, and then were washed twice before use. After removing the epithelium. gut fragments were incubated in 25 ml of PBS containing 2% FCS and 0.15 mgiml collagenase for 75 min while shaking in a 37°C water bath. The supernatant and debris were collected and filtered through a steel
c-kit+ hematopoietic stem cells can generate IEL T cells that are similar to those induced by BM cells. Furthermore, we also discuss the origin of adult liver ckit+ cells and host tolerance after liver transplantation.
Materials and Methods Mice Male CB17/-SCID mice (H-2d) (13), BALBic mice (H-2d) and Ly5.2 C57BLi6 (B6) (H-2’) mice were purchased from CLEA Japan Inc. (Tokyo, Japan). Ly5.1 B6 mice (H-zb) (14) were kindly provided by Dr. K. Kishihara (Medical Institute of Bioregulation, Kyushu University, Fukuoka. Japan). Ly5.2 B6 SCID (H-2b) mice were purchased from the Central Institute for Experimental Animals (Kanagawd, Japan). Cell prepurations All mice were euthanized by exsanguination from the subclavian artery and vein, and then the liver and spleen were removed. The spleen was pressed on a 200-gauge sterile mesh and washed. The pellet was treated with a red blood cell (RBC) lysis solution (I 55 mM NH,CI, 10 mM KHC03, 1 mM EDTA, 170 mM Tris, pH 7.3). Hepatic MNC were prepared as previously described (3). Briefly, the liver was pressed through a stainless steel mesh and suspended in Eagle’s MEM medium supplemented with 5 mM HEPES and 2% FCS. After one washing, the cells were resuspended in an osmolarity-and pH-adjusted
Control
Monocionul Abs (mAbs,i, jlowq~tometric anal~~,sis and cell ,sorting Anti-CD4 (RM4-5). anti-CD8 (53-6.7), anti-CD3 (145-2Cl I), antiaIEL (M290, an IEL specific marker) (15). anti-c-kit (3Cl. a stem cells marker), B220 (RA3%6B2, a B cell marker). anti-Mac-l (Ml/70. a myeloid cell marker), anti-Cr.1 (RA3%8CS. a granulocyte marker).
Liver transfer
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Fig. 1. Induction of DP T ~11s in the tl~ynzus und IEL as 12~11as CD4+ or CDS-+ T cells in every organ of‘ the irradiated CBI 7/-SCID mice by the trmsfer qf rrdult liver MNC or BM cells. Control duta WC’ ,from normnl BA LB/‘c mice.
Liver stem cells and T cells of the intestine
Fig. 2. Induction of yST cells, CD8aa (CDSa’~-) T cells and a-IEL+ T cells in IEL of the irradiated SCID mice by the transfer of adult liver MNC or BM cells.
Control I7- l2.6 .I
Liver transfer
BM transfer
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Small intestine + IEL F I
Small intestine g IEL 8 I CD8a
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intestine
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TERl19 (an erythroid lineage marker) and anti-CD45 (30Fll .l, common leukocyte Ag), Ly5.1 (CD45.1) and Ly5.2 (CD45.2) Abs were all purchased from PharMingen (San Diego, CA, USA). All mAbs were used in an FITC, PE or biotin conjugated form. The biotinylated reagents were developed with FITC or PE-conjugated streptavidin (Be&on-Dickinson Co., Mountain View, CA, USA) or TRI-COLORconjugated streptavidin (CALTAG Lab., San Francisco, CA, USA). To prevent nonspecific staining of mAbs, antiCD32/16 Ab (2.4G2, PharMingen) was added before staining with labeled mAbs. The cell suspensions were stained with mAbs and then were analyzed by FACScan (Becton-Dickinson). Any dead cells were excluded by a forward scatter, side scatter and PI gating. The c-kit+ lineage negative (CD3B220- Mac-lGr-ll TER119-) cells of hepatic MNC and BM cells were sorted by FACS Vantage (Becton-Dickinson).
Cell transfer After 4 Gy-irradiation (4), either 1 x 10’ of BM cells or hepatic MNC from BALBlc mice were injected intravenously into CB17/-SCID mice. Fivex lo4 sorted c-kit+ lineage negative cells of hepatic MNC or BM c-kit+ lineage negative cells were also injected into 4 Gy-irradiated CB17/-SCID mice. In some experiments, 5X lo6 fetal liver cells (day 20) from B6 Ly5.1 mice and 5~ lo6 T-cell depleted adult BM cells from B6 Ly5.2 mice were simultaneously injected into 4 Gyirradiated B6 Ly5.2 SCID mice. The T cell depletion of BM cells was done by staining the BM cells with anti-CD3 Ab and then sorting CD3 negative cells.
Results Liver MNC as well as BM cells reconstituted T cells in IEL of irradiated SCID mice
Four weeks after transferring liver MNC or BM cells from BALB/c mice into 4 Gy irradiated CB17/-SCID mice, the SCID mice were examined. As a result, CD4+ or CD8+ single positive T cells were found to appear in all organs tested, while CD4+8+ DPT cells emerged in the IEL and thymocytes (Fig. 1). In addition, liver MNC were able to develop $T cells, CD8a+P(CD8aa) T cells (conventional CD8+ T cells express CD8atj3+) and a-IEL (an IEL specific marker) positive T cells (Fig. 2). Reconstitution of T cells in IEL of irradiated SCID cells from BALB/c mice FiveX lo4 liver c-kit+ cells or BM c-kit+ cells of BALB/ c mice developed DP T cells (Fig. 3) $T cells (Fig. 4) CD8afpT cells and a-IELf T cells in IEL of CB17/-SCID mice (Fig. 5). Furthermore, liver and BM c-
mice by adult liver c-kit?
683
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et al.
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day 14
day 7 $9.7
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Fig. 3. Induction of DP T cells in IEL by the transfer of 5 X IO4 adult liver ckit+ cells as well as BM c-kit+ cells of irradiated SCID mice. SCID mice were examined 4 weeks ufter cell transfer. The control duta are ,from normal BALB/c mice.
Liver c-kit*cell transfer
BM c-kit’cell transfer
-CD8a------
day 7 r
Control
Liver c-kit+cell transfer
BM c-kit*cell
transfer
684
day 14
day 21
Fig. 4. Induction of uPT cells as well as $T cells in the IEL qf irrudiated SCID mice by the transfer of adult liver c-kit+ cells.
Liver stem cells and T cells of the intestine
day 14 7”i/
day 21
day 14
day 21
-1o”Tq-3q
Control
Liver c-kit*cell transfer
BM c=kit*cell transfer CD3 Fig. 5. Induction of CD8cza (CDSa’~-) liver c-kit+ cells.
T cells and u-IEL+ T cells in IEL of irradiated SCID mice by the transfer of adult
kit+ cells were able to induce IEL-specific T cells within 14 days after cell transfer when thymus could not yet provide T cells into the periphery (4) (Fig. 35), thus indicating that IEL can directly differentiate from stem cells in a thymus independent manner. The transfer of fetal liver cells from B6 Ly.5.1 mice together with BM cells from B6 Ly5.2 mice induced both Ly5.1 and Ly.5.2 c-kit+ cells in the liver of B6 SCID mice When a mixture of 5X IO6fetal liver cells from B6 Ly5.1
mice and the same number of T cell-depleted adult BM cells from B6 mice were transferred into 4 Gy-irradiated B6 SCID (Ly5.2) mice, both Ly5.1 cells and Ly5.2 cells (including T and B cells, not shown) appeared in the liver, thymus and small intestine at 8 weeks after the transfer (Fig. 6). In addition, both Ly5.1 and Ly5.2 ckit+ cells were also recognized in the liver (Fig. 7). Thus, liver c-kit+ cells of irradiated B6 SCID mice were derived from both adult BM and the fetal liver.
Discussion Among mouse T cells, liver IL-2Rj?+ T cells with intermediate TCR (l-4) and certain T cells in IEL (7-12)
can differentiate extrathymically. Extrathymic T cells in IEL reportedly include CD8a+P$T cells, CD8a+j3- @3T cells and DPT cells. Although we recently reported that adult liver hematopoietic stem cells can reconstitute both extrathymic T cells and conventional thymus-derived T cells (4), it is still not known whether or not adult liver stem cells can reconstitute IEL T cells. In addition, if they can reconstitute IEL T cells, it is important to determine whether IEL T cells induced by the liver stem cells’are same as those induced by BM stem cells. The present study has demonstrated that the adult liver contains pluripotent stem cells that can develop not only thymic T cells or peripheral T cells, but also IEL T cells. The IEL T cells induced by liver c-kit+ stem cells were essentially the same as those induced by BM c-kit+ cells. The transfer of both fetal liver hematopoietic cells from Ly5.1 mice and BM cells from adult Ly5.2 mice into irradiated B6 SCID mice induced IEL T cells, thymic T cells, peripheral T cells and B cells in B6 SCID mice. Furthermore, c-kit+ cells in the liver of these SCID mice contained both Ly5.1 c-kit+ cells and Ly5.2 c-kit+ cells, thus suggesting that at least some c-kit+ cells in the livers of adult mice originated from the fetal liver.
68.5
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Fig. 6. Induction of LyS.l+ cells and Ly5.2+ cells in irradiated B6 SCID mice by the transfer of a mixture of Ly5.1i fetal liver hematopoietic cells and L~5.2~ BM cells. 5X IO6fetal liver cells of Ly5.1 86 mice and same number of T cell depleted BM cells ,from adult Ly5.2 86 mice were simultaneously transferred into irradiated B6 SCID (Ly5.2) mice. Eight weeks after the cell transfer, the mice were examined.
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IEL T cells are believed to differentiate extrathymically (7-12). The main reasons for this interpretation are as follows: 1) athymic nude mice have CD8a+p- $T cells, CDXa+p- @I’ cells and DPT cells; 2) the IEL of thymectomized and irradiated mice which received BM cells from normal mice contain T cells; 3) CD801tP- c&I cells in IEL contain potentially autoreactive self-Mls Ag-specific VP cells which are generally deleted during normal thymic T cell development (so-called negative selection) (7-12). Similarly, liver intermediate TCR cells develop in a thymus-independent manner because athymic nude mice have intermediate TCR cells (both $ and q!JTCR cells) in the liver, and liver intermediate @TCR cells also include 686
Fig. 7. Appearance of both Ly5.1 and Ly5.2 c-kit+ cells in irradiated B6 SCID mice by the transfer of the mixture of LyS. I + .fetal liver hematopoietic cells and Ly,5.2+ BM cells. Eight weeks after the cell transfer, the mice were examined. Ly5.1’ cells or L,y5.2’ cells were gated and their CD45 (common leukocyte Ag) and c-kit expressions were anulyzed by a 3-color flowcytometric analvsis.
self-minor lymphocyte stimulatory Ag-specific T cells (2,3). Moreover, thymectomized and irradiated mice which received T cell depleted BM cells can also develop intermediate TCR cells (1). Another notable finding is that adult liver c-kit+ stem cells likely include descendants of fetal liver stem cells. Although we did not rule out the possibility that stem cells of the adult mouse liver might come from BM, our findings suggest that adult liver stem cells are also derived from the fetal liver. In line with these lindings, it was also reported that a number of c-kit+ cells or hematopoietic cells still remain after birth in the liver, while their number gradually decreases with age (l&17). In fact, liver MNC of both neonatal mice and
Liver stem cells and T cells of the intestine
young mice successfully reconstitute lymphoid cells in lethally irradiated hosts (our unpublished observation). Thus, certain stem cells of fetal liver remain in the liver after birth. Therefore, although the degree of hematopoiesis in the liver decreases after birth, it is reasonable to think that liver hematopoiesis continues after birth into adulthood. Since hepatocytes (but not Kupffer cells) are essential for the development of liver intermediate TCR cells (18) and liver perfusion from the portal vein does not affect the number of c-kit’ cells (4), stem cells in the adult liver may indeed reside and differentiate in Disse’s space. We earlier reported that liver MNC gradually increase with age because of the increase in the number of extrathymic intermediate TCR cells (2,3,19). In addition, these intermediate TCR cells gradually increase in the periphery with age, whereas the thymus rapidly involutes with age and therefore the number of the thymus-derived high TCR cells decreases in the periphery (2,3,19). This phenomenon (the increase and activation of liver MNC and the involution of the thymus) is also almost always observed in bacterial infections, malignancies, autoimmune diseases and pregnancy (see review 2). These phenomena can also be regarded as liver hematopoiesis. Furthermore, this study has also shown an additional capacity of the adult liver, namely that it can provide IEL T cell precursors to the intestine. It has also been recognized that a successful liver transplantation facilitates other organ engraftments (20,21), including the kidney, pancreas and the intestine. This phenomenon can be explained by the fact that either donor liver MNC or precursors migrate to other organs and the periphery of the host, and results in a donor-host chimera (the presence of both donor and host leukocytes in the host) (20) inducing resultant host tolerance, and these phenomena are quite consistent with the present results. Liver parenchymal cells also seem to be important because the soluble class I Ags produced by donor liver (but not by other organs) may play a role in inducing host tolerance (21). Bone marrow cells from athymic nude mice generate IEL T cells in thymectomized and irradiated mice, although the number of IEL T cells is considerably smaller than in radiation chimera mice without a thymectomy (12). It has also been suggested that thymic hormones (or cytokines including IL-7) or thymic epithelial cell factors are needed for the effective expansion of extrathymic T cells in the intestine (12,22) and the liver (23,24) of nude mice. Therefore, although liver stem cells from normal mice quickly migrate to the intestine and the liver of SCID mice and generate IEL T cells (as demonstrated in this study) as well as liver intermediate TCR cells (4) within 2 weeks after trans-
fer (before thymic reconstruction), the effective expansion of extrathymic T cells may thereafter be affected by the either thymus or its factors (22-34). Cryptopaches of the intestine were recently reported to have a large number of lymphocytes from 1417 days after birth and thereafter (25). These lymphocytes contain a large fraction of c-kit+ TCR/CD3- IL-7R+ stem cells. Moreover, these intestinal c-kit+ cells from nude mice (which are apparently thymus independent) could also generate IEL T cells without thymic reconstitution when injected into irradiated SCID mice (26). However, it should be noted that liver c-kit+ cells (up to 30% are IL-7R+, our unpublished data), but not intestinal c-kit+ cells, are pluripotent because only the former can generate thymocytes (regular T cells), B cells, erythroid and myeloid lineage cells (4,26,27). Although c-kit+ cells in the intestine are probably provided from the fetal liver and develop in situ into T cells after birth, the possibility cannot be ruled out that intestinal c-kit+ cells originate in the liver and/or the bone marrow after birth. The present results may partly explain why liver transplantation leads to the successful engraftment of the small intestine, since the engrafted liver provides donor leukocytes to the recipients and also creates a donor-host chimera while inducing immune tolerance. Finally, it is interesting to note that bacterial Ags including LPS (endotoxin) are continuously brought from the intestine to the liver via the portal vein (28) and activate liver MNC (29). Therefore, the present results suggest the possibility that lymphoid systems of the liver and the intestine may affect each other.
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S. Yamagiwa et al. 8. Bandeira A, Itohara S, Bonneville M, Burlen-Defranoux 0, Mota-Santos T, Coutinho A, et al. Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor gamma delta. Proc Nat1 Acad Sci USA 1991; 88: 43-7. 9. Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991; 173: 471-81. 10. Rocha B. Vassalli P Guy-Grand D. The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta’ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J Exp Med 1991; 173: 483-86. 11. Poussier P, Edouard P, Lee C, Binnie M, Julius M. Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J Exp Med 1992; 176: 187-99. 12. Rocha B. Vassalli P, Guy-Grand D. Thymic and extrathymic origins of gut intraepithelial lymphocyte populations in mice. J Exp Med 1994; 180: 68 1-6. 13. Bosma GC, Custer RP, Bosma MJ. A severe combined immunodeficiency mutation in the mouse. Nature 1983: 301: 527-30. 14. Morse HC, Shen FW, Hammerling U. Genetic nomenclature for loci controlling mouse lymphocyte antigens. Immunogenetics 1987; 25: 71-8. 15. Kilshaw PJ, Murant SJ. A new surface antigen on intraepithelial lymphocytes in the intestine. Eur J Immunol 1990; 20: 2201-7. 16. Taniguchi H, Toyoshima T. Fukao K, Nakauchi H. Presence of hematopoietic stem cells in the adult liver. Nat Med 1996; 2: 1988 203. 17. Kawamura T, Toyabe S, Moroda T, Iiai T, Takahashi-Iwanaga H, Fukuda M, et al. Neonatal granulocytosis is a postpartum event which is seen in the liver as well as in the blood. Hepatology 1997; 26: 157672. 18. Kawachi Y, Arai K, Moroda T, Kawamura T, Umezu H, Naito M. et al. Supportive cellular elements for hepatic T cell differentiation: T cell expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma, and are lost after hepatocyte damage. Eur J Immunol 1995; 25: 345229. 19. Ohteki T, Okuyama R, Seki S, Abo T, Sugiura K. Kusumi A, et
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