Antigen-Specific Primary Activation of CD8  T Cells Within the Liver 1 Patrick Bertolino, David G. Bowen, Geoffrey W. McCaughan, and Barbara Fazekas de St. Groth 2 It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8  T cells specific for H-2K b were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-K b mice expressing H-2K b in the liver. Activated CD8  cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8  cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8  T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8  T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis. The Journal of Immunology, 2001, 166: 5430 –5438. I t is widely accepted that naive and activated/memory T cells follow different recirculation pathways. Although both cell subsets enter lymphoid organs from the bloodstream, only activated T cells can undergo transendothelial migration into non- lymphoid organ parenchymal tissue (1, 2). Naive T cells become activated in lymph nodes or spleen by interacting with dendritic cells (DCs) 3 expressing peptide/MHC complexes. Primary activa- tion results in blast formation, cytokine secretion, proliferation, and differentiation to a state in which cells express the integrins and chemokine receptors required to undergo transendothelial migration. In contrast to most nonlymphoid tissues, the liver possesses an unusual endothelial barrier consisting of a single layer of fenes- trated endothelial cells that do not form tight junctions (3–7). Moreover, hepatocytes and endothelial cells are not separated by a basement membrane, but define the space of Disse, a compartment communicating directly with the blood vessel lumen. It is therefore possible that naive CD8 + T lymphocytes circulating via the blood can interact directly with hepatocytes through T cell cytoplasmic extensions penetrating the space of Disse. Hepatocyte/T cell con- tact could occur either via the gap between two adjacent endothe- lial cells, or through the endothelial cell fenestrae. Experimental data suggest that liver permeability to naive (8) and differentiated CD8 + T cells (5) differs from that of other organs. However, none of these studies has addressed whether naive CD8 + T cells can be directly activated in the liver. This question has wide relevance with regard to liver immunobiology, viral hepatitis, and liver trans- plantation, as the liver has been implicated in inducing T cell tol- erance (7, 9 –11). To address this issue, we investigated the early events of acti- vation and proliferation of TCR transgenic CD8 + T cells injected into transgenic mice expressing the relevant alloantigen in the liver. Selective retention of autoreactive T cells was seen in the liver within minutes of transfer. Expression of the very early ac- tivation marker CD69 could be detected in the liver, but not blood, as early as 2 h posttransfer. Activated cells started to divide and recirculate via the blood at 24 h. To our knowledge, this is the first report demonstrating that naive T cells can undergo primary acti- vation outside lymphoid organs. This study further challenges the concept that danger and inflammation associated with tissue dam- age are essential for T cell activation and migration into the liver (12, 13). Materials and Methods Mice All mice were maintained in the Centenary Institute animal facility under specific pathogen-free conditions. Met-K b transgenic mice expressing the H-2K b gene in hepatocytes under the control of the sheep metallothionein promoter have been described previously (14) and were a gift of Grant Morahan and J. F. A. P. Miller (Walter and Eliza Hall Institute (WEHI), Melbourne, Australia). No zinc induction of Met-K b mice was performed. The 178.3 transgenic mice expressing the H-2K b gene under the control of its own promoter were provided by William Heath (WEHI) and have been described previously (15). The Des-TCR transgenic mice expressing an H-2K b -specific TCR (16) identifiable by a clonotypic Ab, De ´sire ´ (17), were kindly provided by B. Arnold, G. Scho ¨nrich, and G. J. Ha ¨mmerling (Deut- sches Krebsforschungs Zentrum, Heidelberg, Germany). Met-K b , 178.3, and Des-TCR transgenic mice were maintained by breeding with B10.BR mice obtained from the Animal Resources Center (Perth, Western Austra- lia, Australia). A Ly-5.1 allele was introduced into Met-K b mice by cross- ing with a line of H-2 k Ly-5.1 homozygous mice bred in house from F 2 crosses between B6.SJLPtprc a (H-2 b , Ly-5.1) and B10.BR (H-2 k , Ly-5.2) mice purchased from the Animal Resources Center. Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Australia Received for publication September 28, 2000. Accepted for publication February 22, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Health and Medical Research Council of Australia (NH&MRC). P.B. was supported by a U2000 fellowship from the Univer- sity of Sydney. D.G.B. was supported by an NH&MRC Medical Postgraduate Re- search Scholarship. B.F. is an NH&MRC Principal Research Fellow. 2 Address correspondence and reprint requests to Dr. Barbara Fazekas de St. Groth, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag Number 6, Newtown NSW 2042, Australia. E-mail address: b.fazekas@centenary.usyd.edu.au 3 Abbreviations used in this paper: DC, dendritic cell; FSC, forward scatter; MFI, mean fluorescence intensity; PI, propidium iodide. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00