The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity (original) (raw)
Competition between liver and LNs for primary T cell activation. We have previously examined events following the adoptive transfer of CD8+ Des-TCR T cells, which express an H-2Kb–specific TCR (27), into syngeneic Met-Kb mice on an H-2k background. These studies demonstrated that TCR transgenic CD8+ T cells were specifically retained in the liver of unmanipulated Met-Kb mice within minutes of adoptive transfer and underwent simultaneous activation within both LNs and liver, but not other organs (16). Consistent with these observations, one of the earliest markers of activation, CD69, was expressed on donor transgenic T cells as early as 2 hours after adoptive transfer (16). As Des-TCR LN cells also contain CD8+ T cells previously activated via a second TCR present because of incomplete allelic exclusion of the endogenous α chain gene (28), we thus investigated whether CD69 was expressed within the naive population of CD8+ Des-TCR T cells, which would not have upregulated CD44 at this very early time point. At 2 hours 15 minutes following adoptive transfer, CD44low T cells expressed CD69 in both the LNs and the liver, but not the blood, of Met-Kb mice; a small population of cells with increased but lower CD69 expression was seen in the spleen (Figure 1). This activation was antigen-specific, as CD69 expression by CD8+ Des-TCR+ T cells was not upregulated in nontransgenic recipients (Figure 1). These experiments demonstrate that CD69 expression occurred within the naive T cell population and confirm that the liver is a site of primary activation for CD8+ T cells. Adoptive transfer of Des-TCR donor T cells deficient for RAG-1, in which there is no recombination of the endogenous α chain and all transferred T cells were naive, confirmed these results (data not shown).
Early activation of naive CD8+ Des-TCR T cells occurred independently within the liver and LNs of Met-Kb mice. CFSE-labeled Des-TCR LN cells were adoptively transferred into Met-Kb and control B10.BR mice, and organs were harvested 2 hours 15 minutes later. Lymphocytes isolated from blood, liver, spleen, and LNs were analyzed by flow cytometry. Representative histograms show CD69 expression by CFSE+ CD8+ CD44low cells within a forward and side scatter gate appropriate for lymphocytes.
Interestingly, early antigen-specific T cell retention within the liver was associated with reduced numbers of transgenic CD8+ T cells in the LNs (data not shown and ref. 16), which indicated that T cells retained within the liver were initially withdrawn from the circulation, resulting in a lower number of cells gaining access to the LNs. Primary T cell activation within the liver was associated with loss of the LN-homing receptor CD62L (data not shown), making this process irreversible. These results indicated that antigen-expressing liver competes with LNs for retention and primary T cell activation at early time points.
The occurrence of independent synchronous primary activation in the liver and LNs raised the question of whether the site of primary activation could determine differential outcomes in terms of effector function. To examine the relative contribution of intrahepatic versus intranodal primary activation to liver damage, we investigated whether immune-mediated hepatitis could develop when LN entry by naive CD8+ Des-TCR T cells was prevented.
Anti-CD62L mAb prevented LN entry by CD8+ Des-TCR T cells without affecting intrahepatic T cell activation. LN entry by naive T cells is largely mediated by interactions between L-selectin (CD62L) and peripheral LN addressins expressed by the high endothelial venules of LNs (29). Naive T cells express high levels of CD62L, allowing them to preferentially enter LNs. CD62L is rapidly lost from the surface of cells following primary activation via proteolytic cleavage, with a 90% reduction in expression by 4 hours (30). A number of investigators have effectively blocked the majority of LN entry by both naive CD4+ and CD8+ T cells via the administration of anti-CD62L mAb (31–33). To study events occurring in Met-Kb mice in the absence of LN entry by naive CD8+ Des-TCR T cells, this strategy was therefore adopted in this model.
Intraperitoneal administration of a single dose of 250 μg of the anti-CD62L mAb Mel14 to Met-Kb mice 4 hours prior to adoptive transfer of CD8+ Des-TCR LN cells, a similar protocol to that used in previous studies, led to an approximately 90% fall in the number of CD8+ Des-TCR T cells recovered from the LNs 4 hours after transfer in comparison with control recipients (Figure 2A). Consistent with previous data indicating that entry of naive lymphocytes into the spleen is independent of CD62L (32, 34), the number of CD8+ Des-TCR T cells observed in this organ was unchanged. Interestingly, consistent with the competitive model mentioned above, there was no decrease, but rather an increase, in recruitment of CD8+ Des-TCR T cells to the livers of Met-Kb mice. In addition, antibody treatment did not affect the total number of CD8+ Des-TCR T cells recovered from recipients (Figure 2A). Intrahepatic primary activation of CD8+ Des-TCR T cells in Met-Kb mice was not altered by the prior administration of anti-CD62L antibodies, as assessed by CD69 expression at 4 hours after adoptive transfer (Figure 2B). Furthermore, the administration of anti-CD62L mAb had no effect on the recruitment into division or proliferation of adoptively transferred CD8+ Des-TCR T cells within the livers of Met-Kb mice at 2 days following adoptive transfer, as assessed by decreased CFSE content (Figure 2C). Similar results were obtained using splenectomized mice (Figure 2C), demonstrating that the spleen did not significantly contribute to the pool of proliferating CD8+ Des-TCR T cells observed within the liver. It is important to note that, in the absence of anti-CD62L mAb treatment, T cells activated within the liver and LNs were recruited into division and proliferated at very similar rates, irrespective of whether splenectomy was performed (Figure 2D). These results suggest that in contrast to the situation in the LNs, CD62L is not involved in the retention and subsequent activation and proliferation of naive CD8+ T cells within the liver.
Administration of anti-CD62L antibody reduced LN entry by CD8+ Des-TCR T cells but did not affect their intrahepatic activation or proliferation in Met-Kb mice. (A) Total number of Ly5.1– CD8+ Des-TCR T cells in various organs of Ly5.1+ Met-Kb mice pretreated with anti-CD62L mAb or PBS and injected 4 hours later with Ly5.1– Des-TCR LN cells. Organs were harvested 4 hours after cell transfer. (B) CD69 expression by donor Ly5.1– CD8+ Des-TCR T cells (solid line) versus recipient Ly5.1+ CD8+ T cells (dotted line) within the livers of Met-Kb mice at 4 hours after adoptive transfer. Representative histograms were gated on forward and side scatter gates appropriate for lymphocytes, and Ly5.1– CD8+ PI– cells for CD8+ Des-TCR T cells or Ly5.1+ CD8+ PI– cells for liver resident non–Des-TCR CD8+ T cells. (C) CFSE profiles of donor CD8+ Des-TCR T lymphocytes, at 2.5 days after transfer, isolated from non-splenectomized and splenectomized Met-Kb (shaded plot) and B10.BR (dashed line) mice preadministered control purified rat IgG, or Met-Kb mice preadministered anti-CD62L mAb (solid line). CD8+ Des-TCR T cells did not proliferate in the livers of B10.BR mice administered anti-CD62L (data not shown). (D) CFSE profiles of donor CD8+ Des-TCR lymphocytes isolated from the liver (solid line) and LNs (shaded plot) of Met-Kb mice administered control rat IgG illustrate that donor T cells activated at these 2 sites proliferated at a similar rate.
Interference with LN entry by CD8+ Des-TCR T cells prevented development of hepatitis in Met-Kb mice. To determine the effects of blocking the majority of LN entry by naive CD8+ Des-TCR T cells on the development of hepatitis in Met-Kb mice, anti-CD62L mAb was administered to Met-Kb mice 4 hours prior to adoptive transfer of Des-TCR LN cells, and then daily for 5 days. To exclude the possibility that observed effects may be due to intrasplenic primary activation, experiments were carried out in both intact and splenectomized Met-Kb mice. Consistent with previous results, control recipients developed a significant but self-limited hepatitis, peaking at day 6 (Figure 3A). However, treatment with anti-CD62L mAb led to a marked decrease in hepatitis, based on alanine aminotransferase (ALT) levels, in both unmanipulated and splenectomized animals (Figure 3A). Similar results were obtained when anti-CD62L mAb was administered 4 hours before adoptive transfer of Des-TCR LN cells and then once 24 hours after adoptive transfer (Figure 3B), which suggests that the effects of initial mAb blockade were long-lasting and/or irreversible. Consistent with the marked decrease in ALT levels, immunohistochemistry performed on liver sections using an anti-CD8 mAb demonstrated rare and scattered lobular and portal tract infiltrates in the livers of anti-CD62L–treated animals (data not shown). In sharp contrast, significant lobular and portal infiltrates were present in the livers of control Met-Kb animals treated with control Ig (data not shown).
Administration of anti-CD62L mAb abrogated the development of hepatitis in Met-Kb mice following adoptive transfer of CD8+ Des-TCR T cells. (A) Non-splenectomized and splenectomized Met-Kb mice were preadministered anti-CD62L mAb (solid line) or PBS alone (dotted line) as described in Figure 2A. Further doses of mAb or PBS alone were administered intraperitoneally to mice at daily intervals until day 5 after adoptive transfer. Hepatocyte damage was assessed by measurement of serum levels of ALT. Data points represent means ± SEM for groups of 3 mice. (B) Unmanipulated Met-Kb mice and B10.BR mice were administered anti-CD62L mAb or purified rat IgG. A further dose of antibodies was administered intraperitoneally 24 hours after adoptive transfer. Data points represent serum ALT values from individual mice at day 5.
These results indicate that activation of transgenic T cells within the LNs of Met-Kb mice was required for the development of hepatitis. Moreover, despite efficient primary activation and recruitment into proliferation, T cells activated within the liver did not cause hepatitis.
Alb-Kb mice failed to develop hepatitis. To exclude the possibility that inhibition of hepatitis was due to secondary effects of the anti-CD62L mAb, and to confirm the determinant role of intranodal T cell activation in the development of hepatitis, the contribution of intranodal versus intrahepatic primary activation to hepatitis was investigated using Alb-Kb mice. In contrast to Met-Kb mice, previous studies have indicated that Alb-Kb mice do not develop hepatitis following adoptive transfer of CD8+ Des-TCR T cells in the absence of preexisting inflammation (24). It has been argued that discrepancies between these models may be due to different MHC backgrounds used in each model (H-2k versus H-2dk) and differing levels or sites of transgenic expression (24, 26).
To determine whether the genetic background influenced the response of Des-TCR T cells, Alb-Kb and Met-Kb mice were both bred onto the same H-2dk background and assayed for the development of hepatitis upon transfer of syngeneic Des-TCR (H-2dk) LN cells. Similar to Met-Kb (H-2k) mice, Met-Kb (H-2dk) mice developed hepatitis following adoptive transfer of Des-TCR (H-2dk) LN cells, suggesting that the H-2dk background did not abrogate the occurrence of hepatitis in Met-Kb mice (Figure 4A). In contrast to Met-Kb mice and consistent with previous results, Alb-Kb mice injected with Des-TCR (H-2dk) LN cells did not develop biochemical hepatitis (Figure 4A). A significant difference between Met-Kb and Alb-Kb mice in the development of hepatitis was also observed upon transfer of Des-TCR RAG-1–/– LN cells (data not shown). These results were confirmed at the histological level on day 6, the biochemical peak of hepatitis in the Met-Kb model (data not shown). While Met-Kb (H-2dk) mice developed hepatitis associated with significant CD8+ T cell lobular and portal infiltrates, only minor CD8+ infiltrates were observed in the livers of Alb-Kb (H-2dk) mice (data not shown).
Hepatitis did not occur in Alb-Kb mice on the same genetic background as Met-Kb mice following adoptive transfer of Des-TCR LN cells, despite similar levels of antigen expression by hepatocytes. (A) Serum ALT levels of H-2dk recipient Met-Kb, Alb-Kb, and control mice following adoptive transfer of Des-TCR (H-2dk) LN cells. Data points represent means ± SEM for groups of 4 mice. (B) H-2Kb expression by viable (PI– ) hepatocytes from H-2dk Alb-Kb, Met-Kb, and control mice, analyzed by flow cytometry.
Differences in these models were not explained by higher levels of H-2Kb expression by Met-Kb hepatocytes: Alb-Kb hepatocytes expressed H-2Kb levels that were comparable to C57BL/6 (H-2b) WT levels and 2–3 times higher than the H-2Kb levels expressed by Met-Kb hepatocytes (Figure 4B). Furthermore, H-2Kb expression could not be detected on Kupffer cells, liver DCs, or liver sinusoidal endothelial cells from either Alb-Kb or Met-Kb mice by flow cytometry (Supplemental Figure 1A; supplemental material available at http://www.jci.org/cgi/content/full/114/5/701/DC1). Since levels of intrahepatic H-2Kb expression did not explain why hepatitis occurred in Met-Kb but not in Alb-Kb mice, we investigated whether differences between the 2 models correlated with differences in intranodal H-2Kb expression.
Failure to induce hepatitis in Alb-Kb mice was associated with lack of Des-TCR T cell activation within LNs. To establish whether the lack of hepatitis in the Alb-Kb (H-2dk) model was due to failure to induce Des-TCR T cell activation in the LNs, CFSE-labeled Des-TCR (H-2dk) LN cells were adoptively transferred into Alb-Kb (H-2dk), Met-Kb (H-2dk), and control nontransgenic (H-2dk) recipients, and CD69 expression was analyzed 2 hours 15 minutes after transfer. Consistent with results in Met-Kb (H-2k) mice (Figure 1), CD8+ Des-TCR T cells were found to express CD69 in both the liver and the LNs of Met-Kb (H-2dk) mice (Figure 5A). In common with Met-Kb (H-2dk) mice, CD8+ Des-TCR T cells in the livers of Alb-Kb mice expressed CD69, which suggested they had been activated in situ. However, in contrast to findings in Met-Kb mice, CD69 expression levels of CD8+ Des-TCR T cells in the LNs of Alb-Kb mice were comparable to those in control mice, indicating that in this model, primary activation of Des-TCR T cells did not occur within the LNs (Figure 5A).
CD8+ Des-TCR T cells were activated within the liver and proliferated following adoptive transfer into H-2dk recipient Alb-Kb and Met-Kb transgenic mice. (A) Alb-Kb, Met-Kb, and control mice were injected with CFSE-labeled syngeneic Des-TCR (H-2dk) LN cells (top panels) or Des-TCR RAG-1–/– (H-2k) LN cells (bottom panels). Lymphocytes were purified from LNs, liver, spleen, and blood of recipient mice at 2 hours 15 minutes following adoptive transfer and analyzed by flow cytometry. Representative histograms show CD69 expression by CFSE+ CD8+ CD44low cells within a forward and side scatter gate appropriate for lymphocytes. (B and C) Alb-Kb, Met-Kb, and control mice were injected with CFSE-labeled syngeneic Des-TCR (H-2dk) LN cells containing 5 × 106 transgenic CD8+ T cells. Lymphocytes were purified from LNs, liver, spleen, and blood of recipient mice at 2.5 days (B) or 6 days (C) following adoptive transfer and analyzed by flow cytometry. Histograms represent CFSE division profiles of CD8+ Des-TCR+ PI– cells within a forward and side scatter gate appropriate for lymphocytes. (D and E) Total numbers of Des-TCR+ CD8+ cells expressing upregulated levels of CD43 activation–associated glycoform in various compartments of Alb-Kb, Met-Kb, and B10.BR mice. CFSE-labeled LN cells from Des-TCR mice were adoptively transferred into Alb-Kb, Met-Kb, and B10.BR mice. Organs were harvested at day 2.5 (D) or day 6 (E), and lymphocytes were isolated and analyzed by flow cytometry. Cell numbers for blood represent Des-TCR CD8+ T cells/ml; plots represent means ± SEM of groups of 3–4 mice.
It should be noted that some CD8+ Des-TCR T cells in the livers of both Met-Kb and Alb-Kb mice did not upregulate CD69 expression (Figure 5A). These cells probably represented the CD8+ Des-TCR T cells expressing high levels of endogenous TCR α chain, which consequently did not interact with high enough avidity with cells expressing H-2Kb to trigger activation. To explore this possibility, uniformly transgenic TCR-bearing Des-TCR RAG-1–/– LN cells were adoptively transferred into recipients equivalent to those described above. Consistent with results using Des-TCR (H-2dk) donor LN cells, Des-TCR RAG-1–/– T cells were activated in both the livers and the LNs of Met-Kb (H-2dk) mice, whereas an increased proportion of cells upregulating CD69 were identified only in the livers of Alb-Kb (H-2dk) mice (Figure 5A). However, in contrast to Des-TCR (H-2dk) T cells, the majority of CD8+ Des-TCR RAG-1–/– T cells recovered from these compartments expressed CD69, consistent with the hypothesis that intrahepatic activation was restricted to CD8+ T cells capable of high-avidity interaction with intrahepatic antigen-expressing cells.
Proliferation of Des-TCR T cells occurred following activation within the livers of Alb-Kb mice, as assessed by decreasing CFSE content of donor CD8+ cells at 2.5 and 6 days following adoptive transfer (Figure 5, B and C). Fewer dividing cells were present in the LNs or blood, consistent with primary activation occurring within the liver with subsequent recirculation (16). Confirming significant proliferation, the total number of CD8+ Des-TCR T cells upregulating expression of the CD43 activation–associated glycoform, a marker associated with CTL activity (35) and cell division (data not shown), was increased approximately 25-fold within the livers of Alb-Kb recipients over that observed in control antigen-free recipients at day 6 (Figure 5E). Consistent with activation and proliferation of CD8+ Des-TCR T cells being restricted to the livers of Alb-Kb mice, the number of CD43 activation–associated glycoformhigh CD8+ Des-TCR T cells recovered from other organs of Alb-Kb mice did not differ from background levels in antigen-free control animals at either day 2.5 or day 6 (Figure 5, D and E). Although the total number of effector donor T cells increased significantly in the livers of Alb-Kb mice, it was lower than the number recovered from Met-Kb livers; and while the numbers between the 2 samples differed only 2- to 3-fold at day 2.5 after transfer, this difference increased to 130-fold at day 6. This rising disparity was probably due to the increasing contribution of LN-activated T cells recirculating and accumulating within the livers of Met-Kb mice, whereas, because of the absence of intranodal activation, these cells were absent in Alb-Kb recipients. It is interesting to note that although adoptively transferred CD8+ Des-TCR T cells underwent significant division in both Met-Kb and Alb-Kb mice, a higher proportion of transgenic T cells was recruited into division in Met-Kb liver than in Alb-Kb liver, as judged by the proportion of undivided cells at days 2.5 and 6 (Figure 5, B and C). This difference did vary somewhat from experiment to experiment (compare Figure 5C with Figure 7D) and may be due to higher-avidity interactions resulting from metallothionein promoter–driven transgene upregulation that occurred following infiltration (data not shown). This factor might also contribute to the difference in the total number of effector donor T cells recovered from the livers of Met-Kb and Alb-Kb mice.
CD8+ Des-TCR T cells activated within the liver exhibited defective CTL function. (A) Representative histograms of CD43 activation–associated glycoform expression by CD8+ Des-TCR cells in livers of Met-Kb mice treated with anti-CD62L (thin line) or control IgG (thick line). Organs were harvested 2.5 days after transfer of Des-TCR LN cells. (B) CTL activity of CD8+ Des-TCR cells harvested from the livers of Met-Kb mice treated with anti-CD62L (open squares) or control IgG (filled squares) toward P815-Kb target cells at 2.5 days after transfer. The effector/target (E/T) ratio was calculated by determination of the exact number of transgenic T cells present in the well. CTL activity toward P815 control target cells was not detected (data not shown). (C and D) Representative CD43 activation–associated glycoform histograms (C) and CFSE profiles (D) of CD8+ Des-TCR cells from the livers of Alb-Kb (H-2Kdk) (thin line) and Met-Kb (H-2Kdk) mice (thick line). Organs were harvested 6 days after transfer of Des-TCR (H-2Kdk) LN cells, and lymphocytes were prepared and analyzed by FACS. All plots were gated on forward and side scatter gates appropriate for lymphocytes and CD8+ Des-TCR+ PI– cells. (E) CTL activity of CD8+ Des-TCR cells harvested from the livers and LNs of Alb-Kb (H-2Kdk) (round symbols) and Met-Kb (H-2Kdk) mice (square symbols) incubated with P815 (open symbols) or P815-Kb target cells (filled symbols). Organs were harvested 6 days after transfer of Des-TCR (H-2Kdk) LN cells. The effector/target ratio was calculated by determination of the exact number of transgenic T cells present in each well.
Collectively, these experiments indicate that differences in the site of antigen expression and hence the setting of T cell activation are responsible for the disparity in pathology between the Met-Kb and Alb-Kb models. Combined with the results obtained following the administration of anti-CD62L mAb, these results suggest that naive CD8+ T cells activated within the LNs and the liver follow different pathways of differentiation that result in divergent immune responses. To better understand the mechanisms underlying these pathways, we compared the function and survival of intrahepatically and intranodally activated CD8+ T cells.
CD8+ T cells activated within the liver of Met-Kb mice demonstrated reduced half-life. Results presented above indicated that primary activation of CD8+ Des-TCR T cells within the liver led to an immune response insufficient to cause hepatitis, a phenomenon that may have been due to a quantitative and/or a qualitative defect; that is, due to a deficiency in the number of CTLs generated and/or a defect in CTL function. T cells activated in either Met-Kb or Alb-Kb mice undergo peripheral deletion by 30 days after transfer (16, 36). However, whether T cells activated in the liver and LNs acquire divergent programs of differentiation during this period, leading to variations in half-life, still remained unknown. In order to determine whether T cell activation within the liver or LNs generated different T cell numbers, the number of CD8+ Des-TCR T cells present in various organs of Met-Kb and control B10.BR mice was quantified at 2.5 and 6 days after adoptive transfer, following treatment with anti-CD62L or control Ig. Administration of anti-CD62L mAb to control B10.BR mice did not alter the number of CD8+ Des-TCR T cells recovered from various organs, including LNs (Figure 6, A and B), which indicates that the failure to develop immune-mediated hepatitis in Met-Kb mice was not secondary to anti-CD62L–specific complement-mediated destruction of CD8+ Des-TCR T cells. These results further suggest that despite the initial blocking effect of anti-CD62L mAb at 4 hours after T cell transfer (Figure 2A), naive transgenic T cells were able to again home to the LNs at later time points after mAb treatment; this indicates that the effect of anti-CD62L was not long-lasting when T cells remained naive. In Met-Kb mice, as a result of primary activation at these sites, high numbers of transgenic T cells were recovered from both liver and LNs at 2.5 days after adoptive transfer (Figure 6A). However, in contrast to B10.BR mice, the total number of transgenic T cells recovered from the LNs was reduced in anti-CD62L–treated Met-Kb mice compared with Met-Kb mice treated with control Ig (Figure 6A). This phenomenon was also observed in splenectomized animals (Figure 6A). The apparently long-lasting effects of anti-CD62L mAb treatment in Met-Kb but not B10.BR animals would probably be favored by loss of CD62L expression following activation within the H-2Kb–expressing liver, which would prevent transgenic T cells from homing to the LN.
Intrahepatic activation of CD8+ Des-TCR T cells was associated with reduced half-life. (A and B) Administration of anti-CD62L mAb to Met-Kb mice resulted in reduced CD8+ Des-TCR T cell numbers 2.5 and 6 days after adoptive transfer. Unmanipulated and splenectomized Met-Kb and B10.BR mice were preadministered anti-CD62L mAb or purified rat IgG as described in Methods. A further dose of antibodies was administered intraperitoneally 24 hours after adoptive transfer. Organs were harvested at day 2.5 (A) and day 6 (B), and lymphocytes were analyzed by flow cytometry. Cell numbers for blood represent CD8+ Des-TCR T cells/ml; cell numbers for other organs represent total CD8+ Des-TCR T cells from the organ. Total cell numbers represent the sum of CD8+ Des-TCR T cells retrieved from these organs. Plots represent means ± SEM for groups of 3 mice. (C) T cells activated within the liver are committed to reduced half-life within the first 24 hours following activation. Equal numbers of CFSE-labeled Des-TCR LN cells activated for 24 hours in either liver or LNs of Met-Kb mice were adoptively transferred into a second antigen-free, nontransgenic B10.BR recipient, and 30 days later, lymphocytes from liver, LNs, and spleen were purified as described in Methods. Bars indicate the total number of CD8+ CFSE+ Des-TCR+ cells harvested from the 3 compartments. Plots represent means ± SEM for groups of 3 mice.
The reduced number of CD8+ Des-TCR T cells recovered from the LNs of recipient anti-CD62L–treated Met-Kb mice at 2.5 days was not associated with increased numbers of transgenic T cells at extranodal sites, as CD8+ Des-TCR T cell numbers in livers, blood, and spleens of anti-CD62L–treated animals were comparable to those observed in control Ig–treated animals (Figure 6A). Therefore, the total numbers of transgenic T cells recovered were lower in recipient anti-CD62L–treated Met-Kb mice than in mice treated with control Ig (Figure 2D). It could be argued that in the unmanipulated animal, greater numbers of T cells reaching the LNs than the liver would lead to a higher number of cells proliferating within this organ, thus resulting in the generation of lower total T cell numbers in anti-CD62L–treated recipients, in which this mode of activation is blocked. However, this line of reasoning would hold only if, following anti-CD62L treatment, there was loss of cells that would normally enter the LNs, or if recruitment into division or proliferation rates were different in the organs into which cells were diverted and activated. Analysis soon after cell transfer, before division had occurred, demonstrated that there was no early cell loss associated with antibody treatment (Figure 2A). In addition, despite inhibition of T cell entry into LNs, transgenic T cells recovered from anti-CD62L–treated recipient animals both were recruited into division and proliferated at a rate similar to that of transgenic T cells from animals treated with control Ig (Figure 2C). Furthermore, it should be noted that in the absence of anti-CD62L treatment, recruitment into division of transgenic T cells and their proliferation rate were similar within liver and LNs (Figure 2D). These findings were similar irrespective of whether splenectomy was performed. Overall, these results indicate that most transgenic T cells were diverted and activated within the liver of anti-CD62L–treated animals, where recruitment into division and proliferation rates were similar to those in the LNs. However, despite proliferation, these cells did not accumulate in the recipient animal, suggesting that there was progressive loss of transgenic CD8+ T cells following T cell activation and proliferation within the livers of Met-Kb animals. Statistically significant differences in the proportion of apoptotic donor CD8+ T cells were not detected by flow cytometry analysis of annexin V binding by donor cells from anti-CD62L– and control antibody–treated Met-Kb mice at days 1, 2, and 3 (data not shown); this likely indicates that cell loss was progressive and difficult to visualize in a “snapshot.”
At 6 days after transfer, the number of CD8+ Des-TCR T cells was reduced in anti-CD62L mAb-treated Met-Kb mice compared with that in control Ig-treated Met-Kb mice in all organs examined, rather than in the LNs alone, in contrast to day 2.5 (Figure 6B). Again consistent with progressive cell loss rather than inhibition of cell division, by this time point, all adoptively transferred CD8+ Des-TCR T cells in both anti-CD62L– and control Ig–treated Met-Kb mice had lost detectable CFSE content (data not shown).
Differing half-lives between CD8+ T cells activated within the liver and those activated within the LNs were further demonstrated by transfer of identical numbers of CD8+ Des-TCR+ T cells that were activated within these compartments for 24 hours. Des-TCR LN cells were first adoptively transferred into Met-Kb hosts, then harvested from liver and LNs at 24 hours, and equal numbers of CD8+ Des-TCR T cells from liver and LNs were transferred into separate, antigen-free B10.BR recipients. Thirty days after transfer, lower numbers of donor-derived CD8+ Des-TCR+ T cells were recovered from recipient animals into which intrahepatically activated T cells were transferred than from those into which LN-activated T cells were injected (Figure 6C). The proportion of donor LN-activated CFSE+ CD8+ T cells recovered 30 days after second adoptive transfer (2.1% of transferred donor CD8+ T cells) was very similar to the proportion of CFSE+ CD8– cells (mostly CD4+ and B cells) recovered at this time point (2.8% of transferred donor cells), indicating that, unlike intrahepatically activated T cells, intranodally activated T cells were lost at a rate similar to that of nonactivated lymphocytes.
These results indicate that CD8+ T cells activated within the liver had reduced half-life in comparison with T cells activated within LNs, and that this differentiation program was acquired by T cells within the first 24 hours following activation.
Persisting CD8+ T cells activated within the liver exhibited impaired cytolytic function. Although present in reduced numbers, CD8+ transgenic cells were still detectable in Alb-Kb and anti-CD62L–treated Met-Kb mice. We therefore examined the possibility that in addition to a quantitative defect, T cell activation within the liver resulted in a qualitative defect in CTL function. As an indirect correlate of CTL activity, we first measured the level of CD43 activation–associated glycoform expressed by CD8+ Des-TCR T cells in the livers of anti-CD62L–treated Met-Kb mice at 2.5 days following adoptive transfer. This time point was chosen because previous experiments had demonstrated that, at 48 hours after transfer, CD8+ Des-TCR T cells transferred into Met-Kb mice had divided at least 3 times (Figure 2C), the minimum number of divisions required for their acquisition of specific CTL activity in vitro (37). In addition, since recirculation of dividing cells via the blood was observed at day 2.5 (Figure 5B), cells activated in the LNs could be expected to be present within the livers of non–anti-CD62L–treated Met-Kb mice by this time point. Although upregulation of CD43 activation–associated glycoform was observed, levels expressed by CD8+ Des-TCR+ T cells isolated from the livers of Met-Kb mice treated with anti-CD62L mAb were 2- to 3-fold lower than those expressed by cells from mice treated with control Ig (Figure 7A), which suggested that in mice where the LN contribution was minimized, transgenic T cells activated within the liver exhibited reduced CTL activity. These results were confirmed using a CTL assay in which percentages of specific lysis were corrected for the exact number of transgenic T cells added to the wells. At day 2.5 after transfer, lymphocytes isolated from the liver of Met-Kb mice treated with anti-CD62L displayed lower CTL activity toward H-2Kb–expressing P815 targets than those from the liver of Met-Kb mice treated with control Ig (Figure 7B).
Transgenic T cells adoptively transferred into Alb-Kb mice exhibited a similar phenotype to that observed in Met-Kb mice treated with anti-CD62L mAb. Although upregulation of CD43 activation–associated glycoform was observed, CD8+ Des-TCR+ T cells isolated from the liver of Alb-Kb mice expressed 2- to 3-fold lower levels than transgenic T cells from unmanipulated Met-Kb mice (Figure 7C), consistent with lower CTL activity. This difference in CD43 expression between Alb-Kb and Met-Kb mice was not due to a defect in division-dependent CTL differentiation, as donor T cells exhibited a similar decrease in CFSE content (Figure 7D). To confirm differential CTL activity between unmanipulated Met-Kb and Alb-Kb recipients, we used a classical CTL assay in which percentages of specific lysis were corrected for the exact number of transgenic T cells added to the wells. Organs were harvested at day 6 following T cell transfer, as this was the peak of hepatitis in the Met-Kb model. As shown in Figure 5C, most T cells activated within the LNs of Met-Kb mice recirculated via the blood at day 6 and had already accumulated within the liver, suggesting that most T cells recovered from day 6 Met-Kb livers were of LN origin. In contrast, in the absence of intranodal antigen expression, all activated T cells found in Alb-Kb mice were activated within the liver. Lymphocytes isolated from the livers of Met-Kb mice displayed significantly higher CTL activity toward H-2Kb–expressing P815 targets than those from the livers of Alb-Kb mice, confirming their more potent CTL function (Figure 7E). In contrast to Met-Kb mice, no CTL activity was demonstrated in the LNs of Alb-Kb mice (Figure 7E), a result consistent with lack of transgenic T cell activation and proliferation within this site (Figure 5).
Viewed as a whole, these experiments indicated that the failure to develop a CTL response capable of mediating hepatocellular injury following primary intrahepatic activation is likely to be secondary to a reduction in both CTL activity and CD8+ T cell survival.