The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity

Abstract

Hepatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8+ T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8+ T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

Figure 1

Early activation of naive CD8⁺ Des-TCR T cells occurred independently within the liver and LNs of Met-K^b mice. CFSE-labeled Des-TCR LN cells were adoptively transferred into Met-K^b 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⁺ CD44^low cells within a forward and side scatter gate appropriate for lymphocytes.

Figure 2

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-K^b mice. (A) Total number of Ly5.1^– CD8⁺ Des-TCR T cells in various organs of Ly5.1⁺ Met-K^b 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-K^b 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-K^b (shaded plot) and B10.BR (dashed line) mice preadministered control purified rat IgG, or Met-K^b 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-K^b mice administered control rat IgG illustrate that donor T cells activated at these 2 sites proliferated at a similar rate.

Figure 3

Administration of anti-CD62L mAb abrogated the development of hepatitis in Met-K^b mice following adoptive transfer of CD8⁺ Des-TCR T cells. (A) Non-splenectomized and splenectomized Met-K^b 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-K^b 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.

Figure 4

Hepatitis did not occur in Alb-K^b mice on the same genetic background as Met-K^b mice following adoptive transfer of Des-TCR LN cells, despite similar levels of antigen expression by hepatocytes. (A) Serum ALT levels of H-2^dk recipient Met-K^b, Alb-K^b, and control mice following adoptive transfer of Des-TCR (H-2^dk) LN cells. Data points represent means ± SEM for groups of 4 mice. (B) H-2K^b expression by viable (PI^– ) hepatocytes from H-2^dk Alb-K^b, Met-K^b, and control mice, analyzed by flow cytometry.

Figure 5

CD8⁺ Des-TCR T cells were activated within the liver and proliferated following adoptive transfer into H-2^dk recipient Alb-K^b and Met-K^b transgenic mice. (A) Alb-K^b, Met-K^b, and control mice were injected with CFSE-labeled syngeneic Des-TCR (H-2^dk) LN cells (top panels) or Des-TCR RAG-1^–/– (H-2^k) 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⁺ CD44^low cells within a forward and side scatter gate appropriate for lymphocytes. (B and C) Alb-K^b, Met-K^b, and control mice were injected with CFSE-labeled syngeneic Des-TCR (H-2^dk) 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-K^b, Met-K^b, and B10.BR mice. CFSE-labeled LN cells from Des-TCR mice were adoptively transferred into Alb-K^b, Met-K^b, 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.

Figure 6

Intrahepatic activation of CD8⁺ Des-TCR T cells was associated with reduced half-life. (A and B) Administration of anti-CD62L mAb to Met-K^b mice resulted in reduced CD8⁺ Des-TCR T cell numbers 2.5 and 6 days after adoptive transfer. Unmanipulated and splenectomized Met-K^b 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-K^b 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.

Figure 7

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-K^b 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-K^b mice treated with anti-CD62L (open squares) or control IgG (filled squares) toward P815-K^b 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-K^b (H-2K^dk) (thin line) and Met-K^b (H-2K^dk) mice (thick line). Organs were harvested 6 days after transfer of Des-TCR (H-2K^dk) 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-K^b (H-2K^dk) (round symbols) and Met-K^b (H-2K^dk) mice (square symbols) incubated with P815 (open symbols) or P815-K^b target cells (filled symbols). Organs were harvested 6 days after transfer of Des-TCR (H-2K^dk) LN cells. The effector/target ratio was calculated by determination of the exact number of transgenic T cells present in each well.