Liver damage preferentially results from CD8(+) T cells triggered by high affinity peptide antigens

Abstract

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)-restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4(-)CD8(-)B220(+) T cells occurs more frequently from a CD8(+) precursor than from CD4(+) T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8(+) cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC-restricted TCR. The findings show that CD4(-)CD8(-)B220(+) T cells preferentially derive from a CD8(+) precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.

Figure 1

OVA antigen induces greater increase of liver lymphocytes in OT-1 than in DO.11.10 mice. OT-1 or DO.11.10 mice from experiment no. 2 received two doses of PBS or, respectively, OVA_I and OVA_II at 24-h intervals. Total lymphocyte numbers were determined for each organ on the day indicated after the last injection.

Figure 2

Induction of CD4⁻ CD8⁻Vα2⁺ and CD8⁺B220⁺ phenotype only by high affinity OVA_I peptide. OT-1 mice, three per group, received a single injection of the indicated peptide or PBS. OVA_I confers a high affinity (K _d = 6.5 μM) interaction with the OT-1 TCR when complexed with K^b. R4 and E1 are OVA_I variants that manifest affinities of 57.1 and 22.6 μM, respectively. On day 1 after injections, lymph node cells from each group were pooled and analyzed for expression of CD4, CD8, Vα2, and B220. Numbers in quadrants indicate the percentage of positive cells. The forward and side scatter histograms at the left show that the gates set for this analysis were uniform and did not include small dying cells. Numbers in parentheses represent the percentage of Vα2⁺ cells that express B220. The right hand column displays absolute numbers of CD8⁺Vα2⁺ and CD4⁻CD8⁻ Vα2⁺ cells. The findings are representative of three experiments.

Figure 3

OVA_I induces blast formation of OT-1 T cells followed by the appearance of small T cells that express B220 and are enriched for a CD4⁻CD8⁻Vα2⁺ phenotype. OT-1 mice received two injections of OVA_I or PBS in experiment no. 2. Lymph node cells were analyzed on the indicated days after the last injection. (A) An initial blast population appears on day 2 following OVA_I which then becomes a smaller transition cell population over the next 3 d. Surface phenotype is based on the large and moderate sized cells as shown by the enclosed gate. (B) The small sized population of lymphocytes is enriched for CD4⁻CD8⁻Vα2⁺B220⁺ cells. OVA_I administration also increased the proportion of CD8⁺ cells expressing B220.

Figure 3

OVA_I induces blast formation of OT-1 T cells followed by the appearance of small T cells that express B220 and are enriched for a CD4⁻CD8⁻Vα2⁺ phenotype. OT-1 mice received two injections of OVA_I or PBS in experiment no. 2. Lymph node cells were analyzed on the indicated days after the last injection. (A) An initial blast population appears on day 2 following OVA_I which then becomes a smaller transition cell population over the next 3 d. Surface phenotype is based on the large and moderate sized cells as shown by the enclosed gate. (B) The small sized population of lymphocytes is enriched for CD4⁻CD8⁻Vα2⁺B220⁺ cells. OVA_I administration also increased the proportion of CD8⁺ cells expressing B220.

Figure 4

Cell counts and percentage of B220 expression by the CD8⁺Vα2⁺ and CD4⁻CD8⁻Vα2⁺ subsets of T cells from OT-1 mice after two doses of OVA_I. Analysis is based on the same mice as used in experiment no. 2 in Fig. 1 and phenotypes were determined on the living cells based on flow cytometric size gates. Tissues were pooled from three OT-1 mice per group. (A) Actual lymphocyte counts of subsets in various organs. (B) Percentage of cells in the indicated subset that expressed B220.

Figure 4

Cell counts and percentage of B220 expression by the CD8⁺Vα2⁺ and CD4⁻CD8⁻Vα2⁺ subsets of T cells from OT-1 mice after two doses of OVA_I. Analysis is based on the same mice as used in experiment no. 2 in Fig. 1 and phenotypes were determined on the living cells based on flow cytometric size gates. Tissues were pooled from three OT-1 mice per group. (A) Actual lymphocyte counts of subsets in various organs. (B) Percentage of cells in the indicated subset that expressed B220.

Figure 5

OVA_II does not induce B220 expression or CD4⁻CD8⁻ phenotype by responding CD4⁺ cells from DO.11.10 mice. DO.11.10 mice received OVA_II injections and lymph node cells (top) or liver lymphocytes (bottom) were examined for expression of CD4, CD8, B220, and clonotype TCR using antibody KJ1-26. The right column indicates the absolute numbers of KJ1-26⁺ cells that were CD4⁺ or CD4⁻CD8⁻. Phenotypes were based on gates set for living cells as in Fig. 2.

Figure 6

Some resident liver T lymphocytes in OT-1 mice express B220, but after OVA_I administration the initial influx is of CD8⁺ cells that lack B220. OT-1 mice received two injections of OVA_I in experiment no. 2 and liver lymphocytes were isolated on the indicated days after the last injection. The forward and side scatter histograms at the left show that the gates set for this analysis were uniform and did not include small dying cells.

Figure 7

Liver lymphocytes and lymph node cells are undergoing increased apoptosis in OT-1 mice compared with DO.11.10 mice. Mice are from experiment no. 3 and received three doses of PBS, OVA_I (OT-1 mice), or OVA_II (DO.11.10 mice) at 24-h intervals. Lymphoid cells were isolated at the indicated time points and stained with Annexin V and either anti-CD8 (for OT-1 mice) or anti-CD4 (for DO.11.10 mice). Analysis is gated on CD8⁺ cells from OT-1 mice (A) or on CD4⁺ cells from DO.11.10 mice (B).

Figure 7

Liver lymphocytes and lymph node cells are undergoing increased apoptosis in OT-1 mice compared with DO.11.10 mice. Mice are from experiment no. 3 and received three doses of PBS, OVA_I (OT-1 mice), or OVA_II (DO.11.10 mice) at 24-h intervals. Lymphoid cells were isolated at the indicated time points and stained with Annexin V and either anti-CD8 (for OT-1 mice) or anti-CD4 (for DO.11.10 mice). Analysis is gated on CD8⁺ cells from OT-1 mice (A) or on CD4⁺ cells from DO.11.10 mice (B).

Figure 8

Infiltration of lymphocytes into the liver and resulting hepatocyte damage after OVA administration are both more pronounced in OT-1 mice than DO.11.10 mice. Livers were taken from mice of experiment no. 2 that had received two doses of OVA and were stained with hematoxylin and eosin. Original magnification is ×160 except for J, which is ×1,000. Administration of OVA_I to nontransgenic C57BL/6 mice (A) and OVA_II to nontransgenic BALB/c mice (F) yielded no influx of liver lymphocytes and no hepatocyte damage. Similarly, PBS administration to OT-1 mice (B) or DO.11.10 mice (G) also produced no infiltrates. Beginning on day 2 after administration of OVA_I to OT-1 mice or OVA_II to DO.11.10 mice, periportal lymphocytic infiltrates were observed in both mice but were more intense in OT-1 (C) than DO.11.10 mice (H). By day 3 a massive lymphocytic infiltrate is observed throughout the livers of only OT-1 mice with extensive hepatocyte damage (D). By day 5 the lymphocytic infiltrate in OT-1 livers is resolved (E) and in its wake is observed a dramatic burst of hepatocyte mitotic activity, shown magnified in J. By contrast, DO.11.10 mice never manifested extensive liver sinusoidal lymphocytes as infiltrates were completely gone by day 5 without any evidence of hepatocyte damage or mitotic rebound (I).