Our findings provide evidence that neonatal CD8+ cells play a key effector role in pathogenesis of experimental biliary atresia. Despite the lack of pre-existing immunologic memory, CD8+ cells are able to activate a broad proinflammatory program in response to a viral insult upon exit from the intrauterine environment, injure extrahepatic bile ducts, and drive an experimental phenotype akin to a neonatal human disease: biliary atresia. The Th1-like molecular signature is consistent with the well-recognized ability of neonatal T lymphocytes to activate an adult-like response.24
Although the immune system of neonates differs from adults quantitatively and qualitatively, cytotoxic T lymphocytes (CTL) from neonatal mice have been shown to elicit competent CTL activity in response to strong Th1-cell inducing agents, such as DNA vaccines or oligonucleotides containing CpG motifs.3-5
Here, we show that this response can evolve and injure a target epithelium (bile duct) and disrupt a vital physiological process (bile flow) in response to neonatal challenge with a viral pathogen (rotavirus).
Rotavirus is one of the most frequent causes of childhood diarrhea worldwide, but its role in pathogenesis of biliary atresia in humans remains undefined. While a diarrheal phenotype is observed in mice infected during the first two weeks of life, rotavirus infection soon after birth can produce biliary injury and inflammatory obstruction, suggesting a unique role of the developing neonatal immune system in phenotype determination.11
In the experiments reported herein, we found that among the immune cells neonatal CD8+ T cells emerge as a key determinant of the biliary atresia phenotype, as they are necessary for induction of bile duct injury and obstruction in the early postnatal period. Interestingly, the findings that livers of CD8-depleted mice express Ifng following RRV challenge clearly demonstrate that Ifng is important but not sufficient to promote the biliary atresia phenotype in neonatal mice. Collectively, these data support the existence of a biological setting in which Ifng serves as a molecular signal that promotes the infiltration of injured bile ducts by inflammatory cells, with CD8+ T cells emerging as cellular effectors of luminal obstruction. Therefore, both Ifng and CD8+ T cells constitute two complementary mechanisms that act in concert to produce the atresia phenotype.
The ability of primed-CD8+ cells to recognize diseased and healthy duct epithelium provides new evidence of disordered immunity in pathogenesis of experimental biliary atresia. The long-held view that developmental and immunologic factors play key roles in pathogenesis of biliary atresia in humans is supported by the temporo-spatial features of bile duct injury in the immediate postnatal period6, 25
and mounting evidence of a proinflammatory response at the time of diagnosis in humans.8, 9
In experimental atresia, hepatic and splenic CD4+ cells from RRV-infected mice challenged with inactivated RRV or protein lysates in culture have been shown to be important sources of Ifng.12
While this is in keeping with the recognition of reactive epitopes by CD4+ cells, we found that the in vivo production of Ifng in response to RRV challenge is not lost by depletion of CD4+ cells, but continue to be produced by CD8+ cells along with increased liver expression of other Th1 cytokines. The potential role of T lymphocytes in bile duct injury, perhaps in an autoimmune fashion, was suggested previously by transplantation of CD3+ cells (probably containing CD4+, CD8+ and NK cell subpopulations) into adult SCID mice.12
In these experiments, transplanted cells homed into bile ducts and induced pericholangitis but no luminal obstruction. However, the contribution of individual CD3+ cell subpopulations to targeting the bile duct was not assessed. Here, we show that between CD3/CD4+ and CD3/CD8+ lymphocytes, the recognition of bile ducts is assigned primarily to CD8+ cells, as supported by a halt in disease progression and maintenance of luminal continuity of the bile duct in mice lacking CD8+ cells. The ability of RRV-primed CD8+ cells (but not RRV-primed CD4+ cells) to promote lysis of RRV-infected and naïve cholangiocytes and to home to the bile duct in non-infected newborn recipients is in keeping with a biological model in which CD8+ cells clear RRV-infected cholangiocytes and extend the epithelial injury by targeting adjacent naïve cholangiocytes, perhaps in an autoimmune fashion. While individual molecular mechanisms of cell-mediated injury and of homing are not yet defined, this experimental model appears suitable to future studies investigating candidate circuits such as those controlled by cytokines and integrins.
Autoimmunity has also been proposed as one of the pathogenic mechanisms in infants with biliary atresia based on a prominent tissue infiltration of lymphocytes, surface expression of self-MHC molecules, detection of auto-antibodies, and prevalent HLA haplotypes in specific populations.26
Although primed-CD8+ cells homed into the biliary epithelium following adoptive transfer in our experimental model, they did not induce the complete atresia phenotype of duct obstruction. It is tempting to speculate that the development of the atresia phenotype by transplanted cells requires a much larger number of activated CD8+ T cells or the synergy of other effector cells (such as NK cells) to obstruct the duct lumen. It is also possible that the lack of duct obstruction may result from apoptosis of transferred neonatal cells after transplantation into neonatal mice. This concept is supported by a previous report that the number of lymphocytes undergoing apoptosis is higher following transplantation into neonates than in adult recipients upon re-exposure to antigen.27
Formal answers to these questions are beyond the scope of this report, and will be obtained by future experiments in which CD8+ cells are co-transplanted with other components of the neonatal immune system (such as primed CD4+ and/or NK cells). These unknowns notwithstanding, our data elevate neonatal CD8+ cells to an effector cell position in the pathogenesis of biliary obstruction in experimental atresia, and as a potential therapeutic target to halt progression of disease in humans.