While the phenotypic distribution of human IHLs (11
) and LNLs (27
) is known to differ from that in the periphery, little is known about the compartmentalization of functional HCV-specific T cells in the setting of chronic HCV infection based on direct ex vivo assays. In this study, we used tissues made available as a result of organ transplantation for end-stage liver disease to begin to address this question. Cells were freshly prepared and analyzed without in vitro expansion. Our data indicate that HCV-specific IFN-γ-producing T cells are common in the LNL compartment and uncommon in the PBL and IHL compartments. This may be related to ongoing T-cell activation in the lymph node, even in the setting of end-stage liver disease. Notably, we did not distinguish between CD4 and CD8 responses in these assays, so we cannot comment on whether this compartmentalization encompasses CD4, CD8, or both subsets of T cells. Our demonstration of HCV-specific T-cell responses within the IHL and PBL compartments in under half of the subjects is similar to responses previously found in the PBL compartment of chronic HCV-infected patients with compensated liver function, using the same assay method (47
). This underscores the paucity of IFN-γ-producing T cells previously described for the PBL compartment (16
) and extends the observation to the IHL compartment in the setting of direct ex vivo assays. Importantly, this lack of IFN-γ-secreting effector function in the IHL and PBL compartments appears to be specific to HCV in that T cells with effector function are readily demonstrable at these sites in response to CMV protein antigen and CD8 CEF peptide antigens.
One possible explanation for a paucity of functional T cells in the IHL compartment is a suboptimal environment for T-cell activation. This may be the result of soluble inhibitory factors or inadequate antigen-presenting cell (APC) function. Here, we assayed IHLs in the presence and absence of CD3-depleted PBLs to ascertain if exogenous APCs could correct any identified defect. We found no difference in IFN-γ-producing frequency, proliferation index, or specificity by comparing assays performed in the presence or absence of additional APCs (not shown). This suggests that peripheral APCs are not sufficient to restore any possible IHL compartment APC defect. Notably, we did not perform assays in the presence of LNLs depleted of CD3 cells, which may be more functional than the peripherally derived APCs that have been described as defective in a number of studies (4
Another possible explanation for the paucity of functional HCV-specific T cells within the liver is that liver tissue from subjects with end-stage liver disease may not be reflective of the immune response during earlier stage liver disease. While the pathogenesis of HCV during end-stage liver disease may differ from that during earlier stage disease, assays performed here indicate that functional HCV-specific T cells can be found in perihepatic lymph nodes even in advanced disease. Additionally, the phenotype of IHLs with regard to CD57 expression in earlier stage liver disease (less than grade 4 fibrosis) is similar to that in end-stage disease when liver biopsy specimen lymphocytes are analyzed in comparison to PBLs (data not shown).
Another possible explanation for the paucity of functional HCV-specific T cells in PBL and IHL compartments is terminal differentiation due to chronic antigen exposure (perhaps HCV-specific T cells are present but not detectable in direct ex vivo assays). Notably, based upon in vitro T-cell expansion data, HCV-specific T cells are thought to compartmentalize to the liver, existing there in greater frequency than in the circulation (1
). Perhaps these cells have a reversible state of dysfunction. Our data evaluating the magnitude of the HCV-specific response in the IHL compartment are inadequate for us to comment on the clonal frequencies of HCV-specific T cells in this compartment compared to those in the LNL or PBL compartment, though of the responses identified in this compartment, the magnitude may be greater than that of the responses identified in other compartments. We additionally attempted liver lymphocyte expansions (data not shown) using the method described by Erickson et al. (12
) with samples from five HCV-infected and one disease control subject. While background IFN-γ spot-forming units prohibited interpretation for a number of these samples, we were able to expand cells that were reactive to regions of HCV not identified in the direct ex vivo assays in two instances, consistent with the concept of compartmentalization of dysfunctional cells discussed above.
To investigate the origins of PBL, IHL, and LNL compartmentalization of the viral antigen-specific immune response, we evaluated the lymphocyte phenotypes in parallel. The liver is thought to participate in T-cell trafficking by trapping activated CD8 cells (5
). Therefore, some of the IHL CD8 population present in chronic HCV-infected patients may reflect the non-antigen-specific trapping of activated and differentiated circulating CD8 cells. Alternatively, CD8 T-cell activation and terminal differentiation of HCV-specific T cells may occur within the hepatic parenchyma. To examine the differentiation states of IHLs versus PBLs and LNLs, we measured surface CD57 expression as a marker of terminally differentiated CD8 cells incapable of additional proliferation (8
). We observed reduced expression of CD57 on CD4 and CD8 cells in the LNL compartment compared to that in the PBL compartment. This finding, in combination with our finding of enhanced CMV-, CEF-, and HCV-specific proliferative activities within the LNL compartment, suggests an earlier differentiation state (CD57-negative) central memory cell population existing in the LNL compartment, consistent with normal lymph node biology.
We further found reduced expression of CD57 on cells in the IHL compartment compared to that of CD8 cells in the PBL compartment. This latter finding suggests that either CD57 expression is diminished due to the liver environment itself, a possibility shown in other systems where memory CD8 phenotype has been shown to change in a manner related to tissue localization (28
), or that a portion of intrahepatic CD8 T cells are not terminally differentiated effector CD8 cells, as has been previously proposed (14
). In mouse model systems of prototypical antigen-specific immunity, intrahepatic (as opposed to extrahepatic) antigen presentation results in increased accumulation of memory T cells in the liver, providing direct evidence of intrahepatic T-cell activation (29
). It has therefore been proposed that a suboptimal intrahepatic costimulatory environment for T-cell activation may result in the presence of intrahepatic T cells with differing functions and phenotypes (30
). Our phenotypic data are consistent with such a model.
HCV-specific T-cell immunity has been implicated both in viral replication control/clearance and in hepatocellular damage during HCV infection (9
). Conceivably, the compartmentalization of these lymphocyte subsets within the liver impacts the host response to HCV infection. Our data here indicate that HCV-specific cells with direct ex vivo effector function activity are likely activated or sequestered in the perihepatic lymph node yet may not localize to the IHL compartment or within the context of the liver may be rendered dysfunctional. Future investigation is warranted to confirm that the HCV-specific effector function of intrahepatic T lymphocytes is poor and to identify potential mechanisms.