Esther Unitt

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.

ABSTRACT The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8+ T cells. The role of CD4+CD25+ T regulatory (Treg) cells in priming and expanding virus-specific CD8+ T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8+ T-cell proliferation and gamma interferon (IFN-γ) frequency were analyzed with/without depletion of Treg cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4+CD25+ Treg cells inhibited anti-CD3/CD28 CD8+ T-cell proliferation and perforin expression. Depletion of CD4+CD25+ Treg cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-γ-expressing CD8+ T cells. Although stimulated CD8+ T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4+CD25+ regulatory T cells on CD8+ T-cell proliferation. In conclusion, marked CD4+CD25+ regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8+ T-cell responses and viral persistence.

Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.

Hepatocellular carcinoma (HCC) has a poor prognosis with limited therapeutic options. We propose that local immune responses in patients with HCC are held in check by tumor‐infiltrating CD4+CD25+ T‐regulatory lymphocytes (Treg cells), which suppress the activity and proliferation of effector CD4+ and CD8+ T cells. The phenotype and cell cycle status of tumor‐infiltrating lymphocytes (TILs) in HCC were analyzed via immunohistochemistry of sections from patients undergoing surgery for HCC and via flow cytometry of peripheral blood mononuclear cells and TILs isolated from patients with HCC. Circulating and tumor‐infiltrating T‐cell function and activation status were assessed via proliferation and flow cytometry. More than 96% of TILs were quiescent as measured via Mcm‐2 or Ki‐67 expression, while less than 10% of CD8+ T cells expressed perforin or granzyme B. CD4+CD25+ Treg cells comprised 8.7% (1.4–13.8) of TILs and always exceeded the proportion in distant nontumor tissue (2.4% [1.5–5.6]; P = .014). Treg cells isolated from HCC suppressed proliferation of autologous circulating CD4+CD25− cells and perforin expression and proliferation of autologous CD8+ T cells. The proportion of circulating Treg cells in patients with HCC was similar in healthy controls (7.2% [1.2–23.3] and 9.2% [1.6–30.2], respectively), but the proportion of circulating Treg cells that were also transforming growth factor β1+ was elevated in HCC compared with controls (55.5% [8.2–73.9] and 2.0% [0–4.9], respectively; P = .003). In conclusion, TILs are compromised and contain a subpopulation of suppressive CD4+CD25+Foxp3+ Treg cells. Functional deletion of tumor‐infiltrating Treg cells could enhance tumor‐specific immunotherapy. (HEPATOLOGY 2005;41:722–730.)

Sirolimus-induced pneumonitis following liver transplantation.

Sirolimus‐induced pneumonitis has emerged as a potentially serious complication in renal transplantation but only single case reports of this condition have been described after liver transplantation (LT), where experience with sirolimus is relatively limited. We report our experience, the largest to date, of sirolimus‐induced pneumonitis following LT. Between 1999 and 2006, 186 liver transplant patients received sirolimus‐based immunosuppression, after initial therapy with calcineurin inhibitors (CNIs). All cases of sirolimus‐induced pneumonitis were recorded and a retrospective review of the case notes of such patients was undertaken for the purpose of this analysis. Of 186 liver transplant patients receiving sirolimus, 4 (2.2%) developed pneumonitis that was attributed to the drug; the time from starting sirolimus to presentation was varied (1.5‐30 months). The most common presenting symptoms were dyspnea, cough and fatigue. The median sirolimus level at the time of diagnosis was 9.7 ng/mL (range, 7‐19.5 ng/mL). All patients in the series underwent thoracic computed tomography, which showed similar changes in all patients, and lung biopsy, which revealed features consistent with a drug‐induced pneumonitis. In all 4 patients, sirolimus‐induced pneumonitis resolved following cessation of therapy but took weeks to months for complete recovery. In conclusion, sirolimus‐induced pneumonitis occurred in at least 2% of liver transplant recipients and should be suspected in patients who develop respiratory symptoms while on sirolimus. Although it may be life threatening, early recognition and cessation of sirolimus can lead to complete resolution of pneumonitis. Liver Transpl 13:853–856, 2007.

Minichromosome maintenance protein‐2–positive portal tract lymphocytes distinguish acute cellular rejection from hepatitis C virus recurrence after liver transplantation

Hepatitis C virus (HCV) is a leading indication for liver transplantation worldwide, but graft infection with HCV frequently leads to hepatic fibrosis. Acute cellular rejection (ACR) can be difficult to distinguish confidently from HCV, even with histology, but accurate diagnosis is critical because treatment of ACR may accelerate HCV‐related graft injury. Immunohistochemistry was undertaken on 99 liver biopsies from 31 patients with HCV graft infection, 22 patients with ACR, and 11 patients with HCV infection and unexplained graft dysfunction to investigate whether lymphocyte expression of minichromosome maintenance protein‐2 (Mcm‐2), a marker of licensed cell cycle entry, assessed in a novel semiautomated system could distinguish between ACR and graft infection with HCV. The portal tract area was greater in ACR than in HCV graft infection (P = 0.027), but there was considerable overlap. However, both the number of Mcm‐2–positive lymphocytes per portal tract and the number of Mcm‐2–positive lymphocytes per millimeter squared of portal tract distinguished between ACR and HCV graft infection (P < 0.0001). A cutoff value of 107 positive cells per portal tract had a sensitivity of 81.8% and a specificity of 91.9% (positive predictive value of 66.67% and negative predictive value of 95.75%). Of 11 HCV‐infected patients with an uncertain diagnosis, 7 were deemed ultimately to have HCV graft infection, and 4 had superimposed corticosteroid‐responsive ACR. The number of Mcm‐2–positive cells per portal tract and per millimeter squared of portal tract again distinguished clearly between the groups (P = 0.012). In conclusion, lymphocyte Mcm‐2 expression is a useful adjunct to histology in differentiating between HCV graft infection and ACR. Patients with a low number of Mcm‐2–positive portal tract lymphocytes are less likely to have ACR. Liver Transpl 15:306–312, 2009.

Heterogeneous Inflammatory Changes in Liver Graft Recipients With Normal Biochemistry

Background. Patients with established liver grafts may receive excessive immune suppression. Liver biopsies were analyzed in those with normal liver biochemistry to identify parameters that might identify such cases. Methods. Patients with established grafts (>3 years from engraftment) and normal liver biochemistry (normal alanine transaminase, alkaline phosphatase, and bilirubin) were invited to undergo liver biopsy. Liver tissue was assessed by routine histopathology, a modified Ishak score, and immunohistochemistry for lymphocyte and cell-cycle markers. Circulating and intrahepatic lymphocytes were subjected to flow cytometry. Data were subjected to principal component analysis. Results. Two hundred twenty-five (40%) patients under regular review had an established graft with normal liver biochemistry; liver tissue was obtained in 55. Liver histology was normal in eight cases (14.5%). The most common abnormalities were mild nonspecific hepatitis in 25 (45.4%) and disease recurrence in 14 (25.4%). Principal component analysis identified a cluster of variables that accounted for a significant degree of variation within the dataset. These were lobular inflammation, portal inflammation, interface hepatitis, and fibrosis. Conclusions. Inflammation persisted in established grafted livers in most patients with normal liver biochemistry. Systematic histological and lymphocyte phenotype analysis generated an index that distinguished patient groups. Those with least inflammation and the lowest alanine transaminase may have a reduced requirement for immune suppression.