Jared Klarquist

Upregulation of PD-1 Expression on Circulating and Intrahepatic Hepatitis C Virus-Specific CD8+ T Cells Associated with Reversible Immune Dysfunction

ABSTRACT Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8+ cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1high express higher levels of the senescence marker CD57 than PD-1low CTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4+ T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs.

Acute Hepatitis C in a Contemporary US Cohort: Modes of Acquisition and Factors Influencing Viral Clearance

BACKGROUND Acute hepatitis C virus (HCV) infection is often asymptomatic; thus, its epidemiology and natural history are difficult to define. METHODS Acute HCV infection was identified on the basis of HCV seroconversion within 1 year (n=45), new anti-HCV seropositivity with clinical acute hepatitis (n=21), or HCV strain sequencing after an iatrogenic exposure (n=1). Risk factors were assessed with a baseline questionnaire, and participants were followed up prospectively with serial measurement of viral loads. RESULTS Of 67 persons with acute HCV infection, most were asymptomatic (64%) and injection drug users (66%). Thirteen had an unknown mode of transmission; of these, 11 reported high-risk sexual behavior. Ten acquired acute HCV infection within 3 months of an iatrogenic exposure; 3 had confirmed iatrogenic infection, and 4 had no other risk factors identified. The spontaneous viral clearance rate after 6 months of infection was 18% (95% confidence interval, 11%-31%). The rate of viral clearance varied significantly by sex (34% vs. 3% for women vs. men; P<.001). CONCLUSIONS High-risk sexual or iatrogenic exposures may be important contemporary risk factors for HCV infection. The spontaneous viral clearance rate (18%) in this contemporary study was similar to that reported for past studies of transfusion-associated HCV infection. Women were more likely to clear acute HCV infection than men.

Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence†

Interleukin‐7 (IL‐7) is required for the establishment and maintenance of memory CD4+ and CD8+ T lymphocytes, and cells lacking IL‐7Rα (CD127) demonstrate impaired IL‐2 secretion and have a short life‐span. Chronic HCV is characterized by T cells that are functionally impaired and exhibit an immature phenotype. To investigate the potential role of IL‐7/IL‐7Rα in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n = 24), long‐term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4+ and CD8+ T lymphocytes as compared to normal controls. Total expression was lowest in those patients who subsequently developed persistence and intermediate in those patients with acute‐resolving infection. This reduction affected both naïve and effector/memory T cells. CD127 correlated phenotypically with upregulation of chemokine receptors CCR7 and CXCR4, expression of the anti‐apoptotic molecule B cell leukemia/lymphoma 2 (Bcl‐2), and enhanced IL‐2 production. In six HLA A2‐positive patients, we longitudinally tracked tetramer responses to HCV and CMV epitopes; at baseline, reflecting the expression of CD127 on whole T cell populations, viral‐specific CTLs in patients who became chronic demonstrated lower CD127. In conclusion, CD127 is a useful marker of functional CD4+ and CD8+ T cells and its expression correlates with virologic outcome of acute HCV. These data provide a mechanistic basis for the observation that CTLs generated in early infection rapidly decline as chronicity is established; CD127 expression should be considered in the design of novel immunotherapeutic approaches. (HEPATOLOGY 2006;44:1098–1109.)

Cutting Edge: Programmed Death-1 Expression Is Increased on Immunocytes in Chronic Hepatitis C Virus and Predicts Failure of Response to Antiviral Therapy: Race-Dependent Differences

Up-regulation of programmed death-1 (PD-1) identifies exhausted T cells in various mouse and human viral models including chronic hepatitis C virus (HCV) infection, which is characterized by impaired CTL function. A large proportion of patients fail to eradicate HCV with current IFN-based antiviral therapy; in particular, African Americans are less likely to respond, but the mechanisms for these differences are not fully elucidated. In this study, in 72 treatment-naive patients with persistent HCV we found that PD-1 was significantly up-regulated on CD4+ and CD8+ T cells, HCV-specific CTLs, and NK cells. Increased PD-1 on HCV-specific CTLs was significantly associated with failed early and sustained virologic response to therapy in African American but not Caucasian American patients. Patients with sustained virologic response showed decreases in PD-1 on total CD4+ T cells, HCV-specific CTLs, and the CD56bright NK subset after therapy completion. Collectively, these data indicate that PD-1 is critical in persistent HCV and successful therapy results in global down-regulation of its expression.

Functional Suppression by FoxP3+CD4+CD25high Regulatory T Cells during Acute Hepatitis C Virus Infection

BACKGROUND Infection with hepatitis C virus (HCV) is characterized by impairment of viral effector T cell responses and a high propensity for viral persistence. Previous studies have demonstrated that chronic HCV infection is associated with an increased frequency of regulatory T (T(reg)) cells, compared with that in persons whose infection resolved and in healthy persons. However, all patients in prior analyses had exposures in the distant past, precluding the ability to determine whether T(reg) cells play a causal role in establishing persistence during the earliest stages of infection or whether they are expanded because of viral persistence. METHODS For the first time, we longitudinally analyzed T(reg) cells in patients with acute HCV infection (n = 27). We used a multiparameter approach, including fluorescence-activated cell sorting analysis of cell-surface and intracellular antigens, coculture experiments with highly purified CD4(+)CD25(high) regulatory and CD4(+)CD25(-) responder cell populations, and multiplex analysis of secreted cytokines. RESULTS Forkhead transcription factor 3 (FoxP3) expression and T(reg) cell suppression were greater in patients with acute HCV infection than in healthy control subjects but were not different at the first time point among patients who subsequently developed persistence or resolved HCV infection spontaneously; however, 6 months later, the resolution of disease was associated with a relative loss of functional suppression. CONCLUSIONS Collectively, these data indicate that patients with acute HCV infection who develop chronicity versus spontaneous resolution exhibit temporal changes in T(reg) cell function. It is possible that repetitive viral antigenic stimulation alters the function of T(reg) cells over time.

Spontaneous Recovery in Acute Human Hepatitis C Virus Infection: Functional T-Cell Thresholds and Relative Importance of CD4 Help

ABSTRACT The mechanisms mediating protective immunity to hepatitis C virus (HCV) infection are incompletely understood because early infection in humans is rarely identified, particularly in those individuals who subsequently demonstrate spontaneous virus eradication. We have established a large national network of patients with acute HCV infection. Here, we comprehensively examined total HCV-specific CD4+ and CD8+ T-cell responses and identified functional T-cell thresholds that predict recovery. Interestingly, we found that the presence of HCV-specific cytotoxic T lymphocytes (CTLs) that can proliferate, exhibit cytotoxicity, and produce gamma interferon does not ensure recovery, but whether these CTLs were primed in the presence or absence of CD4+ T-cell help (HCV-specific interleukin-2 production) is a critical determinant. These results have important implications for early prediction of the virologic outcome following acute HCV and for the development of novel immunotherapeutic approaches.

Selective decrease in hepatitis C virus-specific immunity among African Americans and outcome of antiviral therapy.

Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, end‐stage liver disease, and hepatocellular carcinoma throughout the world. Considerable evidence indicates that the risk of viral persistence, natural history, and response to antiviral therapy varies among racial groups, but limited data exist on potential mechanisms to account for these differences. Type 1 helper (Th1) responses to HCV proteins and cytomegalovirus (CMV) antigens were examined using a sensitive interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay in 187 Caucasian American (CA) and 187 African American (AA) patients with chronic genotype 1 infection. ELISPOT responses were examined relative to human leukocyte antigen (HLA) class II alleles and outcome of therapy with pegylated IFN and ribavirin. Th1 responses specific to hepatitis C core protein and combined HCV antigens were significantly lower in AAs compared to CAs, but CMV responses were comparable in the 2 races. The HCV difference in immunity remained after adjusting for gender, serum alanine aminotransferase, histologic severity, and viral level, and was not accounted for by the differential prevalence of human leukocyte antigen class II alleles. Pretreatment total HCV‐specific CD4+ T cell response was associated with sustained virologic response (SVR) to pegylated IFN and ribavirin; 43% of patients who had more than 168 ELISPOTs/106 peripheral blood mononuclear cells (above background) experienced SVR compared to 28% of those who did not (P= 0.007). ELISPOT response was independently associated with SVR by multivariable analysis. Conclusion: Compared to CAs, AAs have weaker HCV‐specific immunity. Pretreatment HCV‐specific immunity is associated with response to combination antiviral therapy. (HEPATOLOGY 2007.)

Prospective analysis of effector and regulatory CD4+ T cells in chronic HCV patients undergoing combination antiviral therapy

BACKGROUND/AIMS The role of HCV-specific CD4(+) T cells and regulatory T cells in influencing the outcome of antiviral therapy is incompletely defined. METHODS CD4(+) IFN-gamma ELISPOT assays (n=58) and flow cytometric analysis of FoxP3-expressing T regulatory cells (n=62) were performed on patients from the Virahep-C study at baseline, during and after cessation of antiviral therapy. RESULTS Total HCV-specific IFN-gamma CD4(+) T cell ELISPOT responses did not increase with therapy, but rather decreased by 8 weeks and remained below baseline 24 weeks after cessation of therapy. There were no statistically significant differences with respect to viral kinetics, race and virologic outcome. In contrast, viral relapse after treatment was associated with a three-fold increase in HCV-specific responses. The frequency and phenotype of regulatory T cells during therapy were not significantly different in terms of race, viral kinetic groups or virologic outcome. CONCLUSIONS A contraction of HCV-specific CD4(+) T cell responses was found during treatment with recovery of responses in patients experiencing virologic relapse after treatment. The levels of FoxP3-expressing regulatory T cells did not vary by race and were not predictive of virologic outcome. Work is ongoing to explore the contribution of mechanisms independent of CD4(+) T cells in therapy-induced viral clearance.

Eya3 promotes breast tumor–associated immune suppression via threonine phosphatase–mediated PD-L1 upregulation

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer–associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

Clonal expansion of vaccine-elicited T cells is independent of aerobic glycolysis

T cell responses to subunit vaccination require both IL-27 and IL-15 to support an aerobic glycolysis–independent metabolic program. Mitochondria fuel vaccine-induced T cells Effective T cell responses to subunit vaccines administered with an adjuvant depend on a different set of signals than natural T cell responses to infectious microbes. Klarquist et al. compared the cytokine and metabolic requirements for clonal expansion of mouse CD8+ T cells responding to adjuvanted subunit vaccination or infection. In contrast to T cells responding to infection, T cells responding to vaccination were compromised in the absence of either IL-27 or IL-15 signaling and used mitochondrial function rather than aerobic glycolysis to support proliferation. These results demonstrate the existence of fundamental metabolic differences in CD8+ T cell responses to vaccines versus infection that need to be considered as part of the process of designing new vaccines. In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine–elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27– or IL-15–deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.