Lack of variant specific CD8+T-cell response against mutant and pre-existing variants leads to outgrowth of particular clones in acute hepatitis C

Background: CTL escape mutations have been described during acute hepatitis C in patients who developed chronic disease later on. Our aim was to investigate the mutual relationship between HCV specific CD8+ T cells and evolution of the viral sequence during early acute HCV infection. Results: We sequenced multiple clones of NS3 1406 epitope in 4 HLA-A{*}02 patients with acute hepatitis C genotype 1b infection. Pentamers specific for the variants were used to monitor the corresponding CD8+ T cell response. We observed outgrowth of mutations, which induced only a weak and thus potentially insufficient CD8+ T cell response. In one patient we observed outgrowth of variant epitopes with similarities to a different genotype rather than de novo mutations most probably due to a lack of responsiveness to these likely pre-existing variants. We could show that in acute hepatitis C CTL escape mutations occur much earlier than demonstrated in previous studies. Conclusions: The adaption of the virus to a new host is characterized by a high and rapid variability in epitopes under CD8+ T cell immune pressure. This adaption takes place during the very early phase of acute infection and strikingly some sequences were reduced below the limit of detection at some time points but were detected at high frequency again at later time points. Independent of the observed variability, HCV-specific CD8+ T cell responses decline and no adaption to different or new antigens during the course of infection could be detected

Inhibitory Phenotype of HBV-Specific CD4⁺ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background: T-cell exhaustion seems to play a critical role in CD8(+) T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4(+) T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4(+) T-cell failure. Methods: The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4(+) T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4(+) T-cell proliferation and cytokine production. Results: CD4(+) T-cell responses during chronic HBV infection was characterized by reduced Tetramer(+)CD4(+) T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-gamma, IL-2 and TNF-alpha secretion as well as enhanced CD4(+) T-cell expansion almost in treated patients with viral control. Conclusion: HBV-specific CD4(+) T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4(+) T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4(+) T-cell functionality with heterogeneous patterns of CD4(+) T-cell rejunivation

Total cardiovascular or fatal events in people with type 2 diabetes and cardiovascular risk factors treated with dulaglutide in the REWIND trail:a post hoc analysis

Abstract Background The Researching cardiovascular Events with a Weekly INcretin in Diabetes (REWIND) double blind randomized trial demonstrated that weekly subcutaneous dulaglutide 1.5 mg, a glucagon like peptide-1 receptor agonist, versus matched placebo reduced the first outcome of major adverse cardiovascular event (MACE), cardiovascular death, nonfatal myocardial infarction or nonfatal stroke (594 versus 663 events) in 9901 persons with type 2 diabetes and either chronic cardiovascular disease or risk factors, and followed during 5.4 years. These findings were based on a time-to-first-event analysis and preclude relevant information on the burden of total major events occurring during the trial. This analysis reports on the total cardiovascular or fatal events in the REWIND participants Methods We compared the total incidence of MACE or non-cardiovascular deaths, and the total incidence of expanded MACE (MACE, unstable angina, heart failure or revascularization) or non-cardiovascular deaths between participants randomized to dulaglutide and those randomized to placebo. Incidences were expressed as number per 1000 person-years. Hazard ratios (HR) were calculated using the conditional time gap and proportional means models. Results Participants had a mean age of 66.2 years, 46.3% were women and 31% had previous cardiovascular disease. During the trial there were 1972 MACE or non-cardiovascular deaths and 3673 expanded MACE or non-cardiovascular deaths. The incidence of total MACE or non-cardiovascular deaths in the dulaglutide and placebo groups was 35.8 and 40.3 per 1000 person-years, respectively [absolute reduction = 4.5 per 1000 person-years; conditional time gap HR 0.90 (95% CI, 0.82–0.98) p = 0.020, and proportional means HR 0.89 (95% CI, 0.80–0.98) p = 0.022]. The incidence of total expanded MACE or non-cardiovascular deaths in the dulaglutide and placebo groups was 67.1 and 74.7 per 1000 person-years, respectively [absolute reduction = 7.6 per 1000 person-years; conditional time gap HR 0.93 (95% CI, 0.87–0.99) p = 0.023, and proportional means HR 0.90 (95% CI, 0.82–0.99) p = 0.028]. Conclusions These findings suggest that weekly subcutaneous dulaglutide reduced total cardiovascular or fatal event burden in people with type 2 diabetes at moderate cardiovascular risk. Clinical Trial Registration: https://www.clinicaltrials.gouv . Unique Identifier NCT01394952)

Dysfunctional CD8(+) T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction

The transcription factor T-bet regulates the production of interferon-γ and cytotoxic molecules in effector CD8 T cells, and its expression correlates with improved control of chronic viral infections. However, the role of T-bet in infections with differential outcome remains poorly defined. Here, we report that high expression of T-bet in virus-specific CD8 T cells during acute hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was associated with spontaneous resolution, whereas T-bet deficiency was more characteristic of chronic evolving infection. T-bet strongly correlated with interferon-γ production and proliferation of virus-specific CD8 T cells, and its induction by antigen and IL-2 stimulation partially restored functionality in previously dysfunctional T-bet–deficient CD8 T cells. However, restoration of a strong interferon-γ response required additional stimulation with IL-12, which selectively induced the phosphorylation of STAT4 in T-bet(+) CD8 T cells. The observation that T-bet expression rendered CD8 T cells responsive to IL-12 suggests a stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of additional transcription factors in the presence of key cytokines. These findings support a critical role of T-bet for viral clearance and suggest T-bet deficiency as an important mechanism behind chronic infection

Effect of PD-L1/2 blockade on CD4⁺ T-cell expansion.

<p>Induction of CD4⁺ T-cell proliferation in chronic HBV (n = 23) from h0 (<i>left</i>) to antigenic stimulation (<i>middle</i>) and PD-L1/2 blockade (<i>right</i>) illustrated as point to point graphs from (<b>A</b>) PD-L1/2 responders (n = 9) and (<b>B</b>) Non-responders (n = 14). Contour plots are shown for each group. (<b>C</b>) Patients treated with nucleosid/nucleotid analogs (<i>black bars</i>) responded to PD-1 blockade using Chi²-test, indicating that viral control let enhance patients susceptibility as (<b>D</b>) treated patients are characterized by low viremia. (<b>E</b>) Decreased PD-1 expression in PD-L1/2 responders (R) (n = 6) versus non-responders (NR) (n = 6). (<b>F</b>) CD4⁺ T-cell frequencies upon antigenic stimulation (<i>black bars</i>) and PD-L1/2 inhibition (<i>grey bars</i>) in relation to PD-1 expression.</p

Inhibitory phenotype of HBV-specific CD4⁺ T-cells.

<p>(<b>A</b>) PD-1 (n = 29), CTLA-4 (n = 8), TIM-3 (n = 14), KLRG1 (n = 12) and CD244 (n = 8) expression is displayed on Tetramer⁺CD4⁺ T-cells during chronic HBV. (<b>B</b>) PD-1 expression in chronic (<i>black points</i>) (n = 29) and acute HBV (<i>black squares</i>) (n = 7), resolved HBV (<i>white squares</i>) (n = 5), EBV (<i>grey points</i>) (n = 15) and Flu (<i>white points</i>) (n = 21) infection. (<b>C</b>) Contour plots illustrate patterns of PD-1 expression during HBV, EBV and Flu infection. (<b>D</b>) CD127 expression on HBV-specific (<i>black points</i>) (n = 11) and Flu-specific CD4⁺ T-cells (<i>white points</i>) (n = 9). (<b>E</b>) Contour plots display CD127 expression on CD14⁻, CD19⁻, Via Probe⁻, Tetramer⁺ T-cells. (<b>F</b>) CD127 defines PD-1 expression as examined in HBV (<i>black bars</i>) and Flu infection (<i>white bar</i>). (<b>G</b>) Influence of viral load on CD127 expression.</p

Baseline Characteristics^*.

<p>*presented as median values.</p><p>**nucleosid/nucleotide analogs.</p><p>***not determined.</p