Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors.

CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.

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.

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-γ, IL-2 and TNF-α 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.

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.

Activation of innate immune defense mechanisms contributes to polyomavirus BK-associated nephropathy.

Polyomavirus-associated nephropathy (PVAN) is a significant complication after kidney transplantation, often leading to premature graft loss. In order to identify antiviral responses of the renal tubular epithelium, we studied activation of the viral DNA and the double-stranded RNA (dsRNA) sensors Toll-like receptor 3 (TLR3) and retinoic acid inducible gene-I (RIG-I) in allograft biopsy samples of patients with PVAN, and in human collecting duct cells in culture after stimulation by the dsRNA mimic polyriboinosinic:polyribocytidylic acid (poly(I:C)), cytokines, or infection with BK virus. Double staining using immunofluorescence for BK virus and TLR3 showed strong signals in epithelial cells of distal cortical tubules and the collecting duct. In biopsies microdissected to isolate tubulointerstitial lesions, TLR3 but not RIG-I mRNA expression was found to be increased in PVAN. Collecting duct cells in culture expressed TLR3 intracellularly, and activation of TLR3 and RIG-I by poly(I:C) enhanced expression of cytokine, chemokine, and IFN-ß mRNA. This inflammatory response could be specifically blocked by siRNA to TLR3. Finally, infection of the collecting duct cells with BK virus enhanced the expression of cytokines and chemokines. This led to an efficient antiviral immune response with TLR3 and RIG-I upregulation without activation of IL-1ß or components of the inflammasome pathway. Thus, PVAN activation of innate immune defense mechanisms through TLR3 is involved in the antiviral and anti-inflammatory response leading to the expression of proinflammatory cytokines and chemokines.

Inhibitory molecules that regulate expansion and restoration of HCV-specific CD4+ T cells in patients with chronic infection.

BACKGROUND & AIMS: Inhibitory receptors such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen (CTLA)-4 mediate CD8+ T-cell exhaustion during chronic viral infection, but little is known about roles in dysfunction of CD4+ T cells. METHODS: We investigated the functions of inhibitory molecules on hepatitis C virus (HCV)-, influenza-, and Epstein-Barr virus (EBV)-specific CD4+ T cells in patients with chronic infections compared with patients with resolved HCV infection and healthy donors. Expression of PD-1, CTLA-4, CD305, and CD200R were analyzed on HCV-specific CD4+ T cells, isolated from peripheral blood using major histocompatibility complex class II tetramers. We investigated the effects of in vitro inhibition of various inhibitory pathways on proliferation and cytokine production by CD4+ T cells, and we compared these effects with those from inhibition of interleukin (IL)-10 and transforming growth factor (TGF)-ß1. RESULTS: PD-1 and CTLA-4 were up-regulated on virus-specific CD4+ T cells from patients with chronic HCV infections. PD-1 expression was lower on influenza- than on HCV-specific CD4+ T cells from subjects with chronic HCV infection, whereas CTLA-4 was expressed at similar levels, independent of their specificity. CD305 and CD200R were up-regulated in HCV resolvers. Blockade of PD-L1/2, IL-10, and TGF-ß1 increased expansion of CD4+ T cells in patients with chronic HCV, whereas inhibition of IL-10 and TGF-ß1 was most effective in restoring HCV-specific production of interferon gamma, IL-2, and tumor necrosis factor α. CONCLUSIONS: We characterized expression of inhibitory molecules on HCV-, influenza-, and EBV-specific CD4+ T cells and the effects of in vitro blockade on CD4+ T-cell expansion and cytokine production. Inhibition of PD-1, IL-10, and TGF-ß1 is most efficient in restoration of HCV-specific CD4+ T cells.