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.

The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function.

UNLABELLED: Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells. CONCLUSION: CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.

Characterization of sequence variations in immunodominant regions of the HBV-nucleocapsid protein as a prerequisite for the development of an epitope-based vaccine.

BACKGROUND/AIMS: In hepatitis B virus infection, viral elimination is dependent on an efficient antiviral T cell response which is not detectable in chronic hepatitis B. Therefore, new therapeutic concepts focus on T cell activation, such as epitope-based T cell-targeted vaccines. However, with the development of peptide-based vaccines in mind, viral mutations frequently described in hepatitis B within known immunodominant helper epitopes may have an influence on peptide selection. METHODS: Mutant peptides within immunodominant epitopes (aa 1-20, aa 91-105, and aa 143-157) at position 12, 14, 93, 97, 147, 151, 153, and 155 were tested with peripheral blood mononuclear and specific clone cells for their ability to induce proliferation, produce cytokines, induce T cell receptor down-regulation or antagonize wild-type activity of the hepatitis B core antigen-specific CD4+ T cell clones. RESULTS: Five variants could not induce T cell proliferation or cytokine production when the variants were presented alone. Coincubation with wild-type epitopes leads to T cell activation showing that the variants do not act as T cell receptor antagonists for hepatitis B virus-specific CD4+ T cells. In contrast, five other variants and wild-type peptides stimulated CD4+ T cell proliferation and production of Th1 cytokines. CONCLUSIONS: Our data demonstrate that frequently occurring mutations within immunodominant epitopes have rather a nonstimulatory than a strengthening effect and thus should not included in a vaccine.

Leucocytapheresis with Adacolumn enhances HCV-specific proliferative responses in patients infected with hepatitis C virus genotype 1.

The most important aim in controlling virus infections is to destroy infected cells. Impaired cellular immunity in HIV and HCV infection leads to chronic infection. This study examined the effect of cytapheresis on the subsequent response to interferon/ribavirin treatment in patients infected with HCV. Adacolumn cytapheresis was carried out once a day for 5 consecutive days in patients who relapsed or did not respond to previous peginterferon and ribavirin combination treatment (n = 14: relapsers = 3, non-responders = 11). Peginterferon and ribavirin combination treatment was started after cytapheresis. During combination treatment, the proliferative response of peripheral blood mononuclear cells to HCV proteins (core, NS3, NS4, and NS5), tetanus toxoid, and phytohemagglutinin was measured, and compared to the early virological response. After treatment by leucocytapheresis, the proliferative response of peripheral blood mononuclear cells to HCV-core and tetanus toxoid increased significantly over the baseline (P < 0.05). A marked increase in the phytohemagglutinin response was observed after peginterferon and ribavirin combination treatment was started (P < 0.01 at week 5 and P < 0.005 at week 13). There were, however, no clear changes in the proliferative response to other antigens. Among the 14 patients, 12 (85.7%) achieved an early virological response by week 13 (12 weeks after the start of combination treatment). After treatment, nine patients (64.3%) had a significant proliferative response to HCV core antigen. Among the nine patients, eight patients (88.9%) achieved early virological response. The results indicate that activation of cellular immunity by leucocytapheresis facilitates an early virological response rate in HCV patients. This new therapy may, therefore, become an additional therapeutic measure for HCV.

A subset of human dendritic cells in the T cell area of mucosa-associated lymphoid tissue with a high potential to produce TNF-alpha.

Recently, a new class of human dendritic cell (DC) precursors has been described in the peripheral blood recognized by the mAb M-DC8. These cells represent approximately 1% of PBMC and acquire several characteristics of myeloid DC upon in vitro culture. In this report we show that M-DC8(+) monocytes secrete in response to LPS >10 times the amount of TNF-alpha as M-DC8(-) monocytes, but produce significantly less IL-10. Consistent with a role in inflammatory responses, we found that M-DC8(+) cells localized in the T cell area of inflamed human tonsils and in the subepithelial dome region of Peyer's patches. In patients with active Crohn's disease, abundant M-DC8(+) cells were detectable in inflamed ileal mucosa, which were entirely depleted after systemic steroid treatment. Our results indicate that M-DC8(+) cells are cells of DC phenotype in inflamed mucosa-associated lymphoid tissue that may contribute to the high level of TNF-alpha production in Crohn's disease. We infer that selective elimination of M-DC8(+) cells in inflammatory diseases has therapeutic potential.

Antibody responses against B-cell epitopes of the hypervariable region 1 of hepatitis C virus in self-limiting and chronic human hepatitis C followed-up using consensus peptides.

A rare collection of serum samples from patients with hepatitis C virus (HCV) infection followed up from the onset of clinical symptoms was acquired. RNA corresponding to the hypervariable region 1 (HVR1) of E2 protein of HCV isolated from nine patients was reverse-transcribed, amplified, sequenced, and HVR1 amino acid sequences were deduced. These sequences and a selection of HVR1 amino acid sequences of matching HCV genotypes from protein and translated DNA sequence databanks were used to create the HVR1 amino acid consensus. The degenerated peptides mimicking N- and C-termini of the consensus were synthesized. Most (76%) of 17 patients followed up for the period from 1 week to a minimum of 7 months from the onset of acute symptoms developed antibodies reacting with peptides representing N- and/or C- termini of HVR1. Antibody recognition of the consensus HVR1 peptides indicates that the variability of HVR1 sequence on the protein level is limited with certain conserved structure(s) being untouched. A tendency was observed for a slower development of anti-HVR1 antibody response in patients developing chronic HCV, as compared to those with self-limiting HCV infection.