Donald D. Anthony

Selective Impairments in Dendritic Cell-Associated Function Distinguish Hepatitis C Virus and HIV Infection

Impaired APC functions may play important roles in chronicity of hepatitis C virus (HCV) and HIV infections. To investigate the separate and combined effects of HCV and HIV infection on immature dendritic cells (DCs), we evaluated myeloid-derived DC (MDC) and plasmacytoid-derived DC (PDC) frequencies and functions, measured by Toll-like receptor ligand-induced IFN-α and IL-12, in healthy controls and subjects with chronic HCV, HIV, and HCV-HIV infection. To evaluate the relation between innate and adaptive immunity, we measured HCV-specific IFN-γ-producing T cell frequency. MDC frequencies tended to be reduced in HIV infection (1.8-fold), while PDC frequencies were minimally reduced in HCV infection (1.4-fold). In contrast, a striking reduction in non-PDC-associated IFN-α production was observed in HIV-infected subjects (17-fold), while PDC-associated IFN-α production was markedly reduced in HCV-infected subjects (20-fold). Both non-PDC and PDC functions were impaired in HCV-HIV coinfection. MDC-associated IL-12 production was markedly reduced in both HCV and HIV-infected subjects (over 10-fold). Functional defects were attenuated with slowly progressive HIV infection. The proportion of subjects with HCV-specific T cell responses, and the number of Ags recognized were reduced in HCV-HIV subjects as compared with HCV singly infected subjects. A positive association was observed between MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-infected subjects. These results indicate that immature DC function is dysregulated in HIV and HCV infections, but differentially, and that these defects are attenuated in slowly progressive HIV infection. These selectively different impairments may contribute to the reduced adaptive immune response to HCV in HCV-HIV coinfection.

T cells reactive to a single immunodominant self-restricted allopeptide induce skin graft rejection in mice.

Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.

Insulin-like growth factor type I biology and targeting in malignant gliomas.

Growth factors such as insulin-like growth factor type I (IGF-I), epidermal growth factor (EGF), vascular-endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) are present during the development of the CNS. When they reappear in the mature brain they are overexpressed in neoplastic glia, participating in the development of the most common human brain malignant tumor, glioblastoma multiforme, which is invariably fatal. Progress in treatment of this disease involves an increase in median survival from 8 to 11 months to an average of 15 months, rarely to 18 months. We do not know any therapy, which can make a complete stop of this neoplasm. To inhibit this process various anti-growth factor therapies have been proposed. We describe actual applications of growth factor inhibitors and antisense approaches. The review highlights results obtained with the promising treatment of glioblastoma multiforme: using inhibitors and antisense targeting growth factors, including IGF-I, their receptors, and their downstream signaling effectors including glycogenesis and oncogenes. The antisense strategies have been the subject of many clinical trials, especially the IGF-I antisense approach. Such antisense therapies, already introduced in clinical trial in the USA, Europe and Asia, will soon become the preferred alternative treatment for human glioblastoma multiforme. The inhibition of signal transduction pathways common to growth factors and glycogenesis appears as a parallel challenge to glioblastoma multiforme inhibition studies.

Interferon-α is the primary plasma type-I IFN in HIV-1 infection and correlates with immune activation and disease markers.

Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.

T-cell epitope mapping using the ELISPOT approach

The ELISPOT assay is particularly well suited to measure both clonal size and effector function of low-frequency antigen-specific T-cell populations directly ex vivo. Typically, an ELISPOT assay is performed with a freshly obtained sample (or cryopreserved sample) using less than 24h of culture. Additionally, this assay allows for the simultaneous analysis of hundreds of variables in parallel from a single tissue specimen. Because of these capabilities, this assay has found widespread use in the context of direct ex vivo immune diagnostic monitoring in humans. Herein we describe the rationale, methodology, and useful hints for performing T-cell epitope mapping using ELISPOT analysis, and review typical results of such an analysis.

Negative regulation of the interferon response by an interferon-induced long non-coding RNA

Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼200 IFN-induced lncRNAs, one transcript showed ∼100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

Immune responses to hepatitis C and non-hepatitis C antigens in hepatitis C virus infected and HIV-1 coinfected patients

ObjectiveTo characterize immune phenotype and function in hepatitis C virus (HCV) infection in the presence and absence of HIV-1 infection. DesignCross-sectional comparison among controls (group A), patients with HCV infection (group B), HCV–HIV-1 coinfected patients (group C), coinfected patients receiving treatment for HIV-1 (group D), and untreated HIV-1 infected patients (group E). MethodsFlow cytometric analysis for lymphocyte phenotypes, lymphocyte proliferation and cytokine production by ELISPOT. ResultsHCV infected patients tended to have an increased percentage of activated (CD38, HLA-DR) CD8 cells (group A, 2 ± 1.4%; group B, 6 ± 3.9%;P = 0.08). Proliferative responses to non-HCV antigens were comparable in group A and group B subjects. A greater proportion of group B patients had stimulation indices (SI) > 3 to the HCV protein NS3 compared to group C and D patients (67%, 0%, and 11% respectively;P < 0.003), but only two patients in group B had SI ⩾ 5. The SI to NS3 was significantly higher in group B patients [median, 4; interquartile range (IQR), 3–9) than in group C (median, 2; IQR, 1–3;P < 0.04) or group D (median, 1; IQR, 1–4;P < 0.009) patients. Plasma HCV RNA levels correlated directly with alanine aminotransferase levels (ρ, 0.52;P < 0.05) and inversely with the number of CD4 lymphocytes (ρ, −0.55;P < 0.009) and proliferation to NS3 (ρ, −0.55;P < 0.009). ConclusionsLymphocytes of HCV infected patients show weak proliferative responses to HCV antigens while responses to other antigens are preserved. Infection with HIV-1 potentiates this deficiency. Poor CD4 T cell responses to HCV are associated with and may determine the failure to control HCV propagation.

TLR Ligand-Dependent Activation of Naive CD4 T Cells by Plasmacytoid Dendritic Cells Is Impaired in Hepatitis C Virus Infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-α or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-γ. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-α did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-α) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.

HIV long-term non-progressors maintain brisk CD8 T cell responses to other viral antigens

Objective To examine the specificity of heightened CD8 cell responses in HIV-1-infected long-term non-progressors. Design Cross-sectional study examining CD8 cell responses to hepatitis C virus (HCV) peptides in HCV–HIV LTNP (n = 6), HCV–HIV progressors (n = 11), HCV singly infected patients (n = 32), HCV singly infected patients with self-limited disease (n = 10), HIV singly infected progressors (n = 7) and HCV-negative, HIV-negative controls (n = 10). Methods The frequency of HCV-reactive interferon γ-producing cells in peripheral blood was assayed by enzyme linked immunospot assay using a panel of 61 HCV-1-derived peptides. Results Five of six HCV–HIV LTNP had HCV-specific CD8 responses. In contrast, responses were observed in 2 of 32 HCV singly infected patients, 2 of the 10 HCV singly infected patients with self-limited disease, and 0 of 11 HCV–HIV progressors (P < 0.001). Both frequency of HCV-specific CD8 cells and number of HCV peptides recognized were greater in HCV–HIV LTNP than in other groups. Conclusions HIV-infected LTNP maintain heightened CD8 cell responses to HCV in addition to heightened HIV specific responses. Common mechanisms may underlie preservation of CD8 immune responses in these individuals. An improved understanding of these mechanisms will help to gain insight into protective antiviral immunity as well as to the means whereby these viruses impair host defenses.

Determinants of Protection among HIV-Exposed Seronegative Persons: An Overview

Both clinical experience and a growing medical literature indicate that some persons who have been exposed to human immunodeficiency virus (HIV) infection remain uninfected. Although in some instances this may represent good fortune, cohorts of uninfected persons have been reported who are considered at high risk for infection. In these cohorts a variety of characteristics have been proposed as mediating protection, but to date only the 32–base pair deletion in the chemokine (C‐C motif) receptor 5 gene, which results in complete failure of cell surface expression of this coreceptor, has been associated with high‐level protection from HIV infection. With this in mind, there are probably many other factors that may individually or in combination provide some level of protection from acquisition of HIV infection. Because some of these factors are probably incompletely protective or inconsistently active, identifying them with confidence will be difficult. Nonetheless, clarifying the determinants of protection against HIV infection is a high priority that will require careful selection of high‐risk uninfected cohorts, who should undergo targeted studies of plausible mediators and broad screening for unexpected determinants of protection.