Emilee R. Knowlton

Serum Levels of Cytokines, and Biomarkers for Inflammation and Immune Activation, and HIV-Associated Non-Hodgkin B cell Lymphoma Risk

Background: HIV infection is associated with a marked increase in risk for non-Hodgkin lymphoma (AIDS-NHL). However, the mechanisms that promote the development of AIDS-NHL are not fully understood. Methods: In this study, serum levels of several cytokines and other molecules associated with immune activation were measured in specimens collected longitudinally during 1 to 5 years preceding AIDS-NHL diagnosis, in 176 AIDS-NHL cases and 176 HIV+ controls from the Multicenter AIDS Cohort Study (MACS). Results: Multivariate analyses revealed that serum levels of immunoglobulin free light chains (FLC), interleukin (IL)-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α were elevated in those HIV+ individuals who went on to develop AIDS-NHL. In addition, the fraction of specimens with detectable IL-2 was increased and the fraction with detectable IL-4 was decreased in these subjects. Conclusions: These results suggest that long-term, chronic immune activation, possibly driven by macrophage-produced cytokines, precedes development of NHL in HIV+ individuals. Impact: FLC, IL-6, IL-10, IP-10/CXCL10, neopterin, and TNF-α may serve as biomarkers for AIDS-NHL. Cancer Epidemiol Biomarkers Prev; 23(2); 343–9. ©2013 AACR.

Monofunctional and polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins.

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. It is postulated that CD8+ T cell responses play an important role in controlling HHV-8 infection and preventing development of disease. In this study, we investigated monofunctional and polyfunctional CD8+ T cell responses to HHV-8 lytic proteins gB (glycoprotein B) and K8.1 and latency proteins LANA-1 (latency-associated nuclear antigen-1) and K12. On the basis of our previous findings that dendritic cells (DC) reveal major histocompatibility complex (MHC) class I epitopes in gB, we used a DC-based system to identify 2 novel epitopes in gB, 2 in K8.1, 5 in LANA-1, and 1 in K12. These new HHV-8 epitopes activated monofunctional and polyfunctional CD8+ T cells that produced various combinations of gamma interferon, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, and cytotoxic degranulation marker CD107a in healthy HHV-8-seropositive individuals. We were also able to detect HHV-8-specific CD8+ T cells in peripheral blood samples using HLA A*0201 pentamer complexes for one gB epitope, one K8.1 epitope, two LANA-1 epitopes, and one K12 epitope. These immunogenic regions of viral lytic and latency proteins could be important in T cell control of HHV-8 infection.

Professional antigen presenting cells in human herpesvirus 8 infection

Professional antigen presenting cells (APC), i.e., dendritic cells (DC), monocytes/macrophages, and B lymphocytes, are critically important in the recognition of an invading pathogen and presentation of antigens to the T cell-mediated arm of immunity. Human herpesvirus 8 (HHV-8) is one of the few human viruses that primarily targets these APC for infection, altering their cytokine profiles, manipulating their surface expression of MHC molecules, and altering their ability to activate HHV-8-specific T cells. This could be why T cell responses to HHV-8 antigens are not very robust. Of these APC, only B cells support complete, lytic HHV-8 infection. However, both complete and abortive virus replication cycles in APC could directly affect viral pathogenesis and progression to Kaposis sarcoma (KS) and HHV-8-associated B cell cancers. In this review, we discuss the effects of HHV-8 infection on professional APC and their relationship to the development of KS and B cell lymphomas.

Human Herpesvirus 8 Induces Polyfunctional B Lymphocytes That Drive Kaposi’s Sarcoma

ABSTRACT Kaposi’s sarcoma (KS) is an unusual neoplasia wherein the tumor consists primarily of endothelial cells infected with human herpesvirus 8 (HHV-8; Kaposi’s sarcoma-associated herpesvirus) that are not fully transformed but are instead driven to excess proliferation by inflammatory and angiogenic factors. This oncogenic process has been postulated but unproven to depend on a paracrine effect of an abnormal excess of host cytokines and chemokines produced by HHV-8-infected B lymphocytes. Using newly developed measures for intracellular detection of lytic cycle proteins and expression of cytokines and chemokines, we show that HHV-8 targets a range of naive B cell, IgM memory B cell, and plasma cell-like populations for infection and induction of interleukin-6, tumor necrosis factor alpha, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, and interleukin-8 in vitro and in the blood of HHV-8/HIV-1-coinfected subjects with KS. These B cell lineage subsets that support HHV-8 infection are highly polyfunctional, producing combinations of 2 to 5 of these cytokines and chemokines, with greater numbers in the blood of subjects with KS than in those without KS. Our study provides a new paradigm of B cell polyfunctionality and supports a key role for B cell-derived cytokines and chemokines produced during HHV-8 infection in the development of KS. IMPORTANCE Kaposi’s sarcoma (KS) is the most common cancer in HIV-1-infected persons and is caused by one of only 7 human cancer viruses, i.e., human herpesvirus 8 (HHV-8). It is unclear how this virus causes neoplastic transformation. Development and outgrowth of endothelial cell lesions characteristic of KS are hypothesized to be dependent on virus replication and multiple immune mediators produced by the KS cells and inflammatory cells, yet the roles of these viral and cell factors have not been defined. The present study advances our understanding of KS in that it supports a central role for HHV-8 infection of B cells inducing multiple cytokines and chemokines that can drive development of the cancer. Notably, HIV-1-infected individuals who developed KS had greater numbers of such HHV-8-infected, polyfunctional B cells across a range of B cell phenotypic lineages than did HHV-8-infected persons without KS. This intriguing production of polyfunctional immune mediators by B cells serves as a new paradigm for B cell function and classification. Kaposi’s sarcoma (KS) is the most common cancer in HIV-1-infected persons and is caused by one of only 7 human cancer viruses, i.e., human herpesvirus 8 (HHV-8). It is unclear how this virus causes neoplastic transformation. Development and outgrowth of endothelial cell lesions characteristic of KS are hypothesized to be dependent on virus replication and multiple immune mediators produced by the KS cells and inflammatory cells, yet the roles of these viral and cell factors have not been defined. The present study advances our understanding of KS in that it supports a central role for HHV-8 infection of B cells inducing multiple cytokines and chemokines that can drive development of the cancer. Notably, HIV-1-infected individuals who developed KS had greater numbers of such HHV-8-infected, polyfunctional B cells across a range of B cell phenotypic lineages than did HHV-8-infected persons without KS. This intriguing production of polyfunctional immune mediators by B cells serves as a new paradigm for B cell function and classification.

Fifty Percent Tissue Culture Infective Dose Assay for Determining the Titer of Infectious Human Herpesvirus 8

ABSTRACT We have developed a human herpesvirus 8 (HHV-8) 50% tissue culture infective dose (TCID50) assay using the T1H6-DC-SIGN cell line. Infection of T1H6-DC-SIGN cells with HHV-8 induces expression of β-galactosidase, which was used to determine TCID50 levels. Validation of TCID50 values was performed by immunofluorescence assay of HHV-8 infection of immature dendritic cells at various TCID50 doses.

Abstract 5075: B-cell expression of AICDA and MME [CALLA, CD10] is predictive of a subsequent non-Hodgkin lymphoma diagnosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Mounting evidence from epidemiologic studies of serum biomarkers highlights the importance of inflammation and immune activation preceding the diagnosis of B-cell non-Hodgkin lymphoma (NHL). Although chronic B-cell hyperactivation is likely involved in the pathogenesis of NHL, an important gap in knowledge remains present as the source and targets of these inflammatory and immune activation associated factors are mainly undefined. Targeted studies of B-cell gene expression can help fill this gap, further define the immune environment from which these tumors arise, and assist in identifying biomarkers for early detection of B-cell NHLs. Methods: This study was based in a large national cohort of HIV+ men, the Multicenter AIDS Cohort Study, who are at increased risk for developing NHL. 144 HIV+ B-cell NHL cases were matched to 144 HIV+ controls on follow-up time and sample availability. B-cells isolated from viably frozen peripheral blood mononuclear cells, collected 2 to 13 years (median 4.7 years) prior to NHL diagnosis or control match date, were used to measure messenger RNA (mRNA) levels of 16 molecules that are associated with B-cell activation, using the Quantigene® Plex 2.0 Assay Kit (Affymetrix) on the Luminex platform. Expression levels were normalized by the geomean of five housekeeping genes. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by conditional logistic regression for gene expression categorized as detectable versus undetectable. Results: Activation-induced cytidine deaminase (AICDA) was detected in 8.3% of cases and 2.8% of controls, OR=3.7, 95% CI=1.0-13.1. Membrane metallo-endopeptidase (MME) was detected in 59.7% of cases and 29.9% of controls, OR=3.9, 95% CI=2.2-6.8. The AICDA and MME associations with NHL were similar across subgroups of site, EBV tumor status, and lag time between sample collection and NHL diagnosis date. AICDA, but not MME, was more strongly associated with NHL among those who were the least immunosuppressed (higher CD4+ T cell count and low HIV RNA levels). In subgroup analyses, CD40 ligand (CD40LG) expression was associated with NHL with a time lag time of <6 years, OR=3.0, 95% CI=1.0-9.3, and Interleukin-6 (IL6) expression was associated with NHL among those with a CD4+ T cell count <300, OR=3.9, 95% CI=0.9-17. Interpretation & Conclusions: AICDA expression is essential for antibody specificity and diversity but its aberrant expression has been implicated in lymphomagenesis. MME is a signature marker of B-cell NHLs of germinal center origin and has previously been associated with NHL survival. We found that AICDA and MME expression precedes the clinical diagnosis of NHL in an HIV+ cohort. Additionally, late CD40LG expression and IL6 expression among the more severely immunosuppressed individuals were associated with NHL risk in this study. These molecules should be studied further for possible early detection and clinical utility. Citation Format: Shehnaz K. Hussain, Marta Epeldegui, Larry I. Magpantay, Elizabeth Crabb Breen, Emilee Knowlton, Steven Wolinksy, Lisa P. Jacobson, Jay H. Bream, Roger Detels, Zuo-Feng Zhang, Otoniel Martinez-Maza. B-cell expression of AICDA and MME [ CALLA, CD10 ] is predictive of a subsequent non-Hodgkin lymphoma diagnosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5075. doi:10.1158/1538-7445.AM2014-5075

Human herpesvirus 8 replicates in primary B lymphocytes and induces polyfunctional cytokine and chemokine responses

Methods B cells were loaded with purified “live” HHV-8, purified UV-light inactivated HHV-8 (UV-HHV-8) or soluble HHV-8 glycoprotein B (gB). HHV-8 replication was measured by real time PCR for viral DNA and intracellular staining (ICS) and flow cytometry for the viral lytic proteins ORF59 and K8.1. ICS was used to assess cellassociated, polyfunctional cytokine-chemokine production, and B cell supernatants were tested for cytokine/ chemokine secretion by Cytometric Bead Array (BD Biosciences).