CD8+ Lymphocytes from Healthy Blood Donors Secrete Antiviral Levels of Interferon-Alpha.

The adaptive immune response to viral infections features the antigen-driven expansion of CD8+ T cells. These cells are widely recognized for their cytolytic activity that is mediated through the secretion of cytokines such as perforin and granzymes. Less appreciated is their ability to secrete soluble factors that restrict virus replication without killing the infected cells. In this study we measured the ability of primary anti-CD3/28-stimulated CD8+ T cells from healthy blood donors to secrete interferon-alpha. Supernatants collected from CD8+ T cell cultures were screened for their ability to suppress HIV-1 replication in vitro and their interferon-alpha concentrations were measured by ELISA. Interferon-alpha concentrations in the CD8+ T cell culture supernatants ranged from undetectable to 28.6 pg/mL. The anti-HIV-1 activity of the cell culture supernatants was observed to be dependent on the presence of interferon-alpha. Appreciable increases in the expression levels of type 1 interferon transcripts were observed following T cell receptor stimulation, suggesting that the secretion of interferon-alpha by CD8+ T cells is an antigen-driven response. In 42-plex cytokine assays, the cultures containing interferon-alpha were also found to contain elevated levels of GM-CSF, IL-10, IL-13, and TNF-alpha. Together, these results demonstrate that the secretion of anti-viral levels of interferon-alpha is a common function of CD8+ T cells. Furthermore, this CD8+ T cell function likely plays broader roles in health and disease.

Analysis of the CD8+ T cell anti-HIV activity in heterologous cell co-cultures reveals the benefit of multiple HLA class I matches.

CD8+ T lymphocytes can reduce the production of human immunodeficiency virus 1 (HIV-1) by CD4+ T cells by cytotoxic and non-cytotoxic mechanisms. To investigate the involvement of human leukocyte antigen (HLA) class I compatibility in anti-HIV responses, we co-cultured primary CD8+ T cells, isolated from the peripheral blood of HIV-1-infected individuals, with panels of autologous and heterologous acutely HIV-1-infected primary CD4+ T cells. Altogether, CD8+ T cell anti-HIV activity was evaluated in more than 200 co-cultures. Marked heterogeneity in HIV-1 replication levels was observed among the co-cultures sharing a common CD8+ T cell source. The co-cultures that exhibited greater than 50% reduction in HIV production were found to have significantly increased numbers of matching HLA class I alleles (Yates chi-square = 54.21; p < 0.001). With CD8+ T cells from HIV controllers and asymptomatic viremic individuals, matching HLA-B and/or HLA-C alleles were more predictive of strong anti-HIV activity than matching HLA-A alleles. Overall, HLA class I genotype matches were more closely associated with CD8+ T cell anti-HIV activity than supertype pairings. Antibodies against HLA class I and CD3 reduced the CD8+ T cell anti-HIV activity. Stimulated CD8+ T cells exhibited increased anti-HIV activity and reduced dependency on HLA compatibility. These findings provide evidence that the maximal suppression of HIV replication by CD8+ T cells requires the recognition of multiple epitopes. These studies provide insight for HIV vaccine development, and the analytic approach can be useful for the functional characterization of HLA class I alleles and tentative HLA class I supertypes.

Recovery and assessment of leukocytes from LR Express filters.

Leukocytes, or white blood cells, are used for a variety of investigational purposes and they offer advantages over laboratory-adapted cell lines. Leukocytes that are typically discarded by blood banks during the collection of red blood cells, platelets, and plasma can often be obtained for research use. However, the available leukocytes are frequently contained within a blood filtration device, such as the Terumo LR Express (TLRE) filter. In this study, procedures were evaluated for the ability to elute viable leukocytes from TLRE filters. The recovered leukocytes were assessed for composition, growth, and functionality. The large majority (>70%) of leukocytes were eluted with a single reverse-elution procedure and the recovered cells contained representative populations of the major leukocyte subsets. Purified T cells exhibited diverse T cell receptor repertoires, characteristic growth upon mitogen stimulation, and CD4+ T cells were able to support HIV-1 propagation. Purified monocytes were able to be differentiated into phenotypically characteristic populations of macrophages and dendritic cells. Overall, TLRE filters offer an attractive source of primary human cells for research and possibly clinical purposes.

Brief Report: Increased Expression of the Type I Interferon Receptor on CD4+ T Lymphocytes in HIV-1-Infected Individuals.

BACKGROUND: Type I interferons (IFN1s; eg, interferon-alpha and interferon-beta) are potent cytokines that inhibit the replication of human immunodeficiency virus-1 (HIV-1) and other viruses. The antiviral and immunoregulatory activities of IFN1 are mediated through ligand-receptor interactions with the IFN1 receptor complex (IFNAR). Variation in the cell-surface density of IFNAR could play a role in HIV-1 pathogenesis. METHODS: In this cross-sectional study of fresh whole blood, we used flow cytometry to evaluate the expression of IFNAR2 on lymphocyte subsets from HIV-1-infected (n = 33) and HIV-1-uninfected (n = 22) individuals. RESULTS: In comparison with healthy blood bank donors, we observed that the HIV-1-infected individuals, particularly those having advanced to disease, exhibited the increased expression of IFNAR2 on CD4 T cells (relative fluorescence intensity 6.9 vs. 9.0; P = 0.027). The CD4:CD4 T-cell IFNAR2 expression-level ratio provides an internally standardized measure of this alteration. The observed increased expression of IFNAR2 was largely restricted to CD4 T cells that expressed the chemokine receptor CXCR4 and lacked the expression of CCR5. CONCLUSIONS: HIV-1-infected individuals exhibit an increased expression of the IFN1 receptor on CD4 T cells. The level of IFNAR2 expression seems to increase with disease progression. These findings provide insight for the immunologic alterations associated with HIV-1 infection and possibly new therapeutic approaches.

Recurrent epimutation of SDHC in gastrointestinal stromal tumors.

Succinate dehydrogenase (SDH) is a conserved effector of cellular metabolism and energy production, and loss of SDH function is a driver mechanism in several cancers. SDH-deficient gastrointestinal stromal tumors (dSDH GISTs) collectively manifest similar phenotypes, including hypermethylated epigenomic signatures, tendency to occur in pediatric patients, and lack of KIT/PDGFRA mutations. dSDH GISTs often harbor deleterious mutations in SDH subunit genes (SDHA, SDHB, SDHC, and SDHD, termed SDHx), but some are SDHx wild type (WT). To further elucidate mechanisms of SDH deactivation in SDHx-WT GIST, we performed targeted exome sequencing on 59 dSDH GISTs to identify 43 SDHx-mutant and 16 SDHx-WT cases. Genome-wide DNA methylation and expression profiling exposed SDHC promoter-specific CpG island hypermethylation and gene silencing in SDHx-WT dSDH GISTs [15 of 16 cases (94%)]. Six of 15 SDHC-epimutant GISTs occurred in the setting of the multitumor syndrome Carney triad. We observed neither SDHB promoter hypermethylation nor large deletions on chromosome 1q in any SDHx-WT cases. Deep genome sequencing of a 130-kbp (kilo-base pair) window around SDHC revealed no recognizable sequence anomalies in SDHC-epimutant tumors. More than 2000 benign and tumor reference tissues, including stem cells and malignancies with a hypermethylator epigenotype, exhibit solely a non-epimutant SDHC promoter. Mosaic constitutional SDHC promoter hypermethylation in blood and saliva from patients with SDHC-epimutant GIST implicates a postzygotic mechanism in the establishment and maintenance of SDHC epimutation. The discovery of SDHC epimutation provides a unifying explanation for the pathogenesis of dSDH GIST, whereby loss of SDH function stems from either SDHx mutation or SDHC epimutation.

CD8(+) lymphocytes suppress human immunodeficiency virus 1 replication by secreting type I interferons.

CD8(+) cells can suppress human immunodeficiency virus 1 (HIV-1) replication by releasing soluble factors. In 26 years of intensive research efforts, the identity of the major CD8(+) cell antiviral factor has remained elusive. To investigate the mechanism for this antiviral immune response, we performed gene expression analyses on primary CD4(+) cells that were exposed to HIV-suppressing CD8(+) cells or CD8(+) cell-conditioned medium having HIV-suppressing activity. These experiments revealed increased levels of multiple genes stimulated by type I interferons (IFN; eg, IFN-α and IFN-ß). Further evaluation revealed that primary CD8(+) cells, particularly those from elite controllers and other asymptomatic HIV-1-infected individuals, secrete IFN, and this response directly contributes to the in vitro suppression of HIV replication in CD4(+) cells. This novel immune response, likely mediated by memory CD8(+) T cells, may play an important role in a wide variety of viral infections, cancers, and autoimmune diseases.

Dual role of autophagy in HIV-1 replication and pathogenesis.

Autophagy, the major mechanism for degrading long-lived intracellular proteins and organelles, is essential for eukaryotic cell homeostasis. Autophagy also defends the cell against invasion by microorganisms and has important roles in innate and adaptive immunity. Increasingly evident is that HIV-1 replication is dependent on select components of autophagy. Fittingly, HIV-1 proteins are able to modulate autophagy to maximize virus production. At the same time, HIV-1 proteins appear to disrupt autophagy in uninfected cells, thereby contributing to CD4+ cell death and HIV-1 pathogenesis. These observations allow for new approaches for the treatment and possibly the prevention of HIV-1 infection. This review focuses on the relationship between autophagy and HIV-1 infection. Discussed is how autophagy plays dual roles in HIV-1 replication and HIV-1 disease progression.

Derivation of non-infectious envelope proteins from virions isolated from plasma negative for HIV antibodies.

Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.

HIV/AIDS: 30 years of progress and future challenges.

Acquired immune deficiency syndrome (AIDS) was first described 30 years ago in a report from the US Centers for Disease Control. Two years later the causative virus was identified and afterwards named the human immunodeficiency virus (HIV). This article reviews the progress made in the three decades since the recognition of AIDS and the discovery of HIV, with respect to the virus, the infected cell, and the host, as well as directions for future studies.

A methyl-deviator epigenotype of estrogen receptor-positive breast carcinoma is associated with malignant biology.

We broadly profiled DNA methylation in breast cancers (n = 351) and benign parenchyma (n = 47) for correspondence with disease phenotype, using FFPE diagnostic surgical pathology specimens. Exploratory analysis revealed a distinctive primary invasive carcinoma subclass featuring extreme global methylation deviation. Subsequently, we tested the correlation between methylation remodeling pervasiveness and malignant biological features. A methyl deviation index (MDI) was calculated for each lesion relative to terminal ductal-lobular unit baseline, and group comparisons revealed that high-grade and short-survival estrogen receptor-positive (ER(+)) cancers manifest a significantly higher MDI than low-grade and long-survival ER(+) cancers. In contrast, ER(-) cancers display a significantly lower MDI, revealing a striking epigenomic distinction between cancer hormone receptor subtypes. Kaplan-Meier survival curves of MDI-based risk classes showed significant divergence between low- and high-risk groups. MDI showed superior prognostic performance to crude methylation levels, and MDI retained prognostic significance (P < 0.01) in Cox multivariate analysis, including clinical stage and pathological grade. Most MDI targets individually are significant markers of ER(+) cancer survival. Lymphoid and mesenchymal indexes were not substantially different between ER(+) and ER(-) groups and do not explain MDI dichotomy. However, the mesenchymal index was associated with ER(+) cancer survival, and a high lymphoid index was associated with medullary carcinoma. Finally, a comparison between metastases and primary tumors suggests methylation patterns are established early and maintained through disease progression for both ER(+) and ER(-) tumors.

Natural suppression of human immunodeficiency virus type 1 replication is mediated by transitional memory CD8+ T cells.

HIV replication is suppressed in vitro by a CD8(+) cell noncytotoxic antiviral response (CNAR). This activity directly correlates with an asymptomatic clinical state. The objective of this study was to identify the phenotype of CD8(+) cell subsets having strong CNAR activity. CD8(+) cell subset frequencies and CNAR levels were measured for human immunodeficiency virus (HIV)-uninfected individuals and three groups of HIV type 1 (HIV-1)-infected individuals: asymptomatic individuals with low-level viremia (vHIV), antiretroviral-drug-treated subjects with undetectable virus levels (TxHIV), and therapy-naïve aviremic elite controllers (EC). CD8(+) cells from the vHIV individuals exhibited the highest HIV-suppressing activity and had elevated frequencies of CD45RA(-) CD27(+) and PD-1(+) (CD279(+)) cells. Functional assessments of CD8(+) cells sorted into distinct subsets established that maximal CNAR activity was mediated by CD45RA(-) CCR7(-) CD27(+) and PD-1(+) CD8(+) cells. T cell receptor (TCR) repertoire profiles of CD8(+) cell subsets having strong CNAR activity exhibited increased perturbations in comparison to those of inactive subsets. Together, these studies suggest that CNAR is driven by HIV replication and that this antiviral activity is associated with oligoclonally expanded activated CD8(+) cells expressing PD-1 and having a transitional memory cell phenotype. The findings better describe the identity of CD8(+) cells showing CNAR and should facilitate the evaluation of this important immune response in studies of HIV pathogenesis, resistance to infection, and vaccine development.

CD8+ cell anti-HIV activity rapidly increases upon discontinuation of early antiretroviral therapy.

INTRODUCTION: CD8+ lymphocytes can suppress HIV replication without killing the infected cells. This CD8+ cell noncytotoxic anti-HIV response (CNAR) is associated with a beneficial clinical course. MATERIALS AND METHODS: In this longitudinal study of 16 participants in the Options Project at UCSF, we measured the ability of CD8+ lymphocytes to suppress HIV replication in CD4+ cells during primary HIV infection, early antiretroviral therapy, and after treatment. RESULTS AND DISCUSSION: CD8+ lymphocytes from subjects with untreated primary HIV-1 infection strongly suppressed HIV replication. Initiation of antiretroviral therapy during primary HIV-1 infection caused a marked decline in this CNAR. CD8+ cells from these subjects regained anti-HIV activity when early therapy was discontinued. The timing of the appearance of CD8+ cell anti-HIV activity directly correlated with the emergence of detectable virus levels. Maximal CNAR activity coincided with a decay in the kinetics of HIV replication. In addition, peak viral loads during treatment interruption were lower than pre-treatment virus levels (median reduction = 0.8 logs, p = 0.005) and CD4+ T cell counts were maintained for a 24-week period of follow-up. CONCLUSION: These results suggest that CNAR plays an important role in suppressing HIV replication in the setting of antiretroviral treatment interruption in HIV-infected individuals.

Large-scale profiling of archival lymph nodes reveals pervasive remodeling of the follicular lymphoma methylome.

Emerging technologies allow broad profiling of the cancer genome for differential DNA methylation relative to benign cells. Herein, bisulfite-modified DNA from lymph nodes with either reactive hyperplasia or follicular lymphoma (FL) were analyzed using a commercial C/UpG genotyping assay. Two hundred fifty-nine differentially methylated targets (DMT) distributed among 183 unique genes were identified in FL. Comparison of matched formalin-fixed, paraffin-embedded and frozen surgical pathology replicates showed the complete preservation of the cancer methylome among differently archived tissue specimens. Analysis of the DMT profile is consistent with a pervasive epigenomic remodeling process in FL that affects predominantly nonlymphoid genes.

Similar changes in plasmacytoid dendritic cell and CD4 T-cell counts during primary HIV-1 infection and treatment.

OBJECTIVES: Reduced dendritic cell (DC) frequencies and functions in individuals with longstanding HIV-1 infection are predictive of opportunistic infections and AIDS. To investigate possible early alterations in DC levels after HIV infection, we prospectively examined plasmacytoid dendritic cell (pDC) and myeloid dendritic cell (mDC) frequencies and plasma IFN-alpha levels in patients undergoing primary HIV-1 infection (PHI). METHODS: Peripheral blood DC frequencies and absolute counts were determined by flow cytometry. Plasma IFN-alpha levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In comparison to uninfected subjects, pDC, but not mDC, levels were reduced (P < 0.001) in subjects with PHI, especially in those with high viral loads or low CD4 T-cell counts. During 24-48 weeks of observation, untreated subjects experienced slight declines in pDC and CD4 T-cell levels. In contrast, subjects initiating early antiretroviral therapy (ART) exhibited increases (P < 0.001) in pDC and CD4 T-cell counts. No effect of treatment on mDC counts was observed. Circulating plasma IFN-alpha was undetectable by ELISA regardless of the duration of HIV-1 infection. CONCLUSION: PHI is characterized by a reduction in pDC and CD4 T-cell counts that correlates with the magnitude of virus replication and is not evidenced by the mDC count or plasma IFN-alpha level. Early ART appears to have similar restorative effects on pDC and CD4 T-cell counts.

The effects of early antiretroviral therapy and its discontinuation on the HIV-specific antibody response.

HIV-specific antibodies become detectable and continue to increase in frequency during primary infection. The effects of early antiretroviral treatment (ART) and its discontinuation on the evolution of this immune response have not been systematically analyzed. To investigate the associations between antibody titer, viral load, and ART, we used a less-sensitive enzyme-linked immunosorbant assay (LS-EIA) to measure changes in HIV-1-specific antibody levels in treated and untreated subjects undergoing primary infection. In this longitudinal study, antibody levels gradually increased in therapy-naive subjects, reaching a plateau approximately 40 weeks postinfection. In contrast, antibody titers remained low among subjects receiving ART. Subjects who discontinued ART exhibited a more rapid rise in antibody titers than therapy-naive subjects, suggesting the presence of an enhanced B cell response. These results demonstrate that early ART prevents the typical evolution of the HIV-1-specific antibody response and can alter the expected kinetics of this response in subjects discontinuing therapy.

Genetic and stochastic influences on the interaction of human immunodeficiency virus type 1 and cytotoxic T lymphocytes in identical twins.

Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8+ T-lymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.

A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses.

The rate of HIV-1 disease progression is influenced by several factors that include pathogen and host genetic variations and the quality of antiviral immune responses. The CD8+ cell non-cytotoxic antiviral response (CNAR) substantially suppresses HIV replication in CD4+ cells and is positively associated with an asymptomatic clinical state. Traditionally, the measurement of CNAR has required several culture procedures and costly reagents. Here we report the development and validation of a screening assay for detection of CNAR that accurately identifies individuals benefiting from this response. Use of the CNAR screening assay should facilitate the evaluation of this important immune parameter in studies of HIV pathogenesis, resistance to infection, and vaccine development.

Clonal breadth of the HIV-1-specific T-cell receptor repertoire in vivo as determined by subtractive analysis.

OBJECTIVE: Although the epitopic breadth of HIV-1-specific CD8 T lymphocyte (CTL) responses has been described, the T cell receptor (TCR) diversity of virus-specific cells remains poorly defined. DESIGN AND METHODS: To address this issue, we applied a novel technique for subtractive analysis of the HIV-1-specific CTL repertoire, combining specific deletion of peptide-specific cells by 5-fluorouracil with TCR spectratyping to identify clonal breadth of CTL recognizing individual peptides. RESULTS: Comprehensive analysis of an infected individual reveals that nine identified HIV-1-specific responses are comprised of at least 38 distinct T-cell clones (ranging from two to 10 distinct clones per epitope). CONCLUSION: Given the potentially crucial role of T-cell receptor breadth for viral recognition and avoidance of escape, this subepitopic analysis of CTL may offer an important measure of cellular immunity for pathogenesis and vaccine studies.

Persistent alterations in the T-cell repertoires of HIV-1-infected and at-risk uninfected men.

OBJECTIVE: We examined the association between immunogenic exposure and T-cell receptor (TCR) diversity to more clearly assess the impact of HIV-1 infection on the T-cell repertoire. METHODS: : To estimate the extent of T-cell clonality attributable to HIV-1 infection, we evaluated T-cell repertoires in low-risk and at-risk seronegative men and HIV-1 seropositive men by assessment of T-cell receptor beta-chain (TCR beta) complimentary determining region 3 (CDR3) lengths. RESULTS: The frequency of T-cell clonality in both HIV-1 infected and at-risk uninfected men was elevated in comparison to low-risk uninfected men. Among low-risk and at-risk seronegative, and HIV-1 seropositive men, clonal expansions were present in 3, 8, and 10% of CD4+ CDR3 lengths, and 18, 22, and 28% of CD8+ CDR3 lengths respectively. In addition, the longitudinal conservation of clonal expansions was observed in at-risk seronegative men. Based on comparisons to at-risk seronegative men, we estimate that at-risk seropositive men with chronic HIV-1 infection exhibit a 27% increase in the number of expanded CD8+ CDR3 lengths. CONCLUSION: These findings provide an approximation of the magnitude of the T-cell response in individuals undergoing chronic HIV-1 infection and demonstrate a significant association between the history of immunogenic challenge and the magnitude of clonality within the T-cell repertoire. In addition, these findings underscore the necessity of selecting controls with similar antigenic exposure histories when investigating T-cell dynamics in HIV-infected individuals.

MaGiK method of T-Cell receptor repertoire analysis.

T-cell receptor diversity enables the cellular immune response to recognize a broad range of viral and other pathogenic agents. An increasingly common method of characterizing T-cell receptor diversity and usage in response to antigenic challenges involves the identification of clonal expansions by PCR amplification of the CDR3 region of distinct TCRVbeta families. Though clonal expansions often appear evident upon visual inspection of the results, a systematic method is needed for the valid enumeration of these expansions. Here, we describe a novel analysis method, termed the MaGiK method, for systematically identifying and enumerating clonal T-cell expansions and for applying the results to investigations of the T-cell receptor repertoire.