DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update).

DAVID is a popular bioinformatics resource system including a web server and web service for functional annotation and enrichment analyses of gene lists. It consists of a comprehensive knowledgebase and a set of functional analysis tools. Here, we report all updates made in 2021. The DAVID Gene system was rebuilt to gain coverage of more organisms, which increased the taxonomy coverage from 17 399 to 55 464. All existing annotation types have been updated, if available, based on the new DAVID Gene system. Compared with the last version, the number of gene-term records for most annotation types within the updated Knowledgebase have significantly increased. Moreover, we have incorporated new annotations in the Knowledgebase including small molecule-gene interactions from PubChem, drug-gene interactions from DrugBank, tissue expression information from the Human Protein Atlas, disease information from DisGeNET, and pathways from WikiPathways and PathBank. Eight of ten subgroups split from Uniprot Keyword annotation were assigned to specific types. Finally, we added a species parameter for uploading a list of gene symbols to minimize the ambiguity between species, which increases the efficiency of the list upload and eliminates confusion for users. These current updates have significantly expanded the Knowledgebase and enhanced the discovery power of DAVID.

Interleukin-2 inhibits HIV-1 replication in some human T cell lymphotrophic virus-1-infected cell lines via the induction and incorporation of APOBEC3G into the virion.

IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.

The CD8+ HLA-DR+ T cells expanded in HIV-1 infection are qualitatively identical to those from healthy controls.

HIV-induced immune activation leads to expansion of a subset of human CD8(+) T cells expressing HLA-DR antigens. Expansion of CD8(+) HLA-DR(+) T cells can be also observed in non-HIV settings including several autoimmune diseases and aging. Although these cells are felt to represent "immune exhaustion" and/or to be anergic, their precise role in host defense has remained unclear. Here, we report that this subset of cells exhibits a restricted repertoire, shows evidence of multiple rounds of division, but lacks markers of recent TCR engagement. Detailed cell cycle analysis revealed that compared with their CD8(+) HLA-DR(-) counterpart, the CD8(+) HLA-DR(+) T-cell pool contained an increased fraction of cells in S-phase with elevated levels of the G2/M regulators: cyclin A2, CDC25C, Cdc2 (CDK1), indicating that these cells are not truly anergic but rather experiencing proliferation in vivo. Together, these data support a hypothesis that antigen stimulation leads to the initial expansion of a CD8(+) pool of cells in vivo that undergo further expansion independent of ongoing TCR engagement. No qualitative differences were noted between CD8(+) HLA-DR(+) cells from HIV(+) and HIV(-) donors, indicating that the generation of CD8(+) HLA-DR(+) T cells is a part of normal immune regulation that is exaggerated in the setting of HIV-1 infection.

Interleukin-27 treated human macrophages induce the expression of novel microRNAs which may mediate anti-viral properties.

Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.

DAVID-WS: a stateful web service to facilitate gene/protein list analysis.

SUMMARY: The database for annotation, visualization and integrated discovery (DAVID), which can be freely accessed at http://david.abcc.ncifcrf.gov/, is a web-based online bioinformatics resource that aims to provide tools for the functional interpretation of large lists of genes/proteins. It has been used by researchers from more than 5000 institutes worldwide, with a daily submission rate of ∼1200 gene lists from ∼400 unique researchers, and has been cited by more than 6000 scientific publications. However, the current web interface does not support programmatic access to DAVID, and the uniform resource locator (URL)-based application programming interface (API) has a limit on URL size and is stateless in nature as it uses URL request and response messages to communicate with the server, without keeping any state-related details. DAVID-WS (web service) has been developed to automate user tasks by providing stateful web services to access DAVID programmatically without the need for human interactions. AVAILABILITY: The web service and sample clients (written in Java, Perl, Python and Matlab) are made freely available under the DAVID License at http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html.

CD4 and CD8 T cell immune activation during chronic HIV infection: roles of homeostasis, HIV, type I IFN, and IL-7.

Immune activation plays an important role in the pathogenesis of HIV disease. Although the causes are not fully understood, the forces that lead to immune dysfunction differ for CD4 and CD8 T cells. In this study, we report that the molecular pathways that drive immune activation during chronic HIV infection are influenced by differences in the homeostatic regulation of the CD4 and CD8 T cell pools. Proliferation of CD4 T cells is controlled more tightly by CD4 T cell numbers than is CD8 T cell proliferation. This difference reflects the importance of maintaining a polyclonal CD4 T cell pool in host surveillance. Both pools of T cells were found to be driven by viral load and its associated state of inflammation. In the setting of HIV-induced lymphopenia, naive CD4 T cells were recruited mainly into the proliferating pool in response to CD4 T cell depletion, whereas naive CD8 T cell proliferation was driven mainly by levels of HIV RNA. RNA analysis revealed increased expression of genes associated with type I IFN and common γ chain cytokine signaling in CD4 T cell subsets and only type I IFN-associated genes in CD8 T cell subsets. In vitro studies demonstrated enhanced STAT1 phosphorylation in response to IFN-α and increased expression of the IFNAR1 transcripts in naive and memory CD4 T cells compared with that observed in CD8 T cells. CD4 T cell subsets also showed enhanced STAT1 phosphorylation in response to exogenous IL-7.

Cutting edge: Ku70 is a novel cytosolic DNA sensor that induces type III rather than type I IFN.

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

HIV infection-associated immune activation occurs by two distinct pathways that differentially affect CD4 and CD8 T cells.

HIV infection is characterized by a brisk immune activation that plays an important role in the CD4 depletion and immune dysfunction of patients with AIDS. The mechanism underlying this activation is poorly understood. In the current study, we tested the hypothesis that this activation is the net product of two distinct pathways: the inflammatory response to HIV infection and the homeostatic response to CD4 T cell depletion. Using ex vivo BrdU incorporation of PBMCs from 284 patients with different stages of HIV infection, we found that CD4 proliferation was better predicted by the combination of CD4 depletion and HIV viral load (R(2) = 0.375, P < 0.001) than by either parameter alone (CD4 T cell counts, R(2) = 0.202, P < 0.001; HIV viremia, R(2) = 0.302, P < 0.001). Interestingly, CD8 T cell proliferation could be predicted by HIV RNA levels alone (R(2) = 0.334, P < 0.001) and this predictive value increased only slightly (R(2) = 0.346, P < 0.001) when CD4 T cell depletion was taken into account. Consistent with the hypothesis that CD4 T cell proliferation is driven by IL-7 as a homeostatic response to CD4 T cell depletion, levels of phosphorylated STAT-5 were found to be elevated in naive subsets of CD4 and CD8 T cells from patients with HIV infection and in the central memory subset of CD4 T cells. Taken together these data demonstrate that at least two different pathways lead to immune activation of T cells in patients with HIV infection and these pathways differentially influence CD4 and CD8 T cell subsets.

Idiopathic CD4+ lymphocytopenia: natural history and prognostic factors.

Idiopathic CD4(+) lymphocytopenia (ICL) is a rare non-HIV-related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome, and prognostic factors of ICL. Thirty-nine patients (17 men, 22 women) 25 to 85 years old with ICL were evaluated between 1992 and 2006, and 36 were followed for a median of 49.5 months. Cryptococcal and nontuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In 32, CD4 T-cell counts remained less than 300/mm(3) throughout the study period and in 7 normalized after an average of 31 months. Overall, 15 (41.6%) developed an opportunistic infection in follow-up, 5 (13.8%) of which were "AIDS-defining clinical conditions," and 4 (11.1%) developed autoimmune diseases. Seven patients died, 4 from ICL-related opportunistic infections, within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (< 180/mm(3)) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (opportunistic infection-related death; P = .003 and .02, respectively).

DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists.

All tools in the DAVID Bioinformatics Resources aim to provide functional interpretation of large lists of genes derived from genomic studies. The newly updated DAVID Bioinformatics Resources consists of the DAVID Knowledgebase and five integrated, web-based functional annotation tool suites: the DAVID Gene Functional Classification Tool, the DAVID Functional Annotation Tool, the DAVID Gene ID Conversion Tool, the DAVID Gene Name Viewer and the DAVID NIAID Pathogen Genome Browser. The expanded DAVID Knowledgebase now integrates almost all major and well-known public bioinformatics resources centralized by the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of diverse gene/protein identifiers and annotation terms from a variety of public bioinformatics databases. For any uploaded gene list, the DAVID Resources now provides not only the typical gene-term enrichment analysis, but also new tools and functions that allow users to condense large gene lists into gene functional groups, convert between gene/protein identifiers, visualize many-genes-to-many-terms relationships, cluster redundant and heterogeneous terms into groups, search for interesting and related genes or terms, dynamically view genes from their lists on bio-pathways and more. With DAVID (http://david.niaid.nih.gov), investigators gain more power to interpret the biological mechanisms associated with large gene lists.

Induction of prolonged survival of CD4+ T lymphocytes by intermittent IL-2 therapy in HIV-infected patients.

HIV infection leads to decreases in the number of CD4 T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27+CD45RO-) as well as central memory (CD27+CD45RO+) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.

DAVID Knowledgebase: a gene-centered database integrating heterogeneous gene annotation resources to facilitate high-throughput gene functional analysis.

BACKGROUND: Due to the complex and distributed nature of biological research, our current biological knowledge is spread over many redundant annotation databases maintained by many independent groups. Analysts usually need to visit many of these bioinformatics databases in order to integrate comprehensive annotation information for their genes, which becomes one of the bottlenecks, particularly for the analytic task associated with a large gene list. Thus, a highly centralized and ready-to-use gene-annotation knowledgebase is in demand for high throughput gene functional analysis. DESCRIPTION: The DAVID Knowledgebase is built around the DAVID Gene Concept, a single-linkage method to agglomerate tens of millions of gene/protein identifiers from a variety of public genomic resources into DAVID gene clusters. The grouping of such identifiers improves the cross-reference capability, particularly across NCBI and UniProt systems, enabling more than 40 publicly available functional annotation sources to be comprehensively integrated and centralized by the DAVID gene clusters. The simple, pair-wise, text format files which make up the DAVID Knowledgebase are freely downloadable for various data analysis uses. In addition, a well organized web interface allows users to query different types of heterogeneous annotations in a high-throughput manner. CONCLUSION: The DAVID Knowledgebase is designed to facilitate high throughput gene functional analysis. For a given gene list, it not only provides the quick accessibility to a wide range of heterogeneous annotation data in a centralized location, but also enriches the level of biological information for an individual gene. Moreover, the entire DAVID Knowledgebase is freely downloadable or searchable at http://david.abcc.ncifcrf.gov/knowledgebase/.

New approaches for monitoring CTL activity in clinical trials.

We have developed a modification of the ELISPOT assay that measures Granzyme B (GrB) release from cytotoxic T lymphocytes (CTLs). The GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytolytic protein. We report that the GrB ELISPOT can be utilized to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells (PBMCs) from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' PBMCs in the GrB ELISPOT, with reactivity in the tetramer, IFN-gamma ELISPOT and chromium (51Cr)-release assays. Differences in immune response over all assays tested were found between patients, and four response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the 51Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. We also optimized the GrB ELISPOT assay to directly measure immune responses against autologous primary tumor cells in vaccinated cancer patients. A perforin ELISPOT assay was also adapted to evaluate peptide-stimulated reactivity of PMBCs from vaccinated melanoma patients. Modifications of the ELISPOT assay described in this chapter allow a more comprehensive evaluation of low-frequency tumor-specific CTLs and their specific effector functions and can provide a valuable insight into immune responses in cancer vaccine trials.

Interleukin-15 (IL-15) Strongly Correlates with Increasing HIV-1 Viremia and Markers of Inflammation.

OBJECTIVE: IL-15 has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. We sought to measure the level of IL-15 in a large, well characterised cohort of HIV-1 infected patients and correlate this with well known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14. DESIGN AND METHODS: IL-15 levels were measured in 501 people (460 patients with HIV-1 infection and 41 uninfected controls). The HIV-1 infected patients were divided into 4 groups based on viral load: <50 copies/ml, 51-10,000 copies/ml, 10,001-100,000 copies/ml and >100,000 copies/ml. The Mann Whitney test (non-parametric) was used to identify significant relationships between different patient groups. RESULTS: IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 ± 1.53 pg/ml) compared to both uninfected controls (1.69 ± 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 ± 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001). CONCLUSIONS: IL-15 levels are significantly elevated in HIV-1 infected patients with viral loads >100,000 copies/ml compared to uninfected controls, with a significant direct correlation noted between IL-15 and HIV-1 viremia and an inverse correlation between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.

Preferential survival of CD4+ T lymphocytes engineered with anti-human immunodeficiency virus (HIV) genes in HIV-infected individuals.

The present study examined the safety and relative in vivo survival of genetically engineered CD4+ T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. Ten pairs of identical twins discordant for HIV infection were recruited, with the uninfected twin serving as the lymphocyte donor. Ten subjects were treated with a total of 19 separate infusions of retroviral vector-transduced CD4+ enriched T cells. Control (neo gene) or anti-HIV gene (antisense trans-activation response [TAR] element and/or trans-dominant Rev)-engineered lymphocytes were monitored in peripheral blood for 3 years, using a vector-specific PCR assay. Data from 9 of the 10 patients (15 of the 19 infusions) demonstrated preferential survival of CD4+ lymphocytes containing the anti-HIV gene(s) in the immediate weeks after infusion. In six of six patients studied long term (>100 weeks), only T cells containing the anti-HIV genes were consistently detected. In addition, a marked survival advantage of anti-HIV gene-containing T cells was observed in a patient treated during a period of high viral load. Thus, these data strongly support the hypothesis that anti-HIV genes afford a survival advantage to T cells and potential benefit to HIV-1+ individuals.

Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry.

BACKGROUND: Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible. METHODS: Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining. RESULTS: Comparable lymphocyte subset percentages in WB or PBMCs were observed regardless of Optliyse method used (all Ps >/= 0.8). However, differences in fluorescence intensity for several markers were observed across procedures. Compared with the standard stain/fix procedures, fix/stain decreased the mean fluorescence intensities for CD4, CD8, CD19 and CD16/56 in WB and PBMCs (P /= 0.13), suggesting that, when the markers of interest are known at the time of field collection, implementation of this procedure might be desirable. Fix/freeze/stain resulted in diminution of intensity in general, but they tended to be more modest than those seen for fix/stain/freeze and therefore might be applicable to field studies in instances when the specific markers of interest cannot be defined upfront. CONCLUSIONS: Freezing of fixed WB and PBMCs before or after cell surface staining is a reliable method for preserving specimens in field sites for later determination of lymphocyte subset percentages, which are commonly assessed in immunodeficient and cancer patients.

Circulating CXCR5⁺CD4⁺ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines.

Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1+ICOS+, PD1+ ICOS-, or PD1-ICOS-. We used these markers to identify different subsets of CXCR5+CD4+ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD-CD38HiCD27+ memory B cells by flow cytometry. PD1+ICOS+ CXCR3+CCR6-CXCR5+CD4+ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1+ICOS+ CXCR3-CCR6-CXCR5+CD4+ (Tfh2-like) cells for both vaccines, and PD1+ICOS+ CXCR3-CCR6+CXCR5+CD4+ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1+ICOS+Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5+CD4+ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.

BACKGROUND: The interferon-gamma (IFN-gamma) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-gamma secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-gamma secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-gamma ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (alphaEN-EBV and alphaJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-gamma ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-gamma secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.

A modified human ELISPOT assay to detect specific responses to primary tumor cell targets.

BACKGROUND: The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). METHODS: Towards this goal, we adapted the IFN-gamma ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. RESULTS: After optimizing several variables, we demonstrated that the modified IFN-gamma ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. CONCLUSIONS: These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets.

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay.

BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-gamma ELISPOT and the standard 51Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3-4 h; in contrast, optimal IFN-gamma secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-gamma production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-gamma ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.