Maria A. Suni

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry

We have recently described a highly sensitive flow cytometric technique, based on the ability to detect single cell expression of cytokines, to simultaneously quantitate and phenotypically characterize antigen-specific memory/effector T cells in PBMC cultures. In this report, we describe a simplified procedural modification which enables the rapid detection of low frequency memory CD4+ and CD8+ T cells expressing cytokines in response to soluble antigen in whole blood. When compared with T cell responses in PBMC cultures, whole blood cultures demonstrated similar but slightly higher percentages of T cells responsive to specific antigen. In addition, T cell responses to cytomegalovirus in whole blood were observed only in sensitized (seropositive) individuals, and CD4+ T cell responses could be blocked by anti-class II MHC antibodies. This procedure may provide a means to examine direct effects of pharmacological drug concentrations on T cell immunity in clinical samples.

Standardization of cytokine flow cytometry assays

BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template.Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

CD4+CD8dim T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV‐1 antigens

CD4+CD8dim T cells represent a minor subset of the total CD3+ T cell population in peripheral blood. Although transient and persistent expansions of these cells havebeen reported in both healthy and diseased individuals, the functional properties of the CD4+CD8dim population are largely unknown. In this study, we examined antigen‐specific cytokine and proliferative responses of the CD4+CD8dim subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV‐1, a significant fraction of the CD4+CD8dim subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4+CD8dim population contained two‐ to eightfold higher frequencies of antigen‐specific cytokine producing cells than the CD4+CD8‐ population. Phenotypic analysis of the cytokine expressing CD4+CD8dim population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4+CD8dim cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4+CD8dim T cells were found to possess higher CMV‐specific cytotoxic activity than purified CD4+CD8– T cells in a standard 51Cr‐release CTL assay. Thus, CD4+CD8dim T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4+ cell populations specific for HCMV and HIV‐1.

Factors Affecting the Efficiency of CD8+ T Cell Cross-Priming with Exogenous Antigens

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, “cross-priming” can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor’s blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual’s APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.

VTX-2337 Is a Novel TLR8 Agonist That Activates NK Cells and Augments ADCC

Purpose: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody–based immunotherapy that includes the activation of natural killer (NK) cells. Experimental Design: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). Results: VTX-2337 selectively activates TLR8 with an EC50 of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). Conclusions: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy. Clin Cancer Res; 18(2); 499–509. ©2011 AACR.

Gamma Interferon Expression in CD8+ T Cells Is a Marker for Circulating Cytotoxic T Lymphocytes That Recognize an HLA A2-Restricted Epitope of Human Cytomegalovirus Phosphoprotein pp65

ABSTRACT Antigen-specific CD8+ T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-γ) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8+ T-cell IFN-γ expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a 51Cr release assay and frequencies of peptide-activated CD8+ T cells expressing IFN-γ at 6 h (r2 = 0.72) or 7 days (r2 = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-γ expression can be a functional surrogate for identification of CTL precursor cells.

Enhancement of HIV type 1 antigen-specific CD4+ T cell memory in subjects with chronic HIV type 1 infection receiving an HIV type 1 immunogen.

We examined HIV-1 specific memory helper T immune responses in chronically HIV-infected subjects who received an immune-based therapy (HIV-1 immunogen, Remune). Subjects in this study exhibited significant increases (p < 0.05) in the frequency of helper T memory cells expressing interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in response to HIV-1 antigens in vitro. The frequencies of HIV-specific memory T cells increased after successive immunizations and exhibited a correlation with the standard tritiated thymidine incorporation lymphocyte proliferation assay (r = 0.72, p < 0.0008). These results support the notion that HIV-specific memory immune responses can be stimulated in subjects with chronic HIV infection. Further investigations are warranted to determine whether the induction of such responses is associated with virologic control.

Performance of plate-based cytokine flow cytometry with automated data analysis.

BackgroundCytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates.ResultsWe have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis.ConclusionWhen combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.

Functional T Cell Responses to Tumor Antigens in Breast Cancer Patients Have a Distinct Phenotype and Cytokine Signature

The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8+ T cells, and a lower proportion of IFN-γ-producing CD4+ and/or CD8+ T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8+ T cells in breast cancer patients was almost completely CD28+CD45RA− (memory phenotype). CMV-responsive CD8+ T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27−CD28−CD45RA+) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.