Exploring the Feasibility of Multi-Site Flow Cytometric Processing of Gut Associated Lymphoid Tissue with Centralized Data Analysis for Multi-Site Clinical Trials

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.     

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4⁺ and CD8⁺ T cells and CD45RA-positive terminal effector CD8⁺ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.     

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250-270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H-thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen-specific T responses, because of the low specific frequencies of the cells. Collagen II (250-270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250-270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3-color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.     

T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

[目的]马传染性贫血病毒(EIAV)弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型.为探讨IFN-γ表达水平与疫苗保护性免疫的关系,本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-γ水平的方法.[方法]我们将分离的马传贫弱毒疫苗免疫马(FDDV)、强毒感染马(LV)和健康马的外周血单核细胞(PBMC),体外分别经病毒(FDDV)和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-γ抗体等过程后,进行流式检测.[结果]疫苗免疫马产生的特异性IFN-γ水平为CD4 1.7(0.9%/CD8 6.1(1.2%,而强毒组则为CD4 0.6(0.1%/CD8 2.4(0.9%.[结论]本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-γ染色和淋巴细胞亚型的方法,具有良好的特异性,稳定性和重复性.为研究EIAV弱毒疫苗免疫保护机制奠定了基础.     

An automated analysis of highly complex flow cytometry-based proteomic data

The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.     

The development of standard samples with a defined number of antigen-specific T cells to harmonize T cell assays: a proof-of-principle study

The validation of assays that quantify antigen-specific T cell responses is critically dependent on cell samples that contain clearly defined measurable numbers of antigen-specific T cells. An important requirement is that such cell samples are handled and analyzed in a comparable fashion to peripheral blood mononuclear cells (PBMC). We performed a proof-of-principle study to show that retrovirally TCR-transduced T cells spiked at defined numbers in autologous PBMC can be used as standard samples for HLA/peptide multimer staining. NY-ESO-1_157–165-specific, TCR-transduced CD8+ T cell batches were successfully generated from PBMC of several HLA-A*0201 healthy donors, purified by magnetic cell sorting on the basis of HLA tetramer (TM) staining and expanded with specific antigen in vitro. When subsequently spiked into autologous PBMC, the detection of these CD3+CD8+TM+ T cells was highly accurate with a mean accuracy of 91.6 %. The standard cells can be preserved for a substantial period of time in liquid nitrogen. Furthermore, TM staining of fresh and cryopreserved standard samples diluted at decreasing concentrations into autologous cryopreserved unspiked PBMC revealed that the spiked CD3+CD8+TM+ T cells could be accurately detected at all dilutions in a linear fashion with a goodness-of-fit of over 0.99 at a frequency of at least 0.02 % among the CD3+CD8+ T cell population. Notably, the CD3+CD8+TM+ cells of the standard samples were located exactly within the gates used to analyze patient samples and displayed a similar scatter pattern. The performance of the cryopreserved standard samples in the hands of 5 external investigators was good with an inter-laboratory variation of 32.9 % and the doubtless identification of one outlier.     

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids.MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously.ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%.ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.     

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF? mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.     

Polychromatic flow cytometry: a rapid method for the reduction and analysis of complex multiparameter data.

BACKGROUND: Recent advances in flow cytometry have resulted in the development of reliable techniques for performing polychromatic (5-17 color) flow cytometry analysis. However, the data reduction and analysis involved in the resolution of hundreds of possible cellular subphenotypes identified, using a single polychromatic flow cytometry staining panel, presents a major obstacle to the successful application of this technology. METHODS: To generate two distinct collections of T cell populations with differentially expressed surface markers, cryopreserved lymph node cells from 5 melanoma patients vaccinated with the modified gp100(209-2M) melanoma peptide were stimulated with cognate peptide and cultured in either IL-21 + low-dose IL-2 or IL-15 + low-dose IL-2. In vitro stimulated (IVS) cells were interrogated using 8-color flow cytometry. Data were analyzed using Winlist Hyperlog and FCOM software, and 32 T cell subsets were resolved for each culture condition. Hierarchical clustering analysis was applied to the relative percentages of each subphenotype for both IVS conditions to determine if unique cell surface marker expression signatures were produced for each IVS culture. RESULTS: Sequential data analysis using Hyperlog and FCOM demonstrated that lymphocytes cultured in IL-21 + IL-2 had a distinctively different set of subphenotype signatures compared to cells grown in IL-15 + IL-2 for all 5 patients. Importantly, subsequent cluster analysis of all 32 subphenotype frequencies in each IVS test condition for all 5 patients reproducibly demonstrated that cellular subphenotypes produced after IL-21 + IL-2 IVS partitioned separately from subphenotypes produced by IL-15 + IL-2 IVS. CONCLUSIONS: The integrated sequential use of Hyperlog and FCOM software with cluster analysis algorithms for the reduction and analysis of polychromatic flow cytometry data produces an effective, rapid technique for the assessment of complex patterns of subphenotype expression between and within multiple test samples. This approach to data analysis may enhance the use of polychromatic flow cytometry for both research and clinical applications.     

Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein

Quantitative evaluation of T cell responses of patients receiving antitumoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-gamma, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-gamma upon stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10(-6). We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1.     

Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA-4 blockade

We have demonstrated previously that the administration of CTLA-4 blockade has mediated objective cancer regression and autoimmunity in patients with metastatic melanoma. To explore the mechanism of these in vivo effects, we have studied the changes in lymphocyte phenotype and function in patients receiving anti-CTLA-4 Ab (MDX-010). Patients with stage IV melanoma or renal cell cancer were treated every 3 wk with an anti-CTLA-4 Ab with or without peptide immunization. Pheresis samples were analyzed using flow cytometry to determine lymphocyte cell surface markers. Gene expression analyses and proliferation assays were conducted on purified T cell subsets. Anti-CTLA-4 Ab did not inhibit the suppressive activity of CD4+CD25+ cells in vitro or in vivo. In addition, there was no decrease in the expression of CD4+CD25+ cells in whole PBMC, nor a decrease in Foxp3 gene expression in the CD4+ or CD4+CD25+ purified cell populations posttreatment. The percentage of CD4+, CD8+, CD4+CD25+, and CD4+CD25- T cells in PBMC expressing the activation marker HLA-DR increased following anti-CTLA-4 Ab administration. Therefore, our results suggest that the antitumor effects of CTLA-4 blockade are due to increased T cell activation rather than inhibition or depletion of T regulatory cells.     

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4⁺ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4⁺ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4⁺ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4⁺ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy

Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50?%. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.     

Endothelial cell suppression of peripheral blood mononuclear cell trafficking in vitro during acute exposure to feline immunodeficiency virus

Trafficking of peripheral blood mononuclear cells (PBMCs) into the brain is a critical step in the initiation of human immunodeficiency virus (HIV)-associated central nervous system disease. To examine potential factors that control trafficking during the earliest stages of infection, PBMC transmigration across a cultured feline brain endothelial cell (BECs) monolayer was measured after selective exposure of various cell types to feline immunodeficiency virus (FIV). Infection of the PBMCs with FIV increased the trafficking of monocytes and CD4 and CD8 T cells. Additional exposure of the BECs to FIV suppressed mean monocyte, CD4 T cell, and CD8 T cell trafficking. B cell trafficking was unaltered by these changing conditions. Subsequent exposure of astrocytes or microglia to FIV altered transmigration of different PBMC subsets in different ways. Treated microglia compared with treated astrocytes decreased monocyte transmigration, whereas B cell transmigration was increased significantly. When both astrocytes and microglia were exposed to FIV, an increase in CD8 T cell transmigration relative to BECs alone, to BECs plus astrocytes, or to BECs plus microglia was demonstrated. Thus, initial exposure of PBMCs to FIV is sufficient to induce a general increase in trafficking, whereas initial exposure of endothelial cells to FIV tends to down-regulate this effect. Selectivity of trafficking of specific PBMC subsets is apparent only after exposure of cells of the central nervous system to FIV in co-culture with the endothelium.     

Influence of calcium ions in the flow cytometric analysis of human CD8-positive cells.

BACKGROUND: The CD8 co-receptor is an important marker used to identify various lymphocyte subsets. A significant decrease in CD8alpha staining intensity was observed in the presence of divalent cation chelators. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were treated with calcium chelators, stained with different anti-human CD8 mAbs, and analyzed by flow cytometry. RESULTS: Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8alpha mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8beta mAb. CONCLUSIONS: Divalent cation chelators are used as cell anti-clumping agents in MACS or FACS applications. Researchers should be aware that such treatment could lead to the almost complete loss of fluorescence with selected anti-human CD8alpha mAbs. Since CD8 staining is used in conjunction with tetramer staining to identify antigen-specific cytotoxic human T cells, the effect of calcium depletion should be taken into account in experimental design.     

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.     

Requirement of monocytes and T-helper cells during development of tumor cell cytotoxicity in targeted T cells

In cocultures of human plancental alkaline phosphatase(PLAP)-positive MO₄ tumor cells and human peripheral blood mononuclear cells (PBMC), also containing a heteroconjugate (7E8-OKT3) synthesized between the anti-PLAP monoclonal antibody 7E8 and the anti-CD3 antibody OKT3, and supplemented with low levels of recombinant interleukin-2 (rIL-2), T cells are progressively activated, resulting in tumor cell lysis. To unravel the contribution of PBMC subsets to the generation of this targetable cytotoxicity, PBMC subsets were studied after their isolation by cell sorting, either from fresh PBMC or from PBMC peractivated with OKTe3 and rIL-2. Whereas no targetable cytotoxicity was found in Fc-receptor-bearing CD3-cells, tumor cells were lysed by CD3⁺ T cells (mostly CD8⁺) isolated from pre-activated PBMC. When isolated from fresh PBMC, neither the CD8⁺ T cell subset, nor the total CD3⁺ T cell population developed significant targetable cytotoxicity, even in the presence of rIL-2. Thus, additional cell types are essential for the CD8⁺ T cell activation. Indeed. CD4⁺ T cells isolated from pre-activated but not from fresh PBMC were capable of eliciting cytotoxicity in fresh CD8⁺ T cells. The non-targeted monocytes were found to be the activators of the CD4⁺ T cells. In summary, targeting T cells to the surface of a tumor cell is not sufficientper se to achieve activation and lysis. The progressive tumor cell lysis by targeted T cells seems to be initiated by non-targeted monocytes activating CD4⁺ T cells, these cells in turn promoting CD8⁺ T cell activation, necessary for the development of cytotoxicity.