Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond.

Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project.

Response definition criteria for ELISPOT assays revisited.

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses.

Evaluation of Elispot assays: influence of method and operator on variability of results.

In this study, a comprehensive comparative analysis of different evaluation methods of Elispot plates was performed. Three investigators using three different evaluation approaches read 50 randomly selected wells at three different time points. The methods were: (1) manual evaluation using a stereomicroscope, (2) automated evaluation using an image analysis reader system with reading parameters established by each investigator, and (3) automated evaluation using a reader system with preset reading parameters using assay-specific controls. We demonstrate that manual evaluation had the highest variability both within the same method and when comparing all methods, followed by automated evaluation with investigator-dependent parameters. The variability was low only when all investigators used the same parameters established using assay-specific controls. This variability was independent of operator or spot number per well. Based on this study, recommendations for standardization and validation procedures of Elispot assay performance and evaluation procedures are presented.

Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: a pilot study.

Heat shock protein (HSP)-peptide complexes isolated from murine cancers elicit protective immunity and T lymphocytes specific for the cancer from which the HSPs are isolated. A pilot study was designed to test the feasibility, immunogenicity and toxicity of such treatment in cancer patients. Sixteen patients with assorted advanced malignancies, which had become refractory to established therapies, were recruited. The gp96 vaccine was prepared for each patient from tumor obtained from that patient. Anti-tumor immune responses were evaluated using Elispot assays of T cells in peripheral blood after minimal in vitro stimulation. No unacceptable vaccine-related toxicities or auto-immune reactions were observed. Immunization with autologous gp96 elicited MHC I-restricted, tumor-specific CD8(+) T lymphocytes in 6/12 patients immunized. In addition, expansion of the NK cell population was seen in 8/13 of patients immunized. These observations are entirely consistent with the murine experience and form a firm basis for future trials with clinical end points, using autologous, patient-specific HSP-peptide vaccines.

Evaluation of CD8(+) T-cell frequencies by the Elispot assay in healthy individuals and in patients with metastatic melanoma immunized with tyrosinase peptide.

The lack of reproducible, quantitative assays for T-cell responses has been a limitation in the development of cancer vaccines to elicit T-cell immunity. We utilized the Elispot assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubations. CD8(+) T-cell reactivity was determined with an interferon (IFN)-gamma Elispot assay detecting T cells at the single cell level that secrete IFN-gamma. We studied both healthy individuals and patients with melanoma. Healthy HLA-A*0201-positive individuals showed a similar mean frequency of CD8(+) cells recognizing a tyrosinase peptide, YMDGTMSQV, when compared with melanoma patients prior to immunization. The frequencies of CD8(+) cells recognizing the tyrosinase peptide remained relatively constant over time in healthy individuals. Nine HLA-A*0201-positive patients with stage IV metastatic melanoma were immunized intradermally with the tyrosinase peptide together with the immune adjuvant QS-21 in a peptide dose escalation study with 3 patients per dose group. Two patients demonstrated a significant increase in the frequency of CD8(+) cells recognizing the tyrosinase peptide during the course of immunization, from approx. 1/16,000 CD8(+) T cells to approx. 1/4,000 in the first patient and from approx. 1/14,000 to approx. 1/2,000 in the second patient. These results demonstrate that modest expansion of peptide-specific CD8(+) T cells can be generated in vivo by immunization with peptide plus QS-21 in at least a subset of patients with melanoma.

Insect cells as HLA-restricted antigen-presenting cells for the IFN-gamma elispot assay.

Measurement of specific cellular immune responses in patients undergoing immunotherapy is difficult. Established approaches, including cytotoxicity (e.g., 51Cr release) and cytokine release assays, require in vitro culturing for several weeks or more of patients' peripheral blood mononuclear cells (PBMC) and the addition of exogenous cytokines. Therefore, the immunological response does not reflect in vivo conditions. To address these disadvantages, we have used an interferon-gamma (IFN-gamma) Elispot assay for detecting peptide-specific CD8(+) lymphocytes in PBMC. A limitation of this assay is the lack of a reproducible source of antigen-presenting cells (APCs). Currently available APCs often lead to significant background levels. It has been shown that transfected insect cells can express empty MHC class I molecules on their surface. We have transfected Drosophila melanogaster S2 cells and the Lepidopteran line Sf9 with the gene encoding human HLA-A2.1. We demonstrate that insect cells expressing a human HLA molecule effectively function as APCs in the IFN-gamma Elispot assay. Initially the feasibility of the assay was assessed using CD8(+) T cells from HLA-A2.1(+) donors with known reactivity against an HLA-A2.1-binding epitope of the influenza matrix protein. Use of insect cells as APCs abrogated background spots, increasing sensitivity. We further observed that a short-term prestimulation of PBMC with peptide-pulsed insect cells markedly enhanced the frequency of peptide-specific T cells that could be measured in the Elispot assay without increasing the background. This approach was then used to measure CD8(+) T cell reactivity to a peptide from tyrosinase, an antigen that is processed and presented by melanoma cells. Insect cells expressing human HLA molecules provide a standard APC for monitoring CD8(+) T cell responses to tumor and viral peptides during immunotherapy.

MHC polymorphism can enrich the T cell repertoire of the species by shifts in intrathymic selection.

The murine class I molecule H-2Kb and its natural gene conversion variant, H-2Kbm8, which differs from H-2Kb solely at 4 aa at the bottom of the peptide-binding B pocket, are expressed in coisogenic mouse strains C57BL/6 (B6) and B6.C-H-2bm8 (bm8). These two strains provide an excellent opportunity to study the effects of Mhc class I polymorphism on the T cell repertoire. We recently discovered a gain in the antiviral CTL repertoire in bm8 mice as a consequence of the emergence of the Mhc class I allele H-2Kbm8. In this report we sought to determine the mechanism behind the generation of this increased CTL diversity. Our results demonstrate that repertoire diversification occurred by a gain in intrathymic positive selection. As previously shown, the emergence of the same Mhc allele also caused a loss in positive selection of T cell repertoire specific for another Ag, OVA-8. This indicates that a reciprocal loss-and-gain pattern of intrathymic selection exists between H-2Kb and H-2Kbm8. Therefore, in the thymus of an individual, a new Mhc allele can select new T cell specificities, while abandoning some T cell specificities selected by the wild-type allele. A byproduct of this repertoire shift is a net gain of T cell repertoire of the species, which is likely to improve its survival fitness.

Generation of tumor-specific cytotoxic T lymphocytes and memory T cells by immunization with tumor-derived heat shock protein gp96.

Tumor-derived heat shock proteins have been shown previously to elicit specific prophylactic immunity to cognate tumors. Here we establish that administration of gp96 results in generation of tumor-specific cytotoxic T lymphocyte response in addition to protective immunity. In one instance, gp96 preparations are shown to elicit an immune response against a tumor, where intact tumor cells are unable to do so. Finally, our results indicate that gp96 preparations elicit a memory T cell response that is capable of being recalled.

Heat shock protein vaccines against cancer.

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Structure of the mRNA and of the gene coding for the rabbit erythroid 15-lipoxygenase.

The complete structure of the rabbit erythroid cell-specific 15-lipoxygenase mRNA and its gene was established by sequencing cDNA and genomic recombinants. The transcription initiation site was obtained by primer-extension sequencing. A presumptive promoter structure was characterized by sequencing 0.5 kb 5' to the transcription initiation site and by transfection experiments using constructs with the chloramphenicol transferase gene. The mRNA codes for a polypeptide of 662 amino acids. Its 3' untranslated region contains an intriguing repeated sequence of 10 copies with the consensus C4PuC3TCTTC4AAG which may be involved in its regulation during reticulocyte maturation. The transcription unit consists of 8.0 kb and is split like the gene for the leukocyte 5' lipoxygenase by 13 introns. Another interesting aspect in the structure of the 15-LOX gene is a highly conserved repeat in intron seven consisting of a unit of 54 nucleotides which is repeated eight times. Comparing the predicted amino acid sequence with those from other lipoxygenases published recently shows that lipoxygenases are a related group of enzymes which may have arisen from a common ancestral gene.

The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase.

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.

The complete sequence of the rabbit erythroid cell-specific 15-lipoxygenase mRNA: comparison of the predicted amino acid sequence of the erythrocyte lipoxygenase with other lipoxygenases.

We report the complete sequence of the rabbit reticulocyte (RBC) 15-lipoxygenase (LOX) mRNA as deduced from (i) sequencing cDNA recombinants isolated by screening cDNA libraries or polymerase-chain-reactions, and (ii) the sequence originating from the transcription start point obtained by primer extension-sequencing reactions. Like the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains a very short 5'-untranslated region with a long 3'-untranslated region. But, unlike the human leukocyte 5-LOX mRNA, the RBC 15-LOX mRNA contains an intriguing repeated sequence (ten copies with the consensus sequence C4PuC3TCTTC4AAG) just after the translational stop codon, which may be involved in its regulation during reticulocyte maturation. Comparison of the RBC 15-LOX mRNA sequence with those of the previously published human 5-LOX mRNA and the soybean 3-LOX gene shows only a few short regions of sequence similarity. However, the predicted amino acid sequences of the encoded LOX enzymes show certain conserved regions that are presumably involved in their catalytic activity, in particular a cluster of five conserved histidines that we predict chelate the iron moiety involved in the active site.