CFSE T Cell Proliferation AssaysProImmune's CFSE T cell proliferation assay can be used to identify epitope sequences that elicit helper T cell proliferation and therefore potentially cause a helper T cell immune response. Unlike traditional assays which are based on radioactive thymidine incorporation, this assay utilizes powerful flow cytometry methods, enabling accurate determination of the percentage of proliferating CD4+ cells and detailed phenotyping of T cell responses, all with significantly improved overall sensitivity. |
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The T cell proliferation assay has been developed to identify the presence or absence of potential T cell epitopes and is useful in preclinical discovery of novel peptides or proteins. The assays can address issues of 'relative immunogenicity' between structurally similar molecules; for example, they can help to distinguish between different candidates, or can give an indication of the success of protein engineering strategies in deimmunization of a potential immunotherapeutic. As part of ProImmune's REVEAL™ Immunogenicity System, results from the T cell proliferation assay can be analyzed alongside the outcome from cell-free HLA-peptide binding assays for more than 50 class II HLA alleles. This gives the customer a detailed profile of the helper T cell immune response to one or more drug leads. T Cell Proliferation Assays in DetailProImmune offers two types of T cell proliferation assay: naïve primary T cell assay for peptide epitope mapping and DC-T cell assay for whole protein screening. Cells are labeled with the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). Those cells that proliferate in response to antigen show a reduction in CFSE fluorescence intensity, which is measured directly by flow cytometry. Since this is a flow cytometric assay, it accurately determines the percentage of proliferating CD4+ cells, enables detailed phenotyping of T cell responses, and is more sensitive than traditional assays based on radioactive thymidine incorporation. Cell proliferation is determined by flow cytometric analysis. For naïve T cell assays, a Cell Division Index is determined for each stimulated sample, and for DC-T cell assays the percent stimulation above background is determined for each stimulated sample, through comparison with results from an unstimulated sample. The lowest and highest values are discarded and the mean and standard deviations from the intermediate 4 results are calculated. A result is considered significantly positive if the Cell Division Index (naïve assay) is greater than 2x background (untreated) plus 2x standard deviation, or if the percent stimulation above background (DC assay) is greater than 0.02% plus 2x standard deviation. For both assays, a response is considered significant if 2 or more donors give a positive result. Naïve Primary T cell Assay for Peptide Epitope ScreeningThis assay is used to identify peptide epitope sequences that can elicit helper CD4+ T cell proliferation and therefore potentially cause a helper T cell immune response that may lead to anti-drug antibody (ADA) responses or other unwanted immunogenicity. CD8+ T cell-depleted and CFSE-labeled donor PBMC are cultured with 5uM of each peptide of interest for 7 days in six replicate wells. Each assay plate includes a set of untreated control wells. The assay also incorporates reference antigen controls, comprising synthetic peptides for known MHC class II antigens, and two potent whole protein antigens.
Figure1: Example staining data from naïve T cell assay, (1) CFSE-labeled T cells cultured in media alone (unstimulated), (2) CFSE-labeled T cells cultured with peptide derived from antigen of interest, (3) CFSE-labeled T cells cultured with control protein (Tuberculin PPD).
Figure 2: Graphical presentation of the results of the naïve primary T cell assay for one donor for 52 peptides and 2 controls. Six peptides and both controls show a significant cell division index (y-axis, >2). DC-T Cell Assays for Whole Protein ScreeningThe Dendritic Cell (DC)-T cell assay for screening whole proteins has wide-ranging applications. It allows for an overall comparison of the T cell driven antigenicity of any number of drug candidates at a pre-clinical stage. Crucially it can also be used for assessing the impact on antigenicity of factors other than protein sequence. Such differences may include a comparison of biosimilars, protein modifications, degradation products, chemical entities given in combination therapies, and other parameters related to manufacturing processes, excipients, drug formulation and stability. Additionally, in some cases it may not be possible to use the antigen to stimulate PBMC directly, particularly if the antigen involved modifies the function of responding T cells. To avoid such assay interference, antigens can be presented using dendritic cells, allowing the relative antigenicity of different leads to be compared directly. Donor PBMC are used as a source of monocytes that are cultured in defined media to generate immature dendritic cells. Dendritic cells are loaded with test antigen (whole protein), and are then induced into a more mature phenotype by further culture in defined media. CD8+ T cell-depleted donor PBMC from the same donor sample are labeled with CFSE then cultured with the antigen-primed DCs for 7 days in six replicate wells. Each DC-T cell culture includes a set of untreated control wells. The assay also incorporates reference antigen controls, comprising two potent whole protein antigens.
Figure 3: Example staining data from DC-T cell assay, (1) CFSE-labeled T cells incubated with DCs that were not co-cultured with antigen, (2) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with whole protein antigen (Drug 1), (3) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with control antigen (Tuberculin PPD). When evaluating immunogenicity, it is appropriate to take account of the frequency of donor cell responses across the study cohort. A positive response (percentage stimulation above background >0.02%) in 2 or more independent donor samples is considered indicative of a potential in vivo T cell response. It is also important to consider the strength of positive donor cell responses. This is determined by taking an average of the percentage stimulation above background obtained across accepted donors for each drug. A Response Index is calculated by multiplying the value of the strength of response by the frequency of the donors responding. This index is more representative of the level of immunogenicity than methods of analysis that rely on the frequency of response alone. If a positive response is observed in a donor sample in either of the two T cell assays, then a proliferative immune response has been mounted through at least one of the six HLA class II alleles presented by that donor. The information gained in the REVEAL™ MHC-peptide binding assays can be compared and validated with these sensitive T cell proliferation assays. See information about ProImmune's REVEAL™ Immunogenicity System.
Figure 4: Graphical representation of results of the DC-T cell assay for 48 donors for 4 whole proteins (antibody drugs) and 2 controls. As can be clearly demonstrated in figure 4, the fully human antibody drug 4 had the lowest response index, indicating the lowest in vitro immunogenicity in the DC-T Cell Assay. As expected, the humanized antibodies (drug 2 and drug 3) showed the next lowest immunogenicity and the chimeric antibody (drug 1) showed the highest immunogenicity. Different therapeutic proteins will yield specific levels of immunogenicity and this can be directly quantified in the ProImmune DC-T cell assay.
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