The Human T Cell Priming Assay (hTCPA)
The induction of allergic contact dermatitis by chemicals depends on their ability to activate a chemical-specific T cell response. This key event that concludes the skin sensitization process is monitored in an in vitro assay, the human T cell priming assay (hTCPA). Naive human T cells are incubated with autologous dendritic cells pulsed with the chemical of interest. After restimulation with the same chemical, the induction of an antigen-specific T cell response can be assessed by the detection of T cell proliferation, cytokine production or surface marker expression. The hTCPA still needs optimization but may nevertheless be a valuable in vitro assay within an integrated testing strategy aiming at replacement of animal testing for contact allergen identification.
KeywordsChemical Contact allergen T cell Skin In vitro assay Animal testing
Allergic contact dermatitis (ACD) as an occupational skin disease causes high socio-economic costs due to the lack of causative treatments . Up to now, the only possibility for sensitized individuals is to symptomatically treat their inflamed skin with corticosteroids or non-steroidal anti-inflammatory drugs—and eventually to avoid contact with the causative chemical. This often necessitates a change in professions, making careful determination of the sensitizing potential of new chemicals mandatory. However, animal testing for the skin sensitizing potential of chemical compounds is considered unacceptable. The European Directive 86/609/EEC aims to reduce the number of animals used for research and other purposes and to promote alternatives to animal testing. In addition, the seventh amendment to the EU Cosmetics Directive prohibits animal testing for skin sensitization potential of chemicals including a complete marketing ban for products that contain ingredients tested on animals. Moreover, the Regulation on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) also aims at a reduction of animal testing. Hence, there is an urgent need for the development of in vitro alternatives.
The previous gold standard for testing the skin sensitizing potential of chemicals was the local lymph node assay (LLNA) (OECD Test Guideline 429). This in vivo assay addresses the proliferation of cells in the lymph nodes of mice after repeated application of a test chemical onto their ear skin . In order to develop in vitro alternatives to the LLNA, the immunological mechanisms underlying the ACD have to be considered, and key events of the sensitization process must be addressed in in vitro assays. Given the complexity of this process, the development of integrated testing strategies (ITS) that combine different assays is the current aim.
Immunologically, contact dermatitis can be of two different types. The irritant contact dermatitis (ICD) is an eczematous skin reaction previously attributed solely to toxic effects of chemicals. However, ICD seems to involve activation of the innate immune system. In contrast, ACD is defined by its activation of both the innate and the adaptive immune system. Here, the initial activation of innate immune responses is necessary to facilitate the activation and migration of skin dendritic cells to the draining lymph node and, subsequently, the efficient priming of antigen-specific T cells. As a classical type IV allergy, ACD results in erythema and eczema formation driven by the cytotoxic effects of different T cell subsets. While in the murine model of ACD—the contact hypersensitivity (CHS) model—CD8+ T cells seem to play a dominant role [3, 4, 5, 6], IL-17 producing Th17/Tc17 cells can also be found. Additionally, Th22 cells can be detected during ACD reactions in the human system .
Several approaches have been proposed to set up a test system for the in vitro identification of the potential allergenicity and the allergenic potency of chemical compounds, mostly analysing various parameters depending on the activation of innate immune responses.
Over the last few years, it has become clear that most probably no single test will be sufficient to provide a standalone assay to determine the sensitizing potential of a chemical. Although in February 2015 the Direct Peptide Reactivity Assay (DPRA) and the ARE-Nrf2 Luciferase Test Method (e.g. Keratinosens™) and more recently the human Cell Line Activation Test (h-CLAT) have been adopted by the OECD as Test No. 442C, 422D and 442E, respectively, in all cases, the use of these assays as part of an IATA (integrated approach to testing and assessment, IATA) was recommended. To this end, the combination of assays addressing different mechanistic aspects of the ACD might prove to be most useful, and different approaches have been evaluated recently [8, 9].
31.2 The hTCPA Principle
We have developed a standard operating procedure (SOP) for a human T cell priming assay in close collaboration with the group of J.F. Nicolas and M. Vocanson in Lyon that enables the differentiation between contact sensitizers and irritants. For this assay, fresh human peripheral blood is initially separated into CD14+ monocytes and CD14- cells. While the CD14- cells are stored until 6–8 days, the monocytes are differentiated with GM-CSF and IL-4 over 5–7 days to immature monocyte-derived dendritic cells (MDDCs). Subsequently, they are incubated with the test chemical for 24 h at a concentration previously determined to result in a cytotoxicity of ~20%. To facilitate a full maturation of the MDDCs regardless of the chemical that is used, the TLR4 agonist LPS is added during this incubation step. On day 7, the CD14- fraction is further purified by depletion of CD25+/CD56+/CD45RO+ and non-T cells to contain only naive T cells. The unbound chemical is removed by washing, and the MDDCs are plated into 96 wells together with the autologous naive T cells. While the optimal MDDC/T cell ratio has to be determined for the specific setup, a ratio of 1:10 usually works best in our hands. After 2 days, IL-7 and IL-15 are added to the culture, after 4 days IL-2, IL-7 and IL-15 are added, and after 6 days the three cytokines are added once again in order to allow for a most efficient T cell priming and proliferation. After 9–10 days of this priming phase, the T cells are restimulated with autologous MDDCs that were either treated with the same chemical as before (antigen specific restimulation), left untreated (background control) or have been treated with an irrelevant chemical (specificity control). As a positive control, a fraction of the T cells can be stimulated by addition of PMA/Ionomycin. Depending on the readout, we usually analyse the T cell activation 6 h after the restimulation step in a multiparametric flow cytometry analysis and detection of intracellular cytokines like IFN-γ or TNF-α. However, other readouts/readout systems can easily be chosen like detection of the cytotoxic activity of the primed T cells via CD107a staining, proliferation via CFSE/Ki67 analysis as well as ELISPOT assays for cytokine and Granzyme B measurement.
31.3 Recent Efforts to Optimize Antigen Detection: Obstacles to Overcome
One of the problems arising with the set-up of a T cell-based assay is the limitation in cell numbers arising from the need to use primary autologous MDDCs and naive T cells from the same blood donor to avoid unspecific activation. This limitation makes a T cell-based assay at best a medium-throughput assay that cannot compete with an assay set-up, for example, with cell lines. However, we have been able to enhance the number of chemicals that can be tested in one approach due to two changes in the original protocol. One change is the use of CD19+ B cells as antigen-presenting cells for the restimulation step. Since the priming of the naive T cells can only be achieved with mature MDDCs, the use of B cells for the restimulation of already primed T cells allows saving the precious MDDCs for the initial step. In addition, the use of alternative readout methods like the ELISPOT to detect IFN-γ or TNF-α production instead of a FACS-based analysis allows reducing the necessary number of DC/T cells by about tenfold. First experiments comparing the efficiency of the ELISPOT-based detection of antigen-specific T cells with the FACS analysis showed comparable results. However, whether or not the important information that can be gained by the multiparametric FACS analysis and that is not provided by the ELISPOT is needed will have to be evaluated in the future.
Another important issue with human T cell priming assays is their ability to detect not only strong sensitizers but also moderate/weak sensitizers with a sufficient sensitivity. While historical protocols developed to analyse innate and adaptive immune responses in vitro used unfractionated human PBMC or murine lymph node cells in the presence or absence of antigens or hapten-loaded MDDCs as antigen-presenting cells, recent protocols have been improved regarding their cellular composition. One approach to enhance the efficiency of lymphocyte reactions was, for example, the use of specific antigen-presenting cell populations modified with haptens. This way the detection of sensitizers like oxazolone that gave no response in assays with unfractionated lymphocytes more closely reflected the in vivo situation where a strong ear swelling response was observed in the CHS model . Especially work by Vocanson et al. and our own results have shown that the presence of CD25+ or CD56+ regulatory T cells hampers the antigen-specific priming of naive T cells. This effect was also described for the induction of antigen-specific responses to viral proteins  and is known to reduce the sensitivity of a T cell priming assay . In addition, Vocanson et al. have recently reported that CD1alow monocyte-derived dendritic cells inhibited T cell activation, while usage of CD1ahigh MDDCs for the priming enhanced T cell activation .
Therefore, using advanced protocols where not only CD45RO+ memory T cells but also CD25+ and CD56+ cells are depleted [14, 15] in combination with a protocol using CD1alow depleted MDDCs for priming should allow to significantly increase the sensitivity of future T cell priming assays.
31.4 Allergenic Potency Determination with the hTCPA: An Opportunity?
A big aim in terms of hazard identification and risk management is the set-up of an assay that not only allows differentiation between sensitizers and non-sensitizers but also enables the classification of contact sensitizers according to their potency as is possible in the LLNA via EC3 values. Several factors may be involved in the determination of the sensitizing potency in vivo. Empirically, the first step in determining whether or not a sensitizer will be strong or weak is its ability to cross the skin barrier and the depth of penetration into the skin that can be reached by the chemical. Another factor is the ability of the chemical to induce the generation of a pro-inflammatory cytokine milieu and the activation of an innate immune response. Without the activation and full maturation of DCs, there will be no priming of naive T cells in the draining lymph nodes. This is an effect particularly to be taken into account for weak allergens—as soon as these are combined with either irritants or mixed with other weak sensitizers, the sensitizing potency of the weak sensitizer is enhanced due to an increased inflammatory immune reaction [16, 17, 18]. Moreover, the efficiency of the activation of counter-regulatory mechanisms like the activation of ICOS+ regulatory T cells can determine the strength of the sensitizing potential of a given chemical .
In this respect, the hTCPA in its current form may not be the optimal assay for potency assessment. As mentioned before, efforts were made to set up an assay that is as sensitive as possible—i.e. all kinds of cells that may dampen the T cell priming were removed, thus possibly removing one of the factors determining allergenic potency. The activation of the MDDCs has been optimized by addition of pro-inflammatory factors like TNF-α or LPS resulting in a full activation of the DCs. In addition, a cytokine supplementation strategy has been worked out to allow for enhanced priming even with weak allergens. This seems to be possible without an increase in background T cell activation. However, all of these optimization steps that improve the sensitivity of the hTCPA remove the factors co-determining the potency of a contact sensitizer in vivo. This precludes potency assessment.
Although further optimisation and evaluation of the most sensitive readout for the human T cell priming assay are still ongoing, this assay may be of relevance as a third-line assay/final validation step in a IATA. The hTCPA addresses the crucial step in the sensitization to contact allergens, i.e. the priming of antigen-specific T cells. The major advantage of the hTCPA protocol is the testing for antigen specificity by an antigen-specific restimulation step—i.e. this assay provides the only protocol, where not only the activation of innate immune cells like the MDDCs but also the extreme specificity of the T cell receptor is taken into account. Chemicals that might provide false-positive results in other assays due to their inherent ability to activate innate immune reactions will not be able to elicit the priming and antigen-specific recognition by a T cell. This makes the hTCPA an assay, that—though not allowing to perform a high-throughput screening of substances—will allow to identify without any doubt the sensitizing potential of a limited number of crucial chemicals. In addition, a comparable protocol has already been successfully implemented to characterize primary T cell responses to drugs .
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