IL-2 Luciferase (IL-2 Luc) Assay Report of thePeer Review Panel

ona JaCVAM co-ordinated study programme addressing the validation
status of the IL-2 Luc assay for the prospective identification ofimmunotoxic substances on T cells

Report completed by the Peer review Panel on June 30th, 2020

Table of Contents
Executive Summary

3

Peer Review Panel Composition

4Background5

IL-2 Luc Test Method Definition

5

Within Laboratory Reproducibility

6Interlaboratory Transferability

6

Between Laboratory Reproducibility

7

Predictive Capacity

7

Applicability Domain

8

Performance Standards

8Additional Comments8Conclusions and Recommendations

9Acknowledgements

9References

9Executive Summary
The IL-2 luciferase (Luc) assay has been proposed as an in vitro alternative to animaltesting. The assay provides information on adverse outcome pathways for immunotoxicity toT cells.
The Peer Review Panel (PRP) found the Validation Management Team’s report presented
the necessary information for an independent review.
The PRP concluded that the IL-2 Luc assay was well defined and has a clear protocol andcriteria for data interpretation. All necessary information, including performance standards,
was sufficiently detailed. Both within- and between-laboratory reproducibility weresatisfactory. The PRP noted that the use of compounds with a better-defined
immunotoxicity mechanism will help to improve the accuracy of the assay. While thepredictive capacity was not satisfactory for a stand-alone method, the IL-2 Luc assay is
acceptable for use in an Integrated Approach to Testing and Assessment.

Peer Review Panel Composition
Fujio Kayama (Chair)

Jichi Medical University, Japan

Henk van Loveren

Maastricht University, The Netherlands

Haley Neff-LaFord

Seattle Genetics, Inc., USA

Barbara Kaplan

Mississippi State University, USAXingchao Geng

National Center for Safety Evaluation ofDrugs (NCSED), China

Takao Ashikaga

JaCVAM, Kawasaki, Japan

Sang-Hyun Kim

Kyungpook National University, Korea

Background
Immune dysregulation can have serious adverse health consequences and it could becaused by many types of chemicals, such as environmental contaminants, food additives, anddrugs. It ranges from reduced resistance to infection and neoplasia to allergic andautoimmune conditions. For many years, to identify such immunotoxic chemicals dependedon animal models. For animal welfare, ethical and scientific reasons there has been a desire
to replace in vivo methods with non-animal alternatives (1). The immune system comprises
innate and adaptive immunity. Both arms of the immune response function differently andare driven by different population of cells. A variety of intracellular signaling pathways also
play roles in innate and adaptive immune responses. Given the complexity of the immune
system, it is unlikely that a single in vitro method will cover all immunotoxicants. Therefore,
Integrated Approaches to Testing and Assessment (IATA) are flexible approaches for safetyassessment based on the integration and translation of the data derived from multiple
methods and sources. For example, target compounds are tested using various approaches
such as the human whole blood cytokine release assay (HWBCRA), lymphocyte
proliferation assay, mixed lymphocyte reaction and fluorescent cell chip assay after theevaluation of myelotoxicity (2). There are many in vitro methods aiming at evaluating
various aspects of molecular and cellular events in the adverse outcome pathway (AOP) forimmunotoxicity. However, no in vitro method is validated formally.
The IL-2 Luc assay was developed to be part of a high-throughput screening system thatenables evaluation of chemical immunotoxicity. This screening system was named MultiImmunoTox assay or MITA (3) (4) (5). The IL-2 Luc assay, using a human cell linetransfected with luciferase genes under control of the IL-2 promoter, identifies the effect ofchemicals on the IL-2 activity in the 2H4 cells in the presence of stimulants (3).
The PRP first met in February 2019 to review a progress report on the IL-2 Luc assay
prepared by the Validation Management Team (VMT). Following commentary on this workby the PRP, the VMT refined the validation report.
The PRP engaged in follow-up telephone conferences in October 2019, November 2019
and June 2020. With the provision of all of the amended, updated and additional material,
including the final VMT report, this PRP Report was prepared.

IL-2 Luc Test Method Definition
The PRP confirmed that the IL-2 Luc assay test method has been fully described in thereport of the VMT and in the associated detailed test protocol. During the validation study,
the test developer changed their prediction model. A clear definition of the 35% thresholdand its reason was explained. The VMT report describes the need for the assay in thecurrent regulatory context (6). Furthermore, a clear rationale for the assay has been given(the rationale for the test method is that drugs and chemicals, environmental contaminants,
food additives, and drugs can target the immune system, resulting in immune
dysregulation). It is known that IL-2 exerts pleiotropic actions on CD4+ T celldifferentiation via its modulation of cytokine receptor expression. It promotes Th1
differentiation by inducing IL-12Rb2 (and IL-12Rb1), promotes Th2 differentiation by
inducing IL-4Ra, inhibits Th17 differentiation by inhibiting gp130 (and IL-6Ra), and drives
Treg differentiation by inducing IL-2Ra. IL-2 also potently represses IL-7Ra, which
decreases survival signals that normally promote cell survival and memory cell development(7). Therefore, it is reasonable that the test developer focused on the regulation of IL-2transcription and attempted to construct an AOP of immunotoxicity with transcriptional
dysregulation of IL-2 as a central key event. The VMT report mentioned that IL-2 Luc assay
will be part of a broader tiered approach to eventually include IL-8 and IL-1β, that
corresponds to the AOPs for immunotoxicity and as such constitutes an IATA.
The PRP agreed that the mechanistic basis of the method and how it related to the T-cell
specific endpoint also was well described in the VMT report.

Within Laboratory Reproducibility
The PRP agreed that the results which emerged have demonstrated a sufficient degree of
within laboratory reproducibility. For achieving such conclusion, the PRP focused on results
obtained with the final protocol and prediction model.
A total of 5 coded chemicals (4 T-cell targeting and 1 non T-cell targeting) were evaluatedby 3 experimental sets. Based on such assumptions, the success criterion of >80% withinlaboratory reproducibility was achieved in each of the three participating laboratories (Lab.
A: 80.0% (4/5), Lab. B: 100% (5/5), Lab. C: 80.0% (4/5)).
The PRP notes that extensive documentation of within-laboratory reproducibility data for
the final and all the development phases of the IL-2 Luc assay has been displayed in theVMT report and its appendices. Taken into account, the data lends support to the view thatthe assay has a sufficient level of reproducibility within laboratories.

Interlaboratory Transferability
The PRP noted that the technical transfer of the IL-2 Luc assay involved training andsuccessful assessment of 3 experiments of 5 test substances (not blinded) by each of theparticipating laboratories. That work was prior to their approval to participate in thesubsequent validation work.

Between Laboratory Reproducibility
With regard to between-laboratory reproducibility, the PRP recognized that the test
results gave 80% (20/25) and met the success criterion of >80% between-laboratory
reproducibility. The number of test chemicals is combined of the Phase I (5) and Phase II(20) .
Again, the PRP notes that extensive and transparent documentation of betweenlaboratory reproducibility data for all phases of the IL-2 Luc assay has been displayed in theVMT report and its appendices.
The PRP concluded that the assay demonstrated successful between-laboratory
reproducibility.

Predictive Capacity
To determine the predictivity of the IL-2 Luc assay, it is crucial to understand theimmunotoxic characteristics of chemicals used in the study. The PRP agreed thatclassification chemicals into those that affect T cell function (T cell-targeting chemicals,TTC) and those that do not directly affect T cell function (non-T cell-targeting chemicals,NTTC) was needed. The PRP confirmed the rationale for classifying immunotoxic
chemicals are clearly described in the validation report.
Demonstration of a test method’s performance should be based on the testing ofrepresentative, preferably coded, reference chemicals. The PRP concluded that thevalidation study used an appropriate level of test chemical coding to ensure fully blindedevaluation. With respect to chemical selection, the PRP confirmed that the criteria forchemical selection were clearly outlined. On the other hand, it should be noted that there isa question as to whether or not the number of true negatives (8/25) in the set was sufficient.
The immunotoxic characteristics of each chemical used in the Phase I and Phase II areshown in the VMT report and based on the criteria total 25 chemicals were classified into 16
positives, 8 negatives and 1 unclassified. According to the classification, accuracy is 75%(18/24), specificity is 75% (6/8) and sensitivity is 75% (12/16). The PRP concluded thepredictive capacity of the test is not sufficient to detect all immunotoxic chemicals if used asa stand-alone test.
The PRP basically agreed with the test developer's opinion that there are at least 2
reasons that the predictivity did not meet the success criterion. First, the classification ofimmunotoxic chemicals is sometimes uncertain, because mechanistic information is oftenlimited. Second, the IL-2 Luc assay does not cover every aspect of the effects of chemicals
on T cell function.
Regarding the second point, the PRP noted again this assay should be used incombination with other assays, as in the context of IATA. With respect to the first point, thePRP noted the use of compounds with a better-defined immunotoxicity mechanism will help
to ultimately improve the accuracy of the validation study and also recommended a Detailed
Review Paper (DRP) on in vitro immunotoxicity testing.
Following phases I and II, the assay was then applied to over 60 chemicals that werepreviously evaluated by the test developer (8). The accuracy was calculated as 82.4%, whichis in line with results obtained in phases I and II.

Applicability Domain
The PRP concluded that the applicability domain should be better defined. First, themethod cannot detect immunotoxicity associated with inhibition of DNA synthesis and celldivision. Second, the assay might not detect compounds that require metabolic activation toa toxic intermediate. Third, the use of PMA/Io as a stimulant bypasses signaling through theT cell receptor. Finally, the IL-2 Luc assay shares limitations common to many suspension
cell-based techniques when testing highly hydrophobic substances.

Performance Standards
The PRP concluded that the list of performance standard (PS) substances placed inAppendix 16 to the VMT report was satisfactory. Mutual Acceptance of Data (MAD) will
only be guaranteed for test methods validated according to the PS, if these test methods
have been reviewed and included in this Test Guideline by the OECD. The PS issupplemented by a list of proficiency chemicals, listed in Appendix 15, to be used as aroutine check on performance of the assay.

Additional Comments
The PRP concluded that the validation study management and conduct met the criteria
set out in OECD GD 34 (2005). The PRP noted that the study was conducted not under
GLP certification but in the spirit of GLP.
The PRP appreciated the transparency with which all the IL-2 Luc assay material waspresented. The PRP noted that during the review they were able to access the raw data files
associated with the IL-2 Luc assay development/validation work.
The PRP also noted that AOP networks and a DRP in this field are essential for theconstruction of an IATA.

Conclusions and Recommendations
The PRP concluded that, even though the predictive capacity was not sufficient to allow
use as a stand-alone test, the IL-2 Luc assay validation has demonstrated that the methodshould be acceptable as a part of IATA for the predictive screening of T-cell targeted
immunotoxicity.

Acknowledgements
The PRP is also grateful to the members of the VMT for supplying the report on thevalidation study of the IL-2 Luc Assay. This research was supported by the WHLW grant.

References
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3) Saito, R., Hirakawa, S., Ohara, H., et al., 2011. Nickel differentially regulates NFAT
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245-255.
4) Takahashi, T., Kimura, Y., Saito, R., et al., 2011. An in vitro test to screen skin sensitizers
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5) Kimura, Y., Fujimura, C., Ito, Y., et al., 2014. Evaluation of the Multi-ImmunoTox Assaycomposed of 3 human cytokine reporter cells by examining immunological effects of drugs.
Toxicol In Vitro 28, 759-768.6) (WHO)/, W.H.O., Meeting, I.P.o.C.S.I.S., 2012. Guidance
for immunotoxicity risk assessment for chemicals. WHO press.
7) Liao, W., Lin, J.X., Wang, L., et al., 2011, Modulation of cytokine receptors by IL-2
broadly regulates differentiation into helper T cell lineages. Nat Immunol. 12: 551-559.
8) Kimura, Y., Fujimura, C., Ito, Y., et al., 2018. Profiling the immunotoxicity of chemicals
based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8
Luc assay. Arch Toxicol 92, 2043-2054.