During the last few years, CIP has been working on the development of tools that have the potential to improve and facilitate immune monitoring. These activities were essentially supported by the Wallace Coulter Foundation.
Cellular reference samples
A main problem of in-vitro T-cell monitoring is the lack of reference samples that contain a controlled number of antigen-specific T cells that can be used to control assay performance over time. Such reference samples are urgently needed for a broad variety of different TCR covering a range of commonly targeted antigens and TCR affinities. In collaboration with the University Medical Clinic in Mainz and TRON, CIP has developed a RNA-based scalable technology which allows the generation of TCR-engineered reference samples (TERS) of selected specificities. TERS are stable reagents that can be used for controlling the performance of HLA-multimer, Elispot and ICS assays over time. In addition, TERS can be produced in-house with the help of a newly developed kit.
Since November 2016, the first TERS kits are proposed by the company jpt.
To learn more:
Bidmon N, Attig S, Rae R, Schröder H, Omokoko TA, Simon P, Kuhn AN, Kreiter S, Sahin U, Gouttefangeas C, van der Burg SH, Britten CM. Generation of TCR-engineered T cells and their use to control the performance of T cell assays. J Immunol. 2015 Jun 15;194(12):6177-89.
Automated analysis of flow cytometry data
As the complexity of flow cytometry data is constantly increasing, the need for unsupervised, automated methods in which cell subsets are quantified by machine algorithms is increasing. A number of such methods are now available, but most of them are not fully available to the immunology community.
⊙ A workshop organized by CIP in 2015 gathered immunologists and developers to discuss the challenges and needs related to the development and the adoption of computational tools.
⊙ In cooperation with Dr. Cliburn Chan from Duke University, CIP is also working on the development of server-based automated tools that can be applied across heterogeneous data sets such as those generated in proficiency panels or multi-centric studies. The Reflow program functions as a repository to upload, manage and annotate data. Reflow also performs similarly to manual analysis for identifying low frequency T cells such as HLA-peptide multimer-positive CD8 T cells. The web platform will soon be tested in the context of a proficiency panel.
To learn more:
Kvistborg P, Gouttefangeas C, Aghaeepour N, Cazaly A, Chattopadhyay PK, Chan C, Eckl J, Finak G, Hadrup SR, Maecker HT, Maurer D, Mosmann T, Qiu P, Scheuermann RH, Welters MJ, Ferrari G, Brinkman RR, Britten CM. Thinking outside the gate: single-cell assessments in multiple dimensions. Immunity. 2015 Apr 21;42(4):591-2
Cron A, Gouttefangeas C, Frelinger J, Lin L, Singh SK, Britten CM, Welters MJ, van der Burg SH, West M, Chan C. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples. PLoS Comput Biol. 2013;9(7):e1003130
White S, Laske K, Welters MJ, Bidmon N, van der Burg SH, Britten CM, Enzor J, Staats J, Weinhold KJ, Gouttefangeas C, Chan C. Managing Multi-center Flow Cytometry Data for Immune Monitoring. Cancer Inform. 2015 Jun 10;13(Suppl 7):111-22
Gouttefangeas C, Chan C, Attig S, Køllgaard TT, Rammensee HG, Stevanović S, Wernet D, thor Straten P, Welters MJ, Ottensmeier C, van der Burg SH, Britten CM. Data analysis as a source of variability of the HLA-peptide multimer assay: from manual gating to automated recognition of cell clusters. Cancer Immunol Immunother. 2015 May;64(5):585-9
Towards a consensus phenotype Ab panel
for defining Tregs
Based on the workshop held in October 2013 and on following discussions, a rationally based ranking list of Treg markers was defined. Subsequently, the proposed Treg markers were tested in order to get an insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. An essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67 and CD45RA and a corresponding robust gating strategy were formulated based on validation experiments on PBMC from healthy donors and cancer patients, as well as in tumor draining lymph nodes and freshly isolated tumors. We have consent on this marker set from all participants of the workshop, 40 gave their approval.
To learn more:
Santegoets SJ, Dijkgraaf EM, Battaglia A, Beckhove P, Britten CM, Gallimore A, Godkin A, Gouttefangeas C, de Gruijl TD, Koenen HJ, Scheffold A, Shevach EM, Staats J, Taskén K, Whiteside TL, Kroep JR, Welters MJ, van der Burg SH. Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry. Cancer Immunol Immunother. 2015,64:1271-86
Harmonization of MDSC phenotyping
There is an increasing interest for monitoring myeloid-derived suppressor cells in cancer patients, but also divergences in their phenotypic definition. After a consultation period, CIP conducted a first proficiency panel with the aim to harmonize MDSC phenotyping. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets tested, especially for the granulocytic subsets. The variability appears to depend on a number of included the gating strategy. Based on these findings we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets.
To learn more:
Mandruzzato S, Brandau S, Britten CM, Bronte V, Damuzzo V, Gouttefangeas C, Maurer D, Ottensmeier C, van der Burg SH, Welters MJ, Walter S. Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study. Mandruzzato S, Brandau S, Britten CM, Bronte V, Damuzzo V, Gouttefangeas C, Maurer D, Ottensmeier C, van der Burg SH, Welters MJ, Walter S. Cancer Immunol Immunother. 2016 Feb;65(2):161-9