The last five years have seen the emergence of two powerful new techniques for immune profiling, cytometry by time of flight (CyTOF) and TCR sequencing. The Center provides access to these two technologies and to the reagents and supportive services necessary for human skin disease researchers to rapidly and effectively use these techniques.
Cytometry by Time of Flight (CyTOF)
- Flow cytometry is a powerful tool but the number of different markers that can be immunostained and detected on a particular cell is limited by spectral overlap of the fluorochomes used.
- Cytometry by time of flight (CyTOF, Fig. 1) combines immunostaining with mass cytometry. Cells are immunostained with antibodies conjugated to rare earth metals and then analyzed by a mass cytometer.
- The outcome of this process is highly quantitative measurement of 45 separate markers, with expansion to 75 labels in the future, with no signal overlap and no need for compensation.
- This is a particularly powerful technique for immune profiling. For example, a panel of surface markers can be used to carefully identify different subpopulations of cells, and these cells can at the same time be analyzed for cytokine production, transcription factor expression, levels of key signaling molecules and DNA content, among other possibilities.
- The Center provides: access to validated CyTOF antibody panels, staining protocols, access to CyTOF instruments to run samples and access to analysis techniques that will allow this technique to be immediately accessible to skin disease researchers regardless of their location or academic affiliation.
Figure 1. Advantages of CyTOF. Conventional flow cytometry (left) utilizes fluorophores that have overlapping spectra. As a result, there is an upper limit to the number of markers that can be studied at one time and increasingly complex compensation is required to separate the signals from different channels. In contrast, CyTOF (right) utilizes antibodies conjugated to rare earth metals. This allows measurement of up to 45 differently labeled antibodies and detection reagents, with expansion to 75 labels in the future, with no overlap and no need for compensation.
High Throughput T Cell Receptor CDR3 sequencing (HTS)
- T cells play a critical role in the etiology of many inflammatory skin diseases, including psoriasis, lupus, dermatomyositis and scleroderma.
- Previously, it was virtually impossible to follow a particular pathogenic T cell (referred to here as a T cell clone) and the daughter cells that arose from it.
- Measuring the TCR diversity in a biologic sample was also either cumbersome (spectratyping) or non-comprehensive (flow cytometry).
- HTS is a remarkable technique that accomplishes three key tasks, measurement of the absolute number of T cells, the diversity of the T cell repertoire, and the ability to track particular pathogenic T cell clones, in a single, one step test.
- This test requires only 150 ng of DNA that can be extracted from blood (including flow sorted purified T cell populations) and both fresh and FFPE tissues specimens.
- HTS is available for human α, β, γ and δ TCR subunits.
- How it works: During T cell development, a unique CDR3 antigen recognition domain is generated by the recombination of the V, D and J regions of the T cell receptor genes and additional diversity is generated at the junctions by insertion of random nucleotides (Figure 2). This process generates a unique CDR3 sequence for each T cell. HTS of the CDR3 regions consists of carrying out multiplex PCR for each of the component V, J and D genes for the chain being analyzed (α, β, γ or δ) followed by precise quantification, correction for PCR bias, normalization of sequences obtained to those obtained by a control plasmid, and normalization to the amount of total DNA input into the assay.
- The output of this process is an exact determination of the i) number of T cells present, ii) diversity of T cells as assessed by the number of different TCR sequences, iii) the relative proportion of a particular T cell sequence in the total population and iv) the exact CDR3 sequences of all T cells present in a particular biologic sample
- HTS has also allowed for the first time the tracking of particular T cell clones in humans, across multiple tissues and over time in the same patient.
- The downsides: designing an experiment correctly is extremely important when using this technique to ensure that this approach will answer the desired question. Also, analysis of thousands of T cell CDR3 sequences can be overwhelming.
- The Center provides: Assistance with experimental design (can your experiment answer the question?), isolation and sorting of cells from human skin, DNA isolation, shipping of samples to Adaptive Biotechnologies (where HTS is carried out) and comprehensive support in the analysis of HTS data.
Figure 2. TCRb CDR3 sequencing assay. (A) During T cell development, multiple V, D and J segments recombine to create a CDR3 region that is unique for each T cell clone. Additional diversity is generated at the junctions by insertion of random nucleotides (shown as N in (B)). (B) The assay uses a multiplex PCR with forward primers in each V segment and reverse primers in each J segment.