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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: Transplantation. 2017 Nov;101(11):2671–2681. doi: 10.1097/TP.0000000000001847

Table 2.

Strategies for tracking alloreactive T cells.

Tracking
Strategy
Which T cells
are
alloreactive
Animal
Models
Human
Studies
Allorecognition
pathways
detected
Advantages Disadvantages References

In vitro restimulation: MLR Cells expressing surface markers of activation and proliferation by flow cytometry Direct and indirect* Simple preparation and readout In vitro stimulation differs from in vivo stimulation conditions 11,14,20,25
Cells with diluted CFSE following CFSE labeling Allows study of polyclonal response to alloantigen Does not reflect effects of immunosuppression
IFNy-producing cells by cytokine flow cytometry Surface markers can be detected within 24 hours of stimulation CFSE dilution requires ≥48 hours for detection

In vitro restimulation: IFNy ELISpot IFNy-secreting cells Direct and indirect* Simple preparation and readout In vitro stimulation differs from in vivo stimulation conditions 1012
Allows study of polyclonal response to alloantigen Does not reflect effects of immunosuppression
More sensitive than intracellular IFNy detection by flow cytometry Underreporting in samples with many IFNy-producing cells

Trans-vivo DTH Cells expressing surface markers of activation and proliferation by flow cytometry Indirect Allows study of polyclonal response to alloantigen Does not fully recapitulate allograft rejection processes 31,32
Cells with diluted CFSE following CFSE labeling Includes influence of factors produced by local cells and other nuances of in vivo stimulation Does not reflect effects of immunosuppression
Labor intensive and requires careful assessment of footpad swelling
IFNy producing cells by cytokine flow cytometry Surface markers can be detected within 24 hours of stimulation CFSE dilution requires ≥48 hours for detection

Transgenic TCR Tg or Rg mouse-derived T cells labeled by CFSE, transgenic fluorescent marker or congenic marker Direct or indirect Allows analysis of naïve or antigen-experienced allospecific T cells Single TCR is not reflective of a polyclonal response 23,33
Allows control of precursor frequency by adoptive transfer Generation of new TCR-Tg mice is slow


Retrogenic TCR** Direct or indirect Faster than generation of TCR-Tg mice Single TCR is not reflective of a polyclonal response 116

pMHC Multimer Multimer-bound T cells Direct or indirect Allows study of a polyclonal response to a single alloantigen specificity Requires knowledge of allopeptide identity 42,43,50,52
Multimer binding to TCR impacts T cell function


pMHC Streptamer** Direct or indirect Lower impact of TCR binding on T cell function than traditional pMHC multimers; allows functional analysis after sorting Requires knowledge of allopeptide identify 56
More costly and difficult to prepare than traditional pMHC multimers
*

In cultures containing host and donor PBMCs, donor or recipient PBMCs can theoretically present alloantigen, although direct responses may predominate. If donor PBMCs are lysed prior to coculture, only PBMCs from the recipient will contain intact antigen-presenting cells and all allopresentation will occur through the indirect pathway.

**

Application of the technique for this purpose has not been reported. However, pMHC Streptamers are structurally and functionally like pMHC multimers and TCR-Rgs are like TCR-Tgs such that many of the applications of TCR-Tgs and pMHC multimers should be possible using TCR-Rgs and pMHC Streptamers, respectively.