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	10–12
					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.