Applying T-cell receptor excision circles and immunoglobulin κ-deleting recombination excision circles to patients with primary immunodeficiency diseases

Figures & data

Table I. Forward and reverse primers for the evaluation of TRECs, KRECs, and TRACs.

	Forward primer	Reverse primer
Construct the plasmid with TRECs, KRECs, and TRACs
TRECs	5’-AAA GAG GGC AGC CCT CTC CAA GGC-3’	5’-GGC TGA TCT TGT CTG ACA TTT GC-3’
KRECs	5’-ccc aag ctt TCA GCG CCC ATT ACG TTT CT-3’	5’-ccc aag ctt GTG AGG GAC ACG CAG CC-3’
TRACs	5’-gac tag tAT GAG ACC GTG ACT TGC CAG-3’	5’-gac tag tGC TGT TGT TGA AGG CGT TTG C-3’
Quantitative evaluation
TRECs	5’-CAC ATC CCT TTC AAC CAT GCT-3’	5’-TGC AGG TGC CTA TGC ATC A-3’
	Probe: 5’-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACTTAMRA-3’
KRECs	5’-TCC CTT AGT GGC ATT ATT TGT ATC ACT-3’	5’-AGG AGC CAG CTC TTA CCC TAG AGT-3’
	Probe: 5’-HEX-TCT GCA CGG GCA GCA GGT TGG-TAMRA-3’
TRACs	5’-TGG CCT AAC CCT GAT CCT CTT-3’	5’-GGA TTT AGA GTC TCT CAG CTG GTA CAC-3’
	Probe: 5’-FAM-TCC CAC AGA TAT CCA GAA CCC TGA CCCTAMRA-3’

Figure 1. The reference standards of KRECs, TRECs, and TRACs (as internal controls) fragments were inserted into DH₅ alpha cells, and each pCR^®2.1-TOPO Vector clone contained the three fragments (each single copy) identified and confirmed by whole-vector PCR. The KRECs and TRECs numbers were shown by extrapolating the respective sample quantities from the standard curve obtained by serial dilutions (10⁷,10⁶, 10⁵, 10⁴,10³, 10², 10¹, and 10⁰) and the linear triple-insert plasmid DNA (A: KRECs, and B: TRECs) that were amplified in each PCR plate. The absolute number of TRECs or KRECs per 250 ng whole blood was calculated from the linear slope by y = –3.327x + 37.373 for TRECs (R² = 0.9947) and y = –3.0727x + 38.526 (R² = 0.9907) for KRECs.

Table II. Normal TRECs and KRECs values from healthy controls (n = 40).

250 ng/whole genomic DNA	TRECs (n = 40)	KRECs (n = 40)
5%–95%	124.0–152.3	157.7–252.4
Range	73.4–258.8	60.9–674.5
Mean ± 2 standard deviation	138.1 ± 44.3	205.0 ± 148.1

Table III. Copy numbers of TRECs, KRECs, and naïve and memory lymphocytes in patients with primary immunodeficiency diseases.

Figure 2. The representative significant linear correlation between A: TRECs versus absolute naïve CD4 + CD45RA+ T cells percentage; B: TRECs versus absolute naïve CD4 + CD45RA+ T cells; C: KRECs versus absolute naïve CD19 + CD27- B cells; D: absolute naïve CD19 + CD27- B cells versus absolute memory CD19 + CD27+ B cells; E: KRECs versus absolute memory CD19 + CD27+ B cells; and F: TRECs versus the ratio of CD4 + plus CD8 + naïve/memory percentages.

Table IV. The copy number of TRECs and naïve and memory CD8 + lymphocytes in patients with primary immunodeficiency diseases.

Table IV. (Continued)

Table V. The relationship of TRECs and KRECs with the respective naïve and memory cells.