Analysis of the Recent Thymic Output Function of 23 TCR Vβ Subfamily Naïve T Cells in Patients with AML.

Regeneration of the T-cell population proceeds normally along two different pathways, in which the thymic-dependent pathway accounts for the more durable reconstitution of the T-cell compartment, and generates a more diverse TCR repertoire. Thus thymus function serves as a direct index to understand cellular immune function and potential of long-term TCR Vβ repertoire reconstitution. The complexity of TCR repertoire is generated in the thymus by regular recombination of series of gene fragments of TCR α or β chains. During these process, by-products of rearrangements are generated in the form of signal joint T-cell receptor excision DNA circles(sjTRECs), as sjTRECs are stable extrachromosomal annular DNA, are not replicated while cell dividing, and their existence suggests functional TCR generation. Thus thymic function can be evaluated by measuring sjTRECs in peripheral blood. At present, the total recent thymic output function is evaluated by quantitating δRec-ψJα sjTRECs, but it can not evaluate particular thymic emigrants of different Vα or Vβ subfamily naïve T cells. As TCR Vα and Vβ naïve T cells include many subfamilies which play different immune roles, and the complexity of TCR repertoire is an important factor for cellular immune reconstitution.

Our previous studies had showed that the recent thymic output function in patients with AML was significant decrease by quantitative detection of δRec-ψJα sjTRECs. To further estimate the recent thymic emigrants of different TCR Vβ subfamily naïve T cells, this study was designed to detect the existence of 23 TCR Vβ subfamily sjTRECs in peripheral blood mononuclear cells (PBMCs) by using 23 Vβ subfamily special primers (including 2Dβ1 sense primers and 23 Vβ subfamily antisense primers). TCR 23 Vβ-Dβ 1 sjTRECs were separately amplified in genomic DNA from 5×10⁴ and 1×10⁴ PBMCs of samples (10 cases of normal individuals and 32 cases of the different FAB subtypes of AML patients) to estimate the frequencies of TCR 23 Vβ-Dβ1 sjTRECs by using semi-nest PCR.

The results indicated that the frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration, and the higher cellular concentration the higher frequency of Vβ subfamily sjTRECs. The number of detectable Vβ subfamily sjTRECs was (5.09±3.28) and (2.59±2.06) in 5×10⁴ and 1×10⁴ PBMCs from AML patients, as compared with (13.7±2.67) and (5.50±2.07) from normal individuals, and the differences were significant (both p=0.000). About 5/23(22%) of Vβ-Dβ1 sjTRECs were detected in 5×10⁴ PBMCs from AML patients, and the frequencies of 13 Vβ subfamily sjTRECs (including Vβ1,Vββ,Vβ3,Vβ4,Vβ5, Vβ9,Vβ10,Vβ12,Vβ13,Vβ14,Vβ17,Vβ22,Vβ24-Dβ1 sjTRECs) were significantly lower than those from normal individuals, among which the lowest were Vβ10 and Vβ14-Dβ1 sjTRECs, the most frequency was Vβ21. But the difference was not significant within the different FAB subtypes of AML patients. It was negative correlation between age and the number of detectable Vβ subfamily sjTRECs in patients with AML, and patients who were < 30 years tended to be higher number of detectable Vβ subfamily sjTRECs than those ≥ 30 years, which became significant at 5×10⁴ PBMCs level (r=−0.481, p=0.005). Taken together, the recent thymic emigrants of 23 Vβ subfamily naïve T cells were absent to a different extent or lower level among patients with AML. These results suggested that AML patients had severe cellular immunodeficiency and the capacity and potential of long-term TCR Vβ repertoire reconstitution were dramatically lowered.