T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/βTREC ratio and thymic naive T cells

Abstract

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and β T-cell receptor excision circles (βTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31(+) thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and βTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and βTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31(+) thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and βTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31(+) thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.

Figure 1.

Gating strategy for the determination of CD3⁺CD4⁺CD45⁺CD31⁺Ki67⁺ cells. After a first gating on the CD3⁺CD4⁺ cell population, these cells were divided into three subpopulations depending on their expression of CD45RA and CD45RO. These fractions were examined for the expression of CD31 and the R2 fraction, as the highest CD31-expressing cell population, was then finally subjected to analysis of Ki67 expression as a marker of proliferation.

Figure 2.

Age dependency of sjTREC and βTREC. Correlation of age and copy number of the target gene in healthy volunteers (HV). The age of the HV ranged from 18 to 92 with a median age of 34 years. There was a negative correlation with age for sjTREC (r=−0.93) (A) as well as for βTREC (r=−0.79) (B) by Spearman’s analysis. In (C) we only included HV up to the age of 55 years and found a regression trend with a slower decline.

Figure 3.

Reconstitution kinetics of TREC and the thymic factor. Pooled data from the 66 patients in the cross-sectional study. The values are given as copy numbers of the respective target gene normalized to 10⁵ CD3⁺ cells. All sjTREC values were measured in duplicate, whereas the βTREC numbers were measured in triplicate. Each point is the mean value of the multiple measurements of the respective sample.

Figure 4.

(A, B, C), Three different reconstitution patterns corresponding to groups A, B and C respectively described in the text. The diagram includes sjTREC (filled squares) and βTREC (open triangles) as well as the thymic factor (filled triangles). The horizontal solid line represents the age-adjusted normal values for sjTREC, while the horizontal dotted line represents the age-adjusted normal values of βTREC. Please note that the age-adjusted TREC standard values decrease as a function of time.