Acute Graft Versus Host Disease Reduces Human Thymic T Cell Differentiation without Acting on Thymocyte Proliferation.

Thymic function is essential for an efficient long term T cell reconstitution after Hematopoietic Stem cell Transplantation (HSCT). We and others have shown that several factors and especially Acute Graft Versus Host Disease (aGVHD) could have an important impact on thymic output as measured by “signal joint T cell Receptor Excision Circles” (sjTREC) quantification. In order to go further in the understanding of aGVHD action on thymic output we quantified the sjTREC (generated just before the T cell Receptor (TCR) alpha chain recombination) but also others TRECs generated during the TCR beta chain recombination, named betaTREC: sj/beta TREC ratio being a marker of thymocyte proliferation. A group of 13 aGVHD Patients was selected first because of a long term follow-up (> 24 months) and compared to a control group of 8 patients that did not have aGVHD, matched for age (mean 29.8 and 28.9 years for aGVHD and non aGVHD patients respectively). Patients received, mainly for leukemia (17 out 21), a Bone Marrow (14) or Peripheral Blood (7) HSCT from a sibling genoidentical donor. TRECs (sj and beta) were quantified by multiplex real-time PCR with albumin gene as standard, using genomic DNA extracted from peripheral blood mononuclear cell samples obtained before HSCT and at 3, 6, 12 and 24 months after. In parallel, absolute number of CD3+ and naïve T cells was measured by flow cytometry using antibodies against CD3, CD4, CD8, CD45RA and CD62L. In our patients, the percentage and absolute number of naïve T cells, as defined by the CD45RA+, CD62L+ phenotype, were not different between the 2 groups showing again the inability of this parameter to measure the recent thymic emigrants in HSCT. Mean sjTREC /150000 cells decreased in both categories of patient 3 months after HSCT. Then, for non aGVHD patients, they started to rise up to reach near normal level at 12 months but not for aGVHD patients where they keeped decreasing until 6 months. At this last time point mean aGVHD patient sjTREC/150000 cells was significantly lower than for non aGVHD patients (255 versus 2402 respectively, p=0.031, Mann-Whitney). This was still true for the mean absolute number of sjTREC (1.44 versus 27.33/mL of blood, p=0.007). Mean betaTREC /150000 cells showed no decrease for control patients but the same pattern than sjTREC for aGVHD patients with also a significant difference at 6 months (55 versus 2302 betaTREC /150000 cells for aGVHD and non aGVHD patients respectively, p=0.010). Finally, there was no significant difference between the 2 groups for the sj/beta ratio. These data confirm that aGVHD patients have a lower thymic output in the first months after HSCT. We also show that the decrease in the thymic output could not be explained by a reduced thymocyte proliferation since the sj/beta TREC ratio is not different between the 2 groups. It is better explained by a decrease in the number of thymocyte precursors that start to differentiate and recombine their TCR beta chain (as measured by beta TRECs). Altogether this suggests that during aGVHD, the allogeneic reaction on the recipient thymic cells could either impair the thymus seeding by the T cell precursors or provoke an inhibition of the signals that trigger the T cell differentiation.