Analysis of T-cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T-cell depleted and unmanipulated grafts

We have studied the repopulation of the T-cell compartment in 27 patients transplanted with bone marrow from an (HLA)-identical sibling. Significant differences were found between recipients of unmanipulated and T-cell depleted grafts. Analysis of the T cells by a method based on amplification of minisatellite DNA regions showed that without depletion > 99.9% of the clones responding to a mitogenic stimulus after transplantation were of donor origin. In contrast, when the graft had been depleted with Campath-1M plus complement, a significant part of the T cells cloned during the first weeks after transplantation comprised of recipient T cells that had survived the preconditioning. This mixed population of low numbers donor and recipient T cells (19 +/- 31/mm3 at day 14) expanded rapidly (predominantly CD8+ T cells) during the first 2 months, without a significant change of the ratio of recipient/donor T cells. In 11 of 17 evaluable patients a late wave ( > 9 months) of donor T cells occurred. As a consequence, T-cell chimerism changed in favor of donor T cells and the CD4/CD8 ratio that had been reversed ( < 0.5) after the first expansion, normalized (1.5 +/- 0.51). Analysis of the T-cell receptor repertoire showed that in recipients of a T-cell depleted graft, the recipient as well as the donor T cells that repopulated the peripheral T-cell pool during the first month, were the progeny of a limited number of precursors. Because without depletion, when larger numbers of donor T cells had been cotransfused with the marrow, the repertoire was much more diverse, these data show that immediately after transplantation, the peripheral pool is repopulated primarily through expansion of circulating T cells.     

Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity

To evaluate the importance of the thymus for the reconstitution of immunity in recipients of a T-cell-depleted bone marrow, we measured the appearance of CD4(+)CD45RA(+)RO(-) naive T cells (thymic rebound), restoration of the diversity of the T-cell-receptor (TCR) repertoire and the response to vaccinations with tetanus toxoid (TT). Repopulation by CD4(+)CD45RA(+)RO(-) thymic emigrants varied among patients, starting at approximately 6 months after transplantation. Young patients reconstituted swiftly, whereas in older patients, the recovery of normal numbers of naive CD4(+) T cells could take several years. Restoration of TCR diversity was correlated with the number of naive CD4(+)CD45RA(+)RO(-) T cells. Moreover, the extent of the thymic rebound correlated with the patient's capacity to respond to vaccinations. Patients without a significant thymic rebound at the moment of vaccination (CD4(+)CD45RA(+)RO(-) T cells less than 30 microL) did not respond, or responded only marginally even after 3 boosts with TT. We conclude that during the first year after transplantation, the absence of an immune response is due mainly to the loss of an adequate T-cell repertoire. Restoration of the repertoire can come only from a thymic rebound that can be monitored by measuring the increase of CD4(+)CD45RA(+)RO(-) naive T cells. This will allow postponing revaccinations to a moment when the patient will be able to respond more effectively. This may be particularly useful in the elderly patient who, owing to low thymic activity, might not yet be able to respond 1 year after transplant when revaccinations are usually scheduled.     

CD45 isoform phenotypes of human T cells: CD4(+)CD45RA(-)RO(+) memory T cells re-acquire CD45RA without losing CD45RO

We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.     

Reconstitution of the T-Cell Compartment After Bone Marrow Transplantation: Restoration of the Repertoire by Thymic Emigrants

We have studied the reconstitution of the T-cell compartment afterbone marrow transplantation (BMT) in five patients who received agraft-versus-host disease (GVHD) prophylaxis consisting ofmethotrexate, cyclosporin, and 10 daily injections (day 4 to day+5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/µL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool wasrepopulated exclusively through expansion of residual T cells with allCD4⁺ T cells expressing the CD45RO-memory marker. In twopatients, the expansion was extensive and within 2 months, the totalnumber of T cells (CD8>>CD4) exceeded 1,000/µL. In the otherthree patients, T cells remained low (87 ± 64 T cells/µL at 6 months) and remained below normal values during the 2 years of thestudy. In all patients, the firstCD4⁺CD45RA⁺RO T cellsappeared after 6 months and accumulated thereafter. In the youngestpatient (age 13), the increase was relatively fast and naiveCD4⁺ T cells reached normal levels (600 T cells/µL) 1 year later. In the four adult patients (age 25 ± 5), the levelsreached at that time-point were significantly lower (71 ± 50 Tcells/µL). In all patients, the T-cell repertoire that had been verylimited, diversified with the advent of theCD4⁺CD45RA⁺RO T cells. Cellsorting experiments showed that this could be attributed to thecomplexity of the T-cell repertoire of theCD4⁺CD45RA⁺RO T cells thatwas comparable to that of a normal individual and that, therefore, itis likely that these cells are thymic emigrants. We conclude that afterBMT, the thymus is essential for the restoration of the T-cellrepertoire. Because the thymic activity is restored with a lag time ofapproximately 6 months, this might explain why, in particular inrecipients of a T-cell-depleted graft, immune recovery is delayed.