T cell repertoire complexity is conserved after LLME treatment of donor lymphocyte infusions

Abstract

Slow reconstitution of the T cell repertoire after allogeneic blood or bone marrow stem cell transplantation is a major risk factor for patient mortality. The delivery of immunocompetent T cells as delayed donor lymphocyte infusions (DLIs) is a potential way of counteracting this problem. The development of graft-versus-host disease (GVHD) is a potential complication of this procedure, however. We previously found that in P-->F1 haploidentical murine models, the ex-vivo treatment of donor lymphocytes with L-leucyl-L-leucine methyl ester (LLME) can prevent the onset of GVHD after DLI, likely by inducing cell death in most of the perforin-positive CD8(+) T cells and in a fraction of CD4(+) T cells. Our previous preclinical studies have formed the basis of an ongoing phase I clinical trial in which patients received LLME-treated DLI from their original donor in an attempt to accelerate T cell reconstitution. To understand how this treatment strategy might affect the complexity of the DLI T cell repertoire, we used T cell receptor Vbeta spectratype analysis to evaluate the DLI product pre-LLME and post-LLME treatment. The results indicated that the LLME-treated DLI product exhibited CDR3-size distribution complexities similar to those of its untreated donor sample. In addition, comparisons of the CD4(+) and CD8(+) T cell repertoire from the donor before LLME treatment with that of the recipient post-DLI demonstrated equal complexity for most of the resolvable Vbeta families. Finally, the in vitro proliferative capacity of LLME-treated DLI product in response to allo-stimulation in a one-way mixed lymphocyte reaction was comparable to that of the untreated product.

Figure 1

CDR3-size spectratype analysis of PBMC enriched from the DLI products of five donors before and after LLME treatment. The spectratype analysis was performed as described in “Materials and Methods.” Three representative Vβ spectratypes are shown for each donor.

Figure 2

CDR3-size spectratype analysis of CD4⁺ T cells enriched from PBMC of three patients after receiving LLME treated DLI. The spectratype analysis was performed as described in “Materials and Methods.” A representative Vβ spectratype is shown for each patient comparing the LLME-treated DLI to the reconstituting patient repertoire, post-transplant.

Figure 3

Proliferative responses of mock- or LLME-treated PBMC. The immunocompetency of mock- and LLME-treated PBMC from four individuals was assessed in vitro by measuring [³H]-TdR incorporation in response to allo-stimulation.