Dept. of Hematology, Leiden University Medical Center, Leiden, The Netherlands.
Haematologica. 2011 Aug;96(8):1204-12. doi: 10.3324/haematol.2010.039099. Epub 2011 May 5.
One of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire.
Antigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers.
Using this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO(+) responder cells resulted in improved priming and expansion of antigen-specific precursor T cells.
In conclusion, we present a robust method for the in-vitro induction and isolation of antigen-specific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.
同种异体干细胞移植的主要挑战之一是在移植物抗宿主病的有害诱导与移植物抗白血病和病原体特异性免疫反应的有益作用之间找到平衡。过继转移具有特定抗白血病或病原体特异性活性的体外生成供体 T 细胞可能是有效的。然而，在许多情况下，这需要从幼稚供体 T 细胞库中体外引发和扩增抗原特异性前体 T 细胞。
通过共培养 CD45RO 耗尽的、调节性 T 细胞耗尽的供体外周血单核细胞与自体肽负载的树突状细胞，然后用肽负载的自体单核细胞进行两次再刺激，生成抗原特异性 CD8 T 细胞。根据 CD137 表达分离反应性 T 细胞，并使用肽/主要组织相容性复合物四聚体进一步纯化。
使用这种方法，我们能够从血清阴性、次要组织相容性抗原阴性供体的幼稚 T 细胞库中重复地产生针对多种病毒抗原和次要组织相容性抗原的功能高亲和力 T 细胞。此外，我们证明通过耗尽 CD45RO(+)应答细胞来减少调节性 T 细胞的频率可导致抗原特异性前体 T 细胞的更好引发和扩增。
总之，我们提出了一种从幼稚库中体外诱导和分离抗原特异性 T 细胞的稳健方法。我们证明成功产生初级免疫反应的可能性取决于免疫反应引发部位的抗原特异性前体 T 细胞数量与调节性 T 细胞数量和激活状态之间的微妙平衡。
