Background

Recently, new advances were made regarding the depletion of CD45RA⁺ naïve T cells from haploidentical grafts as they are suspected to be the most alloreactive.

Methods

Within this project we investigated CD45RA-depletion from G-CSF mobilized PBSC by two different purification strategies according to GMP, specifically direct depletion of CD45RA⁺ cells (one-step approach), or CD34-positive selection followed by CD45RA-depletion (two-step approach).

Results

With log −3.9 and − 3.8 the depletion quality of CD45RA⁺ T cells was equally for both approaches together with a close to complete CD19⁺ B cell depletion. However, due to a high expression of CD45RA the majority of NK cells were lost within both CD45RA depletion strategies. Stem cell recovery after one-step CD45RA-depletion was at median 52.0% (range: 49.7–67.2%), which was comparable to previously published recovery data received from direct CD34 positive selection. Memory T cell recovery including CD4⁺ and CD8⁺ memory T cell subsets was statistically not differing between both purification approaches. The recovery of CD4⁺ and CD8⁺ T cells was as well similar, but overall a higher amount of cytotoxic than T-helper cells were lost as indicated by an increase of the CD4/CD8 ratio.

Conclusions

CD45RA-depletion from G-CSF mobilized PBSC is feasible as one- and two-step approach and results in sufficient reduction of CD45RA⁺ T cells as well as B cells, but also to a co-depletion of NK cells. However, by gaining two independent cell products, the two-step approach enables the highest clinical flexibility in regard to individual graft composition with precise dosage of stem cells and T cells.

背景

最近，关于从半相合移植物中去除 CD45RA ⁺幼稚 T 细胞方面取得了新进展，因为它们被怀疑是最具同种异体反应性的。

方法

在这个项目中，我们根据 GMP 通过两种不同的纯化策略研究了 G-CSF 动员的 PBSC 中的 CD45RA 消耗，特别是 CD45RA ⁺细胞的直接消耗（一步法），或 CD34 阳性选择然后 CD45RA 消耗（两步法）。

结果

对于 log -3.9 和 - 3.8 ，两种方法的 CD45RA ⁺ T 细胞消耗质量相同，并且接近完全的 CD19 ⁺ B 细胞消耗。然而，由于 CD45RA 的高表达，大多数 NK 细胞在两种 CD45RA 消耗策略中都丢失了。一步去除 CD45RA 后的干细胞恢复率中位数为 52.0%（范围：49.7-67.2%），这与先前公布的从直接 CD34 阳性选择中获得的恢复数据相当。包括 CD4 ⁺和 CD8 ⁺记忆 T 细胞亚群在内的记忆 T 细胞恢复在两种纯化方法之间在统计学上没有差异。CD4 ⁺和CD8 ⁺的恢复 T 细胞也很相似，但总体上比 T 辅助细胞丢失了更多的细胞毒性，如 CD4/CD8 比率的增加所示。

结论

从 G-CSF 动员的 PBSC 中去除 CD45RA 是可行的一步和两步方法，并导致 CD45RA ⁺ T 细胞和 B 细胞的充分减少，但也导致 NK 细胞的共同消耗。然而，通过获得两种独立的细胞产品，两步法在具有精确剂量的干细胞和 T 细胞的个体移植组合物方面实现了最高的临床灵活性。