Effects of Various ATG Doses on Rejection Rate and Immune Recovery in Haploidentical T and B Cell Depleted Stem Cell Transplantation

T and B cell depletion of haploidentical stem cells with CD3/CD19 coated magnetic microbeads prevents from GvHD and allows to coinfuse donor NK cells and other accessory cells. Additional in vivo depletion of the graft by serotherapy is not necessary. Thus, a dosing scheme of ATG is needed, which provides a rejection prophylaxis by depleting lymphocyte subsets of the patients but which does not harm the graft, in particular cotransfused NK cells. We retrospectively analyzed the impact of various Grafalon (anti-human T-lymphocyte immunoglobulin = ATLG; formerly ATG-Fresenius) doses given at day -12 on engraftment rate and on posttransplant immune recovery.

A total of 50 pediatric patients received either 3x5 (n=7), 3x10 (n=34) or 3x20mg/kg (n=9) ATLG starting at day -12 to day -9, followed by fludarabine (160 mg/m², d -8 to -5), thiotepa (10 mg/m² d -4) and melphalan (140 mg/m² d -3 to -2). Diagnoses were: ALL/AML n=10, relapsed solid tumors n=33, nonmalignant diseases n=7. Most patients received MMF until day 30-60. ATLG serum levels were measured in 24 patients by flow cytometry (T cell specific rabbit IgG). Apoptosis/necrosis of donor NK cells was assessed with Annexin V/PI staining.

A median number of 16x10⁶ CD34/kg and 65x10⁶ NK cells/kg with 29x10³ T cells/kg were infused. Median time to ANC>500/µl was 9.5 days. Graft rejection occurred in 3/7 patients with 15mg (42%), in 7/34 patients with 30mg (20%) and in 1/9 patients with 60mg ATLG (11%). All rejectors could be rescued by reconditioning and 2^nd stem cell donation or by infusion of autologous back ups. Acute GvHD grade 0-I occurred in 47 patients (94%), GvHD grade II and III-IV was observed in 1 and 2 patients (2%, 4%), respectively. Chronic GvHD occurred in 9 patients (18%). TRM at 1 year was 10% for the entire group. Immune recovery of CD3+ T cells was influenced by the various amounts of ATLG given in the 3 subgroups (15 vs 30 vs 60mg/kg) with a median number of 98 vs 10 vs 2 cells/µl at day +30, 95 vs 100 vs 2 cells/µl at day +90 and 75 vs 218 vs 239 cells/µl at day +180 (figure 1a). Recovery of CD56+ NK cells was fast and reliable and reached peaks of 459 vs 342 vs 155 cells/µl at day +30. Later on NK cells dropped to normal levels of 152 vs 167 vs 104 cells/µl at day +90 and 118 vs 172 vs 99 cells /µl at day +180. ATLG serum levels reached mean peak values of 9.6 µg/ml ±4.7 vs 22.1±13.8 µg/ml vs 53.7 ±13.9 µg/ml at day -8 and dropped to 1.4 ±1µg/ml vs and 3.0 ±1.98 µg/ml and 11.5 ±5.8 µg/ml at day 0 (15 vs 30 vs 60mg/kg, figure 1b). In vitro incubation of isolated NK cells from healthy donors with comparable ATLG doses (1 or 10 µg/ml) resulted in 26 (41)% apoptosis and 0.2 (0.2)% necrosis after 24 hours. Thus, vitality of 73 (59)% of NK cells were preserved.

Conclusions: Our aim was to reduce the rejection rate and to improve immune recovery in patients receiving CD3/19 depleted haploidentical peripheral stem cells, in particular without hampering donor NK cells in the grafts. Administration of 15, 30 or 60 mg/kg ATLG was started at an early time point (day -12) of the regimen. Higher ATLG doses correlated with higher serum levels. Peak levels at day -8 provided a rejection prophylaxis. At day 0, various lower levels were achieved and minimized further in vivo depletion. Increased ATLG doses were associated with a lower rejection rate. On the other hand, recovery of T cells was slower after 60mg/kg ATLG in the early posttransplant phase with lower CD3 counts at day 30 and 90. Since all rejectors were successfully regrafted, it may be reasonable to balance between rejection risk and impaired immune recovery and to use an average dose (30mg). Further studies should investigate if serotherapy might be given even earlier than in this analysis.

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