To the Editor:
We read the article by Nieto et al. with interest . The authors demonstrated that pretransplant nivolumab resulted in a high frequency of IFN-γ-producing effector T cells that might contribute severe graft-versus-host disease (GVHD) after transplant. They also showed that posttransplant cyclophosphamide (PTCy) abrogated the immune activations and could suppress GVHD to the mild levels. The data were comprehensive and convincing, but the analyses of regulatory T cells (Tregs) were limited to just a one-time point. In general, PTCy can contribute to enhancing Treg recovery as well as suppressing alloreactive effector T cells [2,3,4]. Our previous study suggested that programmed cell death 1 (PD-1) plays an important role in Treg homeostasis . Moreover, by using a murine HSCT model, we recently demonstrated that pretransplant PD-1 blockade critically impaired posttransplant Treg recovery, leading to severe GVHD, and PTCy could restore Treg homeostasis and ameliorate GVHD . Based on these findings, we herein analyzed the chronological recovery of Treg after PTCy in patients with or without pretransplant nivolumab therapy and considered the impact of nivolumab on Treg homeostasis after PTCy-based transplant.
We experienced three cases of chemotherapy-refractory classical Hodgkin lymphoma, those were treated by nivolumab as a salvage therapy and followingly received HLA-haploidentical HSCT. The clinical courses of the three patients are described in Supplementary Fig. 1. We compared the detailed immune reconstitutions of patients receiving pretransplant nivolumab followed by PTCy-based HLA-haploidentical HSCT (“Nivo→PTCy”; Cases 1 and 2) and ATG-based HLA-haploidentical HSCT (“Nivo→ATG”; Case 3) with those of patients who received PTCy-based HLA-haploidentical HSCT without ICI (“control-PTCy”; Cases 4–6) and ATG-based HLA-haploidentical HSCT without ICI (“control-ATG”; Cases 7–9), as respective controls. Clinical data and frozen samples were collected from these nine patients. Detailed patient characteristics and transplant outcomes are summarized in supplementary Table 1. All patients provided written informed consent before sample collection.
To understand the homeostasis of each T cell subset post-transplant, we first examined PD-1 expression and cell proliferation of T cells after transplant (Supplementary Fig. 2). PD-1 expression levels in patients without pretransplant nivolumab were elevated within 2 weeks after transplantation, especially in the control-ATG group (Supplementary Fig. 2A). By contrast, the PD-1 expression level in patients who received pretransplant nivolumab was even lower than that of healthy controls. Cell proliferation was assessed based on Ki67-expression in each T cell subset (Supplemental Fig. 2B). In cases without pretransplant nivolumab, cell proliferation was more active in each T cell subset for patients in the control-ATG group than for patients in the control-PTCy group (Supplementary Fig. 2B). Pretransplant nivolumab markedly increased the cell proliferation rates in the case of Nivo→ATG through the examined period, presumably due to the negativity of PD-1 expression. Contrastingly, proliferation of T cell subsets in the Nivo→PTCy group was negatively regulated in the first week and thereafter gradually regained after 2 weeks, although the level was much lower than that of the Nivo→ATG case (Supplementary Fig. 2B). Then, we analyzed the chronological recovery of each T cell subset. Treg and conventional T cells (Tcon) were defined as CD4+Foxp3+ and CD4+Foxp3−, respectively. Of note, the early and vigorous recovery of Treg in the Nivo→PTCy group was observed by day 28 and both the number and the frequency were the highest among the four groups (Fig. 1A). Subsequently, the Treg number in the control-PTCy group increased; however, Treg numbers remained low in the control-ATG and Nivo→ATG groups (Fig. 1A). As Foxp3 is also expressed in activated human Tcon , we evaluated the frequency of the CD25highCD127low population and obtained similar results (data not shown). Increasing Treg were predominantly CD45RA-negative and Helios-positive; these phenotypic characteristics were similar among the four groups (Fig. 1B, C).
These results showed that pretransplant nivolumab and PTCy significantly altered the reconstitution of T cell subsets early after HLA-haploidentical HSCT. Especially, PTCy facilitated Treg recovery in recipients receiving pretransplant nivolumab. The control-ATG group exhibited high PD-1 expression post-HSCT, especially during the first 2 weeks, suggesting that donor lymphocytes after ATG-based GVHD prophylaxis may be more susceptible to nivolumab than those after PTCy-based GVHD prophylaxis. In fact, pretransplant nivolumab resulted in insufficient control of cell proliferation in patients without PTCy (Nivo → ATG). Such prolonged aggressive proliferation might result in unbalanced reconstitution of T cell subsets, leading to refractory GVHD. As Treg are susceptible to apoptosis under conditions of prolonged aggressive proliferation after HSCT , a nivolumab-induced increase in Treg proliferation might promote their susceptibility to apoptosis, thereby predisposing them to homeostatic failure. In contrast, PTCy efficiently eliminated Ki67+ proliferative T cells in the first week, adjusting cell proliferation to an appropriate level by the second week (Nivo → PTCy). This apparently maintained Treg homeostasis, resulting in Treg expansion and contributing to appropriate GVHD control.
Consistent with our preclinical model , the present study indicates that PTCy counteracts the negative impact of nivolumab to promote Treg recovery in clinical situations. Kanakry et al.  showed that PTCy generally preserves Treg via an aldehyde dehydrogenase-dependent mechanism. Our study further suggests that in HSCT after nivolumab treatment, where particularly intense Treg proliferation is predicted, inhibitory regulation of initial Treg proliferation by PTCy is important for Treg preservation.
We acknowledge the limitations of this study. Our results should be interpreted with caution because this study included heterogeneous backgrounds including stem cell source, primary disease, and conditioning intensity. Importantly, the data of Nivo→ATG case were obtained after the rescue transplantation due to engraftment failure following the first transplantation which can affect the post-transplant immune reconstitution.
Despite the small size and exploratory nature of the study precluding a definitive conclusion, based on our results, we can propose a mechanistic hypothesis for the beneficial effect of PTCy to stabilize posttransplant immunity in cases of pretransplant nivolumab administration. Our data suggest that immune activation by pretransplant nivolumab could be safely managed by not only mitigating effector T cell activation, as observed by Nieto et al. , but also by promoting the vigorous recovery of Treg after PTCy leading to immune tolerance. A larger cohort study to validate our mechanistic hypothesis and optimize the prophylactic PTCy effect is warranted.
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The authors would like to thank Kyoko Maeda and Hiromi Nakashima for help with obtaining clinical samples. This work was supported by research funding from the Japan Society for the Promotion of Science KAKENHI (Grant No. 20K08753) to KM.
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Ikegawa, S., Meguri, Y., Mizuhara, K. et al. Pretransplant nivolumab further enhanced Treg expansion after posttransplant cyclophosphamide; another aspect for immune tolerance by PTCy after nivolumab. Leukemia (2021). https://doi.org/10.1038/s41375-021-01167-8