In vivo stimulation of natural killer T cells and downstream regulatory T cells to reduce acute graft-versus-host disease

Allogeneic hematopoietic cell transplantation (HCT) is a curative treatment for many malignant and non-malignant hematological conditions but is limited by the development of acute and chronic graft-versus-host disease (GVHD) (1), which can have long-term impacts on survivorship (2). The addition of post-transplant cyclophosphamide with standard GVHD prophylaxis is increasingly being used in human leukocyte antigen (HLA)-matched HCT recipients receiving reduced intensity conditioning (3). In contrast, improvements in GVHD prophylaxis in HLA-matched HCT recipients requiring myeloablative conditioning remain to be developed. In a new clinical study, DeFilipp, Chen and colleagues used RGI-2001 with standard GVHD prophylaxis to demonstrate its safety and determine its efficacy in reducing acute GVHD in HLA-matched myeloablative HCT patients (4). This provides a potentially improved form of GVHD prophylaxis in this transplant setting.

RGI-2001 is a liposomal formation of KRN7000, a synthetic derivative of α-galactosylceramide, which can bind CD1d (5) on antigen presenting cells to activate invariant natural killer T (iNKT) cells (6). This activation of iNKT cells can lead to the interleukin-4 (IL-4)-dependent proliferation of regulatory T cells (Tregs) (7). In pre-clinical studies, the iNKT cell-mediated expansion of Tregs can mitigate GVHD (7,8), while increased proportions of these cells can be associated with reduced GVHD (9). Notably, both KRN7000 (10) and RGI-2001 (11) can reduce GVHD in pre-clinical studies, with this effect by the latter due to the iNKT cell-mediated expansion of Tregs (11,12). Thus, RGI-2001 affords a potential additional therapy to activate and increase iNKT cells and Tregs to prevent in GVHD in allogeneic HCT recipients.

Following on from an earlier phase 2a trial, in which a single dose of RGI-2001 (1 or 100 μg/kg) administered on the day of allogeneic HCT was deemed to be safe and to potentially reduce GVHD (13), DeFilipp et al. (4) conducted an open-label, single-arm multicenter phase 2b trial with RGI-2001 in 49 blood cancer patients (n=27, 55.1% acute myeloid leukemia) receiving hematopoietic cells (n=40, 81.6% from peripheral blood) from HLA-matched (8/8 alleles) related (n=16, 32.7%) or unrelated (n=32, 65.3%) donors or a HLA-mismatched unrelated (7/8 alleles) donor (n=1, 2%). Each participant received myeloablative conditioning and the calcineurin inhibitor tacrolimus and methotrexate as standard GVHD prophylaxis, along with 6 weekly infusions of RGI-2001 (100 μg/kg) from day 0 of allogeneic HCT.

The study included a safety run-in phase with seven participants. No dose-limiting toxicities were observed. Amongst the entire cohort only standard and expected adverse events occurred, with no cases of graft failure. No serious treatment-related adverse events were observed, but such adverse events of grade ≥3 were noted in 32.7% (n=16) of the cohort. These adverse events included reduced appetite (n=6, 12.2%), thrombocytopenia (n=5, 10.2%), leukopenia (n=5, 10.2%), stomatitis (n=4, 8.2%), mucosal inflammation (n=3, 6.1%), anemia (n=2, 4.1%) and/or neutropenia (n=2, 4.1%); however it was also that these are standard and expected events with myeloablative HCTs. Collectively these observations confirm the feasibility and safety of RGI-2001 in allogeneic HCT recipients.

Amongst the cohort receiving RGI-2001, 24.9% (n=12) of participants developed grade 2 to 4 acute GVHD (median onset on day 46 post-HCT). During the safety run-in phase, the half-life of RGI-2001 was found to be approximately 36 hours, with less than 5% of the maximum concentration detected 7 days after the previous dose. Numbers of blood CD3⁺, CD4⁺ and CD8⁺ T cells increased by day 42, suggesting immune cell reconstitution was not impaired. In line with the mechanism of action of RGI-2001, the numbers of natural killer T (NKT) cells and Tregs also increased over time, with an increased trend in NKT cells and a significant increase in Tregs in those who did not develop GVHD compared to those with GVHD. As acknowledged by the authors, iNKT cells were not directly assessed, with these cells identified simply as CD3⁺CD56⁺CD69⁺ cells (4). Thus, these cells are likely to include other T cell subsets (14,15).

The group receiving RGI-2001 (but excluding the one mismatched donor) was compared to a non-randomized matched control group comprising 207 patients from the same treatment centers, with the following P values based on adjusted hazard ratios. Compared to the control group, the RGI-2001 group had reduced probabilities of Grade 2–4 acute GVHD at days 100 and 180 (P=0.003 and P=0.001, respectively) and Grade 3–4 acute GVHD at days 100 and 180 (P=0.011 and P=0.006, respectively). Compared to the control group, the probability of chronic GVHD was also reduced in the RGI-2001 group at day 180 (P<0.001) but not at 1 year (P=0.56). The authors (4) and others (16) propose that chronic GVHD could be potentially reduced by continued dosing with RGI-2001. Nevertheless, the probability of GVHD-free relapse survival at 1 year was improved in the RGI-2001 group compared to the control group (P=0.004). Importantly, disease relapse was the same between these respective groups (12.5% vs. 16.5%, P=0.196), suggesting that the life-saving graft-versus-leukemia (GVL) response resulting from an allogeneic HCT was not compromised by RGI-2001 infusion. This notion is supported by the study of RGI-2001 in pre-clinical models of GVHD and GVL immunity (11). Another pre-clinical study has shown that iNKT2 and iNKT17 cells can reduce GVHD, while iNKT1 cells have anti-tumor activity (17). Thus, it remains to be resolved which subtypes of iNKT cell subsets are activated by RGI-2001 in allogeneic HCT. It also remains to be determined which cytokines may be involved in this process including IL-4, which mediates the iNKT cell-mediated expansion of Tregs to reduce GVHD in mice (7). Circulating IL-4 measurements were not reported by DeFilipp et al. (4), while the earlier phase 2a trial, with a single dose of RGI-2001, reported low and variable amounts of blood IL-4 (13).

In summary, the clinical study of DeFilipp et al. indicates that RGI-2001 may safely reduce acute GVHD and to a lesser extent chronic GVHD in HLA-matched myeloablative HCT recipients receiving standard GVHD prophylaxis without compromising the GVL response. The study supports a model in which α-galactosylceramide, derived from RGI-2001, is presented by antigen presenting cells via CD1d to iNKT cells inducing their activation and downstream expansion of Tregs to inhibit donor T cells, along with the standard GVHD prophylaxis (tacrolimus and methotrexate) to reduce acute GVHD (Figure 1). Furthermore, the study of DeFilipp et al. supports similar or alternate in vivo strategies to increase iNKT cells and Tregs in the prevention of acute GVHD in allogeneic HCT.

Figure 1 RGI-2001 reduces acute GVHD. α-galactosylceramide, derived from RGI-2001, is presented by antigen presenting cells via CD1d to the invariant T cell receptor of iNKT cells. This induces iNKT cell activation and the downstream expansion of Tregs to inhibit donor T cell activation and proliferation to reduce GVHD. Standard GVHD prophylaxis agents, tacrolimus and methotrexate, which can also impair donor T cell activation and proliferation contribute to a reduction in GVHD. Sluyter R (2026) https://BioRender.com/7o7jay5. APC, antigen presenting cell; α-GalCer, α-galactosylceramide; GVHD, graft-versus-host disease; iNKT, invariant natural killer T cell; MTX, methotrexate; TAC, tacrolimus; Treg, regulatory T cell. Created in BioRender.

Acknowledgments

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Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article did not undergo external peer review.

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-1-0017/coif). The authors have no conflicts of interest to declare.

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