CTLA-4⁺ Effector Regulatory T Cells May Contribute to Maintenance of Treatment-Free Remission in Chronic Myeloid Leukemia

Introduction: Tyrosine kinase inhibitors (TKIs) discontinuation trials have achieved long-term treatment-free remission (TFR) in about half of patients with chronic myelogenous leukemia (CML), and TFR has become a therapeutic goal for CML. On the other hand, about half of TFR-challenged patients show loss of molecular response. Although most patients relapse in 6 months after discontinuation due to inadequate deep response, there are also a few cases of late relapse after discontinuation. In TFR phase, late relapse patients often show so-called “fluctuate” state in which BCR::ABL1-positive cells are detected but MMR is not lost. It is suggesting that fluctuate and late relapse are continuous pathological change and one spectrum. However, there are cases that remain in the “fluctuate” state with MMR for a long period of time. We have analyzed host immunity during the TFR phase and have already reported that regulatory T cells (Treg) may distinguish TFR and relapse patients (Cancers 2021). However, subsequent analysis have not reveal any immune markers that could clearly classify fluctuate and late relapse. This study aims to elucidate the immune mechanisms underlying the persistence of TFR. By focusing on patients who became positive again for minimal residual disease (MRD) positive after TKI discontinuation, we attempted to detect immune markers that could distinguish between fluctuate and late relapse.

Methods: Patients with chronic phase CML who had discontinued TKI therapy at our institution were included. Patients were confirmed to have MR^4.5 or deeper for at least 2 years and were discontinued from TKI. Peripheral blood was collected before discontinuation, 1, 3, 6, 12, 18 months after discontinuation, and at relapse (loss of MMR), and Peripheral blood mononuclear cells (PBMCs) were used for immune profile analysis by multicolor flow cytometry (FCM). In MRD-positive patients who showed loss of DMR (MR^4.5) at 6 months after discontinuation, subsequent molecular genetic changes of IS BCR::ABL1 by quantitative PCR assay and immune profiles obtained by FCM were compared.

Results: Of 60 patients who discontinued TKI at our institution, 7 were eligible for follow-up immunological analysis. 4 of the 7 (57.1%) patients maintained TFR for more than 1 year without loss of MMR (TFR group). The remaining 3 (42.9%) patients lost MMR and received TKI again (Retx group). Six-month follow-up showed no obvious changes in CD4⁺ or CD8⁺ T cells, CD16^dim NK cells, or CD19⁺ B cells, and no differences were observed in comparison between the TFR and the Retx group. On the other hand, FoxP3⁺CD45RA^- effector Treg (eTreg) tended to decrease in TFR group (3/4 patients) and increase in Retx group (3/3 patients)(p = 0.03). Immune co-stimulatory molecule expression in eTreg was also analyze, CTLA-4⁺ eTreg showing the most different variation in the two groups (p < 0.01). ETreg positive for inhibitory molecules such as PD-1, TIM-3, and TIGIT showed a similar trend, whereas eTreg positive for stimulatory molecules such as ICOS, 4-1BB, and GITR showed the opposite trend, with smaller changes between the two groups. Myeloid-derived suppressor cells (MDSCs) showed no difference between the two groups.

Conclusion: Treg are TKI highly sensitive cells (JEM 2020) and show significant changes after TKI discontinuation in CML patients. This change in Treg is a transient fluctuation that does not correlate with Treg function (Cancers 2021). We compared fluctuate and late relapse in MRD-positive patients during TFR. We found that an increase in eTreg, especially CTLA-4⁺ eTreg, may contribute to molecular relapse. This result suggests that host immunopathology may be involved in the maintenance of TFR, especially in fluctuate patients with MRD, and may lead to prognostic biomarkers and new therapeutic targets for successful TFR.

Takahashi:Asahi Kasei: Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono: Research Funding. Ueki:AstraZeneca: Honoraria, Research Funding; GSK: Honoraria; Sanofu: Honoraria; maruho: Research Funding; VIB: Research Funding.