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. 2023 Jul;37(7):1572-1575.
doi: 10.1038/s41375-023-01932-x. Epub 2023 Jun 7.

A pro-inflammatory environment in bone marrow of Treg transplanted patients matches with graft-versus-leukemia effect

Francesco Guardalupi #  1 , Carlo Sorrentino #  1 , Giulia Corradi  1 , Raffaella Giancola  2 , Stefano Baldoni  2 , Francesca Ulbar  1 , Bianca Fabi  1 , Rosa Andres Ejarque  3 , Jessica Timms  3 , Francesco Restuccia  2 , Stella Santarone  2 , Patrizia Accorsi  2 , Paolo Sportoletti  4 , Filomena De Falco  4 , Emanuela Rosati  4 , Alessandra Carotti  4 , Franca Falzetti  4 , Andrea Velardi  4 , Massimo Fabrizio Martelli  4 , Shahram Kordasti  3 , Antonio Pierini  4 , Loredana Ruggeri  4 , Mauro Di Ianni  5   6
Affiliations

A pro-inflammatory environment in bone marrow of Treg transplanted patients matches with graft-versus-leukemia effect

Francesco Guardalupi et al. Leukemia. 2023 Jul.
No abstract available

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Flow cytometry and real-time RT-PCR analysis of DCs, derived from BM or PB of transplanted patients. Flow cytometry analysis of CD4+ and CD8+ T cells and mixed culture of CD3+ T cells with BM- or PB-mDCs.
A The mDC percentage was higher in BM than in PB, in the first month after transplant (*Student’s t-test: p < 0.05). B BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86 (showed as MFI values), compared to PB-derived mDCs (**Student’s t-test: p < 0.01). C, D Real-time RT-PCR showed the gradual decrease of IL-6 and TGF-β expression, after transplantation, in PB-mDCs, while their levels in BM-mDCs remain stable. (*Student’s t-test: p < 0.05). E Real-time RT-PCR showed significantly higher PD-L1 levels in PB-mDCs compared to BM-mDCs (*Student’s t-test: p < 0.05; ***Student’s t-test: p < 0.001). All data (A–E) are represented as mean ± SD. F The percentages of CD3+, CD4+ and CD8+ T cells were comparable (Student’s t-test: p > 0.05) between BM and PB and among early and late follow-up phases (ANOVA: p > 0.05). Data are represented as mean. G Left Panel. BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3% ± 18.8 vs 9.2% ± 4.9; *Student’s t-test: p < 0.05). Data are represented as mean ± SD. Right Panel. The expression of the immune checkpoint inhibitor PD-1 was significantly higher in PB-derived CD4+ (69% ± 29 vs 24% ± 11; *Student’s t-test: p < 0.05) and CD8+ (65% ± 25 vs 4% ± 3; *Student’s t-test: p < 0.05) T cells than in BM-derived T lymphocytes. Data are represented as mean ± SD. H CD3+/CFSE+-mDCs co-cultures showed a T cell proliferation rate that was significantly higher when T cells were cultured in presence of BM-mDCs (25% ± 7.2 vs 6.7% ± 8.7; *Student’s t-test: p < 0.05). Data are represented as mean ± SD of 3 independent experiments.
Fig. 2
Fig. 2. Flow cytometric analysis of CD161+ Treg cells derived from BM or PB of transplanted patients, in vitro generation of CD161+ Treg cells with related functional experiments.
A Upper panel. UMAP plot with T-REX analysis of bone marrow and peripheral blood Tregs based on a statistical threshold of ≥ 95% change in cell phenotypes. Pink and red colour denote regions of phenotypic change identified by T-REX with a higher representation in bone marrow samples. Blue areas are enriched in peripheral blood. (Light colours: 85–95% change, dark colours: ≥95% change). Lower panel. DBSCAN clustering for the areas of ≥95% change. B Heatmap showing MEM enrichment of Treg markers in DBSCAN clusters. C Percentage of Tregs with high expression of CCR4, CCR6, CD161, CD39, HLA-DR in bone marrow and peripheral blood samples. Each patient is represented by a line. *p < 0.05 (two tailed paired Wilcoxon test). D PB-derived Tregs (CD4+CD25highCD127-/low), treated with both IL-6 and TGF-β, showed, after 10 days of culture, a significant higher expression of CD161 (One-way ANOVA, Tukey HSD test: *p < 0.05, **p < 0.01), if compared with cells untreated or treated with IL-6 or TGF-β alone. Untreated = cells only activated with T Cell TransAct™ and IL-2. Data are represented as mean ± SD of 3 independent experiments. E The graphs show the functional activity of CD161+ Tregs. Left panel. Suppression activity of CD161+ Tregs and expanded Tregs, referred as Tregs, on the proliferation of Trans Act stimulated-Tcons in a mixed lymphocyte reaction performed for 4 days. Data are presented as mean ± SD of 3 independent experiments (Two-way ANOVA, Sidak’s multiple comparison test; ****p < 0.0001; ***p = 0.0009). Right panel. Killing of the K562 cell line by Tcons, ratio Effector:Target 1:20. Where indicated, Tcons were co-coltured with CD161+ Tregs and Expanded Tregs, referred as Tregs, at different Tregs:Tcons ratio, 1:2 and 1:5 respectively (mean ± SD of 3 experiments; One-way ANOVA, Tukey’s multiple comparison test: Tcons alone vs + CD161+ Tregs ratio 1:2 **p = 0.001; vs + Tregs ratio 1:2 ****p < 0.0001; vs + CD161+ Tregs ratio 1:5 *p = 0.021; vs + Tregs ratio 1:5 ****p < 0.0001; + CD161+ vs Tregs ratio 1:2 *p = 0.011; + CD161+ vs Tregs ratio 1:5 *p = 0.015). F Flow cytometric analysis of the expression of RORγt, FOXP3, HLA-DR, CCR4, and CCR6 on CD161+ Tregs, defined as CD45+CD3+CD4+CD25highCD127-/lowCD161+ and Expanded Tregs, referred as Tregs and defined as CD45+CD3+CD4+CD25highCD127-/low (mean ± SD of 3 experiments; Two-way ANOVA, Sidak’s multiple comparison test, **p = 0.005; ****p < 0.0001).
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References

    1. Di Ianni M, Falzetti F, Carotti A, Terenzi A, Castellino F, Bonifacio E, et al. Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation. Blood. 2011;117:3921–8. doi: 10.1182/blood-2010-10-311894. - DOI - PubMed
    1. Martelli MF, Di Ianni M, Ruggeri L, Falzetti F, Carotti A, Terenzi A, et al. HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse. Blood. 2014;124:638–44. doi: 10.1182/blood-2014-03-564401. - DOI - PubMed
    1. Pierini A, Ruggeri L, Carotti A, Falzetti F, Saldi S, Terenzi A, et al. Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia. Blood Adv. 2021;5:1199–208. doi: 10.1182/bloodadvances.2020003739. - DOI - PMC - PubMed
    1. Bolton HA, Zhu E, Terry AM, Guy TV, Koh WP, Tan SY, et al. Selective Treg reconstitution during lymphopenia normalizes DC costimulation and prevents graft-versus-host disease. J Clin Investig. 2015;125:3627–41. doi: 10.1172/JCI76031. - DOI - PMC - PubMed
    1. Edinger M, Hoffmann P, Ermann J, Drago K, Fathman CG, Strober S, et al. CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation. Nat Med. 2003;9:1144–50. doi: 10.1038/nm915. - DOI - PubMed

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