Page 244 - 2021_10-Haematologica-web

Letters to the Editor
Figure 2. High-dimensional single-cell profiling reveals the dynamics of the cytomegalovirus-specific T-cell response following haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. (A) Uniform manifold approximation and projection (UMAP) analysis of cytokine-positive CD8+ and CD4+ T cells from peripheral blood mononuclear cell (PBMC) samples stimulated overnight with cytomegalovirus (CMV) pp65 peptide mix. Graphs highlight events belonging to different time points or events positive for a given marker. (B) Heatmaps depict marker expression in normalized integrated median fluores- cence intensity (iMFI) values of the antigen-specific CD8+ or CD4+ T-cell PhenoGraph clusters. Balloon plots show the median cluster frequencies as percentage of total CD8+ or CD4+ T cells in haploidentical hematopoietic stem cell transplantation (haplo-HSCT) patients experiencing post-transplant CMV viremia, after background correction was applied. (C) Polyfunctionality of the CMV-specific T-cell response over time in months post-transplant, as determined by assessment of expression of cytokines IFN-γ, TNF and IL-2, and degranulation marker CD107a by cell clusters identified in (B). Measurements containing <50 CMV-specific cells after background correction were discarded from analysis. Medians are depicted and error bars represent the interquartile range. Significance was deter- mined by Kruskal-Wallis test with post-hoc Dunn’s test (*P<0.05, **P<0.01; ***P<0.001). (D) Balloon plots show the median cluster frequencies as percentage of CMV-specific CD8+ or CD4+ T-cells at month 10-13 compared to that found in the graft, periheral blood (PB) of the donor and PB of unrelated healthy controls, after background correction was applied. Measurements containing <50 CMV-specific cells after background correction were discarded from analysis. Exh: exhausted; HC: healthy control; Mult: multifunctional; Prolif: proliferating; TCM: central memory T cell; TEF: effector T cell; TEM: effector memory T cell; TEMRA: effector memory re-expressing CD45RA T cell; TTE: terminal effector T cell.
Figure 3. Cytomegalovirus viremia control following haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide associates with the development of distinct CD4+ antigen-specific T-cell immunophenotypes. (A) Total cytomegalovirus (CMV)-specific CD8+ or CD4+ T-cell counts and (B) cluster-specific T-cell counts in the blood of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) patients with subclinical (n=6) or clinical (n=13) CMV viremia during the first year post-transplant. Medians with the number of patients per time point are shown and error bars represent interquartile range. Significance was determined by Kruskal-Wallis test and P-values are shown at the upper border of the plot for each time point (*P<0.05). Mult: multifunctional; Prolif: proliferating; TTE: terminal effector.
A
B
CDP, AS, JM, SB and LC collected the patient specimens and clinical data; JJPvB, SP, AR, GG and ES analyzed the data; JJPvB and EL wrote the manuscript; FF, LC, DM and EL supervised the study. All authors contributed to and approved the final manuscript.
Funding: this work was funded by the European Research Council
References
1. Luznik L, O’Donnell P V, Symons HJ, et al. HLA-haploidentical bone marrow transplantation for hematologic malignancies using non- myeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2008;14(6):641-
(ERC-StG-2014 PERSYST #640511 to EL). SP was supported by a 650.
fellowship from the Fondazione Italiana per la Ricerca sul Cancro- Associazione Italiana per la Ricerca sul Cancro (FIRC-AIRC). The purchase of a FACSymphony A5 was defrayed in part by a grant from the Italian Ministry of Health (agreement 82/2015).
2. Crocchiolo R, Bramanti S, Vai A, et al. Infections after T-replete hap- loidentical transplantation and high-dose cyclophosphamide as graft-versus-host disease prophylaxis. Transpl Infect Dis. 2015; 17(2):242-249.
3. Levine JH, Simonds EF, Bendall SC, et al. Data-driven phenotypic
2772
haematologica | 2021; 106(10)