Page 138 - Haematologica Vol. 110 - January 2025

ARTICLE - Immune microenvironment in nodal PTCL
P. Stephan et al.
pression of exhaustion markers by tumor-infiltrating CD8+ T cells, as well as perturbations in the B-cell compartment of patients with AITL.18 In this study, we aimed to bridge this knowledge gap and extensively characterize the phenotype of immune cells recruited in response to PTCL.
Methods
Samples for spectral flow cytometry analyses
Frozen lymph node (LN) cell suspensions from 18 PTCL samples, collected at diagnosis, were obtained through the CeVi_Collection Project from the CALYM Carnot Institute (Lyon, France). Samples were collected as part of an RIPH3 project (ID RCB 2020-A02273-36, validated by the CPP Ile de France V on November 26, 2020; N. 20.07.28.49748). Reactive LN suspensions were obtained from the biologi- cal resource center of Lyon-Sud Hospital (agreement CRB BB-0033-00046). Tissues were assessed by the pathology department, and cancer diagnosis was excluded through pathological evaluation and absence of T or B clonality. All adult patients (aged ≥18 years) gave their written informed consent on the secondary use of their samples for research. The clinical and biological data of the patients were col- lected retrospectively. Their characteristics are presented in Online Supplementary Tables S1 and S2.
Blood samples from healthy volunteers were obtained through the Etablissement Français du Sang (EFS). Pe- ripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation, and red blood cells were lysed with ammonium-chloride-potassium lysis buf- fer. Tonsils obtained anonymously from 5 donors that had undergone tonsillectomy were used as a control (Clinique du Parc, Lyon; agreement N. 1A16502305468). Tonsils were reduced to cell suspensions by mechanical disruption fol- lowed by enzymatic digestion with 2 mg/mL collagenase-D (Roche) and 20 U/mL DNase (Sigma). The resulting cell suspensions were filtered, washed, and frozen.
found in Online Supplementary Table S5. Acquisition was performed on an Aurora Spectral Cytometer (Cytek). Anal- yses were carried out using the OMIQ software (Dotmatics) (www.omiq.ai). Details on the experimental protocol and analyses can be found in the Online Supplementary Methods; the number of cells analyzed in each subset is detailed in Online Supplementary Tables S6 and S7.
Tissue micro-arrays, multi-immunofluorescence protocol, and data analysis
Representative tumor areas were selected on Hematoxylin & Eosin slides by an experienced pathologist. A 4-plex mIF assay (CD3, CD8, CD39 and DAPI) was performed using a modified 7-color TSA protocol template. Slides were imaged using the Vectra Polaris spectral imaging system (Perkin Elmer). Scans were visualized with the Phenochart soft- ware. Cell segmentation and phenotypes were identified by InForm version 2.4.8 (Akoya Biosciences). Parameters were analyzed using R version 4.2.1. More detailed information can be found in the Online Supplementary Methods.
Statistical analyses
Statistics were performed using GraphPad Prism Software v9 and R version 4.2.1. Kruskall-Wallis or two-way ANOVA tests were used for FACS data, unless otherwise stat- ed. Statistical analyses for comparison between clusters established by the FlowSOM algorithm, were carried out using the edgeR package. Progression-free survival (PFS) was calculated from the date of diagnosis to the date of progression, relapse, or death from any cause. OS was calculated from the date of diagnosis to the date of death from any cause. Survival estimates were calculated with the Kaplan-Meier method. Survival distributions were com- pared with the log-rank test, and Cox proportional hazard regression models were used to estimate hazard ratios and associated 95% Confidence Intervals.
Results
Peripheral T-cell lymphoma-specific immune cell populations identified through deep immunophenotyping To document immune phenotypes associated with PTCL, we designed a 33-color flow cytometry panel to identify different innate and adaptive subsets in a single tube, namely DC, macrophages, NK and B cells, and a number of markers to distinguish subsets of T cells. Moreover, ac- tivation, proliferation, and exhaustion of these populations were determined using different intracellular and surface markers (Table 1, Online Supplementary Table S5). We ap- plied this immunophenotyping panel to LN cell suspensions obtained from 18 nodal PTCL patients at diagnosis (11 AITL and 7 PTCL, NOS) (Online Supplementary Tables S1 and S2), as well as 5 non-tumoral reactive LN, 5 tonsil samples, and 5 PBMC samples obtained from healthy donors. Of note,
Samples for multi-immunofluorescence analyses
Forty-three tissue blocks of formalin-fixed and paraf- fin-embedded (FFPE) AITL samples, collected at diagnosis, were obtained from the Department of Pathology at the Lyon-Sud Hospital. This study was performed according to the principles of the Declaration of Helsinki. Patients had provided informed consent. The study was approved by the local ethics committee (MR-004 N. 23-5211). Clinical and biological data of the patients were collected retro- spectively. A summary of the data is presented in Online Supplementary Tables S3 and S4.
Flow cytometry
A total of 1 million live cells were stained through succes- sive incubation steps with dyes and antibodies (Ab). The complete list of Ab and their final concentration can be
Haematologica | 110 January 2025
130