Patients
In total, 43 patients were included in the study. Patients were recruited from the U-CAN consortium of prospectively collected tumor and blood samples at diagnosis and diagnosed during 2013–2016 [
21]. All clinical information was retrieved from patient records. All patients were treated according to National treatment guidelines.
The study was approved by the Regional Ethical Committee (Dnr 2010/198 and Dnr 2013/059). Peripheral blood samples from 15 healthy blood donors were included as controls (mean age 44, range 20–69) with male to female ratio 3:1.
Twenty-four patients were diagnosed with high-grade B-cell lymphoma (HGBCL) most often DLBCL but in some cases the diagnosis was made on core needle biopsies only and a correct subclassification according to the WHO classification [
22] was thus not possible to perform. Nineteen patients were diagnosed with cHL. All diagnoses were reevaluated and for all patients peripheral blood in heparinized tubes at diagnosis was collected and available for multi-color flow cytometry analyses. Ficoll Isopaque-gradient separation of mononuclear cells was done according to manufacturer instructions prior to freezing the cells in DMSO in liquid nitrogen before analyses. The different cell types investigated are summarized in Table
1. Gating strategies of the different cell populations are presented in Additional file
1.
Table 1
Percentages of the different immune cells of viable peripheral blood mononuclear cells in patients with classical Hodgkin lymphoma (cHL), high-grade B-cell lymphoma (HGBCL) and healthy blood donors. Mann-Whitney U test was performed comparing the different groups and p-values refer to all lymphoma patients vs healthy blood donors
T-cells CD3+ | 41.8/45.1 (3.9–65) 15.6 | 40.2/37.9 (14.8–61.4) 11.8 | 50.7/53.4 (36–64.1) 9.5 | 0.03 |
T-regulatory cells CD3+/CD4+/CD25+/CD127low | 4.4/3.8 (0.4–10.9) 2.4 | 5.3/4 (1.2–30.5) 5.9 | 4.1/3.4 (0.4–8) 2.5 | 0.6 |
CD4+/CD8+ ratio | 2/1.6 (0.18–4.9) 1.4 | 2.9/1.9 (0.5–16.2) 3.4 | 2/1.6 (0.53–7.4) | 0.7 |
Monocytes CD33++/CD14+/HLA-DR+ | 20.9/20.6 (4–47) 11.7 | 25.2/22.8 (9.3–66) 11.8 | 18.7/18.5 (3.5–33) 8.8 | 0.2 |
NK cells CD7+/CD3−CD56+/CD16+ | 6.5/4.4 (0.29–27.6) 6.1 | 6.4/5.2 (0.2–24.8) 5.5 | 9/8.9 (0.04–19.4) 4.9 | 0.1 |
NK regulatory cells CD7+/CD3−/CD56++/CD16dim/− | 0.28/0.21 (0.007–0.89) 0.26 | 0.31/0.25 (0.003–0.92) 0.25 | 0.54/0.44 (0.02–1.6) 0.36 | 0.003 |
Monocytic MDSC CD11b+/HLA-DR−/CD33+/CD14+ | 1.1/0.53 (0.043–3.7) 1.1 | 0.61/0.41 (0.024–2.2) 0.57 | 0.49/0.31 (0.015–2.7) 0.66 | 0.07 |
Granulocytic MDSC CD11b+/HLA-DRdim/CD33dim/CD14- | 12.7/8.1 (0.6–83.8) 18.3 | 5.7/4.2 (0.4–19.3) 4.96 | 3.8/1.1 (0.18–17.4) 5.2 | 0.003 |
Flow cytometry
Three different 10-color tubes were used to identify the different cell subtypes. All samples were analyzed on the Navios instrument (Beckman-Coulter). Kaluza Analysis Software 1.2 (Beckman Coulter) and Infinicyte 1.7 flow cytometry software were used for data analysis. Approximately 0.5 × 106 cells/tube were labeled with three different mixes of fluorescent labeled antibodies. All surface antigens were labeled for 10 min. in the dark at 20 °C. The cells in tubes without intracellular staining were washed once and then run immediately in the Navios instrument. For intracellular staining (e.g. FOXP3) the T-reg Detection Kit (Miltenyi Biotec) was used with fixation and permeabilisation according to manufacturer instructions. The following antibodies were used: PD-1- FITC (CD279, clone MIH4, Becton Dickinson), CD25-PE (clone 4E3, Miltenyi Biotec), CD45RA-ECD (clone 2H4LDH11LDB9 (2H4), Beckman Coulter), CD8-PC5.5 (clone SFCI21THy2D3, Beckman Coulter), CCR7-PC7(CD197 clone G043H7, Beckman Coulter), FOXP3-APC (clone 3G3,Miltenyi Biotec), CD127-APC-700 (clone R34.34, Beckman Coulter), CD3-APC-750 (clone UCHT1, Beckman Coulter), CD4-BV421 (clone RPA-T4, Becton Dickinson), CD45-KrO (clone J33, Beckman Coulter), CD161-FITC (clone 191B8, Miltenyi Biotec), Vα24Jα18-PE (clone 6B11, BioLegend), CD56-ECD (clone NKH-1, Beckman Coulter), CD7-PC7 (clone 8H8, Beckman Coulter), CCR7-APC (clone G043H7, BioLegend), CD16-APC-700 (clone 3G8, Beckman Coulter), CD13-FITC (clone SJ1D1, Beckman Coulter), CD115-PE (clone 9-4D2-1E4, BioLegend), CD14-ECD (clone RM052, Beckman Coulter), CD33-PC5.5 (clone D3HL60.251, Beckman Coulter), HLA-DR-PC7 (clone Immu357, Beckman Coulter), CD163-APC (clone GHI/61, BioLegend), CD11b-APC-750 (clone Bear1, Beckman Coulter), CD15-PB (clone 80H5, Beckman Coulter).
In this paper, we did, however, not include the following markers FOXP3, CCR7, CD45RA, PD1, CD115, CD163, CD161, Vα24Jα18.