Published Ahead of Print on December 7, 2016, as doi:10.3324/haematol.2016.151100. Copyright 2016 Ferrata Storti Foundation.
T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers by Marzia Palma, Giusy Gentilcore, Kia Heimersson, Fariba Mozaffari, Barbro Näsman-Glaser, Emma Young, Richard Rosenquist, Lotta Hansson, Anders Österborg, and Håkan Mellstedt Haematologica 2016 [Epub ahead of print] Citation: Palma M, Gentilcore G, Heimersson K, Mozaffari F, Näsman-Glaser B, Young E, Rosenquist R, HanssonL, Österborg A, and Mellstedt H. T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers. Haematologica. 2016; 101:xxx doi:10.3324/haematol.2016.151100 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process.
T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers Marzia Palma1,2, Giusy Gentilcore1, Kia Heimersson1, Fariba Mozaffari1, Barbro NäsmanGlaser1, Emma Young3, Richard Rosenquist3, Lotta Hansson1,2, Anders Österborg1,2 and Håkan Mellstedt1 1
Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centre Karolinska, Karolinska Institutet, Stockholm, Sweden 2
Department of Hematology, Karolinska University Hospital, Stockholm, Sweden
3
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
AÖ and HM contributed equally to this work
Short title for the running head: Immune checkpoints and activation markers in CLL
Correspondence: Marzia Palma, Department of Hematology, Karolinska University Hospital Solna, 171 76 Stockholm, Sweden. E-mail:
[email protected]
word count for abstract: 249 word count for text: 4024 figure/table count: 6 figures/2 tables supplemental files: 1
Acknowledgments The authors thank the patients and donors who consented to the use of their cell samples for this study. The authors thank Ms Leila Relander for excellent secretarial help. This work was supported by grants from The Swedish Cancer Society, the Swedish Research Council, The Cancer Society in Stockholm, King Gustav V Jubilee Fund, The Cancer and Allergy Foundation, The Karolinska Institutet Foundations, The Stockholm County Council and AFA Försäkring.
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Abstract Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T cell defects. T cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared to healthy controls. In general, patients had higher absolute numbers of CD3+ cells and the CD8+ subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4+ and CD8+ cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (p=0.0003 and p=0.001, respectively). A significant increase in antigen-experienced T cells was observed in CLL patients both within the CD4+ and CD8+ subsets, with a significantly higher PD-1 expression. Higher numbers of CD4+ and CD8+ cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67+) and activated (CD69+) CD4+ and CD8+ cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (p60 years (80%) [15]. The research project was approved by the regional Ethics committee (www.epn.se) and conducted in accordance with the Helsinki Declaration. Informed consent was obtained from study participants. Flow cytometric analysis of lymphocyte subsets on whole blood Cells were washed after lysis of red blood cell, resuspended in Cell Staining Buffer (CSB) (BioLegend, San Diego, CA) and stained with CD19-AF488, CD16+56-PE, CD4-PerCp, CD3-PE-Cy7, CD8-APC and CD45-AF700 (Bio-Legend). After incubation and washing, cells were resuspended in CSB and analyzed by FACSCanto II flow cytometer and the FACSDiva version 6.1.3 (BD Biosciences, San Diego, CA) or FlowJo version 8.8.2 (TreeStar, Ashland, OR) softwares. Isolation of peripheral blood mononuclear cells and cell culture conditions Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density gradient centrifugation on a Ficoll-Hypaque gradient (GE Healthcare, Uppsala, Sweden) and washed twice with Dulbecco´s Phosphate-Buffered Saline 0.9% (DPBS) (Gibco, Life Technologies, Carlsbad, CA). Cells were freshly used or stored in liquid nitrogen until usage. After thawing, PBMC were analyzed immediately unless used for stimulation experiments. 3x106 PBMCs were cultured for 72 hours in humidified air with 5% CO2 at 37°C in RPMI 1640 medium (GIBCO, Life Technologies, Carlsbad, CA) supplemented with heat-inactivated autologous serum for fresh samples and pooled normal human AB+ serum for frozen samples in the presence of phytohemagglutinin (10 µg/mL) (PHA-M, Sigma Aldrig, St. Louis, MO). PBMC cultured in medium alone were used as controls.
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Flow cytometric analysis of PBMC PBMC were washed with CSB (BioLegend). The following antibodies were used: CD19AF488 and -PE-Cy7, CD16/CD56-PE, CD4-PerCp, -FITC and -AF700, CD3-PE-Cy7, AF700 and -PerCP, CD8-APC and -AF700, CD45-AF700, CD5-PE and -PerCP, CD45ROFITC, HLA-DR-PerCp, CD25-APC, CD45RA-AF488, PD-1 (CD279)-PE, CD69-AF488, CTLA-4 (CD152)-PE, Ki-67-AF647 (Bio-Legend), CCR6 (CD196)-PE, CCR4 (CD194)-PE, CD127-PE-Cy7, CXCR3 (CD183)-APC, CCR7 (CD197)-AF647, PD-L1 (CD274)-PE (BDBiosciences) and the appropriate isotype controls. Further details are provided in the Online Supplementary Appendix. Sequence analysis of IGHV–IGHD–IGHJ rearrangements IGHV-IGHD-IGHJ rearrangements were determined through PCR amplification, Sanger sequencing and subsequent sequence interpretation following established international guidelines and using the IMGT® databases and the IMGT/V-QUEST tool (http://www.imgt.org), as previously reported [16]. IGHV gene mutational status was defined as either mutated or unmutated based on the clinically relevant 98% cut-off value for identity to the closest germline gene [17, 18]. Subset #2 cases are listed together with unmutated being poor-prognostic [19]. Statistical Analyses Statistical analyses were performed using the GraphPad Prism software 6.0 (GraphPad Software, La Jolla, CA). All tests were two-sided, and p
