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Expanded Antigen-Experienced CD160+CD8+Effector T Cells Exhibit Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-002189 on 30 April 2021. Downloaded from Expanded antigen- experienced CD160+CD8+effector T cells exhibit impaired effector functions in chronic lymphocytic leukemia Najmeh Bozorgmehr,1 Isobel Okoye,1 Olaide Oyegbami,1 Lai Xu,1 Amelie Fontaine,2 Nanette Cox- Kennett,2 Loree M Larratt,3 Mark Hnatiuk,3 Andrei Fagarasanu,2 3 2 1,2,4 Joseph Brandwein, Anthea C Peters, Shokrollah Elahi To cite: Bozorgmehr N, ABSTRACT BACKGROUND Okoye I, Oyegbami O, et al. Background T cell exhaustion compromises antitumor Chronic lymphocytic leukemia (CLL), the Expanded antigen- experienced immunity, and a sustained elevation of co- inhibitory + + most common leukemia among adults in CD160 CD8 effector T cells receptors is a hallmark of T cell exhaustion in solid tumors. exhibit impaired effector Western countries, is identified by the clonal Similarly, upregulation of co- inhibitory receptors has + functions in chronic lymphocytic expansion of CD5 mature B lymphocytes in leukemia. Journal for been reported in T cells in hematological cancers such as chronic lymphocytic leukemia (CLL). However, the the blood, bone marrow (BM), and secondary ImmunoTherapy of Cancer lymphoid organs.1 2 The disease fate is highly 2021;9:e002189. doi:10.1136/ role of CD160, a glycosylphosphatidylinositol-anchored jitc-2020-002189 protein, as one of these co-inhibitor y receptors has been influenced by the advanced clinical stage (ie, contradictory in T cell function. Therefore, we decided to Rai stage), the broad range of genetic factors ► Additional supplemental elucidate how CD160 expression and/or co-expression and remarkable contributions from the tumor material is published online only. with other co- inhibitory receptors influence T cell effector microenvironment.3 Malignant B cells (B- CLL) To view, please visit the journal functions in patients with CLL. impair both innate and adaptive immune online (http:// dx. doi. org/ 10. Methods We studied 56 patients with CLL and 25 age- responses and thus increase susceptibility to 1136/ jitc- 2020- 002189). matched and sex-ma tched healthy controls in this study. infections.4 Several quantitative,5 phenotypic,6 The expression of different co-inhibitor y receptors was and functional7 T cell alterations are reported Accepted 26 March 2021 analyzed in T cells obtained from the peripheral blood or + the bone marrow. Also, we quantified the properties of in patients with CLL. Although CD8 T cells play an important role against the tumor, the extracellular vesicles (EVs) in the plasma of patients with http://jitc.bmj.com/ CLL versus healthy controls. Finally, we measured 29 occurrence of T cell exhaustion compromises + different cytokines, chemokines or other biomarkers in their effector functions. Exhausted CD8 T the plasma specimens of patients with CLL and healthy cells are characterized by the loss of effector © Author(s) (or their employer(s)) 2021. Re- use controls. functions, altered epigenetic and transcrip- permitted under CC BY- NC. No Results We found that CD160 was the most upregulated tional profiles, distinct metabolic style, and commercial re- use. See rights co-inhibitor y receptor in patients with CLL. Its expression 8 the inability to transition to memory T cells. on September 29, 2021 by guest. Protected copyright. and permissions. Published by was associated with an exhausted T cell phenotype. Antigen persistence, soluble mediators, cyto- BMJ. CD160+CD8+ T cells were highly antigen- experienced/ 1 + + kines, and immunoregulatory cells modulate School of Dentistry, Faculty effector T cells, while CD160 CD4 T cells were more 9 of Medicine and Dentistry, heterogeneous. In particular, we identified EVs as a source the severity and the pace of T cell exhaustion. University of Alberta, Edmonton, of CD160 in the plasma of patients with CLL that can be The sustained upregulation of co-inhib - Alberta, Canada itory receptors (co-IRs) is the hallmark of 2 taken up by T cells. Moreover, we observed a dominantly Department of Oncology, proinflammatory cytokine profile in the plasma of patients CD4+ and CD8+ T cell exhaustion in chronic Faculty of Medicine and with CLL. In particular, interleukin-16 (IL-16) was highly 10 Dentistry, University of Alberta, viral infections and cancer. Likewise, it Edmonton, Alberta, Canada elevated and correlated with the advanced clinical stage has been shown that T cells in CLL upreg- 3Division of Hematology, Faculty (Rai). Furthermore, we observed that the incubation of T ulate the expression of programmed cell of Medicine and Dentistry, cells with IL-16 results in the upregulation of CD160. death protein-1 (PD-1), CD160, and 2B4 University of Alberta, Edmonton, Conclusions Our study provides a novel insight into (CD244),11 which results in their impairment Alberta, Canada the influence of CD160 expression/co-expression with 4Medical Microbiology and (eg, proliferation, cytotoxicity, and cytolytic other co- inhibitory receptors in T cell effector functions in 12 13 Immunology, Faculty of Medicine patients with CLL. Besides, IL-16-media ted upregulation functions). Comparable alterations in and Dentistry, Edmonton, of CD160 expression in T cells highlights the importance the gene and protein expression in T cells of Alberta, Canada of IL-16/CD160 as potential immunotherapy targets in TCL1 transgenic CLL mouse model in which Correspondence to patients with CLL. Therefore, our findings propose a adopted T cells acquire the characteristic of T Professor Shokrollah Elahi; significant role for CD160 in T cell exhaustion in patients cell dysfunction on tumor antigen encounter elahi@ ualberta. ca with CLL. have been reported.14 Bozorgmehr N, et al. J Immunother Cancer 2021;9:e002189. doi:10.1136/jitc-2020-002189 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-002189 on 30 April 2021. Downloaded from CD160 as one of these IRs belongs to glycosylphospha- T cells, while CD160+ CD4+ T cells were more heteroge- tidylinositol (GPI)- anchored glycoprotein of the immu- neous. The plasma cytokine profile of patients with CLL noglobulin superfamily.15 It was first identified on the exhibited a proinflammatory phenotype. In particular, membrane of mice natural killer (NK) cells,16 mast cells,17 IL-16 was highly elevated in the plasma of patients with and B- CLL cells but not on normal B cells.18 CD160 can CLL and was correlated with the Rai stage. Finally, we exist in three different forms such as the GPI-anchored, show that isolated EVs from the plasma of patients with transmembrane (TM), and soluble forms. T cells mainly CLL can be a source of CD160. express the GPI but occasionally TM isoforms.19 The soluble isoform composed of the extracellular regions of CD160 that is susceptible to be cleaved from the cell METHODS membrane by a metalloprotease as CD160 shedding has Study population 20 17 been reported from NK and mast cells on activation The peripheral blood and bone marrow (BM) samples and degranulation, respectively. were collected from patients with B- CLL. We recruited Human/mouse CD160 binds weakly to classical 56 patients with CLL for the study (online supplemental and non- classical major histocompatibility complex- I table 1). The diagnosis was based on clinical, morphology, 21 22 (MHC- I) molecules, triggering NK cell cytotoxicity and immuno- phenotyping features. Peripheral blood and the release of proinflammatory cytokines. The inter- from age- matched and sex- matched 25 healthy donors action of CD160 with classical and non-classical MHC- I were obtained for comparison. The staging was done complexes inhibits binding of CD8 to α3 subunits of based on the clinical data using the Rai staging system MHC class I, causing defect in MHC class I- dependent reported elsewhere.32 CD8+ T cell cytotoxicity.20 CD160 and B- T-lymphocyte attenuator (BTLA) interact with the cysteine- rich region Cell isolation and purification of the extracellular domain of herpesvirus entry medi- The peripheral blood mononuclear cells (PBMCs) and ator (HVEM).23 24 The engagement of CD160 with the BM mononuclear cells were isolated using Ficoll-Paque soluble HVEM has a costimulatory effect on human NK gradients (GE Healthcare). CD8+ or CD3+ T cells were cell function.25 Paradoxically, CD160 interaction with isolated by negative selection using the Easysep isola- HVEM receptor delivers an inhibitory signal to activated tion kits (Stem Cell Technologies) with a purity of >90% CD4+ T cells, resulting in diminished cytokine production (online supplemental figure 1a,b). For effector T cell and proliferation.26 However, the role of CD160 in CD8+ (CD8+CCR7-) isolation, CD8+ T cells were stained with T cells is more complex. For example, CD160 exhibits phycoerythrin (PE)- conjugated anti- CCR7 antibody, an inhibitory function in viral infections such as HIV27 followed by anti- PE- conjugated microbeads (Miltenyi) but a stimulatory property in Listeria monocytogenes infec- with a purity of >90% (online supplemental figure 1c). tion28 and the allograft skin reaction.29 The various role B- CLLs were isolated using the human B- CLL Cell Isola- of the CD160 molecule could be related to the expres- tion Kit (Miltenyi) with a purity of >90% (online supple- http://jitc.bmj.com/ sion of its corresponding ligands HVEM or MHC-I. For mental figure 1d). example, the interaction of CD160 with MHC-I induces a stimulatory signal,28 whereas the interaction with HVEM Flow cytometry triggers an inhibitory signal.26 These discrepancies in the The fluorochrome- conjugated antibodies were purchased field warrant further investigations to better understand from BD Biosciences, Thermo Fisher Scientific or the role of CD160 in T cell function in chronic conditions Biolegend including human anti-CD3 (SK7), anti- CD4 on September 29, 2021 by guest. Protected copyright. such as cancer. Moreover, it is unclear how the soluble (RPA- T4), anti- CD8 (RPA- T8), anti- CD5 (UCHT2), anti- CD160 is transported out of immune cells and there is a CD19 (HIB19), anti- CD160 (BY55), anti- HVEM (94801), possibility that extracellular vesicles (EVs) are involved in anti- 2B4 (eBioDM244), anti- TIGIT (MBSA43), anti- PD1 this process.
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