title: CD160 isoforms and regulation of CD4 and CD8 T-cell responses.
abstract: BACKGROUND
  Coexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation.
  
  
  METHODS
  Expression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1.
  
  
  RESULTS
  We first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation.
  
  
  CONCLUSIONS
  Antibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.
authors.affiliation: Boehringer Ingelheim Ltd,, 2100 Rue Cunard, Laval, Quebec, Canada. mohamed.el-far@hotmail.com.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El-Far
authors.forename: Charles
authors.initials: C
authors.lastname: Pellerin
authors.forename: Louise
authors.initials: L
authors.lastname: Pilote
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Fortin
authors.forename: Ivan A D
authors.initials: IA
authors.lastname: Lessard
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peretz
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Wardrop
authors.forename: Patrick
authors.initials: P
authors.lastname: Salois
authors.forename: Richard C
authors.initials: RC
authors.lastname: Bethell
authors.forename: Michael G
authors.initials: MG
authors.lastname: Cordingley
authors.forename: George
authors.initials: G
authors.lastname: Kukolj
mesh_terms: D000328:Adult; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D058851:GPI-Linked Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015658:HIV Infections / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics*; D008297:Male; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D053240:Receptors, Tumor Necrosis Factor, Member 14 / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C114505:CD160 protein, human; D058851:GPI-Linked Proteins; D020033:Protein Isoforms; D011971:Receptors, Immunologic; D053240:Receptors, Tumor Necrosis Factor, Member 14; C506028:TNFRSF14 protein, human