Slides were washed, incubated for 1 hr at RT with anti-HSV-1 gC-FITC (Genway, #20-902-170310) antibody. infected DCs were incubated with T cells isolated from naive C57BL/6-CD28-/- mice at a 11 ratio. As a control some T cells were incubated without DCs (not shown). FACS analyses were carried out using anti-CD3 and anti-PD-1 antibodies. Graphs show the PD-1 staining intensity of CD3+ gated T cells. Number indicates the percent of PD-1+CD3+ expressing T cells per treatment. The left peak represent the PD-1 unfavorable T cells, while the right peak (R9) represent PD-1 positive T cells. Experiments were repeated twice.(PDF) pone.0087617.s002.pdf (49K) GUID:?DB9FD63F-9A7B-4A35-938B-A705A5729CEE Physique S3: Detection of HSV-1 receptors on the surface of DCs. Subconfluent monolayers of DCs isolated from WT C57BL/6 mice were produced on Lab-Tek chamber slides and probed with anti-CD11c/anti-HVEM or anti-CD11c/anti-nectin-1 antibodies. Panels: A and B) Representative Photomicrographs of stained DCs. DAPI is usually shown as a nuclear Purpureaside C counter-stain; and C) Quantification of photomicrographs. Different areas of 3 slides were imaged and the numbers of CD11c+, CD11c+HVEM+, and CD11c+nectin-1+ cells were counted. Each point represents the imply SEM from 24 images.(PDF) pone.0087617.s003.pdf (355K) GUID:?B84CD351-34BA-4E9D-9C2F-FDCD4106DFCD Abstract CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 contamination, we constructed a recombinant HSV-1 computer virus that expresses two copies of the gene in place of the latency associated transcript (LAT). This mutant computer virus (HSV-CD80) expressed high levels of CD80 and experienced similar computer virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental computer virus, this CD80 expressing recombinant computer virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 contamination was mediated by CD80 binding to PD-L1 on DCs. This conversation also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC. Introduction Dendritic cells (DCs) are bone marrow-derived cells that are involved in antigen capture, processing, and presentation and are the most powerful of the antigen presenting cells (APCs), playing a key role in triggering the immune system against infectious brokers [1]C[6]. DCs perform crucial functions in linking innate and adaptive immunity and thus play a key role in triggering the immune system against HSV-1 contamination [7]C[9]. Recently, we showed that although DCs can be infected by HSV-1, DCs do not support HSV-1 replication and are impervious to cell lysis [10]. However, the mechanism of DCs resistance to HSV-1 replication is not known. In addition, we have reported that in contrast to bone marrow (BM)-derived DCs from wild type mice, DCs isolated from transmission transducers and activators of transcription-1 deficient (STAT1-/-) mice were susceptible to HSV-1 replication [10]. Binding of CD28 on T cells to CD80 (B7-1) or CD86 (B7-2) on an APC prospects to T cell proliferation, differentiation, and cytokine secretion [11]. The CD80 and CD86 molecules are expressed by multiple cell types, including B cells, macrophages, DCs, and T cells [12]C[15]. In addition to CD80 and CD86, the B7 pathways comprise the Programmed Death-1 (PD-1) receptor (CD279) and its two ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) [16], [17]. PD-L1 and PD-L2 expression patterns are different; PD-L1 is usually KITH_EBV antibody constitutively expressed on many cell types such as T cells, B cells, macrophages, DCs, and BM-derived mast cells, while PD-L2 expression is Purpureaside C more restricted [18]. Recently we have shown that CD80 binds to PD-L1 and this conversation inhibited T cell proliferation and cytokine production [19]. It was previously shown that DCs were not productively infected despite the fact that DCs express HSV receptors [20]. However, in our hands, few BM-derived DCs expressed HVEM or nectin-1, the two most prominent HSV-1 receptors. The studies presented here utilize a recombinant Purpureaside C HSV-1 computer virus constructed such that it expresses the CD80 gene (HSV-CD80) in an attempt to.