Harnessing cd8⁺cd28⁻ regulatory t cells as a tool to treat autoimmune disease

T regulatory cell therapy presents a novel therapeutic strategy for patients with autoimmune diseases or who are undergoing transplantation. At present, the CD4+ Treg population has been extensively characterized, as a result of defined phenotypic and functional readouts. In this review article, we discuss the development and biology of CD8+ Tregs and their role in murine and human disease indications. A subset of CD8+ Tregs that lack the surface expression of CD28 (CD8+CD28− Treg) has proved efficacious in preclinical models. CD8+CD28− Tregs are present in healthy individuals, but their impaired functionality in disease renders them less effective in mediating immunosuppression. We primarily focus on harnessing CD8+ Treg cell therapy in the clinic to support current treatment for patients with autoimmune or inflammatory conditions

VISTA Deficiency Attenuates Antibody-induced Arthritis and Alters Macrophage Gene Expression in Response to Simulated Immune Complexes

In addition to activated T cells, the immune checkpoint inhibitor “V domain-containing Ig suppressor of T-cell activation” (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated. Methods: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages. Results: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn). Conclusions: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes

VISTA Deficiency Attenuates Antibody-induced Arthritis and Alters Macrophage Gene Expression in Response to Simulated Immune Complexes

In addition to activated T cells, the immune checkpoint inhibitor “V domain-containing Ig suppressor of T-cell activation” (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated. Methods: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages. Results: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn). Conclusions: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes

Concurrent Oral 9 - Rheumatoid Arthritis: Aetiopathogenesis [OP59-OP64]: OP59. The Value of Interleukin-17 Serum Level in Rheumatoid Arthritis Immunopathogenesis

Background: Interleukin (IL)-17 is the main Th-1 cytokine, produced by activated T-lymphocytes. The potential IL-17 value in rheumatoid arthritis (RA) pathogenesis consists of its independent inflammatory response induction and mediated stimulation of proinflammatory factors synthesis resulting in joint destruction. The aim of study was to determine the role of IL-17 in immuno-inflammatory/autoimmune reactions development and to reveal IL-17 serum level associations with clinical and immunological characteristics of RA. Methods: 50 patients with early RA (disease duration >, Russia), anti-CCP antibodies (Axies-Shield Diagnostic, UK) were revealed using ELISA immunoassay. Results: On the base of IL-17 serum level patients were divided in two groups: group1 (n = 28) were patients with normal IL-17 serum level and group2 (n = 22) were those with high IL-17 serum level. In the group2, the rate of patients' pain assessment by visual analogue scale (67.3 ± 7.2 vs 32.8 ± 4.6; P < 0.001), tender (16.7 ± 2.0 vs 8.4 ± 1.1; P < 0.01) and swollen (12.3 ± 2.3 vs 3.9 ± 0.8; P < 0.01) joint count, DAS28 (5.0 ± 0.4 vs 2.8 ± 0.2 P < 0.01) were significantly higher compare to group1. It was found that in group2 the higher T-lymphocyte amount (CD3) was due to CD4 higher quantity, at the same time CD8 amount was significantly lower (22.2 ± 1.5% vs 28.4 ± 1.7%, P < 0.05) compare to group1. This caused the immunoregulative index increasing and indicated in the lost of autoimmune process regulation, including B-lymphocytes (CD19) activation. The CD154 expression was significantly lower in the group2 (3.4 ± 0.4% vs 10.8 ± 2.8%, P < 0.05) compare to group1. The difference in autoimmune reaction indices wasn't significant between groups except antibody-producing B-lymphocytes (13.7 ± 1.5% vs 8.5 ± 1.0%, P < 0.05) and IgM RF serum level (2.9 ± 0.3 U/ml vs 1.6 ± 0.5 U/ml, P < 0.05), which were significantly higher in group1. The IL-17 level had a positive correlative connections with DAS28 (r = 0.7; P < 0.05), circulative immune complex level (r = 0.38; P < 0.05), anti-CCP antibodies (r = 0.4; P < 0.05), IgM RF (r = 0.41; P < 0.05), CD4 (r = 0.38; P < 0.05) and negative correlative connection with CD8 (r = -0.39; P < 0.05). Conclusions: The importance of IL-17 value in immuno-inflammatory and autoimmune reactions development through T-lymphocytes activation in RA pathogenesis was confirmed. Thus the influence on T-depended immuno-inflammatory reaction products synthesis could be a new therapeutic target of RA patients' management. Disclosure statement: All authors have declared no conflicts of interes

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

VISTA Is an Immune Checkpoint Molecule for Human T Cells

VISTA is a potent negative regulator of T cell function that is expressed on hematopoietic cells and leukocytes. VISTA levels are heightened within the tumor microenvironment where its blockade can enhance anti-tumor immune responses in mice. In humans, blockade of the related PD-1 pathway has shown great potential in clinical immunotherapy trials. Here we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells, blunted production of T cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T cell activation, induces Foxp3 expression and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer