Coexpression of TIGIT and FCRL3 identifies Helios+ human memory regulatory T cells.

Two distinct subsets of CD4(+)Foxp3(+) regulatory T (Treg) cells have been described based on the differential expression of Helios, a transcription factor of the Ikaros family. Efforts to understand the origin and biological roles of these Treg populations in regulating immune responses have, however, been hindered by the lack of reliable surface markers to distinguish and isolate them for subsequent functional studies. Using a single-cell cloning strategy coupled with microarray analysis of different Treg functional subsets in humans, we identify the mRNA and protein expression of TIGIT and FCRL3 as a novel surface marker combination that distinguishes Helios(+)FOXP3(+) from Helios(-)FOXP3(+) memory cells. Unlike conventional markers that are modulated on conventional T cells upon activation, we show that the TIGIT/FCRL3 combination allows reliable identification of Helios(+) Treg cells even in highly activated conditions in vitro as well as in PBMCs of autoimmune patients. We also demonstrate that the Helios(-)FOXP3(+) Treg subpopulation harbors a larger proportion of nonsuppressive clones compared with the Helios(+)FOXP3(+) cell subset, which is highly enriched for suppressive clones. Moreover, we find that Helios(-) cells are exclusively responsible for the productions of the inflammatory cytokines IFN-γ, IL-2, and IL-17 in FOXP3(+) cells ex vivo, highlighting important functional differences between Helios(+) and Helios(-) Treg cells. Thus, we identify novel surface markers for the consistent identification and isolation of Helios(+) and Helios(-) memory Treg cells in health and disease, and we further reveal functional differences between these two populations. These new markers should facilitate further elucidation of the functional roles of Helios-based Treg heterogeneity.

Retinal Complications in Patients with Systemic Lupus Erythematosus Treated with Antimalarial Drugs.

OBJECTIVE: Hydroxychloroquine (HCQ) and chloroquine (CQ) are key drugs in systemic lupus (SLE) and related diseases. Retinal toxicity remains the most worrisome complication. We studied factors potentially associated with retinal toxicity, using case-control analyses. METHODS: Within our SLE clinic cohort, we identified patients with retinal changes using the Systemic Lupus International Collaborating Clinics Damage Index. We confirmed HCQ/CQ retinopathy with chart review, and selected up to 3 SLE controls for each case, matched by age at SLE diagnosis and SLE duration. RESULTS: Over an average 12.8 years of followup, within 326 patients exposed to antimalarial drugs, 18 (5.5%) developed retinal toxicity. The minimum number of years of HCQ/CQ exposure before retinopathy developed was 8 years (maximum 33 yrs). Median HCQ/CQ duration was statistically similar in cases [19 yrs, interquartile range (IQR) 14-20] and controls (16 yrs, IQR 11-22), likely due to our matching on SLE duration. Versus controls, cases tended to have more renal disease (cases 22.2%, controls 14.8%) and were slightly less likely to be white (cases 61.1%, controls 74.1%), but neither variable reached statistical significance. Among patients with retinal toxicity, the number previously exposed to CQ was more than 3 times that in controls. CONCLUSION: Just over 5% of patients developed antimalarial retinal complications, over an average of 12.8 years. No cases were detected in the first 5 years of therapy. Past CQ use was more common in cases versus controls. Future studies using larger cohorts are under way to better define the roles of therapy duration, race/ethnicity, and other factors.

Lupus Regulator Peptide P140 Represses B Cell Differentiation by Reducing HLA Class II Molecule Overexpression.

OBJECTIVE: Phosphopeptide P140 (Lupuzor) is an inhibitor of autophagy currently being evaluated in late-stage clinical trials for the treatment of lupus. This study was undertaken to investigate the effect of P140 ex vivo on human T and B cells. METHODS: Human B cells, T cells, and dendritic cells were analyzed by flow cytometry and cellular assays. The expression of autophagy markers was evaluated by immunoblotting and flow cytometry. The levels of B cell receptor (BCR) signaling markers and HLA molecules were assessed by flow cytometry. Toll-like receptor ligands were screened using an assay with transfected HEK 293 cells. P140 cell entry and trafficking were measured by immunofluorescence in the presence of various inhibitors of endosomal pathways. RESULTS: As was previously observed after intravenous injection of the peptide in a mouse model of lupus, P140 entered human B cells by a clathrin coat-dependent endocytosis process and homed into lysosomes. The peptide displayed no direct effect on BCR signaling of memory, naive mature, transitional, and B1 cells. However, it strongly reduced the overexpression of HLA class II molecules on lupus B cells that were acting as antigen-presenting cells, down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG secretion. CONCLUSION: These findings show that P140 down-regulates HLA class II overexpression in human lupus B cells, and also that P140 hampers the differentiation of B cells into autoantibody-secreting plasma cells, likely due to the resulting lack of T cell signaling and activation. This mechanism appears to switch off the downstream events leading to secretion of pathogenic autoantibodies, thus explaining the highly promising results obtained in clinical trials of P140 (Lupuzor) for the treatment of lupus.

CSK regulatory polymorphism is associated with systemic lupus erythematosus and influences B-cell signaling and activation.

The c-Src tyrosine kinase, Csk, physically interacts with the intracellular phosphatase Lyp (encoded by PTPN22) and can modify the activation state of downstream Src kinases, such as Lyn, in lymphocytes. We identified an association of CSK with systemic lupus erythematosus (SLE) and refined its location to the intronic polymorphism rs34933034 (odds ratio (OR) = 1.32; P = 1.04 × 10(-9)). The risk allele at this SNP is associated with increased CSK expression and augments inhibitory phosphorylation of Lyn. In carriers of the risk allele, there is increased B-cell receptor (BCR)-mediated activation of mature B cells, as well as higher concentrations of plasma immunoglobulin M (IgM), relative to individuals with the non-risk haplotype. Moreover, the fraction of transitional B cells is doubled in the cord blood of carriers of the risk allele, due to an expansion of late transitional cells in a stage targeted by selection mechanisms. This suggests that the Lyp-Csk complex increases susceptibility to lupus at multiple maturation and activation points in B cells.

The role of B cells in lupus pathogenesis.

Autoantibodies clearly contribute to tissue inflammation in systemic lupus erythematosus. In order to therapeutically target B cells making pathogenic autoantibodies, it is necessary to identify their phenotype. It is also important to understand the defects in B cell repertoire selection that permit pathogenic autoreactive B cells to enter the immunocompetent B cell repertoire. We present the data that both marginal zone and follicular B cells can produce pathogenic autoantibodies. Moreover, we discuss how B cell survival and maturation are regulated centrally prior to antigen activation and in the periphery after antigen activation to form the repertoire that generates the spectrum of circulating antibodies.