Our studies have addressed several major questions: 1) It has been difficult for researchers to establish a clear connection between human autoimmune disease and abnormalities of Treg numbers and function. Multiple lines of evidence would suggest that Tregs might be reduced in number or function in Systemic Lupus Erythematosis (SLE) due to a relative reduction in T cell IL-2 production and signaling and a high level of interferon-induced gene expression. However, the lack of a single marker or combination of markers to reliably identify human Tregs has made it difficult to correlate Tregs with disease activity in SLE and other autoimmune diseases. In humans, both CD25 (IL2 Receptor alpha-chain) and FoxP3 expression are not limited to Tregs, but may also be expressed in activated conventional T cells, not necessarily conferring a stable regulatory phenotype. Furthermore, CD25 low/negative cells may also contain functional Foxp3+ Tregs. Helios, a member of the Ikaros gene family of transcription factors, has recently been shown by us to be selectively expressed by 70-80% of human FoxP3+ T cells. Studies in the mouse have suggested that Foxp3+Helios+ Treg are thymus-derived, while Foxp3+Helios- T cells are likely induced in peripheral sites. Furthermore, the composition of the Foxp3+Helios- population is complex, as it contains a large number of cytokine producing cells that my represent activated T conventional cells. Our studies of the methylation status of the TSDR of the Foxp3 locus have also suggested that up to 50% of FoxP3+Helios- cells may be activated conventional T cells. We have used expression of Helios combined with FoxP3 to more accurately quantitate peripheral human Tregs in patients with SLE with various disease activities. We have also tested whether FoxP3+Helios+ Tregs in SLE are preferentially low-cytokine producers as compared to FoxP3+Helios- Tregs. The present study shows that the combination of Helios with FoxP3 is a superior method for identifying Treg numbers in SLE patients. By collecting data on absolute CD4 numbers we have been able to calculate the absolute number Foxp3+Helios+ Tregs. We compared the absolute numbers of Foxp3+Helios+ Tregs in healthy donors to SLE patients and included a subset of SLE patients with inactive disease. Whereas patients with inactive disease showed similar percentages of Foxp3+Helios+ Tregs as healthy donors, patients with more active disease had higher percentages of Foxp3+Helios+ Treg when compared to either inactive patients or when compared to healthy donors. Due to a decline in absolute CD4 count in more active SLE patients, the absolute numbers of Foxp3+Helios+ Treg numbers are not significantly changed between SLE patients and healthy donors and are maintained in SLE patients of varying clinical activity. It is still unclear by what mechanism FoxP3+Helios+ Tregs are maintained or even increased in SLE patients with more active disease. It remains possible that FoxP3+Helios+ Tregs are maintained via stimulation by cytokines other than IL-2 or are stimulated by the heightened level of general T cell activation that has been documented in SLE. It also remains unsettled as to whether Tregs are an essential part of the pathophysiology of SLE or just innocent bystanders. In order to justify focusing therapeutic approaches on Tregs in SLE and other autoimmune conditions, it will be necessary to have the best methods for accurately identifying Tregs with the highest suppressive potential. We assert that Helios in combination with FoxP3 is currently the best way to identify the majority of bona fide human Tregs for this purpose. 2) We have used RNA-Seq to analyze human Treg cell gene signatures. Microarray studies comparing hTregs or mTregs with Tconv (with or without prior activation) have provided insights to genes that are differentially expressed in Tregs, the so-called Treg signature. In an independent attempt (in collaboration with the Muljo lab) to find the unique molecules associated with hTregs, we are exploring sequencing of RNA-based libraries (RNA-seq) from human Treg in an attempt to provide a unique digital gene signature. This comprehensive approach guaranties an unbiased transcriptome analysis compared to existing microarray based queries for Tregs. The high sensitivity and accuracy of RNA-seq in detecting genes with very low or very high levels outperforms microarrays across many orders of expression magnitude. Using this approach we compared transcriptome signatures of human Tregs to conventional T cells at various phases of activation. We analyzed the RNA-seq reads using DEGseq, an R package that integrates three different methods (LRT, FET &MARS) to identify differentially expressed genes as well as by GO analysis to define cell membrane associated antigens. The differential expression of a panel of these gene has been validated further in multiple donors under different activation conditions at different time points using nanostring technology. Our results verified all known Treg signature genes including, but not limited to, FoxP3, IKZF2, IFNand CTLA4. A number of other thus far unknown Treg genes were identified and were validated as Treg-specific in multiple healthy donors and cord blood samples under different activation conditions. We are presently performing studies to evaluate the role of several of the candidate genes in the function of human Treg. We are knocking down gene expression using the siRNA technique and then analyzing the effects of decreased gene expression on the function of the Treg in different assays of Treg suppressor function. As a corollary to these studies we are overexpressing by retroviral mediated gene transducition several of the candidate genes in activated T conventional cells and then assaying the transfected for suppressor activity in the suppressor assays. Hoperfully, these approaches will allow us to determine if these genes which have determined are uniquely expressed in human Treg play critical roles in their function and that the products of these genes can ultimately be used to manipulate Treg function. 3) We have developed a CRADA with a group at Boehringer-Ingelheim (BI) Pharmaceuticals for studies to generate novel mAbs that will modulate the function of human Treg cells. We have prepared large numbers of expanded highly purified human Tregs which are used to immunize mice to generate the mAbs. A number of novel screening techniques are being developed by BI to identify mAbs which selectively recognize human Tregs and not activated T conventional cells or non-lymphoid cells. These novel mAbs are in turn being tested by us in a panel of in vitro T suppressor cells that we have developed. We are presently in the process of screening this panel of mAbs. Our goal is to identify mAbs that identify novel antigens that are selectivly expressed on Tregs and more importantly mAbs that can decrease or increase Treg suppressor function in vitro.
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