Our studies have addressed several major questions: 1) We have demonstrated that human Foxp3+ T cells, activated with plate-bound anti-hCD3, are almost as potent inhibitors of the proliferation of mouse CD4+Foxp3- responders (stimulated with soluble anti-mouse CD3 and mouse DC), as mouse CD4+Foxp3+ Treg. This result strongly suggests that at least in vitro a major component of the suppressive function of Foxp3+ Treg is preserved across the species. Human Treg can also inhibit the upregulation of costimulatory molecules on mouse DCs. One advantage of this model is that it allows us to attempt to block the suppressive capacity of the human T cells with mAbs to human cell surface antigens that will not interfere with the activation of the mouse responder cells. We have generated large panels of mAbs to activated human Treg and are in the process of testing these antibodies for their capacity to reverse the inhibitory effects of the human Treg. The antibodies are being are also being tested for their ability to stain different human T cell populations and are being used to characterize the biochemical properties of their target antigen. 2) Although considerable enthusiasm has been generated for adoptive Treg therapy, several major issues remain to be resolved. First, most clinical applications of Treg therapy will require large numbers of cells and optimal methods for Treg expansion are now being explored. Expansion of highly purified populations of human Tregs also frequently results in loss of Foxp3 expression during the expansion process. Secondly, in contrast to studies in the mouse, Foxp3 expression can be readily induced during in vitro stimulation of conventional human T cells, in response to TGF-beta in the serum. Furthermore, T cells induced in vitro to express Foxp3 frequently lack a Treg phenotype, continue to make effector cytokines and lack in vitro suppressive function. Thus, expression of Foxp3 cannot be considered a completely reliable marker for functional human Tregs. Foxp3+ Tregs can be divided into two subpopulations based on expression of the transcription factor, Helios. Foxp3+Helios+ Tregs (70%) are thymic-derived, while Foxp3+Helios- Tregs (30%) are induced in the periphery. Foxp3+Helios+ Tregs differ from Foxp3+Helios- Tregs in terms of epigenetic changes at the Foxp3 locus, their capacity to produce effector cytokines, and their stability of Foxp3 expression upon days to weeks of expansion in vitro. Addition of a 25mer DNA oligonucleotide (ODN) of random composition for a short period during the expansion of Foxp3+ Tregs in vitro results in prolonged stabilization of the Foxp3+Helios+ subpopulation and yields an optimal population for use in cellular biotherapy. Although our studies on the use of ODN have been entirely performed in vitro, interaction of Tregs with DNA released from dying cells during the course of an inflammatory response may also function by a similar pathway to stabilize Foxp3/Helios expression and potentiate Treg function. Conversely, DNA released from dying cells in the tumor microenvironment could also stabilize Foxp3/Helios expression and result in Treg-mediated inhibition of anti-tumor Teff cells. Lastly, as ODN are already available for clinical use and can be removed from the cultures after a few days, expansion of Tregs in the presence of ODN should be safe and readily acceptable for the preparation of clinical-grade Tregs. 3. We have compared the TCR Vbeta repertoire in Foxp3- vs. Foxp3+ CD4+ T cells and also compared the repertoire of the Helios+Foxp3+ and the Helios-Foxp3+ subsets. PBMCs from normal donors were obtained from the DTM at the NIH. PBMCs from cGHVD patients (on average 3 years post HSCT) were obtained from a cohort followed at the NCI. The IOTest Vbeta Kit from Beckman Coulter was used to assess usage of 24 different Vbetas by FACS in combination with co-staining for CD4, FoxP3, and Helios. Overall TCR Vbeta repertoires for FoxP3- vs. FoxP3+ CD4 T cells were similar both in cGVHD patients and in young and older healthy donors. This result is similar to previous published observations. In a number of cGVHD patients and older individuals, there was a striking difference in TCR repertoire usage between the Helios+Foxp3+ and Helios-Foxp3+ subpopulations consistent with the possibility that the Helios-Foxp3+ cells were peripherally induced in response to an environmental or an infectious exposure. Studies are currently under way to identify additional markers that uniquely identify Helios-FoxP3+ cells to facilitate their isolation, functional analysis, and antigen specificity. We have demonstrated that in patients with more clinically severe systemic lupus erythematosus that there is a trend toward a lower ratio between Helios+Foxp3+ Tregs and Helios-FoxP3+ subsets. In addition, we have found that Helios+Foxp3+ Tregs increase in direct correlation with steroid therapy in lupus patients with low to moderate clinical severity. Moreover, we have observed that an inflammatory environment high in Type I interferon inhibits regulatory T cells. Based on these observations, we hypothesize that using Helios in combination with Foxp3 for identification of Tregs provides a more reliable method to evaluate immune disturbance and response to therapy. We also hypothesize that Type I interferon suppresses Treg expansion and activation, which is likely reversed by corticosteroids.
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