Standardization, Evaluation, and Area-Under-Curve Analysis of Human and Murine Treg Suppressive Function.

FOXP3+ T-regulatory (Treg) cells have important roles in immune homeostasis, and alterations in their number and function can predispose to diseases ranging from autoimmunity to allograft rejection and tumor growth. Reliable identification of human Tregs remains a persistent problem due to a lack of specific markers. The most definitive Treg characterization currently involves combined assessment of phenotypic, epigenetic and functional parameters, with the latter typically involving in vitro Treg suppression assays. Unfortunately, suppression assays are frequently performed using differing methods and readouts, limiting comparisons between studies. We provide a perspective on our experience with human and murine Treg suppression assay conditions, including Treg data obtained in clinical transplant studies, Tregs isolated from healthy donors and treated with epigenetically active compounds, and Tregs from standard murine strains (C57BL/6 and BALB/c). We provide detailed descriptions and illustrations of typical problems, shortcomings and troubleshooting; describe new modifications and approaches; and present a new method for calculation of suppressive assay data using a modified area-under-curve (AUC) method. This method allows us to directly compare Treg suppressive function between multiple patients (such as in clinical transplant studies), to reliably track changes in Treg function from the same person over time, or compare effects of Treg-modulating compounds tested with different healthy donors Tregs in separate or combined experimental settings.

Three distinct domains contribute to nuclear transport of murine Foxp3.

Foxp3, a 47-kDa transcription factor, is necessary for the function of CD4+CD25+ regulatory T cells (Tregs), with an essential role in the control of self-reactive T cells and in preventing autoimmunity. Activation of Tregs by TCR engagement results in upregulation of Foxp3 expression, followed by its rapid nuclear transport and binding to chromatin. Here, we identify three distinct Foxp3 domains that contribute to nuclear transport. The first domain (Domain 1) comprises the C-terminal 12 amino acids. The second domain (Domain 2) is located immediately N-terminal to the forkhead domain (FHD), recently reported to be a binding site for the runt-related transcription factor 1/acute myeloid leukemia 1 (Runx1/AML1). The third domain (Domain 3) is located within the N-terminal first 51 amino acids. Unlike the known nuclear localization signals (NLSs), none of these three regions are rich in basic residues and do not bear any similarity to known monopartite or bipartite NLSs that have one or more clusters of basic amino acids. The basic arginine-lysine-lysine-arginine (RKKR) sequence, located 12-aa from the C-terminal end of Foxp3 was previously reported to be a nuclear localization signal (NLS) for several proteins, including for a GFP-Foxp3 hybrid. Evidence is provided here that in the full-length native Foxp3 RKKR does not function as an NLS. The data reported in this study indicates that Foxp3 achieves nuclear transport by binding to other nuclear factors and co-transporting with them to the nucleus.

Deacetylase inhibition increases regulatory T cell function and decreases incidence and severity of collagen-induced arthritis.

Collagen-induced arthritis (CIA) is an established mouse model of disease with hallmarks of clinical rheumatoid arthritis. Histone/protein deacetylase inhibitors (HDACi) are known to inhibit the pathogenesis of CIA and other models of autoimmune disease, although the mechanisms responsible are unclear. Regulatory T cell (Treg) function is defective in rheumatoid arthritis. FOXP3 proteins in Tregs are present in a dynamic protein complex containing histone acetyltransferase and HDAC enzymes, and FOXP3 itself is acetylated on lysine residues. We therefore investigated the effects of HDACi therapy on regulatory T cell function in the CIA model. Administration of an HDACi, valproic acid (VPA), significantly decreased disease incidence (p<0.005) and severity (p<0.03) in CIA. In addition, VPA treatment increased both the suppressive function of CD4(+)CD25(+) Tregs (p<0.04) and the numbers of CD25(+)FOXP3(+) Tregs in vivo. Hence, clinically approved HDACi such as VPA may limit autoimmune disease in vivo through effects on the production and function of FOXP3(+) Treg cells.

Mechanisms underlying blockade of allograft acceptance by TLR ligands.

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4(+)Foxp3(+) T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4(+)Foxp3(+) Tregs.

Allograft rejection requires STAT5a/b-regulated antiapoptotic activity in T cells but not B cells.

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.