Wayne W. Hancock

Deacetylase inhibition promotes the generation and function of regulatory T cells

Histone/protein deacetylases (HDACs) regulate chromatin remodeling and gene expression as well as the functions of more than 50 transcription factors and nonhistone proteins. We found that administration of an HDAC inhibitor (HDACi) in vivo increased Foxp3 gene expression, as well as the production and suppressive function of regulatory T cells (Treg cells). Although Treg cells express multiple HDACs, HDAC9 proved particularly important in regulating Foxp3-dependent suppression. Optimal Treg function required acetylation of several lysines in the forkhead domain of Foxp3, and Foxp3 acetylation enhanced binding of Foxp3 to the Il2 promoter and suppressed endogenous IL-2 production. HDACi therapy in vivo enhanced Treg-mediated suppression of homeostatic proliferation, decreased inflammatory bowel disease through Treg-dependent effects, and, in conjunction with a short course of low-dose rapamycin, induced permanent, Treg-dependent cardiac and islet allograft survival and donor-specific allograft tolerance. Our data show that use of HDACi allows the beneficial pharmacologic enhancement of both the numbers and suppressive function of Foxp3+ Treg cells.

Homeostatic proliferation is a barrier to transplantation tolerance

Despite the ease of inhibiting immune responses by blockade of T-cell costimulation in naive rodent models, it is difficult to suppress those responses in animals with memory cells. Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that extensive T-cell proliferation can occur under conditions of lymphopenia. This process, termed homeostatic proliferation, may induce acquisition of functional memory T cells. Here, using clinically relevant mouse models of peripheral T-cell depletion, we show that residual nondepleted T cells undergo substantial homeostatic expansion. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo. These findings establish the importance of homeostatic proliferation in clinically relevant settings, demonstrate the barrier that homeostatic proliferation can present to the induction of transplantation tolerance, and have important implications for transplantation protocols that use partial or complete peripheral T-cell depletion.

Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor

Although certain chemokines and their receptors guide homeostatic recirculation of T cells and others promote recruitment of activated T cells to inflammatory sites, little is known of the mechanisms underlying a third function, migration of Foxp3+ regulatory T (T reg) cells to sites where they maintain unresponsiveness. We studied how T reg cells are recruited to cardiac allografts in recipients tolerized with CD154 monoclonal antibody (mAb) plus donor-specific transfusion (DST). Real-time polymerase chain reaction showed that intragraft Foxp3 levels in tolerized recipients were ∼100-fold higher than rejecting allografts or allografts associated with other therapies inducing prolonged survival but not tolerance. Foxp3+ cells were essential for tolerance because pretransplant thymectomy or peritransplant depletion of CD25+ cells prevented long-term survival, as did CD25 mAb therapy in well-functioning allografts after CD154/DST therapy. Analysis of multiple chemokine pathways showed that tolerance was accompanied by intragraft up-regulation of CCR4 and one of its ligands, macrophage-derived chemokine (CCL22), and that tolerance induction could not be achieved in CCR4−/− recipients. We conclude that Foxp3 expression is specifically up-regulated within allografts of mice displaying donor-specific tolerance, that recruitment of Foxp3-expressing T reg cells to an allograft tissue is dependent on the chemokine receptor, CCR4, and that, in the absence of such recruitment, tolerizing strategies such as CD154 mAb therapy are ineffectual.

FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression

The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106–190 aa of FOXP3 are required for TIP60–FOXP3, HDAC7–FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression.

Activation of intragraft endothelial and mononuclear cells during discordant xenograft rejection.

Most studies of discordant xenograft rejection have focused on the roles of recipient xenoreactive antibody and complement as mediators of hyperacute rejection; there are essentially no data from in vivo studies as to the contribution of endothelial cell responses to the pathobiology of xenograft rejection. We hypothesized that the mechanism by which xenoreactive natural antibodies and complement of the recipient are involved in rejection of a discordant, immediately vascularized xenograft involves donor organ endothelial cell activation, with the consequences of such activation contributing significantly to the rejection process. We performed a kinetic analysis of rejection of guinea pig hearts by untreated Lewis rats or recipients depleted of complement activity that underwent delayed xenograft rejection. We report that in both hyperacute rejection and delayed xenograft rejection there is widespread evidence of endothelial cell activation, including expression of P-selectin and E-selectin, upregulation of tissue factor, and downregulation of thrombomodulin and antithrombin III expression. Many of these changes occur very early posttransplantation in grafts that are not completely rejected until approximately 3 days. In delayed xenograft rejection, an intense cellular infiltrate is seen that results from progressive accumulation of activated macrophages and natural killer cells. T cell receptor alpha/beta+T cells are present only at relatively low levels. This cellular infiltrate is associated with dense expression of pro-inflammatory cytokines, including interferon gamma, interleukin 1, and tumor necrosis factor-alpha. We conclude that both endothelial cell activation and infiltration by activated macrophages and natural killer cells may play an important role in xenograft rejection. These newly described features of the xenogeneic rejection response may require targeting by future therapeutic regimens aimed at prolonging xenograft survival.

Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection

Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1(+/+) recipients but were accepted permanently by CCR1(-/-) recipients. Second, CCR1(-/-) mice rejected completely class I- and class II-mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1(+/+) mice resulted in permanent allograft acceptance in CCR1(-/-) recipients. These latter allografts showed no sign of chronic rejection 50-200 days after transplantation, and transfer of CD4(+) splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1(+/+) mice conferred no such benefit. Finally, both CCR1(+/+) and CCR1(-/-) allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1(-/-) mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.

A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state

The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis.

Helios Expression Is a Marker of T Cell Activation and Proliferation

Foxp3+ T-regulatory cells (Tregs) normally serve to attenuate immune responses and are key to maintenance of immune homeostasis. Over the past decade, Treg cells have become a major focus of research for many groups, and various functional subsets have been characterized. Recently, the Ikaros family member, Helios, was reported as a marker to discriminate naturally occurring, thymic-derived Tregs from those peripherally induced from naïve CD4+ T cells. We investigated Helios expression in murine and human T cells under resting or activating conditions, using well-characterized molecules of naïve/effector/memory phenotypes, as well as a set of Treg-associated markers. We found that Helios-negative T cells are enriched for naïve T cell phenotypes and vice versa. Moreover, Helios can be induced during T cell activation and proliferation, but regresses in the same cells under resting conditions. We demonstrated comparable findings using human and murine CD4+Foxp3+ Tregs, as well as in CD4+ and CD8+ T cells. Since Helios expression is associated with T cell activation and cellular division, regardless of the cell subset involved, it does not appear suitable as a marker to distinguish natural and induced Treg cells.

TRAF6 Is a Critical Factor for Dendritic Cell Maturation and Development

IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily members are critical for regulating multiple aspects of dendritic cell (DC) biology. Several signaling pathways associated with each family utilize the adapter molecule, TRAF6, but its role in DCs is unclear. By examining TRAF6-deficient mice and bone marrow (BM) chimeras reconstituted with TRAF6-deficient fetal liver cells, we show that proper DC maturation requires TRAF6. In response to either microbial components or CD40L, TRAF6-deficient DCs fail to upregulate surface expression of MHCII and B7.2, or produce inflammatory cytokines. Moreover, LPS-treated TRAF6-deficient DCs do not exhibit an enhanced capacity to stimulate naive T cells. Interestingly, a major population of splenic DCs, the CD4(+)CD8alpha(-) subset, is nearly absent in both TRAF6-deficient mice and BM chimeras. Together these results indicate that TRAF6 regulates the critical processes required for maturation, activation, and development of DCs, the primary cellular bridge between innate and adaptive immunity.

Programmed Death-1 Targeting Can Promote Allograft Survival

The recently identified CD28 homolog and costimulatory molecule programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2, which are homologs of B7, constitute an inhibitory regulatory pathway of potential therapeutic use in immune-mediated diseases. We examined the expression and functions of PD-1 and its ligands in experimental cardiac allograft rejection. In initial studies, we found that most normal tissues and cardiac isografts had minimal expression of PD-1, PD-L1, or PD-L2, but intragraft induction of all three molecules occurred during development of cardiac allograft rejection. Intragraft expression of all three genes was maintained despite therapy with cyclosporin A or rapamycin, but was prevented in the early posttransplant period by costimulation blockade using CD154 or anti-inducible costimulator mAb. We prepared PD-L1.Ig and PD-L2.Ig fusion proteins and showed that each bound to activated PD-1+ T cells and inhibited T cell functions in vitro, thereby allowing us to test the effects of PD-1 targeting on allograft survival in vivo. Neither agent alone modulated allograft rejection in wild-type recipients. However, use of PD-L1.Ig administration in CD28−/− recipients, or in conjunction with immunosuppression in fully MHC-disparate combinations, markedly prolonged cardiac allograft survival, in some cases causing permanent engraftment, and was accompanied by reduced intragraft expression of IFN-γ and IFN-γ-induced chemokines. PD-L1.Ig use also prevented development of transplant arteriosclerosis post-CD154 mAb therapy. These data show that when combined with limited immunosuppression, or in the context of submaximal TCR or costimulatory signals, targeting of PD-1 can block allograft rejection and modulate T and B cell-dependent pathologic immune responses in vivo.