Andrew D. Wells

Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance.

The alloimmune response against fully MHC-mismatched allografts, compared with immune responses to nominal antigens, entails an unusually large clonal size of alloreactive T cells. Thus, induction of peripheral allograft tolerance established in the absence of immune system ablation and reconstitution is a challenging task in transplantation. Here, we determined whether a reduction in the mass of alloreactive T cells due to apoptosis is an essential initial step for induction of stable allograft tolerance with non-lymphoablative therapy. Blocking both CD28–B7 and CD40–CD40 ligand interactions (co-stimulation blockade) inhibited proliferation of alloreactive T cells in vivo while allowing cell cycle-dependent T-cell apoptosis of proliferating T cells, with permanent engraftment of cardiac allografts but not skin allografts. Treatment with rapamycin plus co-stimulation blockade resulted in massive apoptosis of alloreactive T cells and produced stable skin allograft tolerance, a very stringent test of allograft tolerance. In contrast, treatment with cyclosporine A and co-stimulation blockade abolished T-cell proliferation and apoptosis, as well as the induction of stable allograft tolerance. Our data indicate that induction of T-cell apoptosis and peripheral allograft tolerance is prevented by blocking both signal 1 and signal 2 of T-cell activation.

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.

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.

Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response.

A detailed understanding of the effects of costimulatory signals on primary T cell expansion has been limited by experimental approaches that measure the bulk response of a cell population, without distinguishing responses of individual cells. Here, we have labeled live T cells in vitro with a stable, fluorescent dye that segregates equally between daughter cells upon cell division, allowing the proliferative history of any T cell present or generated during a response to be monitored over time. This system permits simultaneous evaluation of T cell surface markers, allowing concomitant assessment of cellular activation and quantitative determination of T cell receptor (TCR) occupancy on individual cells. Through this approach, we find that TCR engagement primarily regulates the frequency of T cells that enter the proliferative pool, but has relatively little effect on the number of times these cells will ultimately divide. In contrast, CD28-costimulation regulates both the frequency of responding cells (particularly at sub-maximal levels of TCR engagement), and more prominently, the number of mitotic events that responding cells undergo. When CD28-stimulation is blocked, provision of IL-2 restores the frequency of responding cells and the normal pattern of mitotic progression, indicating that the other CD28-induced genes are not required for this effect. An unexpected finding was that even at maximal levels of TCR engagement and CD28-mediated costimulation, only 50-60% of the original T cells in culture can be induced to divide. The nondividing cells are heterogeneous for naive versus memory markers, suggesting a more complex relationship between expression of memory markers and the ability to be recruited into the dividing pool. From these studies, we conclude that a stringent checkpoint regulates the participation of activated T cells in clonal expansion, with TCR and CD28 signals having both overlapping and differential effects on the induction and maintenance of T cell responses.

Quantifying the Frequency of Alloreactive T Cells In Vivo: New Answers to an Old Question

Alloreactive T cell precursor frequency was measured in vivo using fluorescent dye labeling in combination with novel models based on lymphocyte activation and recovery. CFSE-labeled C57BL/6 (H-2b) spleen and lymph node cells were adoptively transferred to C57BL/6×DBA F1 (H-2b/d) recipients, a parent→F1 MHC mismatch in which only donor cells respond. Recipients were sacrificed at serial time points to assess engraftment efficiency, and the extent of donor cell activation and proliferation. These data were used to calculate alloreactive T cell frequencies that varied 30-fold (0.71 ± 0.31% to 21.05 ± 3.62%), depending upon whether it was assumed that all donor cells injected became established and were capable of responding, or that only those present at later time points (24–72 h) were available to respond. By measuring the number of cells established in the recipient 24 h after transfer, before proliferation, we calculated an in vivo alloreactive frequency of ∼7%. Using CD69 expression at 48 h to quantify activation, we found that 40–50% of the alloactivated CD4+ donor T cells do not divide. Studies of cotransferred congenic and allogeneic cells demonstrated that bystander proliferation does not occur. We conclude that accurate calculations of alloreactive precursor frequency must account for both proliferation and cell engraftment. When this is done, a high percentage of alloreactive T cells exists across an MHC mismatch, but not all alloreactive cells proliferate in vivo. Bystander proliferation is negligible, revealing exquisite specificity to the alloresponse. These data provide a novel approach to quantify alloreactive T cell responses during specific immunomodulatory strategies in vivo.

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.

T Cell Death and Transplantation Tolerance

In order to achieve central tolerance, long-lasting and massive deletion of donor-specific T cells can be achieved by intense immunosuppression and creation of a mixed donor–recipient chimeric state. To achieve peripheral tolerance, more subtle forms of immunosuppression are required in order to preserve T cell apoptosis. IL-2-dependent AICD is required to deplete alloreactive T cells if nondepletive therapies (e.g., drugs or noncytolytic biologics) are used as the principal treatment modality. In addition, overly intense immunosuppression will block the acquisition of peripheral tolerance because induction of tolerance is an active antigen-driven T cell–dependent process. For example, the combined use of calcineurin inhibitors that block signal 1 plus costimulation blockade that blocks signal 2 causes near total immunosuppression in mice and thereby blocks expression of IL-2, Ag-triggered T cell AICD, and outgrowth of regulatory cells. Following cessation of this intense therapy, acute rejection occurs. In contrast, the use of rapamycin, an agent that blocks the proliferative signals delivered by T cell growth factors but not IL-2-triggered apoptotic signals, in combination with costimulation blockade provides strong synergy as a means to foster peripheral tolerance (Li et al., 1999xBlocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Li, Y., Li, X.C., Zheng, X.X., Wells, A.D., Turka, L.A., and Strom, T.B. Nat. Med. 1999; 5: 1298–1302Crossref | PubMed | Scopus (594)See all References(Li et al., 1999).Anti-CD25 mAb treatment is a useful and safe adjunct to conventional immunosuppression. In some experimental allograft models, targeting CD25 or the high-affinity IL-2R complex with IgM antibodies or toxins can produce transplant tolerance (Strom et al., 1992xInterleukin-2 receptor-directed immunosuppressive therapies (antibody- or cytokine-based targeting molecules) . Strom, T.B., Kelley, V.R., Woodworth, T.G., and Murphy, J.R. Immunol. Rev. 1992; 129: 131–163Crossref | PubMedSee all References(Strom et al., 1992). Tolerance occurs because these specific agents kill many IL-2R+-activated T cells, as nonlytic anti-CD25 mAbs fail to produce tolerance. In clinical practice, the anti-CD25 mAbs available are IL-2 receptor antagonists and have, by comparison with agents used in murine systems, a diminished capacity to directly kill CD25+ T cells. Nonlytic anti-CD25 mAbs block IL-2-triggered apoptosis in a murine model (Demirci et al., 2001xIL-15 and IL-2 (a matter of life and death for T cells in vivo) . Demirci, G., Ferrari-Lacraz, S., Groves, C., Coyle, A., Malek, T.R., Strom, T.B., and Li, X.C. Nat. Med. 2001; 7: 114–118Crossref | PubMed | Scopus (245)See all References(Demirci et al., 2001). In short, the ability of anti-CD25 mAbs to aid the induction of peripheral tolerance hangs on the balance of their (untested) ability to directly kill IL-2R+ T cells versus their capacity to block IL-2-mediated apoptosis.The classical separation between deletional central tolerance and peripheral nondepletive tolerant states may be somewhat artificial. It seems likely that central tolerance is rarely created through complete and total clonal deletion alone but that immunoregulatory processes arise to control “escaped” thymic emigrants. Conversely, in peripheral strategies that are not inherently lymphoablative, clonal depletion may be a prerequisite to allow immunoregulation to emerge and be effective. Thus, in the setting of the allograft response, immunoregulation and clonal depletion are inextricably linked. We believe that the classical definitions of central and peripheral transplant tolerance that appear to form extreme and opposing ends of a spectrum actually embody interactive, not alternative, means of creating transplant tolerance.

The complement inhibitory protein DAF (CD55) suppresses T cell immunity in vivo

Decay-accelerating factor ([DAF] CD55) is a glycosylphosphatidylinositol-anchored membrane inhibitor of complement with broad clinical relevance. Here, we establish an additional and unexpected role for DAF in the suppression of adaptive immune responses in vivo. In both C57BL/6 and BALB/c mice, deficiency of the Daf1 gene, which encodes the murine homologue of human DAF, significantly enhanced T cell responses to active immunization. This phenotype was characterized by hypersecretion of interferon (IFN)-γ and interleukin (IL)-2, as well as down-regulation of the inhibitory cytokine IL-10 during antigen restimulation of lymphocytes in vitro. Compared with wild-type mice, Daf1−/− mice also displayed markedly exacerbated disease progression and pathology in a T cell–dependent experimental autoimmune encephalomyelitis (EAE) model. However, disabling the complement system in Daf1−/− mice normalized T cell secretion of IFN-γ and IL-2 and attenuated disease severity in the EAE model. These findings establish a critical link between complement and T cell immunity and have implications for the role of DAF and complement in organ transplantation, tumor evasion, and vaccine development.

Notch Signaling Augments T Cell Responsiveness by Enhancing CD25 Expression

Notch receptors signal through a highly conserved pathway to influence cell fate decisions. Notch1 is required for T lineage commitment; however, a role for Notch signaling has not been clearly defined for the peripheral T cell response. Notch gene expression is induced, and Notch1 is activated in primary CD4+ T cells following specific peptide-Ag stimulation. Notch activity contributes to the peripheral T cell response, as inhibition of endogenous Notch activation decreases the proliferation of activated T cells in a manner associated with the diminished production of IL-2 and the expression of the high affinity IL-2R (CD25). Conversely, forced expression of a constitutively active Notch1 in primary T cells results in increased surface expression of CD25, and renders these cells more sensitive to both cognate Ag and IL-2, as measured by cell division. These data suggest an important role for Notch signaling during CD4+ T cell responses, which operates through augmenting a positive feedback loop involving IL-2 and its high affinity receptor.

Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation

Regulatory T cells (Treg) express Foxp3, a forkhead family member that is necessary and sufficient for Treg lineage choice and function. Ectopic expression of Foxp3 in non-Treg leads to repression of the interleukin 2 (IL-2) and interferon γ (IFNγ) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNγ loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNγ genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.