Humanized anti-CD25 antibody treatment with daclizumab in multiple sclerosis

Monoclonal antibodies against a variety of receptors and molecules are currently being introduced in clinical medicine. One of these targets is the interleukin-2 receptor alpha-chain CD25. The humanized monoclonal anti-CD25 antibody daclizumab (Zenapax) has been approved several years ago for the prevention of allotransplant rejection and adult T cell leukemia. Following promising observations in uveitis, daclizumab has been tested in a number of small clinical trials in multiple sclerosis based on the rationale that blocking CD25 would prevent the expansion of autoreactive T lymphocytes. The data from this preliminary clinical exploration as well as findings about the mechanism of action of anti-CD25 treatment are summarized in this study.     

Daclizumab Therapy for Multiple Sclerosis

Daclizumab is a humanized monoclonal antibody of IgG1 subtype that binds to the Tac epitope on the interleukin-2 (IL-2) receptor α-chain (CD25), thus, effectively blocking the formation of the high-affinity IL-2 receptor. Because the high-affinity IL-2 receptor signaling promotes expansion of activated T cells in vitro, daclizumab was designed as a therapy that selectively inhibits T-cell activation. Assuming the previous statement, daclizumab received regulatory approval as add-on therapy to standard immunosuppressive regimen for the prevention of acute allograft rejection in renal transplantation. Based on its putative mechanism of action (MOA), daclizumab represented an ideal therapy for T-cell-mediated autoimmune diseases and was subsequently tested in inflammatory uveitis and multiple sclerosis (MS). In both of these diseases, daclizumab therapy significantly inhibited target organ inflammation. Mechanistic studies in MS demonstrated that the MOA of daclizumab is surprisingly broad and that the drug exerts unexpected effects on multiple components of the innate immune system. Specifically, daclizumab dramatically expands and activates immunoregulatory CD56^bright NK cells, which gain access to the intrathecal compartment in MS and can kill autologous activated T cells. Daclizumab also blocks trans-presentation of IL-2 by mature dendritic cells to primed T cells, resulting in profound inhibition of antigen-specific T cells. Finally, daclizumab modulates the development of innate lymphoid cells. In conclusion, daclizumab therapy, which is currently in phase III testing for inflammatory MS, has a unique MOA that does not limit migration of immune cells into the intrathecal compartment, but rather provides multifactorial immunomodulatory effects with resultant inhibition of MS-related inflammation.     

Monocyte-derived dendritic cells in multiple sclerosis: the effect of bacterial infection

We investigated whether monocyte-derived dendritic cells (MDDCs) generated in vitro from bacteria-infected MS patients modified autoreactive T cells activation patterns. T cell clones (TCCs) stimulated with MDDCs from infected MS patients responded with maximal proliferation, inducing IL-12, IL-17 and IFN-gamma secretion, at concentrations significantly lower than after incubation with MDDCs isolated from uninfected individuals and bacterial meningitis (BM) patients. Moreover, infected MDDCs promoted TCCs survival, and secreted more IL-12, IL-18, and IL-23. Finally, MDDCs from infected MS subjects showed higher expression of myeloid differentiation factor 88 (MyD88), as well as of HLA-DR, CD1a, CD80, CD86, CD273, CD40, CD83 and CCR7 when compared to MDDCs from uninfected MS individuals, and BM patients. Thus, activation of the innate immune system by microbial products in MS patients affects the generation MDDCs and their ability to modify autoreactive T cell activation patterns, which may be linked to MS relapse induction during bacterial infections.     

Recombinant Interferon-β blocks proliferation but enhances interleukin-10 secretion by activated human T-cells

Results from recent clinical trials have indicated that recombinant interferon-β (rIFN-β) is a promising drug for the treatment of Multiple Sclerosis (MS), a disease of supposed autoimmune etiology. To gain insight into the immunoregulatory properties of this cytokine, we analyzed effects of interferon-β (IFN-β) on T-cell functions in vitro. Interferon-β inhibited T-cell proliferation, as well as T-cell-dependent immunoglobulin secretion, in a dose-dependent manner. IFN-β did not inhibit upregulation of CD40L on activated T-cells, but blocked induction of CD25 on stimulated T-and B-lymphocytes. Secretion of interferon-gamma (IFN-y), tumour necrosis alpha (TNF-α) and IL-13 was inhibited by the addition of IFN-β, whereas IL-4 secretion was unaffected. Interestingly, IFN-β enhanced secretion of IL-2 about two-fold and secretion of IL-10 nearly four-fold. In summary, these findings suggest that IFN-β may exert direct effects on T-and B-cell function in vivo. In addition, enhanced secretion of IL-10 by activated T-cells may interfere with newly initiated and ongoing inflammatory immune reactions.     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.     

Clinical response to glatiramer acetate correlates with modulation of IFN-gamma and IL-4 expression in multiple sclerosis

OBJECTIVE: To determine whether glatiramer acetate (GA)-induced lymphoproliferation and IFN-gamma and IL-4 modulation correlate with the clinical response in multiple sclerosis (MS). BACKGROUND: GA therapy involves the induction of anti-inflammatory cytokine shifts. However, it is not known whether this response correlates with the clinical outcome. METHODS: Thirty-six relapsing-remitting (RR) MS patients were treated with GA for at least two years, and classified clinically as GA-responders (GA-R=22) or hypo/non-responders (GA-HR/NR = 14). Proliferation of peripheral blood mononuclear cells (PBMC) to GA and Tetanus toxoid (TT), as well as IL-4 and IFN-gamma ELISPOT, were performed. FINDINGS: There was no difference in PBMC proliferation to GA or TT between GA-R and GA-HR/NR before and during treatment (P>0.05). The mean number of IFN-gamma ELISPOTS in unstimulated, TT and anti-CD3/CD28-stimulated PBMC was lower among GA-R (unstimulated: GA-R =10.1+/-6.21 (n=22) versus GA-HR/NR=17.8+/-12.7 (n=14), P=0.04; TT-GA-R =12.2+/-4.06 (n=12) versus GA-HR/NR=26.8+/-21.0 (n=8), P=0.028; anti-CD-3/CD28 GA-R=217.3+/-140.4 (n=22) versus GA-HR/NR=368.5+/-170.1 (n=14), P=0.006). In contrast, the number of IL-4 ELISPOTS remained unchanged in the GA-R group, but was progressively reduced in the GA-HR/NR group during GA therapy (GA-HR/NR IL-4: pre-Rx: 59+/-34 versus 22+/-11 at 12 months (n =6), P=0.0429). The IL-4/ IFN-gamma ratio in anti-CD3/CD28-stimulated PBMC was significantly higher among GA-R compared to GA-HR/NR (P=0.0474). INTERPRETATION: Lymphoproliferation to GA did not differentiate GA-R from GA-HR/NR. However, reduced IFN-gamma expression and stable IL-4 expression in anti-CD3/CD28-stimulated PBMC, and an increased IL-4/IFN-gamma ratio was associated with favorable clinical response. More data are needed to validate the prospective use of IL-4/IFN-gamma expression in PBMC as a biomarker of clinical response to GA for individual patients.     

Single-cell analysis of cytokine production shows different immune profiles in multiple sclerosis patients with active or quiescent disease.

Peripheral blood mononuclear cells of multiple sclerosis (MS) patients were stimulated with myelin basic protein (MBP) together with anti-CD28 monoclonal antibody and staphylococcal enterotoxin B to optimize cytokine production by antigen-specific cells. Type 1 (IL-2, IL-12, IFNgamma) and pro-inflammatory (TNFalpha, IL-1beta, IL-6) cytokines were augmented in CD4+, CD8+, and CD14+ cells of acute MS patients and of patients undergoing disease reactivation. These cytokines were reduced in IFNbeta-treated and in stable MS patients; type 2 cytokines (IL-4, IL-10) were increased in these patients. Similar immune profiles are seen in MS patients in whom remission is naturally or pharmacologically (IFNbeta) achieved. Cytokine alterations are particularly evident in CD14+ cells, underlying their critical role in the modulation of the immune response.     

Treating multiple sclerosis with monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are potent new tools for a molecular targeted approach to modify the course of multiple sclerosis (MS). Besides natalizumab, which was approved in 2006, three other mAbs (alemtuzumab, rituximab and daclizumab) were successfully tested in Phase II MS trials. In this review, introductory notes on the development and systematic nomenclature of therapeutic mAbs in general, set the stage for a detailed discussion of the four mAbs mentioned. We summarize non-MS indications, expression and function of target antigens, scientific rationales for MS therapy, putative modes of action and pharmacological aspects. Particularly, we provide a critical discussion of clinical MS trials, including protocols and interim analyses of trials currently underway. The natalizumab section pays special attention to the clinical handling of safety issues and the diagnostic use of neutralizing antibodies. We finally develop a scenario for how each of the four mAbs might evolve into the market of MS therapeutics within the coming years.     

Defective regulation of IFNgamma and IL-12 by endogenous IL-10 in progressive MS

BACKGROUND: MS is a chronic inflammatory disease of the CNS postulated to be a Th1 type cell-mediated autoimmune disease. There is increased interferon-gamma (IFNgamma) secretion in MS, and IFNgamma administration induces exacerbations of disease. IFNgamma expression is closely regulated by a number of cytokines produced by different cells of the immune system. Interleukin-12 (IL-12) is a major factor leading to Th1-type responses, including IFNgamma secretion, and there is increased secretion of IL-12 in MS. IL-10 is a potent inhibitor of both IL-12 and IFNgamma expression. METHODS: The authors investigated cytokine production and proliferative responses of peripheral blood mononuclear cells stimulated with soluble anti-CD3 in healthy controls and patients with stable relapsing-remitting MS or progressive MS. RESULTS: The authors found that T cell receptor-mediated IFNgamma and IL-10 secretion were increased in progressive MS, whereas IL-4 and IL-2 secretion and lymphocyte proliferative responses were normal. Anti-IL-12 antibody suppressed raised IFNgamma in progressive MS but did not affect raised IL-10. In addition, neutralization of endogenous IL-10 upregulated IFNgamma in controls but not progressive MS. IL-10 was produced by CD4+ cells whereas IFNgamma was produced by both CD4+ and CD8+ cells. There were no differences in IL-10 receptor expression in MS patients. CONCLUSIONS: These abnormalities in IL-10 regulation were not seen in the relapsing-remitting form of MS. Thus, the defect in regulation of both IL-12 and IFNgamma production by endogenous IL-10 in progressive MS could be an important factor involved in the transition of MS from the relapsing to the progressive stage and has implications for treating MS patients with exogenous IL-10.     

Emerging therapies for multiple sclerosis

This review examines the mode of action, safety profile and clinical efficacy of some of the most promising new therapeutic strategies for multiple sclerosis. Autologous hematopoietic stem cell transplantation can regenerate a new and tolerant immune system and is a potentially effective rescue therapy in a subset of patients with aggressive forms of MS refractory to approved immunomodulatory and immunosuppressive agents. High-dose cyclophosphamide without stem cell support is suggested to induce prolonged remissions through similar immunological mechanisms with less toxicity. Fingolimod (FTY720) is a novel oral immunomodulating agent that acts through preventing lymphocyte recirculation from lymphoid organs. Monoclonal antibody therapy has provided scientists and clinicians the opportunity to rationally direct the therapeutic intervention against specific molecules. Targeting molecules of the immune system such as CD52 (alemtuzumab), CD25 (daclizumab), VLA-4 (natalizumab) and CD20 (rituximab) have resulted in potent immunomodulatory effects through sometimes unpredicted mechanisms. The potential of immunoglobulins to induce remyelination in the CNS is being investigated in an attempt to develop therapies promoting tissue repair and functional recovery. The evidence supporting the potential of these emerging immunotherapies suggests that strong progress is being made in the development of effective cures for multiple sclerosis.     

Similar sensitivity of regulatory T cells towards CD95L-mediated apoptosis in patients with multiple sclerosis and healthy individuals

Impaired suppressive function of CD4(+)CD25(high) regulatory T cells (T(reg)) has been reported as a novel pathogenetic mechanism in Multiple sclerosis (MS). We addressed if high apoptosis sensitivity of MS-T(reg) could explain this functional T(reg) defect. T(reg) from treatment-na?ve MS patients showed high sensitivity towards CD95Ligand-mediated apoptosis and exhibited enhanced cell death to IL-2 and TCR-signal deprivation. Since susceptibility of T(reg) to cell death was similar in MS patients and healthy controls, this cannot explain the inhibitory dysfunction of T(reg) associated with MS. Furthermore, as cell death is not enhanced, therapeutic expansion of MS-T(reg)in vitro should be a reasonable and novel therapeutic option.     

Proinflammatory cytokines regulate antigen-independent T-cell Activation by two separate calcium-signaling pathways in multiple sclerosis patients

Central nervous system (CNS) lesions typical of multiple sclerosis (MS) are characterized by demyelinating inflammatory infiltrates that contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenic non-antigen-specific mononuclear cells. Here, we report that in patients with MS the combined action of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), interleukin (IL)-2, and IL-6 leads to the activation of most peripheral T cells (mainly CD4 memory) by promoting a persistent intracellular calcium increase via two independent signaling pathways. The activation of these pathways, one activated by IFNγ and the other by the combination TNFα/IL-2/IL-6, is independent from myelin antigens and precedes by 2 weeks phases of disease activity (eg, clinical relapses and/or appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging scans during 1 year of follow-up). Our results indicate that an appropriate combination of the four cytokines, three with a proinflammatory profile and one necessary for T-cell growth and differentiation, can activate in an antigen-independent fashion most peripheral T cells from MS patients. This mechanism is likely to contribute to the recruitment of nonspecific lymphocytes into the cellular activation processes leading to CNS demyelination and may represent a major target for immune intervention in MS.     

Vaccination with autoreactive T cell clones in multiple sclerosis: overview of immunological and clinical data

Although the etiology and pathogenesis of Multiple Sclerosis (MS) remain elusive, accumulating evidence indicates that MS is a chronic inflammatory disease with an autoimmune component, mediated by autore-active T lymphocytes specific for myelin antigens. The triggering T cell autoantigen has not been identified yet, but recent immunological studies in MS and parallel experiments in experimental allergic encephalomyelitis (EAE), the animal model of MS, have indicated that myelin basic protein (MBP) can be considered as one of the major candidate autoantigens in the pathogenesis of the disease. Based on these observations, several therapeutic strategies have been developed aimed at the specific elimination or inactivation of MBP reactive T cells in MS. One of these approaches involves the immunization of MS patients with autologous attenuated autoreactive T cells to induce an immune response specifically targeted at these autoreactive T cells. This method, termed T cell vaccination, has been shown to prevent and treat EAE. We have recently conducted a pilot trial of T cell vaccination in a limited group of MS patients to evaluate the immunological responses to the injected cells. The data obtained indicate that this type of vaccination induces an effective anti-clonotypic T cell response leading to a specific depletion of circulating MBP reactive T cells. Preliminary data on the clinical effects are promising, encouraging further clinical trials. © 1996 Wiley-Liss, Inc.     

Glatiramer acetate and IFN-beta act on dendritic cells in multiple sclerosis.

Glatiramer acetate (GA; Copolymer 1; Copaxone) and interferon-beta (IFN-beta) modulate the course of multiple sclerosis (MS), but probably by different mechanisms. GA, a mixture of synthetic peptides, is believed to act as an altered peptide ligand with inhibitory effects on autoreactive T cells and promoting Th2 cells. It is unknown whether GA affects dendritic cells (DCs), which, besides strong antigen presenting capacity, orchestrate Th1 and Th2 responses. IFN-beta inhibits IL-12 production by DCs over unknown mechanisms. This study was designed to investigate in vitro effects of GA and IFN-beta on the development and function of DCs from MS patients and healthy controls, and to explore their possible synergistic effects on DCs. DCs were generated from adherent blood mononuclear cells (MNCs). GA or IFN-beta or both, when added at initiation of DC cultures, rapidly promoted the development of adherent MNCs into floating, activated DCs as reflected by up-regulation of HLA-DR and CD86 expression. IFN-beta, but not GA, induced IL-3R expression on DCs. Compared to DCs from healthy controls, MS patients' DCs expressed higher levels of the myeloid DC marker CD1a and produced lower amounts of IL-10. GA reduced IL-12 production by DCs. IFN-beta also reduced IL-12, but increased IL-10 production by DCs from both MS patients and healthy controls. GA and IFN-beta synergistically suppressed CD1a and enhanced CD86 expression on MS DCs. These findings document novel mechanisms of action of GA and IFN-beta at the DC level in MS.     

Bystander modulation of chemokine receptor expression on peripheral blood T lymphocytes mediated by glatiramer therapy

BACKGROUND: Glatiramer acetate therapy is thought to be effective for multiple sclerosis (MS) by promoting T(H)2 cytokine deviation, possibly in the brain, but the exact mechanism and site of action are incompletely understood. Determining the site of action and effect of glatiramer on cell trafficking is of major importance in designing rational combination therapy clinical trials. OBJECTIVE: To determine whether glatiramer therapy will also act in the peripheral blood through bystander modulation of chemokine receptor (CKR) expression and cytokine production on T lymphocytes. DESIGN: Before-and-after trial. SETTING: A university MS specialty center. PATIENTS: Ten patients with relapsing-remitting MS. INTERVENTIONS: Treatment with glatiramer for 12 months and serial phlebotomy. MAIN OUTCOME MEASURES: Cytokine production, CKR expression, and cell migration. RESULTS: The glatiramer-reactive T cells were T(H)2 cytokine biased, consistent with previous studies. We found a significant reduction in the expression of the T(H)1 inflammation associated with the CKRs CXCR3, CXCR6, and CCR5 on glatiramer- and myelin-reactive T cells generated from patients with MS receiving glatiramer therapy vs baseline. Conversely, expression of the lymph node-homing CKR, CCR7, was markedly enhanced on the glatiramer-reactive T cells derived from patients with MS undergoing glatiramer therapy. There was a reduction in the percentage of CD4+ glatiramer-reactive T cells and an increase in the number of CD8+ glatiramer-reactive T cells. CONCLUSIONS: Glatiramer may suppress autoreactive CD4+ effector memory T cells and enhance CD8+ regulatory responses, and bystander modulation of CKRs may occur in the periphery.     

The impact of parasite infections on the course of multiple sclerosis

Previously, we demonstrated that helminth-infected MS patients showed significantly lower number of relapses, reduced disability scores, and lower MRI activity compared to uninfected MS subjects. In the current study, 12 patients with diagnosis of relapsing remitting MS presenting parasite infections were prospectively followed during 90 months; due to exacerbation of helminth-infection symptoms after 63 months of follow-up, 4 patients received anti-parasite treatment. Helminth-infection control was associated with significant increase in clinical and radiological MS activities. Moreover, these patients showed significant increase in the number of IFN-γ and IL-12 producing cells, and a fall in the number of TGF-β and IL-10 secreting cells, as well as CD4+CD25+FoxP3+ Treg cells evident 3 months after anti-helminth treatment began. These new observations on parasite infections associated to MS indicate that parasite regulation of host immunity can alter the course of MS.     

B cells from glatiramer acetate-treated mice suppress experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) thought to be primarily mediated by T cells. However, emerging evidence supports an important role for B cells in the pathogenesis and inhibition of MS. Glatiramer acetate (GA), a Food and Drug Administration-approved drug for the treatment of MS, has a good safety profile. But GA's mechanism of action in MS is still elusive. In this study, we showed that B cells from GA-treated mice increased production of IL-10 and reduced expression of co-stimulatory molecules viz.: CD80 and CD86. B cells from GA-treated mice also diminished proliferation of myelin oligodendrocyte glycoprotein (MOG_35-55) specific T cells. Purified B cells transferred from GA-treated mice suppressed experimental autoimmune encephalomyelitis (EAE) in recipient mice compared with B cells transferred from mice treated with PBS or ovalbumin. The treatment effect of GA in EAE was abrogated in B cell-deficient mice. Transfer of B cells from GA-treated mice inhibited the proliferation of autoreactive T cells as well as the development of Th1 and Th17 cells but promoted IL-10 production in recipient mice. The number of peripheral CD11b⁺ macrophages in recipient mice also decreased after transfer of B cells from GA-treated mice; however, the number of dendritic cells and regulatory T cells remained unaltered. These results suggest that B cells are important to the protective effects of GA in EAE.     

The effect of lymphocytapheresis on multiple sclerosis]

We studied the effect of lymphocytapheresis (LCP) on the expanded disability status scale (EDSS) clinical score, lymphocyte subsets and Interleukin-2 (IL-2) production by peripheral blood mononuclear cell (PBM) in 5 patients with multiple sclerosis (MS). The EDSS clinical score significantly decreased after LCPs (p < 0.05). PBM IL-2 production and CD 4/8 ratio significantly decreased (p < 0.05, p < 0.05), and the number of neutrocytes and CD 11 b+CD 8+ (%) significantly increased immediately after LCP (p < 0.05, p < 0.05). Down-regulation of PBM IL-2 production and CD 4/8 ratio and up-regulation of CD 11 b+CD 8+ may account for therapeutic effect of LCP on MS. However, similar changes were observed in patients with CIDP and MG during immunoadsorbent therapy (IAT). It is possible that down-regulation of PBM IL-2 production and CD 4/8 ratio and up regulation of CD 11 b+CD 8+ and the number of neutrocytes may commonly result from apheresis therapy using extra-corporeal circulation.     

Functional assay for human CD4+CD25+ Treg cells reveals an age-dependent loss of suppressive activity

CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.