Multifaceted effects of synthetic TLR2 ligand and Legionella pneumophilia on Treg-mediated suppression of T cell activation.

BACKGROUND: Regulatory T cells (Treg) play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2) agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff) cells and dendritic cells (DCs) individually and in co-cultures with Tregs. RESULTS: TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs) contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. CONCLUSIONS: These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion.

TLR ligands in the local treatment of established intracerebral murine gliomas.

Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG-oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9(+/+) wild-type and TLR9(-/-) knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN-gamma producing CD4(+) and CD8(+) effector T cells and a marked increase in the ratio of CD4(+) effector T cells to CD4(+)FoxP3(+) regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a therapeutic murine glioma model.

CD25-Treg-depleting antibodies preserving IL-2 signaling on effector T cells enhance effector activation and antitumor immunity.

Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.

Toll-like receptor 2 controls expansion and function of regulatory T cells.

Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4CD25 Treg subset in TLR2 mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2 mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs.

Elimination of regulatory T cells is essential for an effective vaccination with tumor lysate-pulsed dendritic cells in a murine glioma model.

Both melanoma and glioma cells are of neuroectodermal origin and share common tumor associated antigens. In this article, we report that the melanocyte differentiation antigen TRP2 (tyrosinase-related protein 2) is not predominantly involved in the tumor rejection of a syngeneic murine glioma. Although GL261 glioma cells endogenously expressed TRP2 and were lysed by TRP2 specific cytotoxic T cells (CTLs) in vitro, vaccinations with TRP2 peptide-pulsed dendritic cells (DCs) could only induce minor antiglioma responses in a prophylactic setting and failed to work in a stringent setting where vaccine and tumor were administered on the same day. Further analysis revealed that TRP2 is not recognized by bulk CTLs after depletion of regulatory T cells which results in tumor rejections in vivo. In contrast to TRP2 peptide-pulsed DC, tumor lysate-pulsed DCs were more potent as a vaccine and completely protected mice from tumor outgrowth in a prophylactic setting. However, the vaccine efficacy of tumor lysate-pulsed DC was not sufficient to prevent the tumor outgrowth when tumors were inoculated the same day. In this case, Treg depletion before vaccination was essential to boost antiglioma immune responses leading to the rejection of 80% of the mice and long-term immunity. Therefore, we conclude that counteracting the immunosuppressive glioma tumor environment via depletion of regulatory T cells is a prerequisite for successful eradication of gliomas after targeting multiple tumor antigens by using tumor lysate-pulsed DCs as a vaccine in a more stringent setting.

Synergy between in situ cryoablation and TLR9 stimulation results in a highly effective in vivo dendritic cell vaccine.

Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivo-generated, tumor antigen-loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We have previously shown that in situ tumor destruction by ablative treatments efficiently delivers antigens for the in vivo induction of antitumor immunity. In this article, we show that although 20% of the draining lymph node DCs acquire intratumorally injected model antigens after in situ cryoablation, only partial protection against a subsequent tumor rechallenge is observed. However, we also show that a combination treatment of cryoablation plus TLR9 stimulation via CpG-oligodeoxynucleotides is far more effective in the eradication of local and systemic tumors than either treatment modality alone. Analysis of the underlying mechanism revealed that in situ tumor ablation synergizes with TLR9 stimulation to induce DC maturation and efficient cross-presentation in tumor-bearing mice, leading to superior DC function in vivo. Therefore, in situ tumor destruction in combination with CpG-oligodeoxynucleotide administration creates a unique "in situ DC vaccine" that is readily applicable in the clinic.

Ovalbumin-containing core-shell implants suitable to obtain a delayed IgG1 antibody response in support of a biphasic pulsatile release profile in mice.

A single-injection vaccine formulation that provides for both a prime and a boost immunization would have various advantages over a multiple-injection regime. For such a vaccine formulation, it is essential that the booster dose is released after a certain, preferably adjustable, lag time. In this study we investigated whether a core-shell based implant, containing ovalbumin as core material and poly(DL-lactic-co-glycolic acid) of various monomer ratios as shell material can be used to obtain such a booster release. An in vitro release study showed that the lag time after which the ovalbumin was released from the core-shell implant increased with increasing lactic to glycolic acid ratio of the polymer and ranged from 3-6 weeks. Fluorescence spectroscopy showed minimal differences between native ovalbumin and ovalbumin from core-shell implants that were incubated until just before the observed in vitro release. In addition, mice immunized with a subcutaneous inserted core-shell implant containing ovalbumin showed an ovalbumin-specific IgG1 antibody response after a lag time of 4 or 6-8 weeks. Moreover, delayed release of ovalbumin caused higher IgG1 antibody titers than conventional subcutaneous vaccination with ovalbumin dissolved in PBS. Collectively, these findings could contribute to the further development of a single-injection vaccine, making multiple injections of the vaccine superfluous.

In situ tumor ablation creates an antigen source for the generation of antitumor immunity.

Tumor-destructing techniques, like radiofrequency ablation (RFA), allow eradication of large tumors. Potentially, in situ tumor destruction also can provide the immune system with an antigen source for the induction of antitumor immunity. Antigen-presenting cells could take up antigens in the periphery after which they induce specific immune responses. Recent data show that especially antigen-presenting dendritic cells are crucial for the induction of potent immune responses. However, virtually nothing is known regarding the induction of immune responses after in situ tumor destruction in mice or humans. We used the well-defined murine B16-OVA melanoma cell line to develop a novel tumor model to explore: (a). the immunologic consequences of in situ tumor destruction; and (b). the efficacy of a combination approach of tumor destruction and immunostimulation. Applying this model system we demonstrate that following RFA, a weak but detectable immune response develops, directed against OVA, but also against a broader range of B16 antigens. Adoptive transfer experiments further indicate that antitumor reactivity can be transferred to naïve mice by splenocytes. To augment the response observed, we administered a blocking monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 at the time of tumor destruction. Interestingly, this strongly enhanced antitumor immunity, resulting in long-lasting tumor protection. These results illustrate that in situ tumor destruction can provide a useful antigen source for the induction of antitumor immunity, provided that additional immunostimulatory signals are coadministered.

Antitumor efficacy of wild-type p53-specific CD4(+) T-helper cells.

Overexpression of p53 is found in approximately 50% of human cancers, making it an attractive target antigen for immunotherapy of cancer. Research in this area has thus far primarily focused on p53-specific CTLs. Although these CTLs were shown to be highly effective against p53-overexpressing tumors in vivo, immunological tolerance seems to strongly restrict the spectrum of the p53-specific CTL repertoire in p53(+/+) subjects. In view of the emerging role of CD4(+) Th (Th) cells in the antitumor response, we investigated the specificity and antitumor efficacy of the p53-specific Th response in mice. Our data show that high affinity Th cells against the naturally processed epitope p53(108-122) can be elicited in both p53(-/-) and p53(+/+) mice, indicating that the p53-specific T-cell response is not affected by tolerance at the Th level. Furthermore, p53(108-122)-specific Th cells were effective in enabling p53-specific CTLs to control the growth of p53-overexpressing tumors in vivo. Therefore, exploitation of the p53-specific Th response appears to be a highly useful aspect of immunotherapeutic strategies against cancers.

Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity.

Mouse PTP-BL is a large, nontransmembrane protein tyrosine phosphatase of unclear physiological function that consists of a KIND domain, a FERM domain, five PDZ domains, and a COOH-terminal catalytic PTP domain. PTP-BL and its human ortholog PTP-BAS have been proposed to play a role in the regulation of microfilament dynamics, cytokinesis, apoptosis, and neurite outgrowth. To investigate the biological function of PTP-BL enzyme activity, we have generated mice that lack the PTP-BL PTP moiety. These PTP-BL(DeltaP/DeltaP) mice are viable and fertile and do not present overt morphological alterations. Although PTP-BL is expressed in most hematopoietic cell lineages, no alterations of thymocyte development in PTP-BL(DeltaP/DeltaP) mice could be detected. Sciatic nerve lesioning revealed that sensory nerve recovery is unaltered in these mice. In contrast, a very mild but significant impairment of motor nerve repair was observed. Our findings exclude an essential role for PTP-BL as a phosphotyrosine phosphatase and rather are in line with a role as scaffolding or anchoring molecule.

Revival of the regulatory T cell: new targets for drug development.

Compelling new evidence supports the idea that regulatory T cells play a major role in our immune system. Several subsets of these regulators have been identified recently. Differences in the phenotypical and functional characteristics of these subsets have immunological implications. From our growing knowledge of the field of immunology, we could potentially generate a new class of therapeutic agents that target immune-related diseases.

CD25+ regulatory cells from HLA-DQ8 transgenic mice are capable of modulating collagen-induced arthritis.

In the last decade, CD4+CD25+ T regulatory cells have been implicated in the protection against autoimmune diseases. The human DQ8 major histocompatibility complex (MHC) class II molecule is associated with rheumatoid arthritis (RA) and various other autoimmune diseases in humans. The human leukocyte antigen (HLA)-DQ8 transgenic mouse, containing the human DQ8 MHC class II molecule, is predisposed toward collagen-induced arthritis. However, the biologic pathways responsible for DQ8-associated autoimmunity have yet to be defined, including possible defects in the CD4+CD25+ T regulatory cell compartment. To explore this concept, we examined the suppressive capacity of CD4+CD25+ T regulatory cells from DQ8 transgenic mice in vitro and, using CD25-specific depleting antibodies, investigated their influence on collagen-induced arthritis in vivo. CD4+CD25+ T regulatory cells isolated from DQ8 transgenic mice were found to be sufficient suppressors of splenocyte proliferation and interferon (INF)-gamma production. Furthermore, depletion of these cells before immunization led to significant increases in arthritis severity, collagen-specific antibodies, and INF-gamma production. These results indicate that HLA-DQ8 mice contain naturally occurring CD25+ regulatory cells that modulate collagen-induced arthritis and imply that DQ8 expression does not hinder the development of CD25+ T regulatory cells.

Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1.

BACKGROUND: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient. METHODS/PRINCIPAL FINDINGS: NOD/shi-scid/γ(c)null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir. CONCLUSIONS/SIGNIFICANCE: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.

Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients.

PURPOSE: The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody-mediated depletion of CD25(high) regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. EXPERIMENTAL DESIGN: Thirty HLA-A2.1(+) metastatic melanoma patients were vaccinated with mature dendritic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patients were pretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor α-chain (CD25), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parameters were determined. RESULTS: Daclizumab efficiently depleted all CD25(high) immune cells, including CD4(+)FoxP3(+)CD25(high) cells, from the peripheral blood within four days of administration. Thirty days after administration, daclizumab was cleared from the circulation and all CD25(+) cells reappeared. The presence of daclizumab during dendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the presence of antigen-specific T cells. Daclizumab, however, did prevent these CD25(+) T cells from acquiring effector functions. Consequently, significantly less patients pretreated with daclizumab developed functional, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatment had no significant effect on progression-free survival compared with the control group. CONCLUSIONS: Although daclizumab depleted the CD4(+)FoxP3(+)CD25(high) Tregs from the peripheral circulation, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionally suppressed de novo induced CD25(+) effector cells during dendritic cell vaccinations. Our results indicate that for immunotherapeutic benefit of transient Treg depletion, timing and dosing as well as Treg specificity are extremely important.

In vivo colocalization of antigen and CpG [corrected] within dendritic cells is associated with the efficacy of cancer immunotherapy.

Immunostimulatory cytidyl guanosyl (CpG) motifs are of great interest as cancer vaccine adjuvants. They act as potent inducers of Th1 responses, including the activation of cytotoxic CD8(+) T lymphocytes (CTL). Whereas animal models have provided clear evidence that CpG enhances antitumor immunity, clinical trials in humans have thus far been less successful. Applying cryosurgery as an instant in situ tumor destruction technique, we now show that timing of CpG administration crucially affects colocalization of antigen and CpG within EEA-1(+) and LAMP-1(+) compartments within dendritic cells in vivo. Moreover, antigen/CpG colocalization is directly correlated with antigen cross-presentation, the presence of CTL, and protective antitumor immunity. Thus, failure or success of CpG as a vaccine adjuvant may depend on colocalization of antigen/CpG inside DCs and hence on the timing of CpG administration. These data might aid in the design of future immunotherapeutic strategies for cancer patients.

Regulatory T cells and toll-like receptors: regulating the regulators.

Regulatory T cells (Treg) play a crucial role in maintaining control of leucocytes. Several studies have shown that in vivo Treg depletion results in autoimmune syndromes like thyroiditis, gastritis, diabetes mellitus and colitis, but at the same time, may also result in improved anti-tumour vaccination. Although Treg are recognised to maintain peripheral tolerance in healthy individuals, recent research has shown that Treg also suppress immune responses during infections to prevent tissue damage. How the Treg themselves are regulated is still under investigation. Their suppressive activity must be regulated in order to allow for the effective elimination of pathogens. Until recently, this control of Treg function was found to be through modulation via cytokines or by stimulation via co-stimulatory molecules on antigen-presenting cells. It is now demonstrated, however, that the presence of pathogens can be communicated to Treg directly through toll-like receptors (TLRs). Up until now, Treg have been reported to respond to ligands for TLR2, 4, 5 and 8, and different TLRs can have alternative effects on Treg resulting in more suppression or, in contrast, abrogation of suppression. As TLRs can also recognise endogenous proteins, such as heat shock proteins, it is tempting to speculate on the role of these proteins in modulating Treg function during chronic inflammation. In this review, we will discuss the implications of TLR engagement on Treg and any consequences this may have for chronic autoinflammatory diseases like rheumatoid arthritis (RA).

CD4+FoxP3+ regulatory T cells gradually accumulate in gliomas during tumor growth and efficiently suppress antiglioma immune responses in vivo.

The suppressive activity of regulatory T cells (Treg) has been implicated as an important factor limiting immune mediated destruction of tumor cells. However, not much is known about the presence and function of Treg within tumors. Here we show in a syngeneic murine glioma model a time-dependent accumulation of CD4+FoxP3+ Treg in brain tumors. Further analysis revealed a time-dependent upregulation of CD25, CTLA-4, GITR and CXCR4 on intratumoral CD4+FoxP3+ Treg during tumor growth. Moreover, freshly isolated intratumoral Treg were highly suppressive when tested directly ex vivo. Treatment with anti-CD25 monoclonal antibodies (mAbs) significantly reduced the number of these highly suppressive CD4+FoxP3+ cells within the growing tumor and provoked a CD4 and CD8 T cell dependent destruction of the glioma cells. Combining Treg depletion with administration of blocking CTLA-4 mAbs further boosted glioma-specific CD4+ and CD8+ effector T cells as well as antiglioma IgG2a antibody titers resulting in complete tumor eradication without any signs of autoimmunity. These data illustrate that intratumoral accumulation and activation of CD4+FoxP3+ Treg act as a dominant immune escape mechanism for gliomas and underline the importance of controlling tumor-infiltrating Treg in glioma immunotherapy.

Toll-like receptors on regulatory T cells: expanding immune regulation.

Regulatory T (Treg) cells maintain peripheral tolerance and limit effector responses to prevent excessive immune-mediated tissue damage. However, recent research reveals that Treg cells also dampen the induction of immune responses and, thus, must be controlled to enable the effective protection against infections and cancer. Until now, this control of Treg-cell function has been believed to be by communication through cytokines or by stimulation through co-stimulatory molecules on antigen-presenting cells. However, new evidence has demonstrated that Treg cells can also sense pathogens directly through Toll-like receptors (TLRs) and, consequently, modify their behaviour. This review examines the ramifications of TLR engagement on Treg cells and conventional T cells, and discusses the potential role of TLRs on Treg cells and the consequences for disease therapy.

Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells.

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2(-/-) mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2(-/-) macrophages. Although production of the proinflammatory cytokines TNF, IL-1alpha, and IL-1beta is normal, IL-10 release is severely impaired in the TLR2(-/-) mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2(-/-) mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.

CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis.

OBJECTIVE: CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen-induced arthritis (CIA). METHODS: We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII-specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens. RESULTS: CD25+ cell-depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen-specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen. CONCLUSION: These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.