CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness.

CD4(+)CD25(+) regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4(+)CD25(-) T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact-dependent and cytokine independent. The role of TGF-beta1 in CD4(+)CD25(+) suppressor function remains unclear. While most studies have failed to reverse suppression with anti-transforming growth factor (TGF)-beta1 in vitro, one recent study has reported that CD4(+)CD25(+) T cells express cell surface TGF-beta1 and that suppression can be completely abrogated by high concentrations of anti-TGF-beta suggesting that cell-associated TGF-beta1 was the primary effector of CD4(+)CD25(+)-mediated suppression. Here, we have reevaluated the role of TGF-beta1 in CD4(+)CD25(+)-mediated suppression. Neutralization of TGF-beta1 with either monoclonal antibody (mAb) or soluble TGF-betaRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4(+)CD25(+) T cells. Responder T cells from Smad3(-/-) or dominant-negative TGF-beta type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-beta1-induced growth arrest, were as susceptible to CD4(+)CD25(+)-mediated suppression as T cells from wild-type mice. Furthermore, CD4(+)CD25(+) T cells from neonatal TGF-beta1(-/-) mice were as suppressive as CD4(+)CD25(+) from TGF-beta1(+/+) mice. Collectively, these results demonstrate that CD4(+)CD25(+) suppressor function can occur independently of TGF-beta1.

Control of T cell activation by CD4+CD25+ suppressor T cells.

Although the concept of a separate lineage of T cells specifically equipped to suppress immune responses was initially proposed more than 30 years ago, progress in this area of immunoregulation has been hampered by the lack of solid biochemical and molecular data to support the existence of the soluble products of these purported suppressor T cells. Studies over the past 5-10 years have identified a distinct lineage of CD4+CD25+ regulatory or suppressor T cells that control autoreactive effector cells and prevent autoimmunity. The mechanism by which CD4+CD25+ T cells inhibit T cell activation in vivo or in vitro is still poorly defined. While autoreactive effector T cells undergo massive proliferation and expansion following injection into immunocompromised recipients, CD4+CD25+ T cells do not inhibit this lymphopaenia-induced proliferation and act later in the activation process at the site of immune damage in the target organ. The development of in vitro models that partially mimic the in vivo properties of the CD4+CD25+ regulatory T cells has facilitated their characterization. A member of the tumour necrosis receptor family, the GITR is expressed on CD4+CD25+ T cells and after interaction with its ligand down-regulates suppressor activity. Multiple methods of manipulating both the numbers of CD4+CD25+ suppressor T cells and their activation status are now available and will rapidly be applied to therapy of autoimmune, infectious and malignant diseases.

Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8+ T cells in vivo.

The lifespan and survival of dendritic cells (DC) in vivo are potentially critical to the expansion of T cell immune responses. We have previously reported that DC loaded with specific antigen are rapidly eliminated by cytotoxic T lymphocytes (CTL) in vivo, but the site, mechanism, and consequences of DC elimination were not defined. In this article we show that DC elimination in vivo occurs in a perforin-dependent manner and does not require IFN-gamma or the presence of CD4(+)CD25(+) regulatory T cells. Most importantly, failure to eliminate DC had profound consequences on the CTL immune response. Perforin-deficient mice showed a progressive increase in the numbers of antigen-specific CD8(+) T cells after repeated immunizations with DC. In contrast, in control mice the number of antigen-specific CD8(+) T cells did not notably increase with repeated immunizations. Lastly, we also show that CTL-mediated elimination of DC occurs in peripheral tissues but not in the lymph node. Our data suggest that CTL act as "gatekeepers" that control access of antigen-loaded DC into the lymph node, thereby preventing continued expansion of antigen-specific T cells.

Inactivation of CD4+ CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load.

Mycobacterium tuberculosis uses numerous mechanisms to avoid elimination by the infected host. In this study, we investigated the possibility whether, similar to other pathogens, M. tuberculosis exploits natural CD4+ CD25+ T-regulatory cells (Treg) to suppress the effector function of responding host lymphocytes, thus enhancing its survival. During a Mycobacterium bovis bacille calmette guerin (BCG) pulmonary infection, we observed a 2.8-fold increase in forkhead box P3 (Foxp3+) CD25+ Treg in the lung. To inactivate the Treg in vivo, an mAb was given against CD25 (PC61) 3 days before a pulmonary infection with BCG or M. tuberculosis. Following PC61 treatment, we observed significantly decreased CD25 expression on CD4+ T lymphocytes for at least 23 days in the blood, spleen and lung when compared with the control mice. To determine whether Treg inactivation affected the protective antimycobacterial immune response, we measured cytokine production by flow cytometry. We observed small, but significant increases in the percentages of both IFN-gamma-producing and IL-2-producing CD4+ cells from the spleen and the IL-2-producing CD4+ cells from the lungs of PC61-treated BCG-infected mice compared with the infected control mice. Despite this, there was neither a difference between the lung bacterial burdens of PC61-treated mice and control mice, measured until day 44 postinfection, nor was there an effect on infection-induced lung pathology. Together, these data imply that the absence of natural Treg early after infection results in a small increase in cytokine production, but this does not alter the course of either M. tuberculosis or BCG infections. This contrasts with the important role that natural Treg play in the pathogenesis of many other intracellular infectious organisms.

Promiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells.

"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.

Dendritic cells loaded with stressed tumor cells elicit long-lasting protective tumor immunity in mice depleted of CD4+CD25+ regulatory T cells.

Dendritic cell (DC)-based vaccination represents a promising approach to harness the specificity and potency of the immune system to combat cancer. Finding optimal strategies for tumor Ag preparation and subsequent pulsing of DC, as well as improving the immunogenicity of weak tumor Ags remain among the first challenges of this approach. In this report, we use a prophylactic vaccine consisting of DC loaded with whole, nonmanipulated B16-F10 melanoma cells that had been stressed by heat shock and gamma irradiation. Stressed B16-F10 cells underwent apoptosis and were internalized by bone marrow-derived DC during coculture. Surprisingly, coculture of DC with stressed B16-F10 undergoing apoptosis and necrosis did not induce DC maturation. However, a marked retardation in tumor growth was observed in C57BL/6 mice immunized using DC loaded with stressed B16-F10 cells and subsequently challenged with B16-F10 cells. Growth retardation was further increased by treating DC with LPS before in vivo administration. In vivo depletion studies revealed that both CD8(+) and CD4(+) T cells played a critical role in retarding tumor growth. In addition, treatment with anti-CD25 Ab to deplete CD4(+)CD25(+) regulatory T cells before DC vaccination considerably improved the effect of the vaccine and allowed the development of long-lived immune responses that were tumor protective. Our results demonstrate that depletion of regulatory T cells is an effective approach to improving the success of DC-based vaccination against weakly immunogenic tumors. Such a strategy can be readily applied to other tumor models and extended to therapeutic vaccination settings.

Cutting edge: depletion of CD4+CD25+ regulatory T cells is necessary, but not sufficient, for induction of organ-specific autoimmune disease.

Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4(+)CD25(+) regulatory T cells. However, depletion of CD4(+)CD25(+) T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4(+)CD25(-) T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25(+) T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25(+) regulatory T cells.

The role of suppressor T cells in regulation of immune responses.

Suppressor T cells play important roles in the regulation of immune responses and the mediation of dominant immunologic tolerance. Studies of suppressor T-cell function have been hampered until their recent identification as a minor fraction (approximately 10%) of CD4 ( +) T cells that coexpress CD25. CD4(+)CD25(+ ) T cells have been shown to play a critical role in the prevention of organ- specific autoimmunity and allograft rejection. Because tumor antigens are self- antigens, it is not surprising that CD4(+)CD25(+) T cells also inhibit the induction of tumor immunity. The spectrum of activity of CD4(+ ) CD25(+) cells extends to non-self-antigens, including infectious agents. Indeed, T cell-mediated suppression might be responsible for the low level of chronic infection seen with many pathogens. Interestingly, however, this persistent level of infection might be beneficial to the host and needed for maintenance of immunologic memory. Although CD4(+ ) CD25(+) T cells are capable of inhibiting T(H)2 responses, their role in the suppression of allergic responses has not been firmly established. Depending on the desired immune response, enhancement or restraint of suppressor T-cell function might be required. Therefore immunologic or pharmacologic manipulation of regulatory T-cell populations represents an important future approach to immunotherapy of a wide range of immune responses.

Spontaneous organ-specific Th2-mediated autoimmunity in TCR transgenic mice.

CD4(+) T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines of TCR transgenic mice using receptors cloned from pathogenic Th1 or Th2 cells. We previously described spontaneous inflammatory AIG in A23 mice, caused by the transgenic expression of the TCR from a Th1 clone, TXA23. In this study we describe the generation of A51 mouse lines, transgenic for the TCR of a CD4(+) self-reactive Th2 clone, TXA51. A proportion of A51 mice spontaneously develop AIG by 10 wk of age, with a disease characterized by eosinophilic infiltration of the gastric mucosa and Th2 differentiation of transgenic T cells in the gastric lymph node. The Th2 phenotype of this autoimmune response seems to be related to a low availability of MHC class II-self peptide complexes. This in vivo model of spontaneous Th2-mediated, organ-specific autoimmunity provides a unique example in which the clonotypic TCR conveys the Th2 disease phenotype.

Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells.

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR-/- mice, we report that GITR engagement on CD25-, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR-/-CD25- T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25- T cells, rendering them resistant to CD25+ -mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.

CD4(+)CD25(+) immunoregulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effector T cell function. We analyzed CD4(+)CD25(+) and CD4(+)CD25(-) T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4(+)CD25(+) population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4(+)CD25(+) cells, including GITR, a member of the TNF receptor superfamily. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4(+)CD25(+) T cell subset.