David A. Horwitz

OBJECTIVE: Based on a small clinical series and previously published case reports, concordance for systemic lupus erythematosus (SLE) among monozygous (MZ) twins has been reported to be as high as 69%. Using a larger and less biased sample, we provide another estimate of this percentage. METHODS: We established a registry of twins with SLE, based upon self-reports and information provided by the patients' physicians. We used DNA fingerprinting to validate the reported zygosity in a sample of these twins. RESULTS: Of 107 twin pairs meeting the American College of Rheumatology 1982 revised criteria for the diagnosis of SLE, 24% of 45 MZ pairs and 2% of 62 dizygous (DZ) pairs were concordant. The frequency distributions of diagnostic criteria and disease symptoms in the SLE patients were similar to those in other published reports of SLE patients. Zygosity was confirmed by DNA fingerprinting in a subsample of 15 self-described MZ twins and 7 self-described DZ twins. All individuals had correctly predicted their zygosity. CONCLUSION: MZ concordance for SLE is similar to that for other autoimmune diseases and is much lower than previously believed.

Thymus-derived, natural CD4(+)CD25(+) regulatory T cells can educate peripheral CD4(+)CD25(-) cells to develop suppressive activity by poorly understood mechanisms. TGF-beta has IL-2-dependent costimulatory effects on alloactivated naive, human CD4(+) T cells and induces them ex vivo to become potent contact-dependent, cytokine-independent suppressor cells. In this study, we report that CD4(+)CD25(+) cells are the targets of the costimulatory effects of IL-2 and TGF-beta. These cells do not divide, but, instead, greatly increase the numbers of CD4(+)CD25(-) cells that become CD25(+) cytokine-independent suppressor cells. These CD4(+)CD25(+) regulatory cells, in turn, induce other alloactivated CD4(+)CD25(-) cells to become potent suppressor cells by mechanisms that, surprisingly, require both cell contact and TGF-beta and IL-10. The suppressive effects of these secondary CD4(+)CD25(+) cells depend upon TGF-beta and IL-10. Moreover, both the naive CD4(+) cells induced by IL-2 and TGF-beta to become suppressor cells, and the subsequent CD4(+)CD25(-) cells educated by them to become suppressors express FoxP3. We suggest that the long-term effects of adoptively transferred natural-like CD4(+)CD25(+) regulatory cells induced ex vivo are due to their ability to generate new cytokine-producing CD4(+) regulatory T cells in vivo.

IL-2 and TGF-beta both have important roles in the induction and maintenance of immunologic tolerance, but whether these cytokines act separately or together to achieve this effect is poorly understood. Although others have reported that IL-2 can directly enhance forkhead box protein P3 (Foxp3) transcription factor expression by natural CD4(+)CD25(+) regulatory T cells, in this study, we report that the role of IL-2 on the generation of peripheral regulatory CD4(+) cells is indirect. Ab neutralization studies and experiments with IL-2-deficient mice have revealed that IL-2 is required for TGF-beta to induce naive CD4(+)CD25(-) cells to become CD25(+) and express Foxp3, and develop the characteristic properties of CD4(+)CD25(+) regulatory cells. This effect of IL-2 on the generation and expansion of these adaptive Foxp3(+) regulatory cells is nonredundant, but IL-4, IL-7, and IL-15, other common gamma-chain cytokines, could sustain Foxp3 expression. Because subjects with autoimmune diseases often have defects in the production of IL-2 and/or TGF-beta, the generation of autologous T regulatory cells ex vivo with these cytokines for transfer in vivo may have considerable therapeutic potential.

An elusive goal in transplanting organs across histocompatibility barriers has been the induction of specific tolerance to avoid graft rejection. A considerable body of evidence exists that the thymus produces regulatory T cells that suppress the response of other T cells to antigenic stimulation. We report that TGF-beta can induce certain CD4+ T cells in the naive (CD45RA+RO-) fraction in human peripheral blood to develop powerful, contact-dependent suppressive activity that is not antagonized by anti-TGF-beta or anti-IL-10 mAbs. The costimulatory effects of TGF-beta on naive CD4+ T cells up-regulated CD25 and CTLA-4 expression, increased their transition to the activated phenotype, but decreased activation-induced apoptosis. Suppressive activity was concentrated in the CD25+ fraction. These CD4+CD25+ regulatory cells prevented CD8+ T cells from proliferating in response to alloantigens and from becoming cytotoxic effector cells. Moreover, these regulatory cells exerted their suppressive activities in remarkably low numbers and maintained these effects even after they are expanded. Once activated, their suppressive properties were Ag nonspecific. Although <1% of naive CD4+ T cells expressed CD25, depletion of this subset before priming with TGF-beta markedly decreased the generation of suppressive activity. This finding suggests that CD4+CD25+ regulatory T cells induced ex vivo are the progeny of thymus-derived regulatory T cells bearing a similar phenotype. The adoptive transfer of these regulatory T cells generated and expanded ex vivo has the potential to prevent rejection of allogeneic organ grafts.

Previously we reported that TGF-beta has an important role in the generation and expansion of human "professional" CD4(+)CD25(+) regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF-beta induces activated CD4(+)CD25(-) T cells to become Th3 suppressor cells. While stimulating CD4(+) cells with TGF-beta modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF-beta the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF-beta-primed CD4(+) T cells to fresh CD4(+) cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF-beta and was also partially contact dependent. Increased TGF-beta production was associated with a decreased production of IFN-gamma and IL-10. Depletion studies revealed that the precursors of these TGF-beta-producing CD4(+) suppressor cells were CD25 negative. These studies provide evidence that CD4(+)CD25(+) regulatory cells in human blood consist of at least two subsets that have TGF-beta-dependent and independent mechanisms of action. TGF-beta has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4(+) and CD8(+) cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection.