We show that a combination of the immunosuppressive drugs, vitamin D3 and Dexamethasone, induced human and mouse naive CD4+ T cells to differentiate in vitro into regulatory T cells. In contrast to the previously described in vitro derived CD4+ T cells, these cells produced only interleukin (IL)-10, but no IL-5 and interferon (IFN)-γ, and furthermore retained strong proliferative capacity. The development of these IL-10–producing cells was enhanced by neutralization of the T helper type 1 (Th1)- and Th2–inducing cytokines IL-4, IL-12, and IFN-γ. These immunosuppressive drugs also induced the development of IL-10–producing T cells in the absence of antigen-presenting cells, with IL-10 acting as a positive autocrine factor for these T cells. Furthermore, nuclear factor (NF)-κB and activator protein (AP)-1 activities were inhibited in the IL-10–producing cells described here as well as key transcription factors involved in Th1 and Th2 subset differentiation. The regulatory function of these in vitro generated IL-10–producing T cells was demonstrated by their ability to prevent central nervous system inflammation, when targeted to the site of inflammation, and this function was shown to be IL-10 dependent. Generating homogeneous populations of IL-10–producing T cells in vitro will thus facilitate the use of regulatory T cells in immunotherapy.

Genetic differences in immune responses may affect susceptibility to mycobacterial infection, but no specific genes have been implicated in humans. We studied four children who had an unexplained genetic susceptibility to mycobacterial infection and who appeared to have inherited the same recessive mutation from a common ancestor. We used microsatellite analysis, immunofluorescence studies, and sequence analysis to study the affected patients, unaffected family members, and normal controls. A genome search using microsatellite markers identified a region on chromosome 6q in which the affected children were all homozygous for eight markers. The gene for interferon-γ receptor 1 maps to this region. Immunofluorescence studies showed that the receptor was absent on leukocytes from the affected children. Sequence analysis of complementary DNA for the gene for interferon- γ receptor 1 revealed a point mutation at nucleotide 395 that introduces a stop codon and results in a truncated protein that lacks the transmembrane and cytoplasmic domains. Four children with severe mycobacterial infections had a mutation in the gene for interferon-γ receptor 1 that leads to the absence of receptors on cell surfaces and a functional defect in the up-regulation of tumor necrosis factor α by macrophages in response to interferon-γ. The interferon-γ pathway is important in the response to intracellular pathogens such as mycobacteria.

We previously reported that human CD4⁺ Tregs secrete high levels of IL-10 when stimulated in the presence of dexamethasone and calcitriol (vitamin D3). We now show that following stimulation by allergen, IL-10–secreting Tregs inhibit cytokine secretion by allergen-specific Th2 cells in an IL-10–dependent manner. A proportion of patients with severe asthma fail to demonstrate clinical improvement upon glucocorticoid therapy, and their asthma is characterized as glucocorticoid resistant (SR, abbreviation derived from “steroid resistant”). Dexamethasone does not enhance secretion of IL-10 by their CD4⁺ T cells. Addition of vitamin D3 with dexamethasone to cultures of SR CD4⁺ T cells enhanced IL-10 synthesis to levels observed in cells from glucocorticoid-sensitive patients cultured with dexamethasone alone. Furthermore, pretreatment with IL-10 fully restored IL-10 synthesis in these cells in response to dexamethasone. Vitamin D3 significantly overcame the inhibition of glucocorticoid-receptor expression by dexamethasone while IL-10 upregulated glucocorticoid-receptor expression by CD4⁺ T cells, suggesting potential mechanisms whereby these treatments may overcome poor glucocorticoid responsiveness. We show here that administration of vitamin D3 to healthy individuals and SR asthmatic patients enhanced subsequent responsiveness to dexamethasone for induction of IL-10. This strongly suggests that vitamin D3 could potentially increase the therapeutic response to glucocorticoids in SR patients.

Wheezing illnesses begin as early as the first few weeks of life, suggesting that there may be prenatal determinants involved with the genesis of these illnesses. Although wheezing and other asthmalike symptoms are common in the preschool ages, it remains difficult to predict which of these preschoolers will have asthma that persists through midchildhood and onward. Nevertheless, preschool children with wheezing have lung function deficits that persist through later life.^1,2 Thus, prenatal interventions that prevent early recurrent wheezing may be important to reduce the short-term morbidity and to prevent the potential long-term sequelae of reduced lung growth.