Advancements on phenotypic and functional characterization of non-antigen-specific CD8+CD28- regulatory T cells.

Among the different regulatory T lymphocyte (Treg) subpopulations, non-antigen-specific CD8+CD28- Treg (CD8+CD28- Treg) have been characterized for being involved in the pathogenesis of autoimmune diseases and cancer. A better phenotypic and functional characterization of this regulatory T-cell subset could help in identifying modulators of their activity with therapeutic finalities. The results of the present work show that Foxp3, a transcriptional marker of natural CD4+CD25+ Treg, is not expressed by CD8+CD28- Treg, thus indicating different origin and pathways of function for the latter with respect to the former regulatory cell type. Moreover, the results underline that the glucocorticoid induced TNF receptor is involved in generation processes but not in suppressor function of CD8+CD28- Treg. Phenotypic analyses demonstrate that, during their commitment from circulating nonregulatory CD8+CD28- T lymphocytes to Treg (an interleukin-10-dependent process), these cells downmodulate the IL7-receptor, thus differentiating them from long-lived, memory CD8+ T lymphocytes. Interestingly, CD8+CD28- Treg have been found to be resistant to the inhibitory effects of methylprednisolone, one of the most frequently administered corticosteroid drug used in therapy for immunosuppressive purposes.

Endocrine regulation of suppressor lymphocytes: role of the glucocorticoid-induced TNF-like receptor.

Mechanisms responsible for peripheral immune tolerance are currently under investigation in several laboratories, in order to define the role of immune homeostasis in physiological processes and pathologic conditions, such as autoimmunity and cancer. In this context, recent studies attributed a relevant role to the glucocorticoid-induced TNFR-related gene (GITR). GITR is expressed at high levels on CD4(+)CD25(+). T regulatory (Treg) cells, but only at low levels on resting responder T lymphocytes, and is upregulated after activation. GITR triggering induces both pro- and anti-apoptotic effects through different intracellular pathways, abrogates the suppressive activity of Treg cells, and co-stimulates responder T cells. These data hint that GITR triggering overstimulates the immune system. Indeed, in vivo studies demonstrated that GITR stimulation may both induce autoimmune diseases and strengthen anti-virus and anti-tumor immune responses. Therefore, the GITR-GITRL system appears crucial in regulating immunity. Currently, the majority of studies about GITR's role on regulatory cells are focused on CD4(+)CD25(+) Treg cells, while very little is known about the importance of this molecule in other Treg subtypes. We have recently characterized a subpopulation of CD8+ T suppressor lymphocytes able to inhibit both T cell proliferation and cytotoxicity. Preliminary data show that GITR is expressed on such CD8+ T suppressor cells and that its activation by a specific antibody inhibits generation, but not function, of these cells. These early results suggest the importance of GITR in human T suppressor lymphocytes other than CD4(+)CD25(+) Treg cells.

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers.

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.

Different consequences of EGR2 mutants on the transactivation of human Cx32 promoter.

The early growth response 2 (EGR2) transcription factor plays a crucial role in peripheral nerve myelination. Mutations of this gene are associated with a wide variety of demyelinating neuropathies differing from each other in the severity of nerve injury. Although the expression of EGR2 mutants inhibits the transactivation of myelin gene promoters, the exact molecular mechanism by which these mutations cause the alteration of the myelination process is still unknown. Recently, it was reported that EGR2 is directly involved in the transcriptional regulation of Connexin 32, a myelin gene frequently mutated in peripheral neuropathies. Here we describe the differential effect of two EGR2 mutants; while mutant D355V partially induces Cx32 promoter, mutant R381H does not. Furthermore, we show that a sequence located at -216, recognized by the wild-type and the mutant D355V recombinant proteins, is relevant for promoter transactivation.

Search for mutations in the EGR2 corepressor proteins, NAB1 and NAB2, in human peripheral neuropathies.

EGR2/Krox-20, a Cys2-His2 zinc finger transcription factor, plays an essential role in the regulation of myelination in the peripheral nervous system. Dominant and recessive mutations in EGR2 are associated with peripheral myelinopathies, such as Charcot-Marie-Tooth disease type 1, Dejerine-Sottas syndrome, and congenital hypomyelinating neuropathy. One recessive mutation (I268N) is known to affect the inhibitory domain that binds the NAB transcriptional corepressors, NAB1 and NAB2. This mutation abolishes the interaction of EGR2 with the NAB corepressors and thereby increases transcriptional activity. Therefore, we hypothesized that mutations in the EGR2-interacting domains of NAB1 and NAB2 might be associated with the pathogenesis of inherited peripheral neuropathies in currently unexplained cases. However, screening 87 such cases failed to identify any disease-causing mutations within the EGR2-interacting domains of either NAB1 or NAB2. A further mutation analysis of the complete coding regions of NAB1 and NAB2 in these genomic DNA samples did not uncover any disease-causing mutation. Therefore, our analysis indicates that mutations in the human NABI and NAB2 genes are most likely not involved in the pathogenesis of peripheral neuropathies.