Naive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNgamma. T-bet expression correlates with IFNgamma expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNgamma gene and induces endogenous IFNgamma production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNgamma and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs.

Homopolymers or peptides containing a high percentage of cationic amino acids have been shown to have a unique ability to cross the plasma membrane of cells, and consequently have been used to facilitate the uptake of a variety of biopolymers and small molecules. To investigate whether the polycationic character of these molecules, or some other structural feature, was the molecular basis for the effect, the ability of a variety of homopolymers to enter cells was assayed by confocal microscopy and flow cytometry. Polymers of L- or D-arginine containing six or more amino acids entered cells far more effectively than polymers of equal length composed of lysine, ornithine and histidine. Peptides of fewer than six amino acids were ineffective. The length of the arginine side-chain could be varied without significant loss of activity. These data combined with the inability of polymers of citrulline to enter cells demonstrated that the guanidine headgroup of arginine was the critical structural component responsible for the biological activity. Cellular uptake could be inhibited by preincubation of the cells with sodium azide, but not by low temperature (3 degrees C), indicating that the process was energy dependent, but did not involve endocytosis.

Acute graft-versus-host disease (aGVHD) is still a major obstacle in clinical allogeneic bone marrow (BM) transplantation. CD4+CD25+ regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4+CD25− T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD. Here we show that CD4+CD25+ T cells isolated from the spleen or BM of donor C57BL/6 (H-2b) mice that have not been tolerized are still potent inhibitors of the alloresponse in vitro and of lethal aGVHD induced by C57BL/6 CD4+CD25− T cells in irradiated BALB/c (H-2d) hosts in vivo. The addition of the CD4+CD25+ Treg cells at a 1:1 ratio with responder/inducer CD4+CD25− T cells resulted in a >90% inhibition of the mixed leukocyte reaction and marked protection from lethal GVHD. This protective effect depended in part on the ability of the transferred CD4+CD25+ T cells to secrete interleukin 10 and occurred if the Treg cells were of donor, but not host, origin. Our results demonstrate that the balance of donor-type CD4+CD25+ Treg and conventional CD4+CD25− T cells can determine the outcome of aGVHD.

The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells 1 – 3 . Considerable effort has been invested using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary “high affinity” receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ, and γ c 4 – 8 . Naïve T cells express only a low density of IL-2Rβ and γ c , and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ andγ c . Here, using in vitro evolution, we eliminated IL-2’s functional requirement for CD25 expression by engineering an IL-2 “superkine” (termed super-2) with increased binding affinity for IL-2Rβ. Crystal structures of super-2 in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, including a flexible helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in super-2 recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation T cells irrespective of CD25 expression. Compared to IL-2, super-2 induced superior expansion of cytotoxic T cells, leading to improved anti-tumor responses in vivo , and elicited proportionally less expansion of T regulatory cells and reduced pulmonary edema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.