C. Garrison Fathman

A novel transcription factor, T-bet, directs Th1 lineage commitment.

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.

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation.

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) α-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.

Donor-type CD4+CD25+ Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation

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 CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens.

We previously described a mechanism for the maintenance of peripheral self-tolerance. This involves the cross-presentation of tissue-associated antigens by a bone marrow–derived cell type that stimulates the proliferation and ultimate deletion of self-reactive CD8 T cells. This process has been referred to as cross-tolerance. Here, we characterize the elusive cell type responsible for inducing cross-tolerance as a CD8α+ dendritic cell (DC). To achieve this aim, transgenic mice were generated expressing yellow fluorescent protein (YFP) linked to CTL epitopes for ovalbumin and glycoprotein B (gB) of herpes simplex virus under the rat insulin promoter (RIP). Although tracking of YFP was inconclusive, the use of a highly sensitive gB-specific hybridoma that produced β-galactosidase on encounter with antigen, enabled detection of antigen presentation by cells isolated from the pancreatic lymph node. This showed that a CD11c+CD8α+ cell was responsible for cross-tolerance, the same DC subset as previously implicated in cross-priming. These data indicate that CD8α+ DCs play a critical role in both tolerance and immunity to cell-associated antigens, providing a potential mechanism by which cytotoxic T lymphocyte can be immunized to viral antigens while maintaining tolerance to self.

The Subpopulation of CD4+CD25+ Splenocytes That Delays Adoptive Transfer of Diabetes Expresses L-Selectin and High Levels of CCR7

Recently, CD4+CD25+ T cells have been implicated in the control of diabetes, suggesting that the inflamed islets of Langerhans in prediabetic NOD mice are under peripheral immune surveillance. Here we show that CD4+CD25+ splenocytes inhibit diabetes in cotransfer with islet-infiltrating cells. Furthermore, CD62L expression is necessary for this disease-delaying effect of CD4+CD25+ cells in vivo, but not for their suppressor function in vitro. We demonstrate that the CD4+CD25+CD62L+ splenocytes express CCR7 at high levels and migrate toward secondary lymphoid tissue chemokine and ELC (macrophage-inflammatory protein-3β), lymphoid chemokines, whereas CD4+CD25+CD62L− splenocytes preferentially express CCR2, CCR4, and CXCR3 and migrate toward the corresponding inflammatory chemokines. These data demonstrate that CD4+CD25+CD62L+, but not CD4+CD25+CD62L−, splenocytes delay diabetes transfer, and that CD4+CD25+ suppressor T cells are comprised of at least two subpopulations that behave differently in cotransfer in vivo and express distinct chemokine receptor and chemotactic response profiles despite demonstrating equivalent suppressor functions in vitro.

GRAIL: An E3 Ubiquitin Ligase that Inhibits Cytokine Gene Transcription Is Expressed in Anergic CD4+ T Cells

T cell anergy may serve to limit autoreactive T cell responses. We examined early changes in gene expression after antigen-TCR signaling in the presence (activation) or absence (anergy) of B7 costimulation. Induced expression of GRAIL (gene related to anergy in lymphocytes) was observed in anergic CD4(+) T cells. GRAIL is a type I transmembrane protein that localizes to the endocytic pathway and bears homology to RING zinc-finger proteins. Ubiquitination studies in vitro support GRAIL function as an E3 ubiquitin ligase. Expression of GRAIL in retrovirally transduced T cell hybridomas dramatically limits activation-induced IL-2 and IL-4 production. Additional studies suggest that GRAIL E3 ubiquitin ligase activity and intact endocytic trafficking are critical for cytokine transcriptional regulation. Expression of GRAIL after an anergizing stimulus may result in ubiquitin-mediated regulation of proteins essential for mitogenic cytokine expression, thus positioning GRAIL as a key player in the induction of the anergic phenotype.

Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'

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. Considerable effort has been invested in 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 IL-2Rγ. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 ‘superkine’ (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine 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, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. 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 T cell–mediated gene therapy in collagen-induced arthritis

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

Adoptive Immunotherapy of Experimental Autoimmune Encephalomyelitis Via T Cell Delivery of the IL-12 p40 Subunit

CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of “regulatory” cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-γ production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

Autoimmune diseases: genes, bugs and failed regulation

In this Overview, common themes of the accompanying News &Views on RA, SLE, IDDM, thyroiditis and MS are discussed. A unifying concept for the development of these and other autoimmune diseases should incorporate genetic predisposition, environmental factors and immune dysregulation.