James R. Oliver

Late Developmental Plasticity in the T Helper 17 Lineage

Development of T helper (Th) 17 cells requires transforming growth factor (TGF)-beta and interleukin (IL)-6 and is independent of the Th1 pathway. Although T cells that produce interferon (IFN)-gamma are a recognized feature of Th17 cell responses, mice deficient for STAT4 and T-bet-two prototypical Th1 transcription factors-are protected from autoimmunity associated with Th17 pathogenesis. To examine the fate and pathogenic potential of Th17 cells and origin of IFN-gamma-producing T cells that emerge during Th17 immunity, we developed IL-17F reporter mice that identify cells committed to expression of IL-17F and IL-17A. Th17 cells required TGF-beta for sustained expression of IL-17F and IL-17A. In the absence of TGF-beta, both IL-23 and IL-12 acted to suppress IL-17 and enhance IFN-gamma production in a STAT4- and T-bet-dependent manner, albeit with distinct efficiencies. These results support a model of late Th17 developmental plasticity with implications for autoimmunity and host defense.

Regulatory T cell suppression and anergy are differentially regulated by proinflammatory cytokines produced by TLR-activated dendritic cells

CD25+CD4+ regulatory T cells (Tregs) are required for the maintenance of peripheral tolerance to certain self Ags. In this study, the requirements for murine Treg-suppressive activity and proliferation were examined in the context of the maturation of myeloid dendritic cells (DCs). We find that the suppressive function of Tregs is critically dependent on immature DCs and is readily reversed by the maturation of DCs induced by GM-CSF, but does not require TLR activation of either DCs or Tregs. In contrast, reversal of Treg anergy is dependent on TLR activation of DCs, and involves the potentiation of Treg responsiveness to IL-2 by cooperative effects of IL-6 and IL-1, both of which are produced by TLR-activated, mature DCs. Thus, proinflammatory cytokines produced by TLR-activated, mature DCs are required for reversal of Treg anergy, but are not required to overcome Treg suppression.

Memory CD4 T cells emerge from effector T-cell progenitors

A hallmark of adaptive immunity is the generation of memory T cells that confer long-lived, antigen-specific protection against repeat challenges by pathogens. Understanding the mechanisms by which memory T cells arise is important for rational vaccination strategies and improved therapeutic interventions for chronic infections and autoimmune disorders. The large clonal expansion of CD8 T cells in response to some infections has made the development of CD8 T-cell memory more amenable to study, giving rise to a model of memory cell differentiation in which a fraction of fully competent effector T cells transition into long-lived memory T cells. Delineation of CD4 T-cell memory development has proved more difficult as a result of limitations on tracking the smaller populations of CD4 effector T cells generated during a pathogenic challenge, complicating efforts to determine whether CD4 memory T cells are direct descendants of effector T cells or whether they develop by alternative pathways. Here, using two complementary cytokine reporter mouse models to identify interferon (IFN)-γ-positive effector T cells and track their fate, we show that the lineage relationship between effector and memory CD4 T cells resembles that for CD8 T cells responding to the same pathogen. We find that, in parallel with effector CD8 T cells, IFN-γ-positive effector CD4 T cells give rise to long-lived memory T cells capable of anamnestic responses to antigenic rechallenge.

T Helper 1 and T Helper 2 Cells Are Pathogenic in an Antigen-specific Model of Colitis

Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2−/− TCR transgenic mice were transferred into RAG-2−/− recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2−/− T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.

Lineage-specific Effects of 1,25-Dihydroxyvitamin D3 on the Development of Effector CD4 T Cells

Vitamin D deficiency is implicated in autoimmune disease. We therefore evaluated the effects of 1α,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D, on the development of T helper 1 (Th1), Th17, and Th9 cells, which are implicated in the pathogenesis of different types of autoimmunity. 1,25-D3 compromised the development of Th17 and Th9 cells, including IL-22-expressing cells while simultaneously increasing the frequency of IL-10-competent cells. Relative to Th17 and Th9 cells, the effects of 1,25-D3 on Th1 cells were modest, reflecting the significantly reduced levels of the receptor for vitamin D in this lineage. The use of cells deficient in IL-10 or antibodies that block IL-10 signaling abolished the inhibitory effect of 1,25-D3 on Th9 cells but had no effect on inhibition of Th17 cell frequencies. Thus, the induction of IL-10 in cultures of Th9 cells is an important mechanism by which 1,25-D3 compromises Th9 development but does not explain inhibition of Th17 cells. A survey of select representatives of the Th17 transcriptome revealed that the levels of mRNA that encode RORγt, IL-17A, IL-17F, IL-23R, and IL-22, were reduced by 1,25-D3, whereas IL-21 and aryl hydrocarbon receptor mRNA remained unchanged. These data suggest that vitamin D deficiency may promote autoimmunity by favoring the inordinate production of Th17 and Th9 cells at the expense of regulatory IL-10-producing T cells.

Interleukin-2 expression by a subpopulation of primary T cells is linked to enhanced memory/effector function.

Single cell studies have identified intraclonal heterogeneity of cytokine production by activated T cells. To investigate implications of cytokine heterogeneity for cell fate, an interleukin (IL)-2 promoter-green fluorescent protein (GFP) reporter transgenic model was developed to track IL-2+ and IL-2- T cells during differentiation from naive precursors. Antigen-activated IL-2+ and IL-2- cells had comparable proliferative capacities in primary responses. However, T cells that expressed IL-2 in primary responses demonstrated enhanced antigenic sensitivity and increased expression of effector cytokines in secondary responses in vitro and in vivo. Thus, heterogeneity of activation during a primary response translates into heterogeneous secondary responses, in which enhanced memory/effector function is linked to cells that previously exceeded an activation threshold associated with IL-2 gene transcription.

Restricted Clonal Expression of IL-2 By Naive T Cells Reflects Differential Dynamic Interactions with Dendritic Cells

Limited frequencies of T cells express IL-2 in primary antigenic responses, despite activation marker expression and proliferation by most clonal members. To define the basis for restricted IL-2 expression, a videomicroscopic system and IL-2 reporter transgenic model were used to characterize dendritic cell (DC)–T cell interactions. T cells destined to produce IL-2 required prolonged interactions with DCs, whereas most T cells established only transient interactions with DCs and were activated, but did not express IL-2. Extended conjugation of T cells with DCs was not always sufficient to initiate IL-2 expression. Thus, there is intrinsic variability in clonal T cell populations that restricts IL-2 commitment, and prolonged engagement with mature DCs is necessary, but not sufficient, for IL-2 gene transcription.

Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model

BackgroundGene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor.ResultsTo render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCARΔcyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCARΔcyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCARΔcyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCARΔcyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCARΔcyt with high efficiency. Expression of hCARΔcyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCARΔcyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization.ConclusionThe DO11.hCARΔcyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation.

Delayed thromboxane or tumor necrosis factor-α, but not leukotriene inhibition, attenuates prolonged pulmonary hypertension in endotoxemia

ABSTRACT: The early phase of endotoxin‐induced acute hemodynamic disturbances and hypoxemia is mediated by various factors, including eicosanoids and tumor necrosis factor‐&agr; (TNF&agr;). Thromboxane A2 is the major mediator of the early pulmonary hypertension associated with endotoxemia, but the mechanisms underlying the prolonged hemodynamic disturbances observed in ongoing endotoxemia are not well understood. The authors used a chronically instrumented young piglet model to determine the roles of several eicosanoids and of TNF&agr; in the prolonged endotoxin‐induced pulmonary hypertension and other cardiovascular derangements. Animals were given 40 &mgr;g/kg endotoxin intravenously per hour for 30 minutes, followed by 20 &mgr;g/kg per hour. In all animals, persistent pulmonary hypertension, lowered cardiac output, and hypoxemia developed during endotoxin infusion. After 3 hours of endotoxin infusion, randomly ordered infusions of 1 mg/kg dazmegrel (a thromboxane A2 synthesis inhibitor), 5 mg/kg nordihydroguaiaretic acid (a 5‐lipoxygenase inhibitor), and 20 mg/kg pentoxifylline (a TNF&agr; inhibitor) were given intravenously at 30‐ to 60‐minute intervals. Dazmegrel and pentoxifylline lowered pulmonary arterial pressure and resistance and raised arterial oxygen tension. Cardiac output increased significantly after pentoxifylline. These hemodynamic effects persisted for 30‐60 minutes, despite continued endotoxin infusion. The elevated plasma concentrations of thromboxane B2 and TNF&agr; returned toward preendotoxin baseline values after dazmegrel and pentoxifylline treatment, respectively. No beneficial effects were noted after administration of nordihydroguaiaretic acid. Based on these results, both thromboxane A2 and TNF&agr;, but not 5‐lipoxygenase products, play active roles in prolonged endotoxin‐induced pulmonary hypertension and hypoxemia in young piglets. Combined thromboxane A2 and TNF&agr; blockade may be clinically useful in treatment of advanced sepsis in neonates.

Thromboxane synthesis inhibition reverses group B Streptococcus-induced pulmonary hypertension.

Group B Streptococcus (GBS) sepsis in humans may cause the persistent pulmonary hypertension syndrome. Infusions of GBS in animals elevate pulmonary artery pressure (PAP) and resistance and are associated with elevated thromboxane levels. We investigated the hemodynamic effects of the specific thromboxane synthesis inhibitor, dazmegrel, in a piglet model of GBS-induced pulmonary hypertension. PAP rose from 22 ± 6 to 42 ± 11 (SD) mm Hg during infusion of heat-killed GBS; pulmonary vascular resistance increased from 1440 ± 400 to 4000 ± 1040 dyne sec/cm5. No significant changes in cardiac output, mean arterial pressure, or left atrial pressure were noted. Treatment with 1 mg/ kg of dazmegrel resulted in a rapid return of PAP and resistance to control values. No other hemodynamic effects of either bacteria or drug were observed despite continued infusion of GBS.