Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling.

The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge.

The common prophylactic therapy for bowel surgery is ineffective for clearing Bacteroidetes, the primary inducers of systemic inflammation, and causes faster death in response to intestinal barrier damage in mice.

INTRODUCTION AND OBJECTIVE: The role of secreted gut microbial components in the initiation of systemic inflammation and consequences of antibiotic therapies on this inflammatory process are poorly elucidated. We investigate whether peripheral innate cells mount an inflammatory response to gut microbial components, the immune cells that are the primary drivers of systemic inflammation, the bacterial populations that are predominantly responsible, and whether perioperative antibiotics affect these processes. METHOD AND EXPERIMENTAL DESIGN: Conditioned supernatants from gut microbes were used to stimulate murine innate cell types in vitro and in vivo, and proinflammatory responses were characterised. Effects of antibiotic therapies on these responses were investigated using a model of experimental intestinal barrier damage induced by dextran sodium sulfate. RESULTS: Proinflammatory responses in the periphery are generated by components of anaerobes from the Bacteroidetes phylotype and these responses are primarily produced by myeloid dendritic cells. We found that the common prophylactic therapy for sepsis (oral neomycin and metronidazole administered to patients the day prior to surgery) is ineffective for clearing Bacteroidetes from the murine intestine. A point of critical consequence of this result is the increased systemic inflammation and premature death observed in treated mice, and these outcomes appear to be independent of gut bacterial spread in the initial phase of intestinal barrier damage. Importantly, spillage of gut microbial products, rather than dissemination of gut microbes, may underlay the initiation of systemic inflammation leading to death. CONCLUSIONS: Our data further affirm the importance of a balanced gut microflora biodiversity in host immune homeostasis and reinforce the notion that inadequate antibiotic therapy can have detrimental effects on overall immune system.

Skin inflammation arising from cutaneous regulatory T cell deficiency leads to impaired viral immune responses.

Individuals with atopic dermatitis immunized with the small pox vaccine, vaccinia virus (VV), are susceptible to eczema vaccinatum (EV), a potentially fatal disseminated infection. Dysfunction of Forkhead box P3 (FoxP3)-positive regulatory T cells (Treg) has been implicated in the pathogenesis of atopic dermatitis. To test whether Treg deficiency predisposes to EV, we percutaneously VV infected FoxP3-deficient (FoxP3(KO)) mice, which completely lack FoxP3(+) Treg. These animals generated both fewer VV-specific CD8(+) effector T cells and IFN-gamma-producing CD8(+) T cells than controls, had higher viral loads, and exhibited abnormal Th2-polarized responses to the virus. To focus on the consequences of Treg deficiency confined to the skin, we generated mixed CCR4(KO) FoxP3(KO) bone marrow (CCR4/FoxP3) chimeras in which skin, but not other tissues or central lymphoid organs, lack Treg. Like FoxP3(KO) mice, the chimeras had impaired VV-specific effector T cell responses and higher viral loads. Skin cytokine expression was significantly altered in infected chimeras compared with controls. Levels of the antiviral cytokines, type I and II IFNs and IL-12, were reduced, whereas expression of the proinflammatory cytokines, IL-6, IL-10, TGF-beta, and IL-23, was increased. Importantly, infection of CCR4/FoxP3 chimeras by a noncutaneous route (i.p.) induced immune responses comparable to controls. Our findings implicate allergic skin inflammation resulting from local Treg deficiency in the pathogenesis of EV.

Blocking CD27-CD70 costimulatory pathway suppresses experimental colitis.

The pathogenesis of human inflammatory bowel disease (IBD) and most experimental models of IBD is dependent on the activation and expansion of CD4(+) T cells via interaction with mucosal APCs. The costimulatory receptor CD70 is transiently expressed on the surface of conventional dendritic cells, but is constitutively expressed by a unique APC population in the intestinal lamina propria. We used two experimental IBD models to evaluate whether interfering the interaction between CD70 and its T cell ligand CD27 would affect the development of colitis. Adoptive transfer of naive CD27-deficient CD45RB(high) CD4(+) T cells into Rag-1(-/-) mice resulted in significantly less disease than when wild-type CD45RB(high)CD4(+) T cells were used. Moreover, a monoclonal anti-CD70 Ab prevented the disease caused by the transfer of wild-type CD45RB(high) CD4(+) T cells into Rag-1(-/-) mice and the same Ab also ameliorated an established disease. The colitis associated proinflammatory cytokines IL-6, TNF-alpha and IFN-gamma were significantly reduced after anti-CD70 Ab treatment, suggesting an overall reduction in inflammation due to blockade of pathogenic T cell expansion. Anti-CD70 Ab treatment also suppressed trinitrobenzene sulfonic acid-induced colitis in SJL/J mice. Because anti-CD70 Ab treatment suppressed multiple proinflammatory cytokines, this may be a more potent therapeutic approach for IBD than blockade of individual cytokines.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

Skin inflammation in RelB(-/-) mice leads to defective immunity and impaired clearance of vaccinia virus.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory skin disorder occurring in genetically predisposed individuals with a systemic T(H)2 bias. Atopic dermatitis patients exposed to the smallpox vaccine, vaccinia virus (VV), occasionally develop eczema vaccinatum (EV), an overwhelming and potentially lethal systemic infection with VV. OBJECTIVE: To establish a murine model of EV and examine the effects of skin inflammation on VV immunity. METHODS: The skin of RelB(-/-) mice, like that of chronic AD lesions in humans, exhibits thickening, eosinophilic infiltration, hyperkeratosis, and acanthosis. RelB(-/-) and wild-type (WT) control mice were infected with VV via skin scarification. Viral spread, cytokine levels, IgG2a responses and VV-specific T cells were measured. RESULTS: Cutaneously VV-infected RelB(-/-), but not WT mice, exhibited weight loss, markedly impaired systemic clearance of the virus and increased contiguous propagation from the inoculation site. This was associated with a dramatically impaired generation of IFN-gamma-producing CD8(+) vaccinia-specific T cells along with decreased secretion of IFN-gamma by VV-stimulated splenocytes. The T(H)2 cytokines-IL-4, IL-5, IL-13, and IL-10-on the other hand, were overproduced. When infected intraperitoneally, RelB(-/-) mice generated robust T cell responses with good IFN-gamma production. CONCLUSION: Allergic inflammation in RelB(-/-) mice is associated with dysregulated immunity to VV encountered via the skin. We speculate that susceptibility of AD patients to overwhelming vaccinia virus infection is similarly related to ineffective T cell responses. CLINICAL IMPLICATIONS: The susceptibility of patients with AD to EV following cutaneous contact with VV is related to ineffective antiviral immune responses.

Cutting Edge: Distinct NK receptor profiles are imprinted on CD8 T cells in the mucosa and periphery during the same antigen challenge: role of tissue-specific factors.

NK cell receptors (NKRs) modulate T lymphocyte responses by modifying the Ag activation threshold. However, what governs their expression on T cells remains unclear. In this study we show that different NKRs are imprinted on CD8 T cells in the gut mucosa and periphery during the same Ag challenge. After a viral, bacterial, and tumor challenge, most CD8 peritoneal exudate lymphocytes expressed NKG2A but not 2B4. In contrast, most CD8 intraepithelial lymphocytes exhibited 2B4 but not NKG2A. Our data suggest that tissue-specific factors may determine the pattern of NKR expression. In the gut, CD70 licensing appears to promote 2B4 induction on mucosal CD8 T cells. Conversely, retinoic acid produced by the intestinal dendritic cells may suppress NKG2A expression. Thus, tissue-specific factors regulate NKR expression and may confer T cells with differing effector functions in a tissue and site-specific manner.

CD70+ antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa.

One unresolved issue in gut immunity is how mucosal T lymphocytes are activated and which antigen-presenting cell (APC) is critical for the regulation of this process. We have identified a unique population of APCs that is exclusively localized in the lamina propria. These APCs constitutively expressed the costimulatory molecule CD70 and had antigen-presenting functions. After oral infection of mice with Listeria monocytogenes, proliferation and differentiation of antigen-specific T cells occurred in the gut mucosa in situ and blockade of CD70 costimulation abrogated the mucosal T cell proliferation and effector functions. Thus, a potent CD70-dependent stimulation via specialized tissue-specific APCs is required for the proliferation and differentiation of gut mucosal T cells after oral infection.

The gp49B1 inhibitory receptor regulates the IFN-gamma responses of T cells and NK cells.

The magnitude and diversity of Ag-specific T cell effector activity have been proposed to be controlled by an integration of positive signals transduced by the TCR and negative signals originating from inhibitory cell surface molecules. Although the lectin family of NK cell-associated inhibitory receptors has been reported to regulate the function of murine CTLs, gp49B1, the Ig superfamily member is not known to be expressed on T cells. Moreover, the consequences of the lack of an endogenously expressed NK cell-associated inhibitory receptor on T cell functions are not known. We report that gp49B1 is expressed by nearly all activated CD8 and CD4 T cells in addition to NK cells during an immune response to viral, bacterial, or tumor challenge. Kinetics of gp49B1 expression parallel functional capability and subside in the memory phase. Following vaccinia viral infection, IFN-gamma production by both subsets of T cells and NK cells is enhanced in gp49B1-deficient mice compared with gp49B1(+/+) mice. The stimulation threshold for IFN-gamma production is also lower in gp49B1-deficient T cells. In contrast, no significant differences were observed in the cytotoxic responses. We conclude that gp49B1 is a unique inhibitory receptor that is induced in multiple lineages of innate and adaptive immune cells during an infection and controls their IFN-gamma, but not cytotoxic responses.