Timothy P. Moran

Effector CD4+ T cells, the cytokines they generate, and GVHD: something old and something new

GVHD is a syndrome that results from minor and major histocompatibility complex incompatibilities between the donor and recipient. More than 50 years after its initial description, the pathophysiology of GVHD remains poorly understood. Nonetheless, donor T cells have been shown to be critical to the pathophysiology of acute and chronic GVHD, yet precisely how they function remains unclear. The effector mechanisms by which donor T cells mediate tissue inflammation is even less well understood. Identification of several new lineages of CD4(+) T cells made in the past decade and their roles in the pathophysiology of T cell-mediated diseases has shed new light on these effector mechanisms. In this review, we summarize the recent descriptions of these T-cell lineages and the current data supporting their role in acute and to a lesser extent chronic GVHD. Investigations into the activity of these new T-cell lineages may provide more rationale approaches to the treatment or prevention of GVHD.

A Novel Viral System for Generating Antigen-Specific T Cells

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with ∼50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-α treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-α, and IFN-α. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-α-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.

Increased Immunogenicity of a DNA-Launched Venezuelan Equine Encephalitis Virus-Based Replicon DNA Vaccine

ABSTRACT A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.

Is Clinical Tolerance Possible after Allergen Immunotherapy

There is a growing evidence that allergen immunotherapy (AIT) can provide significant and long-lasting clinical benefit for a number of allergic individuals. However, it is less clear if AIT results in clinical tolerance, which is characterized by a persistent state of clinical non-reactivity to allergens after therapy is finished. Addressing this knowledge gap is particularly relevant for patients undergoing AIT for food allergies, as anything less than complete tolerance could have potentially devastating consequences. An increasing number of studies, in particular those involving oral immunotherapy, are attempting to assess tolerance induction following AIT. Clinical tolerance does appear to be achievable in a subset of patients undergoing AIT, but whether this is equivalent to the type of tolerance observed in nonallergic individuals remains unknown. Developing established criteria for assessing tolerance induction, as well as the use of consistent terminology when describing clinical tolerance, will be important for determining the disease-modifying potential of AIT.

The immunosuppressive tumor environment is the major impediment to successful therapeutic vaccination in Neu transgenic mice.

We earlier showed that therapeutic vaccination of FVB/N mice with alphaviral replicon particles expressing rat neuET-VRP induced regression of established neu-expressing tumors. In this study, we evaluated the efficacy of neuET-VRPs in a tolerant mouse model using mice with transgenic expression of neu. Using the same approach that induced regression of 70 mm2 tumors in FVB/N mice, we were unable to inhibit tumor growth in tolerant neu-N mice, despite showing neu-specific B-cell and T-cell responses post vaccination. As neu-N mice have a limited T-cell repertoire specific to neu, we hypothesized that the absence of these T cells led to differences in the vaccine response. However, transfer of neu-specific T cells from vaccinated FVB/N mice was not effective in inducing tumor regression, as these cells did not proliferate in the tumor-draining lymph node. Vaccination given with low-dose cyclophosphamide to deplete regulatory T cells delayed tumor growth but did not result in tumor regression. Finally, we showed that T cells given with vaccination were effective in inhibiting tumor growth, if administered with approaches to deplete myeloid-derived suppressor cells. Our data show that both central deletion of lymphocytes and peripheral immunosuppressive mechanisms are present in neu-N mice. However, the major impediment to successful vaccination is the peripheral tumor-induced immune suppression.

TNF is required for TLR ligand–mediated but not protease-mediated allergic airway inflammation

Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma.

Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 ( Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs.

TH17 Cells, Proteins Associated with TH17 Polarization, and Their Role in Graft vs. Host Disease

Graft vs. host disease (GVHD) is mediated by donor T cells that recognize minor and major MHC disparities in the host and initiate an inflammatory cascade the results in tissue damage in specific GVHD target organs. Over the past 20 years, multiple different T cell subsets under control of specific transcription factors have been described. Of these, the TH1 pathway in which T cells generate IFNγ under control of the transcription factor T-bet has been most closely associated with GVHD. Quite recently, several new T cell subsets have been discovered. TH17 cells are a recently defined subset in which the cells are differentiated in the presence of IL-6, TGF-β1, and IL-1β and expanded by the cytokine IL-23. RORγt and RORα are the critical transcription factors necessary for the generation of TH17 cells. Here, we have reviewed the current data on the roles of TH17 cells and the proteins critical for their generation in the pathogenesis of acute and chronic GVHD. Both data from murine models and clinical studies have been evaluated. While a definitive role for TH17 cells remains to be established, current data suggests that TH1/TH17 cells together may be critical mediators of tissue pathology during GVHD.