Yonghong Wan

TNF-α is a critical negative regulator of type 1 immune activation during intracellular bacterial infection

TNF-alpha has long been regarded as a proimmune cytokine involved in antimicrobial type 1 immunity. However, the precise role of TNF-alpha in antimicrobial type 1 immunity remains poorly understood. We found that TNF-alpha-deficient (TNF(-/-)) mice quickly succumbed to respiratory failure following lung infection with replication-competent mycobacteria, because of apoptosis and necrosis of granuloma and lung structure. Tissue destruction was a result of an uncontrolled type 1 immune syndrome characterized by expansion of activated CD4 and CD8 T cells, increased frequency of antigen-specific T cells, and overproduction of IFN-gamma and IL-12. Depletion of CD4 and CD8 T cells decreased IFN-gamma levels, prevented granuloma and tissue necrosis, and prolonged the survival of TNF(-/-) hosts. Early reconstitution of TNF-alpha by gene transfer reduced the frequency of antigen-specific T cells and improved survival. TNF-alpha controlled type 1 immune activation at least in part by suppressing T cell proliferation, and this suppression involved both TNF receptor p55 and TNF receptor p75. Heightened type 1 immune activation also occurred in TNF(-/-) mice treated with dead mycobacteria, live replication-deficient mycobacteria, or mycobacterial cell wall components. Our study thus identifies TNF-alpha as a type 1 immunoregulatory cytokine whose primary role, different from those of other type 1 cytokines, is to keep an otherwise detrimental type 1 immune response in check.

Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-β

BACKGROUND Administration of donor bone marrow (BM)-derived dendritic cell (DC) progenitors (DCp) that are major histocompatibility complex (MHC) class II+ but costimulatory molecule (CD40, CD80, CD86)-deficient can prolong mouse heart allograft survival This is associated with microchimerism and inhibition of antidonor cytotoxic T lymphocyte (CTL) activity. Genetic modification of these donor antigen-presenting cells to express an immunosuppressive molecule(s) may enhance their in vivo survival and potential tolerogenicity. METHODS The surface phenotype of B10(H-2b) DCp before and after gene transfer using replication-deficient adenoviral (Ad) vectors was determined by monoclonal antibody (mAb) staining and flow cytometry. Transforming growth factor-beta (TGF-beta) production was quantitated by enzyme-linked immunosorbent assay. Allostimulatory activity of the gene-transduced DCp was ascertained by mixed leukocyte reaction (MLR) and CTL induction. To assess their in vivo migratory activity and survival, the transduced cells were injected subcutaneously into one hind footpad of C3H (H-2k) mice. Tissues (draining popliteal lymph nodes [LN], spleens, and thymi) were removed 1, 2, 7, and 14 days later and stained for donor MHC class II using anti-LA(b) mAb in an immunohistochemical procedure. The mean number of IAb+ cells per unit area was determined. RESULTS Transduction with a control Ad vector (Ad-LacZ) at 50 multiplicity of infection slightly increased CD40 and CD86 expression and up-regulated the poor allostimulatory activity of the DCp assessed by MLR and CTL responses. These effects on function were negated in Ad-TGF-beta1-transduced cells. After their injection into mouse footpads, the gene-transduced IAb+ cells were observed in maximal numbers in the popliteal LN at day 1 and in marginal zones and T-dependent areas of spleens (peak at day 7) but were rare in thymi. Transduction with Ad-LacZ reduced the numbers of IAb+ cells identified in both LN and spleens at all time points postinjection, suggesting that the vector alone affected DC life span in allogeneic recipients. TGF-beta1 transgene expression not only fully prevented the reduction in DC induced by Ad transduction alone, but also increased numbers and prolonged the survival of donor cells in the spleen, as shown by a two-to fivefold increase in IAb+ cells at days 2-14 compared with control (Ad-LacZ-transduced) DC. CONCLUSION BM-derived DCp can be transduced efficiently to express TGF-beta1 using an Ad vector. They exhibit very poor allostimulatory activity and similar migration characteristics in vivo to unmodified DCp. Survival of TGF-beta gene-transduced DC, however, is enhanced significantly compared with unmodified and (especially) control Ad-LacZ gene-transduced DC. Genetic engineering of donor DC to express the immunosuppressive molecule TGF-beta promotes their survival in allogeneic hosts and may potentiate their previously reported tolerogenicity.

Adenoviral Vectors Expressing Lymphotactin and Interleukin 2 or Lymphotactin and Interleukin 12 Synergize to Facilitate Tumor Regression in Murine Breast Cancer Models

We have previously demonstrated that intratumoral injection with Ad vectors expressing IL-2 or IL-12 can induce regression in a murine breast cancer model. These IL-2- or IL-12-induced antitumor responses were mainly mediated by Ag-specific T cells. Lymphotactin is a novel lymphocyte chemokine that can cause local accumulation of CD4+, CD8+, and NK cells. We hypothesized that addition of lymphotactin may enhance the antitumor immune responses induced by locally produced IL-2 and IL-12 as we have previously shown. To this end we constructed two double-recombinant adenoviral vectors expressing lymphotactin along with either IL-2 (Ad5 Lym/IL-2) or IL-12 (Ad5 Lym/IL-12). Subcutaneous injection of polyoma middle T (PyMT) or Neu (8142) transgenically derived breast adenocarcinoma cells, in the hind flank of FVB/n mice, results in the formation of tumor nodules in 14-21 days. We show that these constructs elicit potent antitumor responses when administered intratumorally. The antitumor responses are long lasting as determined by rechallenge experiments and hence demonstrate a protective immunity. These observations indicate that by augmenting the antitumor response with adenoviral vectors expressing lymphotactin in combination with IL-2 or IL-12 is a novel way to enhance immunotherapeutic approaches.

Antigen Presentation by Exosomes Released from Peptide-Pulsed Dendritic Cells Is not Suppressed by the Presence of Active CTL

Despite the potency of dendritic cells (DCs) as a vaccine carrier, they are short-lived and sensitive to CTL-mediated elimination. Thus, it is believed that the longevity of Ag presentation by peptide-pulsed DC is limited in vivo. Surprisingly, however, we found that although the majority of injected DCs disappeared from the draining lymph nodes within 7 days, Ag presentation persisted for at least 14 days following DC immunization. This prolonged Ag presentation was not mediated by the remaining injected DCs or through Ag transfer to endogenous APCs. We provide evidence that exosomes released by DCs might be responsible for the persistence of Ag presentation. Functional exosomes could be recovered from the draining lymph nodes of C57BL/6 mice following DC vaccination and, in contrast to DCs, T cell stimulation by exosomes in vivo was not affected by the presence of CTL. Our findings demonstrate that Ag presentation following delivery of DC vaccines persists for longer than expected and indicate that the exosome may play a previously unrecognized role in Ag presentation following DC vaccination. Furthermore, our study reinforces the application of exosomes as a vaccination platform and suggests that exosome-based vaccines may be advantageous for booster immunizations due to their resistance to CTL.

Potentiating Cancer Immunotherapy Using an Oncolytic Virus

Oncolytic viruses (OVs) are highly immunogenic and this limits their use in immune-competent hosts. Although immunosuppression may improve viral oncolysis, this gain is likely achieved at the cost of antitumoral immunity. We have developed a strategy wherein the immune response against the OV leads to enhanced therapeutic outcomes. We demonstrate that immunization with an adenoviral (Ad) vaccine before treatment with an oncolytic vesicular stomatitis virus (VSV) expressing the same tumor antigen (Ag) leads to significantly enhanced antitumoral immunity. Intratumoral replication of VSV was minimally attenuated in Ad-immunized hosts but extending the interval between treatments reduced the attenuating effect and further increased antitumoral immunity. More importantly, our combination approach shifted the immune response from viral Ags to tumor Ags and further reduced OV replication in normal tissues, leading to enhancements in both efficacy and safety. These studies also highlight the benefits of using a replicating, OV to boost a pre-existing antitumoral immune response as this approach generated larger responses versus tumor Ag in tumor-bearing hosts than could be achieved in tumor-free hosts. This strategy should be applicable to other vector combinations, tumor Ags, and tumor targets.

Engineering dendritic cells to enhance cancer immunotherapy.

Cancer immunotherapy aims to establish immune-mediated control of tumor growth by priming T-cell responses to target tumor-associated antigens. Three signals are required for T-cell activation: (i) presentation of cognate antigen in self MHC molecules; (ii) costimulation by membrane-bound receptor-ligand pairs; and (iii) soluble factors to direct polarization of the ensuing immune response. The ability of dendritic cells (DCs) to provide all three signals required for T-cell activation makes them an ideal cancer vaccine platform. Several strategies have been developed to enhance and control antigen presentation, costimulation, and cytokine production. In this review, we discuss progress toward developing DC-based cancer vaccines by genetic modification using RNA, DNA, and recombinant viruses. Furthermore, the ability of DC-based vaccines to activate natural killer (NK) and B-cells, and the impact of gene modification strategies on these populations is described. Clinical trials using gene-modified DCs have shown modest results, therefore, further considerations for DC manipulation to enhance their clinical efficacy are also discussed.

Cigarette Smoke Impairs NK Cell-Dependent Tumor Immune Surveillance

In this study, we investigated the impact of cigarette smoke on tumor immune surveillance and its consequences to lung tumor burden in a murine lung metastasis model. Cigarette smoke exposure significantly increased the numbers of lung metastases following B16-MO5 melanoma challenge. This effect was reversible; we observed significantly fewer tumor nodules following smoking cessation. Using RAG2−/− and RAG2−/−γc−/− mice, we provide strong evidence that increased tumor incidence was NK cell dependent. Furthermore, we show that cigarette smoke suppressed NK activation and attenuated NK CTL activity, without apparent effect on activating or inhibitory receptor expression. Finally, activation of NK cells through bone marrow-derived dendritic cells conferred protection against lung metastases in smoke-exposed mice; however, protection was not as efficacious as in sham-exposed mice. To our knowledge, this is the first experimental evidence showing that cigarette smoke impairs NK cell-dependent tumor immune surveillance and that altered immunity is associated with increased tumor burden. Our findings suggest that altered innate immunity may contribute to the increased risk of cancer in smokers.

Vaccination-induced autoimmune vitiligo is a consequence of secondary trauma to the skin

A major concern for cancer vaccines targeting self-tumor antigens is the risk of autoimmune sequelae. Although antitumor immunity correlates with autoimmune disease in some preclinical models, the mechanism(s) linking antitumor immunity and subsequent autoimmune pathology remain(s) to be determined. In the current study, we demonstrated that intradermal (i.d.) immunization with a recombinant adenovirus (Ad) expressing the murine melanoma antigen tyrosinase-related protein 2 (AdmTrp-2) results in a moderate level of tumor protection against the B16F10 murine melanoma without any vitiligo. Similar immunization with an Ad encoding human Trp-2 (AdhTrp-2) resulted in 50-fold greater protective immunity and produced vitiligo in all of the mice, suggesting that the development of autoimmunity may reflect the potency of the vaccine. Interestingly, delivery of AdhTrp-2 by i.m. injection generated protective immunity comparable with that seen in mice that received the vaccine by the i.d. route, but none of the recipients in the i.m. group developed vitiligo. The cellular and humoral responses in the i.m. immunized mice were greater than in the i.d. group; therefore, the lack of vitiligo was not caused by reduced efficacy of the vaccine. These results led us to hypothesize that vaccine-induced vitiligo was associated with local inflammatory responses. Mice immunized i.m. with AdhTrp-2 did develop vitiligo when they subsequently were injected i.d. with either a control Ad vector or complete Freund’s adjuvant, suggesting that vitiligo is initiated by some form of trauma within the skin. Our data demonstrated that autoimmune pathology is not an unavoidable outcome of effective cancer vaccines directed against self-tumor antigens.

HDAC Inhibition Suppresses Primary Immune Responses, Enhances Secondary Immune Responses, and Abrogates Autoimmunity During Tumor Immunotherapy

Histone deacetylase inhibitors (HDACi) can modulate innate antiviral responses and render tumors more susceptible to oncolytic viruses (OVs); however, their effects on adaptive immunity in this context are largely unknown. Our present study reveals an unexpected property of the HDACi MS-275 that enhances viral vector-induced lymphopenia leading to selective depletion of bystander lymphocytes and regulatory T cells while allowing expansion of antigen-specific secondary responses. Coadministration of vaccine plus drug during the boosting phase focuses the immune response on the tumor by suppressing the primary immune response against the vaccine vector and enhancing the secondary response against the tumor antigen. Furthermore, improvement of T cell functionality was evident suggesting that MS-275 can orchestrate a complex array of effects that synergize immunotherapy and viral oncolysis. Surprisingly, while MS-275 dramatically enhanced efficacy, it suppressed autoimmune pathology, profoundly improving the therapeutic index.

Enterochromaffin cell and 5-hydroxytryptamine responses to the same infectious agent differ in Th1 and Th2 dominant environments

Background/aim: 5-Hydroxytryptamine (5-HT) released from enterochromaffin cells influences intestinal homeostasis by altering gut physiology and is implicated in the pathophysiology of various gut disorders. The mechanisms regulating 5-HT production in the gut remain unclear. This study investigated the T helper (Th) 1/Th2-based immunoregulation of enterochromaffin cell function and 5-HT production in a model of enteric infection. Methods and results: Trichuris muris-infected AKR (susceptible to infection and generates Th1 response), BALB/c (resistant to infection and generates Th2 response), Stat4-deficient (impaired in Th1 response) and Stat6-deficient (impaired in Th2 response) mice were investigated to assess enterochromaffin cells, 5-HT and cytokines. In association with the generation of a Th2 response we observed higher enterochromaffin cell numbers and 5-HT content in the colon of BALB/c mice compared with AKR mice. Numbers of enterochromaffin cells and amount of 5-HT were significantly lower in Stat6-deficient mice after infection compared with Stat4-deficient mice. In addition, enterochromaffin cell numbers and 5-HT content were significantly higher after reconstitution of severe combined immunodeficient mice with in-vitro polarised Th2 cells. Conclusion: The study demonstrated that enterochromaffin cell and 5-HT responses to the same infectious agent are influenced by Th1 or Th2 cytokine predominance and suggests that the immunological profile of the inflammatory response is important in the regulation of enterochromaffin cell biology in the gut. In addition to new data on enterochromaffin cell function in enteric infection and inflammation, this study provides important information on the immuno–endocrine axis in the gut, which may ultimately lead to improved strategies against gut disorders.