Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model

Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.     

Virus-specific CD8+ T cells in the liver: armed and ready to kill

Influenza A virus infection of C57BL/6 mice is a well-characterized model for studying CD8+ T cell-mediated immunity. Analysis of primary and secondary responses showed that the liver is highly enriched for CD8+ T cells specific for the immunodominant H2D(b)NP(366-374) (D(b)NP(366)) epitope. Functional analysis established that these liver-derived virus-specific CD8+ T cells are fully competent cytotoxic effectors and IFN-gamma secretors. In addition, flow cytometric analysis of early apoptotic cells showed that these influenza-specific CD8+ T cells from liver are as viable as those in the spleen, bronchoalveolar lavage, mediastinal lymph nodes, or lung. Moreover, cytokine profiles of the influenza-specific CD8+ T cells recovered from different sites were consistent with the bronchoalveolar lavage, rather than liver population, being the most susceptible to activation-induced cell death. Importantly, adoptively transferred influenza virus-specific CD8+ T cells from the liver survived and were readily recalled after virus challenge. Together, these results show clearly that the liver is not a "graveyard" for influenza virus-specific CD8+ T cells.     

Age‐associated alterations in CD8α+ dendritic cells impair CD8 T‐cell expansion in response to an intracellular bacterium

Age‐associated decline in immunity to infection has been documented across multiple pathogens, yet the relative contributions of the aged priming environment and of lymphocyte‐intrinsic defects remain unclear. To address the impact of the aging environment on T‐cell priming, adult naïve OT‐I TCR transgenic CD8 T cells, specific for the H‐2K^b‐restricted immunodominant OVA_257‐264 epitope, were transferred into adult or old recipient mice infected with the recombinant intracellular bacterium Listeria monocytogenes carrying the chicken ovalbumin protein (Lm‐OVA). We consistently found that adult OT‐I CD8 expansion was reduced in aged recipient mice, and this correlated with numeric, phenotypic, and functional defects selectively affecting CD8α+ dendritic cells (DC). Following Lm‐OVA infection, aged mice failed to accumulate CD8α+ DC in the spleen, and these cells expressed much lower levels of critical costimulatory molecules in the first three days following infection. Further, aged CD8α+ DC showed impaired uptake of the bacteria at very early time points following infection. Treatment of aged mice with Flt3 ligand (Flt3L) improved the number of DC present in the spleen prior to Lm‐OVA infection, and improved, but did not reconstitute, OT‐I expansion to Lm‐OVA infection. These results suggest that age‐associated changes in antigen uptake, pathogen sensing, and/or antigen presentation contribute to impaired adaptive immune responses to microbial pathogens with aging.     

Exploring the Feasibility of Multi-Site Flow Cytometric Processing of Gut Associated Lymphoid Tissue with Centralized Data Analysis for Multi-Site Clinical Trials

The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.     

Perforin-mediated CTL cytolysis counteracts direct cell-cell spread of Listeria monocytogenes

The immune system has evolved various effector cells and functions to combat diverse infectious agents equipped with different virulence strategies. CD8 T cells play a critical role in protective immunity to Listeria monocytogenes (Lm), a bacterium that grows within the host cell cytosol and spreads directly into neighboring cells. The importance of CD8 T cells during Lm infection is currently attributed to the cytosolic niche of this organism, which allows it to evade many aspects of immune surveillance. CTL lysis of infected cells is believed to be an essential protective mechanism, presumably functioning to release intracellular bacteria, although its precise role remains to be fully defined. In this study, we examined the contribution of perforin-mediated CTL cytolysis to protective immunity against recombinant Lm capable of or defective in cell-cell spread. We found that CTL cytolysis is critical for protective immunity to Lm capable of cell-cell spread while protective immunity against spread-defective Lm is largely independent of CTL cytolysis. These results demonstrate that an important function of CTL cytolysis is to counter the microbial virulence strategy of direct cell-cell spread. We propose a model that advances the current view of the role of CTL cytolysis in immunity to intracellular pathogens.     

Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.

Interaction of the B7 molecule on antigen-presenting cells with its receptors CD28 and CTLA-4 on T cells provides costimulatory signals for T cell activation. We have studied the effects of B7 on antitumor immunity to a murine melanoma that expresses a rejection antigen associated with the E7 gene product of human papillomavirus 16. While this E7+ tumor grows progressively in immunocompetent hosts, cotransfection of its cells with B7 led to tumor regression by a B7-dependent immune response mediated by CD8+ cytolytic T lymphocytes. The immune response induced by E7+B7+ tumor cells also caused regression of E7+B7- tumors at distant sites and was curative for established E7+B7- micrometastases. Our findings suggest that increasing T cell costimulation through the CD28 and CTLA-4 receptors may have therapeutic usefulness for generating immunity against tumors expressing viral antigens.     

Liposome-coupled antigens are internalized by antigen-presenting cells via pinocytosis and cross-presented to CD8 T cells

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen-presenting cells (APCs) to CD8+ T cells, and that this process resulted in the induction of antigen-specific cytotoxic T lymphocytes. In the present study, the mechanism by which the liposome-coupled antigens were cross-presented to CD8+ T cells by APCs was investigated. Confocal laser scanning microscopic analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-based liposomes received processing at both MHC class I and class II compartments, while most of the antigens coupled to the surface of saturated-fatty-acid-based liposomes received processing at the class II compartment. In addition, flow cytometric analysis demonstrated that antigens coupled to the surface of unsaturated-fatty-acid-liposomes were taken up by APCs even in a 4°C environment; this was not true of saturated-fatty-acid-liposomes. When two kinds of inhibitors, dimethylamiloride (DMA) and cytochalasin B, which inhibit pinocytosis and phagocytosis by APCs, respectively, were added to the culture of APCs prior to the antigen pulse, DMA but not cytochalasin B significantly reduced uptake of liposome-coupled antigens. Further analysis of intracellular trafficking of liposomal antigens using confocal laser scanning microscopy revealed that a portion of liposome-coupled antigens taken up by APCs were delivered to the lysosome compartment. In agreement with the reduction of antigen uptake by APCs, antigen presentation by APCs was significantly inhibited by DMA, and resulted in the reduction of IFN-γ production by antigen-specific CD8+ T cells. These results suggest that antigens coupled to the surface of liposomes consisting of unsaturated fatty acids might be pinocytosed by APCs, loaded onto the class I MHC processing pathway, and presented to CD8+ T cells. Thus, these liposome-coupled antigens are expected to be applicable for the development of vaccines that induce cellular immunity.     

Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8+ T cell response

Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-gamma, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-gamma expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.     

Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.     

The relative importance of CD4+ and CD8+T cells in immunity to pulmonary paracoccidioidomycosis

Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets has never been investigated. The aim of this study was to characterize the function of CD4+ and CD8+ T cells in the immunity developed by susceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4+ T cells did not alter disease severity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTH reactivity. In resistant mice, both CD4+ and CD8+ T cells control fungal burdens and cytokines although only the former regulate DTH reactions and antibody production. In the intermediate strain, deficiency of whole T and CD8+ T cells but not of CD4+ T or B cells led to increased mortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8+ T cells, whereas antibody production and DTH reactions are regulated by CD4+ T cells; (c) CD4+ T cells play a protective role in the resistant and intermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concomitant CD4+ and CD8+ T cell immunity secreting type 1 and type 2 cytokines.     

Genetic control of immunity to Plasmodium yoelii sporozoites

Using a rodent malaria system, we have shown that protective immunity to the preerythrocytic stages of malaria is genetically controlled by MHC and non-MHC genes. Ten congenic strains of mice were immunized with irradiated sporozoites of Plasmodium yoelii. When challenged with viable sporozoites, only two strains had a high proportion of animals that did not develop blood stage infections. Immunity did not correlate with antisporozoite antibody levels. Two protective mechanisms exist determined by non-H-2 genes, and each mechanism is further controlled by H-2-linked Ir genes. On the BALB background only H-2d mice are protected, and protection is abolished by depleting CD8+ T cells. In contrast, on the B10 background only H-2q mice are strongly protected, and protection is not affected by CD8+ T cell depletion. If similar complex genetic regulation of immunity occurs in the human malarias, it will be a major hurdle for vaccine development.     

Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice

We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.     

5,6-Dimethylxanthenone-4-acetic acid treatment of a non-immunogenic tumour does not synergize with active or passive CD8+ T-cell immunotherapy

The chemotherapeutic drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) inhibits intratumoural blood flow, causing hypoxia, haemorrhagic necrosis, vascular collapse and tumour cell death. Production of TNF-alpha and IFN is also induced, causing local inflammation and activation of immune cells including CD8+ T cells. We used the tumour cell line LL-LCMV, which expresses the gp33 epitope of lymphocytic choriomeningitis virus in a non-immunogenic form, to investigate whether tumour cell death caused by treatment with DMXAA may improve the success of tumour immunotherapy mediated by CD8+ T cells. Treatment with DMXAA was effective at reducing the size of LL-LCMV tumours. However, compared to normal mice, tumour reduction was no more marked or sustained in mice carrying high numbers of naive, tumour-specific CD8+ T cells. The antitumour effect of activated CD8+ T cells was also not affected by DMXAA treatment. Tumour-specific CD8+ T cells activated in vivo by immunization with dendritic cells and specific tumour peptide antigen, or generated in vitro and adoptively transferred into tumour-bearing mice by i.v. injection, did not improve or sustain the reduction in tumour size induced by DMXAA treatment. We conclude that the presence of high numbers of naive CD8+ T cells, or immunotherapies leading to CD8+ T-cell activation, do not synergize with the tumour cell death and local inflammation induced by DMXAA treatment. It is possible that this lack of synergism may result from both treatments inducing activation of CD8+ T cells and that treatments that activate different populations of immune cells may achieve better success.     

Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion

mAb specific for murine CD4+ and CD8+ T cell subsets were utilized to determine the populations participating in delayed-in-time, cutaneous hypersensitivity responses in BALB/c mice. In vivo depletions of these T cell phenotypes revealed that delayed-type hypersensitivity to cellular and protein Ag were mediated by CD4+ effector cells, whereas CD8+ cells down-regulated such responses. Similar depletions in mice prior to sensitization with the hapten 1-fluoro-2,4-dinitrobenzene demonstrated a more complex pattern of cell participation in contact sensitivity (CS) responses. Depletion of CD4+ cells resulted in strikingly enhanced ear swelling, indicating not only an important effector role for CD8+ cells but also a down-regulatory role for some CD4+ cells; depletion of CD8+ cells revealed that some CD4+ cells also act as CS effectors. In vitro depletion of immune lymph node cells with the same mAb before adoptive transfer confirmed CS effector roles for both subsets, and also suggested that at least some CD4+ suppressors act on the efferent limb of the CS response, perhaps by down-regulating the activity of CD8+ effector cells. Partial in vivo depletion with small amounts anti-CD4 mAb and subsequent flow cytometric analysis of residual CD4+ cells was consistent with the hypothesis that CD4+ CS effector cells express a higher density of the CD4 antigen than do CD4+ suppressor cells, raising the possibility that these two functionally distinct CD4+ populations might be separable on the basis of their surface expression of CD4.     

PDL-1 blockade impedes T cell expansion and protective immunity primed by attenuated Listeria monocytogenes

Infection with attenuated Listeria monocytogenes (Lm) is a robust in vivo model for examining how Ag-specific T cells are primed, and subsequent challenge with virulent Lm allows for the protective effects of T cell priming to be quantified. Herein, we investigated the role of programmed death ligand 1 (PDL-1) in T cell priming and immunity conferred after primary infection with Lm DeltaactA followed by virulent Lm challenge. In striking contrast to the inhibitory role of PDL-1 on T cell immunity in other infection models, marked reductions in the magnitude of T cell expansion and the kinetics of T cell proliferation were observed with PDL-1 blockade after primary Lm DeltaactA infection. More importantly, PDL-1 blockade beginning before primary infection and maintained throughout the experiment resulted in delayed bacterial clearance and T cell expansion after secondary challenge with virulent Lm. These results indicate that for immunity to intracellular bacterial infection, PDL-1 plays an important stimulatory role for priming and expansion of protective T cells.     

Psychological stress compromises CD8+ T cell control of latent herpes simplex virus type 1 infections

Recurrent HSV-1 ocular disease results from reactivation of latent virus in trigeminal ganglia, often following immunosuppression or exposure to a variety of psychological or physical stressors. HSV-specific CD8+ T cells can block HSV-1 reactivation from latency in ex vivo trigeminal ganglia cultures through production of IFN-gamma. In this study, we establish that either CD8+ T cell depletion or exposure to restraint stress permit HSV-1 to transiently escape from latency in vivo. Restraint stress caused a reduction of TG-resident HSV-specific CD8+ T cells and a functional compromise of those cells that survive. Together, these effects of stress resulted in an approximate 65% reduction of cells capable of producing IFN-gamma in response to reactivating virus. Our findings demonstrate persistent in vivo regulation of latent HSV-1 by CD8+ T cells, and strongly support the concept that stress induces HSV-1 reactivation from latency at least in part by compromising CD8+ T cell surveillance of latently infected neurons.     

Adoptive transfer of CD8+ T cells from immune animals does not transfer immunity to blood stage Plasmodium yoelii malaria

The malaria parasite, Plasmodium yoelii 17X, causes a self-limited, nonlethal infection characterized, in the blood stage, by preferential invasion of reticulocytes. Previous studies have suggested that immunity to the blood stage infection may be related to enhanced levels of class I MHC Ag on the parasitized reticulocyte surface and can be adoptively transferred to immunodeficient mice by immune CD8+ T cells in the absence of CD4+ T cells. To further examine the mechanisms of CD8+ T cell involvement in immunity to blood stage P. yoelii infection, we performed in vivo CD8 depletion and adoptive transfer experiments. Depletion of CD8+ T cells during primary blood stage infection in BALB/c mice did not diminish the ability of the mice to resolve their infections. Spleen cells from immune BALB/c and C57BL/10 mice were transferred to BALB/c-nu/nu and C57BL/10-nu/nu mice, respectively. The recipient mice were CD4 depleted in vivo to kill any transferred CD4+ T cells. The mice failed to control the infection. Populations of CD4-, CD8+ T cells were transferred from immune CBA/CaJ donors to in vivo CD4-depleted CBA/CaJ recipients. The mice were unable to control the infection. Although immune unfractionated spleen cells transferred rapid protection in all three mouse strains and immune CD4+ T cells transferred immunity in the two mouse strains studied, CD8+ T cells by themselves were neither protective nor did they enhance immunity.     

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.     

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.