Reciprocal effects of Th1 and Treg cell inducing pathogen-associated immunomodulatory molecules on anti-tumor immunity

We have addressed the hypothesis that pathogen-associated immunomodulatory molecules may influence anti-tumor immunity through their pro- and anti-inflammatory activities and abilities to induce effector and regulatory T (Treg) cells. We found that CpG oligonucleotides (CpG) and cholera toxin (CT), which promote Th1 or Th2/Treg cell biased responses, respectively, had differential effects on tumor growth. Therapeutic peritumoral administration of CpG significantly reduced subcutaneous tumor growth and prolonged survival, whereas CT enhanced tumor growth and reduced survival. Peritumoral administration of CpG enhanced the frequency of IFN-γ-secreting and reduced IL-10-secreting CD4⁺ and CD8⁺ T cells, in the tumor and in the draining lymph nodes, whereas, CT significantly enhanced the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells, but reduced IFN-γ-secreting T cells infiltrating the tumor. In contrast to the beneficial effect of CpG in mice with subcutaneous tumors, CpG or CT had no protective effect against tumor growth in the lungs when given therapeutically by the nasal route. However, prophylactic intranasal administration of CpG significantly reduced the number of lung metastases and this was associated with an enhanced frequency of IFN-γ-secreting CD8⁺ T cells in the draining lymph node and enhanced tumor-specific CTL responses. Our findings demonstrate that pathogen-associated molecules can either inhibit or enhance anti-tumor immunity by selectively promoting the induction of effector or regulatory T cells, and that the environment of the growing tumor influences the protective effect. Joanne Lysaght and Andrew G. Jarnicki contributed equally.     

In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

Production of interferons and change of the lymphocyte subpopulation phenotype in peripheral blood at cervical papillomavirus infection

IFN-γ and IFN-α productionin vitro by peripheral blood cells activated by phytohemagglutinin or the Newcastle disease virus was impaired in patients with a benign process, cervical intraepithelial neoplasm and cancerin situ associated with human papillomavirus infection. In case of IFN-γ and IFN-α production impairment following cervical papillomavirus infection, the increased severity of disease was accompanied by remarkable IFN system suppression. The lower synthesis of both IFN correlated with changes of some lymphocyte-subpopulation phenotype in peripheral blood. Lower CD4⁺ and CD3⁺ DR⁺ T cell concentrations were observed in papillomavirus-infected patients with impaired IFN production; impaired IFN-γ production was accompanied by lower CD4/CD8 index.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Objective Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T_reg). To study the role of T_reg in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10⁺ T_reg type 1 (Tr1) was established. Methods An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4⁺CD25⁻ T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) ± 1 nM rapamycin. Autologous iDC and CD4⁺CD25⁻ T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays ± neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-β and IFN-γ in ELISA. Results In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3⁺CD4⁺CD25⁻IL2Rβ⁺IL2Rγ⁺FoxP3⁺CTLA-4⁺IL-10⁺ cells with suppressor activity (mean suppression ± SD = 58 ± 12%). These suppressor cells produced IL-10 (mean ± SD = 535 ± 12 pg/ml) and TGF-β (mean ± SD = 512 ± 38 pg/ml), but no IL-4 or IFN-γ. Suppressor function of co-cultures correlated with the percent of expanding IL-10⁺ Tr1 cells (r ² = 0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent. Conclusion The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4⁺CD25^highFoxP3⁺ nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.     

T cell augments the antitumor activity of tumor-targeting <Emphasis Type="Italic">Salmonella</Emphasis>

Systemic administration of Salmonella to tumor-bearing mice leads to preferential accumulation within tumor sites and retardation of tumor growth. However, the detailed mechanism of Salmonella-induced antitumor immune response via host T cell remains uncertain. Herein, we used wild-type, CD4⁺ T-cell-deficient, and CD8⁺ T-cell-deficient mice to study the role of T cell in the antitumor immune responses induced by Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis). When systemically administered into mice bearing tumors, Salmonella Choleraesuis significantly inhibited tumor growth by 50%. In contrast, in T-cell-deficient mice, there was only 34–42% inhibition of tumor growth. We found that treatment with Salmonella Choleraesuis significantly upregulates interferon-γ in wild-type and CD8⁺ T-cell-deficient mice, but not in CD4⁺ T-cell-deficient mice. Furthermore, immunohistochemical staining of the tumors revealed more infiltration of macrophages and neutrophils in wild-type mice after Salmonella Choleraesuis treatment compared with those in T-cell-deficient mice. The antitumor therapeutic effect mediated by Salmonella Choleraesuis is associated with an inflammatory immune response at the tumor site and a tumor T helper 1-type immune response. In conclusion, these results suggest that tumor-targeted therapy using Salmonella Choleraesuis, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumor.     

Reduced tumorigenesis of EG7 after interleukin-10 gene transfer and enhanced efficacy in combination with intratumorally injection of adenovirus-mediated lymphotactin and the underlying mechanism

Although interleukin-10 (IL-10) is commonly regarded as an immunosuppressive cytokine, a wealth of evidence is accumulating that IL-10 also possesses some immunostimulating antitumor properties. Previous studies demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. In this study, we explored the tumorigenesis of EG7 cells transduced with IL-10 gene. In vivo, IL-10 gene transfer reduced tumorigenic capacity of EG7 cells and prolonged survival of the EG7 tumor-bearing mice. It was found that the cytotoxicities of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) were enhanced. Assessment of the immune status of the animals showed prevalence of a systemic and tumor-specific Th2 response (high levels of IL-4 and IL-10). To improve the therapeutic efficacy, we combined with intratumoral injection of adenovirus-mediated lymphotactin (Ad-Lptn) into the overestablished EG7 tumor model. More significant inhibition of tumor growth were observed in EG7 tumor-bearing mice that received combined treatment with IL-10 and Lptn gene than those of mice treated with IL-10 or Lptn gene alone. The highest NK cells and CTL activity was induced in the combined therapy group, increasing the production of IL-2 and interferon-γ (IFN-γ) significantly but decreasing the expression of immune suppressive cells (CD4⁺Foxp3⁺ Treg cells and Gr1⁺CD11b⁺ MDSCs). The necrosis of tumor cells was markedly observed in the tumor tissues, accompanying with strongest expression of Mig (monokine induced by interferon-gamma) and IP-10 (interferon-inducible protein 10), weakest expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-2 (MMP-2). In vivo, depletion analysis demonstrated that CD8⁺ T cells and NK cells were the predominant effector cell subset responsible for the antitumor effect of IL-10 or Lptn gene. These findings may provide a potential strategy to improve the antitumor efficacy of IL-10 and Lptn.     

Enantiospecific adjuvant activity of cationic lipid DOTAP in cancer vaccine

Commercially available DOTAP is a racemic mixture of two enantiomers. The adjuvanticity of each isomer was examined using a peptide/lipid complex as a therapeutic vaccine in an established murine cervical cancer model. This simple vaccine consists of a cationic lipid (DOTAP) and a major histocompatibility complex (MHC) class I–restricted epitope of the Human Papillomavirus (HPV) 16 protein E7. Dose-dependent tumor regression experiments have been completed for racemic DOTAP/E7, (R)-DOTAP/E7 and (S)-DOTAP/E7. Tumor-bearing mice treated with (R)-DOTAP/E7 complexes have shown tumor regression in a dose-dependent manner comparable to those mice treated with a racemic DOTAP with E7 peptide. These data are supported by IFN-γ production by CD8⁺ splenocytes, in vivo cytotoxic T-lymphocytes (CTL) response, CD8⁺ tumor-infiltrating lymphocytes (TIL), and IFN-γ production by CD8⁺ TIL in (R)-DOTAP/E7-vaccinated mice. When (S)-DOTAP/E7 is delivered, tumor progression is delayed. While IFN-γ production is absent from CD8⁺ splenocytes in mice vaccinated with (S)-DOTAP/E7, IFN-γ production by CD8⁺ TIL is present, supporting our hypothesis that (S)-DOTAP has limited activity. Activation of bone marrow-derived dendritic cells by the enantiomeric formulations has also been evaluated, as well as cytokine production and toxicity with no considerable differences between the groups. The results show the DOTAP enantiomers act differently as adjuvants in vivo, with (R)-DOTAP being more effective at stimulating a CD8⁺ anti-tumor response.     

A B-cell lymphoma vaccine using a depot formulation of interleukin-2 induces potent antitumor immunity despite increased numbers of intratumoral regulatory T cells

Therapeutic vaccination holds great potential as complementary treatment for non-Hodgkin’s lymphoma. Here, we report that a therapeutic whole cell vaccine formulated with IL-2 adsorbed onto aluminum hydroxide as cytokine-depot formulation elicits potent antitumor immunity and induces delayed tumor growth, control of tumor dissemination and longer survival in mice challenged with A20-lymphoma. Therapeutic vaccination induced higher numbers of tumor’s infiltrating lymphocytes (CD4⁺ and CD8⁺ T cells and NK cells), and the production of IFN-γ and IL-4 by intratumoral CD4⁺ T cells. Further, strong tumor antigen-specific cellular responses were detected at systemic level. Both the A20-derived antigenic material and the IL-2 depot formulation were required for induction of an effective immune response that impacted on cancer progression. All mice receiving any form of IL-2, either as part of the vaccine or alone as control, showed higher numbers of CD4⁺CD25^+/highFoxp3⁺ regulatory T cells (Treg) in the tumor, which might have a role in tumor progression in these animals. Nevertheless, for those animals that received the cytokine as part of the vaccine formulation, the overall effect was improved immune response and less disseminated disease, suggesting that therapeutic vaccination overcomes the potential detrimental effect of intratumoral Treg cells. Overall, the results presented here show that a simple vaccine formulation, that can be easily prepared under GMP conditions, is a promising strategy to be used in B-cell lymphoma and may have enough merit to be tested in clinical trials.     

Active hexose correlated compound enhances tumor surveillance through regulating both innate and adaptive immune responses

Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals derived from cocultured mycelia of several species of Basidiomycete mushrooms. AHCC has been implicated to modulate immune functions and plays a protective role against infection. However, the potential role of AHCC in tumor immune surveillance is unknown. In this study, C57BL/6 mice were orally administered AHCC or water, followed by tumor cell inoculation. We showed that compared to pure water-treated mice, AHCC treatment significantly delayed tumor development after inoculation of either melanoma cell line B16F0 or lymphoma cell line EL4. Treatment with AHCC enhanced both Ag-specific activation and proliferation of CD4⁺ and CD8⁺ T cells, increased the number of tumor Ag-specific CD8⁺ T cells, and more importantly, increased the frequency of tumor Ag-specific IFN-γ producing CD8⁺ T cells. Interestingly, AHCC treatment also showed increased cell number of NK and γδ T cells, indicating the role of AHCC in activating these innate-like lymphocytes. In summary, our results demonstrate that AHCC can enhance tumor immune surveillance through regulating both innate and adaptive immune responses.     

Comparison of adjuvant activity of N- and C-terminal domain of gp96 in a Her2-positive breast cancer model

It has been frequently reported that gp96 acts as a strong biologic adjuvant. Some studies have even investigated adjuvant activity of the gp96 C- or N-terminal domain. The controversy surrounding adjuvant activity of gp96 terminal domains prompted us to compare adjuvant activity of gp96 C- or N-terminal domain toward Her2/neu, as DNA vaccine in a Her2/neu-positive breast cancer model. To do so, mice were immunized with DNA vaccine consisting of transmembrane and extracellular domain (TM + ECD) of rat Her2/neu alone or fused to N- or C-terminal domain of gp96. Treatment with Her2/neu fused to N-terminal domain of gp96 resulted in tumor progression, compared to the groups vaccinated with pCT/Her2 or pHer2. Immunological examination revealed that treatment with Her2/neu fused to N-terminal domain of gp96 led to significantly lower survival rates, higher interferon-γ secretion, and induced infiltration of CD4⁺/CD8⁺ cells to the tumor site. However, it could not induce cytotoxic T lymphocyte activity, did not decrease regulatory T cell percentage at the tumor site, and eventually led to tumor progression. Our results reveal that gp96 N-terminal domain does not have adjuvant activity toward Her2/neu. It is also proposed that adjuvant activity and the resultant immune response of gp96 terminal domains may be directed by the antigen applied.     

The Phosphoenolpyruvate Carboxykinase of Mycobacterium Tuberculosis Induces Strong Cell-Mediated Immune Responses in Mice

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes guanosine or adenosine mononucleotide-dependent reversible conversion of oxaloacetate (OAA) and phosphoenolpyruvate (PEP). Mycobacterium (M) tuberculosis possesses a putative GTP-dependent PEPCK. To analyze the immune responses caused by PEPCK, the effects of PEPCK on the induction of CD4⁺ T cells and cytokines such as IFN-γ, IL-12 and TNF-α were evaluated in mice. It was found that the number of CD4⁺ T cells was increased in the PEPCK immunized mice although the change of the number of CD8⁺ T cells was not significant. The cytokines IFN-γ, IL-12 and TNF-α were increased significantly in the mice immunized with PEPCK than those of incomplete adjuvant. These characteristics were further demonstrated in the mice infected by pckA mutated BCG strain. The results indicate that PEPCK can effectively induce cell-mediated immune response by increasing activity of cytokines and PEPCK may be a promising new subunit vaccine candidate for tuberculosis.     

The immunosuppressive tumor microenvironment in hepatocellular carcinoma

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4⁺ T cells, CD8⁺, CD3⁻CD56⁺, CD3⁺CD56⁺, and γδT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3⁺CD56⁺ cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4⁺ cells in the tumor tended to produce more IL-10 but less IFN-γ, whereas CD8⁺ cells showed impaired capacity for the production of both IFN-γ and perforin. Consistent with previous reports, we observed a significant increase of Foxp3⁺ cells in the tumor tissue. Intriguingly, although over 90% of CD4⁺CD25^high cells were found to be Foxp3⁺, the majority of Foxp3⁺ cells were identified in the CD4⁺CD25^medium and CD4⁺CD25⁻ subsets. In support of its role as a negative regulator, CD4⁺CD25^high cells suppressed the proliferation of CD4⁺CD25⁻ cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.     

Impact of aging on immune modulation by tumor

 Tumor development and aging can each alter immune competence. The present study aimed to determine the impact of Lewis lung carcinoma (LLC) presence on immune parameters of middle-aged (averaging 6.5 months) versus aged (averaging 21.3 months) mice. An age-associated decline in the CD4⁺ cell frequency was seen in freshly isolated spleen and lymph node cells, as well as in cultures stimulated with immobilized anti-CD3. This decline was not further exacerbated by tumor presence. What was prominently inhibited by tumor was the capacity of either splenic or lymph node CD4⁺ cells to become stimulated to express IFN-γ. Spleen and lymph node cultures from aged tumor-bearing mice had the lowest frequency of CD4⁺IFN-γ ⁺ cells and the least amount of secreted IFN-γ. CD8⁺ cells were not affected by aging, but tumor presence reduced the induction of CD8⁺IFN-γ ⁺ cells in lymph node cultures. We previously showed that LLC growth stimulates myelopoiesis, as seen by splenomegaly and the mobilization of immune inhibitory CD34⁺ progenitor cells. Tumor presence in middle-aged mice reduced spleen cell blastogenesis, which was mediated by CD34⁺ cells. Aged mice had reduced blastogenesis, and this was further reduced by presence of tumor. However, neither the age-associated immune dysfunction nor the tumor-induced immune suppression in aged mice was due to CD34⁺ progenitor cells. These studies show how tumor presence can further compromise the immune dysfunction that accompanies aging. In addition, they show that aging impacts on the mechanisms by which tumors inhibit T-cell capabilities, with myelopoiesis-associated CD34⁺ cells mediating the immune depression of middle-aged tumor-bearers and an independent mechanism being responsible for the immune depression in aged tumor-bearing mice. Received: 19 March 2001 / Accepted: 4 May 2001     

Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4⁺ memory T cells, whereas CD4⁺ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4⁺ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α. A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.     

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions

Introduction Patients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions. Materials and methods Endothelial cells were pre-treated with LLC tumor-conditioned medium (Endo^T-sup) for 24 h. Control endothelial cells that were exposed to medium (Endo^Media) or epithelial cell-conditioned medium (Endo^Epi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from Endo^Media, Endo^Epi-sup or Endo^T-sup were collected and assayed for immune modulatory products and for immune modulatory activity. Results Supernatant from Endo^T-sup contained increased levels of PGE₂, IL-6 and VEGF as compared to Endo^Media and Endo^Epi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by Endo^Media and Endo^Epi-sup. Exposure of NK cells to supernatants of Endo^T-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by Endo^Media and Endo^Epi-sup. Examination of macrophage functions demonstrated that supernatant from Endo^T-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE₂. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from Endo^T-sup were examined. IFN-γ production by CD8⁺ T-cells was reduced after exposure to Endo^T-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4⁺ T-cells exposed to Endo^T-sup was not altered. Conclusions Taken together, these studies demonstrate that tumors skew endothelial cells to disrupt NK cell, T-cell and macrophages functions, and represents a novel mechanism of tumor-induced immune suppression.     

CD4<Superscript>+</Superscript> T cell response against a non-tumor antigen is unaffected in melanoma-bearing mice

The tumor microenvironment is complex and creates an immunosuppressive network to tolerize tumor-specific immune responses; however, little information is available regarding the response against non-tumor antigens in tumor-bearing individuals. The goal of the present study was to evaluate if tumor burden could influence a CD4⁺ T cell response against a soluble protein, not expressed by the tumor, in the absence of in vitro stimulation. Using an experimental system in which we can compare CD4⁺ T cell responses to the Ea antigen when it is either expressed by B16F10 melanoma cells (B16EaRFP cells) or is an exogenous, non-tumor antigen (soluble EaRFP protein), in immunizations of B16F10 tumor-bearing mice, we observed that the tumor can modulate the CD4⁺ T cell-specific response to the antigen when it is expressed by the tumor cells. TEa cells proliferated poorly and produced less IFN-γ in mice bearing B16F10 melanoma expressing Ea peptide, and tumor growth was impervious to this response. However, in mice bearing 7 days B16F10 tumors, not expressing the Ea antigen, priming of TEa cells was similar to that observed in tumor-free mice, based on the total number of cells recovered and proliferation assessed by CFSE dilution after EaRFP immunization. We also investigated if tumor burden could influence recall responses of already differentiated effector cells. We immunized mice with EaRFP antigen and after a few days injected B16F10 cells. After 10 days of tumor growth, we challenged the mice with the non-tumor antigen. We found that the number of TEa cells producing IFN-γ in tumor-bearing mice was not different compared to tumor-free mice. No differences in antigen presentation, assessed by YAe antibody staining, were verified in the draining lymph node of these two groups. Collectively, our data indicate that tumor burden does not affect immune responses to non-tumor antigens. These results have important implications in the design of anti-cancer therapy.     

Characteristic Immune Response in Peyer’s Patch Cells Induced by Oral Administration of Bifidobacterium Components

We demonstrate immunomodulatory effects, especially those involving murine intestinal IgA secretion, in Peyer's patch cells following oral administration of Bifidobacterium immunomodulator (BIM) derived from sonicated B. pseudocatenulatum 7041. BALB/c mice were administered BIM orally for 7 consecutive days. The PP cells demonstrated upregulated secretion of total IgA including BIM-specific IgA following BIM administration. In observing the response of PP cells co-cultured with BIM, we found enhanced secretion of interferon-γ (IFN-γ) and interleukin (IL)-6 in the CD4⁺ T cells. In contrast, IL-12 secretion by Thy1.2⁻ PP cells was enhanced, but secretion of IFN-γ, IL-5, and IL-6 was not significantly affected. Furthermore, the population of CD4⁺ CD45RB^high T cells in PP increased following oral administration of BIM. These data suggest that CD4⁺ T cells were affected by BIM administration. Overall, the results show that oral administration of BIM induced CD4⁺ PP cells to change their expression of cell surface antigen and cytokine production.