CD95 Ligand expression enhances growth of murine renal cell carcinoma in vivo

The CD95/CD95 ligand (CD95L) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. In this system, two murine CD95L-transfected renca clones and a control renca clone transfected only with the vector were implanted into the subcapsule of the left kidney of Balb/c and Balb/c nude mice. Both CD95L-expressing and control renca clones formed macroscopic tumors in all of the Balb/c and Balb/c nude hosts 14 days after implantation. Growth of tumors of murine CD95L-transfected renca cells was significantly better than that of control renca cells in Balb/c mice, while the growth advantage of CD95L transfectants was not observed in Balb/c nude mice. Lymphocytes underwent apoptosis mainly in the periphery of the CD95L-expressing tumors but not in control tumors grown in Balb/c mice, while lymphocytes undergoing apoptosis were not observed in CD95L-expressing tumors or in control tumors grown in Balb/c nude mice. Neutrophilic recruitment was rarely observed in CD95L-expressing or control tumors. CD95L expressed on renca cells possibly suppressed immune responses against renca tumors by inducing apoptosis of the infiltrating lymphocytes. However, CD95L-expressing renca cells did not form tumors in the renal subcapsule of allogeneic C3H/HeJ mice. Received: 23 July 1998 / Accepted: 23 December 1998     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

Plasmodium chabaudi AS: Distinct CD4+CD25+Foxp3+ regulatory T cell responses during infection in DBA/2 and BALB/c mice

Malaria infections display variation patterns of clinical course and outcome. Although CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. Herein, we compared the disparity in Treg cells response during the course of blood stage Plasmodium chabaudi chabaudi AS (P. c chabaudi AS) infection in DBA/2 and BALB/c mice. BALB/c mice initiated a Th1/Th2 profile respond to P. c chabaudi AS infection, but DBA/2 mice failed to control P. c chabaudi AS infection and almost of them died post-peak parasitemia. At the peak parasitemia, we found that higher proportion of Treg cells with elevated Foxp3 expression in DBA/2 than in BALB/c mice. We used anti-CD25 mAb to deplete Treg cells and found that the survival time and rate were prolonged in DBA/2 mice treated with anti-CD25 mAb. Treatment with anti-CD25 mAb in vivo led to enhanced pro-inflammation responses and Foxp3 expression decline on Treg cells. In contrast, after DBA/2 was treatment with anti-IL-10R mAb, IL-10R blockade in vivo caused excessive pro-inflammation responses and Foxp3 expression loss on CD4⁺CD25⁺ T cells. Earlier death was found in all of DBA/2 mice with anti-IL-10R mAb. It suggested that IL-2 and IL-10 signal involved in maintaining Foxp3 expression on Treg cells. In all, the moderate suppressive activity of Treg cells may facilitate resistance to P. c chabaudi AS infection.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

IL-22 produced by Th22 cells aggravates atherosclerosis development in ApoE−/− mice by enhancing DC-induced Th17 cell proliferation

Th22 cells are a novel subset of CD4⁺ T cells that primarily mediate biological effects through IL-22, with both Th22 cells and IL-22 being closely associated with multiple autoimmune and chronic inflammatory diseases. In this study, we investigated whether and how Th22 cells affect atherosclerosis. ApoE^−/− mice and age-matched C57BL/6J mice were fed a Western diet for 0, 4, 8 or 12 weeks. The results of dynamic analyses showed that Th22 cells, which secrete the majority of IL-22 among the known CD4⁺ cells, play a major role in atherosclerosis. ApoE^−/− mice fed a Western diet for 12 weeks and administered recombinant mouse IL-22 (rIL-22) developed substantially larger plaques in both the aorta and aortic root and higher levels of CD3⁺ T cells, CD68⁺ macrophages, collagen, IL-6, Th17 cells, dendritic cells (DCs) and pSTAT3 but lower smooth muscle cell (SMC) α-actin expression than the control mice. Treatment with a neutralizing anti–IL-22 monoclonal antibody (IL-22 mAb) reversed the above effects. Bone marrow-derived DCs exhibited increased differentiation into mature DCs following rIL-22 and ox-LDL stimulation. IL-17 and pSTAT3 were up-regulated after stimulation with IL-22 and ox-LDL in cells cocultured with CD4⁺ T cells and mature DC supernatant, but this up-regulation was significantly inhibited by IL-6mAb or the cell-permeable STAT3 inhibitor S31-201. Thus, Th22 cell-derived IL-22 aggravates atherosclerosis development through a mechanism that is associated with IL-6/STAT3 activation, DC-induced Th17 cell proliferation and IL-22–stimulated SMC dedifferentiation into a synthetic phenotype.     

Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant

 We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for IFN-γ- and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases. Received: 27 July 2000 / Accepted: 13 March 2001     

Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity

Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires development of type 1 T-helper (Th1) CD4⁺ T cell immunity. Because of their unique capacity to initiate and modulate immune responses, dendritic cells (DCs) are attractive targets for development of novel vaccines. In this study, for the first time, we investigated the capacity of a DC-targeted vaccine to induce protective responses against L. major. To this end, we genetically engineered the N-terminal portion of the stress-inducible 1 protein of L. major (LmSTI1a) into anti-DEC205/CD205 (DEC) monoclonal antibody (mAb) and thereby delivered the conjugated protein to DEC⁺ DCs in situ in the intact animal. Delivery of LmSTI1a to adjuvant-matured DCs increased the frequency of antigen-specific CD4⁺ T cells producing IFN-γ⁺, IL-2⁺, and TNF-α⁺ in two different strains of mice (C57BL/6 and Balb/c), while such responses were not observed with the same doses of a control Ig-LmSTI1a mAb without receptor affinity or with non-targeted LmSTI1a protein. Using a peptide library for LmSTI1a, we identified at least two distinct CD4⁺ T cell mimetopes in each MHC class II haplotype, consistent with the induction of broad immunity. When we compared T cell immune responses generated after targeting DCs with LmSTI1a or other L. major antigens, including LACK (Leishmania receptor for activated C kinase) and LeIF (Leishmania eukaryotic ribosomal elongation and initiation factor 4a), we found that LmSTI1a was superior for generation of IFN-γ-producing CD4⁺ T cells, which correlated with higher protection of susceptible Balb/c mice to a challenge with L. major. For the first time, this study demonstrates the potential of a DC-targeted vaccine as a novel approach for cutaneous leishmaniasis, an increasing public health concern that has no currently available effective treatment.     

ifn-γ-dependent secretion of IL-10 from Th1 cells and microglia/macrophages contributes to functional recovery after spinal cord injury

Transfer of type-1 helper T-conditioned (Th1-conditioned) cells promotes functional recovery with enhanced axonal remodeling after spinal cord injury (SCI). This study explored the molecular mechanisms underlying the beneficial effects of pro-inflammatory Th1-conditioned cells after SCI. The effect of Th1-conditioned cells from interferon-γ (ifn-γ) knockout mice (ifn-γ^−/− Th1 cells) on the recovery after SCI was reduced. Transfer of Th1-conditioned cells led to the activation of microglia (MG) and macrophages (MΦs), with interleukin 10 (IL-10) upregulation. This upregulation of IL-10 was reduced when ifn-γ^−/− Th1 cells were transferred. Intrathecal neutralization of IL-10 in the spinal cord attenuated the effects of Th1-conditioned cells. Further, IL-10 is robustly secreted from Th1-conditioned cells in an ifn-γ-dependent manner. Th1-conditioned cells from interleukin 10 knockout (il-10^−/−) mice had no effects on recovery from SCI. These findings demonstrate that ifn-γ-dependent secretion of IL-10 from Th1 cells, as well as native MG/MΦs, is required for the promotion of motor recovery after SCI.     

Tumor-specific targeting of T helper type 1 (Th1) cells by anti-CD3 × anti-c-ErbB-2 bispecific antibody

T helper type1 (Th1) or type2 (Th2) cells were induced from naive Th cells obtained from ovalbumin-specific T cell receptor (TCR) transgenic mice. Th1 cells producing interferon γ (IFNγ) exhibited stronger antigen-specific cytotoxicity against ovalbumin-(323–339)-peptide-pulsed A20 tumor cells than did Th2 cells. To develop a general method for applying antigen-nonspecific Th1 cells to tumor immunotherapy, we examined the targeting of Th1 cells to tumor cells using a bispecific antibody (bsAb) consisting of anti-(mouse CD3) mAb and anti-(human c-ErbB-2) mAb. When ovalbumin-specific Th1 or Th2 cells were cocultured with c-erbB-2-positive transfectants (CMS7HE), neither type of cell showed significant cytotoxicity or cytokine production in response to tumor cells. However, addition of bsAb resulted in the triggering of both Th1 and Th2 cells. Th1 cells showed higher levels of bsAb-dependent cytotoxicity against CMS7HE tumor cells than did Th2 cells. The targeting of Th1 cells to CMS7HE tumor cells by bsAb also triggered the production of cytokines such as IFNγ, interleukin-2 and tumor necrosis factor α (TNFα). The released TNFα was demonstrated to be a critical cytolytic factor in bsAb-mediated cytotoxicity by Th1 cells. Finally, Th1 cells were demonstrated to show antitumor activity in vivo against human c-erbB-2-positive tumor cells implanted in nude mice. These results suggest that Th1 cells are useful effector cells for the application to adoptive tumor immunotherapy in conjunction with bsAb. Received: 22 April 1999 / Accepted: 2 July 1999     

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that infect humans and livestock¹. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment¹. This model is a powerful tool to examine factors that control CD4⁺ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated cytokines (IL-12, IL-18, IFN-γ) promote chronic infections in genetically susceptible AKR/J mice^2-6. Th2 cytokines promote physiological changes in the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle contraction, all of which have been implicated in worm expulsion^7-15. Here we detail a protocol for propagating Trichuris muris eggs which can be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in the gastrointestinal tract as well as immune effector functions of IECs.     

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Background

The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4⁺ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4⁺ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.

Methodology

Levels of Th17 and the other three CD4⁺ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.

Conclusions

Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.     

Antitumor effects and immunoregulation mechanisms of IL-23 gene in mouse mammary cancer mediated by retrovirus

Background: Interleukin (IL)-23, composed of p19 and p40 subunits, has diverse functions in regulating immune systems, enhancing cell-mediated immunity. In the present study, we investigated whether forced expression of the p19-linked p40 gene in murine mammary cancer cells (MA891) produced antitumor effects in vivo. Tumor growth of MA-891 cells expressing IL-23 (IL-23/MA891) in mice was retarded compared with parental and vector DNA-transduced tumors and survival of the mice inoculated with IL-23/MA-891 cells was prolonged. Expressions of the CD4⁺ T cells and CD8⁺ T cells were up-regulated not only in IL-23/MA-891 tumor specimens but also in spleen cells of mice inoculated with IL-23/MA-891 as compared with those of mice inoculated with parental or vector DNA-transduced tumors. Cytotoxic CD8⁺ T lymphocyte (CTL) activity of spleen cells from mice inoculated with IL-23/MA-891 was also significantly higher than the other two groups. Th1-type cytokines such as interferon-γ, TNF-α and IL-12p70 secreted from spleen cells of mice bearing IL-23/MA-891 tumors were increased while Th2-type cytokine IL-4 was negative regulated. Moreover, we have identified that the quantity of DC in spleen cells of mice bearing IL-23/MA-891 tumors was increased as compared with those mice bearing parental or vector DNA-transfected tumors.     

Elimination of CD4+ T cells in mice bearing an advanced sarcoma augments the antitumor action of interleukin-2

Experiments were undertaken to determine whether the depletion of CD4⁺ T cells from mice bearing an advanced immunogenic SA-1 sarcoma would result in an enhanced ability of interleukin-2 (IL-2) to cause tumor regression. The results show that whereas IL-2 therapy given as a 5-day course starting on day 10 of tumor growth caused complete regression of the tumor, it failed to cause regression if started on day 15 of tumor growth. However, in mice depleted of CD4⁺ T cells by treatment with anti-CD4 monoclonal antibody (mAb), IL-2 therapy started on day 15 resulted in appreciable tumor regression in most animals, and the therapeutic effect was greatly increased if two consccutive courses of anti-CD4 mAb and IL-2 therapy were given. On the other hand, treatment with anti-CD4 mAb alone had no effect on tumor growth. It was shown that the therapeutic action of combination therapy with anti-CD4 mAb and IL-2 was mediated by CD8⁺ T cells, because the therapeutic effect was completely ablated in mice depleted of CD8⁺ T cells with anti-CD8 mAb. Taken together these results suggest that, at a late stage of growth of an immunogenic tumor, depletion of CD4⁺ T cells can enhance the antitumor effect of IL-2 therapy by releasing CD8⁺-T-cell-mediated immunity from T-cell-mediated suppression.     

Superior anti-tumor protection and therapeutic efficacy of vaccination with allogeneic and semiallogeneic dendritic cell/tumor cell fusion hybrids for murine colon adenocarcinoma

Cancer immunotherapy by dendritic cell (DC)/tumor cell fusion hybrids (DC/TC hybrids) has been shown to elicit potent anti-tumor effects via the induction of immune responses against multiple tumor-associated antigens. In the present study, we compared the anti-tumor effects of vaccinating Balb/c mice (H-2^d) with CT26CL25 colon carcinoma cells that had been fused with either syngeneic DCs from Balb/c mice, allogeneic DCs from C57BL/6 mice (H-2^b) or semiallogeneic DCs from B6D2F1 mice (H-2^b/d). Preimmunization with either semiallogeneic or allogeneic DC/TC hybrids induced complete protection from tumor challenge, whereas mice preimmunized with syngeneic DC/TC hybrids were only partially protected (75% tumor rejection). The average number of pulmonary metastases after intravenous tumor injection decreased significantly following immunization with semiallogeneic or allogeneic DC/TC hybrids (8.3 ± 7.9 or 16.3 ± 3.5, mean ± SD) relative to syngeneic DC/TC hybrids (67.8 ± 6.3). These data demonstrate that vaccination with semiallogeneic DC/TC hybrids resulted in the greatest anti-tumor efficacy. Anti-tumor effects showed by in vivo studies were virtually accomplished by the frequency of induced CTLs specific to both gp70 and β-galactosidase assessed by using pentameric assay. Among the fusion vaccines tested, semiallogeneic DC/TC hybrids induced the highest ratio of Th1 cytokine IFN-γ to Th2 cytokine IL-10. In addition, allogeneic or semiallogeneic DC/TC hybrids elicited a significantly stronger NK activity than syngeneic DC/TC hybrids. These findings suggest that in clinical settings, DCs derived from a healthy donor (which are generally characterized as more semiallogeneic than allogeneic) may be more capable than autologous DCs of inducing promising anti-tumor effects in vaccinations with DC/TC hybrids.