Glial TIM-3 Modulates Immune Responses in the Brain Tumor Microenvironment.

T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3), a potential immunotherapeutic target for cancer, has been shown to display diverse characteristics in a context-dependent manner. Thus, it would be useful to delineate the precise functional features of TIM-3 in a given situation. Here, we report that glial TIM-3 shows distinctive properties in the brain tumor microenvironment. TIM-3 was expressed on both growing tumor cells and their surrounding cells including glia and T cells in an orthotopic mouse glioma model. The expression pattern of TIM-3 was distinct from those of other immune checkpoint molecules in tumor-exposed and tumor-infiltrating glia. Comparison of cells from tumor-bearing and contralateral hemispheres of a glioma model showed that TIM-3 expression was lower in tumor-infiltrating CD11b+CD45mid glial cells but higher in tumor-infiltrating CD8+ T cells. In TIM-3 mutant mice with intracellular signaling defects and Cre-inducible TIM-3 mice, TIM-3 affected the expression of several immune-associated molecules including iNOS and PD-L1 in primary glia-exposed conditioned media (CM) from brain tumors. Further, TIM-3 was cross-regulated by TLR2, but not by TLR4, in brain tumor CM- or Pam3CSK4-exposed glia. In addition, following exposure to tumor CM, IFNγ production was lower in T cells cocultured with TIM-3-defective glia than with normal glia. Collectively, these findings suggest that glial TIM-3 actively and distinctively responds to brain tumor, and plays specific intracellular and intercellular immunoregulatory roles that might be different from TIM-3 on T cells in the brain tumor microenvironment. SIGNIFICANCE: TIM-3 is typically thought of as a T-cell checkpoint receptor. This study demonstrates a role for TIM-3 in mediating myeloid cell responses in glioblastoma.

Glial TLR2-driven innate immune responses and CD8+ T cell activation against brain tumor.

Growing interest has been focused on the roles of microglia as sentinels and effector cells that guard diverse pathological milieu in the brain. Here, it has been reported that microglial TLR2 is a crucial molecule that confers innate and adaptive immunity against brain tumor. TLR2 is preferentially expressed on microglia, brain-resident immune cells, in the tumor-bearing cerebral hemisphere of mouse and rat intracranial tumor models. Microglial TLR2 rapidly responds to brain tumor and modulates the inflammation-associated immune responses including phagocytosis, which are markedly decreased in TLR2-deficient mice. We further reveal that TLR2, but not TLR4, is essential for the tumor-triggered increase of MHC I in microglia. in vitro co-culture and in vivo experiments show that the glial TLR2-MHC I axis contributes to the proliferation and activation of CD8+ T cells by brain tumor. In addition, brain tumor-bearing ß2m-/- , Tlr2-/- , or Rag2 -/- γc -/- mice exhibit higher tumor volumes compared with WT mice with tumor. Survival analysis of GL26-bearing MHC I-defective mice also support the contribution of glial TLR2-MHC I axis to brain tumor immunity. Moreover, using publicly available data sets of human brain tumor patients, we find that glioblastoma (GBM) tissues with high TLR2 level have similar co-occurrence patterns with MHC I molecules, and the amounts and activity of infiltrating CD8+ T cells are correlated with TLR2 level in tissues from GBM patients. Collectively, our findings provide the importance of glial TLR2-driven innate and adaptive immune responses in the brain tumor microenvironment.

Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.

Galectin-3, a ß-galactoside-binding lectin, has been proposed to have multifaceted functions in various pathophysiological conditions. However, the characteristics of galectin-3 and its molecular mechanisms of action are still largely unknown. In this study, we show that galectin-3 exerts cytokine-like regulatory actions in rat and mouse brain-resident immune cells. Both the expression of galectin-3 and its secretion into the extracellular compartment were significantly enhanced in glia under IFN-γ-stimulated, inflamed conditions. After exposure to galectin-3, glial cells produced high levels of proinflammatory mediators and exhibited activated properties. Notably, within minutes after exposure to galectin-3, JAK2 and STAT1, STAT3, and STAT5 showed considerable enhancement of tyrosine phosphorylation; thereafter, downstream events of STAT signaling were also significantly enhanced. Treatment of the cells with pharmacological inhibitors of JAK2 reduced the galectin-3-stimulated increases of inflammatory mediators. Using IFN-γ receptor 1-deficient mice, we further found that IFN-γR 1 might be required for galectin-3-dependent activation of the JAK-STAT cascade. However, galectin-3 significantly induced phosphorylation of STATs in glial cells from IFN-γ-deficient mice, suggesting that IFN-γ does not mediate activation of STATs. Collectively, our findings suggest that galectin-3 acts as an endogenous danger signaling molecule under pathological conditions in the brain, providing a potential explanation for the molecular basis of galectin-3-associated pathological events.

Secretion of EGF-like domain of heregulinß promotes axonal growth and functional recovery of injured sciatic nerve.

Neuregulin 1 (NRG1) and epidermal growth factor receptor (ErbB) signaling pathways control Schwann cells during axonal regeneration in an injured peripheral nervous system. We investigated whether a persistent supply of recombinant NRG1 to the injury site could improve axonal growth and recovery of sensory and motor functions in rats during nerve regeneration. We generated a recombinant adenovirus expressing a secreted form of EGF-like domain from Heregulinß (sHRGßE-Ad). This virus, sHRGßE-Ad allowed for the secretion of 30-50 ng of small sHRGßE peptides per 10(7-8) virus particle outside cells and was able to phosphorylate ErbB receptors. Transduction of the concentrated sHRGßE-Ad into an axotomy model of sciatic nerve damage caused an effective promotion of nerve regeneration, as shown by histological features of the axons and Schwann cells, as well as increased expression of neurofilaments, GAP43 and S100 in the distal stump of the injury site. This result is consistent with longer axon lengths and thicker calibers observed in the sHRGßE-Ad treated animals. Furthermore, sensory and motor functions were significantly improved in sHRGßE-Ad treated animals when evaluated by a behavioral test. These results suggest a therapeutic potential for sHRGßE-Ad in treatment of peripheral nerve injury.

Mycophenolate mofetil promotes down-regulation of expanded B cells and production of TNF-alpha in an experimental murine model of colitis.

In this study, we used a murine intestinal inflammation model that mimics immunologic characteristics of human Crohn's disease (CD) to investigate the anti-inflammatory effects of mycophenolate mofetil (MMF) on intestinal injury and tissue inflammation. When these colitic mice were pretreated with MMF, we observed a significant decrease in mortality rates and body weight loss as well as an improvement in both wasting and histopathologic signs of colonic inflammation, relative to untreated colitic mice. To determine the mechanisms of action of MMF, we compared various immunological characteristics of the untreated and MMF-pretreated colitic mice. MMF-pretreated colitic mice showed an 18% decrease in the proportion of CD19+ B cells compared with untreated colitic mice 3 days. As a result, MMF pretreatment increases proportion of apoptotic T and B cells, especially CD19+ B cells. Also, down-regulation of Th1 cytokines (TNF-alpha, IFN-gamma) and augmentation of CD4+CD45RB(low) regulatory T (Treg) cells were observed in MMF-pretreated colitic mice compared with untreated colitic mice. Furthermore, mycophenolic acid (MPA) reduced TNF-alpha-stimulated NF-kappaB activation in HT-29 colon epithelial cells. Also, MMF-pretreated colitic mice significantly reduced expression of MD-1 compared with untreated colitic mice on B cells and dendritic cells (DCs). These studies show that MMF pretreatment can improve experimental colitis by down-regulation of expanded B cells population through apoptosis and augmentation of Treg cells. Through these mechanisms, MMF might also be an effective agent for the treatment of other diseases characterized by mucosal inflammation.

15-deoxyspergualin prevents mucosal injury by inhibiting production of TNF-alpha and down-regulating expression of MD-1 in a murine model of TNBS-induced colitis.

The immunosuppressive drug 15-deoxyspergualin (DSG) is currently being used in clinical trials to prolong graft survival and reverse graft rejection. Here we evaluated whether DSG has a potential for ameliorating diseases characterized by mucosal inflammation. Using a murine model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, we were able to demonstrate that DSG reduced the severity of colitis. Therefore, colitic mice pretreated with DSG showed a striking improvement of the wasting disease compared with colitic mice, as assessed by weight loss as well as clinical, macroscopic and microscopic analysis. Also, we observed the significant change occurred in the CD19(+) B cell subset, which was decreased 15% in DSG pretreated colitic mice compared with colitic mice. However, DSG pretreatment does not influence the apoptotic population of T and B cells. Compared with colitic mice, down-regulation of TNF-alpha production was observed in DSG pretreated colitic mice. In addition, DSG pretreated colitic mice significantly reduced expression of MD-1 compared with colitic mice on B cells and dendritic cells (DCs). Therefore, pretreatment with DSG resulted in a significant histologic improvement, protecting against mucosal ulcerations and reduced inflammatory response by modulating expression of MD-1, which plays a very important role in immune response on B cells and DCs. Also, this improvement was paralleled by a reduction in TNF-alpha levels. Collectively, current results demonstrate that DSG may be an effective agent for the treatment of diseases characterized by mucosal inflammation.

Intratumoral delivery of IL-18 naked DNA induces T-cell activation and Th1 response in a mouse hepatic cancer model.

BACKGROUND: The novel cytokine, interleukin (IL)-18, is a strong interferon-gamma inducer and costimulatory factor in Th1 cell activation. IL-18 triggers IFN-gamma production and enhances cytolytic activity in both T and NK cells. However, the exact mechanism of antitumor action of IL-18 remains to be clarified. To determine the effects of IL-18 plasmid DNA on hepatic cancer in mice, CT26 murine colon adenocarcinoma cells were established in mouse liver. METHODS: Plasmid vectors encoding IL-18 were transferred directly into the liver 7 days after tumor injection to restrict IL-18 expression within the tumor site. The IL-18 protein level was increased in the liver 4 days after plasmid injection, and a marked antitumoral effect was observed at day 7. Antitumor effects were evaluated by measuring tumor regression, immune cell population, and IFN-gamma production. RESULTS: The IL-18 plasmid controlled the growth of hepatic tumors and proliferation of splenic immune cells. Moreover, treatment of CT26 tumors with the IL-18 plasmid significantly enhanced the population of the effector T and NK cells in the spleen and peripheral blood. In spleen, the population of CD4+CD62Low cells was augmented in response to IL-18 on day 7. These results are consistent with the increase in CD4+ T cells secreting IFN-gamma, but not CD8+ T cells. The marked reduction of tumor growth in tumor-bearing mice was associated with the maintenance of IFN-gamma production in spleen in response to IL-18. These antitumoral effects were maintained until 14 days after plasmid injection. CONCLUSION: Our results suggest that direct plasmid DNA transfer of IL-18 with no accompanying reagents to augment transfection efficiency may be useful in tumor immunotherapy.

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation.

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low) and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low) T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two- signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.

Beneficial effects of simultaneous treatment with 15-deoxyspergualin and monoclonal antibodies to CD45RB and CD154 on murine islet transplantation recipients.

BACKGROUND: Treatment of transplant recipients with either 15-deoxyspergualin (DSG) or monoclonal antibodies (mAbs) to T-cell proteins CD45RB and CD154 (a two-signal blockade) has been shown to prolong islet graft survival. Therefore, we investigated the combined effect of DSG, anti-CD45RB, and anti-CD154 in murine islet model. METHODS: Chemically induced diabetic C57BL/6 mice underwent allografting with islets from BALB/c mice or xenografting with rat islets. After transplantation, they were treated with either DSG, the two-signal blockade, or both (the triple treatment). The tolerogenic effects of the posttransplant treatments were measured with an intraperitoneal glucose tolerance test (IPGTT), immunohistology, enzyme-linked immunosorbent assays, and flow cytometry. RESULTS: Blood glucose profiles measured after glucose challenges were improved in all islet recipients. Enhancement of xenograft survival in triple-treated groups was not statistically significant (P = 0.08), compared to graft survival in group received only the two-signal blockade. However, 15 days after transplantation, xenografts in the triple-treated group showed a significant decrease in the proportion of CD4, CD8, and CD4CD45RB T-cells, and in the expression of interleukin-10 and interferon-gamma, relative to grafts in the other treatment groups. In addition, reduced infiltration of the xenografts by CD3 T-cells was observed in groups that had received either the two-signal blockade or the triple treatment. With long-term (>248 days) xenografts, only those in the triple-treated group were free of inflammatory infiltrates. These grafts also exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to grafts in the other treatment groups. CONCLUSION: Triple treatment has a beneficial effect in murine islet xenotransplantation.

Tolerance induction through megadose bone marrow transplantation with two-signal blockade.

BACKGROUND: Induction of mixed chimerism is currently the most promising concept for clinical tolerance induction; however, the toxicity of the required host conditioning for allogeneic bone marrow transplantation (BMT) should be overcome. Therefore, we explored tolerogenic effectiveness of megadose BMT with anti-CD45RB and anti-CD154 mAb (two-signal blockade) in murine recipients without conditioning. MATERIALS AND METHODS: Recipient B6 mice of BALB/c skin allograft received conditioning and an optimal dose (2x10(7) cells) of BMT. For a megadose BMT model, the conditioning was not performed; instead, megadose (2x10(8) cells) of BM was transplanted. The recipients were then treated with anti-CD45RB mAb and anti-CD154 mAb alone or their combination. Flow cytometry was performed to analyze the degree and distribution of donor-derived cells, peripheral deletion of Vbeta5 or Vbeta11 T cells and intrathymic presence of donor MHC class II+ cells. Induction of chimerism-based tolerance to skin allograft was further determined. RESULTS: High levels ( approximately 23.7%) of mixed and multi-lineage chimerism-based tolerance to skin allograft were induced in the recipients (91%) treated with the optimal-dose BMT and the two-signal blockade. The megadose BMT could replace the recipient conditioning and establish low (approximately 10%) and stable multilineage chimerism. Donor-specific tolerance to skin allograft was induced in these chimeras through clonal deletion of donor-reactive cells. CONCLUSIONS: The megadose BMT with the two-signal blockade could effectively establish mixed and multi-lineage chimerism and induce donor-specific tolerance, suggesting its potential for clinical application.

Tolerance induction through simultaneous double bone marrow transplantation with two-signal blockade.

Cotransplantation of donor bone marrow cells (BMCs) in allograft recipients is currently the most promising concept for clinical tolerance induction; however, it still has many difficulties in its successful performance due to the toxicity of the required host conditioning, the risk of engraftment failure, and the problem of graft-versus-host disease (GVHD), as well as the limited accessibility of donor bone marrow cells. Therefore, we performed the studies to determine whether BMCs from multi-donors are simultaneously engrafted and lead to induction of chimerism-based tolerance through the tolerogenic protocol of whom effectiveness we have shown in a previous study. Using a murine model, it was demonstrated that grafted BMCs from BALB/c (H-2(d)) and CBA mice (H-2(k)) establish mixed type and multi-lineage double chimerism and induce immunological donor-specific tolerance to fully MHC-mismatched skin allografts in host C57BL/6 mice (H-2(b)) receiving conditioning with Busulfan and treatment with the two-signal blockade comprised of anti-CD45RB and anti-CD154 monoclonal antibodies.

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of inflammatory bowel disease.

4-1BB (CDw 137), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB monoclonal antibody (mAb) enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of anti-4-1BB mAb reduced the incidence and severity of inflammatory bowel disease. In this study, we investigated the effects of anti-4-1BB mAb in a murine intestinal inflammation model, which induced by the hapten reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) and mimics immunologic characteristics of human Crohn's disease (CD). Colitis was induced by rectal administration of 2mg of TNBS in 35% ethanol using a vinyl catheter positioned 4cm from the anus. All mice were sacrificed 3 and 10 days after the TNBS administration. The disease activity index (DAI), histological changes of the colon and production of cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were evaluated. The surface molecules of T cells in peripheral blood, spleen and mesenteric lymph nodes were analyzed by flow cytometry. When mice were treated with anti-4-1BB mAb, improvement in both wasting and histopathologic signs of colonic inflammation was observed. The increase a number of splenic CD4(+)CD25(+) T cells and decreased synthesis of the Th1 cytokine IL-2 also occurred. Interestingly, increased production of Th1 cytokine IFN-gamma and proportion of CD8(+) T cells were observed in mice treated with anti-4-1BB mAb in comparison to the colitic mice. These studies show, for the first time, that agonistic anti-4-1BB mAb can improve experimental colitis by reduction of IL-2 and augmentation of CD4(+)CD25(+) regulatory T cells. TNBS colitis is Th1-mediated and has similar histologic features and distribution of inflammation to CD. This study suggests that anti-4-1BB mAb therapy could be effective in the treatment of patients with CD.