Glatiramer Acetate Enhances Myeloid-Derived Suppressor Cell Function via Recognition of Paired Ig-like Receptor B.

Glatiramer acetate (GA; Copaxone) is a copolymer therapeutic that is approved by the Food and Drug Administration for the relapsing-remitting form of multiple sclerosis. Despite an unclear mechanism of action, studies have shown that GA promotes protective Th2 immunity and stimulates release of cytokines that suppress autoimmunity. In this study, we demonstrate that GA interacts with murine paired Ig-like receptor B (PIR-B) on myeloid-derived suppressor cells and suppresses the STAT1/NF-κB pathways while promoting IL-10/TGF-ß cytokine release. In inflammatory bowel disease models, GA enhanced myeloid-derived suppressor cell-dependent CD4+ regulatory T cell generation while reducing proinflammatory cytokine secretion. Human monocyte-derived macrophages responded to GA by reducing TNF-α production and promoting CD163 expression typical of alternative maturation despite the presence of GM-CSF. Furthermore, GA competitively interacts with leukocyte Ig-like receptors B (LILRBs), the human orthologs of PIR-B. Because GA limited proinflammatory activation of myeloid cells, therapeutics that target LILRBs represent novel treatment modalities for autoimmune indications.

IL-17 induced NOTCH1 activation in oligodendrocyte progenitor cells enhances proliferation and inflammatory gene expression.

NOTCH1 signalling contributes to defective remyelination by impairing differentiation of oligodendrocyte progenitor cells (OPCs). Here we report that IL-17 stimulation induces NOTCH1 activation in OPCs, contributing to Th17-mediated demyelinating disease. Mechanistically, IL-17R interacts with NOTCH1 via the extracellular domain, which facilitates the cleavage of NOTHC1 intracellular domain (NICD1). IL-17-induced NOTCH1 activation results in the interaction of IL-17R adaptor Act1 with NICD1, followed by the translocation of the Act1-NICD1 complex into the nucleus. Act1-NICD1 are recruited to the promoters of several NOTCH1 target genes (including STEAP4, a metalloreductase important for inflammation and cell proliferation) that are specifically induced in the spinal cord by Th17 cells. A decoy peptide disrupting the IL-17RA-NOTCH1 interaction inhibits IL-17-induced NOTCH1 activation and attenuates Th17-mediated experimental autoimmune encephalitis (EAE). Taken together, these findings demonstrate critical crosstalk between the IL-17 and NOTCH1 pathway, regulating Th17-induced inflammatory and proliferative genes to promote demyelinating disease.

Interferon-Gamma Receptor Signaling Plays an Important Role in Restraining Murine Ovarian Tumor Progression.

Immune cell-derived cytotoxic pathways have been implicated in antitumor immune responses. The goal of this study is to characterize how these cytotoxic pathways influence ovarian cancer development. We have utilized the TgMISIIR-TAg transgenic mouse model which expresses the transforming SV40 TAg in the ovary, leading to spontaneous development of ovarian tumors that closely mimic human epithelial ovarian cancer. To test how perforin (Prf1), granzyme B (GzmB) and interferon-gamma (IFNg) impact tumor occurrence and progression, we bred the TgMISIIR-TAg transgene into Prf1-/-, GzmB-/-, and IFNgR1-/- mice. The transgenic females developed peritoneal tumors at 9-15 weeks and succumbed at 184 ± 37 days of age with 100% penetrance (n=41). Knockout of these cytotoxic genes does not affect tumor occurrence. However, loss of function in the IFNg signaling pathway significantly expedited tumor progression with all of the IFNg R1-/- TgMISIIR-TAg females succumbing to tumor outgrowth at 167 ± 27 days of age (p=0.0074, n=24). In contrast, loss of function of Prf1 or GzmB did not significantly impact tumor progression and host survival. Since tumor cells in the IFNg R1-/- TgMISIIR-TAg mice are IFNg R1 deficient, we used the implantable MOSEC (mouse ovarian surface epithelial cell) tumor line to validate that IFNg R signaling in host immune cells but not in tumor cells impacts tumor progression. Indeed, when the IFNg -responsive MOSEC cells were inoculated, IFNg R1-/- mice exhibited significantly higher tumor burden compared to WT mice. Furthermore, a MOSEC-splenocyte co-culture system confirmed that IFNg R1-/- immune cells were less effective than WT immune cells in controlling MOSEC tumor growth in vitro. Together, these results indicate that the IFNg R signaling pathway plays an important role in restraining murine ovarian tumor progression.

Granzyme B Contributes to the Optimal Graft-Versus-Tumor Effect Mediated by Conventional CD4+ T Cells.

Granzyme B (GzmB) is a key cytotoxic molecule utilized by T cells to kill pathogen-infected cells or transformed tumor cells. Previous studies using allogeneic hematopoietic cell transplantation (allo-HCT) murine models showed that GzmB is required for CD8+ T cells to cause graft-versus-host disease (GVHD). However, our recent study demonstrated that GzmB-mediated damage of CD8+ T cells diminished their graft-versus-tumor (GVT) activity. In this study, we examined the role of GzmB in GVT effect mediated by conventional CD4+CD25- T cells (CD4+ Tcon). GzmB-/-CD4+ Tcon cells exhibited decreased GVT activity compared to wild-type (WT) CD4+ Tcon cells, suggesting that GzmB is required for the optimal GVT activity of CD4+ Tcon cells. On the other hand, GzmB-/- CD4+CD25+ regulatory T cells were as suppressive as WT regulatory T cells in suppressing GVT activity, which is consistent with our previous report showing that GzmB is not required for regulatory T cell-mediated suppression of GVHD. These results demonstrate that GzmB causes opposite impacts on GVT effect mediated by CD4+CD25- versus CD8+ T cells. Interestingly, GzmB-/- total T cells exhibited GVT activity equivalent to that of WT total T cells, suggesting that the opposite impacts of GzmB on the GVT effect of CD4+CD25- versus CD8+ T cells may neutralize each other, which can only be observed when an individual T cell subset is examined. Importantly, these differential roles suggest that targeting GzmB in selective T cell subsets may have the potential to enhance the beneficial GVT effect.

5-Fluorouracil targets thymidylate synthase in the selective suppression of TH17 cell differentiation.

While it is well established that treatment of cancer patients with 5-Fluorouracil (5-FU) can result in immune suppression, the exact function of 5-FU in the modulation of immune cells has not been fully established. We found that low dose 5-FU selectively suppresses TH17 and TH1 cell differentiation without apparent effect on Treg, TH2, and significantly suppresses thymidylate synthase (TS) expression in TH17 and TH1 cells but has a lesser effect in tumor cells and macrophages. Interestingly, the basal expression of TS varies significantly between T helper phenotypes and knockdown of TS significantly impairs TH17 and TH1 cell differentiation without affecting the differentiation of either Treg or TH2 cells. Finally, low dose 5-FU is effective in ameliorating colitis development by suppressing TH17 and TH1 cell development in a T cell transfer colitis model. Taken together, the results highlight the importance of the anti-inflammatory functions of low dose 5-FU by selectively suppressing TH17 and TH1 immune responses.

T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis.

Interleukin 1ß (IL-1ß) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1ß during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1ß production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1ß, whereas ATP stimulation triggered T cell production of IL-1ß via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1ß. Together these data reveal a critical role for IL-1ß produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.

Granzyme B-Mediated Activation-Induced Death of CD4+ T Cells Inhibits Murine Acute Graft-versus-Host Disease.

Granzyme B (GzmB) has previously been shown to be critical for CD8(+) T cell-mediated graft-versus-host disease (GVHD) but dispensable for GVHD mediated by CD4(+) T cells. However, previous studies used high doses of CD4(+) T cells in MHC-mismatched models that caused rapid and lethal GVHD. Because of the hyperacute lethality, it is possible that the role of GzmB was concealed by the system. Therefore, in this study, we have titrated down the T cell dose to precisely determine the contribution of GzmB in GVHD mediated by CD4(+)CD25(-) T cells. Surprisingly, we have found that GzmB(-/-)CD4(+)CD25(-) T cells cause more severe GVHD compared with wild-type CD4(+)CD25(-) T cells in both MHC-matched and mismatched models. Mechanistic analyses reveal that although GzmB does not affect donor T cell engraftment, proliferation or tissue-specific migration, GzmB(-/-) CD4(+)CD25(-) T cells exhibit significantly enhanced expansion because of GzmB-mediated activation-induced cell death of wild-type CD4(+)CD25(-) T cells. As a result of enhanced expansion, GzmB(-/-) T cells produced higher amounts of proinflammatory cytokines (e.g., TNF-α and IFN-γ) that may contribute to the exacerbated GVHD. These results reveal that GzmB diminishes the ability of CD4(+) T cells to cause acute GVHD, which contradicts its established role in CD8(+) T cells. The differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to control GVHD.

A flagellin-derived toll-like receptor 5 agonist stimulates cytotoxic lymphocyte-mediated tumor immunity.

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The "danger context" elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8(+) T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b(+) and CD11c(+) cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes.

Granzyme B-mediated damage of CD8+ T cells impairs graft-versus-tumor effect.

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and other malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the treatment. Using multiple tumor models and diverse donor-host combinations, we have studied the role of granzyme B (GzmB) in GVT effect. We first confirmed previous findings that GzmB deficiency diminished the ability of a high dose of CD8(+) T cells to cause lethal graft-versus-host disease. However, when GVT studies were performed using a moderate cell dose that the hosts could tolerate, GzmB(-/-) CD8(+) T cells demonstrated a significantly enhanced GVT effect. GzmB-mediated, activation-induced cell death in wild-type CD8(+) T cells was found responsible for their reduced GVT activity. Conversely, GzmB(-/-) CD8(+) T cells exhibited enhanced expansion, skewed toward an effector or effector memory phenotype, and produced higher amounts of IFN-γ and Fas ligand that might contribute to GzmB-independent tumor control. These findings demonstrate for the first time, to our knowledge, that GzmB-mediated damage of CD8(+) T cells impairs the desired GVT effect. This study suggests that inhibiting donor-derived GzmB function may represent a promising strategy to improve GVT effect without exacerbating graft-versus-host disease.

A TLR5 agonist enhances CD8(+) T cell-mediated graft-versus-tumor effect without exacerbating graft-versus-host disease.

Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and nonhematologic malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the effectiveness of the transplantation therapy. CBLB502 is a novel agonist for TLR5 derived from Salmonella flagellin. On the basis of TLR5-mediated immunomodulatory function, we examined the effect of CBLB502 on GVT activity. Using two tumor models that do not express TLR5, and thereby do not directly respond to CBLB502, we found that CBLB502 treatment significantly enhanced allogeneic CD8(+) T cell-mediated GVT activity, which was evidenced by decreased tumor burden and improved host survival. Importantly, histopathologic analyses showed that CBLB502 treatment did not exacerbate the moderate graft-versus-host disease condition caused by the allogeneic CD8(+) T cells. Moreover, mechanistic analyses showed that CBLB502 stimulates CD8(+) T cell proliferation and enhances their tumor killing activity mainly indirectly through a mechanism that involves the IL-12 signaling pathway and the CD11c(+) and CD11b(+) populations in the bone marrow cells. This study demonstrates a new beneficial effect of CBLB502, and suggests that TLR5-mediated immune modulation may be a promising approach to improve GVT immunity without exacerbating graft-versus-host disease.

Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses.

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg+alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-gamma and IL-4, but neither IFN-gamma nor IL-4 was detected in the HBsAg+alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.