Combination of anti-CD4 antibody treatment and donor lymphocyte infusion ameliorates graft-versus-host disease while preserving graft-versus-tumor effects in murine allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not only a well-established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo-HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft-versus-host disease (GVHD). CD4+ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8+ T-cell responses. In the present study, we investigated clinically applicable allo-HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo-HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti-CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo-HSCT. Late treatment with anti-CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti-CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo-HSCT followed by anti-CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.

Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8+ T Cells.

The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.

Enhancement of antitumor radiation efficacy and consistent induction of the abscopal effect in mice by ECI301, an active variant of macrophage inflammatory protein-1alpha.

PURPOSE: We studied whether i.v. administration of a chemokine after local tumor site irradiation could prevent remaining, as well as distant, nonirradiated tumor cell growth by leukocyte recruitment. EXPERIMENTAL DESIGN: Tumors were implanted s.c. in the right or both flanks. After local irradiation at the right flank, ECI301, a human macrophage inflammatory protein-1alpha variant was injected i.v. Tumor volumes were measured every 3 days after treatment. RESULTS: In Colon26 adenocarcinoma-bearing BALB/c mice, repeated daily administration (over 3-5 consecutive days) of 2 mug per mouse ECI301 after local irradiation of 6 Gy prolonged survival without significant toxicity, and in about half of the treated mice, the tumor was completely eradicated. Three weekly administrations of ECI301 after local irradiation also led to significant, although less effective, antitumor radiation efficacy. ECI301 also inhibited growth of other syngenic tumor grafts, including MethA fibrosarcoma (BALB/c) and Lewis lung carcinoma (C57BL/6). Importantly, tumor growth at the nonirradiated site was inhibited, indicating that ECI301 potentiated the abscopal effect of radiation. This abscopal effect observed in BALB/c and C57BL/6 mice was tumor-type independent. Leukocyte depletion studies suggest that CD8+ and CD4+ lymphocytes and NK1.1 cells were involved. CONCLUSIONS: Marked inhibition of tumor growth at the irradiated site, with complete tumor eradication and consistent induction of the abscopal effect, was potentiated by i.v. administration of ECI301. The results of this study may offer a new concept for cancer therapy, namely chemokine administration after local irradiation, leading to development of novel therapeutics for the treatment of advanced metastatic cancer.

Combined treatment with HMGN1 and anti-CD4 depleting antibody reverses T cell exhaustion and exerts robust anti-tumor effects in mice.

BACKGROUND: Transient depletion of CD4+ T cells results in tumor suppression and survival benefit in murine models; however, the tumor progression and recurrence still occur over more long-term monitoring of mice. Thus, we explored an additional strategy to enhance endogenous immune responses by an alarmin, high mobility group nucleosome binding protein 1 (HMGN1). METHODS: The anti-tumor effects of HMGN1, anti-CD4 depleting antibody, and their combined treatment were monitored in the Colon26 or the B16F10 subcutaneous murine models. The tumor-infiltrating CD8+ T cell proliferation, differentiation, exhaustion, and its gene expression were determined by flow cytometry, transcriptome analysis, and quantitative real-time PCR. RESULTS: Our results show that a systemic administration of low doses of HMGN1 with an anti-CD4 depleting antibody (HMGN1/αCD4) promoted expansion of CD8+ T cell populations (e.g. CD137+ PD-1+ and CD44hi PD-1+), recruited CCR7+ migratory dendritic cells to the tumor, and reduced co-inhibitory molecules (e.g. PD-1, LAG-3, and TIM-3) to counteract CD8+ T cell exhaustion. CONCLUSION: The HMGN1/αCD4 treatment expanded effector CD8+ T cells and prolonged their anti-tumor activities by rescuing them from exhaustion, thus resulting in tumor regression and even rejection in long-term monitored mice.

Robust Antitumor Effects of Combined Anti-CD4-Depleting Antibody and Anti-PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice.

Depletion of CD4(+) cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4(+) cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25(+) Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8(+) cell depletion. CD4(+) cell depletion led to the proliferation of tumor-specific CD8(+) T cells in the draining lymph node and increased infiltration of PD-1(+)CD8(+) T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies.

Non-steroidal anti-inflammatory drug, nabumetone, prevents indometacin-induced gastric damage via inhibition of neutrophil functions.

Nabumetone is a non-steroidal anti-inflammatory drug (NSAID). It works as a prodrug and is extensively metabolized to an active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). It is well known that neutrophil infiltration and activation are critical in the pathogenesis of NSAID-induced gastric injury, and nabumetone shows less incidence of gastrointestinal irritancy. We examined the effects of nabumetone on neutrophil activation and on indometacin-induced gastric damage. In the indometacin-induced gastric mucosal injury, rats were treated with indometacin and then nabumetone or 6MNA was orally administered. Nabumetone prevented gastric damage accompanied by the reduction of neutrophil infiltration into gastric mucosa, but such an effect was not observed with 6MNA. Nabumetone reduced the formyl methionyl leucyl phenylalanine (fMLP)-induced respiratory burst of human neutrophils to 30% of the control level in-vitro, but 6MNA did not. In addition, nabumetone prevented the fMLP-induced migration of neutrophils. Nabumetone did not inhibit O2- generation in the xanthine-xanthine oxidase system. These results suggest that nabumetone prevents gastric damage induced by the active metabolite, 6MNA, via the suppression of neutrophil activation in gastric mucosa.

Discovery, optimization, and pharmacological characterization of novel heteroaroylphenylureas antagonists of C-C chemokine ligand 2 function.

Through the application of TRAP (target-related affinity profiling), we identified a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-dependent and selective with regard to other chemokines. The compounds, however, did not antagonize the binding of (125)I-labeled CCL2 to the CCR2 receptor nor did they block CCR2-mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC(50) = 80 nM) with excellent oral bioavailability in rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.

Long-term treatment with propagermanium suppresses atherosclerosis in WHHL rabbits.

Macrophages play an important role in the pathogenesis of atherosclerosis, for which monocyte chemoattractant protein (MCP)-1 and CCR2 chemokine receptors may be involved. The authors have recently demonstrated that propagermanium exerts inhibitory effect on the CCR2 receptors. In the current study, the authors examined whether the organic germanium suppresses the MCP-1-induced monocyte migration in vitro and the development of atherosclerosis in WHHL rabbits in vivo. In the in vitro experiment, propagermanium concentration-dependently suppressed the MCP-1-induced migration of THP-1 cells. In the in vivo experiment, 20 WHHL rabbits were randomly divided into two groups; one group was treated with oral administration with propagermanium (9 mg/kg/day) for 3 months, and another group served as a control (n = 10 each). After 3 months, the aorta was isolated and stained with oil red O staining, and neointimal formation was quantified. Macrophage accumulation in the aorta was also evaluated by immunostaining. Long-term treatment with propagermanium did not affect the serum lipid profiles. However, the treatment significantly suppressed the oil red O-positive area of the total aorta (p < 0.05). Similarly, propagermanium significantly suppressed the intimal lesions (maximal intimal thickness and intimal area) and macrophage staining-positive area (all p < 0.05). A significant positive correlation was noted between macrophage staining-positive area and intimal lesions (p < 0.0001). These results indicate that long-term treatment with propagermanium suppresses the development of atherosclerosis in WHHL rabbits, suggesting its usefulness for the treatment of atherosclerotic vascular disease in humans.

Gene therapy expressing amino-terminal truncated monocyte chemoattractant protein-1 prevents renal ischemia-reperfusion injury.

Ischemia-reperfusion is closely associated with tissue damage in various organs, including kidney. Despite clinical investigations, useful therapy for renal ischemia-reperfusion injury is not available so far. This study evaluated therapeutic effects of gene therapy expressing an amino-terminal deletion mutant of MCP-1 called 7ND to inhibit monocyte chemoattractant protein (MCP)-1/CCR2 signaling in vivo on renal ischemia-reperfusion injury. 7ND gene was transferred into the femoral muscle of Balb/c mice. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells were observed, as was marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury in control mice, while these lesions were significantly decreased in 7ND gene-transfected mice. Macrophages in the interstitial region, most of which were CCR2-positive, were markedly decreased in 7ND gene-transfected mice after reperfusion. Although macrophages infiltrated around MCP-1-positive cells in control mice, the smaller number of F4/80-positive cells could infiltrate into the neighbor of MCP-1-positive cells in 7ND-treated mice. These results provide evidence that gene therapy by 7ND is potentially a powerful therapeutic approach to inhibit MCP-1/CCR2 signaling, resulting in rescue from renal ischemia-reperfusion injury.

Blockade of CCR2 ameliorates progressive fibrosis in kidney.

Fibrosis is a hallmark of progressive organ diseases. Monocyte chemoattractant protein (MCP)-1, also termed as macrophage chemotactic and activating factor (MCAF/CCL2) and its receptor, CCR2 are presumed to contribute to progressive fibrosis. However, the therapeutic efficacy of MCP-1/CCR2 blockade in progressive fibrosis remains to be investigated. We hypothesized that blockade of CCR2 may lead to the improvement of fibrosis. To achieve this goal, we investigated renal interstitial fibrosis induced by a unilateral ureteral obstruction in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393, CCR2 inhibitors. Cell infiltrations, most of which were F4/80-positive, were reduced in CCR2 knockout mice. In addition, dual staining revealed that CCR2-positive cells were mainly F4/80-positive macrophages. Importantly, CCR2 blockade reduced renal interstitial fibrosis relative to wild-type mice. Concomitantly, renal transcripts and protein of MCP-1, transforming growth factor-beta, and type I collagen were decreased in CCR2-null mice. Further, this CCR2-dependent loop for renal fibrosis was confirmed by treatment with CCR2 antagonists in a unilateral ureteral obstruction model. These findings suggest that the therapeutic strategy of blocking CCR2 may prove beneficial for progressive fibrosis via the decrease in infiltration and activation of macrophages in the diseased kidneys.

Gene therapy via blockade of monocyte chemoattractant protein-1 for renal fibrosis.

Monocyte chemoattractant protein (MCP)-1, also termed monocyte chemotactic and activating factor (MCAF)/CCL2, plays an important role in progressive organ fibrosis. It was hypothesized that MCP-1, through its cognate receptor, CCR2, regulates the pathogenesis and is therapeutically of importance for renal fibrosis. To achieve this goal, the therapeutic efficacy and efficiency in renal fibrosis induced by a unilateral ureteral obstruction nephropathy model in mice by the blockade of MCP-1/CCR2 signaling was studied. The delivery of N-terminal deletion mutant of the human MCP-1 gene, 7ND, into a skeletal muscle ameliorated renal fibrosis by resulting in decrease in the deposit of type I collagen and in reduced expression of TGF-beta. Concomitantly, gene transfer of 7ND reduced the cell infiltration, most of which were CCR2-positive macrophages, followed by the decrease in MCP-1 expression in the diseased kidneys. These observations suggest that MCP-1 through CCR2 signaling is responsible for Mphi recruitment, which augments downstream events, resulting in renal fibrosis. Moreover, these findings imply that gene therapy against MCP-1/CCR2 signaling via the mutant gene transferred strategy may serve a beneficial therapeutic application for renal fibrosis.

CCR2 signaling contributes to ischemia-reperfusion injury in kidney.

Examined were CCR2-deficient mice to clarify the contribution of macrophages via monocyte chemoattractant protein 1 (MCP-1 or CCL2)/CCR2 signaling to the pathogenesis of renal ischemia-reperfusion injury. Also evaluated was the therapeutic effects via the inhibition of MCP-1/CCR2 signaling with propagermanium (3-oxygermylpropionic acid polymer) and RS-504393. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells and marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury was observed. Ischemia-reperfusion induced the expression of MCP-1 mRNA and protein in injured kidneys, followed by CCR2-positive macrophages in interstitium in wild-type mice. The expression of MCP-1 was decreased in CCR2-deficient mice compared with wild-type mice. The number of interstitial infiltrated macrophages was markedly smaller in the CCR2-deficient mice after ischemia-reperfusion. CCR2-deficient mice decreased the number of interstitial inducible nitric oxide synthase-positive cells after ischemia-reperfusion. The area of tubular necrosis in CCR2-deficient mice was significantly lower than that of wild-type mice after ischemia-reperfusion. In addition, CCR2-deficient mice diminished KC, macrophage inflammatory protein 2, epithelial cell-derived neutrophil-activating peptide 78, and neutrophil-activating peptide 2 expression compared with wild-type mice accompanied with the reduction of interstitial granulocyte infiltration. Similarly, propagermanium and RS-504393 reduced the number of interstitial infiltrated cells and tubular necrosis up to 96 h after ischemia-reperfusion injury. These results revealed that MCP-1 via CCR2 signaling plays a key role in the pathogenesis of renal ischemia-reperfusion injury through infiltration and activation of macrophages, and it offers a therapeutic target for ischemia-reperfusion.