bluuebunny/crossref_metadata_2025 huggingface dataset:
Isoprenol-Induced Neuroprotection in Experimental Multiple Sclerosis
Authors: Ochoa-Reparaz Javier , Strawn Kevin D. , Kirby Trevor O. , Brown Madalyn N. , Walters Dana C. , Gibson K. Michael , Roullet Jean-Baptiste
Published: January 2023

Farnesol is a 15-carbon organic acyclic sesquiterpene alcohol (isoprenol) produced by plants and animals. It is a potent blocker of neuronal voltage-gated Ca2+ found in the human brain. It has potent anti-oxidant and antiinflammatory effects in vitro, and neuroprotection based on the regulation of free radicals production by glial cells was demonstrated in a murine model of neurotoxicity. Because inflammation, oxidative stress and intraneuronal Ca2+ overload are associated with central nervous system (CNS) demyelinating diseases, we sought to determine if farnesol treatment would result in protection against experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). We compared the progression of EAE in C57BL/6 mice treated orally with farnesol (100 mg/kg/daily by gavage in olive oil) versus EAE progression in untreated mice (control) (n = 10/group). Treatment started at EAE induction and continued for 25 days. EAE clinical severity was monitored daily. Factorial repeated measures ANOVA of the scores showed: 1) a significant Day effect (disease onset 9 days after induction), 2) a significant Group effect (approx. 80% reduction in scores in the Farnesol group), and 3) a significant Day * Group interaction with scores being significantly lower in the Farnesol group every day starting one day after disease onset. At day 25, 50% of control mice had been euthanized due to scores ≥ 3.5 vs. none in the Farnesol group. Our results indicate that Farnesol significantly reduces disease onset in EAE. Studies to elucidate the mechanism of action of farnesol, screening of structural analogues of farnesol for anti-EAE activity, and assessment of farnesol therapeutic efficacy in other animal models of MS are being conducted.
Bilirubin Nanoparticle as an anti-inflammatory therapy for graft versus host disease
Authors: PAreek Sumedha , Zhang Mao , Trujillo-Ocampo Abel , Kim Jinyong , Jon Sangyong , Im Jin Seon
Published: January 2023

Graft versus host disease (GvHD) caused by alloreactive donor lymphocytes is a fatal complication of hematopoietic stem cell transplant (HSCT). Conditioning regimen consisting of chemotherapy and/or radiation given prior to HSCT can cause the initial tissue damage, which triggers the cross-presentation of alloantigens to the donor immune cells. Previous studies have shown that water-soluble PEGylated bilirubin nanoparticles (BRNP) selectively accumulate at the site of inflammation and prevent further tissue damage through scavenging reactive oxygen species. Therefore we, hypothesized that BRNP treatment after conditioning regimen can reduce GvHD by abating the initial tissue damage in HSCT, and investigated therapeutic efficacy of BRNP using murine GVHD model. Sublethally irradiated recipient mice (Balb/c) were infused with 4×106 bone marrow and 1×106 splenic T cells from donor mice (C57/B6) on day 1, and with or without BRNP (10mg/kg) on days 0, 2, and 4. GvHD symptoms were monitored for 60 days, and scored for changes in fur, skin, posture, activity, and weight. Untreated recipient mice (n=9) developed significantly worse GVHD (mean GVHD score=4.667) compared to BRNP treated recipient mice (n=9, mean GVHD score=1.556) (p=0.0028). This translated into the significantly better survival of BRNP treated mice with day 60 survival of 65% as compared to the untreated recipient mice with day 60 survival of 11% (p=0.03). In summary, we show that BRNP treatment can ameliorate clinical GvHD symptoms and improve survival in murine GVHD model. In the future, we plan to investigate the role of reactive oxygen species in GVHD and mechanism of action on anti-GVHD effects by BRNP.
Donor-specific antibody mediates chronic, but not acute, kidney allograft rejection in the absence of natural killer cells
Authors: Kish Danielle , Yagisawa Takafumi , Dvorina Nina , Valujskikh Anna , Baldwin William M. , Fairchild Robert L.
Published: January 2023

Dysregulated donor-specific antibody (DSA) responses are induced in B6.CCR5−/− mice transplanted with complete MHC mismatched kidney allografts and NK cells play a critical role in acute graft injury and failure. The current study investigated the consequence of high DSA titers on graft outcomes in the presence vs. absence of NK cell activation within the kidney graft. B6.CCR5−/− recipients of complete MHC mismatched A/J allografts or semi allogeneic (A/J x B6)F1 kidney grafts responded with equivalent serum DSA titers that reached peak by day 14 post-transplant. B6.CCR5−/− recipients rejected A/J allografts between days 15–30, whereas B6 isografts and (A/J x B6)F1 semi allogeneic grafts survived past day 65. On day 7 post-transplant NK cell infiltration into A/J allografts was composed of distinct Ly49b+ cell populations expressing low and high levels of NK1.1 and both NK1.1high and NK1.1low cells increased in A/J allografts with time to rejection. In contrast, NK cell infiltration into (A/J x B6)F1 grafts on day 7 post-transplant was composed entirely of NK1.1high cells that decreased to background/isograft numbers thereafter. (A/J x B6)F1 grafts at day 65 post-transplant had severe chronic injury with prominent interstitial fibrosis, glomerulopathy and arteriopathy that was accompanied by gene expression of profibrogenic factors. These results indicate that NK cells synergize with DSA to cause acute kidney allograft failure whereas high titers of DSA in the absence of NK cell activation cannot cause acute antibody-mediated rejection but induce the indolent development of interstitial fibrosis and glomerular injury that leads to graft dysfunction and failure at later times after transplantation.
Novel Consistently Ex-vivo Expanded CD8+CD25+FoxP3+Treg Cells Prolongs the Allogeneic Islet Survival
Authors: Kim Jung-Sik , Han Kyeo-Rae , Park Chung-Gyu
Published: January 2023

In addition to CD4+ regulatory T cells(Tregs), adoptive transfer of CD8+CD25+FoxP3+T cells is emerging as an alternative adjunctive therapy to diminish current reliance on lifelong, nonspecific immunosuppression after transplantation. Here we evaluated the possible therapeutic application of consistently ex-vivo expanded mouse CD8+CD25+FoxP3+T cells to prevent the rejection of allogeneic islets.

Around 80% of mouse CD8+CD25+T and CD4+CD25+T cells expressed FoxP3+T cells in spleen. Higher level of FoxP3 was expressed in CD8+Tregs compared to CD4+Tregs. Ex-vivo enriched CD8+CD25+Treg cells were successfully and consistently expanded to 600–1,000 times from the initial culture for 12 days with optimized culture conditions. High concentration of IL-2 (>500units) and TGF-beta together with aCD3-and aCD28 stimulation were essential to the massive ex-vivo expansion. Expanded Tregs showed 99% of FoxP3 expression and expressed high level of CD27, CD47 and Lag-3 in their surface. Very high amount of Granzyme B and minor amount of IFN-r and IL-17 were xpressed. Expanded CD8+Tregs successfully inhibits the proliferation and activation of CD4+T cells in vitro. In the allogenic islet transplantation model (Balb.Cà B6), Single injection of ex-vivo expanded CD8+Tregs prolonged islet allograft survival more than 100 days after the reconstitution with CD90.2+T cells in immunodeficient B6 rag2(−/−) mice by inhibiting antigen-specific IFN-r producing cell generation. In this study, we proved that ex-vivo expanded CD8+Treg retain regulatory activity and can protect islet allograft rejection by single administration. These implies that ex-vivo expanded CD8+Tregs has clinical relevance of an adjunctive cellular therapy.
In vitro characteristics and in vivo outcome of α1,3-galactosyltransferase gene-knockout miniature pig cornea in full-thickness corneal xenotransplantation using nonhuman primate
Authors: Yoon Chang Ho , Choi Se Hyun , Lee Hyun Ju , Kim Hong Pyo , Kang Hee Jung , Kim Jong Min , Choi Hyuk Jin , Park Chung-Gyu , Choi Kimyung , Kim Hyunil , Ahn Curie , Kim Mee Kum
Published: January 2023

We investigated the biofunctional profiles of α1, 3-galactosyltransferase gene knockout (GTKO) miniature pig cornea and its outcome in pig-to-rhesus full thickness corneal xenotransplantation (FTCX). Ten eyes of GTKO miniature pigs (n = 5) were evaluated for biofunctional characteristics. Five Chinese rhesus macaques underwent FTCX (mean donor age: 11.3 mo). Systemic tacrolimus, basiliximab, and IVIG were administered as programmed schedules. T cell subsets, antibodies (Ab) in blood, and C3a in aqueous humor (AH) were evaluated. The results were compared with those of the wild type miniature pig corneal grafted group (n = 4, all grafts survived > 24 weeks, using anti-CD154 Ab) as controls. Mean central corneal thickness was 763 μm. Na+/K+ ATPase was well expressed 7 days after preservation. Mean endothelial cell density was 4729/mm2, and was maintained >2800/mm2 through 7 days. Mean doubling time was 30.4 hour which was comparable to that in SNU miniature pigs (30.7 hour, p = .413). Median graft survival was 72 days. Regulatory T cells at rejection decreased from baseline (p = .043) and were lower than those of controls at postoperative (postop) 24 weeks (p = .014), however the other T cell subsets did not change. At rejection, anti-non-αGal Ab increased from baseline (p = .043), however Anti-αGal Ab did not. C3a in AH at postop 4 weeks and at rejection increased from baseline (all p = .043). At postop 4 weeks, AH and plasma C3a were higher than those of controls (p = .016 and .036, respectively). The biofunctional characteristics of GTKO miniature pig cornea are feasible for FTCX. Rejection of GTKO pig-to-rhesus FTCX is related with non-αGal Ab, complement and decrease of regulatory T cells. Addition of B cell suppressing regimen should be considered.
Tc17 cells mediate gut GVHD via IL-22 and dysbiosis
Authors: Song Qingxiao , Wu Xiwei , Zeng Deye , Jenq Robert R. , Van den Brink Marcel R. M. , Riggs Arthur D. , chen Yuanzhong , Zeng Defu
Published: January 2023

Gut GVHD is critical for determining the outcome of allogenic hematopoietic cell transplantation (HCT). Damage of Paneth cells and loss of RegIIIg can lead to dysregulation of intestinal microbiota and gut GVHD pathogenesis. RegIIIg can be produced by Paneth cells and intestinal epithelial cells, and its secretion is augmented by IL-22. However, the circumstances leading to RegIIIg dysregulation and dysbiosis remain largely unknown. Here we show that, in a MHC-mismatched model of C57BL/6 donor to BALB/c recipient, one injection of depleting anti-CD4 effectively prevents acute gut GVHD in recipients given wild-type (WT) transplants but has no effect in recipients given IFN-g−/− transplants. Interestingly, PD-L1−/−recipients given IFN-g−/− transplants showed spontaneous recovery of the disease. Recipients given IFN-g−/− transplants showed expansion of Tc17, IL-22 but not IL-17 from Tc17 was required for the disease induction. Furthermore, host tissue PD-L1 augmented expansion of Tc17 as well as CX3CR1+ DC, CX3CR1+ DC mediate bacteria clearance and prevent bacteria translocation, at the same time, CX3CR1+ DC produce IL-18 BP, which limit Tc17 expansion through IL-18 signaling. In addition, recipients given IFN-g−/− transplants showed no damage of Paneth cells but markedly increased RegIIIg. Gut GVHD in recipients had significant reduction of Barnesiella in feces and significant increase of E. Coli and Lactobacillus. Taken together, these results indicate that 1) in the absence of donor IFN-g, host-tissue PD-L1 mediates expansion of Tc17 through IL-18 signaling and dysregulation of IL-22 functional effect; 2) Dysregualtion of IL-22 from Tc17 cell can lead to dysbiosis and gut GVHD in the absence of Paneth cell damage.
Development of a murine intestinal explant model to examine immune mechanisms of graft versus host disease
Authors: Gao Chunxu , Hitchcock Shannon , Deutsch Heather , Rao Tadimeti , Amuzie Chidozie , Malaviya Ravi
Published: January 2023

Graft-versus host disease (GvHD) is a major course of mortality and morbidity in transplant recipients. Cytokine signaling plays a key role in the development of GvHD. To study the immune mechanisms relevant to intestinal pathology associated with GvHD, we developed a murine ex vivo model on intestinal explants derived from Xeno-GvHD to characterize mouse and human cytokine secretion and signaling. Intestinal tissues from Xeno-GvHD induced by transfer of human PBMC to irradiated NSG mice and control naïve mice were dissected and cultured. Histological analysis of the explants and measurement of lactate dehydrogenase (LDH) in the ex vivo culture system indicate that intestinal explants remain viable in culture for up to 72 hours, with an increased cytotoxicity developing over this time. Release of mouse proinflammatory cytokines including RANTES, IL-6, KC, and GM-CSF were significantly higher in GvHD explants compared to naïve controls. Among the human cytokines, RANTES was a key cytokine detected only from Xeno-GvHD explants. The patterns of cytokine/chemokine release were shown to be consistent in the ex vivo culture over time and on explants from different sites of intestine including ileum and colon. Furthermore, histochemical analysis of GvHD explants revealed much stronger immunoreactivity for RANTES compared to naïve explants. Overall, our results indicate inflammatory milieu in the intestines of GvHD mice leading to sustained production of proinflammatory cytokines in the ex vivo culture. These features indicate that the explant system is a relevant model for studying the mechanism(s) of inflammatory responses in intestinal GvHD.
Activation of host p53 enhances anti-tumor immunity by driving differentiation of immunogenic myeloid DCs in tumors, while simultaneously protecting against collateral autoimmunity against shared self-tumor antigens.
Authors: Sharma Madhav D. , Munn David H.
Published: January 2023

During immunotherapy, we show that large numbers of immunogenic CD103+ DCs can arise in murine tumors via direct differentiation myeloid-lineage (Ly6c+ monocytic) precursors. These “dual-phenotype” Ly6c+CD103+ DCs differentiate from a precursor population of monocytic myeloid-derived suppressor cells (MDSCs) in tumors. Differentiation was controlled by inflammation-induced activation of the transcription factor p53 in MDSCs. Targeted deletion of myeloid p53 selectively ablated the Ly6c+CD103+ DC population, which resulted in abrogation of all response to multiple forms of tumor immunotherapy. Pharmacologically elevating the level of p53 expression in host cells, by administering the p53-agonist (MDM2-inhibitor) drug nutlin-3a, caused a sustained increase in Ly6c+CD103+ DCs in tumors during immunotherapy, and markedly enhanced anti-tumor immunity. Conversely, however, when a nominal tumor antigen (OVA) was also shared by a critical host tissue (Rip-mOVA mice), elevation of host p53 using nutlin-3a was potent immunosuppressive and protective against lethal autoimmunity outside of the tumor, while still driving enhanced immunity – against the same antigen – within the tumor itself. Thus, p53-agonist drugs may represent a novel class immunotherapy agents, with high discrimination between tumor-associated antigens and collateral autoimmunity.
HMGN1 and R848 synergistically activate DCs by multiple signaling pathways
Authors: Alam Md Masud , Yang De , Trivett Anna , Oppenheim Joost J.
Published: January 2023

HMGN1 (High-mobility group nucleosome-binding protein 1, N1) is a non-histone DNA binding protein that acts extracellularly as Th1-polarizing alarmin. N1 promotes dendritic cell (DC) recruitment and maturation primarily via TLR4. Initially various other TLR ligands were tested in combination with N1 to identify the most stimulatory combination. This showed that R848, a synthetic TLR7/8 agonist in combination with N1 synergistically stimulated DC maximally. However, it remained unclear how N1 and R848 synergistically activated human DCs. Here, we show that co-stimulation with N1 and R848 in human monocyte-derived DCs (MoDCs) resulted in marked upregulation of DC surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1β and TNFα. This combination synergistically activated NF-κB and multiple MAPKs including SAPK/JNK, c-Jun, CREB and p38 involved in pro-inflammatory signaling pathways. Moreover, N1 and R848 also synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g. IRF3 and IRF7) and promoted the expression of type I IFN genes including IFNα2, IFNα4 and IFNβ1. Similar signaling pathways were also be induced in mouse bone marrow-derived DCs (BMDC). Overall, our findings show that 1) N1 synergizes with R848 in activating human and mouse DCs and 2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1-lineage immune response mediating tumor rejection in mice.
In situ vaccination improves efficacy of PD-1 blockade in unresponsive lymphoma tumors through induction of a highly efficient cross-presenting dendritic cell subset expressing TLR3
Authors: Hammerich Linda , Dhainaut Maxime , Keler Tibor , Davis Thomas , Salazar Andres M. , Brown Brian D. , Brody Joshua D.
Published: January 2023

B cell lymphomas are generally incurable, with low response rates to anti-PD1 therapy. Here, we tested the combination of in situ vaccination with PD-1 blockade in a pre-clinical mouse model.

A20 lymphoma-bearing mice were treated with a PD1 blocking antibody with or without an in situ vaccine combining intratumoral (it) injections of FMS-like tyrosine kinase-3 ligand (Flt3L), poly-ICLC (pIC) and local irradiation (XRT) of the tumor.

Untreated lymphoma tumors contained few dendritic cells (DC) and anti-PD1 treatment did not induce tumor regression. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC in the tumor. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by it DC. Importantly, tumor antigens were taken up mainly by CD103+ DC and not CD103-DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103-subsets. Combination of Flt3L with XRT and pIC induced tumor-reactive T cells, and delayed tumor growth and improved survival in 40% of mice. In situ vaccination also increased expression of PD-L1 on tumor cells and it DC. Consistently, combination of in situ vaccination with anti-PD1 led to complete tumor regression and increased long-term survival in the majority of mice. PD1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect.

In conclusion, in situ vaccination combining Flt3L, local XRT and pIC improves efficacy of anti-PD1 in checkpoint-unresponsive lymphoma tumors through induction of a highly efficient cross-presenting DC subset.
Cell death-inducing reagents activate cancer immune surveillance via inducing Interleukin-17D in macrophages
Authors: Seelige Ruth , Bui Jack D.
Published: January 2023

We have previously established a role for the cytokine IL-17D during cancer immune surveillance in that its cancer-intrinsic expression recruits NK cells into the tumor microenvironment, thereby leading to cancer regression. We additionally showed that the transcription factor Nrf2 regulates IL-17D and can itself be induced by the chemical tBHQ. Here, we sought to investigate what other endogenous or exogenous signals induce IL-17D not only in cancer cells but also in macrophages that are abundant in the tumor microenvironment. By testing different agonists in vitro, we found that all reagents that led to increased cell death (such as chemotherapy) also induced IL-17D. This effect was moderate in cancer cells but more profound in macrophages and depended on Nrf2. Interestingly, the expression of IL-17D correlated with type I Interferon expression, suggesting that these molecules function in the same pathway. Additionally, the only reagent that induced IL-17D without leading to cell death was poly (dA:dT), a nucleic acid known to induce intracellular DNA sensing pathways such as the Absent in melanoma (AIM)2 or the cGAS/Stimulator of IFNs (STING) pathway. This suggests that ectopic nucleic acids (likely resulting from cell death) are inducers of IL-17D. Remarkably, co-injecting bone marrow-derived macrophages (BMDMs) that had induced IL-17D together with cancer cells increased the number of NK cells in tumors and decreased cancer growth. This effect was dependent on IL-17D because it was abolished if IL-17D deficient BMDMs were injected. We hypothesize that the IL-17D pathway evolved to sense ectopic DNA, likely present due to cell death, and that boosting this pathway in cancer cells or macrophages could serve as cancer immune therapy.
Cell-cell fusion as a mechanism of DNA exchange in cancer
Authors: Searles Stephen , Santosa Endi , Bui Jack D.
Published: January 2023

Cell-cell fusion is a common biological phenomenon involved in both healthy and disease conditions. Fusion occurs when two or more cells combine their plasma membranes and become a single hybrid cell that retains certain genetic information from each parent cell. Here, using a Cre-loxP-based method initially developed to investigate extracellular vesicle targeting, we found that cancer cells spontaneously and rapidly deliver DNA to non-cancer cells in vitro via a cell-cell fusion event. We found that B16 melanoma cells and progressor and regressor fibrosarcomas were capable of spontaneously fusing with immune cells (macrophages) and non-immune cells (fibroblasts) in vitro, with varying degrees of efficiency. Further analysis of hybrid clones resulting from fusion between B16 melanoma cells and mouse embryonic fibroblasts showed that these cells were aneuploid and possessed enhanced clonal diversity and chemoresistance compared to non-hybrid melanoma cells. We also observed cell-cell fusion to occur in vivo between melanoma cells and non-cancer cells of both hematopoietic and non-hematopoietic origin. These findings suggest that cell-cell fusion occurs in murine cancer models and show that this mechanism has the potential to cause massive genomic alterations that are observed in cancer. Furthermore, these findings somewhat contradict recent publications suggesting that the Cre-loxP method measures only extracellular vesicle-mediated intercellular communication.
MyD88-stimulated T cells acquire resistance to MDSC-mediated suppression
Authors: Ciavattone Nicholas G. , Parker Dominique , Joseph Ann Mary , Davila Eduardo
Published: January 2023

The immunosuppressive tumor microenvironment presents a significant challenge to developing effective T cell-based cancer immunotherapies. The heterogeneous population of myeloid-derived suppressor cells (MDSCs) is a major contributor to the suppressive tumor microenvironment in many cancers. Generating tumor-reactive T cells with the capacity to resist MDSC-mediated suppression would help facilitate the production of potent anti-tumor T cell therapies. The activation of the Toll-like receptor-Myeloid differentiation primary response 88 (TLR-MyD88) signaling pathway in CD8+ T cells enhances cell proliferation, cytotoxic function, and survival. Our studies show that TLR-stimulated T cells are resistant to MDSC-mediated suppression from multiple cancer cell lines. MyD88-activated CD8+ T cells co-cultured with tumor-derived MDSCs displayed enhanced proliferation and cytokine production over control T cells. This is recapitulated using synthetic co-receptor CD8a-MyD88 engineered T cells which exhibit greater potential for proliferation, even in the presence of MDSCs. Our future objectives are to understand the mechanisms by which MyD88 stimulated T cells gain resistance properties and observe this phenomenon in vivo. We look to utilize these observations to augment antitumor immune responses.
Ibrutinib has a novel immunomodulatory effect by enhancing DC maturation both in vivo and in a model of inflammation
Authors: Saljoughian Noushin , Varikuti Sanjay , Halsey Gregory , Oghumu Steve , Satoskar Abhay
Published: January 2023

Myeloid derived suppressor cells (MDSCs) can inhibit T cell activation by altering the characteristics and activation of dendritic cells (DCs). We had previously reported that Ibrutinib enhances the expression of co-stimulatory molecules on DCs and promotes DC-mediated T cell activation. In the present study we further determined the mechanisms of DC immunomodulation by Ibrutinib. We evaluated the effect of Ibrutinib on (i) DC maturation and migration from the injection site to draining lymph nodes, (ii) T cell proliferation in vivo, and (iii) monocyte maturation to DCs using a mouse inflammation model. DCs were generated from mouse bone marrow-derived cells in the presence of vehicle or 10μM lbrutinib. CFES-labelled DCs were injected subcutaneously into the footpads of naive mice, and their migration to the regional lymph nodes and maturation profile was analyzed by flow cytometry. To evaluate T cell priming, CFSE-labelled OVA-specific T cells isolated from OT-II mice were injected into the tail vein of syngeneic mice that were prior injected with mature DCs loaded with OVA peptide. After four days, T cell proliferation was evaluated by CFSE dilution. In the inflammation model, C57BL6 mice were treated with vehicle or 6mg/kg/day Ibrutinib orally for a week. A sub-lethal dose of LPS was injected in the dorsal skin and the frequency of inflammatory monocytes and mature DCs was determined. We found that Ibrutinib significantly improves DC maturation and migration, inducing T cell activation and proliferation in vivo. In addition, Ibrutinib enhances monocyte maturation to DCs in the skin. The results suggest a novel effect of Ibrutinib, which may have therapeutic use for cancers characterized by a high MDSC infiltration.
The DNA methyltransferase inhibitor, Guadecitabine, shifts myeloid derived suppressor cell phenotype from immune-suppressive toward immune-stimulatory.
Authors: Luker Andrea , Graham Laura , Tolete Christina , Conrad Daniel , Bear Harry
Published: January 2023

Immune-evasion is a difficult obstacle to overcome in cancer therapy. Through the epigenetic modifications of DNA methylation and the recruitment of immunoregulatory myeloid-derived suppressor cells (MDSCs), tumors are able to dampen the body’s anti-tumor response. Here we show that the DNA methyltransferase inhibitor, Guadecitabine, is an effective tool for altering both tumor and MDSC phenotype. Treatment of 4T1 murine mammary carcinoma cells in vitro with Guadecitabine caused a significant increase in MHC I. In MDSCs, Guadecitabine caused an increased expression of MHC II and CD86, and enhanced antigen uptake. Additionally, Guadecitabine-treated MDSCs had a reduced capacity to block T cell proliferation, at least in part due to diminished nitric oxide production. In vivo, we established 4T1 flank carcinomas for 10 days prior to administering Guadecitabine. The resulting tumor size was markedly reduced and the characteristic splenomegaly from MDSC accumulation was largely reversed. Importantly, Guadecitabine did not affect resident lymphocyte populations in the spleen or infiltration to the tumor. Overall, these results suggest that Guadecitabine may be an effective therapy to boost the patient’s own immune response against cancer.
Tristetraprolin regulates CD8 T cell functions and antitumor immunity through targeting IL-12 family cytokines
Authors: Liu Jianguo , Wang Qinghong , Ning Huan , Hou Rong
Published: January 2023

IFN-γ-producing CD8+T cells (CTLs) are essential for host defense against viral infection and cancer. The mechanisms that regulate the development and function of CTLs still remain largely elusive. Tristetraprolin (TTP) is an RNA-binding protein that promotes decay of the mRNAs encoding cytokines produced in macrophages. We recently found that CD8T cells of TTP deficient mice showed effector phenotype and produced more IFN-γ than CD8 T cells of WT mice. When isolated naïve CD8 T cells were activated in vitro, however, IFN-γ production by WT and Zfp36−/−CD8 T cells was comparable. We found that the levels of IL-27 and IL-12 in serum were increased in Zfp36−/− mice compared to WT mice both at the resting state and after LPS challenge. Macrophages deficient of TTP produced more IL-27 and IL-12. Furthermore, we discovered that TTP inhibited IL-27 by promoting p28 mRNA degradation whereas suppressed IL-12 by affecting NF-kB signaling. Deletion of IL-27 receptor WSX-1 in Zfp36−/−mice (WSX-1/TTP double KO) led to a reduction in CTLs. In a murine mammary gland tumor model, tumor growth was suppressed in Zfp36−/−mice, and this suppression was almost completely abolished in the WSX-1/TTPdouble KO mice and after depleting CD8 T cells. Our study unveils a novel regulatory pathway for CTL function and antitumor immunity regulation mediated by TTP.
The impact of TCR affinity on T cell differentiation and dysfunction in tumors
Authors: Shakiba Mojdeh , Philip Mary , Camara Steven , Socci Nicholas D. , Schietinger Andrea
Published: January 2023

Although tumor-specific CD8 T cells are found in human tumors, cancers progress, indicating that these T cells are dysfunctional; yet the regulatory mechanisms underlying tumor-specific T cell dysfunction remain poorly defined. T cell-mediated immune responses are triggered by T cell receptor (TCR) binding to peptide-major histocompatibility complex (pMHC). In acute infections, affinity of TCR:pMHC interaction is a critical determinant of T cell expansion and effector function. However, little is known about how tumor antigen affinity impacts T cell differentiation and dysfunction in progressing tumors. To investigate the functional and molecular programs determined by affinity, we generated an in vivo tumor model expressing altered peptide ligands (APL) derived from SV40 large T antigen epitope I (TAG) and recognized by TAG-specific transgenic CD8 T cells (TCRTAG) with varying functional avidity. While affinity did not impact T cell activation and differentiation in tumor draining lymph nodes, it drove distinct functional and molecular programs at the tumor site. Interestingly, key transcription factors and effector molecules were regulated by signal strength, preserving a cell-intrinsic functional program in T cells with low-affinity interactions. In contrast, certain hallmarks of T cell dysfunction, including the expression of inhibitory receptors (e.g. PD1 and LAG3), were affinity-independent, revealing that even very weak TCR ligations can induce a typical exhaustion phenotype. Ongoing studies will define the transcriptional and epigenetic programs underlying the distinct dysfunctional T cell states in tumors driven by TCR signal strength.
Regulation of Chitinase-3-like-1 in T cell enhances anti-tumoral T cell responses to suppress lung metastasis
Authors: Kim Do-Hyun , Lim Sangho , Lee Hong-Gyun , Lee Chun Geun , Elias Jack A. , Choi Je-Min
Published: January 2023

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we showed that Chi3l1 expression was increased in Th2 cells while it decreased in Th1 cells by time dependent manner. In addition, Chi3l1-deficient T cells were hyper-responsive to TcR stimulation and were prone to differentiating into Th1 cells, and retroviral transduction of Chi3l1 rescue increased IFNγ expression in Chi3l1-deficient Th1 cells. Chi3l1-deficient Th1 cells showed increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice showed reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, knockdown of Chi3l1 expression in the lung using peptide-siRNA complex efficiently inhibited lung metastasis with enhanced Th1 and CTL infiltration and functions. Collectively, this study demonstrates Chi3l1 is a novel regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.
IL-37 mediates the anti-tumor activity in hepatocellular carcinoma via T cell activation and angiogenesis suppression
Authors: Mei Yu , Teng Wantian , Teo Huey Yee , Liu Haiyan
Published: January 2023

IL-37 has been shown to play an immunosuppressive role against inflammatory and autoimmune diseases. However, the role of IL-37 in tumor development is still controversial. Recent studies demonstrated that IL-37 could recruit NK cells, stimulate CD4+ T cell activation, as well as inhibit tumor angiogenesis to suppress tumor progression in different tumor models. Other studies suggested that IL-37 could affect DC function to suppress adaptive immune response. In the current study, we found that IL-37 could directly inhibit hepatocellular carcinoma (HCC) cell expansion and promote its apoptosis. The in vivo results showed that IL-37 could inhibit HCC development through enhancing the T cell cytokine production and killing capacity in murine orthotopic HCC model and DEN-induced HCC model. This anti-tumor effect was fully abrogated in NOD-SCID mice. Furthermore, we found that IL-37 could also inhibit tumor angiogenesis in peritumoral areas. The in vitro study showed that recombinant IL-37 protein could directly promote the migration, tube formation and IL-1R8, VEGF, MMP2 expression in HUVEC cells. However, the supernatant from IL-37 expressing HCC cells showed an inhibition on the HUVEC cell migration and tube formation, suggesting its secondary role on angiogenesis through tumor cells could be dominant in the tumor microenvironment. Overall, these results showed that IL-37 could suppress HCC development through T cell activation and angiogenesis suppression.
Glucocorticoid receptor signaling during prolonged psychological stress compromises the ability of invariant NKT cells to participate in antitumor immune surveillance
Authors: Rudak Patrick T. , Choi Joshua , Haeryfar S.M. Mansour
Published: January 2023

The nervous system serves critical roles in the regulation of immune responses. Consequently, the neuroimmune functional interface can be disrupted by psychological stress, potentially impeding our ability to combat malignancies. Invariant natural killer T (iNKT) cells are innate-like T cells that, upon activation by glycolipid antigens and/or select environmental cues such as inflammatory cytokines, participate in antitumor immune responses. However, how mediators of stress may impact iNKT cell functions in this context remains unexplored. We subjected BALB/c and/or C57BL/6 mice to prolonged psychological stress, in a well-established model of physical restraint, before administering α-GalCer (a glycolipid agonist of iNKT cells) or a combination of IL-12 and IL-18. We report that stress abrogates IFN-γ production by activated iNKT cells in vivo, an effect that is reversible by blockade of glucocorticoid receptors. Accordingly, α-GalCer-stimulated iNKT cells in stressed mice failed to protect against pulmonary metastasis of B16-F10 melanoma. We also observed that otherwise α-GalCer-transactivated NK cells from stressed mice had a diminished capacity to lyse YAC-1 lymphoma cells. Finally, the suppressive effect of stress on iNKT cells was not due to their apoptotic death. In fact, unlike conventional T cells, iNKT cells were resistant to glucocorticoid-induced apoptosis. Taken together, our findings define a novel mechanism underlying stress-induced immunosuppression, which involves innate-like but not conventional T lymphocytes.
Enhancing the immunotherapeutic Trastuzumab for selective activity in the low pH tumor microenvironment
Authors: Nguyen Annalee W. , Liu Yutong , Maynard Jennifer
Published: January 2023

Highly selective killing of tumor cells is a key requirement for a successful and tolerable intervention in cancer treatment. While immunotherapies have been revolutionary in reducing the systemic impacts of chemotherapy, dramatic and sometimes life-threatening side effects can occur when selectivity is not stringent enough. For example, the popular HER2+ breast cancer therapeutic Trastuzumab has well-documented cardiotoxic effects due to expression of HER2 on cardiomyocytes. To enhance selectivity of immunotherapeutics for tumor cells, we can take advantage of the unique tumor microenvironment and its low pH relative to healthy tissues to bias the activity of antibodies toward tumor tissues. We have engineered antibodies for enhanced binding and efficacy at low pH, which may alleviate the requirement for binding an antigen that is only present on cancer cells while simultaneously reducing side effects due to binding non-tumor cells. Trastuzumab CDRs were mutagenized and screened for binding of HER2 antigen at a tumor pH of 6.5 and reduced binding at the normal tissue pH of 7.4. In a more generalizeable approach, the Fc region of Trastuzumab was also mutagenized and screened for similar pH dependent binding to FcγrIIIA, the interaction responsible for recruitment of key anti-tumor natural killer cells. All library screening took place on the surface of CHO cells, ensuring selection of clones that express well and contain the glycosylation necessary for Fc structure and function. Improved Trastuzumab variants identified through this work have the potential to dramatically improve this popular breast cancer therapy by reducing the cardiotoxic side effects, making the therapy more tolerable and available to more patients.
Agonist Redirected Checkpoint (ARC), SIRPα-Fc-CD40L, for Cancer Immunotherapy
Authors: Fromm George , de Silva Suresh , Patel Arpita , Johannes Kellsey , Hornblower Josiah , Schreiber Taylor H.
Published: January 2023

Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. SIRPα-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain.

The SIRPα end of the ARC binds CD47 at 3.59 nM affinity, but does not cause hemolysis, compared to CD47 mAbs. The CD40L end binds CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates NFκB-functional activity (independent of Fc receptor cross-linking) and IL2 and TNFα secretion in an ex vivo super-antigen (SEB) assay. Furthermore, live cell imaging shows that SIRPα-Fc-CD40L enhanced phagocytosis of tumor cells by primary macrophages. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or their combination. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which may uniquely poise macrophages in the TME for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.
Detection of Immune Cell Markers and Metabolic Profiles in Saliva and Oral Brush Samples from Oropharyngeal Cancer Patients
Authors: Parker Katherine H.
Published: January 2023

Aims

HPV is a predominant cause of oropharyngeal cancers (OPC) in the US. HPV-related OPC incidence is more common in, and rapidly increasing among males. Immunity at the oral cavity in the context of OPC is still unknown. To better understand the oral immune environment in OPC, we assessed immune-cell markers/populations and metabolic profiles in oral specimens from OPC patients and controls.

Method

Fifty newly diagnosed OPC patients, and fifty age-matched controls were recruited at the Moffitt Cancer Center. Flow cytometry was performed on cells in saliva, and those collected with Orcellex® Brushes. Innate-immune and lymphocytic markers were examined within CD45+ cells. Metabolic profiles of saliva supernatants were assessed using ultrahigh performance liquid chromatography-tandem mass spectroscopy at Metabolon.

Results

Percentage of CD1a+, CD14+, CD3+, CD56+, and CD163+ cells within the leukocyte gate were reduced in saliva in cases compared to controls (two-tailed Mann-Whitney test, p < 0.05). Lipid oxidation was elevated in OPC cases, possibly stemming from reduced glycolysis. Moreover, a significant increase in 3-hydroxybutyrylcarnitine, aconitate, and 2-methylcitrate was observed (Welch’s two-sample t-test, p <0.05), as well as a trend in reduction of glucose, pyruvate, lactate, and glycate in cases. Additional proteomic analyses of saliva are under way to better understand protein profiles in OPC. Our findings suggest that immune cell populations are reduced in OPC cases, which may lead to immune suppression. Furthermore, the increase in lipid oxidation is suggestive of increased oxidative stress. Future studies are needed to confirm findings and delineate the role of these markers in cancer development.
Activation of the CD137 Pathway in T cells by a CD137 × 5T4 bispecific ADAPTIR Molecule Requires Co-engagement of CD137 and 5T4
Authors: Blahnik-Fagan Gabriele R. , Bader Robert , Bannink Jeannette , Mitchell Danielle , Misher Lynda , McMahan Cathy , Bienvenue David , Fritzell Sara , Säll Anna , von Schantz Laura , Ellmark Peter , Nelson Michelle , Hernandez-Hoyos Gabriela
Published: January 2023

CD137 (4-1BB) is a key costimulatory immune receptor (member of the TNFR-superfamily) that is expressed primarily on activated CD8+ T and NK cells. Stimulation of CD137 leads to enhanced proliferation, increased survival, intensified cytolytic activity of T cells and induced IFN-γ production. CD137 monoclonal antibody therapies have shown promising anti-tumor effects in the clinic, but systemic immune stimulation have induced dose-limiting hepatic toxicities. A novel bispecific antibody (ALG.APV-527) was developed based on ADAPTIR™ technology to direct the activation of T cells and NK cells to the tumor area, thereby minimizing systemic toxicity. ALG.APV-527 contains binding domains to the costimulatory molecule CD137 and the tumor-associated antigen 5T4. 5T4 is a tumor antigen expressed on a variety of solid tumor types. ALG.APV-527 was generated with binding domains from the Alligator-Gold® human scFv library, and has been optimized for binding and function. ALG.APV-527 increased CD8+ T-cell activation, as measured by IFN-γ production, but only in the presence of 5T4-expressing cells. Additionally, the CD137 ×5T4 bispecific antibody enhanced proliferation of primary T cells and increased potency in an NF-κB reporter assay. Of importance, the binding affinity and function of ALG.APV-527 retained a low Ec50 (nM range) regardless of the level of 5T4 expression on target cells. In conclusion, we have developed a novel ADAPTIR™ bispecific molecule that, based on preclinical data, has potential as a promising therapeutic for the treatment of a variety of 5T4-expressing solid tumors.
Combination of TLR8 and TLR4 agonists reduces the degrading effects of nicotine on DC-NK mediated effector T cells generation
Authors: Nouri-Shirazi Mahyar A. , Tamjidi Saba , Nourishirazi Erika
Published: January 2023

It is recognized that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. Studies suggest that combining more than one TLR agonists enhances the magnitude of immune responses to vaccination. We reported that TLR agonists acted in synergy and promoted human DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector Th1 and Tc1 cells.

In this study, we studied whether the combination of TLR agonists could diminish the degrading effects of nicotine, the immunosuppressive component of cigarette smoke, on human DC-NK mediated effector T cell generation.

We found that none of TLR agonists, single and combined, were able to diminish completely the adverse effects of nicotine on DCs. However, TLR3, TLR4, and TLR8 agonists acted as the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by nicotine-exposed DCs (nicDCs). When combined, TLR3+8 and TLR4+8 synergistically optimized nicDC maturation and IFN-γ secretion from nicotine-exposed NK (nicNK) during co-cultures. Interestingly, in contrast to DC-NK-T, co-cultures of nicDC-nicNK-T treated with TLR3+8 or TLR4+8 agonists produced similar frequency of effector memory CFSE-CCR7− Th1 and Tc1 cells. However, the effector cells from TLR4+8 followed by TLR3+8 treated nicDC-nicNK-T co-cultures produced significantly more IFN-γ when compared with aluminum salt treated co-culture.

Our data suggest that the addition of appropriate TLR agonists to vaccine formulation could potentially augment the vaccination outcome in smokers.
Efficacy of a Prototype Live-vectored Multi-antigen African Swine Fever Virus Vaccine
Authors: Mwangi Waithaka , Lokhandwala Shehnaz , Popescu Luca , Sangewar Neha , Elijah Catherine , Petrovan Vlad , Stoian Ana , Olcha Mathew , Bray Jocelyn , Waghela Surya , Kerrigan Maureen , Rowland Raymond R. R.
Published: January 2023

Subunit vaccine development against some complex pathogens require inclusion of multiple targets to optimize efficacy and or broaden protection against diverse strains. To hasten vaccine development, it is rational to evaluate efficacy of a prototype vaccine in the actual outbred host that require protection. African Swine Fever Virus (ASFV) is a complex high-consequence Transboundary Animal Disease pathogen for which there is no vaccine or treatment available. Current evidence shows that protection against the ASFV can be stimulated with a vaccine since pigs that recover from infection with less virulent isolates are protected against closely related virulent isolates. In addition, there is evidence that ASFV-specific cytotoxic T lymphocytes (CTLs) are required for viral clearance. We have tested protective efficacy of several adenovirus-vectored prototype formulations containing multiple rationally selected ASFV antigens. Homologous prime-boost immunization of commercial pigs with one of the experimental vaccine induced significant and unprecedented antigen-specific CTL responses that were detectable in peripheral blood mononuclear cells (PBMCs). Significant IFN-gamma-secreting cells were also detected in PBMCs post-priming and the memory was recalled upon boost. In addition, the immunogen primed antibody responses that were recalled upon boost. More importantly, the prototype vaccine conferred protection in 5/9 of vaccinees following intranasal challenge with the ASFV Georgia 2007/1 isolate. Notably, these survivors were healthy with low clinical scores. In comparison, only 2/5 of the negative controls were alive 17 days post-challenge and they had high clinical scores that necessitated termination.