bluuebunny/crossref_metadata_2025 huggingface dataset:
TGF-β-induced CD4+Foxp3+ T cells attenuate acute graft-verse-host disease via suppressing expansion and killing of effector CD8+ cells. (TRAN3P.875)
Authors: Zheng Song Guo , Gu Jian , Chen Dongdong , Wang Julie , Olsen Nancy
Published: January 2023

TGF-β-induced CD4+Foxp3+ T cells (iTregs) have been identified as important treating strategies for cell therapy to autoimmune diseases and other disorders. However, the prevention effect of these cells on acute graft-verse-host disease (GVHD) has been unsatisfied because these cells are unstable and less suppressive in the disease. Here we restudied the ability of iTregs to prevent acute GVHD in two different mouse models. Our results showed that iTregs, as long as an appropriate protocol is used, they displayed a strong and consistent ability to control acute GVHD development and increase the survival in two different types of acute GVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8+ cells, the expression of Granzyme A and B and cytotoxic effect of donor CD8+ cells, production of cytokines in T cells in acute GVHD. We therefore conclude that iTreg cells are able to prevent acute GVHD as long as the right methods for generating iTreg cells have been employed.
Crosstalk of BAFF and TLRs after HCMV Infection in renal transplantation recipients (TRAN3P.878)
Authors: He Xiaozhou , Xu Haiyan , Dong Panpan , Xue Dong , Ma Xuyi , Zhang Yanyun , Zhang Xueguang
Published: January 2023

To investigate the correlation of BAFF signal and TLRs activtion after HCMV infection in renal transplant (RTx) recipients. Peripheral blood and urine of RTx recipients (113 cases) were collected. Urine HCMV DNA was detected by quantitative real-time PCR (qPCR); Sera anti-pp65 IgG /IgM antibody were detected with the use of HCMV pp65 antigen slide by indirect immunofluorescence assay; Soluble BAFF (sBAFF) was tested by ELISA; Sera anti-HLA&MICA was determined by single-antigen flow bead assays (One Lambda) on a Luminex platform; The concerned genes selected by Gene Ontology (GO) function classification and KEGG Pathway analysis were tested by qPCR chip assay. In RTx recipients with high copy of urine HCMV DNA (>10,000 copy/ml, 12 cases, positive group), sBAFF, anti-pp65 antibody, and anti-HLA&MICA antibody also increased (P<0.05); The correlation analysis indicated that HCMV DNA level was positively related with sBAFF, anti-pp65 antibody, and anti-HLA&MICA antibody. And the qPCR chip assay results showed that 46 genes were differentially expressed (> 2 times) in all. In positive group BAFF-R and ICAM1 were upregulated, TLR9, NLRC5 and other 20 genes were downregulated, compared with negative group (with low copy of HCMV DNA); IFN-γ and ICAM1 were upregulated and 8 genes were downregulated in positive group, compared with healthy controls. So BAFF signal was enhanced after HCMV infection in RTx recipients, which involved in the decrease of renal allograft function.
Stem cell therapy to promote sensory and motor nerve function recovery in a limb transplant model (TRAN3P.863)
Authors: Fitzpatrick Emilie , DeHart Mary , Tercero Juan , Brown Tommy , Salgar Shashikumar
Published: January 2023

Introduction: Hand and leg transplantation (Tx) in military/civilian populations is increasingly needed to improve the quality of life. Our objective was to investigate whether Mesenchymal Stem Cell (MSC) therapy can improve nerve regeneration and function. Methods: We performed orthotopic syngeneic hind-limb transplants in Lewis (RT1.Al) rats. Following Tx rats received syngeneic MSC (5x106; passage ≤8) or vehicle intravenously and locally. Results: Ex vivo expanded Rat MSCs were CD29+, CD31-, CD34-, CD44+, CD45low, CD90+, MHC Class I+, Class II- and pluripotent. Limb Tx was highly successful (100% survival). Four weeks post-Tx sensory nerve function (determined by cutaneous pain reaction) was <0.3 on a scale of Grade 0 to 3 (0=No function; 3=Normal function). By 8 weeks in vehicle group it was 2.2±0.7(tibial), 1.2±0.5(peroneal) and 1.7±0.9 (sural), and in MSC group it was 2.6±0.4(tibial),1.0±0.9(peroneal) and 1.7±0.9(sural). Overall sensory function was higher but not significant in MSC group compared to vehicle group at 4, 8, 16 and 24 weeks post-Tx. Walking track analysis (for motor nerve function) did not produce clear foot prints to calculate Sciatic Function Index (SFI), so a novel grading system was developed. Gastrocnemius muscle was atrophied (P<0.05) and animals developed flexion-contractures in the transplanted limb. Conclusion: The limb Tx procedure was highly successful, sensory function was recovered (>70%) and MSCs appear to promote nerve function recovery.
DAP12 deficiency in liver allografts results in enhanced donor DC migration, augmented effector T cell responses and abrogation of transplant tolerance (TRAN3P.883)
Authors: Yoshida Osamu , Kimura Shoko , Dou Lei , Matta Benjamin , Geller David , Thomson Angus
Published: January 2023

Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno-adaptor DNAX-activating protein of 12kDa (DAP12) negatively regulated conventional liver DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from B6 wild-type (wt) or DAP12-/- mice into wt C3H recipients. Donor DC (H2-Kb+CD11c+) were quantified in spleens by flow cytometry. Anti-donor T cell reactivity was evaluated by ex vivo CFSE-MLR and in vivo delayed-type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A 3-4-fold increase in donor-derived DC was detected in the spleens of DAP12-/- liver recipients compared with those given wt grafts. Moreover, pro-inflammatory cytokine gene expression in the graft, IFNγ production by graft-infiltrating CD8+ T cells, and systemic levels of IFNγ were all elevated significantly in hosts of DAP12-/- liver recipients. DAP12-/- grafts also exhibited reduced incidences of CD4+Foxp3+ cells and enhanced CD8+ T cell IFNγ secretion in response to donor antigen challenge. Unlike wt grafts, DAP12-/- livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor DC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.
Mild cold stress resulting from standard housing conditions for laboratory mice masks the severity of graft vs. host disease in mouse models of bone marrow transplantation (TRAN3P.887)
Authors: Leigh Nicholas , Kokolus Kathleen , Qiu Jingxin , McCarthy Philip. , Cao Xuefang , Repasky Elizabeth
Published: January 2023

Clinical bone marrow transplants (BMT) yield lethal graft vs. host disease (GVHD) after transplant of un-manipulated bone marrow (BM). This differs from murine GVHD models that typically require addition of spleen or lymph node derived T cells. Recent work has shown that mice housed at mandated sub-thermoneutral ambient temperatures (22°C) are cold stressed, resulting in heat generation activated by the sympathetic nervous system. Since cold stressed mice exhibit weakened immune responses, we hypothesize that baseline GVHD is reduced compared to that seen in mice at thermoneutral temperatures (30°C). We compared GVHD in mice receiving MHC-mismatched un-manipulated BM while housed at 22 vs. 30°C at which cold stress is alleviated. In this BMT model, mice housed at 22°C show no signs of GVHD, however, we found weight loss and lethal GVHD in mice housed at 30°C. The same trend was observed with MHC-matched allogeneic BMT. As housing at 22°C activates the sympathetic nervous system, we blocked its activation by administering propranolol, a commonly prescribed β-blocker. Following BMT, mice at 22°C treated with β-blocker displayed lethal GVHD similar to untreated mice at 30°C. This suggests that T cell responses are dampened by cold stress through a β-adrenergic pathway which is circumvented by thermoneutral housing. This study provides insight into translational discrepancies of clinical and murine BMT and demonstrates lethal GVHD in a mouse model generated by un-manipulated BM.
Origin and function of host-derived dendritic cells in vascularized organ transplants (TRAN3P.889)
Authors: Zhuang Quan , Shen Lin , Zeng Qiang , Williams Amanda , Oberbarnscheidt Martin , Lakkis Fadi
Published: January 2023

Donor DC exit organ grafts after transplantation and are replaced by host DC. The origin and function of these tissue-resident, host DC however is unknown. Using flow cytometry and 2-photon intravital microscopy, we observed rapid replacement of donor DC by host DC in both syngeneic and allogeneic heart and kidney grafts within 3 days after transplantation, and demonstrated that the vast majority of host DC were monocyte-derived (Mono-DC). By day 7, allografts harbored >5-fold more DC than syn grafts and >45-fold more than native tissue. Enhanced DC accumulation in allografts was inhibited by treating recipients with cyclosporine A, indicating a crucial role for the host alloimmune response in DC influx. DC accumulation was only partially dependent on fractalkine receptor (CX3CR1) expression on host monocytes. Host DC that infiltrated grafts localized to perivascular areas, extended dendrites into the vessel lumens, mediated the transmigration of cognate effector T cells (Teff) into the graft, and continued to make stable contacts with the Teff after they had transmigrated. Depletion of host Mono-DC by administering DT to CD11b-DTR mice significantly reduced T cell infiltration and IFN-γ production in the graft. Hence, host Mono-DC amass rapidly in transplanted organs, replacing donor DC, and accelerate rejection by enhancing Teff migration, retention, and activation. Our results provide novel insights into the pathogenesis and treatment of allograft rejection.
Anti-porcine abumin IgG antibodies are elicited in diabetic non-human primate recipients after intraportal porcine islet transplantation (TRAN3P.890)
Authors: Kang Hee Jung , Lee Haneulnari , Park Eun Mi , Park Chung-Gyu , Miyagawa Shuji
Published: January 2023

Xenoreactive antibody responses were investigated in non-human primate (NHP) recipients after porcine islet transplantation (pITx). The levels of anti-αGal and anti-nonGal IgG Abs in serial sera of the recipients were measured by ELISA and flow cytometry using GalT-KO porcine endothelial cells (PECs), respectively. We detected an elicited anti-nonGal IgG response in some sera of the recipients. Through a series of work including EP of protein extracts from GalT-KO PECs, Western blot and MALDI-TOF/TOF mass spectrometry, the putative antigen was identified as bovine albumin. The sera positive of anti-nonGal Ab revealed strong IgG binding to both bovine and porcine albumin, but no binding to human albumin by ELISA. When anti-human, bovine and porcine albumin Abs were measured in pre- and post-pITx sera of 29 NHP recipient, the frequencies of the development of anti-porcine albumin IgG Abs were significantly different according to the types of immunosuppression (IS) regimen: 5/5 (100%) receiving conventional IS regimen, 2/2 (100%) receiving anti-CD154 and sirolimus and 2/21 (9.5%) receiving CD40-CD40L blockade, sirolimus and either anti-ICAM-1 or ATG (P<0.0001 vs. other groups, χ2 test). The anti-bovine albumin IgG was also detected in the sera of 6 (67%) from 9 recipients positive for elicited anti-porcine albumin IgG. These experiments indicate that anti-porcine/bovine albumin Abs are elicited in NHP recipients after porcine ITx, which can be prevented by an adequate IS.
The role of myeloid cells in graft rejection (TRAN3P.893)
Authors: Chain Robert , Chakhtoura Marita , Gallucci Stefania
Published: January 2023

Acceptance of a mismatched graft is achieved through immunosuppression that requires long-term treatment. A variety of methods have been explored to prevent rejection and achieve transplant tolerance in mouse models. The combination of LPS from gram-negative Bacteria, and tissue damage from the transplant procedure has been suggested to prevent rejection and deserves further investigation. We implemented a mouse skin transplant model to determine the effects of LPS and endogenous danger signals released during engraftment, on myeloid cell functions and study their ability to induce rejection or tolerance in transplantation. We used the spontaneous single minor histocompatibility mismatch model of skin graft rejection and performed skin grafts from male mice onto syngeneic female recipients then treated the mice with LPS or PBS. Control mice completely rejected the graft between 24-34 days, while mice treated with LPS did not show graft rejection until an average of 64 days, with 50% graft acceptance. When we studied the graft immune infiltrate, we found that within 48 hours post transplantation, there was an influx of GR-1+ CD11b+ double positive cells. LPS-treated recipients showed a significant decrease in the recruitment of these GR-1+ CD11b+ double positive cells at the graft site. These results indicate that GR-1+ CD11b+ myeloid cells migrate to the site of transplantation following trauma and suggest they play a key role in graft rejection, warranting further study.
Memory CD4 T cell helps alter isotype and specificity of donor-reactive alloantibody (TRAN3P.894)
Authors: Gorbacheva Victoria , Fan Ran , Valujskikh Anna
Published: January 2023

As alloreactive antibody (alloAb) mediate allograft rejection we tested how memory CD4 T cells influence alloAb responses in a murine renal transplantation model. While A/J (H-2a) kidney allografts are spontaneously accepted by naïve B6 (H-2b) recipients, donor-reactive memory CD4 T cells induced rapid rejection through induction of alloAb. We compared serum titers and isotypes of donor-reactive alloAb and the numbers of plasma cells secreting Ab against individual donor MHC antigens in B6 recipients with or without transfer of polyclonal A/J-primed memory CD4 T cells. As anticipated, recipients containing memory CD4 T cells developed faster and stronger alloAb responses than non-sensitized recipients. However, the effects of memory CD4 T cell help were not limited to the accelerated kinetics and higher magnitude of alloAb production. Memory CD4 T cells induced high titers of IgG2c, IgG2b and IgG1 Ab reactive to A/J. In contrast, anti-donor Ab in non-sensitized recipients were exclusively IgG2b. High numbers of plasma cells secreting Ab against donor MHC class I molecule H-2Dd were detected in recipients containing memory CD4 T cells, but not in non-sensitized recipients. Thus, donor-reactive alloAb induced by memory and naive CD4 T cells differ not only in quantity but also in isotype distribution and specificity that in turn may influence their pathogenicity. These findings underscore the importance of targeting helper functions of donor-reactive memory CD4 T cells.
Posttransplant inflammation promotes memory T cell recovery following antibody-mediated lymphoablation (TRAN3P.900)
Authors: Ayasoufi Kathy , Wang Xi , Valujskikh Anna
Published: January 2023

Antibody-mediated depletion is commonly used as an induction therapy in recipients of organ transplants subjected to prolonged cold ischemia. We tested how surgery-induced inflammation and ischemia/reperfusion injury affect the recipient T cell recovery after lymphoablation. We previously reported that preexisting memory T cells rapidly expand and mediate rejection in B6 recipients of BALB/c heart allografts treated with murine antithymocyte globulin (mATG). Memory CD8 T cell recovery was not influenced by specificity for donor alloantigens suggesting a role for posttransplant inflammation. In support of this, CD8 T cells recovered faster in B6 isograft recipients than in non-transplanted mice treated with mATG. The increase in cold ischemic time from 0.5 h to 8 h markedly enhanced the post-depletion expansion of memory CD8 T cells in allograft recipients (10 fold increase at d. 10 posttransplant). Treatment with neutralizing anti-TNFα Ab decreased the numbers of donor-reactive IFNγ producing cells in the spleens of mATG treated heart allograft recipient by 60%, but had little effect on the anti-donor responses in control IgG treated recipients suggesting that TNFα facilitates T cell recovery rather than priming. Our data show that posttransplant inflammation facilitates memory T cell reconstitution following antibody-mediated depletion and that neutralizing proinflammatory cytokines may improve the efficacy of induction therapies in sensitized transplant recipients.
Establishing donor specific tolerance in vascularized composite allotransplantation via local enrichment of endogenous regulatory T cells (TRAN3P.865)
Authors: Fisher James , Jhunjhunwala Siddharth , Unadkat Jignesh , Schweizer Riccardo , Komatsu Chiaki , Thomson Angus , Gorantla Vijay , Little Steven
Published: January 2023

Vascularized composite allotransplantation (VCA)—encompassing transplantation of hands/limbs and face is an emerging field with potential to restore the appearance and function of damaged tissue. Clinically, the process of rejection is suppressed via systemic immunosuppression, which is associated with a host of deleterious side effects. An alternative approach to prevent rejection is to harness the homeostatic mechanisms intrinsic to the immune system. To this end our group has recently developed controlled release microparticle (MP) systems capable of enriching Tregs at given location via synthetic cell constructs referred to as Expansion MP. Accordingly, we hypothesized that Treg enriching systems that release key cytokines, and immunosuppressive agents, can promote long term graft survival in preclinical VCA models. Following fabrication, Expansion MP was tested in an allogeneic rat hind limb transplant model and then in a swine gracilis myocutaneous flap allotransplant model. Following subcutaneous injection of microparticles, rodent hind limbs survived indefinitely (>200 days) and swine allografts survived until the end of the study period (4 weeks) with no signs of rejection. Secondary skin grafts and MLRs in both models demonstrate that ExpansionMP treatment appears to confer donor specific tolerance to recipients. Given these promising results, we believe this technology has the potential to dramatically impact the field of VCA and reconstructive transplantation.
Combination of ATG and rapamycin prolongs the persistence of ex vivo expanded allogeneic Treg transferred in cynomolgus monkeys (TRAN3P.904)
Authors: Zhang Hong , Ezzelarab Mohamed , Lu Lien , Zahorchak Alan , Cooper David , Thomson Angus
Published: January 2023

It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg. In this study, we massively expanded flow-sorted monkey Tregs using artificial APCs. The expanded Tregs maintained expression of Treg signature markers, in vitro suppressive capacity, and demethylation status at the Treg-Specific Demethylation Region. When autologous (auto) and allogeneic (allo) ex vivo expanded Tregs were simultaneously transferred into monkeys, the number of auto-Treg was up to 100 per ml blood for at least 6 days in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml blood for at least 15 days in IS monkeys with the expression of Foxp3 and CD25. The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease.
Defining the mechanism involved in the inhibition of GvHD by azacitidine in vivo through the use of FoxP3 diphtheria toxin receptor (Foxp3DTR) donor T cells. (TRAN3P.911)
Authors: Cooper Matthew , Choi Jaebok , Ritchey Julie , DiPersio John
Published: January 2023

Azacitidine (AzaC) can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how AzaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+), or 2) the direct anti-proliferative and pro-apoptotic effects of AzaC on allogeneic T cells. To determine the importance of AzaC generated Tregs, we designed an MHC-mismatched model of allogeneic bone marrow transplantation in which Tregs could be specifically depleted by Diphtheria toxin (DT) following treatment of mice with AzaC. Mice depleted of nTreg prior to infusion, and treated with AzaC alone had significantly less GvHD and increased overall survival than mice treated with AzaC and depleted of AzaC generated Tregs with DT, indicating an important role of AzaC generated Tregs in GvHD amelioration. Furthermore, mice treated with both AzaC and DT had less GvHD and survived longer than PBS treated control mice suggesting direct cytotoxicity of effector T cells induced by AzaC leads to enhanced survival and reduced GvHD. Our data suggests that increasing FOXP3+ Tregs and AzaC induced cytotoxicity towards allogeneic T cells are both important mechanisms involved in the mitigation of GvHD in vivo.
Pharmacological inhibition of MyD88 prevents dendritic cell maturation and synergizes with CTLA4-Ig to promote transplant survival (TRAN3P.918)
Authors: Raimondi Giorgio , Khalifian Saami , Oh Byoung , Lee W.P. Andrew , Brandacher Gerald , Olson Mark , Saikh Kamal
Published: January 2023

TLRs-induced maturation of dendritic cells (DC) and accumulation of inflammatory cytokines are important contributors to alloreactive T cell activation. We aimed to characterize two new MyD88-specific small molecule inhibitors, EM-163 (EM) and T923 (T9), and determine their synergism with CTLA4-Ig in a mouse skin transplant model. Both EM and T9 reduced upregulation of costimulatory molecules on DC in response to LPS. They also reduced the accumulation of IL-6 in supernatants, though the inhibition was stronger with T9 at equimolar level. Interestingly, both inhibitors promoted high MHC II expression - a favorable scenario for delivery of “Signal 1” with low “Signal 2-3” that could promote induction of T cell tolerance. In vivo analysis in a fully mismatched skin transplantation mouse model (BALB/c to B6) showed that transient administration of T9 (d0-10) combined with CTLA4-Ig doubled skin allograft survival (MST untreated: 9.5d, CTLA4-Ig only: 13d, CTLA4+T9: 19d). Overall, our results demonstrate that pharmacological inhibition of MyD88 is possible and potentiates the protective effect of costimulation blockade. Further elucidation of the mechanisms underlying this synergism and optimization of the protocol in multiple transplant models are underway. These encouraging results also suggest the use of pharmacological inhibition of MyD88 as replacement of CD154 blockade in protocols of costimualtion blockade to promote long term transplant acceptance.
Pediatric liver transplant recipients with operational tolerance exhibit features of immune activation and exhaustion (TRAN3P.869)
Authors: Ramaswami Balathiripurasundari , Yoshida Osamu , Ippolito Renee , Metes Diana , Zeevi Adriana , Thomson Angus , Mazariegos George , Chalasani Geetha
Published: January 2023

Experimental models describe immune exhaustion in liver transplant tolerance. It is not known if immune exhaustion is also occurs in clinical operational tolerance. We tested this in a cross-sectional cohort of pediatric liver transplant recipients with stable allograft function in the absence of immunosuppression (Operationally Tolerant, OT) in comparison to those Weaned off Immunosuppression (WI), continued on Maintenance Immunosuppression (MI) and Healthy Controls (HC). Results: In OT recipients, B cell frequencies (p <0.05) were increased and were enriched in both unswitched (p<0.005) and switched memory B cells (CD19+ IgM+ CD27+ and CD19+ IgG+ CD27+) (p<0.05) compard to MI. CD8 T cells in OT showed an increase in effector memory compared to MI (p<0.05). CD4 Treg frequencies were increased in OT compared to MI (p<0.0005). Both CD4 and CD8 T cells in OT showed an increase in cells expressing CD57, indicative of replicative senescence and these were enriched within CD4 central memory and CD8 effector memory when compared to MI (p<0.05). CD57+ T cells upregulated PD1 and 2B4, markers of exhaustion in OT compared to MI (p<0.05). Upon stimulation, IFNγ expression in CD4 T cells and IL-2 expression in CD8 T cells in OT were decreased compared to MI consistent with exhaustion. Moreover, OT showed decreased CD8 frequencies (p<0.05) and high PDL1 expression on monocytes (p<0.0005) suggesting that this population might contribute to T cell exhaustion in these recipients.
IRF-8 regulates MMP3-dependent mechanisms of tumor progression to metastasis (TUM7P.929)
Authors: Banik Debarati
Published: January 2023

Interferon regulatory factor (IRF-8), originally identified as a tumor suppressor gene in leukemia, was also shown to regulate malignant behavior in solid tumor models. Our previous data revealed that IRF-8 influenced tumor growth, in part, by regulating Fas-mediated signaling. Gene expression profiling further revealed an inverse relationship between IRF-8 and MMP3 expression. Although MMP3 has been implicated in early tumorigenesis, the role of MMP3 beyond the stage of tumor initiation has remained largely unknown. Here, we hypothesized that MMP3 governs later stages of the neoplastic process, namely progression to metastasis, and that MMP3 does so through a novel IRF8-MMP3 regulatory axis. To test this hypothesis, we employed loss- and gain-of-function approaches in multiple tumor models. Overall, we showed that: 1) silencing MMP3 expression in tumor cells reduced their primary growth rate and propensity for lung metastases even in a post-surgical setting; 2) overexpressing MMP3 expression in tumor cells increased their primary tumor growth rates; 3) MMP3 expression impacted malignant behavior at multiple levels, e.g., invasive ability and the generation of active MMP9; 4) IRF-8 downregulated MMP3 expression by binding to an ISRE site within the MMP3 promoter; and 5) modulating IRF8 levels inversely regulated MMP3 expression and, consequently, malignant behavior. Thus, our data define a previously unrecognized role of an IRF8-MMP3 axis in regulating tumor progression.
Immune enhancing effects of fucoidan-agaricus mix under treatment of an immunosuppressive anti-cancer agent (TUM7P.939)
Authors: Miyazaki Yoshiyuki , Nakamizo Masaji , Shibasaki Tetsuya , Kirino Tomomi , Kawahara Kazuhiro , Otsuka Ken , Tachibana Hirofumi , Yamada Koji , Tachikawa Daisuke
Published: January 2023

Fucoidan is a series of sulfated polysaccharides derived from brown algae that mainly consist of L-fucose. Fucoidan have various biological activities, and our investigation with experimental animal models and a healthy human trial demonstrated fucoidan from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with agaricus mycelium extract. In this context, we have reported that feeding of the fucoidan-agaricus mix (FAM) protected mice from immunosuppressive effect of 5-fluorouracil (5-FU). In current study, we confirmed dose-dependent immunoprotective effects of the dietary FAM (at 20 and 40 mg/day) by observing recovery in spleen weight, NK cell activity and IFN-γ production in 5-FU-treated mice. Furthermore, we investigated in vitro effects of FAM against 5-FU on growth and function of primary cultured immune-related cells. As the results with thioglycollate-induced peritoneal exudates cells (PEC), 5-FU dose-dependently suppressed the growth whereas FAM exerted no effects. However, production of cytokines such as IL-12p40 and TNF-α in PEC were stimulated by FAM treatment even in the presence of 5-FU. In the case of bone marrow derived cells differentiated with GM-CSF and M-CSF, FAM markedly enhanced the growth and IL-12 production of these cells. From these results, it was suggested that FAM contributed to amelioration of decline in anti-tumor immune function by 5-FU treatment through activation of innate immune cells.
Activation of innate and adaptive immunity by a TLR5 agonist to prevent and suppress liver metastasis (TUM7P.947)
Authors: Brackett Craig , Kojouharov Bojidar , Veith Jean , Gollnick Sandra , Abrams Scott , Burdelya Lyudmila , Gudkov Andrei
Published: January 2023

Colorectal cancer (CRC) liver metastases are a major cause of mortality of CRC patients providing incentive to develop systemic therapies to prevent and treat this condition. We are exploring the use of a TLR5 agonist Entolimod™ (CBLB502) for immunotherapy against CRC liver metastases. Entolimod is a pharmacologically optimized Salmonella flagellin derivative that is at advanced stage of clinical development as a medical radiation countermeasure. We found that liver is the major primary Entolimod target organ. Entolimod rapidly triggers a TLR5-elicited response in hepatocytes that activates NF-kB and quickly mobilizes immunity to the liver. This includes short-term neutrophilic infiltrate and long-term NK cell accumulation that coordinate CD8 and CD4 T cell immunity against CRC liver metastases. As a result, Entolimod administration dramatically improved long-term survival in a mouse model that mimicked clinically occurring development of post-surgery liver metastases in CRC patients. Importantly, Entolimod induces durable, tumor-specific T cell memory. Unlike other TLR agonists, systemic administration of Entolimod does not induce septic shock-like syndrome due to lack of production of cytokine storm-inducing cytokines upon TLR5 activation. These safe and long-lasting TLR5-elicited changes within liver provide important groundwork for further clinical development of Entolimod, which is currently in clinical trials as a potential immunotherapy against CRC liver metastases.
Natural killer: dendritic cell activation using anti-CD137 mAb improves cetuximab mediated innate and adaptive immune responses in head and neck cancer (TUM7P.948)
Authors: Srivastava Raghvendra , Jie Hyun-bae , Li Jing , Ferris Robert
Published: January 2023

EGFR-specific mAb cetuximab mediated activation of NK by FcγRIIIa cross-linking is important for anti-tumor immunity.Following cetuximab activation,NK express costimulatory receptor CD137, which can be ligated to improve activity of cetuximab-activated NK,which promote DC maturation and CD8+ T-cell priming, leading to tumor antigen spreading and Th1 cytokine release through NK-DC cross-talk. We show that the clinically available CD137 agonist mAb augments cetuximab-mediated ADCC in FcγRIIIa-V/F donors,but not in poor binding FcγRIIIa-F/F.We show that cetuximab-activated NK induce CD137 expression on DC, which can be further stimulated by CD137 mAb, leading to enhanced DC maturation and cross-presentation capacity.In a prospective phase II trial of neoadjuvant cetuximab in stage III/IV HNC patients, intratumoral activated NK cells also upregulated CD137, which was influenced by FcγRIIIa-V/F polymorphism. Moreover, cetuximab treatment induces CD137 expression on intratumoral activated CTL.We show that FcγRIIIaV/F polymorphism on NK cells greatly influences their induction of CD137 expression, as does the EGFR receptor density on HNC.Thus, careful selection of HNC patients in combination immunotherapy using cetuximab plus CD137 mAb may be necessary, using EGFR level of expression and FcgammaRIIIa genotype, to optimize NK:DC priming of tumor antigen specific CTL.Results suggest a beneficial effect of combination of cetuximab and CD137 mAb in currently designed clinical trials.
Organotypic brain slice culture reveals fractalkine signaling alters the kinetics of microglia and medulloblastoma migration in the CNS tumor microenvironment (TUM7P.953)
Authors: Dorand Dixon , Barkauskas Deborah , Nthale Joseph , Myers Jay , Petrositue Agne , Huang Alex
Published: January 2023

Medulloblastoma (MB) is the most common pediatric Central Nervous System (CNS) malignancy and is capable of metastasis to distant sites. Little is known about the interactions between MB and the highly selective CNS immune environment; therefore, we investigated the role of microglia, the endogenous immune cells of the CNS, in MB microenvironment. Microglia reside in the brain parenchyma and help maintain the homeostatic immunosuppressive environment partially due to their expression of CX3CR1 for which Fractalkine (FKN) is the only known ligand. FKN is constitutively expressed by neurons and astrocytes leading to tonic inhibition of microglial activation. Our Enzyme Linked Immunosorbent Assay (ELISA) results demonstrate that MB can produce as much as 2.5 ng/mL/1x106 tumor cells/24hrs of FKN. Organotypic brain slice culture coupled with 2 photon laser scanning microscopy reveals that genetic deletion of CX3CR1 results in an increase basal microglia speed from 0.071 μm/min in CX3CR1+/- mice to 0.123 μm/min in CX3CR1-/- mice. In the presence of MB, microglia speed increases to 0.154 μm/min in CX3CR1+/- mice to 0.203 μm/min in CX3CR1-/- mice. This data supports the role of FKN signaling as an inhibitor of microglial activation. Interestingly, MB tumor cells move faster in brain slices of CX3CR1-/- mice (0.301 μm/min) compared to CX3CR1+/- mice (0.144 μm/min), further suggesting that FKN signaling may play a role in MB invasion as well as microglia movement.
New complement roles revealed by immune gene profiling in endometriosis and endometriosis-associated ovarian cancer (TUM7P.957)
Authors: Vlad Anda , Suryawanshi Swati , Huang Xin , Budiu Raluca , Elishaev Esther , Kim SungHwan , Tseng George , Edwards Robert
Published: January 2023

Endometriosis is a largely benign, chronic inflammatory disease that may predispose to some of the ovarian tumors histotypes, collectively called endometriosis-associated ovarian cancers (EAOC). Although it is widely accepted that chronic inflammation drives cancer, the immune deregulation in chronic precursors of ovarian cancer has not been systematically studied. We performed here the first comprehensive gene profile of the tissue immune microenvironment in normal endometrium, benign endometriosis and EAOC (n=133), using Nanostring and the nCounter® GX Human Immunology Kit comprising probes for 511 immune genes. We found that the complement pathway was most prominently upregulated, suggesting its involvement in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Complement protein expression was confirmed via immunohistochemistry, which identified upregulation of complement in epithelial cells. To explore the roles of complement in early EAOC carcinogenesis, we derived several novel ovarian cell lines with conditional mutations that can be turned on and off, using the Cre-loxP system. Using this in vitro model we demonstrate that genetic instability triggers upregulation of complement genes in epithelial cells, but without an advantage on cell death. These findings reveal an early link between epithelium and innate immune environment and further support a paradigm shift on complement roles in supporting cancer growth.
Anchoring of TLR agonists in the plasma membrane of B cells for enhanced cellular vaccines. (VAC10P.962)
Authors: Szeto Gregory , Van Egeren Debra , Alejandro Brian , Liu Haipeng , Irvine Darrell
Published: January 2023

B cells are a unique lymphocyte subset that can integrate adaptive and innate receptor signaling. B cells can serve as cellular vaccines if appropriately activated (e.g., by TLR ligands) and loaded with antigen. However, activation can also lead to changes in homing receptor expression that abrogate their ability to traffic efficiently to lymphoid organs. To overcome this issue, we present a strategy to provide sustained TLR stimulation to B cells using brief (15 min pulse) exposure to lipid-tailed CpG oligonucleotides (lipoCpGs), which results in stable insertion of the TLR ligand in the plasma membranes of primary murine B cells. LipoCpG internalization was studied by flow cytometry and confocal microscopy, revealing that nearly 100% of lipoCpG was surface-localized for at least 2 h. LipoCpG-pulsed B cells exhibited sustained activation for 3 days in vitro as determined by expression of MHC I, MHC II, CD86 and CD69; expression was equivalent to levels on B cells continuously incubated with soluble CpG. Pulsed B cells did not significantly activate unpulsed B cells in a mixed coculture, indicating that inserted lipoCpG acts in an autocrine fashion. Finally, LipoCpG-pulsed B cells incubated with ovalbumin drove proliferation of OT-I CD8+ T cells in vitro. These data suggest that inserted lipoCpG provides strong activation signaling, potentially enabling rapid cell priming and prolonged in situ activation, facilitating development of B cell-based immunotherapies.
Targeting antigen-loss variant tumors through CD134 plus CD137 dual costimulation-mediated bystander programming (VAC10P.964)
Authors: Mittal Payal , Wang Xi , St. Rose Marie-Clare , Vella Anthony , Adler Adam
Published: January 2023

Tumor-specific cytotoxic CD8 T cell-based therapies can be efficacious in cancer patients. Nevertheless, disease progression often resumes due to outgrowth of antigen-loss variants (ALVs) that lack expression of the targeted MHC class I-restricted epitopes. Co-administration of agonists to the TNF/TNFR costimulatory family members CD134 (OX40) and CD137 (4-1BB) can program antigen-stimulated CD4 T cells to differentiate into cytotoxic Th1 cells, which can directly target MHC II+ tumors and provide classical helper function that facilitates effector differentiation in CD8 T cells responding to a linked epitope. Interestingly, these specific cytotoxic CD4 Th1 cells also imprint antigen-non-responding T cells with a similar ability to express IFN-γ and cytotoxic molecules. Because these bystander-programmed effector T cells are polyclonal, we currently hypothesized that they contain specificities capable of targeting ALVs. In both lymphoma and melanoma models, dual costimulation therapy was markedly enhanced when specific CD4 T cells were engaged to differentiate into cytotoxic Th1 effectors. This augmentation occurred not only when the tumor expressed the MHC class II-restricted epitope recognized by the specific cytotoxic CD4 Th1 cells, but importantly also with tumors modeling ALVs that lack the targeted epitope. Taken together, dual-costimulated tumor-non-specific CD4 T cells provide an alternate form of help that facilitates targeting of ALVs.
Development of an in vitro system for screening adjuvant combinations for induction of specific patterns of CD4 T cell differentiation (VAC10P.967)
Authors: Goldberg Michael , Kadolsky Ulrich , Yates Andrew , Porcelli Steven
Published: January 2023

Adjuvants are a critical component of many vaccines, but their mechanisms of action remain poorly understood. While great progress has been made in defining several pathways that are activated by various adjuvants, their impact on adaptive immunity remains largely unpredictable. Here, we developed an experimental system to screen multiple defined compounds with known adjuvant activity on the strength and quality of antigen-specific murine CD4 T cell priming in vitro. By screening combinations of suboptimal concentrations of 30 adjuvants, we identified combinations that showed remarkable synergy in priming naive CD4 T cells toward TH1, TH2, TH17 or TfH phenotypes. By applying a variety of computational algorithms to data from this in vitro approach, we identified synergy between agonists of different pattern recognition receptor families for induction of specific functional programs in CD4 T cells during priming. These were ranked by strength of polarization and with respect to robustness under different culture conditions. Immunizations in mice showed strong correlations between in vitro ranking and in vivo outcomes of antigen specific CD4 T cell responses. Our approach provides a method for analyzing large numbers of adjuvant combinations to predict their influence on the outcome of CD4 T cell priming in vivo. This should assist in the development of new adjuvants that enable more predictable and functionally polarized CD4 T cell responses following vaccination.
Neutralization of murine myeloid suppressor cells enhances the efficacy of GD2-specific CAR T cells directed against human sarcoma in a xenograft model (VAC11P.1010)
Authors: Highfill Steven , Smith Jillian , Long Adrienne , Cui Yongzhi , Orentas Rimas , Mackall Crystal
Published: January 2023

Genetically engineered T-cells expressing chimeric antigen receptors directly target cell surface antigens on tumor cells. CARs have shown remarkable activity in clinical trials for hematologic malignancies but remain unproven for the treatment of solid tumors. The disialoganglioside GD2 is considered a tumor-associated antigen for neuroblastoma and we have found that GD2 is expressed on pediatric sarcomas. Analysis of primary human tumor tissue samples taken from 18/18 patients with osteosarcoma and alveolar rhabdomyosarcoma (2/15) demonstrate robust expression of GD2 on the cell surface. Based on this finding, we developed assays to test the ability of GD2-CAR T-cells to target human sarcomas. We find that GD2-CAR T-cells specifically lyse GD2-expressing solid tumors in vitro even at low E:T, but fail to induce a response in vivo. In studying these xenografted tumors we discovered an expansion of murine CD11b+Gr1+ MDSC that inhibit human T-cell responses in vitro. This lead us to adopt a combinatorial therapeutic strategy in which we inhibited the suppressive potential of MDSC by administration of all-trans retinoic acid followed by the infusion of GD2-specific CAR T-cells. This resulted in significant improvements in overall survival and tumor growth. The results presented here suggest that ATRA coadministration with CAR T-cell therapy for solid tumors may yield a beneficial additive effect through the modulation of myeloid derived suppressor cells.
Defining the role of the novel CD4+CD26high T cell subset in adoptive cancer immunotherapy (VAC11P.1001)
Authors: Bailey Stefanie , Nelson Michelle , Schwartz Kristina , Bowers Jacob , Paulos Chrystal
Published: January 2023

Th17 cells mediate exquisite tumor eradication upon transfer into mice bearing large, established melanoma. Unfortunately, the polarizing cytokines (TGF-β, IL-6, etc.) required to generate Th17 cells are not yet FDA approved for clinical use. Therefore, we sought to determine if these IL-17-producing CD4+ cells could be enriched from peripheral blood. Herein, we found that functional Th17 cells could be isolated from blood, without the need for polarization, by using the surface marker CD26. Excitingly, very few (5e4 cells) of these CD26-rich cells, termed CD26high T cells, displayed striking antitumor activity and survival upon transfer into melanoma-bearing mice compared to CD26negative or bulk CD4+ T cells. Of translational significance, we found that human CD26high cells had heightened cytotoxicity and caused greater cancer cell death in vitro. Interestingly, both murine and human CD26high T cells boast a unique phenotype compared to their bulk CD4+ or CD26negative counterparts, including: enhanced expression of the cytokines IL-17, IFN-γ, and IL-22, transcription factors RORγ and Tbet, and cytokine receptors CD25, CD122, and CD127. Collectively, these findings describe a novel T cell subset that has promise in cell-based cancer therapies due to its unique polyfunctionality and engraftment. Moreover, enrichment of CD26high T cells from peripheral blood bypasses the need for polarization, thereby enhancing the translational potential for Th17 cells in immunotherapy.
Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for mAb and CAR-modified T cell immunotherapies (VAC11P.1004)
Authors: Lohmueller Jason , Finn Olivera
Published: January 2023

MUC1 is a tumor-associated protein that is abnormally expressed on all human adenocarcinomas. In our recent clinical trial, individuals at-risk for colonic adenoma received a prophylactic MUC1 cancer vaccine. Many individuals responded by producing high-affinity anti-MUC1 antibodies. We isolated a panel of these antibodies and seek to develop them as antibody and CAR T cell cancer immunotherapies. Our collaborators used a novel method combining proteomics and next-gen DNA sequencing to identify and recombinantly produce 12 high-affinity antibodies from the sera of vaccinated individuals. By ELISA we determined that the antibodies bind with high-affinity to diverse epitopes within the MUC1 vaccine peptide. We also found that several of the antibodies can stain MUC1+ pancreas, colon, and breast cancer cell lines but not a cell line expressing normal MUC1. We then determined that the antibodies could stain MUC1+ human pancreas and colon tumor samples but not normal tissues. Having established tumor-specificity we are now assessing the antibodies in a series of functional assays. Finally, we have constructed CAR lentiviral constructs using the variable regions of the antibodies as targeting domains and are testing their ability to mediate MUC1-specific target cell lysis. As the antibodies are human-derived, indication of efficacy in pre-clinical testing has the potential to directly enable clinical trials and hopefully approval of these reagents for therapy of cancer patients.
Reduced ectonucleotidase expression on TGF-β independent Th17 improves tumor control (VAC11P.1006)
Authors: Chatterjee Shilpak , Kesarwani Pravin , Thyagarajan Krishnamurthy , Soloshchenko Myroslawa , Webster Christine , Paulos Chrystal , Mehrotra Shikhar
Published: January 2023

Amongst various effector CD4+ T cells, T helper (Th)-17 subsets hold promise in adoptive T cell transfer therapy for cancer. However, ex vivo programming of Th17 cells in presence of TGF-β increases cell surface expression of ectonucleotidases CD39 and CD73, that in turn increases susceptibility to immunosuppression and reduced effector phenotype. Our data shows that ATP mediated suppression of IFN-γ production by Th17 cells can be overcome either by genetic ablation of CD73 or generating TGF-β independent Th17 in presence of IL-1β. An in vivo comparison of anti-tumor potential between TGF-β dependent and independent Th17 showed that Th17 cells generated in presence of IL-1β are more efficacious than TGF-β dependent Th17 cells in controlling B16 melanoma. IL-1β dependent Th17 cells are also highly polyfunctional and express high level of pathogenic genes, such as T-bet and granzyme B that results in increased cytotoxic potential. It is likely that effector property of IL-1β dependent Th17 is due to their high glycolytic capacity, since generating IL-1β dependent Th17 cells in pyruvate containing media impaired glycolysis and its anti-tumor potential. Thus, our data suggests that owing to TGF-β induced ectonucleotidase expression, ex vivo culture conditions for generating Th17 cells need to be reconsidered in favour of TGF-β independence for exploiting their full potential in adoptive T cell therapy.